U.S. patent application number 10/021758 was filed with the patent office on 2002-07-25 for human beta-1,3-galactosyltransferase.
This patent application is currently assigned to ZymoGenetics, Inc.. Invention is credited to Conklin, Darrell C., Gao, Zeren, Jaspers, Stephen R., Yamamoto, Gayle.
Application Number | 20020098564 10/021758 |
Document ID | / |
Family ID | 26813498 |
Filed Date | 2002-07-25 |
United States Patent
Application |
20020098564 |
Kind Code |
A1 |
Conklin, Darrell C. ; et
al. |
July 25, 2002 |
Human beta-1,3-galactosyltransferase
Abstract
The present invention relates to polynucleotide and polypeptide
molecules for znssp6, a novel member of the galactosyltransferase
family. The polypeptides, and polynucleotides encoding them, are
cell-cell interaction and glycoprotein synthesis modulating and may
be used for delivery and therapeutics. The present invention also
includes antibodies to the znssp6 polypeptides.
Inventors: |
Conklin, Darrell C.;
(Seattle, WA) ; Yamamoto, Gayle; (Seattle, WA)
; Gao, Zeren; (Redmond, WA) ; Jaspers, Stephen
R.; (Edmonds, WA) |
Correspondence
Address: |
Robyn Adams
Patent Department, ZymoGenetics, Inc.
1201 Eastlake Avenue East
Seattle
WA
98102
US
|
Assignee: |
ZymoGenetics, Inc.
|
Family ID: |
26813498 |
Appl. No.: |
10/021758 |
Filed: |
October 22, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10021758 |
Oct 22, 2001 |
|
|
|
09482180 |
Jan 12, 2000 |
|
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60115721 |
Jan 12, 1999 |
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Current U.S.
Class: |
435/193 ;
435/320.1; 435/325; 435/69.1; 536/23.2 |
Current CPC
Class: |
C12N 9/1051
20130101 |
Class at
Publication: |
435/193 ;
435/69.1; 435/325; 435/320.1; 536/23.2 |
International
Class: |
C12N 009/10; C07H
021/04; C12P 021/02; C12N 005/06 |
Claims
What is claimed is:
1. An isolated polypeptide comprising residues 114 to 370 of SEQ ID
NO: 2.
2. The isolated polypeptide according to claim 1 wherein the
polypeptide comprises residues 114 to 378 of SEQ ID NO: 2.
3. The isolated polypeptide according to claim 2 wherein the
polypeptide comprises residues 50 to 378 of SEQ ID NO: 2.
4. The isolated polypeptide according to claim 3 wherein the
polypeptide comprises residues 26 to 378 of SEQ ID NO: 2.
5. The isolated polypeptide according to claim 4 wherein the
polypeptide comprises residues 1 to 378 of SEQ ID NO: 2.
6. An isolated polypeptide selected from the group consisting of:
a) a polypeptide comprising residues 1 to 25 of SEQ ID NO: 2; b) a
polypeptide comprising residues 26 to 49 of SEQ ID NO: 2; c) a
polypeptide comprising residues 50 to 113 of SEQ ID NO: 2; d) a
polypeptide comprising residues 114 to 370 of SEQ ID NO: 2; e) a
polypeptide comprising residues 371 to 378 of SEQ ID NO: 2; and f)
a polypeptide comprising residues 1 to 378 of SEQ ID NO: 2.
7. An isolated polynucleotide encoding a polypeptide wherein the
polypeptide comprises residues 114 to 370 of SEQ ID NO: 2.
8. The isolated polynucleotide according to claim 7, wherein the
polypeptide molecule comprises residues 114 to 378 of SEQ ID NO:
2.
9. The isolated polynucleotide according to claim 8, wherein the
polypeptide molecule comprises residues 50 to 378 of SEQ ID NO:
2.
10. The isolated polynucleotide according to claim 9, wherein the
polypeptide molecule comprises residues 26 to 378 of SEQ ID NO:
2.
11. The isolated polynucleotide according to claim 8, wherein the
polypeptide molecule comprises residues 1 to 378 of SEQ ID NO:
2.
12. An isolated polynucleotide encoding a polypeptide molecule
wherein the polypeptide is selected from the group consisting of:
a) a polypeptide comprising residues 1 to 25 of SEQ ID NO: 2; b) a
polypeptide comprising residues 26 to 49 of SEQ ID NO: 2; c) a
polypeptide comprising residues 50 to 113 of SEQ ID NO: 2; d) a
polypeptide comprising residues 114 to 370 of SEQ ID NO: 2; e) a
polypeptide comprising residues 371 to 378 of SEQ ID NO: 2; and f)
a polypeptide comprising residues 1 to 378 of SEQ ID NO: 2.
13. An expression vector comprising the following operably linked
elements: a transcription promoter; a DNA segment wherein the DNA
segment is a polynucleotide encoding the polypeptide of claim 1;
and a transcription terminator.
14. The expression vector according to claim 13 wherein the DNA
segment contains an affinity tag.
15. A cultured cell into which has been introduced an expression
vector according to claim 13, wherein said cell expresses the
polypeptide encoded by the DNA segment.
16. A method of producing a polypeptide comprising culturing a cell
according to claim 15, whereby said cell expresses the polypeptide
encoded by the DNA segment; and recovering the polypeptide.
17. The polypeptide produced by the method of claim 16.
18. A method of producing an antibody comprising the following
steps in order: inoculating an animal with an epitope bearing
portion of a polypeptide wherein the epitope bearing portion is
selected from the group consisting of: a) a polypeptide comprising
residues 10 to 16 of SEQ ID NO: 2; b) a polypeptide comprising
residues 52 to 61 of SEQ ID NO: 2; c) a polypeptide comprising
residues 52 to 78 of SEQ ID NO: 2; d) a polypeptide comprising
residues 69 to 78 of SEQ ID NO: 2; e) a polypeptide comprising
residues 89 to 94 of SEQ ID NO: 2; f) a polypeptide comprising
residues 89 to 117 of SEQ ID NO: 2; g) a polypeptide comprising
residues 111 to 117 of SEQ ID NO: 2; h) a polypeptide comprising
residues 126 to 134 of SEQ ID NO: 2; i) a polypeptide comprising
residues 126 to 151 of SEQ ID NO: 2; j) a polypeptide comprising
residues 143 to 151 of SEQ ID NO: 2; k) a polypeptide comprising
residues 215 to 220 of SEQ ID NO: 2; l) a polypeptide comprising
residues 215 to 239 of SEQ ID NO: 2; m) a polypeptide comprising
residues 223 to 239 of SEQ ID NO: 2; n) a polypeptide comprising
residues 223 to 257 of SEQ ID NO: 2; o) a polypeptide comprising
residues 251 to 257 of SEQ ID NO: 2; and p) a polypeptide
comprising residues 332 to 337 of SEQ ID NO: 2 wherein the
polypeptide elicits an immune response in the animal to produce the
antibody; and isolating the antibody from the animal.
19. An antibody produced by the method of claim 18, which binds to
a polypeptide comprising residues 114 to 370 of SEQ ID NO: 2.
20. The antibody of claim 19, wherein the antibody is a monoclonal
antibody.
21. The antibody of claim 19 joined to a moiety selected from the
group consisting of: a) an affinity tag; b) a detectable molecule;
c) a cytotoxic molecule; and d) a cytokine.
22. A method of killing cells expressing a polypeptide comprising
residues 114 to 370 of SEQ ID NO: 2, comprising contacting the
cells with the antibody of claim 21, wherein the antibody is joined
to the cytotoxic molecule.
23. A method of modulating cell-cell interactions comprising
contacting the cells with polypeptide of claim 1.
24. A method for modulating cell-cell interactions according to
claim 23, wherein the cells are derived from tissues selected from
the group consisting of: a) tissues from brain; b) tissues from
kidney; and c) tissues from testis.
25. A method for modulating glycoprotein and glycolipid
biosynthesis in cells, cell matrix, or cell culture comprising
contacting the cells, cell culutre, or cell matrix with the
polypeptide of claim 1.
26. A method for modulating glycoprotein and glycolipid
biosynthesis according to claim 25, wherein the cells, cell culture
or cell matrix are derived from tissues selected from the group
consisting of: a) tissues from brain; b) tissues from kidney; and
c) tissues from testis.
27. A method of detecting a znssp6 anti-complementary molecule
comprising contacting a test sample containing the znssp6
anti-complementary molecule with the polypeptide of claim 1.
Description
[0001] REFERENCE TO RELATED APPLICATIONS
[0002] This application is related to Provisional Application
60/115,721 filed on Jan. 12, 1999. Under 35 U.S.C. .sctn.
119(e)(1), this application claims benefit of said Provisional
Application.
BACKGROUND OF THE INVENTION
[0003] Glycosyltransferase molecules transfer carbohydrate
molecules to glycoproteins during biosynthesis. Members of this
family have also been detected on the cell surface where they are
thought to be involved in varying aspects of cell-cell
interactions. This family includes carbohydrate transferring
enzymes, such as sialyltransferases and fucosyltransferases, and
galactosyltransferases. During the formation of O-linked
glycoproteins and the modification of N-linked ones, each sugar
transfer is catalyzed by a different type of glycosyltransferase.
Each glycosyltransferase enzyme is specific for both the donor
sugar nucleotide and the acceptor molecule.
[0004] Galactosyltransferases promote the transfer of an activated
galactose residue in UDP-galactose to the monosaccharide
N-acetylglucosamine. This transfer is a step in the biosynthesis of
the carbohydrate portion of galactose-containing glycoproteins,
such as oligosaccharides and glycolipids, in animal tissues. One
subgroup of the galactosyltransferases is the
beta-1,3-galactosyltransferases, which are characterized by the
elongation of type I oligosaccharide chains. Additionally, the
beta-1,3-galactosyltransferases are found on glycoproteins and
glycolipids, are important precursors of blood group antigens, and
are present in soluble oligosaccharides of human milk. Similar to
other members of galactosytransferases. The
beta-1,3-galactosyltransferases require a divalent cation
(Mn.sup.2+) to function. The beta-1,3-galactosyltransferases seem
to have restricted tissue distributions.
[0005] Some galactosyltransferases are found in the Golgi
apparatus. These Golgi-localized enzymes have structure similarity:
a short N-terminal domain that faces the cytosol, a single
transmembrane a helix, and a large C-terminal domain that faces the
Golgi lumen and that contains the catalytic site. The transmembrane
a. helix is necessary and sufficient to restrict the enzyme to the
Golgi. Of the beta-1,3-galactosyltransferase family two members
(See Amado, M. et al., J. Biol. Chem. 273, 21: 12770-12778, 1998)
have been predicted to have two potentially different initiation
codons, resulting in two different N-terminal cytoplasmic
domains.
[0006] Additionally, galactosyltransferases have been shown to be
expressed on the cell surface, where their function is theorized to
participate in cellular interactions, perhaps as receptors, or
receptor-like complementary molecules as well as secreted ligands.
As a cell surface carbohydrate, galactosyltransferases have been
implicated in varied biology such as cell migration, contact
inhibition, tissue interactions, neuronal specificity,
fertilization, embryonic cell adhesions, limb bud morphogenesis,
mesenchyme development, immune recognition, growth control, and
tumor metastasis.
[0007] See, for example, Shur, B. D., Mol Cell Bioc. 61:143-158,
1984.
[0008] The failure of tumor cell-tumor cell adhesion is believed to
be a contributing factor in tumor metastases. See, for example,
Zetter, Cancer Biology 4:
[0009] 219-29, 1993. Metastases, in turn, are generally associated
with poor prognosis for cancer treatment. The metastatic process
involves a variety of cellular events, including angiogenesis,
tumor cell invasion of the vascular or lymphatic circulation, tumor
cell arrest at a secondary site; tumor cell passage across the
vessel wall into the parenchymal tissue, and tumor cell
proliferation at the secondary site. Thus, both positive and
negative regulation of adhesion are necessary for metastasis. That
is, tumor cells must break away from the primary tumor mass, travel
in circulation and adhere to cellular and/or extracellular matrix
elements at a secondary site. Molecules capable of modulating
cell-cell and cell-matrix adhesion are therefore sought for the
study, diagnosis, prevention and/or treatment of metastases.
[0010] Beta-1,3-galactosyltransferases have limited homology to
each other. In contrast to other glycosyltransferases, they do not
appear to be localized to the same chromosomes. Additionally, a
member of this family has recently been identified in Drosophila.
This molecule, Brainiac (brn), also known as a Neurogenic Secreted
Signaling Peptide (NSSP), is involved in contact and adhesion
between germ-line and follicle cells (Amado, M. et al., J. Biol.
Chem. 273, 21: 12770-12778, 1998). Germline Brainiac activity has
been shown to be essential for development of follicular epithelium
(Goode, S. et al., Dev. Biol. 178:35-50, 1996). Additionally, brn
is required continuously throughout oogenesis, beginning in the
germarium at the time that follicle cells envelop the oocyte-nurse
cell complex and continuing stages when the eggshell is produced.
The expression of brn in the germline continuously throughout
oogenesis is consistent with brn's role in developing the
follicular epithelium around each germline cyst, as well as for
dorsal-ventral patterning of the follicular epithelium during later
phases of oogenesis. See Goode, S. et al., Development. 116:
177-192, 1992.
[0011] A deficiency of beta-1,3-galactosyltransferase enzymes has
been noticed in the Tn-syndrome. This syndrome is a rarely acquired
disorder affecting all hemopoietic lineages, and is characterized
by the expression of the Tn and the sialosyl-Tn antigens on the
cell surface. The Tn is .alpha.N-acetylgalactosamine linked
O-glycosidically to threonine or serine residues of membrane
proteins. These antigens bind naturally occurring serum antibodies
thereby leading to mild hemolytic anemia and pronounced
thrombopenia. Thus, the blood cells in the Tn-syndrome are expected
to carry less sialic acid if galactose can not be transferred to
N-Acetylgalactosamine. The expression of Tn. and sialosyl-Tn
antigens as a consequence of incomplete or disordered. gylcan
biosynthesis has been recognized as a cancer-associated phenomenon.
Tn and sialosyl-Tn antigens are among the most investigated
cancer-associated carbohydrate antigens.
[0012] The present invention provides such polypeptides for these
and other uses that should be apparent to those skilled in the art
from the teachings herein.
SUMMARY OF THE INVENTION
[0013] Within one aspect, the invention provides an isolated
polypeptide comprising residues 114 to 370 of SEQ ID NO: 2. Within
an embodiment, the isolated polypeptide comprises residues 114 to
378 of SEQ ID NO: 2. Within another embodiment, the isolated
polypeptide comprises residues 50 to 378 of SEQ ID NO: 2. Within
another embodiment, the isolated polypeptide comprises residues 26
to 378 of SEQ ID NO: 2. Within another embodiment, the isolated
polypeptide comprises residues 1 to 378 of SEQ ID NO: 2.
[0014] Within another aspect, the invention provides an isolated
polypeptide selected from the group consisting of: a) a polypeptide
comprising residues 1 to 25 of SEQ ID NO: 2; b) a polypeptide
comprising residues 26 to 49 of SEQ ID NO: 2; c) a polypeptide
comprising residues 50 to 113 of SEQ ID NO: 2; d) a polypeptide
comprising residues 114 to 370 of SEQ ID NO: 2; e) a polypeptide
comprising residues 371 to 378 of SEQ ID NO: 2; and f) a
polypeptide comprising residues 1 to 378 of SEQ ID NO: 2.
[0015] Within another aspect, the invention provides an isolated
polynucleotide encoding a polypeptide wherein the polypeptide
comprises residues 114 to 370 of SEQ ID NO: 2. Within another
embodiment, the isolated polynucleotide comprises residues 114 to
378 of SEQ ID NO: 2. Within another embodiment, the isolated
polynucleotide comprises residues 50 to 378 of SEQ ID NO: 2. Within
another embodiment, the isolated polynucleotide comprises residues
26 to 378 of SEQ ID NO: 2. Within another embodiment, the isolated
comprises residues 1 to 378 of SEQ ID NO: 2.
[0016] Within another aspect, the invention provides an isolated
polynucleotide encoding a polypeptide molecule wherein the
polypeptide is selected from the group consisting of: a) a
polypeptide comprising residues 1 to 25 of SEQ ID NO: 2; b) a
polypeptide comprising residues 26 to 49 of SEQ ID NO: 2; c) a
polypeptide comprising residues 50 to 113 of SEQ ID NO: 2; d) a
polypeptide comprising residues 114 to 370 of SEQ ID NO: 2; e) a
polypeptide comprising residues 371 to 378 of SEQ ID NO: 2; and f)
a polypeptide comprising residues 1 to 378 of SEQ ID NO: 2.
[0017] Within another aspect, the invention provides an expression
vector comprising the following operably linked elements: a
transcription promoter; a DNA segment wherein the DNA segment is a
polynucleotide encoding the polypeptide of claim 1; and a
transcription terminator. Within another embodiment, the DNA
segment contains an affinity tag. Within another embodiment, the
invention provides a cultured cell into which has been introduced
the expression vector wherein said cell expresses the polypeptide
encoded by the DNA segment. Within another embodiment, the
invention provides a method of producing a polypeptide comprising
culturing a cell, whereby said cell expresses the polypeptide
encoded by the DNA segment; and recovering the polypeptide. Within
another embodiment, the invention provides the polypeptide produced
by said method of expression.
[0018] Within another aspect, the invention provides a method of
producing an antibody comprising the following steps in order:
inoculating an animal with an epitope bearing portion of a
polypeptide wherein the epitope bearing portion is selected from
the group consisting of: a polypeptide comprising residues 10 to 16
of SEQ ID NO: 2; a polypeptide comprising residues 52 to 61 of SEQ
ID NO: 2; a polypeptide comprising residues 52 to 78 of SEQ ID NO:
2; a polypeptide comprising residues 69 to 78 of SEQ ID NO: 2; a
polypeptide comprising residues 89 to 94 of SEQ ID NO: 2; a
polypeptide comprising residues 89 to 117 of SEQ ID NO: 2; a
polypeptide comprising residues 111 to 117 of SEQ ID NO: 2; a
polypeptide comprising residues 126 to 134 of SEQ ID NO: 2; a
polypeptide comprising residues 126 to 151 of SEQ ID NO: 2; a
polypeptide comprising residues 143 to 151 of SEQ ID NO: 2; a
polypeptide comprising residues 215 to 220 of SEQ ID NO: 2; a
polypeptide comprising residues 215 to 239 of SEQ ID NO: 2; a
polypeptide comprising residues 223 to 239 of SEQ ID NO: 2; a
polypeptide comprising residues 223 to 257 of SEQ ID NO: 2; a
polypeptide comprising residues 251 to 257 of SEQ ID NO: 2;and a
polypeptide comprising residues 332 to 337 of SEQ ID NO: 2; wherein
the polypeptide elicits an immune response in the animal to produce
the antibody; and isolating the antibody from the animal. Within an
embodiment, the antibody produced binds to a polypeptide comprising
residues 114 to 370 of SEQ ID NO: 2. Within another embodiment, the
antibody is a monoclonal antibody. Within another embodiment, the
antibody is joined to a moiety selected from the group consisting
of: a) an affinity tag; b) a detectable molecule; c) a cytotoxic
molecule; and d) a cytokine. Within another embodiment, the
invention provides a method of killing cells expressing a
polypeptide comprising residues 114 to 370 of SEQ ID NO: 2,
comprising contacting the cells with the antibody joined to the
moiety, wherein the antibody is joined to the cytotoxic
molecule.
[0019] Within another aspect, the invention provides a method for
modulating cell-cell interactions by combining a polypeptide
comprising residues 114 to 370 of SEQ ID NO: 2 with cells. Within
another embodiment, the cells are derived from tissues selected
from the group consisting of: a) tissues from brain; b) issues from
kidney; and c) issues from testis.
[0020] Within another aspect, the invention provides a method for
modulating glycoprotein and glycolipid biosynthesis in cells, cell
culture, and cell matrix comprising contacting the cells, cell
culture or cell mattix with the polypeptide comprising residues 114
to 370 of SEQ ID NO: 2. Within an embodiment, the cells are derived
from tissues selected from the group consisting of: a) tissues from
brain; b) issues from kidney; and c) tissues from testis.
[0021] Within another aspect, the invention provides a method of
detecting a znssp6 anti-complementary molecule comprising
contacting a test sample containing the znssp6 anti-complementary
molecule with a polypeptide comprises residues 114 to 370 of SEQ ID
NO: 2.
[0022] Within another aspect, the present invention provides an
isolated polynucleotide molecule encoding a polypeptide wherein
said polynucleotide molecule is selected from the group consisting
of: a) polynucleotide molecules comprising a nucleotide sequence
from nucleotide 474 to nucleotide 1244 of SEQ ID NO: 1; b)
polynucleotide molecules that encode a polypeptide comprising a
sequence of amino acid residues that is at least 70% identical to
amino acid residues 114 to 370 of SEQ ID NO: 2; c) degenerate
nucleotide sequences of a), or b); and d) polynucleotide molecules
that have a complementary sequence to a), b), or c). Within an
embodiment, the polypeptide differs from amino acid residues 114 to
370 of SEQ ID NO: 2 by conservative amino acid substitutions.
Within another embodiment, the polypeptide consists of the sequence
of SEQ ID NO: 2. Within a further embodiment, the polypeptide is
the sequence of amino acid residues 114 to 370 of SEQ ID NO: 2.
Within a related embodiment, is provided an isolated antibody or
antibody fragment that specifically binds to the polypeptide.
[0023] Within another aspect, the present invention provides an
isolated polynucleotide molecule encoding a polypeptide wherein
said polynucleotide molecule is selected from the group consisting
of: a) polynucleotide molecules comprising a nucleotide sequence
from nucleotide 282 to nucleotide 1244 of SEQ ID NO: 1; b)
polynucleotide molecules that encode a polypeptide comprising a
sequence of amino acid residues that is at least 70% identical to
amino acid residues 50 to 370 of SEQ ID NO: 2; c) degenerate
nucleotide sequences of a), or b); and d) polynucleotide molecules
that have a complementary sequence to a), b), or c). Within an
embodiment, the polypeptide differs from amino acid residues 50 to
370 of SEQ ID NO: 2 by conservative amino acid substitutions.
[0024] Within another aspect, the present invention provides an
isolated polynucleotide molecule encoding a polypeptide wherein
said polynucleotide molecule is selected from the group consisting
of: a) polynucleotide molecules comprising a nucleotide sequence
from nucleotide 135 to nucleotide 1268 of SEQ ID NO: 1; b)
polynucleotide molecules that encode a polypeptide comprising a
sequence of amino acid residues that is at least 70% identical to
amino acid residues 1 to 378 of SEQ ID NO: 2; c) degenerate
nucleotide sequences of a), or b); and d) polynucleotide molecules
that have a complementary sequence to a), b), or c). Within an
embodiment, the polypeptide differs from amino acid residues 1 to
370 SEQ ID NO: 2 by conservative amino acid substitutions.
[0025] Within another aspect, the present invention provides, an
expression vector comprising the following operably linked
elements: a transcription promoter; a DNA segment encoding a
polypeptide, wherein the DNA segment is selected from the group
consisting of: a) polynucleotide molecules comprising a nucleotide
sequence from nucleotide 474 to nucleotide 1244 of SEQ ID NO: 1; b)
polynucleotide molecules that encode a polypeptide that is at least
70% identical to amino acid residues 114 to 370 of SEQ ID NO: 2;
and c) degenerate nucleotide sequences of a), or b; and a
transcription terminator. Within an embodiment, the polypeptide
differs from amino acid residues 114 to 370 SEQ ID NO: 2 by
conservative amino acid substitutions. Also is provided a cultured
cell into which has been introduced an expression vector, as
described above, wherein said cell expresses the polypeptide
encoded by the DNA segment.
[0026] Within another aspect, the present invention provides a
method of producing a polypeptide comprising culturing a cell,
whereby said cell expresses the polypeptide encoded by the DNA
segment as described above; and recovering the polypeptide.
[0027] Within another aspect, the present invention provides a
method for modulating cell-cell interactions by combining a
polypeptide, that is at least 70% identical to amino acid residues
114 to 370 SEQ ID NO: 2, with cells in vitro and in vivo.
[0028] Within another aspect, the present invention provides a
pharmaceutical composition comprising a polypeptide, that is at
least 70% identical to amino acid residues 114 to 370 SEQ ID NO: 2,
in combination with a pharmaceutically acceptable vehicle.
[0029] Within another aspect, the present invention provides an
isolated polynucleotide molecule encoding a fusion protein
comprising a beta-1,3-galactosyltransferase domain having the amino
acid sequence of residues 114 to 370 of SEQ ID NO: 2, wherein said
beta-1,3-galactosyltran- sferase domain is operably linked to an
additional polypeptide. Within an embodiment, the additional
polypeptide is selected from the group consisting of: a) affinity
tag polypeptide molecules; b) immunoglobulin heavy chain constant
region polypeptide molecules; c) the hydrophobic region of amino
acid residues 26 to 49 of SEQ ID NO: 2; and d) hydrophobic regions
of other beta-1,3-galactosyltransferase polypeptide molecules.
DETAILED DESCRIPTION OF THE INVENTION
[0030] Prior to setting forth the invention in detail, it may be
helpful to the understanding thereof to define the following
terms:
[0031] The term "affinity tag" is used herein to denote a
polypeptide segment that can be attached to a second polypeptide to
provide for purification of the second polypeptide or provide sites
for attachment of the second polypeptide to a substrate. In
principal, any peptide or protein for which an antibody or other
specific binding agent is available can be used as an affinity tag.
Affinity tags include a poly-histidine tract, protein A (Nilsson et
al., EMBO J. 4:1075, 1985; Nilsson et al., Methods Enzyamol. 198:3,
1991), glutathione S transferase (Smith and Johnson, Gene 67:31,
1988), Glu-Glu affinity tag (Grussenmeyer et al., Proc. Natl. Acad.
Sci. USA 82:7952-4, 1985), substance P, Flag.TM. peptide (Hopp et
al., Biotechnology 6:1204-1210, 1988), streptavidin binding
peptide, maltose binding protein (Guan et al., Gene 67:21-30,
1987), cellulose binding protein, thioredoxin, ubiquitin, T7
polymerase, or other antigenic epitope or binding domain. See, in
general, Ford et al., Protein Expression and Purification 2:
95-107, 1991. DNAs encoding affinity tags and other reagents are
available from commercial suppliers (e.g., Pharmacia Biotech,
Piscataway, N.J.; New England Biolabs, Beverly, Mass.; Eastman
Kodak, New Haven, Conn.).
[0032] The term "allelic variant" is used herein to denote any of
two or more alternative forms of a gene occupying the same
chromosomal locus. Allelic variation arises naturally through
mutation, and may result in phenotypic polymorphism within
populations. Gene mutations can be silent (no change in the encoded
polypeptide) or may encode polypeptides having altered amino acid
sequence. The term allelic variant is also used herein to denote a
protein encoded by an allelic variant of a gene.
[0033] The terms "amino-terminal" and "carboxyl-terminal" are used
herein to denote positions within polypeptides. Where the context
allows, these terms are used with reference to a particular
sequence or portion of a polypeptide to denote proximity or
relative position. For example, a certain sequence positioned
carboxyl-terminal to a reference sequence within a polypeptide is
located proximal to the carboxyl terminus of the reference
sequence, but is not necessarily at the carboxyl terminus of the
complete polypeptide.
[0034] The term "complement/anti-complement pair" denotes
non-identical moieties that form a non-covalently associated,
stable pair under appropriate conditions. For instance, biotin and
avidin (or streptavidin) are prototypical members of a
complement/anti-complement pair. Other exemplary
complement/anti-complement pairs include receptor/ligand pairs,
antibody/antigen (or hapten or epitope) pairs, sense/antisense
polynucleotide pairs, and the like. Where subsequent dissociation
of the complement/anti-complement pair is desirable, the
complement/anti-complem- ent pair preferably has a binding affinity
of <10.sup.9 M.sup.-1.
[0035] The term "complements of a polynucleotide molecule" is a
polynucleotide molecule having a complementary base sequence and
reverse orientation as compared to a reference sequence. For
example, the sequence 5' ATGCACGGG 3' is complementary to 5'
CCCGTGCAT 3'.
[0036] The term "contig" denotes a polynucleotide that has a
contiguous stretch of identical or complementary sequence to
another polynucleotide. Contiguous sequences are said to "overlap"
a given stretch of polynucleotide sequence either in their entirety
or along a partial stretch of the polynucleotide. For example,
representative contigs to the polynucleotide sequence
5'-ATGGAGCTT-3' are 5'-AGCTTgagt-3' and 3'-tcgacTACC-5'.
[0037] The term "degenerate nucleotide sequence" denotes a sequence
of nucleotides that includes one or more degenerate codons (as
compared to a reference polynucleotide molecule that encodes a
polypeptide). Degenerate codons contain different triplets of
nucleotides, but encode the same amino acid residue (i.e., GAU and
GAC triplets each encode Asp).
[0038] The term "expression vector" is used to denote a DNA
molecule, linear or circular, that comprises a segment encoding a
polypeptide of interest operably linked to additional segments that
provide for its transcription. Such additional segments include
promoter and terminator sequences, and may also include one or more
origins of replication, one or more selectable markers, an
enhancer, a polyadenylation signal, etc. Expression vectors are
generally derived from plasmid or viral DNA, or may contain
elements of both.
[0039] The term "isolated", when applied to a polynucleotide,
denotes that the polynucleotide has been removed from its natural
genetic milieu and is thus free of other extraneous or unwanted
coding sequences, and is in a form suitable for use within
genetically engineered protein production systems. Such isolated
molecules are those that are separated from their natural
environment and include cDNA and genomic clones. Isolated DNA
molecules of the present invention are free of other genes with
which they are ordinarily associated, but may include naturally
occurring 5' and 3' untranslated regions such as promoters and
terminators. The identification of associated regions will be
evident to one of ordinary skill in the art (see for example, Dynan
and Tijan, Nature 316:774-78, 1985).
[0040] An "isolated" polypeptide or protein is a polypeptide or
protein that is found in a condition other than its native
environment, such as apart from blood and animal tissue. In a
preferred form, the isolated polypeptide is substantially free of
other polypeptides, particularly other polypeptides of animal
origin. It is preferred to provide the polypeptides in a highly
purified form, i.e. greater than 95% pure, more preferably greater
than 99% pure. When used in this context, the term "isolated" does
not exclude the presence of the same polypeptide in alternative
physical forms, such as dimers or alternatively glycosylated or
derivatized forms.
[0041] The term "joined" when referring to attaching one moiety to
another moiety, indicates that the moieties are directly or
indirectly linked to each other. Thus, such moieties can be joined
by genetic manipulations (such as, for example, fusion proteins),
chemical manipulations (such as, for example, chemical conjugation,
coupling, or chelation), or other means.
[0042] The term "operably linked", when referring to DNA segments,
indicates that the segments are arranged so that they function in
concert for their intended purposes, e.g., transcription initiates
in the promoter and proceeds through the coding segment to the
terminator.
[0043] The term "ortholog" or "species homolog", denotes a
polypeptide or protein obtained from one species that is the
functional counterpart of a polypeptide or protein from a different
species. Sequence differences among orthologs are the result of
speciation.
[0044] "Paralogs" are distinct but structurally related proteins
made by an organism. Paralogs are believed to arise through gene
duplication. For example, a-globin, b-globin, and myoglobin are
paralogs of each other.
[0045] A "polynucleotide" is a single- or double-stranded polymer
of deoxyribonucleotide or ribonucleotide bases read from the 5' to
the 3' end. Polynucleotides include RNA and DNA, and may be
isolated from natural sources, synthesized in vitro, or prepared
from a combination of natural and synthetic molecules. Sizes of
polynucleotides are expressed as base pairs (abbreviated "bp"),
nucleotides ("nt"), or kilobases ("kb"). Where the context allows,
the latter two terms may describe polynucleotides that are
single-stranded or double-stranded. When the term is applied to
double-stranded molecules it is used to denote overall length and
will be understood to be equivalent to the term "base pairs". It
will be recognized by those skilled in the art that the two strands
of a double-stranded polynucleotide may differ slightly in length
and that the ends thereof may be staggered as a result of enzymatic
cleavage; thus all nucleotides within a double-stranded
polynucleotide molecule may not be paired. Such unpaired ends will
in general not exceed 20 nt in length.
[0046] A "polypeptide" is a polymer of amino acid residues joined
by peptide bonds, whether produced naturally or synthetically.
Polypeptides of less than about 10 amino acid residues are commonly
referred to as "peptides".
[0047] The term "promoter" is used herein for its art-recognized
meaning to denote a portion of a gene containing DNA sequences that
provide for the binding of RNA polymerase and initiation of
transcription. Promoter sequences are commonly, but not always,
found in the 5' non-coding regions of genes.
[0048] A "protein" is a macromolecule comprising one or more
polypeptide chains. A protein may also comprise non-peptidic
components, such as carbohydrate groups. Carbohydrates and other
non-peptidic substituents may be added to a protein by the cell in
which the protein is produced, and will vary with the type of cell.
Proteins are defined herein in terms of their amino acid backbone
structures; substituents such as carbohydrate groups are generally
not specified, but may be present nonetheless.
[0049] The term "receptor" denotes a cell-associated protein that
binds to a bioactive molecule (i.e., a ligand) and mediates the
effect of the ligand on the cell. Membrane-bound receptors are
characterized by a multi-domain or multi-peptide structure
comprising an extracellular ligand-binding domain and an
intracellular effector domain that is typically involved in signal
transduction. Binding of ligand to receptor results in a
conformational change in the receptor that causes an interaction
between the effector domain and other molecule(s) in the cell. This
interaction in turn leads to an alteration in the metabolism of the
cell. Metabolic events that are linked to receptor-ligand
interactions include gene transcription, phosphorylation,
dephosphorylation, increases in cyclic AMP production, mobilization
of cellular calcium, mobilization of membrane lipids, cell
adhesion, hydrolysis of inositol lipids and hydrolysis of
phospholipids. In general, receptors can be membrane bound,
cytosolic or nuclear; monomeric (e.g., thyroid stimulating hormone
receptor, beta-adrenergic receptor) or multimeric (e.g., PDGF
receptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor,
G-CSF receptor, erythropoietin receptor and IL-6 receptor).
[0050] The term "secretory signal sequence" denotes a DNA sequence
that encodes a polypeptide (a "secretory peptide") that, as a
component of a larger polypeptide, directs the larger polypeptide
through a secretory pathway of a cell in which it is synthesized.
The larger polypeptide is commonly cleaved to remove the secretory
peptide during transit through the secretory pathway.
[0051] A "segment" is a portion of a larger molecule (e.g.,
polynucleotide or polypeptide) having specified attributes. For
example, a DNA segment encoding a specified polypeptide is a
portion of a longer DNA molecule, such as a plasmid or plasmid
fragment, that, when read from the 5' to the 3' direction, encodes
the sequence of amino acids of the specified polypeptide.
[0052] The term "splice variant" is used herein to denote
alternative forms of RNA transcribed from a gene. Splice variation
arises naturally through use of alternative splicing sites within a
transcribed RNA molecule, or less commonly between separately
transcribed RNA molecules, and may result in several mRNAs
transcribed from the same gene. Splice variants may encode
polypeptides having altered amino acid sequence. The term splice
variant is also used herein to denote a protein encoded by a splice
variant of an MRNA transcribed from a gene.
[0053] Molecular weights and lengths of polymers determined by
imprecise analytical methods (e.g., gel electrophoresis) will be
understood to be approximate values. When such a value is expressed
as "about" X or "approximately" X, the stated value of X will be
understood to be accurate to .+-.10%.
[0054] All references cited herein are incorporated by reference in
their entirety.
[0055] The present invention is based upon the discovery of a novel
cDNA sequence (SEQ ID NO: 1) and corresponding polypeptide (SEQ ID
NO: 2) having homology to a family of proteins, the
beta-1,3-galactosyltransfera- ses. The
beta-1,3-galactosyltransferases are part of the
galactosyltransferases, which in turn, belong in the category of
glycosyltransferases. The beta-1,3-galactosyltransferase family
includes HSY15014 (Kolbinger, F. et al., Journal of Biol. Chem.
273: 433-440, 1998), HSGALT3, HSGALT4, (Amado, M. et al., ibid),
E07739 (Katsutoshi, S. et al., Japanese patent, JP 1994181759-A/1),
and Cardiac and Pancreatic Peptide (Human Genome Sciences, Inc., WO
98/44112). Enzymes in this category are responsible for
transferring galactose to carbohydrate chains during biosynthesis.
It has been predicted that the beta-1,3-galactosyltransferase
family members are in the alpha/beta barrel (TIM barrel) folding
class of enzymes, similar to other glycosyltransferases such as the
alpha-amylases and beta-glycanases (Yuan, Y. et al., Cell 88:9-11,
1997). Another member of the beta-1,3-galactosyltransferase family
is the Drosophila melanogaster locus Brainiac (brn) (Goode, S. et
al., Devel. Biol. 178:35-50, 1996), also known as "putative
neurogenic secreted signaling protein" or NSSP. Brn is required for
epithelial development (Goode, ibid). This activity may be due to
possible cell interactions between the membrane bound
glycosyltransferase and oligosaccharide substrates on adjacent cell
surfaces (Shur, ibid). The beta-1,3-galactosyltransferases family
members are also known as neurogenic secreted signal peptides. See,
for example, Shur, B. D., ibid, and Amado, M. et al., ibid.
[0056] The beta-1,3-galactosyltransferases are predicted to be Type
II transmembrane proteins. An ortholog to E07739, is AF029790
(Hennet, T. et al., Journal of Biol. Chem. 273:58-65, 1998), which
is claimed to be a Type II transmembrane domain based on
hydrophobicity analysis. However, due to the close proximity of
this domain to the initiating methionine and lack of positively
charged residues preceding the domain it is possible that AF029790
is not membrane bound but rather an extracellular secreted
protein.
[0057] The sequence of the novel znssp6 polypeptides of the present
invention was initially identified by searching an EST database for
open reading frames with similarity to brn. The insert of an
expressed sequence tag was obtained and the deduced amino acid
sequence of the insert was determined to be incomplete at the 5'
end. Polymerase chain extensions of this sequence were performed,
and their analysis identified a second EST for which the insert was
obtained and sequenced. Analysis of the nucleotide sequence of the
second insert (SEQ ID NO: 1) revealed an open reading frame
encoding 378 amino acids (SEQ ID NO: 2) which has been designated
as znssp6.
[0058] A representative motif of the beta-1,3-galactosyltransferase
family is described by the following amino acid residue profile:
[D,E] [D] [V] [F,Y] [L,T,V] [G]. The sequence of amino acid
residues from residue 304 to 309 of SEQ ID NO: 2 is representative
of this motif. The core region of similarity to all
beta-1,3-galactosyltransferases begins at residue 114 and ends at
residue 370 of SEQ ID NO: 2. This is believed to be the catalytic,
or anti-complementary molecule binding domain. Conserved negatively
charged amino acid residues 174, 180, 185, 220, 304, and 305 of SEQ
ID NO: 2 are contained within this catalytic/binding domain. Amino
acid residues 50 to 113 of SEQ ID NO: 2 are predicted to form a
stem or linker domain separating the catalytic/binding domain from
the cell membrane. The region from residue 26 to residue 49 of SEQ
ID NO: 2 is strongly hydrophobic and in one form of the znssp6
protein, this region is predicted to form a transmembrane domain
resulting in a membrane bound form of znssp6. In another form of
the znssp6 protein, the hydrophobic domain may act as a secretory
peptide in which case znssp6 is a secreted, soluble protein. Znssp6
shares homology with beta-1,3-galactosyltransfera- ses which are
predicted to be Type II membrane proteins. Znssp6 shows the highest
similarity to Cardiac and Pancreatic Peptide (CAPP, See WO
98/44112, 1998), at 43% amino acid identity. Additionally, znssp6
has 36% identity to brn. Those skilled in the art will recognize
that predicted domain boundaries are approximations based on
primary sequence content, and may vary slightly; however, such
estimates are generally accurate to within.+-.5 amino acid
residues.
[0059] The present invention also provides post translationally
modified polypeptides or polypeptide fragments. A conserved
potential N-linked glycosylation site can be found at amino acid
residue 192 of SEQ ID NO: 2. Other potential N-linked glycosylation
sites are at residues 79, and 104 of SEQ ID NO: 2. Other examples
of post translational modifications include proteolytic cleavage,
disulfide bonding and hydroxylation.
[0060] Analysis of the tissue distribution of znssp6 was performed
by the Northern blotting technique using Human Multiple Tissue and
Master Dot Blots. Strong signals were observed in brain (adult and
fetal), kidney (adult and fetal), and testis. The major transcript
size was about 1.6 kb, although minor transcripts of about 3.0 kb,
and 4.0 kb were also evident. Lower level expression was also
observed in tissues such as spinal cord, colon, prostate, stomach,
ovary, pancreas, pituitary gland, adrenal gland, salivary gland,
mammary gland, liver, small intestine, spleen, thymus, peripheral
leukocyte, lymph node, bone marrow, lung, trachea, placenta, fetal
spleen and fetal lung.
[0061] The highly conserved, negatively charged residues at
positions 174, 180, 185, 220, 304, and 305 of SEQ ID NO: 2 and the
amino acid sequence between 304 and 309 of znssp6 can be used to
identify new family members. For instance, reverse
transcription-polymerase chain reaction (RT-PCR) can be used to
amplify sequences encoding the znssp6 polynucleotide from RNA
obtained from a variety of tissue sources or cell lines. In
particular, highly degenerate primers designed from the znssp6
sequences are useful for this purpose.
[0062] Polynucleotides
[0063] The present invention also provides polynucleotide
molecules, including DNA and RNA molecules, that encode the znssp6
polypeptides disclosed herein. Those skilled in the art will
readily recognize that, in view of the degeneracy of the genetic
code, considerable sequence variation is possible among these
polynucleotide molecules. SEQ ID NO: 3 is a degenerate DNA sequence
that encompasses all DNAs that encode the znssp6 polypeptide of SEQ
ID NO: 2. Those skilled in the art will recognize that the
degenerate sequence of SEQ ID NO: 3 also provides all RNA sequences
encoding SEQ ID NO: 2 by substituting U for T. Thus, znssp6
polypeptide-encoding polynucleotides comprising nucleotide 1 to
nucleotide 1134 of SEQ ID NO: 3 and their RNA equivalents are
contemplated by the present invention. Table 1 sets forth the
one-letter codes used within SEQ ID NO: 3 to denote degenerate
nucleotide positions. "Resolutions" are the nucleotides denoted by
a code letter. "Complement" indicates the code for the
complementary nucleotide(s). For example, the code Y denotes either
C or T, and its complement R denotes A or G, A being complementary
to T, and G being complementary to C.
1 TABLE 1 Nucleotide Resolution Complement Resolution A A T T C C G
G G G C C T T A A R A.vertline.G Y C.vertline.T Y C.vertline.T R
A.vertline.G M A.vertline.C K G.vertline.T K G.vertline.T M
A.vertline.C S C.vertline.G S C.vertline.G W A.vertline.T W
A.vertline.T H A.vertline.C.vertline.T D A.vertline.G.vertline.T B
C.vertline.G.vertline.T V A.vertline.C.vertline.G V
A.vertline.C.vertline.G B C.vertline.G.vertline.T D
A.vertline.G.vertline.T H A.vertline.C.vertline.T N
A.vertline.C.vertline.G.vertline.T N
A.vertline.C.vertline.G.vertline.T
[0064] The degenerate codons used in SEQ ID NO: 3, encompassing all
possible codons for a given amino acid, are set forth in Table
2.
2TABLE 2 One Amino Letter Degenerate Acid Code Codons Codon Cys C
TGC TGT TGY Ser S AGC AGT TCA TCC TCG TCT WSN Thr T ACA ACC ACG ACT
ACN Pro P CCA CCC CCG CCT CCN Ala A GCA GCC GCG GCT GCN Gly G GGA
GGC GGG GGT GGN Asn N AAC AAT AAY Asp D GAC GAT GAY Glu E GAA GAG
GAR Gln Q CAA CAG CAR His H CAC CAT CAY Arg R AGA AGG CGA CGC CGG
CGT MGN Lys K AAA AAG AAR Met M ATG ATG Ile I ATA ATC ATT ATH Leu L
CTA CTC CTG CTT TTA TTG YTN Val V GTA GTC GTG GTT GTN Phe F TTC TTT
TTY Tyr Y TAC TAT TAY Trp W TGG TGG Ter . TAA TAG TGA TRR
Asn.vertline.Asp B RAY Glu.vertline.Gln Z SAR Any X NNN
[0065] One of ordinary skill in the art will appreciate that some
ambiguity is introduced in determining a degenerate codon,
representative of all possible codons encoding each amino acid. For
example, the degenerate codon for serine (WSN) can, in some
circumstances, encode arginine (AGR), and the degenerate codon for
arginine (MGN) can, in some circumstances, encode serine (AGY). A
similar relationship exists between codons encoding phenylalanine
and leucine. Thus, some polynucleotides encompassed by the
degenerate sequence may encode variant amino acid sequences, but
one of ordinary skill in the art can easily identify such variant
sequences by reference to the amino acid sequence of SEQ ID NO: 2.
Variant sequences can be readily tested for functionality as
described herein.
[0066] One of ordinary skill in the art will also appreciate that
different species can exhibit "preferential codon usage." In
general, see, Grantham, et al., Nuc. Acids Res. 8:1893-912, 1980;
Haas, et al. Curr. Biol. 6:315-24, 1996; Wain-Hobson, et al., Gene
13:355-64, 1981; Grosjean and Fiers, Gene 18:199-209, 1982; Holm,
Nuc. Acids Res. 14:3075-87, 1986; Ikemura, J. Mol. Biol.
158:573-97, 1982. As used herein, the term "preferential codon
usage" or "preferential codons" is a term of art referring to
protein translation codons that are most frequently used in cells
of a certain species, thus favoring one or a few representatives of
the possible codons encoding each amino acid (See Table 2). For
example, the amino acid Threonine (Thr) may be encoded by ACA, ACC,
ACG, or ACT, but in mammalian cells ACC is the most commonly used
codon; in other species, for example, insect cells, yeast, viruses
or bacteria, different Thr codons may be preferential. Preferential
codons for a particular species can be introduced into the
polynucleotides of the present invention by a variety of methods
known in the art. Introduction of preferential codon sequences into
recombinant DNA can, for example, enhance production of the protein
by making protein translation more efficient within a particular
cell type or species. Therefore, the degenerate codon sequence
disclosed in SEQ ID NO: 3 serves as a template for optimizing
expression of polynucleotides in various cell types and species
commonly used in the art and disclosed herein. Sequences containing
preferential codons can be tested and optimized for expression in
various species, and tested for functionality as disclosed
herein.
[0067] Within preferred embodiments of the invention the isolated
polynucleotides will hybridize to similar sized regions of SEQ ID
NO: 1, other polynucleotide probes, primers, fragments and
sequences recited herein or sequences complementary thereto.
Polynucleotide hybridization is well known in the art and widely
used for many applications, see for example, Sambrook et al.,
Molecular Cloning: A Laboratory Manual Second Edition, Cold Spring
Harbor, N.Y., 1989;
[0068] Ausubel et al., eds., Current Protocols in Molecular
Biology, John Wiley and Sons, Inc., NY, 1987; Berger and Kimmel,
eds., Guide to Molecular Cloning Techniques, Methods in Enzymology,
volume 152, 1987 and Wetmur, Crit. Rev. Biochem. Mol. Biol.
26:227-59, 1990. Polynucleotide hybridization exploits the ability
of single stranded complementary sequences to form a double helix
hybrid. Such hybrids include DNA-DNA, RNA-RNA and DNA-RNA.
[0069] Hybridization will occur between sequences which contain
some degree of complementarity. Hybrids can tolerate mismatched
base pairs in the double helix, but the stability of the hybrid is
influenced by the degree of mismatch. The T.sub.m of the mismatched
hybrid decreases by 1.degree. C. for every 1-1.5% base pair
mismatch. Varying the stringency of the hybridization conditions
allows control over the degree of mismatch that will be present in
the hybrid. The degree of stringency increases as the hybridization
temperature increases and the ionic strength of the hybridization
buffer decreases. Hybridization buffers generally contain blocking
agents such as Denhardt's solution (Sigma Chemical Co., St. Louis,
Mo.), denatured salmon sperm DNA, milk powders (BLOTTO), heparin or
SDS, and a Na.sup.+source, such as SSC (1.times.SSC: 0.15 M NaCl,
15 mM sodium citrate) or SSPE (1.times.SSPE: 1.8 M NaCl, 10 mM
NaH.sub.2PO.sub.4, 1 mM EDTA, pH 7.7). By decreasing the ionic
concentration of the buffer, the stability of the hybrid is
increased. Typically, hybridization buffers contain from between 10
mM-1 M Na.sup.+. Premixed hybridization solutions are also
available from commercial sources such as Clontech Laboratories
(Palo Alto, Calif.) and Promega Corporation (Madison, Wis.) for use
according to manufacturer's instruction. Addition of destabilizing
or denaturing agents such as formamide, tetralkylammonium salts,
guanidinium cations or thiocyanate cations to the hybridization
solution will alter the T.sub.m of a hybrid. Typically, formamide
is used at a concentration of up to 50% to allow incubations to be
carried out at more convenient and lower temperatures. Formamide
also acts to reduce non-specific background when using RNA
probes.
[0070] Stringent hybridization conditions encompass temperatures of
about 5-25.degree. C. below the thermal melting point .sup.TM of
the hybrid and a hybridization buffer having up to 1 M Na.sup.+.
Higher degrees of stringency at lower temperatures can be achieved
with the addition of formamide which reduces the T.sub.m. of the
hybrid about 1.degree. C. for each 1% formamide in the buffer
solution. Generally, such stringent conditions include temperatures
of 20-70.degree. C. and a hybridization buffer containing 5.times.
to 6.times.SSC and 0-50% formamide. A higher degree of stringency
can be achieved at temperatures of from 40-70.degree. C. with a
hybridization buffer having 3.times. to 4.times.SSC and from 0-50%
formamide. Highly stringent conditions typically encompass
temperatures of 42-70.degree. C. with a hybridization buffer having
up to 2.times.SSC and 0-50% formamide. Different degrees of
stringency can be used during hybridization and washing to achieve
maximum specific binding to the target sequence. Typically, the
washes following hybridization are performed at increasing degrees
of stringency to remove non-hybridized polynucleotide probes from
hybridized complexes.
[0071] The above conditions are meant to serve as a guide and it is
well within the abilities of one skilled in the art to adapt these
conditions for use with a particular polypeptide hybrid. The
T.sub.m for a specific target sequence is the temperature (under
defined conditions) at which 50% of the target sequence will
hybridize to a perfectly matched probe sequence. Those conditions
that influence the T.sub.m include, the size and base pair content
of the polynucleotide probe, the ionic strength of the
hybridization solution, and the presence of destabilizing agents in
the hybridization solution. Numerous equations for calculating
T.sub.m are known in the art, see for example (Sambrook et al.,
ibid.; Ausubel et al., ibid.; Berger and Kimmel, ibid. and Wetmur,
ibid.) and are specific for DNA, RNA and DNA-RNA hybrids and
polynucleotide probe sequences of varying length. Sequence analysis
software such as Oligo 4.0 and Primer Premier, as well as sites on
the Internet, are available tools for analyzing a given sequence
and calculating T.sub.m based on user defined criteria. Such
programs can also analyze a given sequence under defined conditions
and suggest suitable probe sequences. Typically, hybridization of
longer polynucleotide sequences, >50 bp, is done at temperatures
of about 20-25.degree. C. below the calculated T.sub.m. For smaller
probes, <50 bp, hybridization is typically carried out at the
T.sub.m or 5-10.degree. C. below. This allows for the maximum rate
of hybridization for DNA-DNA and DNA-RNA hybrids.
[0072] As previously noted, the isolated polynucleotides of the
present invention include DNA and RNA. Methods for preparing DNA
and RNA are well known in the art. In general, RNA is isolated from
a tissue or cell that produces large amounts of znssp6 RNA. Such
tissues and cells are identified by Northern blotting (Thomas,
Proc. Natl. Acad. Sci. USA 77:5201, 1980), and include brain,
kidney, and testis, spinal cord, colon, prostate, stomach, ovary,
pancreas, pituitary gland, adrenal gland, salivary gland, mammary
gland, liver, small intestine, spleen, thymus, peripheral
leukocyte, lymph node, bone marrow, lung, trachea, placenta, fetal
spleen and fetal lung.
[0073] Total RNA can be prepared using guanidine isothiocyante
extraction followed by isolation by centrifugation in a CsCl
gradient (Chirgwin et al., Biochemistry 18:52-94, 1979). Poly
(A).sup.+ RNA is prepared from total RNA using the method of Aviv
and Leder (Proc. Natl. Acad. Sci. USA 69:1408-12, 1972).
Complementary DNA (cDNA) is prepared from poly(A).sup.+ RNA using
known methods, In the alternative, genomic DNA can be isolated.
Polynucleotides encoding znssp6 polypeptides are then identified
and isolated by, for example, hybridization or PCR.
[0074] A full-length clone encoding znssp6 can be obtained by
conventional cloning procedures. Complementary DNA (cDNA) clones
are preferred, although for some applications (e.g., expression in
transgenic animals) it may be preferable to use a genomic clone, or
to modify a cDNA clone to include at least one genomic intron.
Methods for preparing cDNA and genomic clones are well known and
within the level of ordinary skill in the art, and include the use
of the sequence disclosed herein, or parts thereof, for probing or
priming a library. Expression libraries can be probed with
antibodies to znssp6, or fragments thereof, or other specific
binding partners.
[0075] The invention also provides isolated and purified znssp6
polynucleotide probes. Such polynucleotide probes can be RNA or
DNA. DNA can be either cDNA or genomic DNA. Polynucleotide probes
are single or double-stranded DNA or RNA, generally synthetic
oligonucleotides, but may be generated from cloned cDNA or genomic
sequences and will generally comprise at least 16 nucleotides, more
often from 17 nucleotides to 25 or more nucleotides, sometimes 40
to 60 nucleotides, and in some instances a substantial portion,
domain or even the entire znssp6 gene or cDNA.
[0076] The synthetic oligonucleotides of the present invention have
at least 75% identity to a representative znssp6 DNA sequence (SEQ
ID NO: 1 or 3) or their complements. The invention also provides
oligonucleotide probes or primers comprising at least 14 contiguous
nucleotides of a polynucleotide of SEQ ID NOs: 1 or 3 or a sequence
complementary to SEQ ID NOs: 1 or 3.
[0077] Regions from which to construct probes include the 5' and/or
3' coding sequences, the anti-complementary molecule-binding
regions, the stem domain, and the hydrophobic domain, and the like.
Techniques for developing polynucleotide probes and hybridization
techniques are known in the art, see for example, Ausubel et al.,
eds., Current Protocols in Molecular Biology John Wiley and Sons,
Inc., NY, 1991. For use as probes, the molecules can be labeled to
provide a detectable signal, such as with an enzyme, biotin, a
radionuclide, fluorophore, chemiluminescer, paramagnetic particle
and the like, which are commercially available from many sources,
such as Molecular Probes, Inc., Eugene, Oreg., and Amersham Corp.,
Arlington Heights, Ill., using techniques that are well known in
the art. Such probes can also be used in hybridizations to detect
the presence or quantify the amount of znssp6 gene or mRNA
transcript in a sample. Znssp6 polynucleotide probes could be used
to hybridize to DNA or RNA targets for diagnostic purposes, using
such techniques such as fluorescent in situ hybridization (FISH) or
immunohistochemistry. Polynucleotide probes can be used to identify
genes encoding znssp6-like proteins. For example, znssp6
polynucleotides can be used as primers and/or templates in PCR
reactions to identify other novel members of the
UDP-glycosyltransferase family. Such probes can also be used to
screen libraries for related sequences encoding novel
UDP-glycosyltransferases.
[0078] Such screening would be carried out under conditions of low
stringency which would allow identification of sequences which are
substantially homologous, but not requiring complete homology to
the probe sequence. Such methods and conditions are well known in
the art, see, for example, Sambrook et al., Molecular Cloning: A
Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., 1989.
Such low stringency conditions could include hybridization
temperatures less than 42.degree. C., formamide concentrations of
less than 50% and moderate to low concentrations of salt. Libraries
may be made of genomic DNA or cDNA. Polynucleotide probes are also
useful for Southern, Northern, or dot blots, colony and plaque
hybridization and in situ hybridization. Mixtures of different
znssp6 polynucleotide probes can be prepared which would increase
sensitivity or the detection of low copy number targets, in
screening systems.
[0079] The polynucleotides of the present invention can also be
synthesized using DNA synthesizers. Currently the method of choice
is the phosphoramidite method. If chemically synthesized double
stranded DNA is required for an application such as the synthesis
of a gene or a gene fragment, then each complementary strand is
made separately. The production of short genes (60 to 80 bp) is
technically straightforward and can be accomplished by synthesizing
the complementary strands and then annealing them. For the
production of longer genes (>300 bp), however, special
strategies must be invoked, because the coupling efficiency of each
cycle during chemical DNA synthesis is seldom 100%. To overcome
this problem, synthetic genes (double-stranded) are assembled in
modular form from single-stranded fragments that are from 20 to 100
nucleotides in length. See Glick and Pasternak, Molecular
Biotechnology, Principles & Applications of Recombinant DNA,
(ASM Press, Washington, D.C. 1994); Itakura et al., Annu. Rev.
Biochem. 53: 323-56, 1984 and Climie et al., Proc. Natl. Acad. Sci.
USA 87:633-7, 1990.
[0080] The present invention further provides counterpart
polypeptides and polynucleotides from other species (orthologs).
These species include, but are not limited to mammalian, avian,
amphibian, reptile, fish, insect and other vertebrate and
invertebrate species. Of particular interest are znssp6
polypeptides from other mammalian species, including murine,
porcine, ovine, bovine, canine, feline, equine, and other primate
polypeptides. Orthologs of human znssp6 can be cloned using
information and compositions provided by the present invention in
combination with conventional cloning techniques. For example, a
cDNA can be cloned using mRNA obtained from a tissue or cell type
that expresses znssp6 as disclosed herein. Suitable sources of mRNA
can be identified by probing Northern blots with probes designed
from the sequences disclosed herein. A library is then prepared
from mRNA of a positive tissue or cell line. A znssp6-encoding cDNA
can then be isolated by a variety of methods, such as by probing
with a complete or partial human cDNA or with one or more sets of
degenerate probes based on the disclosed sequences. A cDNA can also
be cloned using the polymerase chain reaction, or PCR (Mullis, U.S.
Pat. No. 4,683,202), using primers designed from the representative
human znssp6 sequence disclosed herein. Within an additional
method, the cDNA library can be used to transform or transfect host
cells, and expression of the cDNA of interest can be detected with
an antibody to znssp6 polypeptide. Similar techniques can also be
applied to the isolation of genomic clones.
[0081] Those skilled in the art will recognize that the sequence
disclosed in SEQ ID NO: 1 represents a single allele of human
znssp6 and that allelic variation and alternative splicing are
expected to occur. Allelic variants of this sequence can be cloned
by probing cDNA or genomic libraries from different individuals
according to standard procedures. Allelic variants of the DNA
sequence shown in SEQ ID NO: 1, including those containing silent
mutations and those in which mutations result in amino acid
sequence changes, are within the scope of the present invention, as
are proteins which are allelic variants of SEQ ID NO: 2. cDNAs
generated from alternatively spliced mRNAs, which retain the
properties of the znssp6 polypeptide are included within the scope
of the present invention, as are polypeptides encoded by such cDNAs
and mRNAs. Allelic variants and splice variants of these sequences
can be cloned by probing cDNA or genomic libraries from different
individuals or tissues according to standard procedures known in
the art.
[0082] The present invention also provides isolated znssp6
polypeptides that are substantially homologous to the polypeptides
of SEQ ID NO: 2 and their orthologs. The term "substantially
homologous" is used herein to denote polypeptides having 50%,
preferably 60%, more preferably at least 80%, sequence identity to
the sequences shown in SEQ ID NO: 2 or their orthologs. Such
polypeptides will more preferably be at least 90% identical, and
most preferably 95% or more identical to SEQ ID NO: 2 or its
orthologs.) Percent sequence identity is determined by conventional
methods. See, for example, Altschul et al., Bull. Math. Bio. 48:
603-16, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA
89:10915-9, 1992. Briefly, two amino acid sequences are aligned to
optimize the alignment scores using a gap opening penalty of 10, a
gap extension penalty of 1, and the "blosum 62" scoring matrix of
Henikoff and Henikoff (ibid.) as shown in Table 3 (amino acids are
indicated by the standard one-letter codes). The percent identity
is then calculated as: 1 Total number of identical matches [length
of the longer sequence plus thenumber of gaps introduced into the
longersequence in order to align the two sequences] .times. 100
3 TABLE 3 A R N D C Q E G H I L K M F P S T W Y V A 4 R -1 5 N -2 0
6 D -2 -2 1 6 C 0 -3 -3 -3 9 Q -1 1 0 0 -3 5 E -1 0 0 2 -4 2 5 G 0
-2 0 -1 -3 -2 -2 6 H -2 0 1 -1 -3 0 0 -2 8 I -1 -3 -3 -3 -1 -3 -3
-4 -3 4 L -1 -2 -3 -4 -1 -2 -3 -4 -3 2 4 K -1 2 0 -1 -3 1 1 -2 -1
-3 -2 5 M -1 -1 -2 -3 -1 0 -2 -3 -2 1 2 -1 5 F -2 -3 -3 -3 -2 -3 -3
-3 -1 0 0 -3 0 6 P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1 -2 -4 7 S 1
-1 1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1 4 T 0 -1 0 -1 -1 -1 -1 -2 -2 -1
-1 -1 -1 -2 -1 1 5 W -3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2 -3 -1 1 -4 -3
-2 11 Y -2 -2 -2 -3 -2 -1 -2 -3 2 -1 -1 -2 -1 3 -3 -2 -2 2 7 V 0 -3
-3 -3 -1 -2 -2 -3 -3 3 1 -2 1 -1 -2 -2 0 -3 -1 4
[0083] Sequence identity of polynucleotide molecules is determined
by similar methods using a ratio as disclosed above.
[0084] Those skilled in the art appreciate that there are many
established algorithms available to align two amino acid sequences.
The "FASTA" similarity search algorithm of Pearson and Lipman is a
suitable protein alignment method for examining the level of
identity shared by an amino acid sequence disclosed herein and the
amino acid sequence of a putative variant znssp6. The FASTA
algorithm is described by Pearson and Lipman, Proc. Nat'l Acad.
Sci. USA 85:2444 (1988), and by Pearson, Meth. Enzymol. 183:63
(1990).
[0085] Briefly, FASTA first characterizes sequence similarity by
identifying regions shared by the query sequence (e.g., SEQ ID NO:
2) and a test sequence that have either the highest density of
identities (if the ktup variable is 1) or pairs of identities (if
ktup=2), without considering conservative amino acid substitutions,
insertions, or deletions. The ten regions with the highest density
of identities are then rescored by comparing the similarity of all
paired amino acids using an amino acid substitution matrix, and the
ends of the regions are "trimmed" to include only those residues
that contribute to the highest score. If there are several regions
with scores greater than the "cutoff" value (calculated by a
predetermined formula based upon the length of the sequence and the
ktup value), then the trimmed initial regions are examined to
determine whether the regions can be joined to form an approximate
alignment with gaps. Finally, the highest scoring regions of the
two amino acid sequences are aligned using a modification of the
Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol.
Biol. 48:444 (1970); Sellers, SIAM J. Appl. Math. 26:787 (1974)),
which allows for amino acid insertions and deletions. Illustrative
parameters for FASTA analysis are: ktup=1, gap opening penalty=10,
gap extension penalty=1, and substitution matrix=BLOSUM62. These
parameters can be introduced into a FASTA program by modifying the
scoring matrix file ("SMATRIX"), as explained in Appendix 2 of
Pearson, Meth. Enzymol. 183:63 (1990).
[0086] FASTA can also be used to determine the sequence identity of
nucleic acid molecules using a ratio as disclosed above. For
nucleotide sequence comparisons, the ktup value can range between
one to six, preferably from four to six.
[0087] The present invention includes nucleic acid molecules that
encode a polypeptide having one or more conservative amino acid
changes, compared with the amino acid sequence of SEQ ID NO: 2. The
BLOSUM62 table is an amino acid substitution matrix derived from
about 2,000 local multiple alignments of protein sequence segments,
representing highly conserved regions of more than 500 groups of
related proteins (Henikoff and Henikoff, Proc. Nat'l Acad. Sci. USA
89:10915 (1992)). Accordingly, the BLOSUM62 substitution
frequencies can be used to define conservative amino acid
substitutions that may be introduced into the amino acid sequences
of the present invention. As used herein, the language
"conservative amino acid substitution" refers to a substitution
represented by a BLOSUM62 value of greater than -1. For example, an
amino acid substitution is conservative if the substitution is
characterized by a BLOSUM62 value of 0, 1, 2, or 3. Conservative
amino acid substitutions are characterized by a BLOSUM62 value of
at least 1 (e.g., 1, 2 or 3), while more conservative amino acid
substitutions are characterized by a BLOSLUM62 value of at least 2
(e.g., 2 or 3).
[0088] Variant znssp6 polypeptides or substantially homologous
znssp6 polypeptides are characterized as having one or more amino
acid substitutions, deletions or additions. These changes are
preferably of a minor nature, that is conservative amino acid
substitutions (see Table 4) and other substitutions that do not
significantly affect the folding or activity of the polypeptide;
small deletions, typically of one to about 30 amino acids; and
small amino- or carboxyl-terminal extensions, such as an
amino-terminal methionine residue, a small linker peptide of up to
about 20-25 residues, or an affinity tag. The present invention
thus includes polypeptides of from 378 to 410 amino acid residues
that comprise a sequence that is at least 70%, preferably at least
80%, and more preferably 90% or more identical to the corresponding
region of SEQ ID NO: 2. Polypeptides comprising affinity tags can
further comprise a proteolytic cleavage site between the znssp6
polypeptide and the affinity tag. Preferred such sites include
thrombin cleavage sites and factor Xa cleavage sites.
4TABLE 4 Conservative amino acid substitutions Basic: arginine
lysine histidine Acidic: glutamic acid aspartic acid Polar:
glutamine asparagine Hydrophobic: leucine isoleucine valine
Aromatic: phenylalanine tryptophan tyrosine Small: glycine alanine
serine threonine methionine
[0089] The present invention further provides a variety of other
polypeptide fusions and related multimeric proteins comprising one
or more polypeptide fusions. For example, a znssp6 polypeptide can
be prepared as a fusion to a dimerizing protein as disclosed in
U.S. Pat. Nos. 5,155,027 and 5,567,584. Preferred dimerizing
proteins in this regard include immunoglobulin heavy chain constant
region domains.
[0090] Immunoglobulin-znssp6 polypeptide fusions can be expressed
in genetically engineered cells to produce a variety of multimeric
znssp6 analogs wherein the Fc portions are disulfide bonded to each
other and two non-Ig polypeptides are arrayed in closed proximity
to each other. Fusions of this type can be used to evaluate
specific donor/acceptor molecules, affinity purify ligands, or use
as an in vitro assay tool. This fusion can also be used to
determine the homodimerization potential for znssp6. For use in
assays, the chimeras are bound to a support via the Fc region and
used in an ELISA format.
[0091] Auxiliary domains can be fused to znssp6 polypeptides to
target them to specific cells, tissues, or macromolecules (e.g.,
brain, kidney, and testis, for example).
[0092] For example, a protease, or ablation antibody polypeptide or
protein could be targeted to a predetermined cell type by fusing a
said protease, or ablation antibody polypeptide to a ligand that
specifically binds to a receptor or receptor-like complementary
molecule on the surface of the target cell, such as, brain, kidney,
and testis. In this way, polypeptides and proteins can be targeted
for therapeutic or diagnostic purposes. Such
beta-1,3-galactosyltransferase polypeptides can be fused to two or
more moieties, such as an affinity tag for purification and a
targeting domain. Polypeptide fusions can also comprise one or more
cleavage sites, particularly between domains. See, Tuan et al.,
Connective Tissue Research 34:1-9, 1996.
[0093] Polypeptide fusions of the present invention will generally
contain not more than about 1,700 amino acid residues, not more
than about 1,200 residues, or not more than about 1,000 residues,
and will in many cases be considerably smaller. For example,
residues of znssp6 polypeptide can be fused to E. coli
.beta.-galactosidase (1,021 residues; see Casadaban et al., J.
Bacteriol. 143:971-980, 1980), a 10-residue spacer, and a 4-residue
factor Xa cleavage site. In a second example, residues of znssp6
polypeptide can be fused to maltose binding protein (approximately
370 residues), a 4-residue cleavage site, and a 6-residue
polyhistidine tag.
[0094] Some proteins in the .beta.3Gal-T family been shown to be
expressed intracellulary and are involved in intracellular
glycoprotein and glycolipid processing. Other members of this
family have been shown to be extracellularly expressed and are
involved in glycoprotein and glycolipid processing (such as in the
case of the Tn antigen). Other members of the family are expressed
extracellularly and are involved in cell-cell interactions and
intracellular signaling. Thus, molecules of the present invention
can function as an enzyme both intracellularly and extracellulary,
in which case its anti-complementary molecule is a substrate.
Additionally, molecules of the present invention can function
extracellularly and modulate cell-cell interactions. The
extracellular binding of znssp6 to its anti-complementary molecule
can cause a cellular event in the cell that is expressing it (i.e.
znssp6 acts as a receptor or receptor-like molecule), or in the
cell expressing the anti-complementary molecule to which it binds
(i.e., znssp6 acts as a ligand). Additionally, znssp6 can function
extracellularly as a soluble enzyme, ligand, receptor or
receptor-like molecule. Similarly, as an extracellulary expressed
znssp6 enzyme, the processing of its anti-complementary substrate
can result in a cellular response (similar to intracellular
signaling) in the cell expressing the substrate. Also as an
extracellularly expressed protein, znssp6 can function to form a
"bridge" between cells maintaining their proximity to each
other.
[0095] Thus, for the purposes of this application, znssp6 is
referred to as a complementary molecule and its cognate binding
partner is referred to as an anti-complementary molecule.
[0096] The invention also provides soluble znssp6 polypeptides,
used to form fusion or chimeric proteins with human Ig, as
His-tagged proteins, or FLAG.TM.-tagged proteins. One such
construct is comprises residues 50 to 370 of SEQ ID NO: 2, fused to
human Ig. znssp6 or znssp6-Ig chimeric proteins are used, for
example, to identify the znssp6 anti-complementary molecule,
including the natural anti-complementary molecule, as well as
agonists and antagonists of the natural anti-complementary
molecule. Using labeled soluble znssp6, cells expressing the
anti-complementary molecule are identified by fluorescence
immunocytometry or immunohistochemistry. The soluble fusion
proteins or soluble Ig fusion protein is useful in studying the
distribution of the anti-complementary molecule on tissues or
specific cell lineages, and to provide insight into complementary
molecule-anti-complementary molecule biology.
[0097] In an alternative approach, a soluble znssp6 extracellular
anti-complementary molecule-binding region can be expressed as a
chimera with immunoglobulin heavy chain constant regions, typically
an Fc fragment, which contains two constant region domains and a
hinge region, but lacks the variable region. Such fusions are
typically secreted as multimeric molecules, wherein the Fc portions
are disulfide bonded to each other and two znssp6 polypeptides are
arrayed in close proximity to each other. Fusions of this type can
be used to affinity purify the cognate anti-complementary molecule
from solution, as an in vitro assay tool, to block signals in vitro
by specifically titrating out anti-complementary molecule, and as
antagonists in vivo by administering them to block the
anti-complementary molecule stimulation. To purify
anti-complementary molecule, a znssp6-Ig fusion protein (chimera)
is added to a sample containing the anti-complementary molecule
under conditions that facilitate complementary
moleucle-anti-complementary molecule binding (typically
near-physiological temperature, pH, and ionic strength). The
chimera-anti-complementary molecule complex is then separated by
the mixture using protein A, which is immobilized on a solid
support (e.g., insoluble resin beads). The anti-complementary
molecule is then eluted using conventional chemical techniques,
such as with a salt or pH gradient. In the alternative, the chimera
itself can be bound to a solid support, with binding and elution
carried out as above. For use in assays, the chimeras are bound to
a support via the F.sub.c region and used in an ELISA format.
[0098] The present invention also includes "functional fragments"
of znssp6 polypeptides and nucleic acid molecules encoding such
functional fragments. Routine deletion analyses of nucleic acid
molecules can be performed to obtain functional fragments of a
nucleic acid molecule that encodes a znssp6 polypeptide. As an
illustration, DNA molecules having the nucleotide sequence of SEQ
ID NO: 1 can be digested with Bal31 nuclease to obtain a series of
nested deletions. The fragments are then inserted into expression
vectors in proper reading frame, and the expressed polypeptides are
isolated and tested for cell-cell interactions, or for the ability
to bind anti-znssp6 antibodies. One alternative to exonuclease
digestion is to use oligonucleotide-directed mutagenesis to
introduce deletions or stop codons to specify production of a
desired fragment. Alternatively, particular fragments of an znssp6
gene can be synthesized using the polymerase chain reaction.
[0099] Standard methods for identifying functional domains are
well-known to those of skill in the art. For example, studies on
the truncation at either or both termini of interferons have been
summarized by Horisberger and Di Marco, Pharmac. Ther. 66:507
(1995). Moreover, standard techniques for functional analysis of
proteins are described by, for example, Treuter et al., Molec. Gen.
Genet. 240:113 (1993), Content et al., "Expression and preliminary
deletion analysis of the 42 kDa 2-5A synthetase induced by human
interferon," in Biological Interferon Systems, Proceedings of
ISIR-TNO Meeting on Interferon Systems, Cantell (ed.), pages 65-72
(Nijhoff 1987), Herschman, "The EGF Enzyme," in Control of Animal
Cell Proliferation, Vol. 1, Boynton et al., (eds.) pages 169-199
(Academic Press 1985), Coumailleau et al., J. Biol. Chem. 270:29270
(1995); Fukunaga et al., J. Biol. Chem. 270:25291 (1995); Yamaguchi
et al., Biochem. Pharmacol. 50:1295 (1995), and Meisel et al.,
Plant Molec. Biol. 30:1 (1996).
[0100] The present invention also contemplates functional fragments
of an znssp6 gene that has amino acid changes, compared with the
amino acid sequence of SEQ ID NO: 2. A variant znssp6 gene can be
identified on the basis of structure by determining the level of
identity with nucleotide and amino acid sequences of SEQ ID NOs: 1
and 2, as discussed above. An alternative approach to identifying a
variant gene on the basis of structure is to determine whether a
nucleic acid molecule encoding a potential variant znssp6 gene can
hybridize to a nucleic acid molecule having the nucleotide sequence
of SEQ ID NO: 1, as discussed above.
[0101] The proteins of the present invention can also comprise
non-naturally occurring amino acid residues. Non-naturally
occurring amino acids include, without limitation,
trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline,
trans-4-hydroxyproline, N-methylglycine, allo-threonine,
methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine,
nitroglutamine, homoglutamine, pipecolic acid, thiazolidine
carboxylic acid, dehydroproline, 3- and 4-methylproline,
3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenylalanine,
3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine.
Several methods are known in the art for incorporating
non-naturally occurring amino acid residues into proteins. For
example, an in vitro system can be employed wherein nonsense
mutations are suppressed using chemically aminoacylated suppressor
tRNAs. Methods for synthesizing amino acids and aminoacylating tRNA
are known in the art. Transcription and translation of plasmids
containing nonsense mutations is carried out in a cell-free system
comprising an E. coli S30 extract and commercially available
enzymes and other reagents. Proteins are purified by
chromatography. See, for example, Robertson et al., J. Am. Chem.
Soc. 113:2722, 1991; Ellman et al., Methods Enzymol. 202:301, 1991;
Chung et al., Science 259:806-9, 1993; and Chung et al., Proc.
Natl. Acad. Sci. USA 90:10145-9, 1993). In a second method,
translation is carried out in Xenopus oocytes by microinjection of
mutated mRNA and chemically aminoacylated suppressor tRNAs
(Turcatti. et al., J. Biol. Chem. 271:19991-8, 1996). Within a
third method, E. coli cells are cultured in the absence of a
natural amino acid that is to be replaced (e.g., phenylalanine) and
in the presence of the desired non-naturally occurring amino
acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine,
4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturally
occurring amino acid is incorporated into the protein in place of
its natural counterpart. See, Koide et al., Biochem. 33:7470-6,
1994. Naturally occurring amino acid residues can be converted to
non-naturally occurring species by in vitro chemical modification.
Chemical modification can be combined with site-directed
mutagenesis to further expand the range of substitutions (Wynn and
Richards, Protein Sci. 2:395-403, 1993).
[0102] A limited number of non-conservative amino acids, amino
acids that are not encoded by the genetic code, non-naturally
occurring amino acids, and unnatural amino acids may be substituted
for znssp6 amino acid residues.
[0103] Essential amino acids in the polypeptides of the present
invention can be identified according to procedures known in the
art, such as site-directed mutagenesis or alanine-scanning
mutagenesis (Cunningham and Wells, Science 244: 1081-5, 1989; Bass
et al., Proc. Natl. Acad. Sci. USA 88:4498-502, 1991). In the
latter technique, single alanine mutations are introduced at every
residue in the molecule, and the resultant mutant molecules are
tested for biological activity as disclosed below to identify amino
acid residues that are critical to the activity of the molecule.
See also, Hilton et al., J. Biol. Chem. 271:4699-708, 1996. Sites
of galactosyltransferase, or znssp6 anti-complementary molecule
binding activity can also be determined by physical analysis of
structure, as determined by such techniques as nuclear magnetic
resonance, crystallography, electron diffraction or photoaffinity
labeling, in conjunction with mutation of putative contact site
amino acids. See, for example, de Vos et al., Science 255:306-12,
1992; Smith et al., J. Mol. Biol. 224:899-904, 1992; Wlodaver et
al., FEBS Lett. 309:59-64, 1992. The identities of essential amino
acids can also be inferred from analysis of homologies with related
galactosyltransferase molecules.
[0104] Multiple amino acid substitutions can be made and tested
using known methods of mutagenesis and screening, such as those
disclosed by Reidhaar-Olson and Sauer (Science 241:53-7, 1988) or
Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-6, 1989).
Briefly, these authors disclose methods for simultaneously
randomizing two or more positions in a polypeptide, selecting for
functional polypeptide, and then sequencing the mutagenized
polypeptides to determine the spectrum of allowable substitutions
at each position. Other methods that can be used include phage
display (e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner et
al., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO 92/06204)
and region-directed mutagenesis (Derbyshire et al., Gene 46:145,
1986; Ner et al., DNA 7:127, 1988).
[0105] Variants of the disclosed znssp6 DNA and polypeptide
sequences can be generated through DNA shuffling as disclosed by
Stemmer, Nature 370:389-91, 1994, Stemmer, Proc. Natl. Acad. Sci.
USA 91:10747-51, 1994 and WIPO Publication WO 97/20078. Briefly,
variant DNAs are generated by in vitro homologous recombination by
random fragmentation of a parent DNA followed by reassembly using
PCR, resulting in randomly introduced point mutations. This
technique can be modified by using a family of parent DNAs, such as
allelic variants or DNAs from different species, to introduce
additional variability into the process. Selection or screening for
the desired activity, followed by additional iterations of
mutagenesis and assay provides for rapid "evolution" of sequences
by selecting for desirable mutations while simultaneously selecting
against detrimental changes.
[0106] Mutagenesis methods as disclosed herein can be combined with
high-throughput, automated screening methods to detect activity of
cloned, mutagenized polypeptides in host cells. Mutagenized DNA
molecules that encode active polypeptides (e.g.,
galactosyltransferase activity as evidenced by glycoprotein
synthesis, or cell-cell interactions) can be recovered from the
host cells and rapidly sequenced using modem equipment. These
methods allow the rapid determination of the importance of
individual amino acid residues in a polypeptide of interest, and
can be applied to polypeptides of unknown structure.
[0107] Regardless of the particular nucleotide sequence of a
variant znssp6 gene, the gene encodes a polypeptide that is
characterized by its anti-complementary molecule binding activity,
or by the ability to bind specifically to an anti-znssp6 antibody.
More specifically, variant znssp6 genes encode polypeptides which
exhibit greater than 75, 80, or 90%, of the activity of polypeptide
encoded by the human znssp6 gene described herein.
[0108] Using the methods discussed herein, one of ordinary skill in
the art can identify and/or prepare a variety of polypeptide
fragments or variants of SEQ ID NO: 2 or that retain the
galactsyltransferase properties, or cell-cell interactions of the
wild-type znssp6 protein. Such polypeptides may include additional
amino acids from, for example, an extracellular anti-complementary
molecule-binding domain of another member of the
galactosyltransferase family as well as part or all of the
transmembrane and intracellular domains. Additionally fragments of
znssp6 may include additional amino acids from the catalytic site
of the galactosyltransferase domains of other family members.
Additional amino acids from affinity tags and the like may also be
included.
[0109] For any znssp6 polypeptide, including variants and fusion
proteins, one of ordinary skill in the art can readily generate a
fully degenerate polynucleotide sequence encoding that variant
using the information set forth in Tables 1 and 2 above.
[0110] The znssp6 polypeptides of the present invention, including
full-length polypeptides, biologically active fragments, and fusion
polypeptides, can be produced in genetically engineered host cells
according to conventional techniques. Suitable host cells are those
cell types that can be transformed or transfected with exogenous
DNA and grown in culture, and include bacteria, fungal cells, and
cultured higher eukaryotic cells. Eukaryotic cells, particularly
cultured cells of multicellular organisms, are preferred.
Techniques for manipulating cloned DNA molecules and introducing
exogenous DNA into a variety of host cells are disclosed by
Sambrook et al., Molecular Cloning: A Laboratory Manual, 2.sup.nd
ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,
1989, and Ausubel et al., eds., Current Protocols in Molecular
Biology, John Wiley and Sons, Inc., NY, 1987.
[0111] In general, a DNA sequence encoding a znssp6 polypeptide is
operably linked to other genetic elements required for its
expression, generally including a transcription promoter and
terminator, within an expression vector. The vector will also
commonly contain one or more selectable markers and one or more
origins of replication, although those skilled in the art will
recognize that within certain systems selectable markers may be
provided on separate vectors, and replication of the exogenous DNA
may be provided by integration into the host cell genome. Selection
of promoters, terminators, selectable markers, vectors and other
elements is a matter of routine design within the level of ordinary
skill in the art. Many such elements are described in the
literature and are available through commercial suppliers.
[0112] To direct a znssp6 polypeptide into the secretory pathway of
a host cell, a secretory signal sequence (also known as a leader
sequence, prepro sequence or pre sequence) is provided in the
expression vector. The secretory signal sequence may be that of
znssp6, or may be derived from another secreted protein (e.g.,
t-PA) or synthesized de novo. The secretory signal sequence is
operably linked to the znssp6 DNA sequence, i.e., the two sequences
are joined in the correct reading frame and positioned to direct
the newly synthesized polypeptide into the secretory pathway of the
host cell. Secretory signal sequences are commonly positioned 5' to
the DNA sequence encoding the polypeptide of interest, although
certain secretory signal sequences may be positioned elsewhere in
the DNA sequence of interest (see, e.g., Welch et al., U.S. Pat.
No. 5,037,743; Holland et al., U.S. Pat. No. 5,143,830).
[0113] The native secretory signal sequence of the polypeptides of
the present invention is used to direct other polypeptides into the
secretory pathway. The present invention provides for such fusion
polypeptides. A signal fusion polypeptide can be made wherein a
secretory signal sequence derived from a znssp6 polypeptide is
operably linked to another polypeptide using methods known in the
art and disclosed herein. The secretory signal sequence contained
in the fusion polypeptides of the present invention is preferably
fused amino-terminally to an additional peptide to direct the
additional peptide into the secretory pathway. Such constructs have
numerous applications known in the art. For example, these novel
secretory signal sequence fusion constructs can direct the
secretion of an active component of a normally non-secreted
protein, such as a receptor. Such fusions may be used in vivo or in
vitro to direct peptides through the secretory pathway.
[0114] Cultured mammalian cells are suitable hosts within the
present invention. Methods for introducing exogenous DNA into
mammalian host cells include calcium phosphate-mediated
transfection (Wigler et al., Cell 14:725, 1978; Corsaro and
Pearson, Somatic Cell Genetics 7:603, 1981: Graham and Van der Eb,
Virology 52:456, 1973), electroporation (Neumann et al., EMBO J.
1:841-5, 1982), DEAE-dextran mediated transfection (Ausubel et al.,
ibid.), and liposome-mediated transfection (Hawley-Nelson et al.,
Focus 15:73, 1993; Ciccarone et al., Focus 15:80, 1993, and viral
vectors (Miller and Rosman, BioTechniques 7:980-90, 1989; Wang and
Finer, Nature Med. 2:714-6, 1996). The production of recombinant
polypeptides in cultured mammalian cells is disclosed, for example,
by Levinson et al., U.S. Pat. No. 4,713,339; Hagen et al., U.S.
Pat. No. 4,784,950; Palmiter et al., U.S. Pat. No. 4,579,821; and
Ringold, U.S. Pat. No. 4,656,134. Suitable cultured mammalian cells
include the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651),
BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC
No. CRL 1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) and
Chinese hamster ovary (e.g. CHO-K1; ATCC No. CCL 61) cell lines.
Additional suitable cell lines are known in the art and available
from public depositories such as the American Type Culture
Collection, Rockville, Md. In general, strong transcription
promoters are preferred, such as promoters from SV-40 or
cytomegalovirus. See, e.g., U.S. Pat. No. 4,956,288. Other suitable
promoters include those from metallothionein genes (U.S. Pat. Nos.
4,579,821 and 4,601,978) and the adenovirus major late
promoter.
[0115] Drug selection is generally used to select for cultured
mammalian cells into which foreign DNA has been inserted. Such
cells are commonly referred to as "transfectants". Cells that have
been cultured in the presence of the selective agent and are able
to pass the gene of interest to their progeny are referred to as
"stable transfectants." A preferred selectable marker is a gene
encoding resistance to the antibiotic neomycin. Selection is
carried out in the presence of a neomycin-type drug, such as G-418
or the like. Selection systems can also be used to increase the
expression level of the gene of interest, a process referred to as
"amplification." Amplification is carried out by culturing
transfectants in the presence of a low level of the selective agent
and. then increasing the amount of selective agent to select for
cells that produce high levels of the products of the introduced
genes. A preferred amplifiable selectable marker is dihydrofolate
reductase, which confers resistance to methotrexate. Other drug
resistance genes (e.g. hygromycin resistance, multi-drug
resistance, puromycin acetyltransferase) can also be used.
Alternative markers that introduce an altered phenotype, such as
green fluorescent protein, or cell surface proteins such as CD4,
CD8, Class I MHC, placental alkaline phosphatase may be used to
sort transfected cells from untransfected cells by such means as
FACS sorting or magnetic bead separation technology.
[0116] Other higher eukaryotic cells can also be used as hosts,
including plant cells, insect cells and avian cells. The use of
Agrobacterium rhizogenes as a vector for expressing genes in plant
cells has been reviewed by Sinkar et al., J. Biosci. (Bangalore)
11:47-58, 1987. Transformation of insect cells and production of
foreign polypeptides therein is disclosed by Guarino et al., U.S.
Pat. No. 5,162,222 and WIPO publication WO 94/06463. Insect cells
can be infected with recombinant baculovirus, commonly derived from
Autographa californica nuclear polyhedrosis virus (AcNPV). See,
King, L. A. and Possee, R. D., The Baculovirus Expression System: A
Laboratory Guide, London, Chapman & Hall; O'Reilly, D. R. et
al., Baculovirus Expression Vectors: A Laboratory Manual, New York,
Oxford University Press., 1994; and, Richardson, C. D., Ed.,
Baculovirus Expression Protocols. Methods in Molecular Biology,
Totowa, N.J., Humana Press, 1995. A second method of making
recombinant znssp6 baculovirus utilizes a transposon-based system
described by Luckow (Luckow, V. A, et al., J. Virol. 67:4566-79,
1993). This system, which utilizes transfer vectors, is sold in the
Bac-to-Bac.TM. kit (Life Technologies, Rockville, Md.). This system
utilizes a transfer vector, pFastBac1.TM. (Life Technologies)
containing a Tn7 transposon to move the DNA encoding the znssp6
polypeptide into a baculovirus genome maintained in E. coli as a
large plasmid called a "bacmid." The pFastBac1.TM. transfer vector
utilizes the AcNPV polyhedrin promoter to drive the expression of
the gene of interest, in this case znssp6. However, pFastBac1.TM.
can be modified to a considerable degree. The polyhedrin promoter
can be removed and substituted with the baculovirus basic protein
promoter (also known as Pcor, p6.9 or MP promoter) which is
expressed earlier in the baculovirus infection, and has been shown
to be advantageous for expressing secreted proteins. See,
Hill-Perkins, M. S. and Possee, R. D., J. Gen. Virol. 71:971-6,
1990; Bonning, B. C. et al., J. Gen. Virol. 75:1551-6, 1994; and,
Chazenbalk, G. D., and Rapoport, B., J. Biol. Chem. 270:1543-9,
1995. In such transfer vector constructs, a short or long version
of the basic protein promoter can be used. Moreover, transfer
vectors can be constructed which replace the native znssp6
secretory signal sequences with secretory signal sequences derived
from insect proteins. For example, a secretory signal sequence from
Ecdysteroid Glucosyltransferase (EGT), honey bee Melittin
(Invitrogen, Carlsbad, Calif.), or baculovirus gp67 (PharMingen,
San Diego, Calif.) can be used in constructs to replace the native
znssp6 secretory signal sequence. In addition, transfer vectors can
include an in-frame fusion with DNA encoding an epitope tag at the
C- or N-terminus of the expressed znssp6 polypeptide, for example,
a Glu-Glu epitope tag (Grussenmeyer, T. et al., Proc. Natl. Acad.
Sci. 82:7952-4, 1985). Using a technique known in the art, a
transfer vector containing znssp6 is transformed into E. coli, and
screened for bacmids which contain an interrupted lacZ gene
indicative of recombinant baculovirus. The bacmid DNA containing
the recombinant baculovirus genome is isolated, using common
techniques, and used to transfect Spodoptera frugiperda cells, e.g.
Sf9 cells. Recombinant virus that expresses znssp6 is subsequently
produced. Recombinant viral stocks are made by methods commonly
used in the art.
[0117] The recombinant virus is used to infect host cells,
typically a cell line derived from the fall armyworm, Spodoptera
frugiperda. See, in general, Glick and Pasternak, Molecular
Biotechnology: Principles and Applications of Recombinant DNA, ASM
Press, Washington, D.C., 1994. Another suitable cell line is the
High FiveO.TM. cell line (Invitrogen) derived from Trichoplusia ni
(U.S. Pat. No. 5,300,435). Commercially available serum-free media
are used to grow and maintain the cells. Suitable media are Sf900
II.TM. (Life Technologies) or ESF 921.TM. (Expression Systems) for
the Sf9 cells; and Ex-cellO405TM (JRH Biosciences, Lenexa, Kans.)
or Express FiveO.TM. (Life Technologies) for the T. ni cells. The
cells are grown up from an inoculation density of approximately
2-5.times.10.sup.5 cells to a density of 1-2.times.10.sup.6 cells
at which time a recombinant viral stock is added at a multiplicity
of infection (MOI) of 0.1 to 10, more typically near 3. Procedures
used are generally described in available laboratory manuals (King,
L. A. and Possee, R. D., ibid.; O'Reilly, D. R. et al., ibid.;
Richardson, C. D., ibid.). Subsequent purification of the znssp6
polypeptide from the supernatant can be achieved using methods
described herein.
[0118] Fungal cells, including yeast cells, can also be used within
the present invention. Yeast species of particular interest in this
regard include Saccharomyces cerevisiae, Pichia pastoris, and
Pichia methanolica. Methods for transforming S. cerevisiae cells
with exogenous DNA and producing recombinant polypeptides therefrom
are disclosed by, for example, Kawasaki, U.S. Pat. No. 4,599,311;
Kawasaki et al., U.S. Pat. No. 4,931,373; Brake, U.S. Pat. No.
4,870,008; Welch et al., U.S. Pat. No. 5,037,743; and Murray et
al., U.S. Pat. No. 4,845,075. Transformed cells are selected by
phenotype determined by the selectable marker, commonly drug
resistance or the ability to grow in the absence of a particular
nutrient (e.g., leucine). A preferred vector system for use in
Saccharomyces cerevisiae is the POTI vector system disclosed by
Kawasaki et al. (U.S. Pat. No. 4,931,373), which allows transformed
cells to be selected by growth in glucose-containing media.
Suitable promoters and terminators for use in yeast include those
from glycolytic enzyme genes (see, e.g., Kawasaki, U.S. Pat. No.
4,599,311; Kingsman et al., U.S. Pat. No. 4,615,974; and Bitter,
U.S. Pat. No. 4,977,092) and alcohol dehydrogenase genes. See also
U.S. Pat. Nos. 4,990,446; 5,063,154; 5,139,936 and 4,661,454.
Transformation systems for other yeasts, including Hansenula
polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis,
Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia
methanolica, Pichia guillermondii and Candida maltosa are known in
the art. See, for example, Gleeson et al., J. Gen. Microbiol.
132:3459-65, 1986 and Cregg, U.S. Pat. No. 4,882,279. Aspergillus
cells may be utilized according to the methods of McKnight et al.,
U.S. Pat. No. 4,935,349. Methods for transforming Acremonium
chrysogenum are disclosed by Sumino et al., U.S. Pat. No.
5,162,228. Methods for transforming Neurospora are disclosed by
Lambowitz, U.S. Pat. No. 4,486,533.
[0119] The use of Pichia methanolica as host for the production of
recombinant proteins is disclosed in WIPO Publications WO 97/17450,
WO 97/17451, WO 98/02536, and WO 98/02565. DNA molecules for use in
transforming P. methanolica will commonly be prepared as
double-stranded, circular plasmids, which are preferably linearized
prior to transformation. For polypeptide production in P.
methanolica, it is preferred that the promoter and terminator in
the plasmid be that of a P. methanolica gene, such as a P.
methanolica alcohol utilization gene (AUG1 or AUG2). Other useful
promoters include those of the dihydroxyacetone synthase (DHAS),
formate dehydrogenase (FMD), and catalase (CAT) genes. To
facilitate integration of the DNA into the host chromosome, it is
preferred to have the entire expression segment of the plasmid
flanked at both ends by host DNA sequences. A preferred selectable
marker for use in Pichia methanolica is a P. methanolica ADE2 gene,
which encodes phosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC
4.1.1.21), which allows ade2 host cells to grow in the absence of
adenine. For large-scale, industrial processes where it is
desirable to minimize the use of methanol, it is preferred to use
host cells in which both methanol utilization genes (AUG1 and AUG2)
are deleted. For production of secreted proteins, host cells
deficient in vacuolar protease genes (PEP4 and PRB1) are preferred.
Electroporation is used to facilitate the introduction of a plasmid
containing DNA encoding a polypeptide of interest into P.
methanolica cells. It is preferred to transform P. methanolica
cells by electroporation using an exponentially decaying, pulsed
electric field having a field strength of from 2.5 to 4.5 kV/cm,
preferably about 3.75 kV/cm, and a time constant (t) of from 1 to
40 milliseconds, most preferably about 20 milliseconds.
[0120] Prokaryotic host cells, including strains of the bacteria
Escherichia coli, Bacillus and other genera are also useful host
cells within the present invention. Techniques for transforming
these hosts and expressing foreign DNA sequences cloned therein are
well known in the art (see, e.g., Sambrook et al., ibid.). When
expressing a znssp6 polypeptide in bacteria such as E. coli, the
polypeptide may be retained in the cytoplasm, typically as
insoluble granules, or may be directed to the periplasmic space by
a bacterial secretion sequence. In the former case, the cells are
lysed, and the granules are recovered and denatured using, for
example, guanidine isothiocyanate or urea. The denatured
polypeptide can then be refolded and dimerized by diluting the
denaturant, such as by dialysis against a solution of urea and a
combination of reduced and oxidized glutathione, followed by
dialysis against a buffered saline solution. In the latter case,
the polypeptide can be recovered from the periplasmic space in a
soluble and functional form by disrupting the cells (by, for
example, sonication or osmotic shock) to release the contents of
the periplasmic space and recovering the protein, thereby obviating
the need for denaturation and refolding.
[0121] Transformed or transfected host cells are cultured according
to conventional procedures in a culture medium containing nutrients
and other components required for the growth of the chosen host
cells. A variety of suitable media, including defined media and
complex media, are known in the art and generally include a carbon
source, a nitrogen source, essential amino acids, vitamins and
minerals. Media may also contain such components as growth factors
or serum, as required. The growth medium will generally select for
cells containing the exogenously added DNA by, for example, drug
selection or deficiency in an essential nutrient which is
complemented by the selectable marker carried on the expression
vector or co-transfected into the host cell. P. methanolica cells
are cultured in a medium comprising adequate sources of carbon,
nitrogen and trace nutrients at a temperature of about 25.degree.
C. to 35.degree. C. Liquid cultures are provided with sufficient
aeration by conventional means, such as shaking of small flasks or
sparging of fermentors. A preferred culture medium for P.
methanolica is YEPD (2% D-glucose, 2% Bacto.TM. Peptone (Difco
Laboratories, Detroit, Mich.), 1% Bacto.TM. yeast extract (Difco
Laboratories), 0.004% adenine and 0.006% L-leucine).
[0122] It is preferred to purify the polypeptides of the present
invention to .gtoreq.80% purity, more preferably to .gtoreq.90%
purity, even more preferably .gtoreq.95% purity, and particularly
preferred is a pharmaceutically pure state, that is greater than
99.9% pure with respect to contaminating macromolecules,
particularly other proteins and nucleic acids, and free of
infectious and pyrogenic agents. Preferably, a purified polypeptide
is substantially free of other polypeptides, particularly other
polypeptides of animal origin.
[0123] Expressed recombinant znssp6 polypeptides (or chimeric
znssp6 polypeptides) can be purified using fractionation and/or
conventional purification methods and media. Ammonium sulfate
precipitation and acid or chaotrope extraction may be used for
fractionation of samples. Exemplary purification steps may include
hydroxyapatite, size exclusion, FPLC and reverse-phase high
performance liquid chromatography. Suitable chromatographic media
include derivatized dextrans, agarose, cellulose, polyacrylamide,
specialty silicas, and the like. PEI, DEAE, QAE and Q derivatives
are preferred. Exemplary chromatographic media include those media
derivatized with phenyl, butyl, or octyl groups, such as
Phenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas,
Montgomeryville, Pa.), Octyl-Sepharose (Pharmacia) and the like; or
polyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the
like. Suitable solid supports include glass beads, silica-based
resins, cellulosic resins, agarose beads, cross-linked agarose
beads, polystyrene beads, cross-linked polyacrylamide resins and
the like that are insoluble under the conditions in which they are
to be used. These supports may be modified with reactive groups
that allow attachment of proteins by amino groups, carboxyl groups,
sulfhydryl groups, hydroxyl groups and/or carbohydrate moieties.
Examples of coupling chemistries include cyanogen bromide
activation, N-hydroxysuccinimide activation, epoxide activation,
sulfhydryl activation, hydrazide activation, and carboxyl and amino
derivatives for carbodiimide coupling chemistries. These and other
solid media are well known and widely used in the art, and are
available from commercial suppliers. Methods for binding receptor
and receptor-like complementary polypeptides to support media are
well known in the art. Selection of a particular method is a matter
of routine design and is determined in part by the properties of
the chosen support. See, for example, Affinity Chromatography:
Principles & Methods, Pharmacia LKB Biotechnology, Uppsala,
Sweden, 1988.
[0124] The polypeptides of the present invention can be isolated by
a combination of procedures including, but not limited to, anion
and cation exchange chromatography, size exclusion, and affinity
chromatography. For example, immobilized metal ion adsorption
(IMAC) chromatography can be used to purify histidine-rich
proteins, including those comprising polyhistidine tags. Briefly, a
gel is first charged with divalent metal ions to form a chelate
(Sulkowski, Trends in Biochem. 3:1-7, 1985). Histidine-rich
proteins will be adsorbed to this matrix with differing affinities,
depending upon the metal ion used, and will be eluted by
competitive elution, lowering the pH, or use of strong chelating
agents. Other methods of purification include purification of
glycosylated proteins by lectin affinity chromatography and ion
exchange chromatography (Methods in Enzymol., Vol. 182, "Guide to
Protein Purification", M. Deutscher, (ed.), Acad. Press, San Diego,
1990, pp.529-39). Within additional embodiments of the invention, a
fusion of the polypeptide of interest and an affinity tag (e.g.,
maltose-binding protein, an immunoglobulin domain) may be
constructed to facilitate purification.
[0125] To direct the export of a znssp6 polypeptide from the host
cell, the znssp6 DNA is linked to a second DNA segment encoding a
secretory peptide, such as a t-PA secretory peptide or a znssp6
secretory peptide. To facilitate purification of the secreted
znssp6 polypeptide, a C-terminal extension, such as a
poly-histidine tag, substance P, Flag peptide (Hopp et al.,
Bio/Technology 6:1204-1210, 1988; available from Eastman Kodak Co.,
New Haven, Conn.) or another polypeptide or protein for which an
antibody or other specific binding agent is available, can be fused
to the znssp6 polypeptide.
[0126] Moreover, using methods described in the art, polypeptide
fusions, or hybrid znssp6 proteins, are constructed using regions
or domains of the inventive znssp6 in combination with those of
other human galactosyltransferase family proteins (e.g. HSGALT3,
HSGALT4, .beta.3Gal-T2, and .beta.3Gal-T3, or human homologs to the
human ortholog of Brainiac), or heterologous proteins (Sambrook et
al., ibid., Altschul et al., ibid., Picard, Cur. Opin. Biology,
5:511-5, 1994, and references therein). These methods allow the
determination of the biological importance of larger domains or
regions in a polypeptide of interest. Such hybrids may alter
reaction kinetics, binding, constrict or expand the
anti-complementary molecule specificity, or alter tissue and
cellular localization of a polypeptide, and can be applied to
polypeptides of unknown structure.
[0127] Fusion proteins can be prepared by methods known to those
skilled in the art by preparing each component of the fusion
protein and chemically conjugating them. Alternatively, a
polynucleotide encoding both components of the fusion protein in
the proper reading frame can be generated using known techniques
and expressed by the methods described herein. For example, part or
all of a domain(s) conferring a biological function may be swapped
between znssp6 of the present invention with the functionally
equivalent domain(s) from another family member, such as the human
species ortholog of Brainiac, or other galactosyltransferases, etc.
Such domains include, but are not limited to, the hydrophobic
region thought to be a putative secretory signal sequence or
transmembrane domain (residues 26 to 49 of SEQ ID NO: 2), them stem
or linker domain (residues 50 to 113 of SEQ ID NO: 2), and other
conserved motifs such as the beta-1,3-galactosyltransferase
homology region (residues 114 to 378 of SEQ ID NO: 2), and
significant domains or regions in this family. Such fusion proteins
would be expected to have a biological functional profile that is
the same or similar to polypeptides of the present invention or
other known galactosyltransferase family proteins (e.g. HSGALT3,
HSGALT4, and Brainiac), depending on the fusion constructed.
Moreover, such fusion proteins may exhibit other properties as
disclosed herein.
[0128] Znssp6 polypeptides or fragments thereof may also be
prepared through chemical synthesis. Znssp6 polypeptides may be
monomers or multimers; glycosylated or non-glycosylated; pegylated
or non-pegylated; and may or may not include an initial methionine
amino acid residue.
[0129] Znssp6 polypeptides of the present invention can also be
synthesized by exclusive solid phase synthesis, partial solid phase
methods, fragment condensation or classical solution synthesis. The
polypeptides are preferably prepared by solid phase peptide
synthesis, for example as described by Merrifield, J. Am. Chem.
Soc. 85:2149, 1963. The synthesis is carried out with amino acids
that are protected at the alpha-amino terminus. Trifunctional amino
acids with labile side-chains are also protected with suitable
groups to prevent undesired chemical reactions from occurring
during the assembly of the polypeptides. The alpha-amino protecting
group is selectively removed to allow subsequent reaction to take
place at the amino-terminus. The conditions for the removal of the
alpha-amino protecting group do not remove the side-chain
protecting groups.
[0130] The alpha-amino protecting groups are those known to be
useful in the art of stepwise polypeptide synthesis. Included are
acyl type protecting groups (e.g., formyl, trifluoroacetyl,
acetyl), aryl type protecting groups (e.g., biotinyl), aromatic
urethane type protecting groups [e.g., benzyloxycarbonyl (Cbz),
substituted benzyloxycarbonyl and 9-fluorenylmethyloxy-carbonyl
(Fmoc)], aliphatic urethane protecting groups [e.g.,
t-butyloxycarbonyl (tBoc), isopropyloxycarbonyl,
cyclohexloxycarbonyl] and alkyl type protecting groups (e.g.,
benzyl, triphenylmethyl). The preferred protecting groups are tBoc
and Fmoc, thus the peptides are said to be synthesized by tBoc and
Fmoc chemistry, respectively.
[0131] The side-chain protecting groups selected must remain intact
during coupling and not be removed during the deprotection of the
amino-terminus protecting group or during coupling conditions. The
side-chain protecting groups must also be removable upon the
completion of synthesis using reaction conditions that will not
alter the finished polypeptide. In tBoc chemistry, the side-chain
protecting groups for trifunctional amino acids are mostly benzyl
based. In Fmoc chemistry, they are mostly tert-butyl or trityl
based.
[0132] In tBoc chemistry, the preferred side-chain protecting
groups are tosyl for arginine, cyclohexyl for aspartic acid,
4-methylbenzyl (and acetamidomethyl) for cysteine, benzyl for
glutamic acid, serine and threonine, benzyloxymethyl (and
dinitrophenyl) for histidine, 2-Cl-benzyloxycarbonyl for lysine,
formyl for tryptophan and 2-bromobenzyl for tyrosine. In Fmoc
chemistry, the preferred side-chain protecting groups are
2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc) or
2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) for
arginine, trityl for asparagine, cysteine, glutamine and histidine,
tert-butyl for aspartic acid, glutamic acid, serine, threonine and
tyrosine, tBoc for lysine and tryptophan.
[0133] For the synthesis of phosphopeptides, either direct or
post-assembly incorporation of the phosphate group is used. In the
direct incorporation strategy, the phosphate group on serine,
threonine or tyrosine may be protected by methyl, benzyl, or
tert-butyl in Fmoc chemistry or by methyl, benzyl or phenyl in tBoc
chemistry. Direct incorporation of phosphotyrosine without
phosphate protection can also be used in Fmoc chemistry. In the
post-assembly incorporation strategy, the unprotected hydroxyl
groups of serine, threonine or tyrosine are derivatized on solid
phase with di-tert-butyl-, dibenzyl- or
dimethyl-N,N'-diisopropylphosphoramidite and then oxidized by
tert-butylhydroperoxide.
[0134] Solid phase synthesis is usually carried out from the
carboxyl-terminus by coupling the alpha-amino protected (side-chain
protected) amino acid to a suitable solid support. An ester linkage
is formed when the attachment is made to a chloromethyl,
chlortrityl or hydroxymethyl resin, and the resulting polypeptide
will have a free carboxyl group at the C-terminus. Alternatively,
when an amide resin such as benzhydrylamine or
p-methylbenzhydrylamine resin (for tBoc chemistry) and Rink amide
or PAL resin (for Fmoc chemistry) are used, an amide bond is formed
and the resulting polypeptide will have a carboxamide group at the
C-terminus. These resins, whether polystyrene- or polyamide-based
or polyethyleneglycol-grafted, with or without a handle or linker,
with or without the first amino acid attached, are commercially
available, and their preparations have been described by Stewart et
al., "Solid Phase Peptide Synthesis" (2.sup.nd Edition), (Pierce
Chemical Co., Rockford, Ill., 1984) and Bayer & Rapp Chem.
Pept. Prot. 3:3 (1986); and Atherton et al., Solid Phase Peptide
Synthesis: A Practical Approach, IRL Press, Oxford, 1989.
[0135] The C-terminal amino acid, protected at the side chain if
necessary, and at the alpha-amino group, is attached to a
hydroxylmethyl resin using various activating agents including
dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide
(DIPCDI) and carbonyldiimidazole (CDI). It can be attached to
chloromethyl or chlorotrityl resin directly in its cesium
tetramethylammonium salt form or in the presence of triethylamine
(TEA) or diisopropylethylamine (DIEA). First amino acid attachment
to an amide resin is the same as amide bond formation during
coupling reactions.
[0136] Following the attachment to the resin support, the
alpha-amino protecting group is removed using various reagents
depending on the protecting chemistry (e.g., tBoc, Fmoc). The
extent of Fmoc removal can be monitored at 300-320 nm or by a
conductivity cell. After removal of the alpha-amino protecting
group, the remaining protected amino acids are coupled stepwise in
the required order to obtain the desired sequence.
[0137] Various activating agents can be used for the coupling
reactions including DCC, DIPCDI, 2-chloro-1,3-dimethylimidium
hexafluorophosphate (CIP),
benzotriazol-1-yl-oxy-tris-(dimethylamino)-phosphonium
hexafluoro-phosphate (BOP) and its pyrrolidine analog (PyBOP),
bromo-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBroP),
O-(benzotriazol-1-yl)-1,1,3,3-tetramethyl-uronium
hexafluorophosphate (HBTU) and its tetrafluoroborate analog (TBTU)
or its pyrrolidine analog (HBPyU),
O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl-uronium
hexafluorophosphate (HATU) and its tetrafluoroborate analog (TATU)
or its pyrrolidine analog (HAPyU). The most common catalytic
additives used in coupling reactions include
4-dimethylaminopyridine (DMAP),
3-hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine (HODhbt),
N-hydroxybenzotriazole (HOBt) and 1-hydroxy-7-azabenzotriazole
(HOAt). Each protected amino acid is used in excess (>2.0
equivalents), and the couplings are usually carried out in
N-methylpyrrolidone (NMP) or in DMF, CH2Cl2 or mixtures thereof.
The extent of completion of the coupling reaction can be monitored
at each stage, e.g., by the ninhydrin reaction as described by
Kaiser et al., Anal. Biochem. 34:595, 1970. In cases where
incomplete coupling is found, the coupling reaction is extended and
repeated and may have chaotropic salts added. The coupling
reactions can be performed automatically with commercially
available instruments such as ABI model 430A, 431A and 433A peptide
synthesizers.
[0138] After the entire assembly of the desired peptide, the
peptide-resin is cleaved with a reagent with proper scavengers. The
Fmoc peptides are usually cleaved and deprotected by TFA with
scavengers (e.g., H20, ethanedithiol, phenol and thioanisole). The
tBoc peptides are usually cleaved and deprotected with liquid HF
for 1-2 hours at -5 to 0.degree. C., which cleaves the polypeptide
from the resin and removes most of the side-chain protecting
groups. Scavengers such as anisole, dimethylsulfide and
p-thiocresol are usually used with the liquid HF to prevent cations
formed during the cleavage from alkylating and acylating the amino
acid residues present in the polypeptide. The formyl group of
tryptophan and the dinitrophenyl group of histidine need to be
removed, respectively by piperidine and thiophenyl in DMF prior to
the HF cleavage. The acetamidomethyl group of cysteine can be
removed by mercury(II)acetate and alternatively by iodine,
thallium(III)trifluoroacetate or silver tetrafluoroborate which
simultaneously oxidize cysteine to cystine. Other strong acids used
for tBoc peptide cleavage and deprotection include
trifluoromethanesulfonic acid (TFMSA) and
trimethylsilyltrifluoroacetate (TMSOTf).
[0139] The activity of molecules of the present invention can be
measured using a variety of assays that measure, for example,
cell-cell interactions, glycolipid and glycoprotein biosynthesis,
development, and other biological functions associated with
galactosyltransferase family members. Of particular interest are
changes in the transfer of galactosyl molecules in glycoprotein
synthesis and in cell-cell interactions in brain, kidney, and
testis tissue and cell lines derived from these tissues. Such
assays are well known in the art. For a general reference, see
Kolbinger, F. et al., J. Biol. Chem. 273: 433-440, 1998; Amado, M.
et al., J. Biol. Chem. 273:12770-12778, 1998; Hennet, T. et al., J.
Biol. Chem. 273:58-65, 1998; and Ram B. P., and Munjal, D. D., CRC
Crit. Rev. Biochem. 17:257-311, 1985. Specific assays include, but
are not limited to bioassays measuring cell migration, contact
inhibition, tissue interactions, neuronal specificity,
fertilization, embryonic cell adhesions, limb bud morphogenesis,
mesenchyme development, immune recognition, growth control, tumor
metastasis and suppression, and glycoprotein and glycolipid
biosynthesis.
[0140] Additional activities likely associated with the
polypeptides of the present invention include proliferation of
cells of the brain, kidney, and testis directly or indirectly
through other growth factors; action as a chemotaxic factor; and as
a factor for expanding neuronal and epithelial stem cell and
precursor populations.
[0141] Another assay of interest measures or detects changes in
proliferation, differentiation, and development. Proliferation can
be measured using cultured primary brain, kidney, and testis cells,
ex plant tissues, or in vivo by administering molecules of the
claimed invention to the appropriate cells, tissues, or animal
models. Generally, proliferative effects are observed as an
increase in cell number and therefore, may include inhibition of
apoptosis, as well as mitogenesis. Likewise, a decrease in cell
number and cell migration could be analyzed. Assays measuring cell
proliferation are well known in the art. For example, assays
measuring proliferation include such assays as chemosensitivity to
neutral red dye (Cavanaugh et al., Investigational New Drugs
8:347-354, 1990,), incorporation of radiolabelled nucleotides (Cook
et al., Analytical Biochem. 179:1-7, 1989,), incorporation of
5-bromo-2'-deoxyuridine (BrdU) in the DNA of proliferating cells
(Porstmann et al., J. Immunol. Methods 82:169-179, 1985), and use
of tetrazolium salts (Mosmann, J. Immunol. Methods 65:55-63, 1983;
Alley et al., Cancer Res. 48:589-601, 1988; Marshall et al., Growth
Reg. 5:69-84, 1995; and Scudiero et al., Cancer Res. 48:4827-4833,
1988).
[0142] Proliferation of bone marrow and peripheral blood lymphocyte
cells can be assayed by harvesting these cells from mice,
suspending the mononuclear cells in a base medium, and measuring
proliferation in the presence of znssp6 protein. Similarly,
clonogenic assays can be performed.
[0143] To determine if znssp6 is a chemotractant in vivo, znssp6
can be given by intradermal or intraperitoneal injection.
Characterization of the accumulated leukocytes at the site of
injection can be determined using lineage specific cell surface
markers and fluorescence immunocytometry or by immunohistochemistry
(Jose, J. Exp. Med. 179:881-87, 1994). Release of specific
leukocyte cell populations from bone marrow into peripheral blood
can also be measured after znssp6 injection.
[0144] Differentiation is a progressive and dynamic process,
beginning with pluripotent stem cells and ending with terminally
differentiated cells. Pluripotent stem cells that can regenerate
without commitment to a lineage express a set of differentiation
markers that are lost when commitment to a cell lineage is made.
Progenitor cells express a set of differentiation markers that may
or may not continue to be expressed as the cells progress down the
cell lineage pathway toward maturation. Differentiation markers
that are expressed exclusively by mature cells are usually
functional properties such as cell products, enzymes to produce
cell products and receptors and receptor-like complementary
molecules. The stage of a cell population's differentiation is
monitored by identification of markers present in the cell
population. For example, myocytes, osteoblasts, adipocytes,
chrondrocytes, fibroblasts and reticular cells are believed to
originate from a common mesenchymal stem cell (Owen et al., Ciba
Fdn. Symp. 136:42-46, 1988). Markers for mesenchymal stem cells
have not been well identified (Owen et al., J. of Cell Sci.
87:731-738, 1987), so identification is usually made at the
progenitor and mature cell stages.
[0145] There is evidence to suggest that factors that stimulate
specific cell types down a pathway towards terminal differentiation
or dedifferentiation affect the entire cell population originating
from a common precursor or stem cell. Thus, znssp6 polypeptides may
stimulate inhibition or proliferation of endocrine and exocrine
cells of the brain, kidney and testis, as well as, cells associated
with the spinal cord, colon, prostate, stomach, ovary, pancreas,
pituitary gland, adrenal gland, salivary gland, mammary gland,
liver, small intestine, spleen, thymus, peripheral leukocyte, lymph
node, bone marrow, lung, trachea, placenta, fetal spleen and fetal
lung. The znssp6 molecules of the present invention may, while
stimulating proliferation or differentiation of brain, kidney and
testis cells, inhibit proliferation or differentiation of other
tissues, by virtue of their effect on common precursor/stem cells.
The novel polypeptides of the present invention are useful to study
neural and epithelial stem cells and brain, kidney and testis
progenitor cells, both in vivo and ex vivo.
[0146] Assays measuring differentiation include, for example,
measuring cell-surface markers associated with stage-specific
expression of a tissue, enzymatic activity, functional activity or
morphological changes (Watt, FASEB, 5:281-284, 1991; Francis,
Differentiation 57:63-75, 1994; Raes, Adv. Anim. Cell Biol.
Technol. Bioprocesses, 161-171, 1989).
[0147] The znssp6 polypeptides of the present invention can be used
to study brain, kidney, and testis proliferation or
differentiation. Such methods of the present invention generally
comprise incubating cells derived from these tissues in the
presence and absence of znssp6 polypeptide, monoclonal antibody,
agonist or antagonist thereof and observing changes in cell
proliferation or differentiation.
[0148] Proteins, including alternatively spliced peptides, and
fragments, of the present invention are useful for cell-cell
interactions, neuronal specificity, fertilization, morphogenesis,
development, immune recognition, growth control, tumor suppression,
and glycoprotein and glycolipid biosynthesis. Znssp6 molecules,
variants, and fragments can be applied in isolation, or in
conjunction with other molecules (growth factors, cytokines, etc.)
in brain, kidney, and testis. Alternative splicing of znssp6 may be
cell-type specific and confer activity to specific tissues.
[0149] Cell lines of brain, kidney, and testis are available from
commercial manufacturers, such as the American Type Culture
Collection. One skilled in the art would know how to order and
establish such cell lines, and perform assays as described herein.
Exemplary cell lines from ATCC include ATCC# CRL-7773, a brain
ganglioneuroblastoma; ATCC# HTB-148, a brain neuoglioma; ATCC#
CRL-7540, a kidney hypemephroma; ATCC# CRL-7192, a kidney
carcinoma; and ATCC# CRL-7800, a testis seminoma, (ATCC, Manassas,
Va.).
[0150] Other assays to measure the effects of znssp6 include
proliferation assays (i.e., of brain, kidney, and testis) by
testing tissue and cells from healthy volunteers with znssp6
protein, or a znssp6-free negative control for the ability of the
tissue and cells to proliferate.
[0151] Proteins of the present invention are useful for delivery of
therapeutic agents such as, but not limited to, radionuclides,
chemotherapy agents, and small molecules. The effects of znssp6 can
be measured in vitro using cultured cells, ex vivo on tissue
slices, or in vivo by administering molecules of the claimed
invention to the appropriate animal model. For instance, znssp6
transfected (or co-transfected) expression host cells may be
embedded in an alginate environment and injected (implanted) into
recipient animals. Alginate-poly-L-lysine microencapsulation,
permselective membrane encapsulation and diffusion chambers have
been described as a means to entrap transfected mammalian cells or
primary mammalian cells. These types of non-immunogenic
"encapsulations" or microenvironments permit the transfer of
nutrients into the microenvironment, and also permit the diffusion
of proteins and other macromolecules secreted or released by the
captured cells across the environmental barrier to the recipient
animal. Most importantly, the capsules or microenvironments mask
and shield the foreign, embedded cells from the recipient animal's
immune response. Such microenvironments can extend the life of the
injected cells from a few hours or days (naked cells) to several
weeks (embedded cells).
[0152] Alginate threads provide a simple and quick means for
generating embedded cells. The materials needed to generate the
alginate threads are readily available and relatively inexpensive.
Once made, the alginate threads are relatively strong and durable,
both in vitro and, based on data obtained using the threads, in
vivo. The alginate threads are easily manipulable and the
methodology is scalable for preparation of numerous threads. In an
exemplary procedure, 3% alginate is prepared in sterile H.sub.2O,
and sterile filtered. Just prior to preparation of alginate
threads, the alginate solution is again filtered. An approximately
50% cell suspension (containing about 5.times.10.sup.5 to about
5.times.10.sup.7 cells/ml) is mixed with the 3% alginate solution.
One ml of the alginate/cell suspension is extruded into a 100 mM
sterile filtered CaCl.sub.2 solution over a time period of
.about.15 min, forming a "thread". The extruded thread is then
transferred into a solution of 50 mM CaCl.sub.2, and then-into a
solution of 25 mM CaCl.sub.2. The thread is then rinsed with
deionized water before coating the thread by incubating in a 0.01%
solution of poly-L-lysine. Finally, the thread is rinsed with
Lactated Ringer's Solution and drawn from solution into a syringe
barrel (without needle attached). A large bore needle is then
attached to the syringe, and the thread is intraperitoneally
injected into a recipient in a minimal volume of the Lactated
Ringer's Solution.
[0153] An alternative in vivo approach for assaying proteins of the
present invention involves viral delivery systems. Exemplary
viruses for this purpose include adenovirus, herpesvirus,
lentivirus, vaccinia virus and adeno-associated virus (AAV).
[0154] Adenovirus, a double-stranded DNA virus, is currently the
best studied gene transfer vector for delivery of heterologous
nucleic acid (for a review, see T. C. Becker et al., Meth. Cell
Biol. 43:161-89, 1994; and J. T. Douglas and D. T. Curiel, Science
& Medicine 4:44-53, 1997). The adenovirus system offers several
advantages: adenovirus can (i) accommodate relatively large DNA
inserts; (ii) be grown to high-titer; (iii) infect a broad range of
mammalian cell types; and (iv) be used with a large number of
available vectors containing different promoters. Also, because
adenoviruses are stable in the bloodstream, they can be
administered by intravenous injection.
[0155] By deleting portions of the adenovirus genome, larger
inserts (up to 7 kb) of heterologous DNA can be accommodated. These
inserts can be incorporated into the viral DNA by direct ligation
or by homologous recombination with a co-transfected plasmid. In an
exemplary system, the essential E1 gene has been deleted from the
viral vector, and the virus will not replicate unless the E1 gene
is provided by the host cell (the human 293 cell line is
exemplary). When intravenously administered to intact animals,
adenovirus primarily targets the liver. If the adenoviral delivery
system has an E1 gene deletion, the virus cannot replicate in the
host cells. However, the host's tissue (e.g., liver) will express
and process (and, if a secretory signal sequence is present,
secrete) the heterologous protein. Secreted proteins will enter the
circulation in the highly vascularized liver, and effects on the
infected animal can be determined.
[0156] The adenovirus system can also be used for protein
production in vitro. By culturing adenovirus-infected non-293 cells
under conditions where the cells are not rapidly dividing, the
cells can produce proteins for extended periods of time. For
instance, BHK cells are grown to confluence in cell factories, then
exposed to the adenoviral vector encoding the secreted protein of
interest. The cells are then grown under serum-free conditions,
which allows infected cells to survive for several weeks without
significant cell division. Alternatively, adenovirus vector
infected 293S cells can be grown in suspension culture at
relatively high cell density to produce significant amounts of
protein (see Garnier et al., Cytotechnol. 15:145-55, 1994). With
either protocol, an expressed, secreted heterologous protein can be
repeatedly isolated from the cell culture supernatant. Within the
infected 293S cell production protocol, non-secreted proteins may
also be effectively obtained.
[0157] In view of the tissue distribution (i.e., brain, kidney, and
testis and various other tissues) observed for znssp6, agonists
(including the natural anti-complementary molecule) and antagonists
have enormous potential for both in vitro and in vivo applications.
Compounds identified as znssp6 agonists are useful for studying
galactosylation of cell surface antigens as well as cell-cell
interactions in vitro and in vivo. For example, znssp6 and agonist
compounds are useful as components of defined cell culture media,
and may be used alone or in combination with other cytokines and
hormones to replace serum that is commonly used in cell culture.
Agonists are thus useful in specifically promoting the growth
and/or development of brain, kidney, and testis in culture.
Alternatively, znssp6 polypeptides and znssp6 agonist polypeptides
are useful as a research reagent, particularly for the growth and
expansion of neural and epithelial cells. Znssp6 polypeptides are
added to tissue culture media for these cell types.
[0158] Additionally, molecules of the present invention can be used
in vitro to modify glycoproteins. Expression of proteins which will
be used as therapeutics can result in aberrant glycosylation.
Znssp6 molecules can be added in vitro to production or reagent
grade proteins to modify the improper galactosylation of
proteins.
[0159] Antagonists of znssp6 molecules are also useful as research
reagents for characterizing sites of interactions between member of
complement/anti-complement pairs as well as site of
galactosyltransferase catalysis.
[0160] Inhibitors of znssp6 activity (znssp6 antagonists) include
anti-znssp6 antibodies and soluble znssp6 polypeptides, as well as
other peptidic and non-peptidic agents (including ribozymes).
[0161] The invention also provides antagonists, which either bind
to znssp6 polypeptides or, alternatively, to a anti-complementary
molecule to which znssp6 polypeptides bind, thereby inhibiting or
eliminating the function of znssp6. Such znssp6 antagonists would
include antibodies; polypeptides which bind either to the znssp6
polypeptide or to its anti-complementary molecule or natural or
synthetic analogs of znssp6 anti-complementary molecule which
retain the ability to bind the anti-complementary molecule but do
not result in glycoprotein or glycolipid synthesis or cell-cell
interactions. Such analogs could be peptides or peptide-like
compounds.
[0162] Natural or synthetic small molecules which bind to znssp6
polypeptides and prevent glyprotein or glycolipid synthesis or
cell-cell interactions are also contemplated as antagonists. Also
contemplated are soluble znssp6 polypeptides. As such, znssp6
antagonists would be useful as therapeutics for treating certain
disorders where blocking glycosylation or binding of the
znssp6-anti-complementary molecule would be beneficial.
[0163] Znssp6 polypeptides may be used within diagnostic systems to
detect the presence of znssp6 anti-complementary molecule
polypeptides. Antibodies or other agents that specifically bind to
znssp6 or its anti-complementary molecule may also be used to
detect the presence of circulating znssp6 or anti-complementary
molecule polypeptides. Such detection methods are well known in the
art and include, for example, enzyme-linked immunosorbent assay
(ELISA) and radioimmunoassay.
[0164] Immunohistochemically labeled znssp6 antibodies can be used
to detect znssp6 and/or znssp6 anti-complementary molecule in
tissue samples. znssp6 levels can also be monitored by such methods
as RT-PCR, where znssp6 mRNA can be detected and quantified. The
information derived from such detection methods would provide
insight into the significance of znssp6 polypeptides in various
diseases, and as such would serve as diagnostic tools for diseases
for which altered levels of znssp6 are significant.
[0165] Altered levels of znssp6 polypeptides may be indicative of
pathological conditions including, for example, cancer, auto-immune
diseases, digestive disorders and inflammatory disorders.
[0166] A "soluble protein" is a protein that is not bound to a cell
membrane. Soluble znssp6 proteins are most commonly
anti-complementary molecule-binding polypeptides that lack
transmembrane and cytoplasmic domains. Soluble proteins can
comprise additional amino acid residues, such as affinity tags that
provide for purification of the polypeptide or provide sites for
attachment of the polypeptide to. a anti-complementary molecule, or
immunoglobulin constant region sequences. Many cell-surface
proteins have naturally occurring, soluble counterparts that are
produced by proteolysis or translated from alternatively spliced
mRNAs. Proteins are said to be substantially free of transmembrane
and intracellular polypeptide segments when they lack sufficient
portions of these segments to provide membrane anchoring or signal
transduction, respectively.
[0167] Soluble forms of znssp6 polypeptides can be generated by
removing the hydrophobic region between residues 26 and 49 of SEQ
ID NO: 2. Soluble zassp6 polypeptides are useful in studying the
effects of the present invention in vivo and in vitro. Such soluble
molecules comprise the anti-complmentary moleucle binding domain
comprising reisudes 114 to 370 of SEQ ID NO: 2. Thus, soluble forms
of znssp6 can also include polypeptides selected from the rest of
the molecule excluding the hydrophobic region. Some exemplary forms
of znssp6 polypeptides include: the polypeptide from residue 114 to
370 fo SEQ ID NO: 2; the polypeptide from residue 114 to residue
378 of SEQ ID NO: 2; the polypeptide from residue 50 to residue 370
of SEQ ID NO: 2; and the polypeptide from residue 50 to residue 378
of SEQ ID NO: 2.
[0168] Soluble forms of znssp6 polypeptides may act as antagonists
to or agonists of znssp6 polypeptides, and would be useful to
modulate the effects of znssp6 in brain, kidney and testis. Since
polypeptides of this nature are not anchored to the membrane, they
can act at sites distant from the tissues in which they are
expressed. Thus, the activity of the soluble form of znssp6
polypeptides can be more wide spread than its membrane-anchored
counterpart. Both isoforms would be useful in studying the effects
of the present invention in vitro an in vivo.
[0169] Znssp6 can also be used to identify inhibitors (antagonists)
of its activity. Test compounds are added to the assays disclosed
herein to identify compounds that inhibit the activity of znssp6.
In addition to those assays disclosed herein, samples can be tested
for inhibition of znssp6 activity within a variety of assays
designed to measure binding or the stimulation/inhibition of
znssp6-dependent cellular responses. For example, znssp6-responsive
cell lines can be transfected with a reporter gene construct that
is responsive to a znssp6-stimulated cellular pathway. Reporter
gene constructs of this type are known in the art, and will
generally comprise a znssp6-DNA response element operably linked to
a gene encoding an assayable protein, such as luciferase. DNA
response elements can include, but are not limited to, cyclic AMP
response elements (CRE), hormone response elements (HRE) insulin
response element (IRE) (Nasrin et al., Proc. Natl. Acad. Sci. USA
87:5273-7, 1990) and serum response elements (SRE) (Shaw et al.
Cell 56: 563-72, 1989). Cyclic AMP response elements are reviewed
in Roestler et al., J. Biol. Chem. 263 (19):9063-6; 1988 and
Habener, Molec. Endocrinol. 4 (8):1087-94; 1990. Hormone response
elements are reviewed in Beato, Cell 56:335-44; 1989. Candidate
compounds, solutions, mixtures or extracts are tested for the
ability to inhibit the activity of znssp6 on the target cells as
evidenced by a decrease in znssp6 stimulation of reporter gene
expression. Assays of this type will detect compounds that directly
block znssp6 binding to cell-surface anti-complementary molecule,
as well as compounds that block processes in the cellular pathway
subsequent to complementary molecule/anti-complementary molecule
binding. In the alternative, compounds or other samples can be
tested for direct blocking of znssp6 binding to its
anti-complementary molecule using znssp6 tagged with a detectable
label (e.g., 125I, biotin, horseradish peroxidase, FITC, and the
like). Within assays of this type, the ability of a test sample to
inhibit the binding of labeled znssp6 to the anti-complementary
molecule is indicative of inhibitory activity, which can be
confirmed through secondary assays. Anti-complementary molecules
used within binding assays may be cellular or isolated,
immobilized, or receptor-like complementary molecules.
[0170] Assays measuring the inhibition of galactosyltransferase
activity in glycoprotein synthesis are listed in Ram, B. P.,
(ibid).
[0171] Also, znssp6 polypeptides, agonists or antagonists thereof
may be therapeutically useful for promoting wound healing, for
example, in brain, kidney and testis tissues. To verify the
presence of this capability in znssp6 polypeptides, agonists or
antagonists of the present invention, such znssp6 polypeptides,
agonists or antagonists ar& evaluated with respect to their
ability to facilitate wound healing according to procedures known
in the art. If desired, znssp6 polypeptide performance in this
regard can be compared to growth factors, such as EGF, NGF,
TGF-.alpha., TGF-.beta., insulin, IGF-I, IGF-II, fibroblast growth
factor (FGF) and the like. In addition, znssp6 polypeptides or
agonists or antagonists thereof may be evaluated in combination
with one or more growth factors to identify synergistic
effects.
[0172] A znssp6 polypeptide can be expressed as a fusion with an
immunoglobulin heavy chain constant region, typically an Fc
fragment, which contains two constant region domains and lacks the
variable region. Methods for preparing such fusions are disclosed
in U.S. Pat. Nos. 5,155,027 and 5,567,584. Such fusions are
typically secreted as multimeric molecules wherein the Fc portions
are disulfide bonded to each other and two non-Ig polypeptides are
arrayed in closed proximity to each other. Fusions of this type can
be used to evaluate specific donor/acceptor molecules, affinity
purify ligands, or use as an in vitro assay tool. This fusion can
also be used to determine the homodimerization potential for
znssp6. For use in assays, the chimeras are bound to a support via
the F.sub.c region and used in an ELISA format.
[0173] A znssp6 polypeptide can also be used for purification of
its anti-complementary molecule. The polypeptide is immobilized on
a solid support, such as beads of agarose, cross-linked agarose,
glass, cellulosic resins, silica-based resins, polystyrene,
cross-linked polyacrylamide, or like materials that are stable
under the conditions of use. Methods for linking polypeptides to
solid supports are known in the art, and include amine chemistry,
cyanogen bromide activation, N-hydroxysuccinimide activation,
epoxide activation, sulfhydryl activation, and hydrazide
activation. The resulting medium will generally be configured in
the form of a column or chip, and fluids containing the
anti-complementary molecule are passed through the column or chip
one or more times to allow the znssp6 polypeptide to bind to the
anti-complementary molecule. The anti-complementary molecule is
then eluted using changes in salt concentration, chaotropic agents
(guanidine HCl), or pH to disrupt znssp6-anti-complementary
molecule binding.
[0174] An assay system that uses a ligand-binding receptor (or an
antibody, one member of a complement/anti-complement pair) or a
binding fragment thereof, and a commercially available
biosensor--instrument (BIAcore, Pharnacia Biosensor, Piscataway,
N.J.) may be advantageously employed. Such receptor, antibody,
member of a complement/anti-complement pair or fragment is
immobilized onto the surface of a receptor chip. Use of this
instrument is disclosed by Karlsson, J. Immunol. Methods
145:229-40, 1991 and Cunningham and Wells, J. Mol. Biol.
234:554-63, 1993. A receptor, antibody, member or fragment is
covalently attached, using amine or sulfhydryl chemistry, to
dextran fibers that are attached to gold film within the flow cell.
A test sample is passed through the cell. If a ligand, epitope, or
opposite member of the complement/anti-complement pair is present
in the sample, it will bind to the immobilized receptor, antibody
or member, respectively, causing a change in the refractive index
of the medium, which is detected as a change in surface plasmon
resonance of the gold film. This system allows the determination of
on- and off-rates, from which binding affinity can be calculated,
and assessment of stoichiometry of binding.
[0175] Znssp6 polypeptides can also be used within other assay
systems known in the art. Such systems include Scatchard analysis
for determination of binding affinity (see Scatchard, Ann. NY Acad.
Sci. 51: 660-72, 1949) and calorimetric assays (Cunningham et al.,
Science 253:545-48, 1991; Cunningham et al., Science 245:821-25,
1991).
[0176] Within the polypeptides of the present invention are
polypeptides that comprise an epitope-bearing portion of a protein
as shown in SEQ ID NO: 2. An "epitope" is a region of a protein to
which an antibody can bind. See, for example, Geysen et al., Proc.
Natl. Acad. Sci. USA 81:3998-4002, 1984. Epitopes can be linear or
conformational, the latter being composed of discontinuous regions
of the protein that form an epitope upon folding of the protein.
Linear epitopes are generally at least 6 amino acid residues in
length. Relatively short synthetic peptides that mimic part of a
protein sequence are routinely capable of eliciting an antiserum
that reacts with the partially mimicked protein. See, Sutcliffe et
al., Science 219:660-666, 1983. Antibodies that recognize short,
linear epitopes are particularly useful in analytic and diagnostic
applications that employ denatured protein, such as Western
blotting (Tobin, Proc. Natl. Acad. Sci. USA 76:4350-4356, 1979), or
in the analysis of fixed cells or tissue samples. Antibodies to
linear epitopes are also useful for detecting fragments of znssp6,
such as might occur in body fluids or cell culture media.
[0177] Antigenic, epitope-bearing polypeptides of the present
invention are useful for raising antibodies, including monoclonal
antibodies, that specifically bind to a znssp6 protein. The znssp6
polypeptide or a fragment thereof serves as an antigen (immunogen)
to inoculate an animal and elicit an immune response. One of skill
in the art would recognize that antigenic, epitope-bearing
polypeptides contain a sequence of at least six, or at least nine,
or from 15 to about 30 contiguous amino acid residues of a znssp6
protein (e.g., SEQ ID NO: 2). Polypeptides comprising a larger
portion of a znssp6 protein, i.e. from 30 to 100 residues up to the
entire sequence, are included. Antigens or immunogenic epitopes can
also include attached tags, adjuvants and carriers, as described
herein. Suitable antigens include the znssp6 polypeptides encoded
by SEQ ID NO: 2 from amino acid number 1 to amino acid number 378,
or a contiguous 9 to 378 amino acid fragment thereof. Such regions
include the catalytic domain, the stem domain, or the hydrophobic
domain of znssp6 and fragments thereof. Polypeptides in this regard
include those comprising residues 1 to 25 of SEQ ID NO: 2; residues
26 to 49 of SEQ ID NO: 2; residues 50 to 113 of SEQ ID NO: 2;
residues 114 to 378 of SEQ ID NO: 2; and residues 371 to 378 of SEQ
ID NO: 2.
[0178] The present invention also provides polypeptide fragments or
peptides comprising an epitope-bearing portion of an znssp6
polypeptide described herein. Such fragments or peptides may
comprise an "immunogenic epitope," which is a part of a protein
that elicits an antibody response when the entire protein is used
as an immunogen. Immunogenic epitope-bearing peptides can be
identified using standard methods (see, for example, Geysen et al.,
Proc. Nat'l Acad. Sci. USA 81:3998 (1983)).
[0179] In contrast, polypeptide fragments or peptides may comprise
an "antigenic epitope," which is a region of a protein molecule to
which an antibody can specifically bind. Certain epitopes consist
of a linear or contiguous stretch of amino acids, and the
antigenicity of such an epitope is not disrupted by denaturing
agents. It is known in the art that relatively short synthetic
peptides that can mimic epitopes of a protein can be used to
stimulate the production of antibodies against the protein (see,
for example, Sutcliffe et al., Science 219:660 (1983)).
Accordingly, antigenic epitope-bearing peptides and polypeptides of
the present invention are useful to raise antibodies that bind with
the polypeptides described herein.
[0180] Antigenic epitope-bearing peptides and polypeptides contain
at least four to ten amino acids, or at least ten to fifteen amino
acids, or 15 to 30 amino acids of SEQ ID NO: 2. Such
epitope-bearing peptides and polypeptides can be produced by
fragmenting an znssp6 polypeptide, or by chemical peptide
synthesis, as described herein. Moreover, epitopes can be selected
by phage display of random peptide libraries (see, for example,
Lane and Stephen, Curr. Opin. Immunol. 5:268 (1993), and Cortese et
al., Curr. Opin. Biotechnol. 7:616 (1996)). Standard methods for
identifying epitopes and producing antibodies from small peptides
that comprise an epitope are described, for example, by Mole,
"Epitope Mapping," in Methods in Molecular Biology, Vol. 10, Manson
(ed.), pages 105-116 (The Humana Press, Inc. 1992), Price,
"Production and Characterization of Synthetic Peptide-Derived
Antibodies," in Monoclonal Antibodies: Production, Engineering, and
Clinical Application, Ritter and Ladyman (eds.), pages 60-84
(Cambridge University Press 1995), and Coligan et al. (eds.),
Current Protocols in Immunology, pages 9.3.1-9.3.5 and pages
9.4.1-9.4.11 (John Wiley & Sons 1997).
[0181] As an illustration, potential antigenic sites in znssp6
polypeptides (SEQ ID NO: 2) were identified using the Jameson-Wolf
method, Jameson and Wolf, CABIOS 4:181, (1988), as implemented by
the PROTEAN program (version 3.14) of LASERGENE (DNASTAR; Madison,
Wis.). Default parameters were used in this analysis.
[0182] Suitable antigens of znssp6 polypeptides include residue 10
to residue 16 of SEQ ID NO: 2; residue 52 to residue 61 of SEQ ID
NO: 2; residue 52 to residue 78 of SEQ ID NO: 2; residue 69 to
residue 78 of SEQ ID NO: 2; residue 89 to residue 94 of SEQ ID NO:
2; residue 89 to residue 117 of SEQ ID NO: 2; residue 111 to
residue 117 of SEQ ID NO: 2; residue 126 to residue 134 of SEQ ID
NO: 2; residue 126 to residue 151 of SEQ ID NO: 2; residue 143 to
residue 151 of SEQ ID NO: 2; residue 215 to residue 220 of SEQ ID
NO: 2; residue 215 to residue 239 of SEQ ID NO: 2; residue 223 to
residue 239 of SEQ ID NO: 2; residue 223 to residue 257 of SEQ ID
NO: 2; residue 251 to residue 257 of SEQ ID NO: 2;and residue 332
to residue 337 of SEQ ID NO: 2; or a portion thereof which contains
a 4 to 10 amino acid segment. Hydrophilic peptides, such as those
predicted by one of skill in the art from a hydrophobicity plot are
also immonogenic. Znssp6 hydrophilic peptides include peptides
comprising amino acid sequences selected from the group consisting
of: residue 6 to residue 20 of SEQ ID NO: 2; residue 7 to residue
15 of SEQ ID NO: 2; residue 52 to residue 82 of SEQ ID NO: 2;
residue 52 to residue 69 of SEQ ID NO: 2; residue 54 to residue 62
of SEQ ID NO: 2; residue 62 to residue 82 SEQ ID NO: 2; residue 69
to residue 79 of SEQ ID NO: 2; residue 89 to residue 104 of SEQ ID
NO: 2; residue 97 to residue 104 of SEQ ID NO: 2; residue 126 to
residue 153 of SEQ ID NO: 2; residue 129 to residue 134 of SEQ ID
NO: 2; residue 126 to residue 140 of SEQ ID NO: 2 ; residue 135 to
residue 140 of SEQ ID NO: 2; residue 141 to residue 153 of SEQ ID
NO: 2; residue 172 to residue 181 SEQ ID NO: 2 ; residue 172 to
residue 189 of SEQ ID NO: 2 ; residue 175 to residue 181 of SEQ ID
NO: 2; residue 175 to residue 189 of SEQ ID NO: 2; residue 213 to
residue 218 of SEQ ID NO: 2 ; residue 233 to residue 238 of SEQ ID
NO: 2 ; residue 249 to residue 261 of SEQ ID NO: 2; residue 249 to
residue 257 of SEQ ID NO: 2; residue 251 to residue 257 of SEQ ID
NO: 2 ; residue 251 to residue 261 of SEQ ID NO: 2 ; residue 266 to
residue 272 SEQ ID NO: 2; residue 266 to residue 277 of SEQ ID NO:
2; residue 328 to residue 336 of SEQ ID NO: 2; and residue 373 to
residue 378 of SEQ ID NO: 2; or a portion thereof which contains a
4 to 10 amino acid segment. Additionally, antigens can be generated
to portions of the polypeptide which are likely to be on the
surface of the folded protein. These antigens include: residue 3 to
residue 8 SEQ ID NO: 2; residue 3 to residue 15 of SEQ ID NO: 2;
residue 10 to residue 15 of SEQ ID NO: 2; residue 51 to residue 62
of SEQ ID NO: 2; residue 51 to residue 77 of SEQ ID NO: 2; residue
68 to residue 77 SEQ ID NO: 2; residue 129 to residue 135 of SEQ ID
NO: 2; residue 173 to residue 178 of SEQ ID NO: 2; residue 250 to
residue 258 of SEQ ID NO: 2; residue 250 to residue 274 SEQ ID NO:
2; and residue 265 to residue 274 of SEQ ID NO: 2; or a portion
thereof which contains a 4 to 10 amino acid segment.
[0183] Antibodies generated from this immune response can be
isolated and purified as described herein. Methods for preparing
and isolating polyclonal and monoclonal antibodies are well known
in the art. See, for example, Current Protocols in Immunology,
Cooligan, et al. (eds.), National Institutes of Health, John Wiley
and Sons, Inc., 1995; Sambrook et al., Molecular Cloning: A
Laboratory Manual, Second Edition Cold Spring Harbor, N.Y., 1989;
and Hurrell, J. G. R., Ed., Monoclonal Hybridoma Antibodies:
Techniques and Applications, CRC Press, Inc., Boca Raton, Fla.,
1982.
[0184] As would be evident to one of ordinary skill in the art,
polyclonal antibodies can be generated from inoculating a variety
of warm-blooded animals such as horses, cows, goats, sheep, dogs,
chickens, rabbits, mice, and rats with a znssp6 polypeptide or a
fragment thereof. The immunogenicity of a znssp6 polypeptide may be
increased through the use of an adjuvant, such as alum (aluminum
hydroxide) or Freund's adjuvant. Polypeptides useful for
immunization also include fusion polypeptides, such as fusions of
znssp6 or a portion thereof with an immunoglobulin polypeptide or
with maltose binding protein. The polypeptide immunogen may be a
full-length molecule or a portion thereof. If the polypeptide
portion is "hapten-like", such portion may be advantageously joined
or linked to a macromolecular carrier (such as keyhole limpet
hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid) for
immunization.
[0185] As used herein, the term "antibodies" includes polyclonal
antibodies, affinity-purified polyclonal antibodies, monoclonal
antibodies, and antigen-binding fragments, such as F(ab').sub.2 and
Fab proteolytic fragments. Genetically engineered intact antibodies
or fragments, such as chimeric antibodies, Fv fragments, single
chain antibodies and the like, as well as synthetic antigen-binding
peptides and polypeptides, are also included. Non-human antibodies
may be humanized by grafting non-human CDRs onto human framework
and constant regions, or by incorporating the entire non-human
variable domains (optionally "cloaking" them with a human-like
surface by replacement of exposed residues, wherein the result is a
"veneered" antibody). In some instances, humanized antibodies may
retain non-human residues within the human variable region
framework domains to enhance proper binding characteristics.
Through humanizing antibodies, biological half-life may be
increased, and the potential for adverse immune reactions upon
administration to humans is reduced.
[0186] Alternative techniques for generating or selecting
antibodies useful herein include in vitro exposure of lymphocytes
to znssp6 protein or peptide, and selection of antibody display
libraries in phage or similar vectors (for instance, through use of
immobilized or labeled znssp6 protein or peptide). Genes encoding
polypeptides having potential znssp6 polypeptide binding domains
can be obtained by screening random peptide libraries displayed on
phage (phage display) or on bacteria, such as E. coli. Nucleotide
sequences encoding the polypeptides can be obtained in a number of
ways, such as through random mutagenesis and random polynucleotide
synthesis. These random peptide display libraries can be used to
screen for peptides which interact with a known target which can be
a protein or polypeptide, such as a ligand or receptor, a
biological or synthetic macromolecule, or organic or inorganic
substances. Techniques for creating and screening such random
peptide display libraries are known in the art (Ladner et al., U.S.
Pat. No. 5,223,409; Ladner et al., U.S. Pat. No. 4,946,778; Ladner
et al., U.S. Pat. No. 5,403,484 and Ladner et al., U.S. Pat. No.
5,571,698) and random peptide display libraries and kits for
screening such libraries are available commercially, for instance
from Clontech (Palo Alto, Calif.), Invitrogen Inc. (San Diego,
Calif.), New England Biolabs, Inc. (Beverly, Mass.) and Pharmacia
LKB Biotechnology Inc. (Piscataway, N.J.). Random peptide display
libraries can be screened using the znssp6 sequences disclosed
herein to identify proteins which bind to znssp6. These "binding
proteins" which interact with znssp6 polypeptides can be used for
tagging cells; for isolating homolog polypeptides by affinity
purification; they can be directly or indirectly conjugated to
drugs, toxins, radionuclides and the like. These binding proteins
can also be used in analytical methods such as for screening
expression libraries and neutralizing activity. The binding
proteins can also be used for diagnostic assays for determining
circulating levels of polypeptides; for detecting or quantitating
soluble polypeptides as marker of underlying pathology or disease.
These binding proteins can also act as znssp6 "antagonists" to
block znssp6 binding and signal transduction in vitro and in vivo.
These anti-znssp6 binding proteins would be useful for mediating
galactosyltransferase activity extracellularly, therefore,
mediating cell-cell interactions, such as, for example, tumor
formation and metastasis, proliferation and differentiation, as
well as glycoprotein synthesis.
[0187] As used herein, the term "binding proteins" additionally
includes antibodies to znssp6 polypeptides, the cognate
anti-complementary molecule of znssp6 polypeptides, proteins useful
for purification of znssp6 polypeptides, and proteins associated
with the catalytic domain (residues 114 to 378 of SEQ ID NO:
2).
[0188] Antibodies are determined to be specifically binding if: 1)
they exhibit a threshold level of binding activity, and/or 2) they
do not significantly cross-react with related polypeptide
molecules. First, antibodies herein specifically bind if they bind
to a znssp6 polypeptide, peptide or epitope with a binding affinity
(K.sub.a) of 10.sup.6 M.sup.-1 or greater, preferably 10.sup.7
M.sup.-1 or greater, more preferably 10.sup.8 M.sup.-1 or greater,
and most preferably 10.sup.9 M.sup.-1 or greater. The binding
affinity of an antibody can be readily determined by one of
ordinary skill in the art, for example, by Scatchard analysis
(Scatchard, G., Ann. NY Acad. Sci. 51: 660-672, 1949).
[0189] Second, antibodies are determined to specifically bind if
they do not significantly cross-react with related polypeptides.
Antibodies do not significantly cross-react with related
polypeptide molecules, for example, if they detect znssp6 but not
known related polypeptides using a standard Western blot analysis
(Ausubel et al., ibid.). Examples of known related polypeptides are
orthologs, proteins from the same species that are members of a
protein family (e.g. IL-16), znssp6 polypeptides, and non-human
znssp6. Moreover, antibodies may be "screened against" known
related polypeptides to isolate a population that specifically
binds to the inventive polypeptides. For example, antibodies raised
to znssp6 are adsorbed to related polypeptides adhered to insoluble
matrix; antibodies specific to znssp6 will flow through the matrix
under the proper buffer conditions. Such screening allows isolation
of polyclonal and monoclonal antibodies non-crossreactive to
closely related polypeptides (Antibodies: A Laboratory Manual,
Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988;
Current Protocols in Immunology, Cooligan, et al. (eds.), National
Institutes of Health, John Wiley and Sons, Inc., 1995). Screening
and isolation of specific antibodies is well known in the art. See,
Fundamental Immunology, Paul (eds.), Raven Press, 1993; Getzoff et
al., Adv. in Immunol. 43: 1-98, 1988; Monoclonal Antibodies:
Principles and Practice, Goding, J. W. (eds.), Academic Press Ltd.
1996; Benjamin et al., Ann. Rev. Immunol. 2: 67-101, 1984.
[0190] A variety of assays known to those skilled in the art can be
utilized to detect antibodies which specifically bind to znssp6
proteins or peptides. Exemplary assays are described in detail in
Antibodies: A Laboratory Manual Harlow and Lane (Eds.), Cold Spring
Harbor Laboratory Press, 1988. Representative examples of such
assays include: concurrent immunoelectrophoresis, radioimmunoassay,
radioimmuno-precipitation, enzyme-linked immunosorbent assay
(ELISA), dot blot or Western blot assay, inhibition or competition
assay, and sandwich assay. In addition, antibodies can be screened
for binding to wild-type versus mutant znssp6 protein or
polypeptide.
[0191] Antibodies to znssp6 may be used for tagging or labeling
cells that express znssp6; for isolating znssp6 by affinity
purification; for diagnostic assays for determining circulating
levels of znssp6 polypeptides; for detecting or quantitating
soluble znssp6 as marker of underlying pathology or disease,; in
analytical methods employing FACS; for screening expression
libraries; for generating anti-idiotypic antibodies; and as
neutralizing antibodies or as antagonists to block znssp6 in vitro
and in vivo. Suitable direct tags or labels include radionuclides,
enzymes, substrates, cofactors, inhibitors, fluorescent markers,
chemiluminescent markers, magnetic particles and the like; indirect
tags or labels may feature use of biotin-avidin or other
complement/anti-complement pairs as intermediates. Antibodies
herein may also be directly or indirectly conjugated to drugs,
toxins, radionuclides and the like, and these conjugates used for
in vivo diagnostic or therapeutic applications. Moreover,
antibodies to znssp6 or fragments thereof may be used in vitro to
detect denatured znssp6 or fragments thereof in assays, for
example, Western Blots or other assays known in the art.
[0192] The soluble znssp6 is useful in studying the distribution of
its anti-complentary molecule in tissues or specific cell lineages,
and to provide insight into complementary molecule-anti-complentary
molecule biology. Using labeled znssp6, cells expressing the
anti-complentary molecule are identified by fluorescence
immunocytometry or immunocytochemistry. Application may also be
made of the specificity of UDP-glycosyltransferases for their
substrates.
[0193] Antibodies can be made to soluble, znssp6 polypeptides which
are His or FLAG.TM. tagged. Alternatively, such polypeptides form a
fusion protein with Human Ig. In particular, antiserum containing
polypeptide antibodies to His-tagged, or FLAG.TM.-tagged soluble
znssp6 can be used in analysis of tissue distribution of znssp6 by
immunohistochemistry on human or primate tissue. These soluble
znssp6 polypeptides can also be used to immunize mice in order to
produce monoclonal antibodies to a soluble human znssp6
polypeptide. Monoclonal antibodies to a soluble human znssp6
polypeptide can also be used to mimic anti-complentary molecule
coupling, resulting in activation or inactivation of the
complementary molecule-anti-complentary molecule pair. For
instance, it has been demonstrated that cross-linking anti-soluble
CD40 monoclonal antibodies provides a stimulatory signal to B cells
that have been sub-optimally activated with anti-IgM or LPS, and
results in proliferation and immunoglobulin production. These same
monoclonal antibodies act as antagonists when used in solution by
blocking activation of the receptor. Monoclonal antibodies to
znssp6 can be used to determine the distribution, regulation and
biological interaction of the znssp6 and its anti-complentary
molecule pair on specific cell lineages identified by tissue
distribution studies.
[0194] Antibodies or polypeptides herein can also be directly or
indirectly conjugated to drugs, toxins, radionuclides and the like,
and these conjugates used for in vivo diagnostic or therapeutic
applications. For instance, polypeptides or antibodies of the
present invention can be used to identify or treat tissues or
organs that express a corresponding anti-complementary molecule.
More specifically, znssp6 polypeptides or anti-znssp6 antibodies,
or bioactive fragments or portions thereof, can be coupled to
detectable or cytotoxic molecules and delivered to a mammal having
cells, tissues or organs that express the anti-complementary
molecule.
[0195] Suitable detectable molecules may be directly or indirectly
attached to the polypeptide or antibody, and include radionuclides,
enzymes, substrates, cofactors, inhibitors, fluorescent markers,
chemiluminescent markers, magnetic particles and the like. Suitable
cytotoxic molecules may be directly or indirectly attached to the
polypeptide or antibody, and include bacterial or plant toxins (for
instance, diphtheria toxin, Pseudomonas exotoxin, ricin, abrin and
the like), as well as therapeutic radionuclides, such as
iodine-131, rhenium-188 or yttrium-90 (either directly attached to
the polypeptide or antibody, or indirectly attached through means
of a chelating moiety, for instance). Polypeptides or antibodies
may also be conjugated to cytotoxic drugs, such as adriamycin. For
indirect attachment of a detectable or cytotoxic molecule, the
detectable or cytotoxic molecule can be conjugated with a member of
a complementary/anticomplementary pair, where the other member is
bound to the polypeptide or antibody portion. For these purposes,
biotin/streptavidin is an exemplary complementary/anticomplementary
pair.
[0196] In another embodiment, polypeptide-toxin fusion proteins or
antibody-toxin fusion proteins can be used for targeted cell or
tissue inhibition or ablation (for instance, to treat cancer cells
or tissues). Alternatively, if the polypeptide has multiple
functional domains (i.e., an activation domain or a ligand binding
domain, plus a targeting domain), a fusion protein including only
the targeting domain may be suitable for directing a detectable
molecule, a cytotoxic molecule or a complementary molecule to a
cell or tissue type of interest. In instances where the domain only
fusion protein includes a complementary molecule, the
anti-complementary molecule can be conjugated to a detectable or
cytotoxic molecule. Such domain-complementary molecule fusion
proteins thus represent a generic targeting vehicle for
cell/tissue-specific delivery of generic
anti-complementary-detectable/cytotoxic molecule conjugates.
[0197] In another embodiment, znssp6-cytokine fusion proteins or
antibody-cytokine fusion proteins can be used for enhancing in vivo
killing of target tissues (for example, brain, kidney, testis, and
other tissues), if the znssp6 polypeptide or anti-znssp6 antibody
targets, for example, the hyperproliferative brain, kidney, testis
(See, generally, Hornick et al., Blood 89:4437-47, 1997). They
described fusion proteins which enable targeting of a cytokine to a
desired site of action, thereby providing an elevated local
concentration of cytokine. Suitable znssp6 polypeptides or
anti-znssp6 antibodies target an undesirable cell or tissue (i.e.,
a tumor or a leukemia), and the fused cytokine mediated improved
target cell lysis by effector cells. Suitable cytokines for this
purpose include interleukin 2 and granulocyte-macrophage
colony-stimulating factor (GM-CSF), for instance.
[0198] The bioactive polypeptide or antibody conjugates described
herein can be delivered intravenously, intraarterially or
intraductally, or may be introduced locally at the intended site of
action.
[0199] znssp6 polynucleotides and/or polypeptides may be useful for
regulating the maturation of UDP-glycosyltransferase
substrate-bearing cells, such as fibroblasts, lymphocytes and
hematopoietic cells. znssp6 polypeptides will also find use in
mediating metabolic or physiological processes in vivo. The effects
of a compound on proliferation and differentiation can be measured
in vitro using cultured cells. Bioassays and ELISAs are available
to measure cellular response to znssp6, in particular are those
which measure changes in cytokine production as a measure of
cellular response (see for example, Current Protocols in Immunology
ed. John E.
[0200] Coligan et al., NIH, 1996). Assays to measure other cellular
responses, including glycoprotein and glycolipid biosynthesis and
metabolism, and cell-cell interactions are known in the art.
[0201] The activity of znssp6 or a peptide to which znssp6 binds,
can be measured by a silicon-based biosensor microphysiometer which
measures the extracellular acidification rate or proton excretion
associated with such protein interactions and subsequent
physiologic cellular responses. An exemplary device is the
Cytosensor.TM. Microphysiometer manufactured by Molecular Devices,
Sunnyvale, Calif. A variety of cellular responses, such as cell
proliferation, ion transport, energy production, inflammatory
response, regulatory and enzyme or enzyme activation, and the like,
can be measured by this method. See, for example, McConnell, H. M.
et al., Science 257:1906-1912, 1992; Pitchford, S. et al., Meth.
Enzymol. 228:84-108, 1997; Arimilli, S. et al., J. Immunol. Meth.
212:49-59, 1998; Van Liefde, I. et al., Eur. J. Pharmacol.
346:87-95, 1998. The microphysiometer can be used for assaying
adherent or non-adherent eukaryotic or prokaryotic cells. By
measuring extracellular acidification changes in cell media over
time, the microphysiometer directly measures cellular responses to
various stimuli, including znssp6 proteins, their agonists, and
antagonists. The microphysiometer can be used to measure responses
of a znssp6-responsive eukaryotic cell, compared to a control
eukaryotic cell that does not respond to znssp6 polypeptide.
znssp6-responsive eukaryotic cells comprise cells into which a
polynucleotide for znssp6 has been transfected creating a cell that
is responsive to znssp6; or cells containing endogenous znssp6
polynucleotides. Differences, measured by a change in the response
of cells exposed to znssp6 anti-complentary molecule, relative to a
control not exposed to znssp6 anti-complentary molecule, directly
measure the znssp6-modulated cellular responses. Moreover, such
znssp6-modulated responses can be assayed under a variety of
stimuli. The present invention provides a method of identifying
agonists and antagonists of znssp6 protein, comprising providing
cells responsive to a znssp6 polypeptide, culturing a first portion
of the cells in the absence of a test compound, culturing a second
portion of the cells in the presence of a test compound, and
detecting a measurable change in a cellular response of the second
portion of the cells as compared to the first portion of the cells.
Moreover, culturing a third portion of the cells in the presence of
znssp6 anti-complementary molecule and the absence of a test
compound provides a positive control for the znssp6-responsive
cells, and a control to compare the agonist activity of a test
compound with that of the znssp6 anti-complementary molecule.
Antagonists of znssp6 can be identified by exposing the cells to
znssp6 anti-complementary molecule in the presence and absence of
the test compound, whereby a reduction in znssp6-modulated activity
is indicative of antagonist activity in the test compound.
[0202] Moreover, znssp6 can be used to identify cells, tissues, or
cell lines which respond to a znssp6-modulated pathway. The
microphysiometer, described above, can be used to rapidly identify
cells responsive to znssp6 of the present invention. Cells can be
cultured in the presence or absence of znssp6 polypeptide. Those
cells which elicit a measurable change in extracellular
acidification in the presence of znssp6 are responsive to znssp6.
Such cell lines, can be used to identify znssp6 anti-complentary
molecule, antagonists and agonists of znssp6 polypeptide as
described above.
[0203] Molecules of the present invention can be used to identify
and isolate receptors, ligands, or members of
complement/anti-complement pairs involved in cell-cell
interactions, and glycoprotein synthesis. For example, proteins and
peptides of the present invention can be immobilized on a column
and membrane preparations run over the column (Immobilized Affinity
Ligand Techniques, Hermanson et al., eds., Academic Press, San
Diego, Calif., 1992, pp.195-202). Proteins and peptides can also be
radiolabeled (Methods in Enzymol., vol. 182, "Guide to Protein
Purification", M. Deutscher, ed., Acad. Press, San Diego, 1990,
721-37) or photo affinity labeled (Brunner et al., Ann. Rev.
Biochem. 62:483-514, 1993 and Fedan et al., Biochem. Pharmacol.
33:1167-80, 1984) and specific cell-surface proteins can be
identified.
[0204] As a reagent, the polynucleotide encoding the amino acid
residues from residue 304 to 309 of SEQ ID NO: 2, and the
degenerate polynucleotide of SEQ ID NO: 3, can be used to identify
new family members. This would be useful in finding new
galactosyltransferase and putative neurogenic secreted signaling
peptides from the same or other tissues.
[0205] The polypeptides, nucleic acid and/or antibodies of the
present invention can be used in treatment of disorders associated
with cell migration, contact inhibition, tissue interactions,
neuronal specificity, fertilization, embryonic cell adhesions, limb
bud morphogenesis, mesenchyme development, immune recognition,
growth control, tumor metastasis, and glycoprotein and glycolipid
biosynthesis. The molecules of the present invention can be used to
modulate glycoprotein synthesis and/or cell-cell interactions or to
treat or prevent development of pathological conditions in such
diverse tissue as brain, kidney, testis, spinal cord, colon,
prostate, stomach, ovary, pancreas, pituitary gland, adrenal gland,
salivary gland, mammary gland, liver, small intestine, spleen,
thymus, peripheral leukocyte, lymph node, bone marrow, lung,
trachea, placenta, fetal spleen and fetal lung. In particular,
certain neuronal and epithelial deficiencies and malignancies may
be amenable to such diagnosis, treatment or prevention.
[0206] Polynucleotides encoding znssp6 polypeptides are useful
within gene therapy applications where it is desired to increase or
inhibit znssp6 activity. If a mammal has a mutated or absent znssp6
gene, the znssp6 gene can be introduced into the cells of the
mammal. In one embodiment, a gene encoding a znssp6 polypeptide is
introduced in vivo in a viral vector. Such vectors include an
attenuated or defective DNA virus, such as, but not limited to,
herpes simplex virus (HSV), papillomavirus, Epstein Barr virus
(EBV), adenovirus, adeno-associated virus (AAV), and the like.
[0207] Defective viruses, which entirely or almost entirely lack
viral genes, are preferred. A defective virus is not infective
after introduction into a cell. Use of defective viral vectors
allows for administration to cells in a specific, localized area,
without concern that the vector can infect other cells. Examples of
particular vectors include, but are not limited to, a defective
herpes simplex virus 1 (HSV1) vector (Kaplitt et al., Molec. Cell.
Neurosci. 2:320-30, 1991); an attenuated adenovirus vector, such as
the vector described by Stratford-Perricaudet et al., J. Clin.
Invest. 90:626-30, 1992; and a defective adeno-associated virus
vector (Samulski et al., J. Virol. 61:3096-101, 1987; Samulski et
al., J. Virol. 63:3822-8, 1989).
[0208] In another embodiment, a znssp6 gene can be introduced in a
retroviral vector, e.g., as described in Anderson et al., U.S. Pat.
No. 5,399,346; Mann et al. Cell 33:153, 1983; Temin et al., U.S.
Pat. No. 4,650,764; Temin et al., U.S. Pat. No. 4,980,289;
Markowitz et al., J. Virol. 62:1120, 1988; Temin et al., U.S. Pat.
No. 5,124,263; International Patent Publication No. WO 95/07358,
published Mar. 16, 1995 by Dougherty et al.; and Kuo et al., Blood
82:845, 1993.
[0209] Alternatively, the vector can be introduced by lipofection
in vivo using liposomes. Synthetic cationic lipids can be used to
prepare liposomes for in vivo transfection of a gene encoding a
marker (Felgner et al., Proc. Natl. Acad. Sci. U.S.A 84:7413-7,
1987; Mackey et al., Proc. Natl. Acad. Sci. U.S.A 85:8027-31,
1988). The use of lipofection to introduce exogenous genes into
specific organs in vivo has certain practical advantages. Molecular
targeting of liposomes to specific cells represents one area of
benefit. More particularly, directing transfection to particular
cells represents one area of benefit. For instance, directing
transfection to particular cell types would be particularly
advantageous in a tissue with cellular heterogeneity, such as the
pancreas, liver, kidney, and brain. Lipids may be chemically
coupled to other molecules for the purpose of targeting. Targeted
peptides (e.g., hormones or neurotransmitters), proteins such as
antibodies, or non-peptide molecules can be coupled to liposomes
chemically. Similarly, the znssp6 polynucleotide itself can be used
to target specific tissues.
[0210] It is possible to remove the target cells from the body; to
introduce the vector as a naked DNA plasmid; and then to re-implant
the transformed cells into the body. Naked DNA vectors for gene
therapy can be introduced into the desired host cells by methods
known in the art, e.g., transfection, electroporation,
microinjection, transduction, cell fusion, DEAE dextran, calcium
phosphate precipitation, use of a gene gun or use of a DNA vector
transporter. See, e.g., Wu et al., J. Biol. Chem. 267:963-7, 1992;
Wu et al., J. Biol. Chem. 263:14621-4, 1988.
[0211] Various techniques, including antisense and ribozyme
methodologies, can be used to inhibit znssp6 gene transcription and
translation, such as to inhibit cell proliferation in vivo.
Polynucleotides that are complementary to a segment of a
znssp6-encoding polynucleotide (e.g., a polynucleotide as set forth
in SEQ ID NO: 1) are designed to bind to znssp6-encoding mRNA and
to inhibit translation of such mRNA. Such antisense polynucleotides
are used to inhibit expression of znssp6 polypeptide-encoding genes
in cell culture or in a subject.
[0212] The present invention also provides reagents which will find
use in diagnostic applications. For example, the znssp6 gene, a
probe comprising znssp6 DNA or RNA or a subsequence thereof can be
used to determine if the znssp6 gene is present on chromosome
12q24.31 or if a mutation has occurred. Detectable chromosomal
aberrations at the znssp6 gene locus include, but are not limited
to, aneuploidy, gene copy number changes, insertions, deletions,
restriction site changes and rearrangements. Such aberrations can
be detected using polynucleotides of the present invention by
employing molecular genetic techniques, such as restriction
fragment length polymorphism (RFLP) analysis, short tandem repeat
(STR) analysis employing PCR techniques, and other genetic linkage
analysis techniques known in the art (Sambrook et al., ibid.;
Ausubel et. al., ibid.; Marian, Chest 108:255-65, 1995).
[0213] In general, these diagnostic methods comprise the steps of
(a) obtaining a genetic sample from a patient; (b) incubating the
genetic sample with a polynucleotide probe or primer as disclosed
above, under conditions wherein the polynucleotide will hybridize
to complementary polynucleotide sequence, to produce a first
reaction product; and (iii) comparing the first reaction product to
a control reaction product. A difference between the first reaction
product and the control reaction product is indicative of a genetic
abnormality in the patient. Genetic samples for use within the
present invention include genomic DNA, cDNA, and RNA. The
polynucleotide probe or primer can be RNA or DNA, and will comprise
a portion of SEQ ID NOs: 1 or 3, the complement of SEQ ID NOs: 1 or
3, or an RNA equivalent thereof. Suitable assay methods in this
regard include molecular genetic techniques known to those in the
art, such as restriction fragment length polymorphism (RFLP)
analysis, short tandem repeat (STR) analysis employing PCR
techniques, ligation chain reaction (Barany, PCR Methods and
Applications 1:5-16, 1991), ribonuclease protection assays, and
other genetic linkage analysis techniques known in the art
(Sambrook et al., ibid.; Ausubel et. al., ibid.; Marian, Chest
108:255-65, 1995). Ribonuclease protection assays (see, e.g.,
Ausubel et al., ibid., ch. 4) comprise the hybridization of an RNA
probe to a patient RNA sample, after which the reaction product
(RNA-RNA hybrid) is exposed to RNase. Hybridized regions of the RNA
are protected from digestion. Within PCR assays, a patient's
genetic sample is incubated with a pair of polynucleotide primers,
and the region between the primers is amplified and recovered.
Changes in size or amount of recovered product are indicative of
mutations in the patient. Another PCR-based technique that can be
employed is single strand conformational polymorphism (SSCP)
analysis (Hayashi, PCR Methods and Applications 1:34-8, 1991).
[0214] In addition, such polynucleotide probes could be used to
hybridize to counterpart sequences on individual chromosomes.
Chromosomal identification and/or mapping of the znssp6 gene could
provide useful information about gene function and disease
association. Many mapping techniques are available to one skilled
in the art, for example, mapping somatic cell hybrids, and
fluorescence in situ hybridization (FISH). One method is radiation
hybrid mapping. Radiation hybrid mapping is a somatic cell genetic
technique developed for constructing high-resolution, contiguous
maps of mammalian chromosomes (Cox et al., Science 250:245-50,
1990). Partial or full knowledge of a gene's sequence allows one to
design PCR primers suitable for use with chromosomal radiation
hybrid mapping panels. Radiation hybrid mapping panels are
commercially available which cover the entire human genome, such as
the Stanford G3 RH Panel and the GeneBridge 4 RH Panel (Research
Genetics, Inc., Huntsville, Ala.). These panels enable rapid,
PCR-based chromosomal localizations and ordering of genes,
sequence-tagged sites (STSs), and other nonpolymorphic and
polymorphic markers within a region of interest. This includes
establishing directly proportional physical distances between newly
discovered genes of interest and previously mapped markers. The
precise knowledge of a gene's position can be useful for a number
of purposes, including: 1) determining if a sequence is part of an
existing contig and obtaining additional surrounding genetic
sequences in various forms, such as YACs, BACs or cDNA clones; 2)
providing a possible candidate gene for an inheritable disease
which shows linkage to the same chromosomal region; and 3)
cross-referencing model organisms, such as mouse, which may aid in
determining what function a is particular gene might have.
[0215] Sequence tagged sites (STSs) can also be used independently
for chromosomal localization. An STS is a DNA sequence that is
unique in the human genome and can be used as a reference point for
a particular chromosome or region of a chromosome. An STS is
defined by a pair of oligonucleotide primers that are used in a
polymerase chain reaction to specifically detect this site in the
presence of all other genomic sequences. Since STSs are based
solely on DNA sequence they can be completely described within an
electronic database, for example, Database of Sequence Tagged Sites
(dbSTS), GenBank, (National Center for Biological Information,
National Institutes of Health, Bethesda, Md.
http://www.ncbi.nlm.nih.gov), and can be searched with a gene
sequence of interest for the mapping data contained within these
short genomic landmark STS sequences.
[0216] Transgenic mice, engineered to express the znssp6 gene, and
mice that exhibit a complete absence of znssp6 gene function,
referred to as "knockout mice" (Snouwaert et al., Science 257:1083,
1992), may also be generated (Lowell et al., Nature 366:740-42,
1993). These mice may be employed to study the znssp6 gene, cells
affected by the znssp6 gene product, and the protein encoded
thereby in an in vivo system.
[0217] Znssp6 polypeptides, variants, and fragments thereof, may be
useful as replacement therapy for disorders associated with
glycoprotein synthesis and cell-cell interactions.
[0218] Brn is thought to be a signaling molecule for a novel
receptor, thus regulating adhesion between germ and follicle cells.
It is also theorized that brn may be required for lateral
inhibition during early neurogenesis, maintaining epithelial
structure within the neurogenic ectoderm during neuroblast
segregation, and is necessary for epithelial maintenance. See
Goode, S. et al., Dev. Biol. 178:35-50, 1996. Since the Drosophila
follicular epithelium has developmental, morphological, and
molecular properties of vertebrate epithelia, (Goode, S. et al.,
Development 122:3863-3879, 1996) it is considered likely that its
human orthologue would be involved in epithelial development.
[0219] The protein of the present invention has 36% identity to
brn. High expression of znssp6 is observed in fetal and adult
brain. This suggests that znssp6 plays a role in epithelial and
neuronal development and maintenance. It's role in embryonic
development as well as differentiation and stability throughout
life may be critical.
[0220] Additionally, it has been proposed (Goode, et al., 1996,
ibid) that brn and another neurogenic secreted signaling peptide,
Egghead, collaborate with yet a third neurogenic secreted signaling
peptide, Notch, on the apical surface of follicle cells to mediate
germline follicle cell adhesion. Thus, znssp6 may act on cell-cell
contact alone, or in collaboration with other surface
molecules.
[0221] Mutants of these neurogenic secreted signaling peptides,
brn, Egghead, and Notch, result in follicle cells which over
proliferate (Goode, S. et al., ibid). Thus, these peptides, and the
genes encoding them can play a role in tumor suppression. It is
likely that homologous proteins, such as znssp6, can mediate tumor
suppression as well.
[0222] The protein of the present invention has 43% homology to
Cardiac and Pancreatic Protein (WO 98/44112) which is contemplated
to exert an effect on the differentiation of cells in early stages
of cell and tissue development, and possibly serves to aid in the
differentiation of embryonic cells into heart and pancreas
cells.
[0223] Similarly, znssp6 may affect the differentiation of cells in
the early stages of cell and tissue development. Znssp6 may also
affect the differentiation of embryonic cells into brain, kidney,
and testis cells, or the cells and tissues such as spinal cord,
colon, prostate, stomach, ovary, pancreas, pituitary gland, adrenal
gland, salivary gland, mammary gland, liver, small intestine,
spleen, thymus, peripheral leukocyte, lymph node, bone marrow,
lung, trachea, placenta, fetal spleen and fetal lung.
[0224] A less widely appreciated determinant of tissue
morphogenesis is the process of cell rearrangement: Both cell
motility and cell-cell adhesion are likely to play central roles in
morphogenetic cell rearrangements. Cells need to be able to rapidly
break and probably simultaneously remake contacts with neighboring
cells. See Gumbiner, B. M., Cell 69:385-387, 1992. As a secreted
protein in brain, spinal cord, kidney, and testes, znssp6 can play
a role in intercellular rearrangement in these and other
tissues.
[0225] The znssp6 polypeptide is expressed in tissues of the brain,
kidney and testis. Thus, the polypeptides of the present invention
are useful in studying cell adhesion and the role thereof in
metastasis and may be useful in preventing metastasis, in
particular metastasis in tumors of the brain, kidney and testis.
Similarly, polynucleotides and polypeptides of znssp6 may be used
to replace their defective counterparts in tumor or diseased
tissues. Thus, znssp6 polypeptide pharmaceutical compositions of
the present invention may be useful in prevention or treatment of
disorders associated with pathological regulation or the expansion
of these tissues. The polynucleotides of the present invention may
also be used in conjunction with a regulatable promoter, thus
allowing the dosage of delivered protein to be regulated.
[0226] Moreover, the activity and effect of znssp6 on tumor
progression and metastasis can be measured in vivo. Several
syngeneic mouse models have been developed to study the influence
of polypeptides, compounds or other treatments on tumor
progression. In these models, tumor cells passaged in culture are
implanted into mice of the same strain as the tumor donor. The
cells will develop into tumors having similar characteristics in
the recipient mice, and metastasis will also occur in some of the
models. Tumor models include the Lewis lung carcinoma (ATCC No.
CRL-1642) and B16 melanoma (ATCC No. CRL-6323), amongst others.
These are both commonly used tumor lines, syngeneic to the C57BL6
mouse, that are readily cultured and manipulated in vitro. Tumors
resulting from implantation of either of these cell lines are
capable of metastasis to the lung in C57BL6 mice. The Lewis lung
carcinoma model has recently been used in mice to identify an
inhibitor of angiogenesis (O'Reilly MS, et al. Cell 79:
315-328,1994). C57BL6/J mice are treated with an experimental agent
either through daily injection of recombinant protein, agonist or
antagonist or a one time injection of recombinant adenovirus. Three
days following this treatment, 10.sup.5 to 10.sup.6 cells are
implanted under the dorsal skin. Alternatively, the cells
themselves may be infected with recombinant adenovirus, such as one
expressing znssp6, before implantation so that the protein is
synthesized at the tumor site or intracellularly, rather than
systemically. The mice normally develop visible tumors within 5
days. The tumors are allowed to grow for a period of up to 3 weeks,
during which time they may reach a size of 1500-1800 mm.sup.3 in
the control treated group. Tumor size and body weight are carefully
monitored throughout the experiment. At the time of sacrifice, the
tumor is removed and weighed along with the lungs and the liver.
The lung weight has been shown to correlate well with metastatic
tumor burden. As an additional measure, lung surface metastases are
counted. The resected tumor, lungs and liver are prepared for
histopathological examination, immunohistochemistry, and in situ
hybridization, using methods known in the art and described herein.
The influence of the expressed polypeptide in question, e.g.,
znssp6, on the ability of the tumor to recruit vasculature and
undergo metastasis can thus be assessed. In addition, aside from
using adenovirus, the implanted cells can be transiently
transfected with znssp6. Moreover, purified znssp6 or
znssp6-conditioned media can be directly injected in to this mouse
model, and hence be used in this system. Use of stable znssp6
transfectants as well as use of induceable promoters to activate
znssp6 expression in vivo are known in the art and can be used in
this system to assess znssp6 induction of metastasis. For general
reference see, O'Reilly M S, et al. Cell 79:315-328, 1994; and
Rusciano D, et al. Murine Models of Liver Metastasis. Invasion
Metastasis 14:349-361, 1995.
[0227] Tn-syndrome, also called Permanent Mixed-Field
Polyagglutinability, is a very rare acquired disorder affecting all
hematopoietic lineages. This syndrome is characterized by the
expression of the Tn and sialosyl-Tn antigens on the cell surface.
The Tn antigen has been identified as an unsubstituted
.alpha.-linked N-acetyl-galactosamine linked O-glycosidically to
threonine or serine residues of membrane proteins. In healthy
blood, this sugar is substituted by galactose and sialic acid to
form a tetrasaccharide. This Tn antigen may be a result of a
deficiency in beta-1,3-galactosyltransferase. Expression of the Tn
antigen along with the sialosyl-Tn antigen and a TF antigen
(characterized by a deficiency in alpha-2,3-sialyltransferase) have
been recognized as a cancer-associated phenomenon for many years.
See Berger, E. G. et al., Transfus. Clin. Biol. 2:103-108,
1994.
[0228] Thus, the study of this syndrome has been useful in
elucidating the biology of carbohydrate glycosylation disorders and
the appearance of crypt antigens on the cell surface, and cancer.
Highly specific and complex tumor glycan antigens are likely of
great interest in studying tissue specific tumors and znssp6 can be
useful for studying tumors in brain, kidney, and testis
tissues.
[0229] The expression of these cryptantigens in tissues from
normal, chronic pancreatitic, and pancreatic cancer patients was
studied by Itzkowitz, et al, Gastroenterology 100:1691-1700, 1991.
The sialosyl-Tn antigen is expressed in 97% of malignant, but 0% of
normal tissues. The authors suggest that normal pancreas tissue is
preferentially galactosylated resulting in less silaosyl-Tn
antigen. In malignant tissue, conditions favor the sialylation of
Tn antigens thereby accounting for enhanced expression of sialosyl
Tn over T anitgens. A similar sialylation of tissues in the brain,
kidney, and testis could be associated with disease in these
tissues.
[0230] In view of the high expression of znssp6 in the in normal
tissues of the brain, kidney, and testis, a defect in the znssp6
gene may result in defective galactosylation of cell surface
carbohydrates of brain, kidney and testis tissues, leading to over
sialylation of the Tn antigen. Thus, znssp6 polypeptides would be
useful as a brain, kidney or testis beta-1,3-galactosyltransferase
replacement therapy for pre-cancerous and cancer tissues. To verify
the presence of such activity in znssp6 containing normal cell
lines and tumor cell lines, such cell lines are evaluated with
respect to the presence of the Tn antigen according to procedures
known in the art. See, for example, Berger et al., ibid., Itzkowitz
et al., ibid. and the like.
[0231] Additionally, the lack of conditions favoring proper
galactosylation may result in an increase in sialosyl Tn antigens
in tissues expressing znssp6, which may cause an auto-immune
reaction resulting in an immune attack on the brain, kidney, and
testis. In these cases, znssp6 molecules may be used to encourage
proper galactosylation and limit the antigenic recognition in
tissues over expressing the sialosyl Tn antigen.
[0232] Similarly, a defective znssp6 gene may result in improper
glycoslation of the surface carbohydrates of the tissues of brain,
kidney, and testis, thus affecting cell-cell interactions and
possibly cell cycle regulation. Such cases could be treated by
administering polypeptides of znssp6 to mammals with such a
defective gene.
[0233] Znssp6 gene may be useful as a probe to identify humans who
have a defective znssp6 gene. The strong expression of znssp6 in
brain, kidney, and testis suggests that znssp6 polynucleotides or
polypeptides are down-regulated in tumor or malignant tissues.
Thus, polynucleotides and polypeptides of znssp6, and mutations to
them, can be used a diagnostic indicators of cancer in these
tissues. Thus, polynucleotides and polypeptides of znssp6, and
mutations to them, can be used a indicators of pancreatic and
colonic cancer, and disease, in diagnosis.
[0234] The polypeptides, nucleic acid, and/or antibodies of the
present invention may be used in treatment of disorders associated
with brain, kidney, and testis activity and in disorders associated
with glycoprotein synthesis. The molecules of the present invention
may used to modulate or to treat or prevent development of
pathological conditions in such diverse tissue as brain, kidney,
testis, spinal cord, colon, prostate, stomach, ovary, pancreas,
pituitary gland, adrenal gland, salivary gland, mammary gland,
liver, small intestine, spleen, thymus, peripheral leukocyte, lymph
node, bone marrow, lung, trachea, placenta, fetal spleen and fetal
lung. In particular, certain syndromes or diseases may be amenable
to such diagnosis, treatment or prevention.
[0235] The znssp6 polypeptide is expressed in the brain, kidney,
and testis. Thus, znssp6 polypeptide pharmaceutical compositions of
the present invention may be useful in prevention or treatment of
disorders associated with pathological regulation or the expansion
of brain, kidney, and testis tissues.
[0236] The polynucleotides of the present invention may also be
used in conjunction with a regulatable promoter, thus allowing the
dosage of delivered protein to be regulated.
[0237] The localization of znssp6 to chromosome 12q24 maps this
gene to the same region as a disease known as Adult Spinal Muscular
Atrophy (also known as Spinal Muscular Atrophy IV (SMA IV);
Hereditary Motor Neuropathy, Distal Included (HMN, included); and
HMN, Distal, Type II, Included (HMN2, included)). This disease is
characterized by rapid progression of a form of Spinal Muscular
Atrophy beginning between the end of the fourth and sixth decades.
More information on this disease can be found on the internet on
the Online Mendelian Inheritance of Man home page
(http://www3.ncbi.nlm.nih.gov/Omim/). Thus, znssp6 could be the
gene causing Adult Spinal Muscular Atrophy. In such case, therapy
with znssp6 as replacement of the polypeptide or polynucleotide
would be useful.
[0238] For pharmaceutical use, the proteins of the present
invention can be administered orally, rectally, parenterally,
intracisternally, intravaginally, intraperitoneally, topically (as
powders, ointments, drops or transdermal patch) bucally, or as a
pulmonary or nasal inhalant. Intravenous administration will be by
bolus injection or infusion over a typical period of one to several
hours. In general, pharmaceutical formulations will include a
znssp6 protein in combination with a pharmaceutically acceptable
vehicle, such as saline, buffered saline, 5% dextrose in water or
the like. Formulations may further include one or more excipients,
preservatives, solubilizers, buffering agents, albumin to prevent
protein loss on vial surfaces, etc. Methods of formulation are well
known in the art and are disclosed, for example, in Remington: The
Science and Practice of Pharmacy Gennaro, ed., Mack Publishing Co.,
Easton, Pa., .sub.19th ed., 1995. Therapeutic doses will generally
be in the range of 0.1 to 100 .mu.g/kg of patient weight per day,
preferably 0.5-20 mg/kg per day, with the exact dose determined by
the clinician according to accepted standards, taking into account
the nature and severity of the condition to be treated, patient
traits, etc.
[0239] Determination of dose is within the level of ordinary skill
in the art. The proteins may be administered for acute treatment,
over one week or less, often over a period of one to three days or
may be used in chronic treatment, over several months or years. In
general, a therapeutically effective amount of znssp6 is an amount
sufficient to produce a clinically significant change in brain,
kidney and testis tissues. Similarly, a therapeutically effective
amount of znssp6 is an amount sufficient to produce a clinically
significant change in disorders associated with spinal cord, colon,
prostate, stomach, ovary, pancreas, pituitary gland, adrenal gland,
salivary gland, mammary gland, liver, small intestine, spleen,
thymus, peripheral leukocyte, lymph node, bone marrow, lung,
trachea, placenta, fetal spleen and fetal lung. The invention is
further illustrated by the following non-limiting examples.
EXAMPLES
Example 1
[0240] Extension of EST Sequence
[0241] The polynucleotide sequence of the novel polypeptides of the
present invention was initially identified by querying an EST
database. A cDNA clone, corresponding to an EST was obtained and
the deduced amino acid sequence of the insert was determined to be
incomplete at the 5' terminal. Nested 5' RACE polymerase chain
reactions were performed using human kidney marathon cDNAs. The
first RACE used primers ZC9739(SEQ ID NO: 4) and ZC17164 (SEQ ID
NO: 5) and thermalcycler conditions as follows: one cycle of
94.degree. C. for 2 minutes; followed by thirty cycles of
94.degree. C. for 20 seconds, 72.degree. C. for 1 minute; followed
by one cycle of 72.degree. C. for 2 minutes. The second, nested,
RACE reaction used diluted product from the first reaction and
primers ZC9719 (SEQ ID NO: 6) and ZC17165 (SEQ ID NO: 7) and
thermalcycler conditions as follows: one cycle of 94.degree. C. for
2 minutes; followed by five cycles of 94.degree. C. for 20 seconds,
66.degree. C. for 30 seconds, 72.degree. C. for 1 minute; followed
by twenty-five cycles of 94.degree. C. for 20 seconds, 64.degree.
C. for 30 seconds, 72.degree. C. for 1 minute; followed by one
cycle at 72.degree. C. for 7 minutes. Comparison of the 5'
extension of the EST sequence with other family members indicated
that the 5' RACE product was still not full length. A REX analysis
performed on the newly generated 5' sequence data, identified a
second EST which overlaps with the 5' RACE sequence. The insert
corresponding to this second EST was determined to be full-length
znssp6.
[0242] One skilled in the art would be able to isolate the
full-length polynucleotide in the following manner: Sense and
antisense oligonucleotides can be designed to encompass the 5' and
3' ends of the polynucleotide sequence, respectively. Exemplary
oligonucleotides would be a sense primer (SEQ. ID NO: 8), and an
antisense primer (SEQ. ID NO: 9). cDNA from a kidney library can be
used as template, and the following thermalcycler conditions can be
used: 94 degrees for 2 minutes; followed by thirty cycles of 94
degrees for 20 seconds, 72 degrees for 1 minute; followed by a
final extension of 72 degrees for 7 minutes. The resulting PCR
product can be subcloned and sequenced.
[0243] Alternatively, one can order the clone corresponding to the
second EST, number 188406, from IMAGE Consortium
(info@image.llnl.gov).
Example 2
Tissue Distribution
[0244] Analysis of tissue distribution was performed by the
Northern blotting technique using Human Multiple Tissue and Master
Dot Blots (Clontech, Palo Alto, Calif.). A probe of about 430 base
pairs was obtained by PCR of the original EST template using
primers ZC17161 (SEQ ID NO: 10) and ZC17160 (SEQ ID NO: 11).
Thermalcycler conditions were as follows: one cycle of 94.degree.
C. for 2 minutes; followed by thirty-five cycles of 94.degree. C.
for 20 seconds, 65.degree. C. for 30 seconds, 72.degree. C. for 30
seconds; followed by one cycle of 72.degree. C. for 2 minutes. The
PCR product was gel-purified and random prime labeled with 32p
using a commercially available kit (Rediprime DNA Labeling System;
Amersham Corp., Arlington Heights, Ill.) according to the
manufacturer's direction. The probe was then purified using a
NucTrap.RTM. probe purification column (Stratagene, La Jolla,
Calif.). ExpressHyb.TM. Hybridization Solution (Clontech, Palo
Alto, Calif.) was used for pre-hybridization and hybridization.
Hybridization took place overnight at 65.degree. C., and the blots
were then washed three times in 2.times.SSC and 0.05% SDS at
55.degree. C., followed by two washes in 0.1.times.SSC and 0.1% SDS
at 55.degree. C. The blots were then exposed to autoradiograph
film, which was then developed. Strong signals were observed in
brain (adult and fetal), kidney (adult and fetal), and testis. The
major transcript size was about 1.6 kb while minor transcripts of
about 3.0 kb, and 4.0 kb were also evident. Lower level expression
was also observed in tissues such as spinal cord, colon, prostate,
stomach, ovary, pancreas, pituitary gland, adrenal gland, salivary
gland, mammary gland, liver, small intestine, spleen, thymus,
peripheral leukocyte, lymph node, bone marrow, lung, trachea,
placenta, fetal spleen and fetal lung.
Example 3
Chromosomal Assignment and Placement of Znssp6
[0245] Znssp6 was mapped to chromosome 12 using the commercially
available version of the "Stanford G3 Radiation Hybrid Mapping
Panel" (Research Genetics, Inc., Huntsville, Ala.). The "Stanford
G3 RH Panel" contains DNA from each of 83 radiation hybrid clones
of the whole human genome, plus two control DNAs (the RM donor and
the A3 recipient). A publicly available WWW server
(http://shgc-www.stanford.edu) allows chromosomal localization of
markers.
[0246] For the mapping of znssp6 with the "Stanford G3 RH Panel",
20 .mu.l reactions were set up in a 96-well microtiter plate
compatible for PCR (Stratagene, La Jolla, Calif.) and used in a
"RoboCycler Gradient 96" thermal cycler (Stratagene). Each of the
85 PCR reactions consisted of 2 .mu.l 10.times.KlenTaq PCR reaction
buffer (CLONTECH Laboratories, Inc., Palo Alto, Calif.), 1.6 .mu.l
dNTPs mix (2.5 mM each, PERKIN-ELMER, Foster City, Calif.), 1 .mu.l
sense primer, ZC 19,220 (SEQ ID NO: 12), 1 .mu.l antisense primer,
ZC 19,221 (SEQ ID NO: 13), 2 .mu.l "RediLoad" (Research Genetics,
Inc., Huntsville, Ala.), 0.4 .mu.l 50.times.Advantage KlenTaq
Polymerase Mix (Clontech Laboratories, Inc.), 25 ng of DNA from an
individual hybrid clone or control and distilled water for a total
volume of 20 .mu.l. The reactions were overlaid with an equal
amount of mineral oil and sealed. The PCR cycler conditions were as
follows: one cycle of 94.degree. C. for 5 minutes; followed by 35
cycles of 94 for 45 seconds, 68.degree. C. for 45 seconds, and
72.degree. C. for 1 minute, 15 seconds; followed by a final
extension of 7 minutes at 72.degree. C. The reactions were
separated by electrophoresis on a 2% agarose gel (Life
Technologies, Gaithersburg, Md.).
[0247] The results showed linkage of znssp6 to the framework marker
SHGC-13898 with a LOD score of >9 and at a distance of 19.43
cR.sub.--10000 from the marker. The use of surrounding genes and/or
markers positions znssp6 in the 12q24.
Example 4
Construction of znssp6 Glu-Glu-Tagged Expression Vectors for Pichia
methanolica
[0248] Expression of znssp6 in Pichia methanolica utilizes the
expression system described in co-assigned WIPO publication WO
97/17450. An expression plasmid containing all or part of a
polynucleotide encoding znssp6 is constructed via homologous
recombination. The expression vector is built from pCZR190, which
contains the AUG1 promoter, followed by the alpha factor prepro
(aFpp) leader sequence, followed by an amino-terminal Glu-Glu tag,
a blunt-ended Sma I restriction site for insertion of the gene
sequence of interest, a translational stop codon, followed by the
AUG1 terminator, the ADE2 selectable marker, and finally the AUG1
3' untranslated region. Also included in this vector are the URA3
and CEN-ARS sequences required for selection and replication in S.
cerevisiae, and the AmpR and colE1 ori sequences required for
selection and replication in E. coli. The znssp6 sequence inserted
into this vector begins at residue 50 (Arg) of the znssp6 amino
acid sequence (SEQ ID NO: 2).
[0249] Gene expression constructs are prepared by PCR and
homologously recombined into the yeast expression vector pCZR190.
For the amino terminal tagged protein, the N-terminal primer,
ZC25565 (SEQ ID NO: 14), comprises 40 base pairs containing the
.alpha.Fpp coding sequence joined to a nucleotide sequence encoding
a Glu-Glu tag followed by 25 base pairs of nucleotide sequence
encoding a portion of the amino-terminus from the ectodomain of the
znssp6 sequence. The C-terminal primer, ZC25561 (SEQ ID NO: 15),
comprises about 25 base pairs of carboxy terminus coding sequence
of the znssp6 joined with 40 base pairs of AUG1 terminator
sequence. The polymerase chain reaction contains 1 ng znssp6
template (i.e., from SEQ ID NO: 1), 100 pmol of each primer, 10
.mu.l of 10.times.PCR buffer, 1 .mu.l Pwo polymerase (Boehringer
Mannheim, Indianapolis, Ind.), 10 .mu.l of 0.25 mM nucleotide
triphosphate mix (Perkin Elmer, Foster City, Calif.) and dH.sub.2O
to a total volume of 100 .mu.l. PCR conditions are as follows: 25
cycles of 30 seconds at 94.degree. C, 1 minute at 55.degree. C and
1 minute at 72.degree. C.
[0250] Similarly, for the carboxyl terminal tagged protein, the
N-terminal primer, ZC25563 (SEQ ID NO: 16), comprises 40 base pairs
containing the aFpp coding sequence joined to 25 base pairs of
nucleotide sequence encoding a portion of the amino-terminus from
the ectodomain of the znssp6 sequence. The C-terminal primer,
ZC25564 (SEQ ID NO: 17), comprises about 25 base pairs of carboxy
terminus coding sequence of the znssp6 joined to a nucleotide
sequence encoding a Glu-Glu tag followed by 40 base pairs of AUG1
terminator sequence. Ploymerase chain reaction volumes and
conditions are the same as those listed for the amino terminal
tagged gene construct.
[0251] An untagged gene expression construct is also prepared: the
N-terminal primer, ZC25562 (SEQ ID NO: 18), comprises 40 base pairs
containing the .alpha.Fpp coding sequence joined to 25 base pairs
of nucleotide sequence encoding a portion of the amino-terminus
from the ectodomain of the znssp6 sequence. The C-terminal primer,
ZC25566 (SEQ ID NO: 19), comprises about 25 base pairs of carboxy
terminus coding sequence of the znssp6 joined to 40 base pairs of
AUG1 terminator sequence. Ploymerase chain reaction volumes and
conditions are the same as those listed for the amino terminal
tagged gene construct.
[0252] The NEE-, CEE- and untagged znssp6 plasmids are made by
homologously recombining 100 ng of Sma I digested pCRZ204 acceptor
vector, and 1 .mu.g of Eco RI-Xho I znssp.sup.6 gene constructs
made above into S. cerevisiae. One hundred microliters of competent
yeast cells (S. cerevisiae) are combined with 10 .mu.l of each of
the fragments and transferred to a 0.2 cm electroporation cuvette.
The yeast/DNA mixture is electropulsed at 0.75 kV (5 kV/cm),
.infin. ohms, 25 .mu.F. To the cuvette is added 600 .mu.l of 1.2 M
sorbitol and 300 .mu.l aliquots of the yeast/sorbitol mixture are
plated onto two URA D plates and incubated at 30.degree. C.
[0253] After about 48 hours the Ura+yeast transformants from a
single plate are resuspended in 2.5 ml H.sub.2O and spun briefly to
pellet the yeast cells. The cell pellet is resuspended in 1 ml of
lysis buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris, pH
8.0, 1 mM EDTA). Five hundred microliters of the lysis mixture was
added to an Eppendorf tube containing 300 .mu.l acid washed glass
beads and 200 .mu.l phenol-chloroform, vortexed for 1 minute
intervals two or three times, followed by a 5 minute spin in a
Eppendorf centrifuge at maximum speed. Three hundred microliters of
the aqueous phase is transferred to a fresh tube and the DNA
precipitated with 600 .mu.l ethanol (EtOH), followed by a
centrifugation at 14,000 RPM of 10 minutes at 40C. The DNA pellet
is resuspended in 100 .mu.l H.sub.2O.
[0254] Five microliters of the resuspended DNA prep is used to
transform 40 .mu.l of electrocompetent E. coli cells (DH10B, Gibco
BRL). The cells are electropulsed at 2.0 kV and 400 ohms. Following
electroporation, 1 ml SOC (2% Bacto.TM. Tryptone (Difco, Detroit,
Mich.), 0.5% yeast extract (Difco), 10 mM NaCl, 2.5 mM KCl, 10 mM
MgCl.sub.2, 10 mM MgSO.sub.4, 20 mM glucose) is added and the cells
are allowed to recover for 1 hour at 37.degree. C. prior to plating
250 .mu.l aliquots on four LB AMP plates (LB broth (Lennox), 1.8%
Bacto.TM. Agar (Difco), 100 mg/L Ampicillin).
[0255] Individual clones harboring the correct expression construct
are identified by PCR screening. The primers used to amplify the
N-tagged znssp6 clone are ZC25565 (SEQ ID NO: 14) and ZC25561(SEQ
ID NO: 15). The insert sequence of positive clones, identified by a
1071 bp fragment, are verified by sequence analysis. The primers
used to amplify the C-tagged znssp6 clone are ZC25563 (SEQ ID NO:
16) and ZC25564(SEQ ID NO: 17). The insert sequence of positive
clones, identified by a 1067 bp fragment, are verified by sequence
analysis. The primers used to amplify the untagged znssp6 clone are
ZC25562 (SEQ ID NO: 18) and ZC25566(SEQ ID NO: 19). The insert
sequence of positive clones, identified by a 1066 bp fragment, are
verified by sequence analysis. Larger scale plasmid DNA is isolated
using Qiagen maxi kits (Qiagen, Valencia, Calif.) and the DNA is
digested with Not I to liberate the Pichia-znssp6 expression
cassette from the vector backbone. The Not I DNA fragment is then
transformed into the Pichia methanolica expression host, PMAD16.
This is done by mixing 100 .mu.l of prepared competent PMAD16 cells
with 10 ng of Not I digested tagged or untagged znssp6 fragment and
transferred to a 0.2 cm electroporation cuvette. The yeast/DNA
mixture is electropulsed at 0.75 kV, 25 .mu.F, infinite ohms. To
the cuvette is added 1 ml of IX Yeast Nitrogen Base and 500 ml
aliquots are plated onto two ADE DS (0.056% -Ade -Trp -Thr powder,
0.67% yeast nitrogen base without amino acids, 2% D-glucose, 0.5%
200.times.tryptophan, threonine solution, and 18.22% D-sorbitol)
plates for selection and incubated at 30.degree. C. Transformants
are then picked and screened via Western blot for high-level
expression and subjected to large scale fermentation.
[0256] From the foregoing, it will be appreciated that, although
specific embodiments of the invention have been described herein
for purposes of illustration, various modifications may be made
without deviating from the spirit and scope of the invention.
Accordingly, the invention is not limited except as by the appended
claims.
Sequence CWU 1
1
20 1 1420 DNA Homo sapiens CDS (135)...(1271) 1 cacgagctcc
acgcccgtac cccggcgtca cgctcagccc gcggtgctcg cacacctgag 60
actcatctcg cttcgacccc gccgccgccg ccgcccggca tcctgagcac ggagacagtc
120 tccagctgcc gttc atg ctt cct ccc cag cct tcc gca gcc cac cag gga
170 Met Leu Pro Pro Gln Pro Ser Ala Ala His Gln Gly 1 5 10 agg ggc
ggt agg agt ggc ctt tta cca aag gga ccg gcg atg ctc tgc 218 Arg Gly
Gly Arg Ser Gly Leu Leu Pro Lys Gly Pro Ala Met Leu Cys 15 20 25
agg ctg tgc tgg ctg gtc tcg tac agc ttg gct gtg ctg ttg ctc ggc 266
Arg Leu Cys Trp Leu Val Ser Tyr Ser Leu Ala Val Leu Leu Leu Gly 30
35 40 tgc ctg ctc ttc ctg agg aag gcg gcc aag ccc gca gga gac ccc
acg 314 Cys Leu Leu Phe Leu Arg Lys Ala Ala Lys Pro Ala Gly Asp Pro
Thr 45 50 55 60 gcc cac cag cct ttc tgg gct ccc cca aca ccc cgt cac
agc cgg tgt 362 Ala His Gln Pro Phe Trp Ala Pro Pro Thr Pro Arg His
Ser Arg Cys 65 70 75 cca ccc aac cac aca gtg tct agc gcc tct ctg
tcc ctg cct agc cgt 410 Pro Pro Asn His Thr Val Ser Ser Ala Ser Leu
Ser Leu Pro Ser Arg 80 85 90 cac cgt ctc ttc ttg acc tat cgt cac
tgc cga aat ttc tct atc ttg 458 His Arg Leu Phe Leu Thr Tyr Arg His
Cys Arg Asn Phe Ser Ile Leu 95 100 105 ctg gag cct tca ggc tgt tcc
aag gat acc ttc ttg ctc ctg gcc atc 506 Leu Glu Pro Ser Gly Cys Ser
Lys Asp Thr Phe Leu Leu Leu Ala Ile 110 115 120 aag tca cag cct ggt
cac gtg gag cga cgt gcg gct atc cgc agc acg 554 Lys Ser Gln Pro Gly
His Val Glu Arg Arg Ala Ala Ile Arg Ser Thr 125 130 135 140 tgg ggc
agg gtg ggg gga tgg gct agg ggc cgg cag ctg aag ctg gtg 602 Trp Gly
Arg Val Gly Gly Trp Ala Arg Gly Arg Gln Leu Lys Leu Val 145 150 155
ttc ctc cta ggg gtg gca gga tcc gct ccc cca gcc cag ctg ctg gcc 650
Phe Leu Leu Gly Val Ala Gly Ser Ala Pro Pro Ala Gln Leu Leu Ala 160
165 170 tat gag agt agg gag ttt gat gac atc ctc cag tgg gac ttc act
gag 698 Tyr Glu Ser Arg Glu Phe Asp Asp Ile Leu Gln Trp Asp Phe Thr
Glu 175 180 185 gac ttc ttc aac ctg acg ctc aag gag ctg cac ctg cag
cgc tgg gtg 746 Asp Phe Phe Asn Leu Thr Leu Lys Glu Leu His Leu Gln
Arg Trp Val 190 195 200 gtg gct gcc tgc ccc cag gcc cat ttc atg cta
aag gga gat gac gat 794 Val Ala Ala Cys Pro Gln Ala His Phe Met Leu
Lys Gly Asp Asp Asp 205 210 215 220 gtc ttt gtc cac gtc ccc aac gtg
tta gag ttc ctg gat ggc tgg gac 842 Val Phe Val His Val Pro Asn Val
Leu Glu Phe Leu Asp Gly Trp Asp 225 230 235 cca gcc cag gac ctc ctg
gtg gga gat gtc atc cgc caa gcc ctg ccc 890 Pro Ala Gln Asp Leu Leu
Val Gly Asp Val Ile Arg Gln Ala Leu Pro 240 245 250 aac agg aac act
aag gtc aaa tac ttc atc cca ccc tca atg tac agg 938 Asn Arg Asn Thr
Lys Val Lys Tyr Phe Ile Pro Pro Ser Met Tyr Arg 255 260 265 gcc acc
cac tac cca ccc tat gct ggt ggg gga gga tat gtc atg tcc 986 Ala Thr
His Tyr Pro Pro Tyr Ala Gly Gly Gly Gly Tyr Val Met Ser 270 275 280
aga gcc aca gtg cgg cgc ctc cag gct atc atg gaa gat gct gaa ctc
1034 Arg Ala Thr Val Arg Arg Leu Gln Ala Ile Met Glu Asp Ala Glu
Leu 285 290 295 300 ctc tcc att gat gat gtc ttt gtg ggt atg tgc ctg
agg agg ctg ggg 1082 Leu Ser Ile Asp Asp Val Phe Val Gly Met Cys
Leu Arg Arg Leu Gly 305 310 315 ctg agc cct atg cac cat gct ggc ttc
aag aca ttt gga atc cgg cgg 1130 Leu Ser Pro Met His His Ala Gly
Phe Lys Thr Phe Gly Ile Arg Arg 320 325 330 ccc ctg gac ccc tta gac
ccc tgc ctg tat agg ggg ctc ctg ctg gtt 1178 Pro Leu Asp Pro Leu
Asp Pro Cys Leu Tyr Arg Gly Leu Leu Leu Val 335 340 345 cac cgc ctc
agc ccc ctc gag atg tgg acc atg tgg gca ctg gtg aca 1226 His Arg
Leu Ser Pro Leu Glu Met Trp Thr Met Trp Ala Leu Val Thr 350 355 360
gat gag ggg ctc aag tgt gca gct ggc ccc ata ccc cag cgc tga 1271
Asp Glu Gly Leu Lys Cys Ala Ala Gly Pro Ile Pro Gln Arg * 365 370
375 agggtgggtt gggcaacagc ctgagagtgg actcagtgtt gattctctat
cgtgatgcga 1331 aattgatgcc tgctgctcta cagaaaatgc caacttggtt
ttttaactcc tctcaccctg 1391 ttagctctga ttaaaaacac tgcaaccca 1420 2
378 PRT Homo sapiens 2 Met Leu Pro Pro Gln Pro Ser Ala Ala His Gln
Gly Arg Gly Gly Arg 1 5 10 15 Ser Gly Leu Leu Pro Lys Gly Pro Ala
Met Leu Cys Arg Leu Cys Trp 20 25 30 Leu Val Ser Tyr Ser Leu Ala
Val Leu Leu Leu Gly Cys Leu Leu Phe 35 40 45 Leu Arg Lys Ala Ala
Lys Pro Ala Gly Asp Pro Thr Ala His Gln Pro 50 55 60 Phe Trp Ala
Pro Pro Thr Pro Arg His Ser Arg Cys Pro Pro Asn His 65 70 75 80 Thr
Val Ser Ser Ala Ser Leu Ser Leu Pro Ser Arg His Arg Leu Phe 85 90
95 Leu Thr Tyr Arg His Cys Arg Asn Phe Ser Ile Leu Leu Glu Pro Ser
100 105 110 Gly Cys Ser Lys Asp Thr Phe Leu Leu Leu Ala Ile Lys Ser
Gln Pro 115 120 125 Gly His Val Glu Arg Arg Ala Ala Ile Arg Ser Thr
Trp Gly Arg Val 130 135 140 Gly Gly Trp Ala Arg Gly Arg Gln Leu Lys
Leu Val Phe Leu Leu Gly 145 150 155 160 Val Ala Gly Ser Ala Pro Pro
Ala Gln Leu Leu Ala Tyr Glu Ser Arg 165 170 175 Glu Phe Asp Asp Ile
Leu Gln Trp Asp Phe Thr Glu Asp Phe Phe Asn 180 185 190 Leu Thr Leu
Lys Glu Leu His Leu Gln Arg Trp Val Val Ala Ala Cys 195 200 205 Pro
Gln Ala His Phe Met Leu Lys Gly Asp Asp Asp Val Phe Val His 210 215
220 Val Pro Asn Val Leu Glu Phe Leu Asp Gly Trp Asp Pro Ala Gln Asp
225 230 235 240 Leu Leu Val Gly Asp Val Ile Arg Gln Ala Leu Pro Asn
Arg Asn Thr 245 250 255 Lys Val Lys Tyr Phe Ile Pro Pro Ser Met Tyr
Arg Ala Thr His Tyr 260 265 270 Pro Pro Tyr Ala Gly Gly Gly Gly Tyr
Val Met Ser Arg Ala Thr Val 275 280 285 Arg Arg Leu Gln Ala Ile Met
Glu Asp Ala Glu Leu Leu Ser Ile Asp 290 295 300 Asp Val Phe Val Gly
Met Cys Leu Arg Arg Leu Gly Leu Ser Pro Met 305 310 315 320 His His
Ala Gly Phe Lys Thr Phe Gly Ile Arg Arg Pro Leu Asp Pro 325 330 335
Leu Asp Pro Cys Leu Tyr Arg Gly Leu Leu Leu Val His Arg Leu Ser 340
345 350 Pro Leu Glu Met Trp Thr Met Trp Ala Leu Val Thr Asp Glu Gly
Leu 355 360 365 Lys Cys Ala Ala Gly Pro Ile Pro Gln Arg 370 375 3
1134 DNA Artificial Sequence degenerate sequence 3 atgytnccnc
cncarccnws ngcngcncay carggnmgng gnggnmgnws nggnytnytn 60
ccnaarggnc cngcnatgyt ntgymgnytn tgytggytng tnwsntayws nytngcngtn
120 ytnytnytng gntgyytnyt nttyytnmgn aargcngcna arccngcngg
ngayccnacn 180 gcncaycarc cnttytgggc nccnccnacn ccnmgncayw
snmgntgycc nccnaaycay 240 acngtnwsnw sngcnwsnyt nwsnytnccn
wsnmgncaym gnytnttyyt nacntaymgn 300 caytgymgna ayttywsnat
hytnytngar ccnwsnggnt gywsnaarga yacnttyytn 360 ytnytngcna
thaarwsnca rccnggncay gtngarmgnm gngcngcnat hmgnwsnacn 420
tggggnmgng tnggnggntg ggcnmgnggn mgncarytna arytngtntt yytnytnggn
480 gtngcnggnw sngcnccncc ngcncarytn ytngcntayg arwsnmgnga
rttygaygay 540 athytncart gggayttyac ngargaytty ttyaayytna
cnytnaarga rytncayytn 600 carmgntggg tngtngcngc ntgyccncar
gcncayttya tgytnaargg ngaygaygay 660 gtnttygtnc aygtnccnaa
ygtnytngar ttyytngayg gntgggaycc ngcncargay 720 ytnytngtng
gngaygtnat hmgncargcn ytnccnaaym gnaayacnaa rgtnaartay 780
ttyathccnc cnwsnatgta ymgngcnacn caytayccnc cntaygcngg nggnggnggn
840 taygtnatgw snmgngcnac ngtnmgnmgn ytncargcna thatggarga
ygcngarytn 900 ytnwsnathg aygaygtntt ygtnggnatg tgyytnmgnm
gnytnggnyt nwsnccnatg 960 caycaygcng gnttyaarac nttyggnath
mgnmgnccny tngayccnyt ngayccntgy 1020 ytntaymgng gnytnytnyt
ngtncaymgn ytnwsnccny tngaratgtg gacnatgtgg 1080 gcnytngtna
cngaygargg nytnaartgy gcngcnggnc cnathccnca rmgn 1134 4 27 DNA
Artificial Sequence oligonucleotide primer 4 ccatcctaat acgactcact
atagggc 27 5 23 DNA Artificial Sequence oligonucleotide primer 5
cggatagccg cacgtcgctc cac 23 6 23 DNA Artificial Sequence
oligonucleotide primer 6 actcactata gggctcgagc ggc 23 7 23 DNA
Artificial Sequence oligonucleotide primer 7 tgaccaggct gtgacttgat
ggc 23 8 24 DNA Artificial Sequence oligonucleotide primer 8
cttggcacga ggcacgagct ccac 24 9 24 DNA Artificial Sequence
oligonucleotide primer 9 ctcaggctgt tgcccaaccc accc 24 10 25 DNA
Artificial Sequence oligonucleotide primer 10 gtcttgaagc cagcatggtg
catag 25 11 25 DNA Artificial Sequence oligonucleotide primer 11
gggagtttga tgacatcctc cagtg 25 12 18 DNA Artificial Sequence
oligonucleotide primer 12 gccaagcccg caggagac 18 13 18 DNA
Artificial Sequence oligonucleotide primer 13 acggctaggc agggacag
18 14 66 DNA Artificial Sequence Oligonucleotide primer 14
aaattataaa aatatccaaa cacgcagccc tagaatacta gtcatctctg gggtatgggg
60 ccagct 66 15 65 DNA Artificial Sequence Oligonucleotide primer
15 ggacaagaga gaagaagaat acatgccaat ggaaggtggt aggaaggctg
ccaaacccgc 60 aggag 65 16 65 DNA Artificial Sequence
Oligonucleotide primer 16 cattgctgct aaagaagaag gtgtaagctt
ggacaagaga aggaaagcgg ctaagcccgc 60 aggag 65 17 65 DNA Artificial
Sequence Oligonucleotide primer 17 actaggaatt ctactccata ggcatatact
cctcgcctcc gcgttggggt atggggccag 60 ctgca 65 18 65 DNA Artificial
Sequence Oligonucleotide primer 18 cattgctgct aaagaagaag gtgtaagctt
ggacaagaga aggaaggcgg ctaagcccgc 60 aggag 65 19 120 DNA Artificial
Sequence Oligonucleotide primer 19 catagtttct ttcttaacag atatgggcag
aagaaatggc tgaatgcctc tggccatagc 60 ggccggcccc taggatccga
attctagaag ctttgtgtct caaaatctct gatgttacat 120 20 6 PRT Artificial
Sequence amino acid motif 20 Xaa Asp Val Xaa Xaa Gly 1 5
* * * * *
References