U.S. patent application number 10/177079 was filed with the patent office on 2003-09-18 for beta-1,3-galactosyltransferase homologs.
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 | 20030175922 10/177079 |
Document ID | / |
Family ID | 26809148 |
Filed Date | 2003-09-18 |
United States Patent
Application |
20030175922 |
Kind Code |
A1 |
Conklin, Darrell C. ; et
al. |
September 18, 2003 |
Beta-1,3-galactosyltransferase homologs
Abstract
The present invention relates to polynucleotide and polypeptide
molecules for znssp2, 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 znssp2 polypeptides.
Inventors: |
Conklin, Darrell C.;
(Seattle, WA) ; Yamamoto, Gayle; (Seattle, WA)
; Jaspers, Stephen R.; (Edmonds, WA) ; Gao,
Zeren; (Redmond, WA) |
Correspondence
Address: |
Robyn Adams
Patent Department
ZymoGenetics, Inc.
1201 Eastlake Avenue East
Seattle
WA
98102
US
|
Assignee: |
ZymoGenetics, Inc.
|
Family ID: |
26809148 |
Appl. No.: |
10/177079 |
Filed: |
June 21, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10177079 |
Jun 21, 2002 |
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09459133 |
Dec 10, 1999 |
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6416988 |
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60111697 |
Dec 10, 1998 |
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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; 435/320.1; 435/325; 536/23.2 |
International
Class: |
C07H 021/04; C12N
009/10; C12P 021/02; C12N 005/06 |
Claims
What is claimed is:
1. An isolated polypeptide comprising residues 148 to 397 of SEQ ID
NO:2.
2. The isolated polypeptide according to claim 1 wherein the
polypeptide comprises residues 19 to 397 of SEQ ID NO:2.
3. The isolated polypeptide according to claim 2 wherein the
polypeptide comprises residues 1 to 397 of SEQ ID NO:2.
4. An isolated polypeptide selected from the group consisting of:
a) a polypeptide comprising residues 1 to 18 of SEQ ID NO:2; b) a
polypeptide comprising residues 19 to 147 of SEQ ID NO:2; c) a
polypeptide comprising residues 148 to 397 of SEQ ID NO:2; d) a
polypeptide comprising residues 19 to 397 of SEQ ID NO:2; and e) a
polypeptide comprising residues 1 to 397 of SEQ ID NO:2.
5. An isolated polynucleotide encoding a polypeptide wherein the
polypeptide comprises residues 148 to 397 of SEQ ID NO:2.
6. The isolated polynucleotide according to claim 5, wherein the
polypeptide molecule comprises residues 19 to 397 of SEQ ID
NO:2.
7. The isolated polynucleotide according to claim 5, wherein the
polypeptide molecule comprises residues 1 to 397 of SEQ ID
NO:2.
8. An isolated polynucleotide encoding a polypeptide molecule
wherein the polypeptide is selected from the group consisting of:
a) a polypeptide comprising residues 1 to 18 of SEQ ID NO:2; b) a
polypeptide comprising residues 19 to 147 of SEQ ID NO:2; c) a
polypeptide comprising residues 148 to 397 of SEQ ID NO:2; d) a
polypeptide comprising residues 19 to 397 of SEQ ID NO:2; and e) a
polypeptide comprising residues 1 to 397 of SEQ ID NO:2.
9. 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.
10. The expression vector according to claim 9 wherein the DNA
segment contains an affinity tag.
11. A cultured cell into which has been introduced an expression
vector according to claim 9, wherein said cell expresses the
polypeptide encoded by the DNA segment.
12. A method of producing a polypeptide comprising culturing a cell
according to claim 11, whereby said cell expresses the polypeptide
encoded by the DNA segment; and recovering the polypeptide.
13. A method of producing an antibody comprising the following
steps in order: inoculating an animal with a polypeptide selected
from the group consisting of: a) polypeptide comprising residues 1
to 18 of SEQ ID NO:2; b) a polypeptide comprising residues 19 to
147 of SEQ ID NO:2; c) a polypeptide comprising residues 148 to 397
of SEQ ID NO:2; d) a polypeptide comprising residues 19 to 397 of
SEQ ID NO:2; and e) a polypeptide comprising residues 1 to 397 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.
14. An antibody produced by the method of claim 13, which binds to
a residues 1 to 397 of SEQ ID NO:2.
15. The antibody of claim 14, wherein the antibody is a monoclonal
antibody.
16. An antibody which specifically binds to a polypeptide of claim
3.
17. 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 consisting
of residues 1 to 6 of SEQ ID NO:2; b) a polypeptide consisting of
residues 26 to 54 of SEQ ID NO:2; c) a polypeptide consisting of
residues 82 to 94 of SEQ ID NO:2; d) a polypeptide consisting of
residues 110 to 117 of SEQ ID NO:2; e) a polypeptide consisting of
residues 110 to 127 of SEQ ID NO:2; f) a polypeptide consisting of
residues 122 to 127 of SEQ ID NO:2; g) a polypeptide consisting of
residues 122 to 136 of SEQ ID NO:2; h) a polypeptide consisting of
residues 131 to 136 of SEQ ID NO:2; i) a polypeptide consisting of
residues 131 to 146 of SEQ ID NO:2; j) a polypeptide consisting of
residues 139 to 146 of SEQ ID NO:2; k) a polypeptide consisting of
residues 154 to 177 of SEQ ID NO:2; l) a polypeptide consisting of
residues 187 to 197 of SEQ ID NO:2; m) a polypeptide consisting of
residues 187 to 207 of SEQ ID NO:2; n) a polypeptide consisting of
residues 202 to 207 of SEQ ID NO:2; o) a polypeptide consisting of
residues 282 to 289 of SEQ ID NO:2; p) a polypeptide consisting of
residues 282 to 301 of SEQ ID NO:2; q) a polypeptide consisting of
residues 295 to 301 of SEQ ID NO:2; r) a polypeptide consisting of
residues 358 to 365 of SEQ ID NO:2; s) a polypeptide consisting of
residues 358 to 397 of SEQ ID NO:2; and t) a polypeptide consisting
of residues 387 to 397 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.
18. An antibody produced by the method of claim 17, which binds to
a residues 1 to 397 of SEQ ID NO:2.
19. The antibody of claim 18, wherein the antibody is a monoclonal
antibody.
20. A method for modulating cell-cell interactions by combining the
polypeptide according to claim 1, with cells in vivo and in
vitro.
21. A method for modulating cell-cell interactions according to
claim 20, whereby the cells are derived from tissues selected from
the group consisting of: a) tissues from pancreas; b) tissues from
colon; c) tissues from small intestine; d) tissues from bladder; e)
tissues from prostate; f) tissues from myometrium; and g) tissues
from breast.
22. A method for modulating glycoprotein and glycolipid
biosynthesis by combining the polypeptide according to claim 1,
with cells in vivo and in vitro.
23. A method for modulating cell-cell interactions according to
claim 22, whereby the cells are derived from tissues selected from
the group consisting of: a) tissues from pancreas; b) tissues from
colon; c) tissues from small intestine; d) tissues from bladder; e)
tissues from prostate; f) tissues from myometrium; and g) tissues
from breast.
24. A method of detecting a molecule which binds to a polypeptide
comprising contacting the polypeptide with a test sample containing
the molecule wherein the polypeptide comprises residues 148 to 397
of SEQ ID NO:2 and whereby the molecule binds the polypeptide.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application is related to Provisional Application No.
60/111,697 filed on Dec. 10, 1998. Under 35 U.S.C. .sctn.119(e)(1),
this application claims benefit of said Provisional
Application.
BACKGROUND OF THE INVENTION
[0002] Beta-1,3-galactosyltransferase molecules are classified in
the family of glycosyltransferases. In addition to transferring
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.
[0003] 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. The
Beta-1,3-galactosyl-transferases are characterized by the
elongation of type I oligosaccharide chains, and the
Beta-1,4-galactosyl-transferases are characterized by the
elongation of type II oligosaccharide chains. Both types of
carbohydrate structures are present in soluble oligosaccharides of
human milk, and are also found on glycoproteins and glycolipids,
and are important precursors of blood group antigens. Both
galactosyltransferases require a divalent cation (Mn.sup.2+) to
function. Beta-1,4-galactosyltransferases are expressed in various
cell types and tissues, while the Beta-1,3-galactosyltransferases
seem to have more restricted tissue distributions.
[0004] 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
.alpha. 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.
[0005] 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 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. See, for
example, Shur, B. D., Mol Cell Bioc. 61:143-158, 1984.
[0006] The failure of tumor cell-tumor cell adhesion is believed to
be a contributing factor to tumor metastases. See, for example,
Zetter, Cancer Biology, 4: 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 or treatment of
metastases.
[0007] .beta.1.fwdarw.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, is involved in contact and
adhesion between germ-line and follicle cells (Amado, M. et al., J.
Biol. Chem. 273, 21: 12770-12778, 1998).
[0008] 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 imcomplete or disordered gylcan
biosynthesis has been recognized as a cancer-associated phenomenon.
Tn and sialosyl-Tn antigens are among the most investigated
cancer-associated carbohydrates antigens.
[0009] 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
[0010] Within one aspect, the present invention provides an
isolated polypeptide comprising residues 148 to 397 of SEQ ID NO:2.
Within an embodiment, the isolated polypeptide comprises residues
19 to 397 of SEQ ID NO:2. Within another embodiment, the isolated
polypeptide comprises residues 1 to 397 of SEQ ID NO:2.
[0011] Within another aspect, the present invention provides an
isolated polypeptide selected from the group consisting of: a
polypeptide comprising residues 1 to 18 of SEQ ID NO:2; a
polypeptide comprising residues 19 to 147 of SEQ ID NO:2; a
polypeptide comprising residues 148 to 397 of SEQ ID NO:2; a
polypeptide comprising residues 19 to 397 of SEQ ID NO:2; and a
polypeptide comprising residues 1 to 397 of SEQ ID NO:2.
[0012] Within another aspect, the present invention provides an
isolated polynucleotide encoding a polypeptide wherein the
polypeptide comprises residues 148 to 397 of SEQ ID NO:2. Within an
embodiment, the polypeptide molecule comprises residues 19 to 397
of SEQ ID NO:2. Within anothe embodiment, the polypeptide molecule
comprises residues 1 to 397 of SEQ ID NO:2.
[0013] Within another aspect, the present invention provides an
isolated polynucleotide encoding a polypeptide molecule wherein the
polypeptide is selected from the group consisting of: a polypeptide
comprising residues 1 to 18 of SEQ ID NO:2; a polypeptide
comprising residues 19 to 147 of SEQ ID NO:2; a polypeptide
comprising residues 148 to 397 of SEQ ID NO:2; a polypeptide
comprising residues 19 to 397 of SEQ ID NO:2; and a polypeptide
comprising residues 1 to 397 of SEQ ID NO:2. Within an embodiment
is provided an expression vector comprising the following operably
linked elements: a) a transcription promoter; b) 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 the cell, whereby said cell
expresses the polypeptide encoded by the DNA segment; and
recovering the polypeptide.
[0014] Within another aspect is provided a method of producing an
antibody comprising the following steps in order: inoculating an
animal with a polypeptide selected from the group consisting of: a
polypeptide comprising residues 1 to 18 of SEQ ID NO:2; a
polypeptide comprising residues 19 to 147 of SEQ ID NO:2; a
polypeptide comprising residues 148 to 397 of SEQ ID NO:2; a
polypeptide comprising residues 19 to 397 of SEQ ID NO:2; and a
polypeptide comprising residues 1 to 397 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 residues 1 to 397 of
SEQ ID NO:2. Within another embodiment the antibody is a monoclonal
antibody. Within another embodiment the antibody specifically binds
to a polypeptide of residues 1 to 397 of SEQ ID NO:2.
[0015] Within another apsect is provided 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 consisting of residues 1 to 6 of SEQ ID NO:2; a
polypeptide consisting of residues 26 to 54 of SEQ ID NO:2; a
polypeptide consisting of residues 82 to 94 of SEQ ID NO:2; a
polypeptide consisting of residues 110 to 117 of SEQ ID NO:2; a
polypeptide consisting of residues 110 to 127 of SEQ ID NO:2; a
polypeptide consisting of residues 122 to 127 of SEQ ID NO:2; a
polypeptide consisting of residues 122 to 136 of SEQ ID NO:2; a
polypeptide consisting of residues 131 to 136 of SEQ ID NO:2; a
polypeptide consisting of residues 131 to 146 of SEQ ID NO:2; a
polypeptide consisting of residues 139 to 146 of SEQ ID NO:2; a
polypeptide consisting of residues 154 to 177 of SEQ ID NO:2; a
polypeptide consisting of residues 187 to 197 of SEQ ID NO:2; a
polypeptide consisting of residues 187 to 207 of SEQ ID NO:2; a
polypeptide consisting of residues 202 to 207 of SEQ ID NO:2; a
polypeptide consisting of residues 282 to 289 of SEQ ID NO:2; a
polypeptide consisting of residues 282 to 301 of SEQ ID NO:2; a
polypeptide consisting of residues 295 to 301 of SEQ ID NO:2; a
polypeptide consisting of residues 358 to 365 of SEQ ID NO:2; a
polypeptide consisting of residues 358 to 397 of SEQ ID NO:2; and a
polypeptide consisting of residues 387 to 397 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 which binds to a residues 1 to
397 of SEQ ID NO:2. Within another embodiment, the antibody is a
monoclonal antibody.
[0016] Within another aspect the invention provides a method for
modulating cell-cell interactions by combining the polypeptide of
residues 148 to 397, with cells in vivo and in vitro. Within an
embodiment the cells are derived from tissues selected from the
group consisting of: a) tissues from pancreas; b) tissues from
colon; c) tissues from small intestine; d) tissues from bladder; e)
tissues from prostate; f) tissues from myometrium; and g) tissues
from breast.
[0017] Within another aspect the invention provides a method for
modulating glycoprotein and glycolipid biosynthesis by combining
the polypeptide according to claim 1, with cells in vivo and in
vitro. Within an embodiment the cells are derived from tissues
selected from the group consisting of: a) tissues from pancreas; b)
tissues from colon; c) tissues from small intestine; d) tissues
from bladder; e) tissues from prostate; f) tissues from myometrium;
and g) tissues from breast.
[0018] Within another aspect, the invention provides a ethod of
detecting a molecule which binds to a polypeptide comprising
contacting the polypeptide with a test sample containg the molecule
wherein the polypeptide comprises residues 148 to 397 of SEQ ID
NO:2 and whereby the molecule binds the polypeptide.
[0019] These and other aspects of the invention will become evident
upon reference to the following detailed description of the
invention and attached drawings.
DETAILED DESCRIPTION OF THE INVENTION
[0020] Prior to setting forth the invention in detail, it may be
helpful to the understanding thereof to define the following
terms:
[0021] 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 Enzymol. 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 comrnercial suppliers (e.g., Pharmacia Biotech,
Piscataway, N.J.; New England Biolabs, Beverly, Mass.; Eastman
Kodak, New Haven, Conn.).
[0022] 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.
[0023] 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.
[0024] 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.
[0025] 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'.
[0026] 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'.
[0027] 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).
[0028] 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.
[0029] 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).
[0030] 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.
[0031] "Operably linked" means that two or more entities are joined
together such that they function in concert for their intended
purposes. When referring to DNA segments, the phrase indicates, for
example, that coding sequences are joined in the correct reading
frame, and transcription initiates in the promoter and proceeds
through the coding segment(s) to the terminator. When referring to
polypeptides, "operably linked" includes both covalently (e.g., by
disulfide bonding) and non-covalently (e.g., by hydrogen bonding,
hydrophobic interactions, or salt-bridge interactions) linked
sequences, wherein the desired function(s) of the sequences are
retained.
[0032] 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.
[0033] "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.
[0034] 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.
[0035] 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".
[0036] 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.
[0037] 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.
[0038] 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).
[0039] 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.
[0040] 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.
[0041] 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.
[0042] 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%.
[0043] All references cited herein are incorporated by reference in
their entirety.
[0044] 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.fwdarw.3
galactosyltransferases (.beta.1.fwdarw.3GalTases).
.beta.1.fwdarw.3GalTases are the .beta.3 subfamily of human
galactosyltransferases (.beta.3Gal-T family) which includes
HSY15014 (Kolbinger, F. et al., Journal of Biol. Chem. 273:
433-440, 1998), HSGALT3, HSGALT4, (Amado, M. et al., ibid) and
E07739 Katsutoshi, S. et al., Japanese patent, JP 1994181759-A/1).
.beta.1.fwdarw.3GalTases are responsible for transferring galactose
to carbohydrate chains during biosynthesis. It has been predicted
that .beta.1.fwdarw.3GalTases 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). Also in the .beta.3Gal-T
family is the Drosophila melanogaster Brainiac, (BRN) (Goode, S. et
al., Devel. Biol. 178:35-50, 1996), known as "putative neurogenic
secreted signaling protein" or NSSP. BRN is required for epithelial
development. This activity may be due to possible cell interactions
between the membrane bound glycosyltransferase and oligosaccharide
substrates on adjacent cell surfaces (Shur, ibid). Thus,
.beta.3Gal-T family members are also known as neurogenic secreted
signal peptides. See, for example, Shur, B. D., ibid, and Amado, M.
et al., ibid. This novel polypeptide and its polynucleotides have
been designated znssp2.
[0045] The .beta.3Gal-Ts 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 a extracellular secreted protein.
[0046] The novel znssp2 polypeptide-encoding polynucleotides of the
present invention were initially identified by searching an EST
database for open reading frames with similarity to BRN. The insert
of an expressed sequence tag corresponding to nucleotides 673 to
1532 of SEQ ID NO:1 was used to obtain a clone that had been
isolated from a bone marrow library. Analysis of the DNA encoding a
znssp2 polypeptide (SEQ ID NO: 1) revealed an open reading frame
encoding 397 amino acids (SEQ ID NO: 2). Znssp2 shares homology
with .beta.3Gal-T's which are predicted to be Type II membrane
proteins. Znssp2 shows the highest similarity to HSGALT3, at 34%
amino acid identity over the region of amino acids from residue 148
to residue 348 of SEQ ID NO:2. Amino acid residues 19 to 147 of SEQ
ID NO:2 are predicted to form a "stem" domain, and amino acid
residues 148 to 397 of SEQ ID NO:2 are predicted to form a
"catalytic" domain.
[0047] Due to the close proximity of the hydrophobic domain
(residues 1 to 18 of SEQ ID NO:2) to the initiation methionine, and
the lack of positively charged residues preceding this domain, it
is possible that znssp2 is a secreted protein comprising a signal
peptide of 18 amino acid residues (residues 1-18 of SEQ ID NO:2)
and a mature polypeptide of 379 amino acids (residues 19 to 397 of
SEQ ID NO:2). Conserved negatively charged amino acid residues 202,
208, 216, 248, 333, and 334 of SEQ ID NO:2 are contained within the
catalytic domain. Additionally, the sequence of amino acid residues
from residue 333 to 338 is representative of a peptide motif of
this family. This motif is further described by the following amino
acid residue profile: [D,E] [D] [V] [F,Y] [L,T,V] [G]. 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.
[0048] The present invention also provides post translationally
modified polypeptides or polypeptide fragments. A potential
N-linked glycosylation site can be found at amino acid residue 220
of SEQ ID NO:2. Post translational modifications in members of the
.beta.3Gal-T family may regulate whether the protein is expressed
in the Golgi or on the surface of the cell. Other examples of post
translational modifications include proteolytic cleavage, disulfide
bonding and hydroxylation.
[0049] Additionally, znssp2 has 29% homology to the BRN gene.
[0050] The present invention also includes the murine ortholog of
znssp2 (znssp2-m) which was identified in a mouse EST database. The
polynucleotide, polypeptide, and degenerate sequences of znssp2-m
are shown in SEQ ID NOs:12, 13, and 14, respectively.
[0051] Analysis of the tissue distribution of znssp2 was performed
by the Northern blotting technique using Human Multiple Tissue and
Master Dot Blots. A very strong signal of 1.2 kb was seen in
pancreas. A strong signal was seen in colon; with less strong
signals seen in spinal cord, bone marrow, small intestine, and
peripheral blood leukocytes. Fainter signals were seen in heart,
lung, spleen, prostate, stomach, thyroid, trachea, placenta,
skeletal muscle, kidney and lymph node.
[0052] The highly conserved, negatively charged residues at
positions 202, 208, 216, 248, 333, and 334 of SEQ ID NO:2 and the
amino acid sequence between 333 and 338 of znssp2 can be used as a
tool to identify new family members. For instance, reverse
transcription-polymerase chain reaction (RT-PCR) can be used to
amplify sequences encoding the znssp2 polynucleotide from RNA
obtained from a variety of tissue sources or cell lines. In
particular, highly degenerate primers designed from the znssp2
sequences are useful for this purpose. The present invention
further provides polynucleotide molecules, including DNA and RNA
molecules, encoding znssp2 proteins. The polynucleotides of the
present invention include the sense strand; the anti-sense strand;
and the DNA as double-stranded, having both the sense and
anti-sense strand annealed together by their respective hydrogen
bonds. Representative DNA sequences encoding znssp2 proteins are
set forth in SEQ ID NOs:1, 3, 12 and 14. DNA sequences encoding
other znssp2 proteins can be readily generated by those of ordinary
skill in the art based on the genetic code.
[0053] The present invention also provides polynucleotide
molecules, including DNA and RNA molecules, that encode the znssp2
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 znssp2 polypeptide of SEQ
ID NO:2. SEQ ID NO:14 is a degenerate DNA sequence that encompasses
all DNAs that encode the znssp2 polypeptide of SEQ ID NO:13. 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 and in the same manner, all the degenerate
sequences of SEQ ID NO:14 also provides all RNA sequences encoding
SEQ ID NO:13. Thus, znssp2 polypeptide-encoding polynucleotides
comprising nucleotide 1 to nucleotide 1191 of SEQ ID NO:3 and their
RNA equivalents, and the polypeptide-encoding polynucleotides
comprising nucleotide 1 to nucleotide 1167 of SEQ ID NO:14 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.
1TABLE 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.vertlin- e.T N
A.vertline.C.vertline.G.vertline.T
[0054] The degenerate codons used in SEQ ID NOs:3 and 14,
encompassing all 5 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
[0055] 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 NOs:2
or 13. Variant sequences can be readily tested for functionality as
described herein.
[0056] 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 commronly 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 sequences
disclosed in SEQ ID NOs:3 and 14 serve as a templates 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.
[0057] Within preferred embodiments of the invention the isolated
polynucleotides will hybridize to similar sized regions of SEQ ID
NOs:1 or 12, 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; 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.
[0058] 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 1C 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 to 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.
[0059] Stringent hybridization conditions encompass temperatures of
about 5-25.degree. C. below the thermal melting point (T.sub.m) 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.
[0060] 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.
[0061] 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.
[0062] 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 znssp2 RNA. Such
tissues and cells are identified by Northern blotting (Thomas,
Proc. Natl. Acad. Sci. USA 77:5201, 1980), and include pancreas,
colon, spinal cord, small intestine, heart, lung, spleen, kidney,
prostate, peripheral blood leukocytes, stomach, thyroid, and
trachea. 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 znssp2 polypeptides are then identified
and isolated by, for example, hybridization or PCR.
[0063] A full-length clone encoding znssp2 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 znssp2, or fragments thereof, or other specific
binding partners.
[0064] The invention also provides isolated and purified znssp2
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 znssp2 gene or cDNA. The synthetic
oligonucleotides of the present invention have at least 75%
identity to a representative znssp2 DNA sequence (SEQ ID NOs:1, 3,
12 or 14) 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, 3, 12, or 14 or a
sequence complementary to SEQ ID NOs: 1, 3, 12 or 14.
[0065] Regions from which to construct probes include the 5' and/or
3' coding sequences, substrate binding regions, and signal
sequences, 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 znssp2 gene or mRNA transcript in a sample. Znssp2
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
znssp2-like proteins. For example, znssp2 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. 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 znssp2 polynucleotide probes
can be prepared which would increase sensitivity or the detection
of low copy number targets, in screening systems.
[0066] 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.
[0067] 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 znssp2
polypeptides from other mammalian species, including murine,
porcine, ovine, bovine, canine, feline, equine, and other primate
polypeptides. Orthologs of human znssp2 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 znssp2 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 znssp2-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 znssp2 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 znssp2 polypeptide. Similar techniques can also be
applied to the isolation of genomic clones.
[0068] Those skilled in the art will recognize that the sequences
disclosed in SEQ ID NOd:1, and 12 represent single alleles of human
and mouse znssp2, respectively 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 NOs:1 and 12,
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 NOs:2 and 13. cDNAs generated from alternatively
spliced mRNAs, which retain the properties of the znssp2
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.
[0069] The present invention also provides isolated znssp2
polypeptides that are substantially homologous to the polypeptides
of SEQ ID NOs:2 and 12 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 NOs:2 and 13 or their orthologs. Such
polypeptides will more preferably be at least 90% identical, and
most preferably 95% or more identical to SEQ ID NOs:2 and 13 or
their 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 the number of gaps
introduced into the longer sequence 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 -2 -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
[0070] Sequence identity of polynucleotide molecules is determined
by similar methods using a ratio as disclosed above.
[0071] 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 znssp2. 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).
[0072] 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).
[0073] 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.
[0074] The present invention includes nucleic acid molecules that
encode a polypeptide having one or more conservative amino acid
changes, compared with the amino acid sequences of SEQ ID NOs:2 and
13. 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 BLOSUM62 value of at least 2
(e.g., 2 or 3).
[0075] Variant znssp2 polypeptides or substantially homologous
znssp2 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 397 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 znssp2
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 tyrosme Small: glycine alanine
serine threonine methiomne
[0076] The present invention further provides a variety of other
polypeptide fusions and related multimeric proteins comprising one
or more polypeptide fusions. For example, a znssp2 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 constant region
domains. Immunoglobulin-znssp2 polypeptide fusions can be expressed
in genetically engineered cells to produce a variety of multimeric
znssp2 analogs. Auxiliary domains can be fused to znssp2
polypeptides to target them to specific cells, tissues, or
macromolecules (e.g., pancreas, colon, spinal cord, bone marrow,
heart, and small intestine etc.). 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, pancreas, colon, spinal cord, or bone
marrow. 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.
[0077] 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 znssp2 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 znssp2
polypeptide can be fused to maltose binding protein (approximately
370 residues), a 4-residue cleavage site, and a 6-residue
polyhistidine tag.
[0078] 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 znssp2 to its anti-complementary molecule
can cause a cellular event in the cell that is expressing it (i.e.
znssp2 acts as a receptor or receptor-like molecule), or in the
cell expressing the anti-complementary molecule to which it binds
(i.e., znssp2 acts as a ligand). Additionally, znssp2 can function
extracellularly as a soluble enzyme, ligand, receptor or receptor
like molecule. Similarly, as an extracellulary expressed znssp2
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, znssp2 can function to form a "bridge" between
cells maintaining their proximity to each other. Thus, for the
purposes of this application, znssp2 is referred to as a
complementary molecule and its cognate binding partner is referred
to as an anti-complementary molecule.
[0079] The invention also provides soluble znssp2 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 19 to 397 of SEQ ID NO:2, fused to
human Ig. znssp2 or znssp2-Ig chimeric proteins are used, for
example, to identify the znssp2 anti-complementary molecule,
including the natural anti-complementary molecule, as well as
agonists and antagonists of the natural anti-complementary
molecule. Using labeled soluble znssp2, 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.
[0080] In an alternative approach, a soluble znssp2 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 enzyme polypeptides are
arrayed in close proximity to each other. Fusions of this type can
be used to affinity purify the cognate substrate 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 anti-complementary molecule
stimulation. To purify anti-complementary molecule, a znssp2-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-substrate 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 Fc
region and used in an ELISA format.
[0081] The present invention also includes "functional fragments"
of znssp2 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 an znssp2 polypeptide. As an
illustration, DNA molecules having the nucleotide sequences of SEQ
ID NOs:1 and 12 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-znssp2 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 znssp2 gene can be synthesized using the polymerase
chain reaction.
[0082] 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).
[0083] The present invention also contemplates functional fragments
of an znssp2 gene that has amino acid changes, compared with the
amino acid sequences of SEQ ID NOs:2 and 13. A variant znssp2 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, 2, 12 and 13, 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 znssp2 gene can hybridize to a nucleic acid molecule having
the nucleotide sequence of SEQ ID NO: 1, as discussed above.
[0084] 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, teri-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).
[0085] 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 znssp2 amino acid residues.
[0086] 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 activity and cell-cell interactions 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.
[0087] 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).
[0088] Variants of the disclosed znssp2 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.
[0089] 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, such as intracellular
signaling) 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.
[0090] Regardless of the particular nucleotide sequence of a
variant znssp2 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-znssp2 antibody.
More specifically, variant znssp2 genes encode polypeptides which
exhibit greater than 75, 80, or 90%, of the activity of polypeptide
encoded by the human znssp2 gene described herein.
[0091] 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 znssp2 protein. Such polypeptides may include additional
amino acids from, for example, an extracellular ligand-binding
domain of another member of the galactosyltransferase family as
well as part or all of the transmembrane and intracellular domains.
Additionally fragments of znssp2 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.
[0092] For any znssp2 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. Moreover,
those of skill in the art can use standard software to devise
znssp2 variants based upon the nucleotide and amino acid sequences
described herein. Accordingly, the present invention includes a
computer-readable medium encoded with a data structure that
provides at least one of the following sequences: SEQ ID NO:1, SEQ
ID NO:2, SEQ ID NO:3, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14.
Suitable forms of computer-readable media include magnetic media
and optically-readable media. Examples of magnetic media include a
hard or fixed drive, a random access memory (RAM) chip, a floppy
disk, digital linear tape (DLT), a disk cache, and a ZIP disk.
Optically readable media are exemplified by compact discs (e.g.,
CD-read only memory (ROM), CD-rewritable (RW), and CD-recordable),
and digital versatile/video discs (DVD) (e.g., DVD-ROM, DVD-RAM,
and DVD+RW).
[0093] The znssp2 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, 2nd 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.
[0094] In general, a DNA sequence encoding a znssp2 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.
[0095] To direct a znssp2 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
znssp2, 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 znssp2 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).
[0096] 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 znssp2 polypeptide is be
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.
[0097] 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, Bio Techniques 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, Manassas, Va. 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.
[0098] 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.
[0099] 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 znssp2 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.).
[0100] This system utilizes a transfer vector, pFastBac1.TM. (Life
Technologies) containing a Tn7 transposon to move the DNA encoding
the znssp2 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 znssp2. 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 znssp2 secretory signal sequences with secretory signal
sequencesz 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 znssp2 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 znssp2
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 znssp2 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
znssp2 is subsequently produced. Recombinant viral stocks are made
by methods commonly used the art.
[0101] 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-cellO405.TM. (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 znssp2
polypeptide from the supernatant can be achieved using methods
described herein.
[0102] 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 POT1 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 30 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.
[0103] 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.
[0104] 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 znssp2 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.
[0105] 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).
[0106] 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.
[0107] Expressed recombinant znssp2 polypeptides (or chimeric
znssp2 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.
[0108] 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.
[0109] To direct the export of a receptor polypeptide from the host
cell, the receptor DNA is linked to a second DNA segment encoding a
secretory peptide, such as a t-PA secretory peptide or a znssp2
secretory peptide. To facilitate purification of the secreted
receptor 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 receptor polypeptide.
[0110] Moreover, using methods described in the art, polypeptide
fusions, or hybrid znssp2 proteins, are constructed using regions
or domains of the inventive znssp2 in combination with those of
other human galactosyltransferase family proteins (e.g. HSGALT3,
HSGALT4, P3Gal-T2, and p3Gal-T3, or the human species 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 substrate specificity, or alter
tissue and cellular localization of a polypeptide, and can be
applied to polypeptides of unknown structure.
[0111] 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 znssp2 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 1 to 18 of SEQ ID NO:2), and other
conserved motifs such as the .beta.1.fwdarw.3 GalTase homology
region (residues 148 to 397 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.
[0112] Znssp2 polypeptides or fragments thereof may also be
prepared-through chemical synthesis. Znssp2 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.
[0113] Znssp2 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.
[0114] 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.
[0115] 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.
[0116] 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.
[0117] 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.
[0118] 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" (2nd 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.
[0119] 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.
[0120] 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 nim 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.
[0121] 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.
[0122] 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., H2O, 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).
[0123] 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 pancreas, colon, or
small intestine tissue 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. Of additional interest are
differences in cellular expression of znssp2 in diseased versus
non-diseased tissues. Specific assays include but are not limited
to bioassays measuring cell migration, contact inhibition, tissue
interactions, and metastasis. Additional assays would measure
neuronal specificity, fertilization, embryonic cell adhesions, limb
bud morphogenesis, mesenchyme development, immune recognition,
growth control, tumor metastasis and suppression, and intracellular
and extracellular glycoprotein and glycolipid biosynthesis.
[0124] Additional activities likely associated with the
polypeptides of the present invention include proliferation of
cells of the pancreas, colon, spinal cord, bone marrow, small
intestine, peripheral leukocytes, bladder, prostate, myometrium,
and breast directly or indirectly through other growth factors;
action as a chemotaxic factor; and as a factor for expanding
pancreas and mesenchymal stem cell and precursor populations.
[0125] Another assay of interest measures or detects changes in
proliferation, differentiation, and development. Proliferation can
be measured using cultured primary pancreas 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. Established cell lines can be
established by one skilled in the art and are available from
American Type Culture Collection (Manasas, Va.). 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).
[0126] 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 znssp2 protein. Similarly,
clonogenic assays can be performed.
[0127] To determine if znssp2 is a chemotractant in vivo, znssp2
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 znssp2 injection.
[0128] 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. 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. The novel polypeptides of the present invention are useful
for studies to isolate mesenchymal stem cells and pancreas
progenitor cells, both in vivo and ex vivo.
[0129] 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, znssp2 polypeptides may
stimulate inhibition or proliferation of endocrine and exocrine
cells of the pancreas, as well as, cells associated with the colon,
spinal cord, bone marrow, heart, small intestine, and peripheral
leukocytes. Molecules of the present invention may, while
stimulating proliferation or differentiation of pancreas cells,
inhibit proliferation or differentiation of other tissues, by
virtue of their effect on common precursor/stem cells.
[0130] 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).
[0131] The znssp2 polypeptides of the present invention can be used
to study pancreatic cell proliferation or differentiation. Such
methods of the present invention generally comprise incubating a
cells, .alpha. cells, .beta. cells, .delta. cells and acinar cells
in the presence and absence of znssp2 polypeptide, monoclonal
antibody, agonist or antagonist thereof and observing changes in
cell proliferation or differentiation. An exemplary model system to
study the formation of pancreatic endocrine cells in vitro uses
AR42J cells which are derived from acinar cells. Mashima, H. et
al., Endocrinology 137:3969-3976, 1996.
[0132] Proteins, including alternatively spliced peptides, and
fragments, of the present invention are useful for modulating
cell-cell interactions, neuronal specificity, fertilization,
morphogenesis, development, inflammation, tumorigenesis, immune
recognition, growth control, tumor suppression, and glycoprotein
and glycolipid biosynthesis. Znssp2 molecules, variants, and
fragments can be applied in isolation, or in conjunction with other
molecules (growth factors, cytokines, etc.) in pancreas, colon,
spinal cord, bone marrow, heart, small intestine, and peripheral
leukocytes. Alternative splicing of znssp2 may be cell-type
specific and confer activity to specific tissues.
[0133] As exemplary cell line of the pancreas to test the activity
of znssp2 is CRL-1682, an human pancreas adenocarcinoma cell line,
(ATCC, Manassas, Va.).
[0134] Other assays to measure the effects of znssp2 include
proliferation assays (i.e., of pancreas, bone marrow, spinal cord,
colon, or small intestine) by testing tissue and cells from healthy
volunteers with znssp2 protein, or a znssp2-free negative control
for the ability of the tissue and cells to proliferate.
[0135] 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 znssp2 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, znssp2
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).
[0136] 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 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.
[0137] 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).
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.
[0138] 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.
[0139] 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.
[0140] In view of the tissue distribution (i.e., pancreas, colon,
spinal cord, bone marrow, small intestine, peripheral leukocytes,
and various other tissues) observed for znssp2, agonists (including
the natural ligand/substrate/cofactor/etc.) and antagonists have
enormous potential in both in vitro and in vivo applications.
Compounds identified as znssp2 agonists are useful for studying
galactosylation of cell surface antigens as well as cell-cell
interactions in vitro and in vivo. For example, znssp2 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 pancreas, colon, spinal cord, bone marrow,
small intestine, and peripheral leukocytes in culture.
Alternatively, znssp2 polypeptides and znssp2 agonist polypeptides
are useful as a research reagent, particularly for the growth and
expansion of pancreas, colon or small intestine cells. Znssp2
polypeptides are added to tissue culture media for these cell
types.
[0141] Additionally, molecules of the present invention can be used
in vitro to modify glycoproteins. Aberrant glycosylation can be
modified by the application of the proteins of the present
invention. Znssp2 molecules can be added in vitro to production or
reagent grade proteins to modify the improper galactosylation of
proteins. Additionally, molecules of the present invention can be
used in the production of properly glycosylated saccharide
chains.
[0142] Antagonists 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.
[0143] Inhibitors of znssp2 activity (znssp2 antagonists) include
anti-znssp2 antibodies and soluble znssp2 molecules, as well as
other peptidic and non-peptidic agents (including ribozymes).
[0144] The invention also provides antagonists, which either bind
to znssp2 polypeptides or, alternatively, to a anti-complementary
molecule to which znssp2 polypeptides bind, thereby inhibiting or
eliminating the function of znssp2. Such znssp2 antagonists would
include antibodies; polypeptides which bind either to the znssp2
polypeptide or to its anti-complementary molecule or natural or
synthetic analogs of znssp2 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. Natural or synthetic small molecules which bind to
znssp2 polypeptides and prevent glyprotein or glycolipid synthesis
or cell-cell interactions are also contemplated as antagonists.
Also contemplated are soluble znssp2 polypeptides. As such, znssp2
antagonists would be useful as therapeutics for treating certain
disorders where blocking glycosylation or binding of the
znssp2-anti-complementary molecule would be beneficial.
[0145] Znssp2 polypeptides may be used within diagnostic systems to
detect the presence of znssp2 anti-complementary molecule
polypeptides. Antibodies or other agents that specifically bind to
znssp2 or its anti-complementary molecule may also be used to
detect the presence of circulating znssp2 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. Immunohistochemically labeled znssp2
antibodies can be used to detect znssp2 and/or znssp2
anti-complementary molecule in tissue samples. znssp2 levels can
also be monitored by such methods as RT-PCR, where znssp2 mRNA can
be detected and quantified. The information derived from such
detection methods would provide insight into the significance of
znssp2 polypeptides in various diseases, and as such would serve as
diagnostic tools for diseases for which altered levels of znssp2
are significant. Altered levels of znssp2 polypeptides may be
indicative of pathological conditions including, for example,
cancer, auto-immune diseases, digestive disordersm and inflammatory
disorders.
[0146] A "soluble protein" is a protein that is not bound to a cell
membrane. Soluble 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 substrate, 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.
[0147] Soluble forms of znssp2 polypeptides may act as antagonists
to or agonists of znssp2 polypeptides, and would be useful to
modulate the effects of znssp2 in pancreas, colon and small
intestine. The soluble form of znssp2 does not contain a
transmembrane domain (i.e., the polypeptide of residues 19 to 397
of SEQ ID NO:2) and may act as an agonist or antagonist of znssp2
activity. 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
znssp2 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.
[0148] Znssp2 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 znssp2.
In addition to those assays disclosed herein, samples can be tested
for inhibition of znssp2 activity within a variety of assays
designed to measure complementary molecule-anti-complementary
molecule binding or the stimulation/inhibition of znssp2-dependent
cellular responses. For example, znssp2-responsive cell lines can
be transfected with a reporter gene construct that is responsive to
a znssp2-stimulated cellular pathway. Reporter gene constructs of
this type are known in the art, and will generally comprise a
znssp2-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 znssp2 on the target cells as evidenced by
a decrease in znssp2 stimulation of reporter gene expression.
Assays of this type will detect compounds that directly block
znssp2 binding to anti-complementary molecules, as well as
compounds that block processes in the cellular pathway subsequent
to this binding. In the alternative, compounds or other samples can
be tested for direct blocking of znssp2 binding to its
anti-complementary molecule using znssp2 tagged with a detectable
label (e.g., .sup.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 znssp2 to the anti-complementary
molecule is indicative of inhibitory activity, which can be
confirmed through secondary assays. Complementary molecules used
within binding assays may be cellular complementary molcuels or
isolated, immobilized complementary molecules, or receptor-like
complementary molecules.
[0149] Assays measuring the inhibition of galactosyltransferase
activity in glycoprotein synthesis are listed in Ram, B. P.,
(ibid).
[0150] Also, znssp2 polypeptides, agonists or antagonists thereof
may be therapeutically useful for promoting wound healing, for
example, in the pancreas. To verify the presence of this capability
in znssp2 polypeptides, agonists or antagonists of the present
invention, such znssp2 polypeptides, agonists or antagonists are
evaluated with respect to their ability to facilitate wound healing
according to procedures known in the art. If desired, znssp2
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,
znssp2 polypeptides or agonists or antagonists thereof may be
evaluated in combination with one or more growth factors to
identify synergistic effects.
[0151] A znssp2 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
znssp2. For use in assays, the chimeras are bound to a support via
the F.sub.c region and used in an ELISA format.
[0152] A znssp2 ligand-binding polypeptide can also be used for
purification of ligand. 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 ligand are
passed through the column or chip one or more times to allow ligand
to bind to the receptor or receptor-like complementary polypeptide.
The ligand is then eluted using changes in salt concentration,
chaotropic agents (guanidine HCl), or pH to disrupt ligand-receptor
binding.
[0153] 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, Pharmacia 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 sulflhydryl
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.
[0154] Znssp2 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).
[0155] Within the polypeptides of the present invention are
polypeptides that comprise an epitope-bearing portion of a protein
as shown in SEQ ID NOs:2 and 13. 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 znssp2, such as might occur in
body fluids or cell culture media.
[0156] Antigenic, epitope-bearing polypeptides of the present
invention are useful for raising antibodies, including monoclonal
antibodies, that specifically bind to a znssp2 protein. The znssp2
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 znssp2
protein (e.g., SEQ ID NO:2). Polypeptides comprising a larger
portion of a znssp2 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 znssp2 polypeptides 25
encoded by SEQ ID NO:2 from amino acid number 1 to amino acid
number 397, or a contiguous 9 to 397 amino acid fragment thereof.
Such regions include secretory sequence, the catalytic domain, or
the transmembrane domain of znssp2 and fragments thereof.
Polypeptides in this regard include those comprising residues 1 to
18 of SEQ ID NO:2; residues 19 to 147 of SEQ ID NO:2; residues 148
to 397 of SEQ ID NO:2; and residues 19 to 397 of SEQ ID NO:2.
[0157] The present invention also provides polypeptide fragments or
peptides comprising an epitope-bearing portion of an znssp2
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)).
[0158] 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.
[0159] 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 NOs:2 or 13. Such
epitope-bearing peptides and polypeptides can be produced by
fragmenting an znssp2 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).
[0160] As an illustration, potential antigenic sites in human (SEQ
ID NO:2) and mouse (SEQ ID NO:13) znssp2 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.
[0161] Suitable antigens of the human sequence include residue 1 to
residue 6 of SEQ ID NO:2; residue 26 to residue 54 of SEQ ID NO:2;
residue 82 to residue 94 of SEQ ID NO:2; residue 110 to residue 117
of SEQ ID NO:2; residue 122 to residue 127 of SEQ ID NO:2; residue
131 to residue 136 of SEQ ID NO:2; residue 139 to residue 146 of
SEQ ID NO:2; residue 154 to residue 177 of SEQ ID NO:2; residue 187
to residue 197 of SEQ ID NO:2; residue 202 to residue 207 of SEQ ID
NO:2; residue 282 to residue 289 of SEQ ID NO:2; residue 295 to
residue 301 of SEQ ID NO:2; residue 358 to residue 365 of SEQ ID
NO:2; and residue 387 to residue 397 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. Znssp2 hydrophilic
peptides include peptides comprising amino acid sequences selected
from the group consisting of: residue 24 to residue 53 of SEQ ID
NO:2; residue 72 to residue 81 of SEQ ID NO:2; residue 85 to
residue 94 of-SEQ ID NO:2; residue 109 to residue 115 of SEQ ID
NO:2; residue 128 to residue 134 of SEQ ID NO:2; residue 156 to
residue 173 of SEQ ID NO:2; residue 200 to residue 209 of SEQ ID
NO:2; residue 281 to residue 291 of SEQ ID NO:2; residue 297 to
residue 306 of SEQ ID NO:2; residue 359 to residue 367 of SEQ ID
NO:2; and residue 387 to residue 397 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 25 to residue 54 of SEQ ID NO:2; residue 57 to
residue 62 of SEQ ID NO:2; residue 72 to residue 78 of SEQ ID NO:2;
residue 84 to residue 93 of SEQ ID NO:2; residue 108 to residue 115
of SEQ ID NO:2; residue 155 to residue 167 SEQ ID NO:2; residue 202
to residue 207 of SEQ ID NO:2; residue 218 to residue 233 of SEQ ID
NO:2; residue 281 to residue 287 of SEQ ID NO:2; residue 358 to
residue 363 of SEQ ID NO:2; and residue 388 to residue 393 of SEQ
ID NO:2; or a portion thereof which contains a 4 to 10 amino acid
segment.
[0162] Suitable antigens based on the Jameson-Wolf method for the
mouse sequence include residue 1 to residue 7 of SEQ ID NO:13;
residue 26 to residue 52 of SEQ ID NO:13; residue 83 to residue 88
of SEQ ID NO:13; residue 125 to residue 132 of SEQ ID NO:13;
residue 133 to residue 139 of SEQ ID NO:13; residue 146 to residue
152 of SEQ ID NO:13; residue 158 to residue 163 of SEQ ID NO:13;
residue 183 to residue 189 of SEQ ID NO:13; residue 193 to residue
200 of SEQ ID NO:13; residue 209 to residue 215 of SEQ ID NO:13;
residue 237 to residue 242 of SEQ ID NO:13; residue 273 to residue
281 of SEQ ID NO:13; residue 288 to residue 295 of SEQ ID NO:13;
residue 351 to residue 360 of SEQ ID NO:13; and residue 369 to
residue 374 of SEQ ID NO:13; 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. znssp2 hydrophilic peptides include peptides
comprising amino acid sequences selected from the group consisting
of: residue 1 to residue 6 of SEQ ID NO:13; residue 24 to residue
53 of SEQ ID NO:13; residue 68 to residue 78 of SEQ ID NO:13;
residue 148 to residue 154 of SEQ ID NO:13; residue 156 ti residue
164 of SEQ ID NO:13; residue 192 to residue 200 of SEQ ID NO:13;
residue 208 to residue 215 of SEQ ID NO:13; residue 273 to residue
280 of SEQ ID NO:13; residue 288 to residue 298 of SEQ ID NO:13;
and residue 351 to residue 359 of SEQ ID NO: 13; 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 25 to residue 37 of SEQ ID NO:13; residue 42 to
residue 52 of SEQ ID NO:13; residue 69 to residue 76 of SEQ ID
NO:13; residue 157 to residue 163 of SEQ ID NO:13; residue 193 to
residue 198 of SEQ ID NO:13; residue 210 to residue 215 SEQ ID
NO:13; residue 271 to residue 278 of SEQ ID NO:13; and residue 350
to residue 356 of SEQ ID NO:13; or a portion thereof which contains
a 4 to 10 amino acid segment.
[0163] Antibodies from an immune response generated by inoculation
of an animal with the antigens listed above can be isolated and
purified as described herein.
[0164] 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. 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.
[0165] 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 znssp2 polypeptide or a
fragment thereof. The immunogenicity of a znssp2 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
znssp2 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.
[0166] 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.
[0167] Alternative techniques for generating or selecting
antibodies useful herein include in vitro exposure of lymphocytes
to znssp2 protein or peptide, and selection of antibody display
libraries in phage or similar vectors (for instance, through use of
immobilized or labeled znssp2 protein or peptide). Genes encoding
polypeptides having potential znssp2 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 znssp2 sequences disclosed
herein to identify proteins which bind to znssp2. These "binding
proteins" which interact with znssp2 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 znssp2 "antagonists" to
block znssp2 binding and signal transduction in vitro and in vivo.
These anti-znssp2 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 and glycolipid synthesis.
[0168] As used herein, the term "binding proteins" additionally
includes antibodies to znssp2 polypeptides, the cognate
anti-complementary molecule of znssp2 polypeptides, proteins useful
for purification of znssp2 polypeptides, and proteins associated
with the catalytic (residues 19 to 397 of SEQ ID NO:2).
[0169] 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 znssp2 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).
[0170] 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 znssp2 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, znssp2 polypeptides, and non-human znssp2.
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 znssp2
are adsorbed to related polypeptides adhered to insoluble matrix;
antibodies specific to znssp2 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; Getzoffet
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.
[0171] A variety of assays known to those skilled in the art can be
utilized to detect antibodies which specifically bind to znssp2
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
znssp2 protein or polypeptide.
[0172] Antibodies to znssp2 may be used for tagging cells that
express znssp2; for isolating znssp2 by affinity purification; in
analytical methods employing FACS; for screening expression
libraries; for generating anti-idiotypic antibodies; and as
neutralizing antibodies or as antagonists to block znssp2 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 znssp2 or fragments thereof may be used in vitro to
detect denatured znssp2 or fragments thereof in assays, for
example, Western Blots or other assays known in the art.
[0173] The soluble znssp2 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 znssp2, cells expressing the
anti-complentary molecule are identified by fluorescence
immunocytometry or immunocytochemistry. Application may also be
made of 7 the specificity of UDP-glycosyltransferases for their
substrates.
[0174] Antibodies can be made to soluble, znssp2 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
znssp2 can be used in analysis of tissue distribution of znssp2 by
immunohistochemistry on human or primate tissue. These soluble
znssp2 polypeptides can also be used to immunize mice in order to
produce monoclonal antibodies to a soluble human znssp2
polypeptide. Monoclonal antibodies to a soluble human znssp2
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
znssp2 can be used to determine the distribution, regulation and
biological interaction of the znssp2 and its anti-complentary
molecule pair on specific cell lineages identified by tissue
distribution studies.
[0175] 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
(receptor, enzyme, receptor-like complementary molecule or antigen,
respectively, for instance). More specifically, znssp2 polypeptides
or anti-znssp2 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.
[0176] 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.
[0177] 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 or
diseased 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/cy- totoxic molecule conjugates.
[0178] In another embodiment, znssp2-cytokine fusion proteins or
antibody-cytokine fusion proteins can be used for enhancing in vivo
killing of target tissues (for example, pancreas, colon, spinal
cord, bone marrow, small intestine and peripheral leukocyte
cancers), if the znssp2 polypeptide or anti-znssp2 antibody
targets, for example, the hyperproliferative pancreas, colon,
spinal cord, bone marrow, small intestine and peripheral leukocyte
cells (See, generally, Hornick et al., Blood 89:4437-47, 1997).
They described fusion proteins enable targeting of a cytokine to a
desired site of action, thereby providing an elevated local
concentration of cytokine. Suitable znssp2 polypeptides or
anti-znssp2 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.
[0179] In yet another embodiment, if the znssp2 polypeptide or
anti-znssp2 antibody targets vascular cells or tissues, such
polypeptide or antibody may be conjugated with a radionuclide, and
particularly with a beta-emitting radionuclide, to reduce
restenosis. Such therapeutic approach poses less danger to
clinicians who administer the radioactive therapy. 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.
[0180] 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.
[0181] znssp2 polynucleotides and/or polypeptides may be useful for
regulating the maturation of UDP-glycosyltransferase
substrate-bearing cells, such as fibroblasts, lymphocytes and
hematopoietic cells. znssp2 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 znssp2, 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. 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.
[0182] The activity of znssp2 or a peptide to which znssp2 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 znssp2 proteins, their agonists, and
antagonists. The microphysiometer can be used to measure responses
of a znssp2-responsive eukaryotic cell, compared to a control
eukaryotic cell that does not respond to znssp2 polypeptide.
znssp2-responsive eukaryotic cells comprise cells into which a
polynucleotide for znssp2 has been transfected creating a cell that
is responsive to znssp2; or cells containing endogenous znssp2
polynucleotides. Differences, measured by a change in the response
of cells exposed to znssp2 anti-complentary molecule, relative to a
control not exposed to znssp2 anti-complentary molecule, directly
measure the znssp2-modulated cellular responses. Moreover, such
znssp2-modulated responses can be assayed under a variety of
stimuli. The present invention provides a method of identifying
agonists and antagonists of znssp2 protein, comprising providing
cells responsive to a znssp2 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
znssp2 substrate and the absence of a test compound provides a
positive control for the znssp2-responsive cells, and a control to
compare the agonist activity of a test compound with that of the
znssp2 substrate. Antagonists of znssp2 can be identified by
exposing the cells to znssp2 substrate in the presence and absence
of the test compound, whereby a reduction in znssp2-modulated
activity is indicative of antagonist activity in the test
compound.
[0183] Moreover, znssp2 can be used to identify cells, tissues, or
cell lines which respond to a znssp2-modulated pathway. The
microphysiometer, described above, can be used to rapidly identify
cells responsive to znssp2 of the present invention. Cells can be
cultured in the presence or absence of znssp2 polypeptide. Those
cells which elicit a measurable change in extracellular
acidification in the presence of znssp2 are responsive to znssp2.
Such cell lines, can be used to identify znssp2 anti-complentary
molecule, antagonists and agonists of znssp2 polypeptide as
described above.
[0184] 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 and glycolipid 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 photoaffinity 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.
[0185] As a reagent, the polynucleotide encoding the amino acid
residues from residue 333 to 338 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.
[0186] 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,
inflammation, tumorigenesis, growth control, tumor metastasis, and
intracellular and extracellular 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 pancreas, colon, spinal cord, bone marrow, heart,
lung, spleen, prostate, small intestine, peripheral blood
leukocytes, stomach, thyroid, trachea, placenta, skeletal muscle,
kidney, lymph node, bladder, prostate, myometrium, spleen, and
breast. In particular, certain pancreatic enzymatic deficiencies
and malignancies, and pancreatic-cell mediated deficiencies may be
amenable to such diagnosis, treatment or prevention.
[0187] Polynucleotides encoding znssp2 polypeptides are useful
within gene therapy applications where it is desired to increase or
inhibit znssp2 activity. If a mammal has a mutated or absent znssp2
gene, the znssp2 gene can be introduced into the cells of the
mammal. In one embodiment, a gene encoding a znssp2 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.
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).
[0188] In another embodiment, a znssp2 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.
[0189] 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. USA 84:7413-7, 1987;
Mackey et al., Proc. Natl. Acad. Sci. USA 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 znssp2 polynucleotide itself can be used
to target specific tissues.
[0190] 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.
[0191] Various techniques, including antisense and ribozyme
methodologies, can be used to inhibit znssp2 gene transcription and
translation, such as to inhibit cell proliferation in vivo.
Polynucleotides that are complementary to a segment of a
znssp2-encoding polynucleotide (e.g., a polynucleotide as set froth
in SEQ ID NOs:1 or 12) are designed to bind to znssp2-encoding mRNA
and to inhibit translation of such mRNA. Such antisense
polynucleotides are used to inhibit expression of znssp2
polypeptide-encoding genes in cell culture or in a subject.
[0192] The present invention also provides reagents which will find
use in diagnostic applications. For example, the znssp2 gene, a
probe comprising znssp2 DNA or RNA or a subsequence thereof can be
used to determine if the znssp2 gene is present on chromosome
19q13.2 or if a mutation has occurred. Detectable chromosomal
aberrations at the znssp2 gene locus include, but are not limited
to, aneuploidy, gene copy number changes, insertions, deletions,
restriction site changes and rearrangements. These aberrations can
occur within the coding sequence, within introns, or within
flanking sequences, including upstream promoter and regulatory
regions, and may be manifested as physical alterations within a
coding sequence or changes in gene expression level. 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).
[0193] 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., ibi; 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).
[0194] In addition, such polynucleotide probes could be used to
hybridize to counterpart sequences on individual chromosomes.
Chromosomal identification and/or mapping of the znssp2 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 particular gene might have.
[0195] 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.
[0196] Transgenic mice, engineered to express the znssp2 gene, and
mice that exhibit a complete absence of znssp2 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 znssp2 gene and the
protein encoded thereby in an in vivo system.
[0197] Znssp2 polypeptides, variants, and fragments thereof, may be
useful as replacement therapy for disorders associated with
glycoprotein synthesis, functions of the digestive system, and
cell-cell interactions.
[0198] 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 tissues of the pancreas, colon, small intestine, etc.,
znssp2 can play a role in intercellular rearrangement in these and
other tissues.
[0199] The znssp2 polypeptide is expressed in tissues of the
pancreas, colon, spinal cord, bone marrow, small intestine, and
peripheral leukocytes. 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 pancreas, colon, spinal
cord, bone marrow, small intestine, and peripheral leukocytes.
Similarly, polynucleotides and polypeptides of znssp2 may be used
to replace their defective counterparts in tumor or diseased
tissues. Thus, znssp2 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.
[0200] Moreover, the activity and effect of znssp2 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 B 16 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 M S, 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 znssp2, 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.,
znssp2, 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 znssp2. Moreover, purified znssp2 or
znssp2-conditioned media can be directly injected in to this mouse
model, and hence be used in this system. Use of stable znssp2
transfectants as well as use of induceable promoters to activate
znssp2 expression in vivo are known in the art and can be used in
this system to assess znssp2 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.
[0201] 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.13,galactosyltransferase. Expression of the Tn
antigen along with the sialosyl-Tn antigen and a TF antigen
(characterized by a deficiency in .alpha.,23,sialyl-transferase)
have been recognized as a cancer-associated phenomenon for many
years. See Berger, E. G. et al., Transfus. Clin. Biol. 2:103-108,
1994.
[0202] Thus, the study of this syndrome has been useful in
elucidating the biology of carbohydrate glycosylation disorders and
the appearance of cryptantigens on the cell surface, and cancer.
Highly specific and complex tumor glycan antigens are likely of
great interest in studying tissue specific tumors and znssp2 can be
useful for studying tumors of the pancreas, colon, spinal cord,
bone marrow, small intestine, and peripheral leukocytes.
[0203] Itzkowitz, et al., looked at the expression of these
cryptantigens in tissues from normal, chronic pancreatitic, and
pancreatic cancer patients. 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.
[0204] In view of the high expression of znssp2 in the pancreas,
and colon in normal tissue, a defect in the znssp2 gene may result
in defective galactosylation of cell surface carbohydrates of
pancreatic cells, leading to over sialylation of the Tn antigen, or
over galactosylation of cellular antigens, in general. Thus, znssp2
polypeptides would be useful as a pancreas- or colon-specific
.beta.,13, galactosyltransferase replacement therapy for
pre-cancerous and cancer tissues. To verify the presence of such
activity in znssp2 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.
[0205] Additionally, the lack of conditions favoring proper
galactosylation may result in an increase in sialosyl Tn antigens
in tissues expressing znssp2, which may cause an auto-immune
reaction resulting in an immune attack on the pancreas, colon,
spinal cord, bone marrow, small intestine, and peripheral
leukocytes. In these cases, znssp2 molecules may be used to
encourage proper galactosylation and limit the antigenic
recognition in tissues over expressing the sialosyl Tn antigen.
[0206] Similarly, a defective znssp2 gene may result in improper
glycoslation of the surface carbohydrates of the tissues of
pancreas, colon, spinal cord, bone marrow, small intestine, and
peripheral leukocytes, thus affecting cell-cell interactions and
possibly cell cycle regulation. Such cases could be treated by
administering polypeptides of znssp2 to mammals with such a
defective gene.
[0207] Exocrine cells of the pancreas are important for the
production of necessary enzymes involved in digestion. Persons
defective in the znssp2 gene may be unable to properly digest food
and and nutrients. Polynucleotides of znssp2 may be useful in
treating a defective pancreatic specific
.beta.,13,galactosyl-transferase gene by gene therapy. Likewise,
polypeptides of the present invention could be administered to a
mammal as replacement therapy for a defective digestive enzyme.
[0208] Znssp2 gene may be useful to as a probe to identify humans
who have a defective pancreatic or colonic specific .beta.,13,
galactosyltransferase gene. The strong expression of znssp2 in
pancreas, and colon suggests that znssp2 polynucleotides or
polypeptides are down-regulated in tumor, malignant, or
immune-responding tissues. Thus, polynucleotides and polypeptides
of znssp2, and mutations to them, can be used a indicators of
pancreatic and colonic cancer, and disease, in diagnosis.
[0209] As a protein showing strong expression in the pancreas and
colon, additional applications are to modulate gastric secretion in
the treatment of acute pancreatitis and gastrointestinal
disorders.
[0210] The polypeptides, nucleic acid, and/or antibodies of the
present invention may be used in treatment of disorders associated
with pancreas, diabetes, hypoglycemia; digestive systems including
pancreas, colon, and small intestine; neuronal tissues, bone
marrow, and peripheral leukocytes; 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 pancreas, colon, spinal cord,
heart and bone marrow. In particular, certain syndromes or diseases
may be amenable to such diagnosis, treatment or prevention.
[0211] The znssp2 polypeptide is expressed in the pancreas. Thus,
znssp2 polypeptide pharmaceutical compositions of the present
invention may be useful in prevention or treatment of pancreatic
disorders associated with pathological regulation of the expansion
of neuroendocrine and exocrine cells in the pancreas, such as IDDM,
pancreatic cancer, pathological regulation of blood glucose levels,
insulin expression, insulin resistance or digestive function.
[0212] The znssp2 polypeptide of the present invention may act in
the neuroendocrine/exocrine cell fate decision pathway and may
therefore be capable of regulating the expansion of neuroendocrine
and exocrine cells in the pancreas. One such regulatory use is that
of islet cell regeneration. Also, it has been hypothesized that the
autoimmunity that triggers IDDM starts in utero, and znssp2
polypeptide is a developmental gene involved in cell partitioning.
Assays and animal models are known in the art for monitoring the
exocrine/neuroendocrine cell lineage decision, for observing
pancreatic cell balance and for evaluating znssp2 polypeptide,
fragment, fusion protein, antibody, agonist or antagonist in the
prevention or treatment of the conditions set forth above.
[0213] For pharmaceutical use, the proteins of the present
invention are formulated for parenteral, particularly intravenous
or subcutaneous, delivery according to conventional methods.
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 znssp2 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., 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. 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 znssp2
is an amount sufficient to produce a clinically significant change
in disorders such as, diabetes, autoimmunity, cancer, as well as
disorders of the digestive system including, Crohn's disease,
ulcerative colitis, pancreatitis, digestive enzymatic disfunction,
and cancer suppression and ablation. Similarly, a therapeutically
effective amount of znssp2 is an amount sufficient to produce a
clinically significant change in disorders accociated with heart,
placenta, lung, skeleton muscle, kidney, spleen, prostate, small
intestine, colon, peripheral leukocyte, stomach, thyroid, spinal
cord, lymph node, trachea, bone marrow, bladder, breast, prostate,
and myometrium.
[0214] The invention is further illustrated by the following
non-limiting examples.
EXAMPLES
Example 1
[0215] Extension of EST Sequence
[0216] The novel znssp2 polypeptide-encoding polynucleotides of the
present invention were initially identified by querying an EST
database. A cDNA clone, corresponding to an EST was obtained and
the deduced amino acid sequence was determined to be incomplete at
the 5' terminal. Nested 5' RACE polymerase chain reactions were
performed. The first RACE used primers ZC9719 (SEQ ID NO:8) and
ZC17035 (SEQ ID NO:9) and thermolcycler conditions as follows: one
cycle at 94.degree. C. for 2 minutes; followed by twenty-five
cycles at 94.degree. C. for 20 seconds, 65.degree. C. for 30
seconds, 72.degree. C. for 45 seconds, followed by one cycle at
72.degree. C. for 2 minutes. Bone marrow marathon cDNA was used as
a template. The second, nested, RACE reaction used primers ZC9739
(SEQ ID NO:10) and ZC17036 (SEQ ID NO:11) and thermolcycler
conditions as follows: one cycle at 94.degree. C. for 2 minutes;
followed by five cycles at 94.degree. C. for 20 seconds, 69.degree.
C. for 45; followed by twenty-eight cycles 94.degree. C. for 20
seconds, 64.degree. C. for 30 seconds, 72.degree. C. for 45
seconds, followed by one cycle at 72.degree. C. for 7 minutes.
Thus, the 5' terminal of the polynucleotide sequence was
elucidated. In order to subclone the 5' portion of the
polynucleotide in to a pCR2.1 cloning vector, TA Cloning Kit
(Invitrogen, Carlsbad, Calif.), oligonucleotides ZC 18227 (SEQ ID
NO:4) and ZC17035 (SEQ ID NO:5), designed to the final 5' terminal
of the polynucleotide sequence and the original EST clone,
respectively, were used as primers. Marathon cDNA prepared from
bone marrow was used as a template. Thermocycler conditions were as
follows: one cycle at 94.degree. C. for 2 minutes; followed by
thirty cycles at 94.degree. C. for 20 seconds, 65.degree. C. for 30
seconds, 72.degree. C. for 30 seconds, followed by one cycle at
72.degree. C. for 5 minutes. The resulting PCR products were
gel-purified, subcloned, and sequenced. A consensus sequence was
generated by combining the sequence from the PCR products with the
cDNA sequence from the EST clone.
[0217] Polymorphisms were evident in the consensus sequence at the
following positions: R at nucleotide positions 172 and 509, and M
at nucleotide position 700; where R is G or A, and M is C or A. A
version of the consensus sequence, with a G chosen at position 172
and an A at position 700, was joined to the original EST clone.
Thus, amino acid residue 137 of SEQ ID NO:2 is either glycine or
serine.
Example 2
[0218] Tissue Distribution
[0219] Analysis of tissue distribution was performed by the
Northern blotting technique using Human Multiple Tissue and Master
Dot Blots (Clontech, Palo Alto, Calif.). A 134 bp probe was
obtained by PCR of the original EST template using primers ZC16893
(SEQ ID NO:6) and ZC16894 (SEQ ID NO:7). Thermocycler conditions
were as follows: one cycle at 94.degree. C. for 2 minutes; followed
by thirty cycles at 94.degree. C. for 20 seconds, 65.degree. C. for
30 seconds, 72.degree. C. for 30 seconds, followed by one cycle at
72.degree. C. for 2 minutes. The PCR product was random prime
labeled with .sup.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 four
times in 2.times.SSC and 0.05% SDS at 50.degree. C., followed by
washing twice in 0.1.times.SSC and 0.1% SDS at 50.degree. C., and
developed. Transcripts of about 1.2 kb, 2.8 kb, 4.5 kb and 7.0 kb
showed very strong signals in pancreas. Longer exposure indicates
that mRNA (of about 1.8 kb) is also present in tissues including
heart, placenta, lung, skeleton muscle, kidney, spleen, prostate,
small intestine, colon, peripheral leukocyte, stomach, thyroid,
spinal cord, lymph node, trachea, and bone marrow.
Example 3
[0220] Chromosomal Assignment and Placement of znssp2.
[0221] Znssp2 was mapped to chromosome 19 using the commercially
available version of the "Stanford G3 Radiation Hybrid Mapping
Panel" (Research Genetics, Inc., Huntsville, Ala.). The "Stanford
G3 RH Panel" contains PCRable DNAs 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.
[0222] For the mapping of znssp2 with the "Stanford G3 RH Panel",
20 .mu.l reactions were set up in a 96-well microtiter plate used
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 19,140 (SEQ ID NO:17), 1 .mu.l antisense primer, ZC
19,141 (SEQ ID NO:18), 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 ddH.sub.2O 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: an
initial 1 cycle 5 minute denaturation at 94.degree. C., 35 cycles
of a 45 seconds denaturation at 94.degree. C., 45 seconds annealing
at 64.degree. C. and 1 minute AND 15 seconds extension at
72.degree. C., followed by a final 1 cycle extension of 7 minutes
at 72.degree. C. The reactions were separated by electrophoresis on
a 2% agarose gel (Life Technologies, Gaithersburg, Md.).
[0223] The results showed linkage of mssp2 to the framework marker
SHGC-33470 with a LOD score of >15 and at a distance of 3.85
cR.sub.--10000 from the marker.
[0224] The use of surrounding markers positions znssp2 in the 19q13
region on the integrated LDB chromosome 19 map (The Genetic
Location Database, University of Southhampton, WWW server:
http://cedar.genetics. soton.ac.uk/public_html/).
Example 4
[0225] Construct for generating znssp2h Transgenic Mice
[0226] Oligonucleotides were designed to generate a PCR fragment
containing a consensus Kozak sequence and the exact znssp2 human
(znssp2h) coding region (nucleotides 101 to 1294 of SEQ ID NO:1).
These oligonucleotides were designed with an PmeI site at the 5'
end and an AscI site at the 3' end to facilitate cloning into
pTg12-8, our standard transgenic vector. PTg12-8 contains the mouse
MT-1 promoter and a 5' rat insulin II intron upstream of the PmeI
site.
[0227] A full-length clone of znssp2h was generated by the
ligation: A sequencing vector containing a znssp2h DNA segment from
nucleotide 1 to nucleotide 696 of SEQ ID NO:1, (with the addition
of the EcoRi site at the 5' end) was digested from the sequencing
vector as an EcoRI/SpeI fragment. Similarly, a sequencing vector
containing a znssp2h DNA segment from nucleotide 691 to 1359 of SEQ
ID NO:1 was digested from the sequencing vector as a SpeI/NaeI
fragment. These digested fragments were ligated to a pre-digested
(EcoRI/HincII) pUC19 cloning vector. The NaeI-HincII sites were
destroyed in this ligation. A glycine residue was used at position
137 of SEQ ID NO:2.
[0228] About one microliter of the ligation reaction was
electroporated into DHIOB ElectroMax.TM. competent cells (GIBCO
BRL, Gaithersburg, Md.) according to manufacturer's direction and
plated onto LB plates containing 100 .mu.g/ml ampicillin, and
incubated overnight. Colonies were picked and grown in LB media
containing 100 .mu.g/ml ampicillin. Miniprep DNA was prepared from
the picked clones and screened for the znssp2h insert by
restriction digestion with EcoRI, and subsequent agarose gel
electrophoresis.
[0229] A polymerase chain reaction using the this full-length
sequence as template (200 ng) was used to add a PmeI restriction
site and a Kozak sequence (oligonucleotide ZC20336, SEQ ID NO:15)
to the 5' end of the znssp2h sequence and an AscI site to the 3'
end (oligonucleotide ZC20316, SEQ ID NO:16). PCR reaction
conditions were as follows: 95.degree. C. for 5 minutes, wherein
Advantage cDNA polymerase (Clontech) was added; 15 cycles of
95.degree. C. for 60 seconds, 62.degree. C. for 60 seconds, and
72.degree. C for 90 seconds; and 72.degree. C. for 7 minutes. PCR
products were separated by agarose gel electrophoresis and purified
using a QiaQuick.TM. (Qiagen) gel extraction kit. The isolated,
1194 bp, DNA fragment was digested with PmeI and AscI (New England
Biolabs), ethanol precipitated and ligated into pTg12-8 that was
previously digested with PmeI and AscI. The pTg12-8 plasmid,
designed for expression of a gene of interest in transgenic mice,
contains an expression cassette flanked by 10 kb of MT-1 5' DNA and
7 kb of MT-1 3' DNA. The expression cassette comprises the MT-1
promoter, the rat insulin II intron, a polylinker for the insertion
of the desired clone, and the human growth hormone poly A
sequence.
[0230] About one microliter of the ligation reaction was
electroporated as described above. Colonies were picked and grown
in LB media containing 100 .mu.g/ml ampicillin. Miniprep DNA was
prepared from the picked clones and screened for the znssp2h insert
by restriction digestion with EcoRI. Maxipreps of the correct
pTg12-8-znssp2h construct were performed and digested with SalI.
The SalI fragment fragment containing with 5' and 3' flanking
sequences, the MT-1 promoter, the rat insulin II intron, znssp2h
cDNA and the human growth hormone poly A sequence was prepared to
be used for microinjection into fertilized murine oocytes.
Example 5
[0231] Cloning of the Mouse Ortholog
[0232] The human znssp2 gene was used to query the mouse EST
database for orthologs. A cDNA clone corresponding to the human
znssp2 sequence was obtained and the deduced amino acid sequence
was determined to be full-length and a murine ortholog of human
znssp2 (znssp2-m). The polynucleotide and polypeptide sequences of
the mouse ortholog are shown in SEQ ID NOs:12 and 13. The
degenerate sequence for the mouse ortholog is shown in SEQ ID NO:
14.
Example 6
[0233] Identification of Cells Expressing znssp2 Using in situ
Hybridization
[0234] Human pancreas tissues were isolated and screened for znssp2
expression by in situ hybridization. The human tissues prepared,
sectioned and subjected to in situ hybridization included
pancreases from normal and pancreatitis patients. The tissues were
fixed in 10% buffered formalin and blocked in paraffin using
standard techniques. Tissues were sectioned at 4 to 8 microns.
Tissues were prepared using a standard protocol ("Development of
non-isotopic in situ hybridization" at
http://dir.niehs.nih.gov/dirlep/ish.html). Briefly, tissue sections
were deparaffinized with HistoClear (National Diagnostics, Atlanta,
Ga.) and then dehydrated with ethanol. Next they were digested with
Proteinase K (50 .mu.g/ml) (Boehringer Diagnostics, Indianapolis,
Ind.) at 37.degree. C. for 3 to 5 minutes. This step was followed
by acetylation and re-hydration of the tissues.
[0235] An in situ probe generated by PCR was designed against the
human znssp2 sequence. A set of oligos were designed to generate
probes for separate regions of the znssp2 cDNA: Oligos ZC25,177
(SEQ ID NO: 19) and ZC25,232 (SEQ ID NO:20) were used to generate a
551 bp probe for znssp2. The antisense oligo from the PCR primer
set also contained the working sequence for the T7 RNA polymerase
promoter to allow for easy transcription of antisense RNA probes
from these PCR products. The PCR reaction conditions were as
follows: 35 cycles at 94.degree. C. for 30 sec, 45.degree. C. for 1
min., 72.degree. C. for 1 min with 5% Dimethyl Sulpnoxide (DMSO)
(Sigma Chemical Co, MO). The PCR product was purified by Qiagen
spin columns followed by phenol/chloroform extraction and ethanol
precipitation. The probe was subsequently labeled with digoxigenin
(Boehringer) or biotin (Boehringer) using an In Vitro transcription
System (Promega, Madison, Wis.) as per manufacturer's
instruction.
[0236] In situ hybridization was performed with a digoxigenin- or
biotin-labeled znssp2 probe (above). The probe was added to the
slides at a concentration of 1 to 5 pmol/ml for 12 to 16 hours at
55-60.degree. C. Slides were subsequently washed in 2.times.SSC and
0.1.times.SSC at 50-55.degree. C. The signals were amplified using
tyramide signal amplification (TSA) (TSA, in situ indirect kit;
NEN) and visualized with Vector Red substrate kit (Vector Lab) as
per manufacturer's instructions. The slides were then
counter-stained with hernatoxylin (Vector Laboratories, Burlingame,
Calif.).
[0237] A signal was seen in both normal and pancreatitis pancreas.
The positive-staining cells appeared to be acinar and related
cells.
[0238] 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 0
0
* * * * *
References