U.S. patent application number 12/216077 was filed with the patent office on 2008-12-04 for beta1,3-n-acetyl-d-galactosamine transferase protein, nucleic acid encoding the same and method of examining canceration using the same.
This patent application is currently assigned to National Institute of Advanced Industrial Science and Technology. Invention is credited to Toru Hiruma, Niro Inaba, Yasuko Ishizuka, Hisashi Narimatsu, Akira Togayachi.
Application Number | 20080299615 12/216077 |
Document ID | / |
Family ID | 32776807 |
Filed Date | 2008-12-04 |
United States Patent
Application |
20080299615 |
Kind Code |
A1 |
Narimatsu; Hisashi ; et
al. |
December 4, 2008 |
Beta1,3-N-acetyl-D-galactosamine transferase protein, nucleic acid
encoding the same and method of examining canceration using the
same
Abstract
The N-acetyl-D-galactosamine transferase protein of the present
invention is characterized by transferring N-acetyl-D-galactosamine
to N-acetyl-D-glucosamine with .beta.1,3 linkage, and it preferably
has the amino acid sequence shown in SEQ ID NO: 2 or 4. The
canceration assay according to the present invention uses a nucleic
acid for measurement which hybridizes under stringent conditions to
the nucleotide sequence shown in SEQ ID NO: 1 or 3 or a nucleotide
sequence complementary to at least one of them.
Inventors: |
Narimatsu; Hisashi;
(Tsukuba-shi, JP) ; Togayachi; Akira;
(Tsukuba-shi, JP) ; Inaba; Niro; (Tokyo, JP)
; Hiruma; Toru; (Tsukuba-shi, JP) ; Ishizuka;
Yasuko; (Tsukuba-shi, JP) |
Correspondence
Address: |
NIXON & VANDERHYE, PC
901 NORTH GLEBE ROAD, 11TH FLOOR
ARLINGTON
VA
22203
US
|
Assignee: |
National Institute of Advanced
Industrial Science and Technology
Tokyo
JP
|
Family ID: |
32776807 |
Appl. No.: |
12/216077 |
Filed: |
June 30, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10539450 |
Dec 23, 2005 |
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PCT/JP2004/000608 |
Jan 23, 2004 |
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12216077 |
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Current U.S.
Class: |
435/69.1 ;
536/55.2 |
Current CPC
Class: |
C12N 9/1051
20130101 |
Class at
Publication: |
435/69.1 ;
536/55.2 |
International
Class: |
C12P 21/02 20060101
C12P021/02; C07H 5/06 20060101 C07H005/06 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 23, 2003 |
JP |
14792/2003 |
Aug 1, 2003 |
JP |
2852310/2003 |
Nov 21, 2003 |
JP |
392555/2003 |
Claims
1. A method for producing a .beta.1,3-N-acetyl-D-galactosamine
transferase protein, which comprises growing a transformant
containing a vector carrying a nucleic acid to express the protein
and collecting the protein from the transformant, wherein the
nucleic acid is selected from the group consisting of the following
i) to vi): i) a nucleic acid consisting of a nucleotide sequence
encoding a polypeptide having an amino acid sequence shown in SEQ
ID NO: 2 or 4; or a polypeptide having an amino acid sequence with
substitution, deletion or insertion of one or more amino acids in
the amino acid sequence shown in SEQ ID NO: 2 or 4 and which
transfers N-acetyl-D-galactosamine to N-acetyl-D-glucosamine with
.beta.1,3 linkage, or a nucleotide sequence complementary thereto;
ii) a nucleic acid consisting of the nucleotide sequence shown in
SEQ ID NO: 1 or 3 or a nucleotide sequence complementary to at
least one of them; iii) a nucleic acid consisting of a nucleotide
sequence covering nucleotides 565 to 1503 shown in SEQ ID NO: 1 or
a nucleotide sequence complementary thereto; iv) a nucleic acid
consisting of a nucleotide sequence covering nucleotides 106 to
1503 shown in SEQ ID NO: 1 or a nucleotide sequence complementary
thereto; v) a nucleic acid consisting of a nucleotide sequence
covering nucleotides 103 to 1512 shown in SEQ ID NO: 3 or a
nucleotide sequence complementary thereto; and vi) a nucleic acid
according to any one of i) to v), which is DNA.
2. A method for transferring N-acetyl-D-galactosamine to
N-acetyl-D-glucosamine with .beta.-1,3 linkage, using the
.beta.1,3-N-acetyl-D-galactosamine transferase protein produced by
the method according to claim 1.
Description
TECHNICAL FIELD
[0001] The present invention relates to a novel
.beta.1,3-N-acetyl-D-galactosaminyltransferase protein and a
nucleic acid encoding the same, as well as a canceration assay
using the same, etc.
BACKGROUND ART
[0002] Recent attention has been focused on the in vivo roles of
sugar chains and/or complex carbohydrates. For example, factors for
determining blood types are glycoproteins, and it is glycolipids
that are involved in the functions of the nervous system. Thus,
enzymes having the ability to synthesize sugar chains constitute an
extremely important key to analyzing physiological activities
provided by various sugar chains.
[0003] For example, N-acetyl-D-galactosamine (hereinafter also
referred to as "GalNAc") is among the components constituting
glycosaminoglycans, as well as being a sugar residue found in
various sugar chain structures such as glycosphingolipids and
mucin-type sugar chains. Thus, an enzyme transferring GalNAc will
serve as an extremely important tool in analyzing the roles of
sugar chains in various tissues in vivo.
[0004] As described above, attention has been focused on the in
vivo roles of sugar chains, but it cannot be said that sufficient
headway has been made in analyzing in vivo sugar chain synthesis.
This is in part because the mechanism of sugar chain synthesis and
the in vivo localization of sugar synthesis have not been fully
analyzed. In analyzing the mechanism of sugar chain synthesis, it
is necessary to analyze glycosylation enzymes (particularly
glycosyltransferases) and to analyze what kind of sugar chains are
synthesized by means of the enzymes. To this end, there is a strong
demand for searching novel glycosyltransferases and analyzing their
functions.
[0005] There are some reports of glycosyltransferases having the
ability to transfer GalNAc (Non-patent Documents 1 to 4). For
example, among human GalNAc transferases, enzymes transferring
GalNAc with ".beta.1,4 linkage" are known (Non-patent Document 1)
and enzymes using "galactose" as their acceptor substrate are known
as enzymes transferring GalNAc with .beta.1,3 linkage (Non-patent
Document 2) (".beta.1,3" or ".beta.3" as used herein refers to a
glycosidic linkage between an .alpha.-hydroxyl group at the
1-position of a sugar residue in an acceptor substrate and a
hydroxyl group at the 3-position of a sugar residue to be
transferred and linked thereto).
[0006] On the other hand, in higher organisms like humans, no
enzyme is known to transfer GalNAc with ".beta.1,3 linkage" to
"N-acetylglucosamine" (hereinafter also referred to as
"GlcNAc").
[0007] Although there is a report showing that the sugar chain
structure in which GalNAc and GlcNAc are linked in a .beta.1,3
fashion was confirmed in sugar chains on neutral glycolipids of
fly, a kind of arthropod (Non-patent Document 5), it has been
believed that such a sugar chain structure is not present in
mammals, particularly in humans, to begin with.
Patent Document 1
[0008] International Patent Publication No. WO 01/79556
Non-patent Document 1
[0009] Cancer Res. 1993 Nov. 15; 53(22):5395-400: Yamashiro S, Ruan
S, Furukawa K, Tai T, Lloyd K O, Shiku H, Furukawa K. Genetic and
enzymatic basis for the differential expression of GM2 and GD2
gangliosides in human cancer cell lines.
Non-Patent Document 2
[0010] Biochim Biophys Acta. 1995 Jan. 3; 1254(1):56-65: Taga S,
Tetaud C, Mangeney M, Tursz T, Wiels J. Sequential changes in
glycolipid expression during human B cell, differentiation:
enzymatic bases.
Non-patent Document 3
[0011] Proc Natl Acad Sci USA. 1996 Oct. 1; 93(20):10697-702:
Haslam D B, Baenziger J U. Related Articles, Links, Expression
cloning of Forssman gly colipid synthetase: a novel member of the
histo-blood group ABO gene family.
Non-patent Document 4
[0012] J Biol. Chem. 1997 Sep. 19; 272(38): 23503-14: Wandall H H,
Hassan H, Mirgorodskaya E, Kristensen A K, Roepstorff P. Bennett E
P, Nielsen P A, Hollingsworth M A, Burchell J, Taylor-Papadimitriou
J, Clausen H. Substrate specificities of three members of the
human, UDP-N-acetyl-alpha-D-galactosamine: Polypeptide
N-acetylgalactosaminyltransferase family, GalNAc-T1, -T2, and
-T3.
Non-Patent Document 5
[0013] J. Biochem. (Tokyo) 1990 June; 107(6); 899-903: Sugita M.
Inagaki F, Naito H, Hori T., Studies on glycosphingolipids in
larvae of the green-bottle fly, Lucilia Caesar: two neutral
glycosphingolipids having large straight oligosaccaride chains with
eight and nine sugars.
DISCLOSURE OF THE INVENTION
[0014] A problem to be solved by the present invention is to
provide a polypeptide which is a mammal-derived (particularly
human-derived) glycosyltransferase and which has a novel
transferase activity to transfer GalNAc with .beta.1,3 linkage to
GlcNAc, as well as a nucleic acid encoding such a polypeptide,
etc.
[0015] Another problem to be solved by the present invention is to
provide a transformant expressing the nucleic acid in host cells, a
method for producing the encoded protein by allowing the
transformant to produce the protein and then collecting the
protein, and an antibody recognizing the protein.
[0016] On the other hand, since sugar chain synthesis may be
affected by canceration, the identification and expression analysis
of such a glycosylation enzyme can be expected to provide an index
useful for cancer diagnosis, etc. The present invention also
provides detailed procedures and criteria useful for canceration
assay or the like by analyzing and comparing, at the tissue or cell
line level, the transcription level of such a protein which varies
in correlation with canceration or malignancy.
BRIEF DESCRIPTION OF DRAWINGS
[0017] FIG. 1 is a diagram showing changes in the activity of the
G34 enzyme protein according to this example, plotted against the
reaction time.
[0018] FIG. 2A shows the results of NMR measurement, used for
analysis of the sugar chain structure synthesized by the G34 enzyme
protein according to this example.
[0019] FIG. 2B shows a partial magnified view of the NMR results in
FIG. 2A.
[0020] FIG. 3 is a table summarizing NOE in NMR shown in FIG. 2.
Various conditions for the data in Table 1 are as follows: 1.08 mM,
298K, D.sub.2O, CH.sub.2 (high)=4.557 ppm for non-marked data,
chemical shifts for data marked with * are CH.sub.2 (low)=4.778
ppm, phenyl(ortho)=7.265 ppm, phenyl(meta)=7.354 ppm and
phenyl(para)=7.320 ppm, calculated from the 1D spectrum.
[0021] FIG. 4 is a table summarizing relevant data (tentative NOE)
for each pyranose with respect to NMR shown in FIG. 2 (s: strong,
m: medium, w: weak, vw: very weak, A: GlcNAc, B: GalNAc).
[0022] FIG. 5 shows a comparison of amino acid sequences between
G34 enzyme protein according to this example and known .beta.3Gal
transferases.
[0023] FIG. 6 shows a comparison of motifs involved in the
.beta.3-linking activity between G34 enzyme protein according to
this example and various known .beta.3-linking
glycosyltransferases. "b3" represents a .beta.1-3 linkage and "Gn"
represents GlcNAc.
[0024] FIG. 7 is a diagram showing the pH dependence of the
activity of the G34 enzyme protein according to this example.
[0025] FIG. 8 is a diagram showing ion requirement for the activity
of the G34 enzyme protein according to this example.
[0026] FIG. 9 presents graphs showing the expression levels of the
G34 enzyme protein according to this example in human cell
lines.
[0027] FIG. 10 shows amino acid sequence alignment between mouse
G34 according to this example (upper) and human G34 (lower).
[0028] FIG. 11 shows the result of in situ hybridization performed
on a mouse testis sample using the mG34 nucleic acid according to
this example.
DETAILED DESCRIPTION OF THE INVENTION
[0029] To solve the problems stated above, the inventors of the
present invention have attempted to isolate and purify a nucleic
acid of interest, which may have high sequence identity, on the
basis of the nucleotide sequence of an enzyme gene functionally
similar to the intended enzyme. More specifically, first, the
sequence of a known glycosyltransferase .beta.3
galactosyltransferase 6 (.beta.3GalT6) was used as a query for a
BLAST search to thereby find a sequence with homology (GenBank No.
AX285201). It should be noted that this nucleotide sequence was
known as the sequence of SEQ ID NO: 1006 disclosed in International
Publication No. WO 01/79556 (Patent Document 1 listed above), but
its activity remained unknown.
[0030] First, the inventors of the present invention have
independently cloned the above gene by PCR, have determined its
nucleotide sequence (SEQ ID NO: 1) and putative amino acid sequence
(SEQ ID NO: 2), and have succeeded in identifying a certain
biological activity of a polypeptide encoded by the nucleic acid,
thus completing the present invention. Moreover, when using the
sequence as a query to search mouse genes, the inventors have found
the nucleotide sequence of SEQ ID NO: 3 and its putative amino acid
sequence (SEQ ID NO: 4).
[0031] The gene having the nucleotide sequence of SEQ ID NO: 1 and
the protein having the amino acid sequence of SEQ ID NO: 2 were
designated human G34, while the gene having the nucleotide sequence
of SEQ ID NO: 3 and the protein having the amino acid sequence of
SEQ ID NO: 4 were designated mouse G34.
[0032] According to the studies of the inventors, the above G34
protein uses an N-acetyl-D-galactosamine residue as a donor
substrate and an N-acetyl-D-glucosamine residue as an acceptor
substrate. As detailed later in Example 2, the G34 protein was
found to retain three motifs in its amino acid sequence, which are
well conserved in the enzyme family transferring various sugars
(e.g., galactose, N-acetyl-D-glucosamine) in the linking mode of
.beta.1,3. In light of these points, the G34 protein was
unexpectedly believed to have transferase activity to synthesize a
novel sugar chain structure "GalNAc-.beta.-1,3-GlcNAc," for which
no report has been made for mammals, particularly humans. The
linking mode was actually confirmed by NMR.
[0033] Namely, the present invention relates to a
.beta.1,3-N-acetyl-D-galactosaminyltransferase protein which
transfers N-acetyl-D-galactosamine to N-acetyl-D-glucosamine with
.beta.1,3 linkage.
[0034] An enzyme protein according to a preferred embodiment of the
present invention may have at least one or any combination of the
following properties (a) to (c).
(a) Acceptor Substrate Specificity
[0035] When using an oligosaccharide as an acceptor substrate, the
enzyme protein shows transferase activity toward Bz-.beta.-GlcNAc,
GlcNAc-.beta.1-4-GlcNAc-.beta.-Bz, Gal-.beta.1-3 (GlcNAc-.beta.1-6)
GalNAc-.alpha.-pNp, GlcNAc-.beta.1-3 GalNAc-.alpha.-pNp and
GlcNAc-.beta.1-6GalNAc-.alpha.-pNp ("GlcNAc" represents an
N-acetyl-D-glucosamine residue, "GalNAc" represents an
N-acetyl-D-galactosamine residue, "Bz" represents a benzyl group,
"pNp" represents a p-nitrophenyl group, and "-" represents a
glycosidic linkage. Numbers in these formulae each represent the
carbon number in the sugar ring where a glycosidic linkage is
present, and ".alpha." and ".beta." represent anomers of the
glycosidic linkage at the 1-position of the sugar ring. An anomer
whose positional relationship with CH.sub.2OH or CH.sub.3 at the
5-position is trans and cis is represented by ".alpha." and
".beta.", respectively).
[0036] Preferably, the enzyme protein is substantially free from
transferase activity toward Bz-.alpha.-GlcNAc and Gal .beta.1-3
GlcNAc-.beta.-pNp.
(b) Reaction pH
[0037] The activity is lower in a pH range of 6.2 to 6.6 than in
other pH ranges.
(c) Divalent Ion Requirement
[0038] Although the above activity is enhanced at least in the
presence of Mn.sup.2+, Co.sup.2+ or Mg.sup.2+, the
Mn.sup.2+-induced enhancement of the activity is almost completely
eliminated in the presence of Cu.sup.2+.
[0039] Moreover, in a preferred embodiment of the above
glycosyltransferase protein, the glycosyltransferase protein of the
present invention comprises the following polypeptide (A) or
(B):
(A) a polypeptide which has the amino acid sequence shown in SEQ ID
NO: 2 or 4; or (B) a polypeptide which has an amino acid sequence
with substitution, deletion or insertion of one or more amino acids
in the amino acid sequence shown in SEQ ID NO: 2 or 4 and which
transfers N-acetyl-D-galactosamine to N-acetyl-D-glucosamine with
.beta.1,3 linkage.
[0040] Moreover, in a more preferred embodiment of the above
glycosyltransferase protein, the above polypeptide (A) is a
glycosyltransferase protein consisting of a polypeptide having an
amino acid sequence covering amino acids 189 to 500 shown in SEQ ID
NO: 2. Likewise, in an even more preferred embodiment of the above
glycosyltransferase protein, the above polypeptide (A) is a
glycosyltransferase protein consisting of a polypeptide having an
amino acid sequence covering amino acids 36 to 500 shown in SEQ ID
NO: 2.
[0041] In addition, other embodiments of the glycosyltransferase
protein of the present invention encompass proteins consisting of
polypeptides having amino acid sequences sharing at least more than
30% identity, preferably at least 40% identity, and more preferably
at least 50% identity with an amino acid sequence covering amino
acids 189 to 500 shown in SEQ ID NO: 2 or amino acids 35 to 504
shown in SEQ ID NO: 4.
[0042] In another aspect, the present invention provides a nucleic
acid consisting of a nucleotide sequence encoding any one of the
above polypeptides or a nucleotide sequence complementary
thereto.
[0043] In a preferred embodiment, the nucleic acid encoding the
protein of the present invention is a nucleic acid consisting of
the nucleotide sequence shown in SEQ ID NO: 1 or 3 or a nucleotide
sequence complementary to at least one of them. More preferably, in
the case of human origin, such a nucleic acid consists of a
nucleotide sequence covering nucleotides 565 to 1503 shown in SEQ
ID NO: 1 or a nucleotide sequence complementary thereto, and most
preferably consists of a nucleotide sequence covering nucleotides
106 to 1503 shown in SEQ ID NO: 1 or a nucleotide sequence
complementary thereto. In the case of mouse origin, such a nucleic
acid consists of a nucleotide sequence covering nucleotides 103 to
1512 shown in SEQ ID NO: 3 or a nucleotide sequence complementary
thereto.
[0044] Embodiments of the above nucleic acids according to the
present invention encompass DNA.
[0045] The present invention further provides a vector carrying any
one of the above nucleic acids and a transformant containing the
vector.
[0046] In yet another aspect, the present invention provides a
method for producing a
.beta.1,3-N-acetyl-D-galactosaminyltransferase protein, which
comprises growing the above transformant to express the above
glycosyltransferase protein and collecting the glycosyltransferase
protein from the grown transformant.
[0047] In yet another aspect, the present invention provides an
antibody recognizing any one of the above
.beta.1,3-N-acetyl-D-galactosaminyltransferase proteins.
[0048] On the other hand, in response to the discovery of the above
G34, the inventors of the present invention have clarified that the
expression level of G34 mRNA is increased significantly in
cancerous tissues and cell lines.
[0049] Thus, the present invention also provides a nucleic acid for
measurement, which is useful as an index of canceration or
malignancy and which hybridizes under stringent conditions to the
nucleotide sequence shown in SEQ ID NO: 1 or 3 or a nucleotide
sequence complementary to at least one of them.
[0050] The nucleic acid for measurement of the present invention
may typically consist of a nucleotide sequence covering at least a
dozen contiguous nucleotides in the nucleotide sequence shown in
SEQ ID NO: 1 or 3 or a nucleotide sequence complementary
thereto.
[0051] In a preferred embodiment, the nucleic acid for measurement
of the present invention encompasses a probe consisting of the
nucleotide sequence shown in SEQ ID NO: 16 or a nucleotide sequence
complementary thereto, as well as a primer set consisting of the
following nucleotide sequences (1) or (2):
(1) a pair of the nucleotide sequences shown in SEQ ID NOs: 14 and
15; or (2) a pair of the nucleotide sequences shown in SEQ ID NOs:
17 and 18.
[0052] Also, the nucleic acid for measurement of the present
invention may be used as a tumor marker.
[0053] The present invention further provides a method for assaying
canceration in a biological sample, which comprises:
(a) using any one of the above nucleic acids to measure the
transcription level of the nucleic acid in the biological sample;
and (b) determining whether the measured value is significantly
higher than that of a normal biological sample.
[0054] In a preferred embodiment, the canceration assay of the
present invention includes cases where the measurement of the
transcription level is made by hybridization or PCR targeted at the
above biological sample and using any one of the above nucleic
acids.
[0055] In a further aspect of the canceration assay of the present
invention, the present invention provides a method for assaying the
effectiveness of treatment in cancer therapy, which comprises using
any one of the above nucleic acids to measure the transcription
level of the nucleic acid in a biological sample treated by cancer
therapy, and determining whether the measured value is
significantly lower than that obtained before treatment or than
that of an untreated sample.
[0056] In particular, the above biological sample may be derived
from the large intestine (colon) or lung.
MODE FOR CARRYING OUT THE INVENTION
[0057] The mode for carrying out the present invention will be
described in detail below.
(1) Nucleic Acid Encoding the G34 Enzyme Protein of the Present
Invention
[0058] Based upon the above discovery, the inventors of the present
invention expressed the G34 enzyme protein encoded by the nucleic
acid, isolated and purified the protein, and further identified its
enzymatic activity. When focusing on the fact that an amino acid
sequence having the desired enzymatic activity was identified, the
nucleotide sequence of SEQ ID NO: 1 or 3 is one embodiment of a
nucleic acid encoding the isolated polypeptide having the enzymatic
activity. This means that the nucleic acid of the present invention
encompasses all, but a limited number of, nucleic acids having
degenerate nucleotide sequences capable of encoding the same amino
acid sequence for the G34 enzyme protein.
[0059] The present invention also provides a nucleic acid encoding
the full-length or a fragment of a polypeptide consisting of a
novel amino acid sequence as mentioned above. A typical nucleic
acid encoding such a novel polypeptide may have the nucleotide
sequence shown in SEQ ID NO: 1 or 3 or a nucleotide sequence
complementary to at least one of them.
[0060] The nucleic acid of the present invention also encompasses
both single-stranded and double-stranded DNA and their
complementary RNA. Examples of DNA include naturally-occurring DNA,
recombinant DNA, chemically-bound DNA, PCR-amplified DNA, and
combinations thereof. However, DNA is preferred in terms of
stability during vector and/or transformant preparation.
[0061] The nucleic acid of the present invention may be prepared in
the following manner, by way of example.
[0062] First, the known sequence under GenBank No. AX285201 or a
part thereof may be used to perform nucleic acid amplification on a
cDNA library in a routine manner using basic procedures for genetic
engineering (e.g., hybridization, nucleic acid amplification),
thereby cloning the nucleic acid of the present invention. Since
the nucleic acid may be obtained, e.g., as a DNA fragment of
approximately 1.5 kbp as a PCR product, the fragment may be
separated using techniques for screening DNA fragments based on
their molecular weight (e.g., agarose gel electrophoresis) and
isolated in a routine manner, e.g. using techniques for excising a
specific band.
[0063] Moreover, according to the putative amino acid sequence (SEQ
ID NO: 2 or 4) of the isolated nucleic acid, the nucleic acid may
be estimated to have a hydrophobic transmembrane region at its
N-terminal end. By preparing a region of a nucleotide sequence
encoding a polypeptide free from this transmembrane region, it is
also possible to obtain the nucleic acid of the present invention
that encodes a soluble form of the polypeptide.
[0064] Based on the nucleotide sequence of the nucleic acid
disclosed herein, it is easy for those skilled in the art to create
appropriate primers from nucleotide sequences located at both ends
of a nucleic acid of interest or a region thereof to be prepared
and to use the primers thus created for nucleic acid amplification
to amplify and prepare the region of interest.
[0065] The above nucleic acid amplification includes, for example,
reactions requiring thermal cycling such as polymerase chain
reaction (PCR) [Saiki R. K., et al., Science, 230, 1350-1354
(1985)], ligase chain reaction (LCR) [Wu D. Y., et al., Genomics,
4, 560-569 (1989); Barringer K. J., et al., Gene, 89, 117-122
(1990); Barany F., Proc. Natl. Acad. Sci. USA, 88, 189-193 (1991)]
and transcription-based amplification [Kwoh D. Y., et al., Proc.
Natl. Acad. Sci. USA, 86, 1173-1177 (1989)], as well as isothermal
reactions such as strand displacement amplification (SDA) [Walker
G. T., et al., Proc. Natl. Acad. Sci. USA, 89, 392-396 (1992);
Walker G. T., et al., Nuc. Acids Res., 20, 1691-1696 (1992)],
self-sustained sequence replication (3SR) [Guatelli J. C., Proc.
Natl. Acad. Sci. USA, 87, 1874-1878 (1990)] and Q.beta. replicase
system [Lizardi et al., BioTechnology 6, p. 1197-1202 (1988)]. It
is also possible to use other reactions, e.g., nucleic acid
sequence-based amplification (NASBA) through competitive
amplification between a target nucleic acid and a mutated sequence,
found in European Patent No. 0525882. Preferred is PCR.
[0066] The use of the nucleic acid of the present invention also
enables the expression of the intended enzyme protein or the
provision of probes and antisense primers for the purpose of
medical research or gene therapy, as described later.
[0067] Those skilled in the art will be able to obtain a nucleic
acid as useful as the sequence of SEQ ID NO: 1 or 3 by preparing a
nucleic acid consisting of a nucleotide sequence sharing a certain
homology with the nucleotide sequence of SEQ ID NO: 1 or 3. For
example, the homologous nucleic acid of the present invention
encompasses nucleic acids encoding proteins which share homology
with the amino acid sequence shown in SEQ ID NO: 2 or 4 and which
have the ability to transfer N-acetyl-D-galactosamine to
N-acetyl-D-glucosamine with .beta.1,3 linkage.
[0068] To identify the range of nucleic acids encoding such
homologous proteins according to the present invention, an identity
search is performed for the nucleic acid sequence shown in SEQ ID
NO: 1 or 3 of the present invention, indicating that the nucleic
acid sequence shares 40% identity with the nucleic acid sequence of
a known .beta.1,4GalNAc transferase showing the highest homology
(Non-patent Document 1 listed above) and also shares 40% identity
with the nucleic acid sequence of a known .beta.1,3Gal transferase
showing the highest homology (Non-patent Document 2 listed above).
In light of these points, a preferred nucleic acid sequence
encoding the homologous protein of the present invention typically
shares more than 40% identity, more preferably at least 50%
identity, and particularly preferably at least 60% identity with
any one of the entire nucleotide sequence of SEQ ID NO: 1 or 3,
preferably a partial nucleotide sequence consisting of nucleotides
106 to 1503 in SEQ ID NO: 1, preferably a partial nucleotide
sequence consisting of nucleotides 103 to 1512 in SEQ ID NO: 3, or
nucleotide sequences complementary to these sequences.
[0069] Likewise, the nucleotide sequences shown in SEQ ID NOs: 1
and 3 share 86% identity with each other. In light of this point, a
preferred nucleic acid sequence encoding the homologous protein of
the present invention can be defined as sharing at least 86%,
preferably 90% identity with any one of the entire nucleotide
sequence of SEQ ID NO: 1, preferably nucleotides 106 to 1503, or a
nucleotide sequence complementary thereto.
[0070] The above percentage of identity may be determined by visual
inspection and mathematical calculation. Alternatively, the
percentage of identity between two nucleic acid sequences may be
determined by comparing sequence information using the GAP computer
program, version 6.0, described by Devereux et al., Nucl. Acids
Res. 12: 387, 1984 and available from the University of Wisconsin
Genetics Computer Group (UWGCG). The preferred default parameters
for the GAP program include: (1) a unary comparison matrix
(containing a value of 1 for identities and 0 for non-identities)
for nucleotides, and the weighted comparison matrix of Gribskov and
Burgess, Nucl. Acids Res. 14:6745, 1986, as described by Schwartz
and Dayhoff, eds., Atlas of Protein Sequence and Structure, pp.
353-358, National Biomedical Research Foundation, 1979; (2) a
penalty of 3.0 for each gap and an additional 0.10 penalty for each
symbol in each gap; and (3) no penalty for end caps. It is also
possible to use other sequence comparison programs used by those
skilled in the art.
[0071] Other nucleic acids homologous as the structural gene of the
present invention typically include nucleic acids which hybridize
under stringent conditions to a nucleotide consisting of a
nucleotide sequence within SEQ ID NO: 1 or 3, preferably a
nucleotide sequence consisting of nucleotides 106 to 1503 of SEQ ID
NO: 1, preferably a nucleotide sequence consisting of nucleotides
103 to 1512 of SEQ ID NO: 3, or a nucleotide sequence complementary
thereto and which encode polypeptides having the ability to
transfer N-acetyl-D-galactosamine to N-acetyl-D-glucosamine with
.beta.1,3 linkage.
[0072] As used herein, "under stringent conditions" means that a
nucleic acid hybridizes under conditions of moderate or high
stringency. More specifically, conditions of moderate stringency
may readily be determined by those having ordinary skill in the
art, e.g., depending on the length of DNA. Primary conditions can
be found in Sambrook et al., Molecular Cloning: A Laboratory
Manual, 3rd edition, Vol. 1, 7.42-7.45 Cold Spring Harbor
Laboratory Press, 2001 and include the use of a prewashing solution
for nitrocellulose filters 5.times.SSC, 0.5% SDS, 1.0 mM EDTA (pH
8.0), hybridization conditions of about 50% formamide, 2.times.SSC
to 6.times.SSC at about 40-50.degree. C. (or other similar
hybridization solutions, such as Stark's solution, in about 50%
formamide at about 42.degree. C.) and washing conditions of about
60.degree. C., 0.5.times.SSC, 0.1% SDS. Conditions of high
stringency can also be readily determined by those skilled in the
art, e.g., depending on the length of DNA. In general, such
conditions include hybridization and/or washing at a higher
temperature and/or at a lower salt concentration than that required
under conditions of moderate stringency and, for example, are
defined as hybridization conditions as above and with washing at
about 68.degree. C., 0.2.times.SSC, 0.1% SDS. Those skilled in the
art will recognize that the temperature and washing solution salt
concentration can be adjusted as necessary according to factors
such as the length of nucleotide sequences.
[0073] As described above, those skilled in the art will readily
determine and achieve conditions of suitably moderate or high
stringency on the basis of common knowledge about hybridization
conditions which are known in the art, as well as on the empirical
rule which will be obtained through commonly used experimental
means.
(2) Vector and Transformant of the Present Invention
[0074] The present invention provides a recombinant vector carrying
the above nucleic acid. Procedures for integrating a DNA fragment
of the nucleic acid into a vector (e.g., a plasmid) include those
described in Sambrook, J. et al., Molecular Cloning, A Laboratory
Manual (3rd edition), Cold Spring Harbor Laboratory, 1.1 (2001).
For convenience, a commercially available ligation kit (e.g., a
product of TaKaRa Shuzo Co., Ltd., Japan) may be used.
[0075] The recombinant vector (e.g., recombinant plasmid) thus
obtained may be introduced into host cells (e.g., E. coli
DH5.alpha., TB1, LE392, or XL-LE392 or XL-1Blue). Procedures for
introducing the plasmid into host cells include those described in
Sambrook, J. et al., Molecular Cloning, A Laboratory Manual (3rd
edition), Cold Spring Harbor Laboratory, 16.1 (2001), exemplified
by the calcium chloride method or the calcium chloride/rubidium
chloride method, electroporation, electroinjection, chemical
treatment (e.g., PEG treatment), and the gene gun method.
[0076] A vector which can be used may be prepared readily by
linking a desired gene to a recombination vector available in the
art (e.g., plasmid DNA) in a routine manner. Specific examples of a
vector to be used include, but are not limited to, E. coli-derived
plasmids such as pDONR201, pBluescript, pUC18, pUC19 and
pBR322.
[0077] Those skilled in the art will be able to select appropriate
restriction ends to fit into the intended expression vector. The
expression vector may be selected appropriately by those skilled in
the art such that the vector is suitable for host cells where the
enzyme of the present invention is to be expressed. Moreover, the
expression vector is preferably constructed to allow regions
involved in gene expression (e.g., promoter region, enhancer region
and operator region) to be properly located to ensure expression of
the above nucleic acid in target host cells, so that the nucleic
acid is properly expressed.
[0078] The type of expression vector is not limited in any way as
long as the vector allows expression of a desired gene in various
prokaryotic and/or eukaryotic host cells and has the function of
producing a desired protein. Preferred examples include pQE-30,
pQE-60, pMAL-C2, pMAL-p2 and pSE420 for E. coli expression, pYES2
(Saccharomyces) and pPIC3.5K, pPIC9K and pAO815 (all Pichia) for
yeast expression, as well as pFastBac, pBacPAK8/9, pBK283, pVL1392
and pBlueBac4.5 for insect expression.
[0079] To construct the expression vector, a Gateway system
(Invitrogen Corporation) may be used which does not require
restriction treatment and ligation operation. The Gateway system is
a site-specific recombination system which allows cloning while
maintaining the orientation of PCR products and also allows
subcloning of a DNA fragment into a properly modified expression
vector. More specifically, this system prepares an expression clone
corresponding to the intended expression system by creating an
entry clone from a PCR product and a donor vector by the action of
a site-specific recombinase BP clonase and then transferring the
PCR product to a destination vector which allows recombination with
this clone by the action of another recombinase LR clonase. One
feature of this system is that a time- and labor-consuming
subcloning step which requires treatment with restriction enzymes
and/or ligases can be eliminated when an entry clone is created to
begin with.
[0080] The above expression vector carrying the nucleic acid of the
present invention may be integrated into host cells to give a
transformant for producing the polypeptide of the present
invention. In general, host cells used for obtaining the
transformant may be either eukaryotic cells (e.g., mammalian cells,
yeast, insect cells) or prokaryotic cells (e.g., E. coli, Bacillus
subtilis). Also, cultured cells of human origin (e.g., HeLa, 293T,
SH-SY5Y) or mouse origin (e.g., Neuro2a, NIH3T3) may be used for
this purpose. All of these host cells are known and commercially
available (e.g., from Dainippon Pharmaceutical Co., Ltd., Japan),
or available from public research institutions (e.g., RIKEN Cell
Bank). Alternatively, it is also possible to use embryos, organs,
tissues or non-human individuals.
[0081] Since the nucleic acid of the present invention was found
from human genome libraries, it is believed that when eukaryotic
cells are used as host cells, the G34 enzyme protein of the present
invention may have properties close to native proteins (e.g.,
embodiments where glycosylation occurs). In light of this point, it
is preferable to select eukaryotic cells, particularly mammalian
cells, as host cells. Specific examples of mammalian cells include
animal cells of mouse, Xenopus laevis, rat, hamster, monkey or
human origin or cultured cell lines established from these cells.
E. coli, yeast or insect cells available for use as host cells are
specifically exemplified by E. coli (e.g., DH5.alpha., M15, JM109,
BL21), yeast (e.g., INVSc1 (Saccharomyces), GS115, KM71 (both
Pichia)) or insect cells (e.g., Sf21, BmN4, silkworm larva).
[0082] In general, an expression vector can be prepared by linking
at least a promoter, an initiation codon, a gene encoding a desired
protein, a termination codon and a terminator region to an
appropriate replicable unit to give a continuous loop. In this
case, if desired, it is also possible to use an appropriate DNA
fragment (e.g., linkers, other restriction enzyme sites) through
routine techniques such as digestion with a restriction enzyme
and/or ligation using T4 DNA ligase. When bacterial (particularly
E. coli) cells are used as host cells, an expression vector is
generally composed of at least a promoter/operator region, an
initiation codon, a gene encoding a desired protein, a termination
codon, a terminator and a replicable unit. When yeast cells, plant
cells, animal cells or insect cells are used as host cells, it is
generally preferred that an expression vector comprises at least a
promoter, an initiation codon, a gene encoding a desired protein, a
termination codon and a terminator. In this case, the vector may
also comprise DNA encoding a signal peptide, an enhancer sequence,
5'- and 3'-terminal untranslated regions of the desired gene, a
selective marker region or a replicable unit, as appropriate.
[0083] A replicable unit refers to DNA having the ability to
replicate its entire DNA sequence in host cells and includes a
native plasmid, an artificially modified plasmid (i.e., a plasmid
prepared from a native plasmid) and a synthetic plasmid. Examples
of a preferred plasmid include plasmid pQE30, pET or pCAL or an
artificially modified product thereof (i.e., a DNA fragment
obtained from pQE30, pET or pCAL by treatment with an appropriate
restriction enzyme) for E. coli cells, plasmid pYES2 or pPIC9K for
yeast cells, as well as plasmid pBacPAK8/9 for insect cells.
[0084] A methionine codon (ATG) may be given as an example of an
initiation codon preferred for the vector of the present invention.
Examples of a termination codon include commonly used termination
codons (e.g., TAG, TGA, TAA). As for enhancer and terminator
sequences, it is also possible to use those commonly used by those
skilled in the art, such as SV40-derived enhancer and terminator
sequences.
[0085] As a selective marker, a commonly used one can be used in a
routine manner. Examples include antibiotic resistance genes such
as those resistant to tetracycline, ampicillin, or kanamycin or
neomycin, hygromycin or spectinomycin.
[0086] The introduction (also referred to as transformation or
transfection) of the expression vector according to the present
invention into host cells may be accomplished by using
conventionally known techniques. Transformation may be
accomplished, for example, by the method of Cohen et al. [Proc.
Natl. Acad. Sci. USA, 69, 2110 (1972)], the protoplast method [Mol.
Gen. Genet., 168, 111 (1979)] or the competent method [J. Mol.
Biol., 56, 209 (1971)] for bacterial cells (e.g., E. coli, Bacillus
subtilis) and by the method of Hinnen et al. [Proc. Natl. Acad.
Sci. USA, 75, 1927 (1978)] or the lithium method [J. B. Bacteriol.,
153, 163 (1983)] for Saccharomyces cerevisiae. Transformation may
also be accomplished, for example, by the leaf disk method
[Science, 227, 129 (1985)] or electroporation [Nature, 319, 791
(1986)] for plant cells, by the method of Graham et al. [Virology,
52, 456 (1973)] for animal cells, and by the method of Summer et
al. [Mol. Cell. Biol., 3, 2156-2165 (1983)] for insect cells.
(3) G34 Enzyme Protein of the Present Invention
[0087] As illustrated in the Example section described later, a
polypeptide having a novel enzymatic activity can be isolated and
purified, for example, by integrating a nucleic acid having the
nucleotide sequence of SEQ ID NO: 1 or 3 into an expression vector
and then expressing the nucleic acid.
[0088] First, in light of the above point, a typical embodiment of
the protein of the present invention is an isolated G34 enzyme
protein consisting of the putative amino acid sequence shown in SEQ
ID NO: 2 or 4. More specifically, this enzyme protein has the
activities shown below.
Catalytic Reaction
[0089] The enzyme protein allows transfer of
"N-acetyl-D-galactosamine (GalNAc)" from its donor substrate to an
acceptor substrate containing "N-acetyl-D-glucosamine (GlcNAc)."
Examination of motif sequences in the amino acid sequence indicates
that the linking mode between N-acetylgalactosamine and
N-acetylglucosamine is a .beta.1,3 glycosidic linkage (see Example
2).
Donor Substrate Specificity:
[0090] The above N-acetyl-D-galactosamine donor substrate
encompasses sugar nucleotides having N-acetylgalactosamine, such as
uridine diphosphate-N-acetylgalactosamine (UDP-GalNAc), adenosine
diphosphate-N-galactosamine (ADP-GalNAc), guanosine
diphosphate-N-acetylgalactosamine (GDP-GalNAc) and cytidine
diphosphate-N-acetylgalactosamine (CDP-GalNAc). A typical donor
substrate is UDP-GalNAc.
[0091] Namely, the G34 enzyme protein of the present invention
catalyzes a reaction of the following scheme:
[0092] UDP-GalNAc+GlcNAc-R.fwdarw.UDP+GalNAc-.beta.1,3-GlcNAc-R
(wherein R represents, e.g., a glycoprotein, glycolipid,
oligosaccharide or polysaccharide having the GlcNAc residue).
Acceptor Substrate Specificity:
[0093] An acceptor substrate of the above GalNAc is
N-acetyl-D-glucosamine, typically an N-acetyl-D-glucosamine residue
of glycoproteins, glycolipids, oligosaccharides or polysaccharides,
etc.
[0094] When using an oligosaccharide as an acceptor substrate, the
human G34 protein obtained in Example 1 described later (typically
having a region covering amino acid 36 to the C-terminal end of SEQ
ID NO: 2) shows transferase activity toward Bz-.beta.-GlcNAc,
GlcNAc-.beta.1-4-GlcNAc-.beta.-Bz, pNp-core2
(core2=Gal-.beta.1-3-(GlcNAc-.beta.1-6) GalNAc-.alpha.-pNp; the
same applying hereinafter), pNp-core3 (core3=GlcNAc-.beta.1-3
GalNAc-.alpha.-pNp; the same applying hereinafter) and pNp-core6
(core6=GlcNAc-.beta.1-6-GalNAc-.alpha.-pNp; the same applying
hereinafter). Preferably, the human G34 protein is free from
transferase activity toward Bz-.alpha.-GlcNAc and Gal-.beta.1-3
GlcNAc-.beta.-pNp. Moreover, when the activity is compared between
these substrates, the transferase activity is very high in
transferring to pNp-core2 and Bz-.beta.-GlcNAc, particularly
highest in transferring to pNp-core2. The transferase activity is
relatively low in transferring to
GlcNAc-.beta.1-4-GlcNAc-.beta.-Bz, pNp-core3 and pNp-core6.
[0095] Likewise, the mouse G34 protein obtained in Example 4
described later (typically having an active region covering amino
acid 35 to the C-terminal end of SEQ ID NO: 4) shows transferase
activity toward Bz-.beta.-GlcNAc, pNp-.beta.-Glc,
GlcNAc-.beta.1-4-GlcNAc-.beta.-Bz, pNp-core2, pNp-core3 and
pNp-core6. When the activity is compared between these substrates,
the transferase activity is highest in transferring to
Bz-.beta.-GlcNAc, followed by core2-pNp, core6-pNp, core3-pNp,
pNp-.beta.-Glc and GlcNAc-.beta.1-4-GlcNAc-.beta.-Bz in the order
named.
[0096] As used herein, "GlcNAc" represents an
N-acetyl-D-glucosamine residue, "GalNAc" represents an
N-acetyl-D-galactosamine residue, "Glc" represents a glucosamine
residue, "Bz" represents a benzyl group, "pNp" represents a
p-nitrophenyl group, "oNp" represents a o-nitrophenyl group, and
"-" represents a glycosidic linkage. Numbers in these formulae each
represent the carbon number in the sugar ring where the above
glycosidic linkage is present. Likewise, ".alpha." and ".beta."
represent anomers of the above glycosidic linkage at the 1-position
of the sugar ring. An anomer whose positional relationship with
CH.sub.2OH or CH.sub.3 at the 5-position is trans and cis is
represented by ".alpha." and ".beta.", respectively.
Optimum Buffer and Optimum pH (Table 3 and FIG. 4):
[0097] Examination of the human G34 protein indicates that the
protein has the above catalytic effect in each of the following
optimum buffers: MES (2-morpholinoethanesulfonic acid) buffer,
sodium cacodylate buffer or HEPES
(N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid])
buffer.
[0098] The pH dependence of the activity in each buffer is as
follows: in MES buffer, the activity is highest around a pH of at
least 5.50 to 5.78 and second highest around pH 6.75; in sodium
cacodylate buffer, the activity increases with decrease in pH from
around 6.2 to around 5.0 and is highest around pH 5.0, while the
activity also increases in a pH-dependent manner between around pH
6.2 and 7.0 and nearly plateaus around pH 7.4; and in HEPES buffer,
the activity is highest around a pH of 7.4 to 7.5. Among them,
HEPES buffer at a pH of about 7.4 to about 7.5 results in the
strongest activity. In all the buffers, the activity is lower in a
pH range of 6.2 to 6.6 than in other pH ranges.
Divalent Ion Requirement (Table 4 and FIG. 5):
[0099] The activity of the human G34 protein is enhanced in the
presence of a divalent metal ion, particularly Mn.sup.2+, Co.sup.2+
or Mg.sup.2+. The influence of each metal ion concentration on the
activity is as follows: in the case of Mn.sup.2+ and Co.sup.2+, the
activity increases in a concentration-dependent manner up to around
5.0 nM and then nearly plateaus at higher concentrations, while in
the case of Mg.sup.2+, the activity increases in a
concentration-dependent manner up to around 2.5 nM and then nearly
plateaus at higher concentrations. However, the Mn.sup.2+-induced
enhancement of the activity is completely eliminated in the
presence of Cu.sup.2+.
[0100] As described above, the G34 enzyme protein of the present
invention can transfer a GalNAc residue to a GlcNAc residue with
.beta.1-3 glycosidic linkage under given enzymatic reaction
conditions as mentioned above and is useful for such sugar chain
synthesis or modification reactions targeted at glycoproteins,
glycolipids, oligosaccharides or polysaccharides, etc.
[0101] Secondly, having disclosed herein the amino acid sequences
shown in SEQ ID NOs: 2 and 4 which are given as typical examples of
the primary structure of the above enzyme protein, the present
invention provides all proteins which can be produced on the basis
of these amino acid sequences through genetic engineering
procedures well known in the art (hereinafter also referred to as
"mutated proteins" or "modified proteins"). Namely, according to
common knowledge in the art, the enzyme protein of the present
invention is not limited only to a protein consisting of the amino
acid sequence of SEQ ID NO: 2 or 4 estimated from the nucleotide
sequence of each cloned nucleic acid, and is also intended to
include, for example, a protein consisting of a non-full-length
polypeptide having, e.g., a partial N-terminal deletion of the
amino acid sequence, or a protein homologous to such an amino acid
sequence, each of which has properties inherent to the protein, as
illustrated below.
[0102] First, the human G34 enzyme protein of the present invention
may preferably have an amino acid sequence covering amino acid 189
to the C-terminal end of SEQ ID NO: 2, more preferably an amino
acid sequence covering amino acid 36 to the C-terminal end as
obtained in the Example section described later. Likewise, the
mouse G34 enzyme protein of the present invention may preferably
have an amino acid sequence covering amino acid 35 to the
C-terminal end of SEQ ID NO: 4.
[0103] Moreover, in proteins usually having physiological
activities equivalent to enzymes, it is well known that the
physiological activities are maintained even when their amino acid
sequences have substitution, deletion, insertion or addition of one
or more amino acids. It is also known that among
naturally-occurring proteins, there are mutated proteins which have
gene mutations resulting from differences in the species of source
organisms and/or differences in ecotype or which have one or more
amino acid mutations resulting from the presence of closely
resembling isozymes, etc. In light of this point, the protein of
the present invention also encompasses mutated proteins which have
an amino acid sequence with substitution, deletion, insertion or
addition of one or more amino acids in each amino acid sequence
shown in SEQ ID NO: 2 or 4 and which have the ability to transfer a
GalNAc residue to a GlcNAc residue with .beta.1-3 glycosidic
linkage under given enzymatic reaction conditions as mentioned
above. Moreover, particularly preferred are modified proteins
having amino acid sequences with substitution, deletion, insertion
or addition of one or several amino acids in each amino acid
sequence shown in SEQ ID NO: 2 or 4.
[0104] The expression "one or more amino acids" found above means
preferably 1 to 200 amino acids, more preferably 1 to 100 amino
acids, even more preferably 1 to 50 amino acids, and most
preferably 1 to 20 amino acids. In general, in a case where amino
acid substitution occurs as a result of site-specific mutagenesis,
the number of amino acids which can be substituted while
maintaining the activities inherent to the original protein is
preferably 1 to 10.
[0105] The modified protein of the present invention also includes
those obtained by substitution between functionally equivalent
amino acids. Namely, it is generally well known to those skilled in
the art that recombinant proteins having a desired mutation(s) can
be prepared by procedures involving introduction of substitution
between functionally equivalent amino acids (e.g., replacement of
one hydrophobic amino acid with another hydrophobic amino acid,
replacement of one hydrophilic amino acid with another hydrophilic
amino acid, replacement of one acidic amino acid with another
acidic amino acid, or replacement of one basic amino acid with
another basic amino acid). The modified proteins thus obtained
often have the same properties as the original protein. In light of
this point, modified proteins having such amino acid substitutions
also fall within the scope of the present invention.
[0106] Moreover, the modified protein of the present invention may
be a glycoprotein having sugar chains attached to the polypeptide
as long as it has such an amino acid sequence as defined above and
has an enzymatic activity inherent to the intended enzyme.
[0107] To identify the range of the homologous protein of the
present invention, an identity search using GENETYX software
(Genetyx Corporation, Japan) is performed for the amino acid
sequence shown in SEQ ID NO: 2 or 4 of the present invention,
indicating that the amino acid sequence shares 14% identity with a
known .beta.1,4GalNAc transferase showing the highest homology
(Non-patent Document 1 listed above) and also shares 30% identity
with a known .beta.1,3Gal transferase showing the highest homology
(Non-patent Document 2 listed above). In light of these points, a
preferred amino acid sequence for the homologous protein of the
present invention preferably shares more than 30% identity, more
preferably at least 40% identity, and particularly preferably at
least 50% identity with the amino acid sequence shown in SEQ ID NO:
2 or 4.
[0108] Likewise, the amino acid sequences shown in SEQ ID NOs: 2
and 4 share 88% identity with each other. In light of this point, a
preferred amino acid sequence for the homologous protein of the
present invention can be defined as sharing at least 88%, more
preferably 90% identity with the amino acid sequence within SEQ ID
NO: 2.
[0109] The above GENETYX is genetic information processing software
for nucleic acid/protein analysis and enables standard analyses of
homology and multialignment, as well as signal peptide prediction,
promoter site prediction and secondary structure prediction. The
homology analysis program used herein employs the Lipman-Pearson
method (Lipman, D. J. & Pearson, W.R., Science, 277, 1435-1441
(1985)) frequently used as a rapid and sensitive method. In the
present invention, the percentage of identity may be determined by
comparing sequence information using, e.g., the BLAST program
described by Altschul et al. (Nucl. Acids. Res., 25. 3389-3402
(1997)) or the FASTA program described by Pearson et al. (Proc.
Natl. Acad. Sci. USA, 2444-2448 (1988)). These programs are
available on the Internet at the web site of the National Center
for Biotechnology Information (NCBI) or the DNA Data Bank of Japan
(DDBJ). The details of various conditions (parameters) for each
identity search using each program are shown on these web sites,
and default values are commonly used for these searches although
part of the settings may be changed as appropriate. It is also
possible to use other sequence comparison programs used by those
skilled in the art.
[0110] Thirdly, the isolated protein of the present invention may
be administered as an immunogen to an animal to produce an antibody
against the protein, as described later. Such an antibody may be
used for immunoassays to measure and quantify the enzyme. Thus, the
present invention is also useful in preparing such an immunogen. In
light of this point, the protein of the present invention also
includes a polypeptide fragment, mutant or fusion protein thereof,
which contains an antigenic determinant or epitope for eliciting
antibody formation.
(4) Isolation and Purification of the G34 Enzyme Protein of the
Present Invention
[0111] The enzyme protein of the present invention may be isolated
and purified in the following manner.
[0112] Recent studies have established genetic engineering
procedures which involve culturing and growing a transformant and
isolating and purifying a substance of interest from the resulting
culture or grown transformant. The enzyme protein of the present
invention may also be expressed (produced), e.g., by culturing in a
nutrient medium a transformant containing an expression vector
carrying the nucleic acid of the present invention.
[0113] A nutrient medium used for transformant culturing preferably
contains a carbon source, an inorganic nitrogen source or an
organic nitrogen source required for host cell (transformant)
growth. Examples of a carbon source include glucose, dextran,
soluble starch, sucrose and methanol. Examples of an inorganic or
organic nitrogen source include ammonium salts, nitrate salts,
amino acids, corn steep liquor, peptone, casein, meat extracts,
soybean meal and potato extracts. If desired, the medium may
contain other nutrients such as inorganic salts (e.g., sodium
chloride, calcium chloride, sodium dihydrogen phosphate, magnesium
chloride), vitamins, and antibiotics (e.g., tetracycline, neomycin,
ampicillin, kanamycin). Culturing may be accomplished in a manner
known in the art. Culture conditions such as temperature, medium pH
and culture period may be appropriately selected such that the
protein according to the present invention is produced in a large
quantity.
[0114] The enzyme protein of the present invention may be obtained
from the above culture or grown transformant as follows. Namely, in
a case where a protein of interest is accumulated in host cells,
the host cells may be collected by manipulations such as
centrifugation or filtration, suspended in an appropriate buffer
(e.g., Tris buffer, phosphate buffer, HEPES buffer or MES buffer at
a concentration around 10 to 100 mM, the pH of which will vary from
buffer to buffer, but desirably falls within the range of 5.0 to
9.0), and then crushed in a manner suitable for the host cells
used, followed by centrifugation to obtain the contents of the host
cells. On the other hand, in a case where a protein of interest is
secreted from host cells, the host cells and the medium are
separated from each other by manipulations such as centrifugation
or filtration to obtain a culture filtrate. The crushed host cell
solution or culture filtrate may be provided directly or may be
treated by ammonium sulfate precipitation and dialysis before being
provided for isolation and purification of the protein.
[0115] Isolation and purification of a protein of interest may be
accomplished in the following manner. Namely, in a case where the
protein is labeled with a tag such as 6.times. histidine, GST or
maltose-binding protein, the isolation and purification may be
accomplished by affinity chromatography suitable for each of the
commonly used tags. On the other hand, in a case where the protein
according to the present invention is produced without being
labeled with such a tag, the isolation and purification may be
accomplished, e.g., by ion exchange chromatography, which may
further be combined with gel filtration, hydrophobic
chromatography, isoelectric chromatography, etc.
[0116] Moreover, an expression vector may be constructed to
facilitate isolation and purification. In particular, the isolation
and purification is facilitated if an expression vector is
constructed to express a fusion protein of a polypeptide having an
enzymatic activity with a labeling peptide and the enzyme protein
is prepared in a genetic engineering manner. An example of the
above identification peptide is a peptide having the function of
facilitating secretion, separation, purification or detection of
the enzyme according to the present invention from the grown
transformant by allowing the enzyme to be expressed as a fusion
protein in which the identification peptide is attached to a
polypeptide having an enzymatic activity when the enzyme according
to the present invention is prepared by gene recombination
techniques.
[0117] Examples of such an identification peptide include peptides
such as a signal peptide (a peptide composed of 15 to 30 amino acid
residues, which is present at the N-terminal end of many proteins
and is functional in cells for protein selection in the
intracellular membrane permeation mechanism; e.g., OmpA, OmpT,
Dsb), protein kinase A, Protein A (a protein with a molecular
weight of about 42,000, which is a component constituting the
Staphylococcus aureus cell wall), glutathione S transferase, His
tag (a sequence consisting of 6 to 10 histidine residues in
series), myc tag (a 13 amino acid sequence derived from cMyc
protein), FLAG peptide (an analysis marker composed of 8 amino acid
residues), T7 tag (composed of the first 11 amino acid residues of
the gene 10 protein), S tag (composed of pancreas RNase A-derived
15 amino acid residues), HSV tag, pelb (a 22 amino acid sequence
from the E. coli external membrane protein pelB), HA tag (composed
of hemagglutinin-derived 10 amino acid residues), Trx tag
(thioredoxin sequence), CBP tag (calmodulin-binding peptide), CBD
tag (cellulose-binding domain), CBR tag (collagen-binding domain),
.beta.-lac/blu (.beta.-lactamase), .beta.-gal
(.beta.-galactosidase), luc (luciferase), HP-Thio (His-patch
thioredoxin), HSP (heat shock peptide), Lny (laminin
.gamma.-peptide), Fn (fibronectin partial peptide), GFP (green
fluorescent peptide), YFP (yellow fluorescent peptide), CFP (cyan
fluorescent peptide), BFP (blue fluorescent peptide), DsRed, DsRed2
(red fluorescent peptides), MBP (maltose-binding peptide), LacZ
(lactose operator), IgG (immunoglobulin G), avidin and Protein G,
any of which can be used.
[0118] Among them, particularly preferred are the signal peptide,
protein kinase A, Protein A, glutathione S transferase, His tag,
myc tag, FLAG peptide, T7 tag, S tag, HSV tag, pelB and HA tag
because they facilitate expression and purification of the enzyme
according to the present invention through genetic engineering
procedures. In particular, it is preferable to obtain the enzyme as
a fusion protein with FLAG peptide
(Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) because it is very easy to
handle. The above FLAG peptide is extremely antigenic and provides
an epitope capable of reversible binding of a specific monoclonal
antibody, thus enabling rapid assay and easy purification of the
expressed recombinant protein. A mouse hybridoma called 4E11
produces a monoclonal antibody which binds to FLAG peptide in the
presence of a certain divalent metal cation, as described in U.S.
Pat. No. 5,011,912 (incorporated herein by reference). A 4 .mu.l
hybridoma cell line has been deposited under Accession No. HB 9259
with the American Type Culture Collection. The monoclonal antibody
binding to FLAG peptide is available from Eastman Kodak Co.,
Scientific Imaging Systems Division, New Haven, Conn.
[0119] pFLAG-CMV-1 (SIGMA) can be presented as an example of a
basic vector which can be expressed in mammalian cells and enables
obtaining the enzyme protein of the present invention as a fusion
protein with the above FLAG peptide. Likewise, examples of a vector
which can be expressed in insect cells include, but are not limited
to, pFBIF (i.e., a vector prepared by integrating the region
encoding FLAG peptide into pFastBac (Invitrogen Corporation); see
the Example section described later). Those skilled in the art will
be able to select an appropriate basic vector depending on, e.g.,
the host cell, restriction enzyme and identification peptide to be
used for expression of the enzyme.
(5) Antibody Recognizing the G34 Enzyme Protein of the Present
Invention
[0120] The present invention provides an antibody which is
immunoreactive to the G34 enzyme protein. Such an antibody is
capable of specifically binding to the enzyme protein via the
antigen-binding site of the antibody (as opposed to non-specific
binding). More specifically, a protein having the amino acid
sequence of SEQ ID NO: 2 or 4 or a fragment, mutant or fusion
protein thereof may be used as an immunogen for producing an
antibody immunoreactive to each of them.
[0121] More specifically, such a protein, fragment, mutant or
fusion protein contains an antigenic determinant or epitope for
eliciting antibody formation. These antigenic determinant and
epitope may be either linear or conformational (discontinuous). The
antigenic determinant or epitope can be identified by any technique
known in the art. Thus, the present invention also relates to an
antigenic epitope of the G34 enzyme protein. Such an epitope is
useful in preparing an antibody, particularly a monoclonal
antibody, as described in more detail below.
[0122] The epitope of the present invention can be used in assays
and as a research reagent for purifying a specific binding antibody
from materials such as polyclonal sera or supernatants from
cultured hybridomas. Such an epitope or a variant thereof may be
prepared using techniques known in the art (e.g., solid phase
synthesis, chemical or enzymatic cleavage of a protein) or using
recombinant DNA technology.
[0123] The enzyme protein of the present invention may be used to
derive any embodiment of an antibody. If the entire or partial
polypeptide of or an epitope of the protein has been isolated, both
polyclonal and monoclonal antibodies can be prepared using
conventional techniques. See, e.g., Kennet et al. (eds.),
Monoclonal Antibodies, Hybridomas: A New Dimension in Biological
Analyses, Plenum Press, New York, 1980.
[0124] The present invention also provides a hybridoma cell line
producing a monoclonal antibody specific to the G34 enzyme protein.
Such a hybridoma can be produced and identified by conventional
techniques. One method for producing such a hybridoma cell line
involves immunizing an animal with the enzyme protein of the
present invention, collecting spleen cells from the immunized
animal, fusing the spleen cells with a myeloma cell line to give
hybridoma cells, and identifying a hybridoma cell line which
produces a monoclonal antibody binding to the enzyme. The resulting
monoclonal antibody may be collected by conventional
techniques.
[0125] The monoclonal antibody of the present invention encompasses
chimeric antibodies, for example, humanized mouse monoclonal
antibodies. Such a humanized antibody is advantageous in reducing
immunogenicity when administered to a human subject.
[0126] The present invention also provides an antigen-binding
fragment of the above antibody. Examples of an antigen-binding
fragment which can be produced by conventional techniques include,
but are not limited to, Fab and F(ab').sub.2 fragments. The present
invention also provides an antibody fragment and derivative which
can be produced by genetic engineering techniques.
[0127] The antibody of the present invention can be used in assays
to detect the presence of the G34 enzyme protein of the present
invention or a polypeptide fragment thereof, either in vitro or in
vivo. The antibody of the present invention may also be used in
purifying the G34 enzyme protein or a polypeptide fragment thereof
by immunoaffinity chromatography.
[0128] Moreover, the antibody of the present invention may also be
provided as a blocking antibody capable of blocking the binding of
the above glycosyltransferase protein to its binding partner (e.g.,
acceptor substrate), thus inhibiting the enzyme's biological
activity resulting from such binding. Such a blocking antibody may
be identified using any suitable assay procedure, for example, by
testing the antibody for the ability to inhibit the binding of the
protein to certain cells expressing an acceptor substrate.
[0129] Alternatively, the blocking antibody may also be identified
in assays for the ability to inhibit a biological effect resulting
from the enzyme protein bound to its binding partner in target
cells. Such an antibody may be used in an in vitro procedure or
administered in vivo to inhibit a biological activity mediated by
the entity that generated the antibody. Thus, the present invention
also provides an antibody for treating disorders which are caused
or exacerbated by either direct or indirect interaction between the
G34 enzyme protein and its binding partner. Such therapy will
involve in vivo administration of the blocking antibody to a mammal
in an amount effective for inhibiting a binding partner-mediated
biological activity. For use in such therapy, monoclonal antibodies
are preferred and, in one embodiment, an antigen-binding antibody
fragment is used.
(6) Nucleic Acid of the Present Invention for Canceration assay
[0130] In response to the discovery of the above G34 enzyme
protein, the inventors of the present invention have confirmed that
mRNA encoding this protein is widely found in cancerous tissues and
cell lines and that the expression level of the mRNA is
significantly increased particularly in cancerous tissues. Thus,
the G34 nucleic acid is useful as a tumor marker that is useful
for, e.g., cancer diagnosis targeted at biological samples
containing transcription products. In this aspect, the present
invention provides a nucleic acid for measurement, which is capable
of hybridizing under stringent conditions to a nucleic acid defined
by the nucleotide sequence shown in SEQ ID NO: 1 or 3.
[0131] In one embodiment, the nucleic acid for measurement of the
present invention is a primer or probe targeting the G34 nucleic
acid in a biological sample and having a nucleotide sequence
selected from the nucleotide sequence of SEQ ID NO: 1 or 3. In
particular, since the nucleotide sequence of SEQ ID NO: 1 is
derived from mRNA encoding a structural gene and contains the
entire open reading frame (ORF) of the G34 gene, full-length or
nearly full-length sequences of SEQ ID NO: 1 or 3 are usually found
in transcription products from a biological sample. In light of
this point, the primer or probe according to the present invention
has a desired partial sequence selected from each nucleotide
sequence of SEQ ID NO: 1 or 3 (either homologous or complementary
to the selected sequence depending on the intended use) and hence
can be provided as a nucleic acid capable of specifically
hybridizing to the target sequence.
[0132] Typical examples of such a primer or probe include a native
DNA fragment derived from a nucleic acid having at least a part of
the nucleotide sequence shown in SEQ ID NO: 1 or 3, a DNA fragment
synthesized to have at least a part of the nucleotide sequence
shown in SEQ ID NO: 1 or 3, or complementary strands of these
fragments.
[0133] Such a primer or probe as mentioned above may be used to
detect and/or quantify the target nucleic acid in a biological
sample, as described later. Since sequences on the genome can also
be targeted, the nucleic acid of the present invention may also be
used as an antisense primer for medical research or gene
therapy.
(A) Probe of the Present Invention
[0134] In a preferred embodiment, the nucleic acid for measurement
of the present invention is a probe targeting a nucleic acid having
the nucleotide sequence of SEQ ID NO: 1 or 3 or a complementary
strand of at least one of them. The probe contains an
oligonucleotide composed of at least a dozen nucleotides,
preferably at least 15 nucleotides, preferably at least 17
nucleotides, and more preferably at least 20 nucleotides selected
from the nucleotide sequences of SEQ ID NOs: 1 and 3, or a
complementary strand of the oligonucleotide, or full-length cDNA of
its ORF region or a complementary strand of the cDNA.
[0135] In a case where the nucleic acid for measurement of the
present invention is provided as an oligonucleotide probe, it is
understood that a length of a dozen nucleotides (e.g., 15
nucleotides, preferably 17 nucleotides) may be sufficient for the
nucleic acid to specifically hybridize under stringent conditions
to its target nucleic acid. Namely, those skilled in the art will
be able to select an appropriate partial sequence composed of at
least 15 to 20 nucleotides from the nucleotide sequence of SEQ ID
NO: 1 or 3 in accordance with known various strategies for
oligonucleotide probe design. In this case, the amino acid sequence
information shown in SEQ ID NO: 2 or 4 is helpful in selecting a
unique sequence that may be suitable as a probe.
[0136] Likewise, in the case of a cDNA probe, for example, a probe
with a high molecular weight is generally difficult to handle when
used as a reagent or diagnostic agent for medical research. In
light of this point, the probe of the present invention intended
for medical research includes a nucleic acid composed of 50 to 500
nucleotides, more preferably 60 to 300 nucleotides selected from
each nucleotide sequence of SEQ ID NO: 1 or 3.
[0137] The term "stringent conditions" found above means conditions
of moderate or high stringency as explained earlier. Those skilled
in the art will be able to readily determine and achieve conditions
of moderate or high stringency suitable for the selected probe, on
the basis of common knowledge and empirical rule about known
procedures for various probe designs and hybridization
conditions.
[0138] Although depending on, e.g., the nucleotide length to be
selected and the hybridization conditions to be applied, a
relatively short oligonucleotide probe can serve as a probe even
when it has a mismatch of one or several nucleotides, particularly
one or two nucleotides, in comparison with the nucleotide sequence
of SEQ ID NO: 1 or 3. Likewise, a relatively long cDNA probe can
also serve as a probe even when it has a mismatch of 50% or less,
preferably 20% or less, in comparison with the nucleotide sequence
of SEQ ID NO: 1 or a nucleotide sequence complementary thereto.
[0139] The probe of the present invention thus designed can be used
as a labeled probe having a label such as a fluorescent label, a
radioactive label or a biotin label, in order to detect or confirm
a hybrid formed with a target sequence in G34.
[0140] For example, the labeled probe of the present invention may
be used for confirmation or quantification of PCR amplification
products from the G34 nucleic acid. In this case, it is preferable
to use a probe targeting the nucleotide sequence located in a
region between a pair of primer sequences used for PCR. An example
of such a probe may be an oligonucleotide consisting of the
nucleotide sequence shown in SEQ ID NO: 16 (corresponding to a
complementary strand against nucleotides 525 to 556 in SEQ ID NO:
1) (see Example 3).
[0141] The probe of the present invention may be included in a kit
such as a diagnostic DNA probe kit or may be immobilized on a chip
such as a DNA microarray chip.
(B) Primers of the Present Invention
[0142] In a preferred embodiment, the primers obtained from the
nucleic acid for the canceration assay of the present invention are
oligonucleotide primers. To prepare oligonucleotide primers, two
regions may be selected from the ORF region of the nucleotide
sequence shown in SEQ ID NO: 1 or 3 in such a manner as to satisfy
the following conditions:
a) the length of each region is at least several tens of
nucleotides, particularly at least 15 nucleotides, preferably at
least 17 nucleotides, more preferably at least 20 nucleotides, and
at most 50 nucleotides; and b) the G+C content in each region is
40% to 70%.
[0143] In actual fact, oligonucleotide primers may be prepared as
single-stranded DNAs having nucleotide sequences identical or
complementary to the two regions thus selected, or may be prepared
as single-stranded DNAs modified not to lose the binding
specificity to these nucleotide sequences. Although each primer of
the present invention preferably has a sequence that is completely
complementary to the selected target sequence, a mismatch of one or
two nucleotides may be permitted.
[0144] Examples of the pair of primers according to the present
invention include a pair of oligonucleotides consisting of SEQ ID
NOs: 14 and 15 (corresponding to complementary strands against
nucleotides 481-501 and 562-581 in SEQ ID NO: 1, respectively) for
human G34, and a pair of oligonucleotides consisting of SEQ ID NOs:
17 and 18 (corresponding to complementary strands against
nucleotides 481-501 and 562-581 in SEQ ID NO: 3, respectively) for
mouse G34.
(7) Canceration Assay According to the Present Invention
[0145] As described earlier, the G34 nucleic acid of the present
invention was confirmed to show a significant increase in the
expression level (i.e., transcription level of the gene from the
genome into mRNA) in a cancerous biological sample when compared to
a normal biological sample. The G34 nucleic acid of the present
invention was demonstrated to be useful at least in a canceration
assay for large intestine (colon) cancer or lung cancer (see
Example 3).
[0146] According to detailed embodiments of the canceration assay
of the present invention, transcription products extracted from a
biological sample or a nucleic acid library derived therefrom may
be used as a test sample and measured for the amount of the G34
nucleic acid (typically the amount of its mRNA) using the above
probe or primer to determine whether the measured value is
significantly higher than that of a normal biological sample. In
this case, if the measured value of the test biological sample is
significantly higher than the reference value of the normal
biological sample, the test biological sample is determined as
being cancerous or having a high grade of malignancy.
[0147] In the canceration assay of the present invention, the
reference value for a normal biological sample used as a control
may be a value measured for a control site (typically a normal
site) in the same tissue of the same patient or may be a value
normalized from known data obtained in a control site, e.g., the
mean value of mRNA levels in normal tissues.
[0148] According to the measurement of expression levels using the
nucleic acid for measurement of the present invention, human G34 is
found to be expressed at a high level in the brain, skeletal
muscle, pancreas, adrenal gland, testis and prostate when measured
in normal sites, and there is also significant expression in other
sites, although at a relatively low level. This indicates that
human G34 expression is widely found over various tissues and that
the expression level of human G34 is significantly increased even
in tissues with a relatively low expression level, such as large
intestine (colon) and lung tissues. Once these data have been
provided, those skilled in the art will recognize the actual
utility and effect of the nucleic acid for measurement of the
present invention.
[0149] In this assay, whether the measured value for a test sample
is significantly higher than that of a normal sample may be
determined by the criteria that are set depending on the accuracy
(positive rate) required for the assay or the grade of malignancy
to be determined. The criteria may be freely set depending on the
intended purpose; for example, the reference value to be determined
as positive may be set to a lower value for the purpose of
detecting tissues with a high grade of malignancy or may be set to
a higher value for the purpose of comprehensively detecting test
samples with signs or risk of canceration.
[0150] Examples will be given below of hybridization and PCR assays
to illustrate the canceration assay of the present invention.
(A) Hybridization Assay
[0151] Embodiments of this assay include those using a probe
obtained from the nucleic acid of the present invention, e.g.,
methods using various hybridization assays well known to those
skilled in the art, exemplified by Southern blotting, Northern
blotting, dot blotting or colony hybridization. In the case of
requiring amplification and/or quantification of the detected
signal, these methods may further be combined with immunoassay.
[0152] According to typical hybridization assays, a nucleic acid
extracted from a biological sample or an amplification product
thereof may be immobilized on a solid phase and hybridized with a
labeled probe under stringent conditions. After washing, the label
attached to the solid phase may be measured.
[0153] Extraction and purification of transcription products from a
biological sample may be accomplished by using any method known to
those skilled in the art.
(B) PCR Assay
[0154] In a preferred embodiment, the canceration assay of the
present invention includes PCR methods based on nucleic acid
amplification using the primers of the present invention. The
details of PCR are as explained earlier. In this subsection, a
detailed PCR-based embodiment of this assay will be explained.
[0155] G34 mRNA in transcription products to be assayed can be
amplified by PCR using a pair of primers located at both ends of a
given region selected from the nucleotide sequence of G34. In this
step, if even trace amounts of G34 nucleic acid fragments are
present in an analyte, these fragments will serve as templates to
replicate and amplify the nucleic acid region between the primer
pair. After repeating a given number of PCR cycles, the nucleic
acid fragments serving as templates are each amplified to a desired
concentration. Under the same amplification conditions, the
amplification product will be obtained in proportion to the amount
of G34 mRNA present in the analyte. Then, the above probe or the
like targeting the amplified region may be used to confirm whether
the amplification product is the nucleic acid of interest and also
quantify the same. Likewise, the nucleic acid in a normal tissue
may also be measured in the same manner. In this case, a nucleic
acid of a gene that is widely and usually present in the same
tissue or the like (e.g., a nucleic acid encoding
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or .beta.-actin)
may be used as a control to remove variations among individuals.
The measured value for the transcription level of G34 is provided
for comparison to assay the presence of canceration or the grade of
malignancy, as described above.
[0156] A nucleic acid sample provided for PCR methods may be either
total mRNA extracted from a biological sample (e.g., a test tissue
or cell) or total cDNA reverse transcribed from mRNA. In a case
where mRNA is amplified, the NASBA method (3SR method, TMA method)
using the primer pair mentioned above may be employed. Since the
NASBA method per se is well known and kits for this method are
commercially available, the method may be readily accomplished by
using the primer pair of the present invention.
[0157] To detect or quantify the above amplification product, the
reaction solution after amplification may be electrophoresed and
the resulting bands may be stained with ethidium bromide or the
like, or alternatively, the electrophoresed amplification product
may be immobilized onto a solid phase (e.g., a nylon membrane),
hybridized with a labeled probe specifically hybridizing to a test
nucleic acid (e.g., a probe having the nucleotide sequence of SEQ
ID NO: 16) and washed, followed by detection of the label.
[0158] Examples of PCR methods preferred for this assay include
quantitative PCR, especially kinetic RT-PCR or quantitative
real-time PCR. In particular, quantitative real-time RT-PCR
targeted at mRNA libraries is preferred in view that it allows
direct purification of a target to be measured from a biological
sample and directly reflects the transcription level. However, the
nucleic acid quantification in this assay is not limited to
quantitative PCR. Other known quantitative DNA assays (e.g.,
Northern blotting, dot blotting, DNA microarray) using the above
probe may also be applied to the PCR products.
[0159] Moreover, when performed using a quencher fluorescent dye
and a reporter fluorescent dye, quantitative RT-PCR also enables
quantification of a target nucleic acid in an analyte. In
particular, it may be readily performed since kits for quantitative
RT-PCR are commercially available.
[0160] Moreover, a target nucleic acid may also be semi-quantified
based on the intensity of the corresponding electrophoretic
band.
(C) Assay for Therapeutic Effect on Cancer
[0161] Other embodiments of the canceration assay of the present
invention include an assay for determining the effect of curing or
alleviating cancer. For example, targets of this assay include all
treatments such as administration of an anticancer agent and
radiation therapy, and targets of these treatments include in vitro
cancer cells or cancer tissues derived from cancer patients or
experimental animal models for carcinogenesis.
[0162] According to this assay, in a case where a biological sample
is subjected to a certain treatment, it is possible to know the
therapeutic effect of the treatment on cancer by determining
whether the transcription level of the G34 nucleic acid in the
biological sample is reduced due to the treatment. This assay is
not limited to a determination whether the transcription level is
reduced, and the result may also be evaluated as effective when an
increase in the transcription level is significantly prevented. The
transcription level may not only be compared with that of an
untreated tissue, but also traced over time after the
treatment.
[0163] The assay of the present invention for therapeutic effect on
cancer includes, for example, a determination whether a candidate
substance for an anticancer agent is effective for cancerous
tissues, whether resistance is developed to an anticancer agent in
cancer patients receiving the agent, or whether a candidate
substance for an anticancer agent is effective for diseased tissues
or the like in experimental animal models. Test tissues from
experimental animal models are not limited to in vitro samples, and
also include in vivo or ex vivo samples.
(8) Creation of Genetically Engineered Animal
[0164] As described earlier, the inventors of the present invention
have identified the presence of mouse G34 and its nucleic acid
sequence (SEQ ID NO: 3). The present invention also relates to a
means for expression and functional analysis of G34 at the animal
level on the basis of various gene conversion techniques using
fertilized eggs or ES cells, typically relates to creating
transgenic animals into which the G34 gene is introduced and
knockout mice which are deficient in mouse G34, etc.
[0165] For example, the creation of knockout mice may be
accomplished in accordance with routine techniques in the art (see,
e.g., Newest Technique for Gene Targeting, edited by Takeshi Yagi,
Yodosha Co., Ltd., Japan; Gene targeting, translated and edited by
Tetsuo Noda, Medical Science International, Ltd., Japan). Namely,
those skilled in the art will be able to obtain G34 homologous
recombinant ES cells in accordance with known gene targeting
techniques using sequence information of the mouse G34 nucleic acid
disclosed herein, thus creating G34 knockout mice using these cells
(see Example 7).
[0166] Recently, a method has been developed to prevent gene
expression by small interfering RNA (T. R. Brummelkamp et al.,
Science, 296, 550-553 (2002)); it is also possible to create G34
knockout mice in accordance with such a known method.
[0167] The provision of G34 knockout mice will be helpful in
elucidating the involvement of the G34 gene in certain vital
phenomena, i.e., information on redundancy of the gene, the
relationship between deficiency of the gene and phenotype at the
animal level (including any type of abnormality affecting motor,
mental and sensory functions), as well as functions of the gene
during the animal life cycle including development, growth and
ageing. More specifically, the knockout mice thus obtained may be
used to detect a carrier of sugar chains synthesized by G34 and
mG34 and to examine their relationship with physiological functions
or diseases, etc. For example, glycoproteins and glycolipids may be
extracted from each tissue derived from the knockout mice and
compared with those of wild-type mice by techniques such as
proteomics (e.g., two-dimensional electrophoresis, two-dimensional
thin-layer chromatography, mass spectrometry) to identify a carrier
of the synthesized sugar chains. Moreover, the relationship with
physiological functions or diseases may be estimated by comparing
phenotypes (e.g., fetal formation, growth process, spontaneous
behavior) between knockout mice and wild-type mice.
Definitions of Terms
[0168] As used herein to describe the transcription level of a
nucleic acid, the term "measured value" or "expression level"
refers to the amount of the nucleic acid present in transcription
products from a fixed amount of a biological sample, i.e., the
concentration of the nucleic acid. Moreover, since the assay of the
present invention relies on the comparison of such measured values,
even when a nucleic acid is amplified, e.g., by PCR for the purpose
of quantification or even when signals from a probe label are
amplified, these amplified values may also be provided for relative
comparison. Thus, the "measured value for a nucleic acid" can also
be understood as the amount of the nucleic acid after amplification
or the signal level after amplification.
[0169] As used herein, the term "target nucleic acid" or "the
nucleic acid" encompasses all types of nucleic acids, regardless of
in vivo or in vitro, including of course G34 mRNA, as well as those
obtained using the mRNA as a template. It should be noted that the
term "nucleotide sequence" used herein also includes a
complementary sequence thereof, unless otherwise specified.
[0170] As used herein, the term "biological sample" refers to an
organ, tissue or cell, as well as an experimental animal-derived
organ, tissue, cell or the like, preferably refers to a tissue or
cell. Examples of such a tissue include the brain, fetal brain,
cerebellum, medulla oblongata, submandibular gland, thyroid gland,
trachea, lung, heart, skeletal muscle, esophagus, duodenum-, small
intestine, large intestine (colon), rectum, colon, liver, fetal
liver, pancreas, kidney, adrenal gland, thymus, bone marrow,
spleen, testis, prostate, mammary gland, uterus and placenta, with
the large intestine (colon) and lung being more preferred.
[0171] As used herein, the term "measure", "measurement" or "assay"
encompasses all of detection, amplification, quantification and
semi-quantification. In particular, the assay according to the
present invention relates to a canceration assay for a biological
sample, as described above, and hence can be applied to, e.g.,
cancer diagnosis and treatment in the medical field. The term
"canceration assay" used herein includes an assay as to whether a
biological sample becomes cancer, as well as an assay as to whether
the grade of malignancy is high. The term "cancer" used herein
typically encompasses malignant tumors in general and also includes
disease conditions caused by the malignant tumors. Thus, targets of
the assay according to the present invention include, but are not
necessarily limited to, neuroblastoma, glioma, lung cancer,
esophageal cancer, gastric cancer, pancreatic cancer, liver cancer,
kidney cancer, duodenal cancer, small intestine cancer, large
intestine (colon) cancer, rectal cancer, colon cancer and leukemia,
with large intestine (colon) cancer and lung cancer being
preferred.
[0172] The present invention will now be illustrated in more detail
by way of the following examples.
EXAMPLES
Example 1
Cloning and Expression of Human G34 Gene, as Well as Purification
of the Expressed Protein
[0173] .beta.3 galactosyltransferase 6 (.beta.3GalT6) was used as a
query for a BLAST search to thereby find a nucleic acid sequence
with homology (SEQ ID NO: 1). The open reading frame (ORF)
estimated from the nucleic acid sequence is composed of 1503 bp,
i.e., 500 amino acids (SEQ ID NO: 2) when calculated as an amino
acid sequence. The product encoded by these nucleic acid and amino
acid sequences was designated human G34.
[0174] The amino acid sequence of G34 has a hydrophobic amino acid
region characteristic of glycosyltransferases at its N-terminal end
and shares a homology of 47% (nucleic acid sequence) and 28% (amino
acid sequence) with the above .beta.3GalT6. The amino acid sequence
of G34 also retains all of the three motifs conserved in the P3GalT
family.
[0175] In this example, G34 was not only confirmed for its
expression in mammalian cells, but also allowed to be expressed in
insect cells for further examination of its activity.
[0176] For activity confirmation, it would be sufficient to express
at least an active region covering amino acid 189 to the C-terminal
end of SEQ ID NO: 1, which is relatively homologous to
.beta.3GalT6. In this example, however, an active region covering
amino acid 36 to the C-terminal end was attempted to be
expressed.
Confirmation of Human G34 Gene Expression in Mammalian Cells
[0177] The active region covering amino acid 36 to the C-terminal
end of G34 was genetically introduced into a mammalian cell line
expression vector pFLAG-CMV3 using a FLAG Protein Expression system
(Sigma-Aldrich Corporation).
[0178] Since pFLAG-CMV3 has a multicloning site, a gene of interest
can be introduced into pFLAG-CMV3 when the gene and pFLAG-CMV3 are
treated with restriction enzymes and then subjected to ligation
reaction.
[0179] Kidney-derived cDNA (Clontech, Marathon-ready cDNA) was used
as a template and subjected to PCR using a 5'-primer (G34-CMV-F1;
SEQ ID NO: 5) and a 3'-primer (G34-CMV-R1; SEQ ID NO: 6) to obtain
a DNA fragment of interest. PCR was performed under conditions of
25 cycles of 98.degree. C. for 10 seconds, 55.degree. C. for 30
seconds, and 72.degree. C. for 2 minutes. The PCR product was then
electrophoresed on an agarose gel and isolated in a standard manner
after gel excision. This PCR product has restriction enzyme sites
HindIII and BamHI at the 5' and 3' sides, respectively.
[0180] After this DNA fragment and pFLAG-CMV3 were each treated
with restriction enzymes HindIII and BamHI, the reaction solutions
were mixed together and subjected to ligation reaction, so that the
DNA fragment was introduced into pFLAG-CMV3. The reaction solution
was purified by ethanol precipitation and then mixed with competent
cells (E. coli DH5.alpha.). After heat shock treatment (42.degree.
C., 30 seconds), the cells were seeded on ampicillin-containing LB
agar medium.
[0181] On the next day, the resulting colonies were confirmed by
direct PCR for the DNA of interest. For more reliable results,
after sequencing to confirm the DNA sequence, the vector
(pFLAG-CMV3-G34A) was extracted and purified.
[0182] Human kidney cell-derived cell line 293T cells
(2.times.10.sup.6) were suspended in 10 ml antibiotic-free DMEM
medium (Invitrogen Corporation) supplemented with 10% fetal bovine
serum, seeded in a 10 cm dish and cultured for 16 hours at
37.degree. C. in a CO.sub.2 incubator. pFLAG-CMV3-G34A (20 ng) and
Lipofectamin 2000 (30 .mu.l, Invitrogen Corporation) were each
mixed with 1.5 ml OPTI-MEM (Invitrogen Corporation) and incubated
at room temperature for 5 minutes. These two solutions were further
mixed gently and incubated at room temperature for 20 minutes. This
mixed solution was added dropwise to the dish and cultured for 48
hours at 37.degree. C. in a CO.sub.2 incubator.
[0183] The supernatant (10 ml) was mixed with NaN.sub.3 (0.05%),
NaCl (150 mM), CaCl.sub.2 (2 mM) and anti-FLAG-M1 resin (100 .mu.l,
SIGMA), followed by overnight stirring at 4.degree. C. On the next
day, the supernatant was centrifuged (3000 rpm, 5 minutes,
4.degree. C.) to collect a pellet fraction. After addition of 2 mM
CaCl.sub.2-TBS (900 .mu.l), centrifugation was repeated (2000 rpm,
5 minutes, 4.degree. C.) and the resulting pellet was suspended in
200 .mu.l of 1 mM CaCl.sub.2-TBS for use as a sample for activity
measurement (G34 enzyme solution). A part of this sample was
electrophoresed by SDS-PAGE and Western blotted using anti-FLAG
M2-peroxidase (SIGMA) to confirm the expression of the G34 protein
of interest.
[0184] As a result, a band was detected at a position of about 60
kDa, thus confirming the expression of the G34 protein.
Insertion of Human G34 Gene into Insect Cell Expression Vector
[0185] The active region covering amino acid 36 to the C-terminal
end of G34 was integrated into pFastBac (Invitrogen Corporation) in
a GATEWAY system (Invitrogen Corporation). Moreover, a Bac-to-Bac
system (Invitrogen Corporation) was also used to construct a
bacmid.
(1) Creation of entry clone
[0186] Kidney-derived cDNA (Clontech, Marathon-ready cDNA) was used
as a template and subjected to PCR using a 5'-primer (G34-GW-F1;
SEQ ID NO: 7) and a 3'-primer (G34-GW-R1; SEQ ID NO: 8) to obtain a
DNA fragment of interest. PCR was performed under conditions of
25-cycles of 98.degree. C. for 10 seconds, 55.degree. C. for 30
seconds, and 72.degree. C. for 2 minutes. The PCR product was then
electrophoresed on an agarose gel and isolated in a standard manner
after gel excision.
[0187] This product was integrated into pDONR201 (Invitrogen
Corporation) through BP clonase reaction to create an "entry
clone." The reaction was accomplished by incubating the DNA
fragment of interest (5 .mu.l), pDONR201 (1 .mu.l, 150 ng),
reaction buffer (2 .mu.l) and BP clonase mix (2 .mu.l) at
25.degree. C. for 1 hour. The reaction was stopped by addition of
proteinase K (1 .mu.l) and incubation at 37.degree. C. for 10
minutes. The above reaction solution (1 .mu.l) was then mixed with
100 .mu.l competent cells (E. coli DH5.alpha., TOYOBO). After heat
shock treatment, the cells were seeded in a kanamycin-containing LB
plate.
[0188] On the next day, colonies were collected and confirmed by
direct PCR for the DNA of interest. For more reliable results,
after sequencing to confirm the DNA sequence, the vector
(pDONR-G34A.) was extracted and purified.
(2) Creation of Expression Clone
[0189] At both sides of the insertion site, the above entry clone
has attL recombination sites for excision of lambda phage from E.
coli. When the entry clone is mixed with LR clonase (a mixture of
lambda phage recombination enzymes Int, IHF and Xis) and a
destination vector, the insertion site is transferred to the
destination vector to give an expression clone. Detailed steps are
as shown below.
[0190] First, the entry clone (1 .mu.l), pFBIF (0.5 .mu.l, 75 ng),
LR reaction buffer (2 .mu.l), TE (4.5 .mu.l) and LR clonase mix (2
.mu.l) were reacted at 25.degree. C. for 1 hour. The reaction was
stopped by addition of proteinase K (1 .mu.l) and incubation at
37.degree. C. for 10 minutes (this recombination reaction results
in pFBIF-G34A). pFBIF is a pFastBacl vector modified to have a IgK
signal sequence (SEQ ID NO: 9) and a FLAG peptide for purification
(SEQ ID NO: 10). The IgK signal sequence is inserted for the
purpose of converting the expressed protein into a secretion form,
while the FLAG peptide is inserted for the purpose of purification.
To insert the FLAG peptide, a DNA fragment obtained from OT3 (SEQ
ID NO: 11) as a template using primers OT20 (SEQ ID NO: 12) and
OT21 (SEQ ID NO: 13) was inserted with Bam H1 and Eco R1. Further,
to insert a Gateway sequence, a Gateway Vector Conversion system
(Invitrogen Corporation) was used to introduce a Conversion
cassette.
[0191] Subsequently, the whole volume of the above mixed solution
(11 .mu.l) was mixed with 100 .mu.l competent cells (E. coli
DH5.alpha.). After heat shock treatment, the cells were seeded in
an ampicillin-containing LB plate. On the next day, colonies were
collected and confirmed by direct PCR for the DNA of interest, and
the vector (pFBIF-G34A) was extracted and purified.
(3) Construction of Bacmid by Bac-to-Bac System
[0192] Next, a Bac-to-Bac system (Invitrogen Corporation) was used
to cause recombination between the above pFBIF- and pFastBac, so
that G34 and other sequences were inserted into a bacmid capable of
growing in insect cells.
[0193] This system utilizes a Tn7 recombination site and allows a
gene of interest to be incorporated into a bacmid through a
recombinant protein produced from a helper plasmid when pFastBac
carrying the inserted gene of interest is merely introduced into
bacmid-containing E. coli (DH10BAC, Invitrogen Corporation). In
addition, such a bacmid contains the lacZ gene and allows selection
based on the classical blue (not inserted)/white (inserted) colony
screening.
[0194] Namely, the vector purified above (pFB1H-G34A) was mixed
with 50 .mu.l competent cells (E. coli DH10BAC). After heat shock
treatment, the cells were seeded in a LB plate containing
kanamycin, gentamicin, tetracycline, Bluo-gal and IPTG. On the next
day, white single colonies were further cultured to collect the
bacmid.
Introduction of Human G34 Gene-Containing Bacmid into Insect
Cells
[0195] After confirming that the sequence of interest was inserted
into the bacmid obtained from the above white colonies, this bacmid
was introduced into insect cells (Sf21, commercially available from
Invitrogen Corporation).
[0196] Namely, Sf21 cells were added to a 35 mm dish at
9.times.10.sup.5 cells/2 ml antibiotic-containing Sf-900SFM
(Invitrogen Corporation) and cultured at 27.degree. C. for 1 hour
to allow cell adhesion. (Solution A) Purified bacmid DNA (5 .mu.l)
diluted with 100 .mu.l antibiotic-free Sf-900SFM. (Solution B)
CellFECTIN Reagent (6 .mu.l, Invitrogen Corporation) diluted with
100 .mu.l antibiotic-free Sf-900SFM. Solutions A and B were then
mixed carefully and incubated for 45 minutes at room temperature.
After confirming cell adhesion, the culture solution was aspirated
and replaced by antibiotic-free Sf-900SFM (2 ml). The solution
prepared by mixing Solutions A and B (lipid-DNA complexes) was
diluted and mixed carefully with antibiotic-free Sf900II (800
.mu.l). The culture solution was aspirated from the cells and
replaced by the diluted solution of lipid-DNA complexes, followed
by incubation at 27.degree. C. for 5 hours. The transfection
mixture was then removed and replaced by antibiotic-containing
Sf-900SFM culture solution (2 ml), followed by incubation at
27.degree. C. for 72 hours. At 72 hours after transfection, the
cells were released by pipetting and collected together with the
culture solution, followed by centrifugation at 3000 rpm for 10
minutes. The resulting supernatant was stored in another tube
(which was used as a first virus solution).
[0197] Sf21 cells were introduced into a T75 culture flask at
1.times.10.sup.7 cells/20 ml Sf-900SFM (antibiotic-containing) and
incubated at 27.degree. C. for 1 hour. After the cells were
adhered, the first virus (800 .mu.l) was added and cultured at
27.degree. C. for 48 hours. After 48 hours, the cells were released
by pipetting and collected together with the culture solution,
followed by centrifugation at 3000 rpm for 10 minutes. The
resulting supernatant was stored in another tube (which was used as
a second virus solution).
[0198] Moreover, Sf21 cells were introduced into a T75 culture
flask at 1.times.10.sup.7 cells/20 ml Sf-900SFM
(antibiotic-containing) and incubated at 27.degree. C. for 1 hour.
After the cells were adhered, the second virus solution (100 .mu.l)
was added and cultured at 27.degree. C. for 72 hours. After
culturing, the cells were released by pipetting and collected
together with the culture solution, followed by centrifugation at
3000 rpm for 10 minutes. The resulting supernatant was stored in
another tube (which was used as a third virus solution). In
addition, Sf21 cells were introduced into a 100 ml spinner flask at
a concentration of 6.times.10.sup.5 cells/ml in a volume of 100 ml.
The third virus solution (1 ml) was added and cultured at
27.degree. C. for about 96 hours. After culturing, the cells and
the culture solution were collected and centrifuged at 3000 rpm for
10 minutes. The resulting supernatant was stored in another tube
(which was used as a fourth virus solution).
Resin Purification of G34
[0199] The pFLAG-G34 supernatant of the above fourth virus solution
(10 ml) was mixed with NaN.sub.3 (0.05%), NaCl (150 mM), CaCl.sub.2
(2 mM) and anti-FLAG-M1 resin (100 .mu.l, SIGMA), followed by
overnight stirring at 4.degree. C. On the next day, the mixture was
centrifuged (3000 rpm, 5 minutes, 4.degree. C.) to collect a pellet
fraction. After addition of 2 mM CaCl.sub.2-TBS (900 .mu.l),
centrifugation was repeated (2000 rpm, 5 minutes, 4.degree. C.) and
the resulting pellet was suspended in 200 .mu.l of 1 mM
CaCl.sub.2-TBS for use as a sample for activity measurement (G34
enzyme solution). A part of this sample was electrophoresed by
SDS-PAGE and Western blotted using anti-FLAG M2-peroxidase (SIGMA)
to confirm the expression of the G34 protein of interest. As a
result, a plurality of bands were detected broadly around a
position of about 60 kDa (which would be due to differences in
post-translational modifications such as glycosylation), thus
confirming the expression of the G34 protein.
Example 2
Search for Glycosyltransferase Activity of Human
G34 Protein
(1) Screening of GalNAc Transferase Activity
[0200] The G34 protein was examined for its substrate specificity,
optimum buffer, optimum pH and divalent ion requirement in its
.beta.1,3-N-acetylgalactosaminyltransferase activity.
[0201] The following reaction system was used for examining the G34
enzyme protein for its acceptor substrate specificity in its GalNAc
transfer activity.
[0202] In the reaction solutions shown below, each of the following
was used at 10 nmol as an acceptor substrate: pNp-.alpha.-Gal,
oNp-.beta.-Gal, Bz-.alpha.-GlcNAc, pNp-.beta.-GlcNAc,
Bz-.alpha.-GalNAc, pNp-.beta.-GalNAc, pNp-.alpha.-Glc,
pNp-.beta.-Glc, pNp-.beta.-GlcA, pNp-.alpha.-Fuc, pNp-.alpha.-Xyl,
pNp-.beta.-Xyl and pNp-.alpha.-Man (all purchased from SIGMA),
wherein "Gal" represents a D-galactose residue, "Xyl" represents a
D-xylose residue, "Fuc" represents a D-fucose residue, "Man"
represents a D-mannose residue and "GlcA" represents a glucuronic
acid residue.
[0203] Each reaction solution was prepared as follows (final
concentrations in parentheses): each substrate (10 nmol), MES
(2-morpholinoethanesulfonic acid) (pH 6.5, 50 mM), MnCl.sub.2 (10
mM), Triton X-100 (trade name) (0.1%), UDP-GalNAc (2 mM) and
UDP-[.sup.14C]GlcNAc (40 nCi) were mixed and supplemented with 5
.mu.l G34 enzyme solution, followed by dilution with H.sub.2O to a
total volume of 20 .mu.l (see Table 1).
TABLE-US-00001 TABLE 1 Composition of reaction solutions (.mu.l)
E(+), D(+) X8 E(-), D(+) E(+), D(-) Enzyme solution 5 40 0 5 140 mM
HEPES 2 16 2 2 pH 7.4 100 mM UDP-GalNAc 0.5 4 0.5 0 200 mM
MnCl.sub.2 1 8 1 1 10% Triton CF-54 0.6 4.8 0.6 0.6 H.sub.2O 5.9
47.2 10.9 6.4 10 nmol/.mu.l Acceptor 5 40 5 5 Total 20 20 20
[0204] The above reaction mixtures were each reacted at 37.degree.
C. for 16 hours. After completion of the reaction, 200 .mu.l
H.sub.2O was added and each mixture was lightly centrifuged to
obtain the supernatant. The supernatant was passed through a
Sep-Pak plus C18 Cartridge (Waters), which had been washed once
with 1 ml methanol and twice with 1 ml H.sub.2O and then
equilibrated, to allow the substrate and product in the supernatant
to adsorb to the cartridge. After washing the cartridge twice with
1 ml H.sub.2O, the adsorbed substrate and product were eluted with
1 ml methanol. The eluate was mixed with 5 ml liquid scintillator
ACSII (Amersham Biosciences) and measured for the amount of
radiation with a scintillation counter (Beckman Coulter).
[0205] As a result, the G34 protein was identified to be GalNAc
transferase having the ability to transfer GalNAc to
pNp-.beta.-GlcNAc. The enzymatic activity was linearly increased at
least over the course of the reaction time between 0 and 16 hours
when UDP-GlcNAc was used as a donor substrate and Bz-.beta.-GlcNAc
was used as an acceptor substrate (see Table 2 and FIG. 1).
TABLE-US-00002 TABLE 2 Reaction time Area (%) 1 hour 0 2 hours
2.388 4 hours 6.195 16 hours 13.719
Determination of Linking Mode
[0206] NMR was performed to analyze the linking mode of the sugar
chain structure synthesized by the G34 enzyme protein.
[0207] First, the reaction solution (final concentrations in
parentheses) was prepared by adding Bz-.beta.-GlcNAc (640 nmol) as
an acceptor substrate, HEPES buffer (pH 7.4, 14 mM), Triton CF-54
(trade name) (0.3%), UDP-GalNAc (2 mM), MnCl.sub.2 (10 mM) and 500
.mu.l G34 enzyme solution, followed by dilution with H.sub.2O to a
total volume of 2 ml. This reaction solution was reacted at
37.degree. C. for 16 hours. The reaction solution was heated for 5
minutes at 95.degree. C. to stop the reaction and then purified by
filtration through an Ultrafree-MC (Millipore Corporation).
[0208] In one development, 50 .mu.l of the filtrate was analyzed by
high performance liquid chromatography (HPLC) using a
reversed-phase column ODS-80Ts QA (4.6.times.250 mm, Tosoh
Corporation, Japan). The developing solvent used was an aqueous 9%
acetonitrile-0.1% trifluoroacetic acid solution. The elution
conditions were set to 1 ml/minute at 40.degree. C. Absorbance at
210 nm was used as an index for elution peak detection using an
SPD-10A.sub.vp (Shimadzu Corporation, Japan). As a result, a new
elution peak was observed, which was not detected in the control.
This peak was separated and lyophilized for use as an NMR
sample.
[0209] NMR was performed using a DMX750 (Bruker Daltonics). As a
result, the sample was determined as having a .beta.1-3 linkage
between GalNAc and GlcNAc-.beta.1-o-Bz (see FIGS. 2A and 2B). The
reasons for this determination are as follows (see FIGS. 2A and 2B,
along with FIGS. 3 and 4): a) two residues (referred to as A and B)
both have a piston coupling constant of 8.4 Hz for the signal at
position 1, suggesting that two pyranoses are in .beta.-form; b)
the spin coupling constants given in FIG. 3 indicate that A shows a
spin coupling constant characteristic of glucose, while B shows a
spin coupling constant characteristic of galactose; c) it is A that
is linked to the benzyl because NOE was observed between methylene
proton of the benzyl and A1 proton; d) there are two signals
resulting from the methyl of N-acetyl and hence both residues are
identified as N-acetylated sugars; and e) NOESY indicates the
presence of NOE in B1-A3.
[0210] On the other hand, examination was also performed on motif
sequences involved in the above enzymatic activity.
[0211] FIG. 5 shows the putative amino acid sequence of the G34
protein (SEQ ID NO: 2) compared with the amino acid sequences of
various human .beta.1-3Gal transferases (.beta.3Gal-T1 to -T6). In
FIG. 5, the boxed regions indicate the motifs common to Gal
transferases. Among them, three motifs indicated with M1 to M3 are
common to .beta.1,3-linking glycosyltransferases. In this figure,
the amino acid residues indicated with * are conserved among the
compared sequences.
[0212] FIG. 6 shows a comparison of three motifs involved in the
ability to form .beta.1,3 linkages (corresponding to the M1 to M3
motifs in FIG. 5) among various .beta.1-3GlcNAc transferases
(.beta.3Gn-T2 to -T5) and human Gal transferases T1 to T3, T5 and
T6. In this figure, the amino acid residues indicated with * are
conserved among the compared sequences.
[0213] As shown in FIGS. 5 and 6, it was indicated that the amino
acid sequence of the G34 protein was conserved enough to have all
the motifs (M1 to M3) involved in .beta.1,3 linkages, upon
comparison with the amino acid sequences of known various
.beta.1,3-linking glycosyltransferases.
[0214] Thus, this motif examination also supported the conclusion
that the G34 protein has the ability to transfer GalNAc to GlcNAc
with .beta.1,3 glycosidic linkage.
Optimum Buffer and Optimum pH
[0215] The following reaction system was used for examining the
optimum buffer and pH for the GalNAc transferase activity of G34.
The acceptor substrate used was pNp-.beta.-GlcNAc.
[0216] Any one of the following buffers was used (final
concentrations in parentheses): MES (2-morpholinoethanesulfonic
acid) buffer (pH 5.5, 5.78, 6.0, 6.5 and 6.75, 50 mM), sodium
cacodylate buffer (pH 5.0, 5.6, 6.0, 6.2, 6.6, 6.8, 7.0, 7.2, 7.4
and 7.5, 25 mM) and
N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES)
buffer (pH 6.75, 7.00, 7.30, 7.40 and 7.50, 14 mM). The substrate
(10 nmol), MnCl.sub.2 (10 mM), Triton CF-54 (trade name) (0.3%),
UDP-GalNAc (2 mM) and UDP-[.sup.14C]GlcNAC (40 nCi) were mixed and
supplemented with 5 .mu.l G34 enzyme solution, followed by dilution
with H.sub.2O to a total volume of 20 .mu.l.
[0217] The above reaction mixtures were each reacted at 37.degree.
C. for 16 hours. After completion of the reaction, 200 .mu.l
H.sub.2O was added and each mixture was lightly centrifuged to
obtain the supernatant. The supernatant was passed through a
Sep-Pak plus C18 Cartridge (Waters), which had been washed once
with 1 ml methanol and twice with 1 ml H.sub.2O and then
equilibrated, to allow the substrate and product in the supernatant
to adsorb to the cartridge. After washing the cartridge twice with
1 ml H.sub.2O, the adsorbed substrate and product were eluted with
1 ml methanol. The eluate was mixed with 5 ml liquid scintillator
ACSII (Amersham Biosciences) and measured for the amount of
radiation with a scintillation counter (Beckman Coulter).
[0218] As indicated by the results (see Table 3 and FIG. 7), in MES
buffer, G34 showed the same strong activity around pH 5.50 and pH
5.78 within the examined range and its activity decreased in a
pH-dependent manner until pH 6.5, but became strong again at pH
6.75. In sodium cacodylate buffer, the activity was highest at pH
5.0 within the examined range and the activity decreased in a
pH-dependent manner until pH 6.2, increased in a pH-dependent
manner until pH 7.0, and then plateaued until pH 7.4. In HEPES
buffer, the activity increased in a pH-dependent manner and reached
the highest value at pH 7.4 to 7.5 within the examined range. Among
them, HEPES buffer at pH 7.4 to 7.5 resulted in the strongest
activity.
TABLE-US-00003 TABLE 3 PH + - Sodium cacodylate 5.0 6042 204 5838
5.6 3353 159 3194 6.0 2689 260 2429 6.2 907 138 769 6.6 1093 136
957 6.8 2488 258 2230 7.0 4965 259 4706 7.2 4377 309 4068 7.4 4930
304 4626 pH + - MES 5.50 3735 197 3538 5.78 3755 184 3571 6.00 2514
141 2373 6.50 1981 734 1247 6.75 3289 136 3153 pH + - HEPES 6.75
4894 149 4745 7.00 4912 121 4791 7.30 4294 127 4167 7.40 6630 120
6510 7.50 6895 240 6655
[0219] The following reaction system was used for examining the
divalent ion requirement. The acceptor substrate used was
Bz-.beta.-GlcNAc.
[0220] The reaction solution (final concentrations in parentheses)
was prepared by adding the substrate (10 nmol), HEPES buffer (pH
7.4, 14 mM), Triton CF-54 (trade name) (0.3%), UDP-GalNAc (2 mM),
UDP-[.sup.14C]GlcNAC (40 nCi) and 5 .mu.l G34 enzyme solution and
further adding MnCl.sub.2, MgCl.sub.2 or CoCl.sub.2 at 2.5 mM, 5
mM, 10 mM, 20 mM or 40 mM, followed by dilution with H.sub.2O to a
total volume of 20 .mu.l.
[0221] The above reaction mixture was reacted at 37.degree. C. for
16 hours. After completion of the reaction, 200 .mu.l H.sub.2O was
added and the mixture was lightly centrifuged to obtain the
supernatant. The supernatant was passed through a Sep-Pak plus C18
Cartridge (Waters), which had been washed once with 1 ml methanol
and twice with 1 ml H.sub.2O and then equilibrated, to allow the
substrate and product in the supernatant to adsorb to the
cartridge. After washing the cartridge twice with 1 ml H.sub.2O,
the adsorbed substrate and product were eluted with 1 ml methanol.
The eluate was mixed with 5 ml liquid scintillator ACSII (Amersham
Biosciences) and measured for the amount of radiation with a
scintillation counter (Beckman Coulter).
[0222] The results (see Table 4 and FIG. 8) indicated that the
activity was enhanced by the addition of each divalent ion and
confirmed that the G34 protein was an enzyme requiring divalent
ions. Its activity nearly plateaued at 5 nM or higher concentration
of Mn or Co and at 10 nM or higher concentration of Mg. Moreover,
the Mn-induced enhancement of the activity was completely
eliminated by addition of Cu.
TABLE-US-00004 TABLE 4 RI assay (divalent ion requirement) Metal
ion Concentration (mM) DPM Mn 2.5 7260.09 5 8270.23 10 7748.77 20
7515.86 40 4870.48 40 371.53 Co 2.5 10979.99 5 9503.91 10 10979.99
20 8070.47 40 7854.92 Mg 2.5 4800.03 5 8692.15 10 8980.56 20
6726.32 40 5592.88 none -- 2427.39 EDTA 20 149.32 Mn + Cu 10 + 10
239 none -- 155.64
Substrate Specificity to Oligosaccharides
[0223] The following reaction system was used for examining the
acceptor substrate specificity to oligosaccharides. The acceptor
substrates used were pNp-.alpha.-Gal, oNp-.beta.-Gal,
Bz-.alpha.-GlcNAc, Bz-.beta.-GlcNAc, Bz-.alpha.-GalNAc,
pNp-.beta.-GalNAc, pNp-.alpha.-Glc, pNp-.beta.-Glc,
pNp-.beta.-GlcA, pNp-.alpha.-Fuc, pNp-.alpha.-Xyl, pNp-.beta.-Xyl,
pNp-.alpha.-Man, lactoside-Bz, Lac-ceramide, Gal-ceramide,
paragloboside, globoside, Gal-.beta.1-4 GalNAc-.alpha.-pNp,
Gal-.beta.1-3-GlcNAc-.beta.-pNp, GlcNAc-.beta.1-4 GlcNAc-.beta.-Bz,
pNp-core1 (Gal-.beta.1-3 GalNAc-.alpha.-pNp), pNp-core2
(Gal-.beta.1-3 (GlcNAc-.beta.1-6) GalNAc-.alpha.-pNp), pNp-core3
(GlcNAc-.beta.1-3 GalNAc-.alpha.-pNp) and pNp-core6
(GlcNAc-.beta.1-6 GalNAc-.alpha.-pNp). "Lac" represents a D-lactose
residue.
[0224] Each reaction solution (final concentrations in parentheses)
was prepared by adding each substrate (50 nmol), HEPES buffer (pH
7.4, 14 mM), Triton CF-54 (trade name) (0.3%), UDP-GalNAc (2 mM),
MnCl.sub.2 (10 mM), UDP-[.sup.3H]GlcNAc and 5 .mu.l G34 enzyme
solution, followed by dilution with H.sub.2O to a total volume of
20 .mu.l.
[0225] The above reaction mixtures were each reacted at 37.degree.
C. for 2 hours. After completion of the reaction, 200 .mu.l
H.sub.2O was added and each mixture was lightly centrifuged to
obtain the supernatant. The supernatant was passed through a
Sep-Pak plus C18 Cartridge (Waters), which had been washed once
with 1 ml methanol and twice with 1 ml H.sub.2O and then
equilibrated, to allow the substrate and product in the supernatant
to adsorb to the cartridge. After washing the cartridge twice with
1 ml H.sub.2O, the adsorbed substrate and product were eluted with
1 ml methanol. The eluate was mixed with 5 ml liquid scintillator
ACSII (Amersham Biosciences) and measured for the amount of
radiation with a scintillation counter (Beckman Coulter).
[0226] The results thus measured were compared assuming that the
radioactivity obtained using Bz-.beta.-GlcNAc as a substrate was
set to 100% (see Table 5). When used as a substrate, pNp-core2
showed the largest increase in radioactivity. Bz-.beta.-GlcNAc,
GlcNAc-.beta.1-4-GlcNAc-.beta.-Bz, pNp-core6 and pNp-core3 also
showed increases in radioactivity in the order named. The other
substrates showed no increase in radioactivity.
TABLE-US-00005 TABLE 5 No. Acceptor substrate % 1 pNp-.alpha.-Gal
N.D. 2 oNp-.beta.-Gal N.D. 3 Bz-.alpha.-GlcNAc N.D. 4
Bz-.beta.-GlcNAc 100 5 Bz-.alpha.-GalNAc N.D. 6 pNp-.beta.-GalNAc
N.D. 7 pNp-.alpha.-Glc N.D. 8 pNp-.beta.-Glc N.D. 9 pNp-.beta.-GlcA
N.D. 10 pNp-.alpha.-Fuc N.D. 11 pNp-.alpha.-Xyl N.D. 12
pNp-.beta.-Xyl N.D. 13 pNp-.alpha.-Man N.D. 14 Lactoside-Bz N.D. 15
Lac-ceramide N.D. 16 Gal-ceramide N.D. 17 Paragloboside N.D. 18
Globoside N.D. 19 Gal.beta.1-4GalNAc-.alpha.-pNp N.D. 20
Gal.beta.1-3GlcNAc-.beta.-pNp N.D. 21
GlcNAc.beta.1-4GlcNAc-.beta.-Bz 29 22 core1-pNp N.D. 23 core2-pNp
185 24 core3-pNp 8 25 core6-pNp 19 N.D.: Not determined due to no
radioactivity core1: Gal-.beta.1-3-GalNAc-.alpha.-pNp core2:
Gal-.beta.1-3-(GlcNAc-.beta.1-6)GalNAc-.alpha.-pNp core3:
GlcNAc-.beta.1-3-GalNAc-.alpha.-pNp core6:
GlcNAc-.beta.1-6-GalNAc-.alpha.-pNp
(2) Confirmation of Activity by HPLC Analysis
[0227] Using uridine diphosphate-N-acetylgalactosamine (UDP-GalNAc;
Sigma-Aldrich Corporation) as a sugar residue donor substrate and
Bz-.beta.-GlcNAc as a sugar residue acceptor substrate, the
enzymatic activity of G34 was analyzed by high performance liquid
chromatography (HPLC).
[0228] The reaction solution (final concentrations in parentheses)
was prepared by adding Bz-.beta.-GlcNAc (10 nmol), HEPES buffer (pH
7.4, 14 mM), Triton CF-54 (trade name) (0.3%), UDP-GalNAc (2 mM),
MnCl.sub.2 (10 mM) and 10 .mu.l G34 enzyme solution, followed by
dilution with H.sub.2O to a total volume of 20 .mu.l. This reaction
solution was reacted at 37.degree. C. for 16 hours. The reaction
was stopped by addition of H.sub.2O (100 .mu.l) and the reaction
solution was purified by filtration through an Ultrafree-MC
(Millipore Corporation).
[0229] The filtrate (10 .mu.l) was analyzed by high performance
liquid chromatography (HPLC) using a reversed-phase column ODS-80Ts
QA (4.6.times.250 mm, Tosoh Corporation, Japan). The developing
solvent used was an aqueous 9% acetonitrile-0.1% trifluoroacetic
acid solution. The elution conditions were set to 1 ml/minute at
40.degree. C. Absorbance at 210 nm was used as an index for elution
peak detection using an SPD-10A.sub.vp (Shimadzu Corporation,
Japan).
[0230] As a result, a new elution peak was observed, which was not
detected in the control.
(3) Analysis of Reaction Product by Mass Spectrometry
[0231] The above peak was collected and the reaction product was
analyzed by mass spectrometry. Matrix-associated laser desorption
ionization-time of flight/mass spectrometry (MALDI-TOF-MS) was
performed using a Reflex IV (Bruker Daltonics). The sample at 10
.mu.mol was dried and dissolved in 1 .mu.l distilled water for use
as a MALDI-TOF-MS sample.
[0232] As a result, a peak at 538.194 m/z was observed.
[0233] This peak corresponded to the molecular weight of
GalNAc-GlcNAc-Bz (sodium salt).
[0234] This result also indicated that the G34 enzyme protein
transfers GalNAc to Bz-.beta.-GlcNAc.
Example 3
Measurement for mRNA Expression Level of Human G34
(1) Expression Levels in Various Human Normal Tissues
[0235] Quantitative real-time PCR was used for comparing the mRNA
expression levels of G34 in human normal tissues. Quantitative
real-time PCR is a PCR method using a sense primer and an antisense
primer in combination with a fluorescently-labeled probe. When a
gene is amplified by PCR, a fluorescent label of the probe will be
released to produce fluorescence. The fluorescence intensity is
amplified in correlation with gene amplification and thus used as
an index for quantification.
[0236] RNA of each human normal tissue (Clontech) was extracted
with an RNeasy Mini Kit (QIAGEN) and converted into single strand
DNA by the oligo(dT) method using a Super-Script First-Strand
Synthesis System (Invitrogen Corporation). This DNA was used as a
template and subjected to quantitative real-time PCR in an ABI
PRISM 7700 (Applied Biosystems Japan Ltd.) using a 5'-primer (SEQ
ID NO: 14), a 3'-primer (SEQ ID NO: 15) and a TaqMan probe (SEQ ID
NO: 16). PCR was performed under conditions of 50.degree. C. for 2
minutes and 95.degree. C. for 10 minutes, and then under conditions
of 50 cycles of 95.degree. C. for 15 seconds and 60.degree. C. for
1 minute. To prepare a calibration curve, plasmid DNA obtained by
introducing a partial sequence of G34 into pFLAG-CMV3 (Invitrogen
Corporation) was used as a template and subjected to PCR as
described above.
[0237] The results confirmed that high-level expression was
observed specifically in the testis, followed by skeletal muscle
and prostate in the order named (Table 6).
TABLE-US-00006 TABLE 6 G34 mRNA expression levels in human normal
tissues Copy number Tissue (.times.10000/.mu.g, total RNA) Standard
error Brain 5.0 1.1 Fetal brain 10.3 0.7 Cerebellum 2.8 0.3 Medulla
oblongata 4.9 0.3 Submandibular gland 6.7 0.4 Thyroid gland 1.8 0.6
Trachea 3.9 0.3 Lung 0.4 0.1 Heart 0.1 0.1 Skeletal muscle 25.8 1.1
Small intestine 5.1 0.3 Large intestine (colon) 0.6 0.3 Liver 0.3
0.1 Fetal liver 0.7 0.3 Pancreas 4.2 1.1 Kidney 1.6 0.3 Adrenal
gland 10.8 1.3 Thymus 4.8 0.2 Bone marrow 3.1 0.4 Spleen 4.2 0.3
Testis 115.5 2.0 Prostate 14.6 1.5 Mammary gland 5.2 0.2 Uterus 5.0
0.2 Placenta 1.4 0.4
(2) Expression Levels in Human Cancer Cell Lines
[0238] Quantitative real-time PCR as mentioned above was used for
comparing the mRNA expression levels of G34 in various
cancer-derived human cell lines. After cells of each human cell
line were collected, RNA was extracted with an RNeasy Mini Kit
(QIAGEN) and converted into single strand DNA by the oligo(dT)
method using a Super-Script First-Strand Synthesis System
(Invitrogen Corporation). This DNA was used as a template and
subjected to quantitative real-time PCR in an ABI PRISM 7700
(Applied Biosystems Japan Ltd.) using a 5'-primer (SEQ ID NO: 14),
a 3'-primer (SEQ ID NO: 15) and a TaqMan probe (SEQ ID NO: 16). PCR
was performed under conditions of 50.degree. C. for 2 minutes and
95.degree. C. for 10 minutes, and then under conditions of 50
cycles of 95.degree. C. for 15 seconds and 60.degree. C. for 1
minute.
[0239] As a result, the expression was observed in all the human
cell lines (Table 7, FIG. 9).
TABLE-US-00007 TABLE 7 G34 mRNA expression levels in human cell
lines Copy Copy number number Cell (.times.10.sup.4/.mu.g,
(.times.10.sup.4/.mu.g, line total RNA) Cell line total RNA) Neuro-
SCCH-26 7.9 0.6 Esophageal ES1 23.0 2.5 blastoma NAGAI 19.5 1.5
cancer ES2 16.1 0.6 NB-9 40.6 2.3 ES6 42.8 3.0 SK-N-SH 14.9 0.7
Gastric MKN1 6.2 1.1 SK-N-MC 5.8 0.5 cancer MKN28 8.6 1.0 NB-1 20.9
0.5 MKN7 9.7 0.1 IMR32 21.0 0.2 MKN74 3.5 0.8 Glioma T98G 6.2 0.2
MKN-45 7.3 2.1 YKG-1 3.9 0.0 HSC-43 42.8 1.7 A172 13.4 0.9 KATOIII
6.4 0.4 GI-1 13.7 1.3 TMK-1 10.8 1.2 U118MG 6.8 0.5 Large LSC 11.8
0.6 U251 28.9 1.9 intestine LSB 4.9 0.3 KG-1-C 9.1 0.6 (colon)
SW480 10.1 0.4 Lung cancer Lu130 6.8 0.4 cancer SW1116 24.1 1.4
Lu134A 30.3 1.2 Colo201 10.4 0.4 Lu134B 6.8 0.4 Colo205 6.8 0.9
Lu135 7.2 1.3 C1 21.9 1.2 Lu139 10.7 0.5 WiDr 1.2 0.0 Lu140 15.4
1.8 HCT8 82.2 6.2 SBC-1 2.5 0.2 HCT15 12.1 1.0 PC-7 9.1 0.2 Others
A204 67.9 4.4 PC-9 22.4 0.1 A-431 30.6 2.5 HAL-8 15.2 1.2 SW1736
11.9 1.1 HAL-24 20.8 1.7 HepG2 2.3 0.3 ABC-1 10.3 0.9 Capan-2 19.4
1.2 RERF-LC- 22.8 2.2 293T 55.1 8.3 MC PA-1 3.5 0.6 EHHA-9 20.3 7.9
Leukemia HL-60 2.1 0.1 PC-1 2.1 0.2 K-562 17.1 1.8 EBC-1 4.4 0.2
Lymphoma Daudi 2.4 0.2 PC-10 118.8 4.9 Namalwa 13.0 1.2 A549 27.1
2.6 KHM-IB 16.4 0.4 LX-1 30.7 2.1 Ramos 9.5 0.7 Raji 11.6 1.3
Jurkat 42.7 1.9
(3) Expression Levels in Cancerous Tissues
[0240] Quantitative real-time PCR as mentioned above was used for
comparing the mRNA expression levels of G34 in cancer tissues and
their surrounding normal tissues derived from patients with large
intestine (colon) cancer and lung cancer.
[0241] From cancer and normal tissues of the same patient, RNA was
extracted with an RNeasy Mini Kit (QIAGEN) and converted into
single strand DNA by the oligo(dT) method using a Super-Script
First-Strand Synthesis System (Invitrogen Corporation). This DNA
was used as a template and subjected to quantitative real-time PCR
in an ABI PRISM 7700 (Applied Biosystems Japan Ltd.) using a
5'-primer (SEQ ID NO: 14), a 3'-primer (SEQ ID NO: 15) and a TaqMan
probe (SEQ ID NO: 16). PCR was performed under conditions of 50
cycles of 50.degree. C. for 2 minutes, 95.degree. C. for 10
minutes, 95.degree. C. for 15 seconds, and 60.degree. C. for 1
minute. To correct variations among individuals, the resulting data
were divided by the value of .beta.-actin (internal standard gene)
quantified using a kit of Applied Biosystems Japan before being
compared.
[0242] The results indicated that the mRNA expression level of the
G34 gene was significantly increased in these cancerous tissues
(Table 8, Table 9).
TABLE-US-00008 TABLE 8 G34 mRNA expression levels in tissues from
large intestine cancer patients Patient Normal Standard Cancer
Standard No. tissue error tissue error % Change 1 0.15 0.04 0.35
0.07 2.3 2 0.15 0.07 8.63 0.65 58.0 3 0.07 0.02 1.55 0.15 23.5 4
0.08 0.05 1.82 0.26 22.0 5 0.08 0.02 0.60 0.07 7.2 6 1.04 0.08 1.92
0.21 1.8 7 0.07 0.02 5.37 1.06 81.3 8 1.54 0.27 8.30 0.96 5.4 9
0.05 0.04 1.70 0.37 34.3 10 0.05 0.04 0.10 0.04 2.0 11 0.60 0.29
10.23 1.47 17.2 12 0.17 0.13 2.36 0.43 14.3 13 0.18 0.09 1.70 0.27
9.4 14 0.18 0.08 2.76 0.23 15.2 15 0.18 0.05 3.49 0.34 19.2 16 0.20
0.15 1.84 0.25 9.3 17 0.28 0.05 7.41 0.51 26.4 18 0.05 0.04 5.92
0.38 119.3 19 0.15 0.11 4.68 0.67 31.4 20 0.13 0.06 4.61 2.22 34.9
21 0.02 0.02 8.40 1.65 508.0 22 0.20 0.07 3.57 0.43 18.0 23 0.55
0.27 2.33 1.23 4.3 Average 0.25 0.07 3.97 0.55 15.6 Copy number
(.times.10000/.mu.g, total RNA)
TABLE-US-00009 TABLE 9 G34 mRNA expression levels in tissues from
lung cancer patients Patient Normal Standard Cancer Standard No.
tissue error tissue error % Change 1 0.48 0.06 2.03 0.27 4.2 3 0.00
0.00 0.55 0.21 -- 4 2.43 0.40 6.13 0.17 2.5 5 0.10 0.04 2.74 0.32
27.7 6 1.69 0.28 3.11 0.69 1.8 7 0.60 0.16 2.76 0.35 4.6 8 2.30
0.38 6.23 0.21 2.7 9 1.26 0.27 2.51 0.10 2.0 10 1.47 0.18 4.76 0.57
3.2 11 0.64 0.00 1.14 0.11 1.8 12 0.56 0.06 0.69 0.04 1.2 13 1.32
0.02 1.98 0.15 1.5 14 0.17 0.02 0.66 0.02 4.0 15 0.71 0.05 2.71
0.13 3.8 16 1.07 0.13 15.64 1.11 14.6 17 1.03 0.12 8.27 0.73 8.1 18
0.13 0.02 1.95 0.09 14.8 Average 0.94 0.71 3.76 3.64 4.0 Copy
number (.times.10000/.mu.g, total RNA)
Example 4
Cloning and Expression of Mouse G34 Gene
[0243] The human G34 sequence obtained in Example 1 was used as a
query for a search against the mouse gene sequence serela (Applied
Biosystems) to thereby find a corresponding nucleic acid sequence
with high homology. The open reading frame (ORF) estimated from
this nucleic acid sequence is composed of 1515 bp (SEQ ID NO: 3),
i.e., 504 amino acids (SEQ ID NO: 4) when calculated as an amino
acid sequence, and has a hydrophobic amino acid region
characteristic of glycosyltransferases at its N-terminal end. This
sequence shares a homology of 86% (nucleic acid sequence) and 88%
(amino acid sequence) with human G34 (SEQ ID NOs: 1 and 2) (see
FIG. 10). Moreover, the sequence retains all of the three motifs
conserved in the .beta.3GalT family. The product encoded by the
nucleic acid sequence of SEQ ID NO: 3 and the amino acid sequence
of SEQ ID NO: 4 was designated mouse G34 (mG34).
[0244] To examine the activity of mG34, G34 was allowed to be
expressed in a mammalian cell line. In this example, the active
region covering amino acid 35 to the C-terminal end of mG34 was
genetically introduced into a mammalian cell line expression vector
pFLAG-CMV3 using a FLAG Protein Expression system (Sigma-Aldrich
Corporation).
[0245] The expression in mouse tissues was confirmed by PCR. Each
mouse tissue (brain., thymus, stomach, small intestine, large
intestine (colon), liver, pancreas, spleen, kidney, testis or
skeletal muscle) was used as a template and subjected to PCR using
a 5'-primer (mG34-CMV-F1; SEQ ID NO: 17) and a 3'-primer
(mG34-CMV-R1; SEQ ID NO: 18). PCR was performed under conditions of
25 cycles of 98.degree. C. for 10 seconds, 55.degree. C. for 30
seconds, and 72.degree. C. for 2 minutes. The PCR product was
electrophoresed on an agarose gel to confirm a band of
approximately 1500 bp. As a result, as shown in Table 10, the
expression level was highest in the testis, followed by spleen and
skeletal muscle in the order named.
TABLE-US-00010 TABLE 10 mG34 mRNA expression levels in mouse
tissues Tissue Expression level Brain .+-. Thymus - Stomach + Small
intestine - Large intestine + (colon) Liver + Pancreas - Spleen -
Kidney ++ Testis +++ Skeletal muscle ++
[0246] Mouse testis-derived cDNA was used as a template and
subjected to PCR using a 5'-primer (mG34-CMV-F1; SEQ ID NO: 17) and
a 3'-primer (mG34-CMV-R1; SEQ ID NO: 18) to obtain a DNA fragment
of interest. PCR was performed under conditions of 25 cycles of
98.degree. C. for 10 seconds, 55.degree. C. for 30 seconds, and
72.degree. C. for 2 minutes. The PCR product was then
electrophoresed on an agarose gel and isolated in a standard manner
after gel excision. This PCR product has restriction enzyme sites
HindIII and NotI at the 5' and 3' sides, respectively.
[0247] After this DNA fragment and pFLAG-CMV3 were each treated
with restriction enzymes HindIII and NotI, the reaction solutions
were mixed together and subjected to ligation reaction, so that the
DNA fragment was introduced into pFLAG-CMV3. The reaction solution
was purified by ethanol precipitation and then mixed with competent
cells (E. coli DH5.alpha.). After heat shock treatment (42.degree.
C., 30 seconds), the cells were seeded on ampicillin-containing LB
agar medium.
[0248] On the next day, the resulting colonies were confirmed by
direct PCR for the DNA of interest. For more reliable results,
after sequencing to confirm the DNA sequence, the vector
(pFLAG-CMV3-mG34A) was extracted and purified.
[0249] Human kidney cell-derived cell line 293T cells
(2.times.10.sup.6) were suspended in 10 ml antibiotic-free DMEM
medium (Invitrogen Corporation) supplemented with 10% fetal bovine
serum, seeded in a 10 cm dish and cultured for 16 hours at
37.degree. C. in a CO.sub.2 incubator. pFLAG-CMV3-mG34A (20 ng) and
Lipofectamin 2000 (30 .mu.l, Invitrogen Corporation) were each
mixed with 1.5 ml OPTI-MEM (Invitrogen Corporation) and incubated
at room temperature for 5 minutes. These two solutions were further
mixed gently and incubated at room temperature for 20 minutes. This
mixed solution was added dropwise to the dish and cultured for 48
hours at 37.degree. C. in a CO.sub.2 incubator.
[0250] The supernatant (10 ml) was mixed with NaN.sub.3 (0.05%),
NaCl (150 mM), CaCl.sub.2 (2 mM) and anti-M1 resin (100 .mu.l,
SIGMA), followed by overnight stirring at 4.degree. C. On the next
day, the supernatant was centrifuged (3000 rpm, 5 minutes,
4.degree. C.) to collect a pellet fraction. After addition of 2 mM
CaCl.sub.2-TBS (900 .mu.l), centrifugation was repeated (2000 rpm,
5 minutes, 4.degree. C.) and the resulting pellet was suspended in
200 .mu.l of 1 mM CaCl.sub.2-TBS for use as a sample for activity
measurement (mouse G34 enzyme solution). A part of this sample was
electrophoresed by SDS-PAGE and Western blotted using anti-FLAG
M2-peroxidase (SIGMA) to confirm the expression of the mG34 protein
of interest. As a result, a band was detected at a position of
about 60 kDa, thus confirming the expression of the mG34
protein.
Example 5
Search for Glycosyltransferase Activity of Mouse G34
[0251] The following reaction system was used for examining mouse
G34 for its substrate specificity in its
.beta.1,3-N-acetylgalactosamine transferase activity. In the
reaction solutions shown below, each of the following was used at
10 nmol as an "acceptor substrate": pNp-.alpha.-Gal,
oNp-.beta.-Gal, Bz-.alpha.-GlcNAc, Bz-.beta.-GlcNAc,
Bz-.alpha.-GalNAc, pNp-.beta.-GalNAc, pNp-.alpha.-Glc,
pNp-.beta.-Glc, pNp-.beta.-GlcA, pNp-.alpha.-Fuc, pNp-.alpha.-Xyl,
pNp-.beta.-Xyl, pNp-.alpha.-Man, lactoside-Bz, Lac-ceramide,
Gal-ceramide, Gb3, globoside, Gal-.beta.1-4GalNAc-.alpha.-pNp,
Gal.beta.1-3GlcNAc-.beta.-Bz, GlcNAc-.beta.1-4-GlcNAc-.beta.-Bz,
core1-pNp, core2-pNp, core3-pNp and core6-pNp (all purchased from
SIGMA).
[0252] Each reaction solution was prepared as follows (final
concentrations in parentheses): each substrate (10 nmol), HEPES
(N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]) (pH 7.4,
14 mM), MnCl.sub.2 (10 mM), Triton CF-54 (trade name) (0.3%),
UDP-GalNAc (2 mM) and UDP-[.sup.14C]GlcNAC (40 nCi) were mixed and
supplemented with 5 .mu.l mouse G34 enzyme solution, followed by
dilution with H.sub.2O to a total volume of 20 .mu.l.
[0253] The above reaction mixtures were each reacted at 37.degree.
C. for 16 hours. After completion of the reaction, 200 .mu.l
H.sub.2O was added and each mixture was lightly centrifuged to
obtain the supernatant. The supernatant was passed through a
Sep-Pak plus C18 Cartridge (Waters), which had been washed once
with 1 ml methanol and twice with 1 ml H.sub.2O and then
equilibrated, to allow the substrate and product in the supernatant
to adsorb to the cartridge. After washing the cartridge twice with
1 ml H.sub.2O, the adsorbed substrate and product were eluted with
1 ml methanol. The eluate was mixed with 5 ml liquid scintillator
ACSII (Amersham Biosciences) and measured for the amount of
radiation with a scintillation counter (Beckman Coulter).
[0254] The results thus measured were compared assuming that the
radioactivity obtained using Bz-.beta.-GlcNAc as a substrate was
set to 100% (Table 11). When used as a substrate, Bz-.beta.-GlcNAc
showed the largest increase in radioactivity. core2-pNp, core6-pNp,
core3-pNp, pNp-.beta.-Glc and GlcNAc-.beta.1-4-GlcNAc-.beta.-Bz
also showed high radioactivity in the order named. The other
substrates showed no increase in radioactivity.
TABLE-US-00011 TABLE 11 Acceptor substrate % pNp-.alpha.-Gal ND
oNp-.beta.-Gal ND Bz-.alpha.-GlcNAc ND Bz-.beta.-GlcNAc 100
Bz-.alpha.-GalNAc ND pNp-.beta.-GalNAc ND pNp-.alpha.-Glc ND
pNp-.beta.-Glc 12 pNp-.beta.-GlcA ND pNp-.alpha.-Fuc ND
pNp-.alpha.-Xyl ND pNp-.beta.-Xyl ND pNp-.alpha.-Man ND
Lactoside-Bz ND Lac-ceramide ND Gal-ceramide ND Gb3 ND Globoside ND
Gal.beta.1-4GalNAc-.alpha.-pNp ND Gal.beta.1-3GlcNAc-.beta.-pNp ND
GlcNAc.beta.1-4GlcNAc-.beta.-Bz 10 core1-pNp ND core2-pNp 25
core3-pNp 14 core6-pNp 18
Example 6
In Situ Hybridization on Mouse Testis
[0255] In situ hybridization using mG34 was performed on a mouse
testis-derived sample to confirm the expression of mG34 in the
mouse testis sample (see FIG. 11).
Example 7
Creation of G34 Knockout Mouse
[0256] A targeting vector (pBSK-mG34-KOneo) is constructed in which
pBluescript II SK(-) (TOYOBO) is inserted with a chromosomal
fragment (about 10 kb) primarily composed of an approximately 10 kb
fragment covering exons (i.e., Exons 3 to 12 (1242 bp) within the
ORF region of mG34) containing activation domains of the gene
(mG34) to be knocked out. pBSK-mG34-KOneo is also designed to have
the drug resistance gene neo (neomycin resistance gene) introduced
into Exons 7 to 9 which are putative GalNAc transferase active
regions of mG34. As a result, Exons 7 to 9 of mG34 are deleted and
replaced by neo. The pBSK-mG34-KOneo thus obtained is linearized
with a restriction enzyme NotI, 80 .mu.g of which is then
transfected (e.g., by electroporation) into ES cells (derived from
E14/129Sv mice) to select G418-resistant colonies. The
G418-resistant colonies are transferred to 24-well plates and then
cultured. After a part of the cells are frozen and stored, DNA is
extracted from the remaining ES cells and around 120 colonies of
recombinant clones are selected by PCR. Further, Southern blotting
or other techniques are performed to confirm whether recombination
occurs as expected, finally selecting around 10 clones of
recombinants. ES cells from two of the selected clones are injected
into C57BL/6 mouse blastocysts. The mouse embryos injected with the
ES cells are transplanted into the uteri of recipient mice to
generate chimeric mice, followed by germline transmission to obtain
heterozygous knockout mice.
Sequence CWU 1
1
2711503DNAHomo sapiens 1atgcgaaact ggctggtgct gctgtgcccg tgtgtgctcg
gggccgcgct gcacctctgg 60ctgcggctgc gctccccgcc gcccgcctgc gcctccgggg
ccggccctgc agatcagttg 120gccttatttc ctcagtggaa atctactcac
tatgatgtgg tagttggcgt gttgtcagct 180cgcaataacc atgaacttcg
aaacgtgata agaagcacct ggatgagaca tttgctacag 240catcccacat
taagtcaacg tgtgcttgtg aagttcataa taggtgctca tggctgtgaa
300gtgcctgtgg aagacaggga agatccttat tcctgtaaac tactcaacat
cacaaatcca 360gttttgaatc aggaaattga agcgttcagt ctgtccgaag
acacttcatc ggggctgcct 420gaggatcgag ttgtcagcgt gagtttccga
gttctctacc ccatcgttat taccagtctt 480ggagtgttct acgatgccaa
tgatgtgggt ttccagagga acatcactgt caaactttat 540caggcagaac
aagaggaggc cctcttcatt gctcgcttca gtcctccaag ctgtggtgtg
600caggtgaaca agctgtggta caagcccgtg gaacaattca tcttaccaga
gagctttgaa 660ggtacaatcg tgtgggagag ccaagacctc cacggccttg
tgtcaagaaa tctccacaaa 720gtgacagtga atgatggagg gggagttctc
agagtcatta cagctgggga gggtgcattg 780cctcatgaat tcttggaagg
tgtggaggga gttgcaggtg gttttatata tactattcag 840gaaggtgatg
ctctcttaca caaccttcat tctcgccctc aaagacttat tgatcatata
900aggaatctcc atgaggaaga tgccttactg aaggaggaaa gcagcatcta
tgatgatatt 960gtttttgtgg atgttgtcga cacttatcgt aatgttcctg
caaaattatt gaacttctat 1020agatggactg tggaaacaac gagcttcaat
ttgttgctga agacagatga tgactgttac 1080atagacctcg aagctgtatt
taataggatt gtccaaaaga atctggatgg gcctaatttt 1140tggtggggaa
atttcagact gaattgggca gttgaccgaa ccggaaagtg gcaggagttg
1200gagtacccga gccccgctta ccctgccttt gcatgtgggt caggatatgt
gatctccaag 1260gacatcgtca agtggctggc aagcaactcg gggaggttaa
agacctatca gggtgaagat 1320gtaagcatgg gcatctggat ggctgccata
ggacctaaaa gataccagga cagtctgtgg 1380ctgtgtgaga agacctgtga
gacaggaatg ctgtcttctc ctcagtattc tccgtgggaa 1440ctgacggaac
tgtggaaact gaaggaacgg tgcggtgatc cttgtcgatg tcaagcaaga 1500taa
15032500PRTHomo sapiens 2Met Arg Asn Trp Leu Val Leu Leu Cys Pro
Cys Val Leu Gly Ala Ala1 5 10 15Leu His Leu Trp Leu Arg Leu Arg Ser
Pro Pro Pro Ala Cys Ala Ser 20 25 30Gly Ala Gly Pro Ala Asp Gln Leu
Ala Leu Phe Pro Gln Trp Lys Ser 35 40 45Thr His Tyr Asp Val Val Val
Gly Val Leu Ser Ala Arg Asn Asn His 50 55 60Glu Leu Arg Asn Val Ile
Arg Ser Thr Trp Met Arg His Leu Leu Gln65 70 75 80His Pro Thr Leu
Ser Gln Arg Val Leu Val Lys Phe Ile Ile Gly Ala 85 90 95His Gly Cys
Glu Val Pro Val Glu Asp Arg Glu Asp Pro Tyr Ser Cys 100 105 110Lys
Leu Leu Asn Ile Thr Asn Pro Val Leu Asn Gln Glu Ile Glu Ala 115 120
125Phe Ser Leu Ser Glu Asp Thr Ser Ser Gly Leu Pro Glu Asp Arg Val
130 135 140Val Ser Val Ser Phe Arg Val Leu Tyr Pro Ile Val Ile Thr
Ser Leu145 150 155 160Gly Val Phe Tyr Asp Ala Asn Asp Val Gly Phe
Gln Arg Asn Ile Thr 165 170 175Val Lys Leu Tyr Gln Ala Glu Gln Glu
Glu Ala Leu Phe Ile Ala Arg 180 185 190Phe Ser Pro Pro Ser Cys Gly
Val Gln Val Asn Lys Leu Trp Tyr Lys 195 200 205Pro Val Glu Gln Phe
Ile Leu Pro Glu Ser Phe Glu Gly Thr Ile Val 210 215 220Trp Glu Ser
Gln Asp Leu His Gly Leu Val Ser Arg Asn Leu His Lys225 230 235
240Val Thr Val Asn Asp Gly Gly Gly Val Leu Arg Val Ile Thr Ala Gly
245 250 255Glu Gly Ala Leu Pro His Glu Phe Leu Glu Gly Val Glu Gly
Val Ala 260 265 270Gly Gly Phe Ile Tyr Thr Ile Gln Glu Gly Asp Ala
Leu Leu His Asn 275 280 285Leu His Ser Arg Pro Gln Arg Leu Ile Asp
His Ile Arg Asn Leu His 290 295 300Glu Glu Asp Ala Leu Leu Lys Glu
Glu Ser Ser Ile Tyr Asp Asp Ile305 310 315 320Val Phe Val Asp Val
Val Asp Thr Tyr Arg Asn Val Pro Ala Lys Leu 325 330 335Leu Asn Phe
Tyr Arg Trp Thr Val Glu Thr Thr Ser Phe Asn Leu Leu 340 345 350Leu
Lys Thr Asp Asp Asp Cys Tyr Ile Asp Leu Glu Ala Val Phe Asn 355 360
365Arg Ile Val Gln Lys Asn Leu Asp Gly Pro Asn Phe Trp Trp Gly Asn
370 375 380Phe Arg Leu Asn Trp Ala Val Asp Arg Thr Gly Lys Trp Gln
Glu Leu385 390 395 400Glu Tyr Pro Ser Pro Ala Tyr Pro Ala Phe Ala
Cys Gly Ser Gly Tyr 405 410 415Val Ile Ser Lys Asp Ile Val Lys Trp
Leu Ala Ser Asn Ser Gly Arg 420 425 430Leu Lys Thr Tyr Gln Gly Glu
Asp Val Ser Met Gly Ile Trp Met Ala 435 440 445Ala Ile Gly Pro Lys
Arg Tyr Gln Asp Ser Leu Trp Leu Cys Glu Lys 450 455 460Thr Cys Glu
Thr Gly Met Leu Ser Ser Pro Gln Tyr Ser Pro Trp Glu465 470 475
480Leu Thr Glu Leu Trp Lys Leu Lys Glu Arg Cys Gly Asp Pro Cys Arg
485 490 495Cys Gln Ala Arg 50031515DNAMouse 3atgcgaaact ggctggtgct
gctgtgccct tgcgtgctcg gggccgcgct gcacctctgg 60cacctctggc tccgttcccc
gccggacccc cacaacaccg ggcccagcgc ggcagatcaa 120tcagccttat
ttcctcactg gaaatttagc cactatgatg tggtagttgg tgtgttatca
180gctcgaaata accacgaact tcgaaatgtg ataaggaaca cctggctgaa
gaatttgctg 240catcatccta cattaagtca acgtgtgctt gtgaagttca
taataggtgc ccgtggctgt 300gaagtgcctg tggaagacag ggaggatcct
tactcctgcc gactgctcaa catcaccaat 360ccagttttga atcaagaaat
tgaggcattc agctttcctg aagatgcctc ctcatctaga 420ctctctgaag
accgagttgt cagcgtgagc ttcagagttc tctacccaat cgtgattacc
480agtcttggag tgttctacga tgccagtgat gttggttttc aaaggaacat
cacagtcaag 540ttgtatcaga cagagcagga ggaggccctt ttcatcgccc
gattcagtcc tccaagttgt 600ggcgtacaag tgaacaagct ctggtataag
cccgtggaac agttcatctt accagagagc 660tttgaaggta caatcgtgtg
ggaaagccaa gatctccatg gcctcgtgtc cagaaacctg 720cacagagtga
cagtgaatga tggagggggt gttctcagag tccttgcagc tggggaaggg
780gcactgcctc atgaattcat ggaaggtgtg gagggagttg cgggtggctt
tatctacact 840gttcaggaag gtgatgcact attaagaagc ctttattctc
ggccccagag acttgcagat 900cacatacagg atctgcaggt ggaagatgcc
ttactgcagg aggaaagcag tgtccatgac 960gacattgtct tcgtggatgt
tgtggatact taccggaatg ttcctgcaaa attactgaac 1020ttctatagat
ggactgtgga atccaccagc ttcgatttgc tgctcaagac agatgacgac
1080tgttatatag acttagaagc tgtgtttaat agaattgctc agaagaatct
agatgggcct 1140aatttttggt ggggaaattt caggttgaat tgggcagtgg
acagaaccgg aaaatggcag 1200gagctggaat acccgagccc ggcttaccct
gcctttgcat gtgggtcagg gtatgtgatc 1260tccaaggata tcgttgactg
gctggcaggc aactccagaa ggttaaagac ctatcagggt 1320gaagatgtca
gcatgggcat ttggatggca gccataggac ctaaaagaca ccaggacagc
1380ctgtggctgt gtgagaaaac ctgtgagaca ggaatgctgt cttctcctca
gtactcacca 1440gaagagctga gcaaactctg ggaactgaag gagctgtgtg
gggatccttg tcagtgtgaa 1500gcaaaagtac gatga 15154504PRTMouse 4Met
Arg Asn Trp Leu Val Leu Leu Cys Pro Cys Val Leu Gly Ala Ala1 5 10
15Leu His Leu Trp His Leu Trp Leu Arg Ser Pro Pro Asp Pro His Asn
20 25 30Thr Gly Pro Ser Ala Ala Asp Gln Ser Ala Leu Phe Pro His Trp
Lys 35 40 45Phe Ser His Tyr Asp Val Val Val Gly Val Leu Ser Ala Arg
Asn Asn 50 55 60His Glu Leu Arg Asn Val Ile Arg Asn Thr Trp Leu Lys
Asn Leu Leu65 70 75 80His His Pro Thr Leu Ser Gln Arg Val Leu Val
Lys Phe Ile Ile Gly 85 90 95Ala Arg Gly Cys Glu Val Pro Val Glu Asp
Arg Glu Asp Pro Tyr Ser 100 105 110Cys Arg Leu Leu Asn Ile Thr Asn
Pro Val Leu Asn Gln Glu Ile Glu 115 120 125Ala Phe Ser Phe Pro Glu
Asp Ala Ser Ser Ser Arg Leu Ser Glu Asp 130 135 140Arg Val Val Ser
Val Ser Phe Arg Val Leu Tyr Pro Ile Val Ile Thr145 150 155 160Ser
Leu Gly Val Phe Tyr Asp Ala Ser Asp Val Gly Phe Gln Arg Asn 165 170
175Ile Thr Val Lys Leu Tyr Gln Thr Glu Gln Glu Glu Ala Leu Phe Ile
180 185 190Ala Arg Phe Ser Pro Pro Ser Cys Gly Val Gln Val Asn Lys
Leu Trp 195 200 205Tyr Lys Pro Val Glu Gln Phe Ile Leu Pro Glu Ser
Phe Glu Gly Thr 210 215 220Ile Val Trp Glu Ser Gln Asp Leu His Gly
Leu Val Ser Arg Asn Leu225 230 235 240His Arg Val Thr Val Asn Asp
Gly Gly Gly Val Leu Arg Val Leu Ala 245 250 255Ala Gly Glu Gly Ala
Leu Pro His Glu Phe Met Glu Gly Val Glu Gly 260 265 270Val Ala Gly
Gly Phe Ile Tyr Thr Val Gln Glu Gly Asp Ala Leu Leu 275 280 285Arg
Ser Leu Tyr Ser Arg Pro Gln Arg Leu Ala Asp His Ile Gln Asp 290 295
300Leu Gln Val Glu Asp Ala Leu Leu Gln Glu Glu Ser Ser Val His
Asp305 310 315 320Asp Ile Val Phe Val Asp Val Val Asp Thr Tyr Arg
Asn Val Pro Ala 325 330 335Lys Leu Leu Asn Phe Tyr Arg Trp Thr Val
Glu Ser Thr Ser Phe Asp 340 345 350Leu Leu Leu Lys Thr Asp Asp Asp
Cys Tyr Ile Asp Leu Glu Ala Val 355 360 365Phe Asn Arg Ile Ala Gln
Lys Asn Leu Asp Gly Pro Asn Phe Trp Trp 370 375 380Gly Asn Phe Arg
Leu Asn Trp Ala Val Asp Arg Thr Gly Lys Trp Gln385 390 395 400Glu
Leu Glu Tyr Pro Ser Pro Ala Tyr Pro Ala Phe Ala Cys Gly Ser 405 410
415Gly Tyr Val Ile Ser Lys Asp Ile Val Asp Trp Leu Ala Gly Asn Ser
420 425 430Arg Arg Leu Lys Thr Tyr Gln Gly Glu Asp Val Ser Met Gly
Ile Trp 435 440 445Met Ala Ala Ile Gly Pro Lys Arg His Gln Asp Ser
Leu Trp Leu Cys 450 455 460Glu Lys Thr Cys Glu Thr Gly Met Leu Ser
Ser Pro Gln Tyr Ser Pro465 470 475 480Glu Glu Leu Ser Lys Leu Trp
Glu Leu Lys Glu Leu Cys Gly Asp Pro 485 490 495Cys Gln Cys Glu Ala
Lys Val Arg 500537DNAArtificial SequenceDescription of Artificial
Sequence 5' primer for PCR 5cccaagcttg ggcctgcaga tcagttggcc
ttatttc 37642DNAArtificial SequenceDescription of Artificial
Sequence 3' primer for PCR 6aacgcggatc cgcgctgtta tcttgcttga
catcgacaag ga 42756DNAArtificial SequenceDescription of Artificial
Sequence 5' primer for PCR 7ggggacaagt ttgtacaaaa aagcaggctt
ccctgcagat cagttggcct tatttc 56858DNAArtificial SequenceDescription
of Artificial Sequence 3' primer for PCR 8ggggaccact ttgtacaaga
aagctgggtc ctgttatctt gcttgacatc gacaagga 58922PRTArtificial
SequenceDescription of Artificial Sequence Igkappa signal sequence
9Met His Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser1 5
10 15Val Ile Met Ser Arg Gly20108PRTArtificial SequenceDescription
of Artificial Sequence FLAG peptide 10Asp Tyr Lys Asp Asp Asp Asp
Lys1 51194DNAArtificial SequenceDescription of Artificial Sequence
primer OT3 11gatcatgcat tttcaagtgc agattttcag cttcctgcta atcagtgcct
cagtcataat 60gtcacgtgga gattacaagg acgacgatga caag
941226DNAArtificial SequenceDescription of Artificial Sequence
primer OT20 12cgggatccat gcattttcaa gtgcag 261325DNAArtificial
SequenceDescription of Artificial Sequence primer OT21 13ggaattcttg
tcatcgtcgt ccttg 251421DNAArtificial SequenceDescription of
Artificial Sequence 5' primer for PCR 14ggagtgttct acgatgccaa t
211520DNAArtificial SequenceDescription of Artificial Sequence 3'
primer for PCR 15ctgaagcgag caatgaagag 201632DNAArtificial
SequenceDescription of Artificial Sequence TaqMan Probe
16cactgtcaaa ctttatcagg cagaacaaga gg 321737DNAArtificial
SequenceDescription of Artificial Sequence 5' primer for PCR
17cccaagcttg ggagcgcggc agatcaatca gccttat 371853DNAArtificial
SequenceDescription of Artificial Sequence 3' primer for PCR
18ttttcctttt gcggccgctt ttttcctttc atcgtacttt tgcttcacac tga
5319248PRTHomo sapiensb3Gal-T1 19Phe Leu Val Ile Leu Ile Ser Thr
Thr His Lys Glu Phe Asp Ala Arg1 5 10 15Gln Ala Ile Arg Glu Thr Trp
Gly Asp Glu Asn Asn Phe Lys Gly Ile 20 25 30Lys Ile Ala Thr Leu Phe
Leu Leu Gly Lys Asn Ala Asp Pro Val Leu 35 40 45Asn Gln Met Val Glu
Gln Glu Ser Gln Ile Phe His Asp Ile Ile Val 50 55 60Glu Asp Phe Ile
Asp Ser Tyr His Asn Leu Thr Leu Lys Thr Leu Met65 70 75 80Gly Met
Arg Trp Val Ala Thr Phe Cys Ser Lys Ala Lys Tyr Val Met 85 90 95Lys
Thr Asp Ser Asp Ile Phe Val Asn Met Asp Asn Leu Ile Tyr Lys 100 105
110Leu Leu Lys Pro Ser Thr Lys Pro Arg Arg Arg Tyr Phe Thr Gly Tyr
115 120 125Val Ile Asn Gly Gly Pro Ile Arg Asp Val Arg Ser Lys Trp
Tyr Met 130 135 140Pro Arg Asp Leu Tyr Pro Asp Ser Asn Tyr Pro Pro
Phe Cys Ser Gly145 150 155 160Thr Gly Tyr Ile Phe Ser Ala Asp Val
Ala Glu Leu Ile Tyr Lys Thr 165 170 175Ser Leu His Thr Arg Leu Leu
His Leu Glu Asp Val Tyr Val Gly Leu 180 185 190Ser Leu His Thr Arg
Leu Leu His Leu Glu Asp Val Tyr Val Gly Leu 195 200 205His Trp Lys
Met Ala Tyr Ser Leu Cys Arg Tyr Arg Arg Val Ile Thr 210 215 220Val
His Gln Ile Ser Pro Glu Glu Met His Arg Ile Trp Asn Asp Met225 230
235 240Ser Ser Lys Lys His Leu Arg Cys 24520271PRTHomo
sapiensb3Gal-T2 20Phe Leu Ile Leu Leu Ile Ala Ala Glu Pro Gly Gln
Ile Glu Ala Arg1 5 10 15Arg Ala Ile Arg Gln Thr Trp Gly Asn Glu Ser
Leu Ala Pro Gly Ile 20 25 30Gln Ile Thr Arg Ile Phe Leu Leu Gly Leu
Ser Ile Lys Leu Asn Gly 35 40 45Tyr Leu Gln Arg Ala Ile Leu Glu Glu
Ser Arg Gln Tyr His Asp Ile 50 55 60Ile Gln Gln Glu Tyr Leu Asp Thr
Tyr Tyr Asn Leu Thr Ile Lys Thr65 70 75 80Leu Met Gly Met Asn Trp
Val Ala Thr Tyr Cys Pro His Ile Pro Tyr 85 90 95Val Met Lys Thr Asp
Ser Asp Met Phe Val Asn Thr Glu Tyr Leu Ile 100 105 110Asn Lys Leu
Leu Lys Pro Asp Leu Pro Pro Arg His Asn Tyr Phe Thr 115 120 125Gly
Tyr Leu Met Arg Gly Tyr Ala Pro Asn Arg Asn Lys Asp Ser Lys 130 135
140Trp Tyr Met Pro Pro Asp Leu Tyr Pro Ser Glu Arg Tyr Pro Val
Phe145 150 155 160Cys Ser Gly Thr Gly Tyr Val Phe Ser Gly Asp Leu
Ala Glu Lys Ile 165 170 175Phe Lys Val Ser Leu Gly Ile Arg Arg Leu
His Leu Glu Asp Val Tyr 180 185 190Val Gly Ile Cys Leu Ala Lys Leu
Arg Ile Asp Pro Val Pro Pro Pro 195 200 205Asn Glu Phe Val Phe Asn
His Trp Arg Val Ser Tyr Ser Ser Cys Lys 210 215 220Tyr Ser His Leu
Ile Thr Ser His Gln Phe Gln Pro Ser Glu Leu Ile225 230 235 240Lys
Tyr Trp Asn His Leu Gln Gln Asn Lys His Asn Ala Cys Ala Asn 245 250
255Ala Ala Lys Glu Lys Ala Gly Arg Tyr Arg His Arg Lys Leu His 260
265 27021253PRTHomo sapiensb3Gal-T3 21Phe Leu Val Ile Leu Val Thr
Ser His Pro Ser Asp Val Lys Ala Arg1 5 10 15Gln Ala Ile Arg Val Thr
Trp Gly Glu Lys Lys Ser Trp Trp Gly Tyr 20 25 30Glu Val Leu Thr Phe
Phe Leu Leu Gly Gln Glu Ala Glu Lys Glu Asp 35 40 45Lys Met Leu Ala
Leu Ser Leu Glu Asp Glu His Leu Leu Tyr Gly Asp 50 55 60Ile Ile Arg
Gln Asp Phe Leu Asp Thr Tyr Asn Asn Leu Thr Leu Lys65 70 75 80Thr
Ile Met Ala Phe Arg Trp Val Thr Glu Phe Cys Pro Asn Ala Lys 85 90
95Tyr Val Met Lys Thr Asp
Thr Asp Val Phe Ile Asn Thr Gly Asn Leu 100 105 110Val Lys Tyr Leu
Leu Asn Leu Asn His Ser Glu Lys Phe Phe Thr Gly 115 120 125Tyr Pro
Leu Ile Asp Asn Tyr Ser Tyr Arg Gly Phe Tyr Gln Lys Thr 130 135
140His Ile Ser Tyr Gln Glu Tyr Pro Phe Lys Val Phe Pro Pro Tyr
Cys145 150 155 160Ser Gly Leu Gly Tyr Ile Met Ser Arg Asp Leu Val
Pro Arg Ile Tyr 165 170 175Glu Met Met Gly His Val Lys Pro Ile Lys
Phe Glu Asp Val Tyr Val 180 185 190Gly Ile Cys Leu Asn Leu Leu Lys
Val Asn Ile His Ile Pro Glu Asp 195 200 205Thr Asn Leu Phe Phe Leu
Tyr Arg Ile His Leu Asp Val Cys Gln Leu 210 215 220Arg Arg Val Ile
Ala Ala His Gly Phe Ser Ser Lys Glu Ile Ile Thr225 230 235 240Phe
Trp Gln Val Met Leu Arg Asn Thr Thr Cys His Tyr 245 25022253PRTHomo
sapiensb3Gal-T5 22Phe Leu Val Leu Leu Val Thr Ser Ser His Lys Gln
Leu Ala Glu Arg1 5 10 15Met Ala Ile Arg Gln Thr Trp Gly Lys Glu Arg
Met Val Lys Gly Lys 20 25 30Gln Leu Lys Thr Phe Phe Leu Leu Gly Thr
Thr Ser Ser Ala Ala Glu 35 40 45Thr Lys Glu Val Asp Gln Glu Ser Gln
Arg His Gly Asp Ile Ile Gln 50 55 60Lys Asp Phe Leu Asp Val Tyr Tyr
Asn Leu Thr Leu Lys Thr Met Met65 70 75 80Gly Ile Glu Trp Val His
Arg Phe Cys Pro Gln Ala Ala Phe Val Met 85 90 95Lys Thr Asp Ser Asp
Met Phe Ile Asn Val Asp Tyr Leu Thr Glu Leu 100 105 110Leu Leu Lys
Lys Asn Arg Thr Thr Arg Phe Phe Thr Gly Phe Leu Lys 115 120 125Leu
Asn Glu Phe Pro Ile Arg Gln Pro Phe Ser Lys Trp Phe Val Ser 130 135
140Lys Ser Glu Tyr Pro Trp Asp Arg Tyr Pro Pro Phe Cys Ser Gly
Thr145 150 155 160Gly Tyr Val Phe Ser Gly Asp Val Ala Ser Gln Val
Tyr Asn Val Ser 165 170 175Lys Ser Val Pro Tyr Ile Lys Leu Glu Asp
Val Phe Val Gly Leu Cys 180 185 190Leu Glu Arg Leu Asn Ile Arg Leu
Glu Glu Leu His Ser Gln Pro Thr 195 200 205Phe Phe Pro Gly Gly Leu
Arg Phe Ser Val Cys Leu Phe Arg Arg Ile 210 215 220Val Ala Cys His
Phe Ile Lys Pro Arg Thr Leu Leu Asp Tyr Trp Gln225 230 235 240Ala
Leu Glu Asn Ser Arg Gly Glu Asp Cys Pro Pro Val 245 25023272PRTHomo
sapiensb3Gal-T6 23Phe Leu Ala Val Leu Val Ala Ser Ala Pro Arg Ala
Ala Glu Arg Arg1 5 10 15Ser Val Ile Arg Ser Thr Trp Leu Ala Arg Arg
Gly Ala Pro Gly Asp 20 25 30Val Trp Ala Arg Phe Ala Val Gly Thr Ala
Gly Leu Gly Ala Glu Glu 35 40 45Arg Arg Ala Leu Glu Arg Glu Gln Ala
Arg His Gly Asp Leu Leu Leu 50 55 60Leu Pro Ala Leu Arg Asp Ala Tyr
Glu Asn Leu Thr Ala Lys Val Leu65 70 75 80Ala Met Leu Ala Trp Leu
Asp Glu His Val Ala Phe Glu Phe Val Leu 85 90 95Lys Ala Asp Asp Asp
Ser Phe Ala Arg Leu Asp Ala Leu Leu Ala Glu 100 105 110Leu Arg Ala
Arg Glu Pro Ala Arg Arg Arg Arg Leu Tyr Trp Gly Phe 115 120 125Phe
Ser Gly Arg Gly Arg Val Lys Pro Gly Gly Arg Trp Arg Glu Ala 130 135
140Ala Trp Gln Leu Cys Asp Tyr Tyr Leu Pro Tyr Ala Leu Gly Gly
Gly145 150 155 160Tyr Val Leu Ser Ala Asp Leu Val His Tyr Leu Arg
Leu Ser Arg Asp 165 170 175Tyr Leu Arg Ala Trp His Ser Glu Asp Val
Ser Leu Gly Ala Trp Leu 180 185 190Ala Pro Val Asp Val Gln Arg Glu
His Asp Pro Arg Phe Asp Thr Glu 195 200 205Tyr Arg Ser Arg Gly Cys
Ser Asn Gln Tyr Leu Val Thr His Lys Gln 210 215 220Ser Leu Glu Asp
Met Leu Glu Lys His Ala Thr Leu Ala Arg Glu Gly225 230 235 240Arg
Leu Cys Lys Arg Glu Val Gln Leu Arg Leu Ser Tyr Val Tyr Asp 245 250
255Trp Ser Ala Pro Pro Ser Gln Cys Cys Gln Arg Arg Glu Gly Ile Pro
260 265 27024255PRTHomo sapiensb3GnT2 24Phe Leu Leu Leu Ala Ile Lys
Ser Leu Thr Pro His Phe Ala Arg Arg1 5 10 15Gln Ala Ile Arg Glu Ser
Trp Gly Gln Glu Ser Asn Ala Gly Asn Gln 20 25 30Thr Val Val Arg Val
Phe Leu Leu Gly Gln Thr Pro Pro Glu Asp Asn 35 40 45His Pro Asp Leu
Ser Asp Met Leu Lys Phe Glu Ser Glu Lys His Gln 50 55 60Asp Ile Leu
Met Trp Asn Tyr Arg Asp Thr Phe Phe Asn Leu Ser Leu65 70 75 80Lys
Glu Val Leu Phe Leu Arg Trp Val Ser Thr Ser Cys Pro Asp Thr 85 90
95Glu Phe Val Phe Lys Gly Asp Asp Asp Val Phe Val Asn Thr His His
100 105 110Ile Leu Asn Tyr Leu Asn Ser Leu Ser Lys Thr Lys Ala Lys
Asp Leu 115 120 125Phe Ile Gly Asp Val Ile His Asn Ala Gly Pro His
Arg Asp Lys Lys 130 135 140Leu Lys Tyr Tyr Ile Pro Glu Val Val Tyr
Ser Gly Leu Tyr Pro Pro145 150 155 160Tyr Ala Gly Gly Gly Gly Phe
Leu Tyr Ser Gly His Leu Ala Leu Arg 165 170 175Leu Tyr His Ile Thr
Asp Gln Val His Leu Tyr Pro Ile Asp Asp Val 180 185 190Tyr Thr Gly
Met Cys Leu Gln Lys Leu Gly Leu Val Pro Glu Lys His 195 200 205Lys
Gly Phe Arg Thr Phe Asp Ile Glu Glu Lys Asn Lys Asn Asn Ile 210 215
220Cys Ser Tyr Val Asp Leu Met Leu Val His Ser Arg Lys Pro Gln
Glu225 230 235 240Met Ile Asp Ile Trp Ser Gln Leu Gln Ser Ala His
Leu Lys Cys 245 250 25525265PRTHomo sapiensb3GnT3 25Phe Leu Leu Leu
Val Ile Lys Ser Ser Pro Ser Asn Tyr Val Arg Arg1 5 10 15Glu Leu Leu
Arg Arg Thr Trp Gly Arg Glu Arg Lys Val Arg Gly Leu 20 25 30Gln Leu
Arg Leu Leu Phe Leu Val Gly Thr Ala Ser Asn Pro His Glu 35 40 45Ala
Arg Lys Val Asn Arg Leu Leu Glu Leu Glu Ala Gln Thr His Gly 50 55
60Asp Ile Leu Gln Trp Asp Phe His Asp Ser Phe Phe Asn Leu Thr Leu65
70 75 80Lys Gln Val Leu Phe Leu Gln Trp Gln Glu Thr Arg Cys Ala Asn
Ala 85 90 95Ser Phe Val Leu Asn Gly Asp Asp Asp Val Phe Ala His Thr
Asp Asn 100 105 110Met Val Phe Tyr Leu Gln Asp His Asp Pro Gly Arg
His Leu Phe Val 115 120 125Gly Gln Leu Ile Gln Asn Val Gly Pro Ile
Arg Ala Phe Trp Ser Lys 130 135 140Tyr Tyr Val Pro Glu Val Val Thr
Gln Asn Glu Arg Tyr Pro Pro Tyr145 150 155 160Cys Gly Gly Gly Gly
Phe Leu Leu Ser Arg Phe Thr Ala Ala Ala Leu 165 170 175Arg Arg Ala
Ala His Val Leu Asp Ile Phe Pro Ile Asp Asp Val Phe 180 185 190Leu
Gly Met Cys Leu Glu Leu Glu Gly Leu Lys Pro Ala Ser His Ser 195 200
205Gly Ile Arg Thr Ser Gly Val Arg Ala Pro Ser Gln His Leu Ser Ser
210 215 220Phe Asp Pro Cys Phe Tyr Arg Asp Leu Leu Leu Val His Arg
Phe Leu225 230 235 240Pro Tyr Glu Met Leu Leu Met Trp Asp Ala Leu
Asn Gln Pro Asn Leu 245 250 255Thr Cys Gly Asn Gln Thr Gln Ile Tyr
260 26526260PRTHomo sapiensb3GnT4 26Phe Leu Leu Leu Ala Ile Lys Ser
Gln Pro Gly His Val Glu Arg Arg1 5 10 15Ala Ala Ile Arg Ser Thr Trp
Gly Arg Val Gly Gly Trp Ala Arg Gly 20 25 30Arg Gln Leu Lys Leu Val
Phe Leu Leu Gly Val Ala Gly Ser Ala Pro 35 40 45Pro Ala Gln Leu Leu
Ala Tyr Glu Ser Arg Glu Phe Asp Asp Ile Leu 50 55 60Gln Trp Asp Phe
Thr Glu Asp Phe Phe Asn Leu Thr Leu Lys Glu Leu65 70 75 80His Leu
Gln Arg Trp Val Val Ala Ala Cys Pro Gln Ala His Phe Met 85 90 95Leu
Lys Gly Asp Asp Asp Val Phe Val His Val Pro Asn Val Leu Glu 100 105
110Phe Leu Asp Gly Trp Asp Pro Ala Gln Asp Leu Leu Val Gly Asp Val
115 120 125Ile Arg Gln Ala Leu Pro Asn Arg Asn Thr Lys Val Lys Tyr
Phe Ile 130 135 140Pro Pro Ser Met Tyr Arg Ala Thr His Tyr Pro Pro
Tyr Ala Gly Gly145 150 155 160Gly Gly Tyr Val Met Ser Arg Ala Thr
Val Arg Arg Leu Gln Ala Ile 165 170 175Met Glu Asp Ala Glu Leu Phe
Pro Ile Asp Asp Val Phe Val Gly Met 180 185 190Cys Leu Arg Arg Leu
Gly Leu Ser Pro Met His His Ala Gly Phe Lys 195 200 205Thr Phe Gly
Ile Arg Arg Pro Leu Asp Pro Leu Asp Pro Cys Leu Tyr 210 215 220Arg
Gly Leu Leu Leu Val His Arg Leu Ser Pro Leu Glu Met Trp Thr225 230
235 240Met Trp Ala Leu Val Thr Asp Glu Gly Leu Lys Cys Ala Ala Gly
Pro 245 250 255Ile Pro Gln Arg 26027290PRTHomo sapiensb3GnT5 27Leu
Leu Leu Leu Phe Val Lys Thr Ala Pro Glu Asn Tyr Asp Arg Arg1 5 10
15Ser Gly Ile Arg Arg Thr Trp Gly Asn Glu Asn Tyr Val Arg Ser Gln
20 25 30Leu Asn Ala Asn Ile Lys Thr Leu Phe Ala Leu Gly Thr Pro Asn
Pro 35 40 45Leu Glu Gly Glu Glu Leu Gln Arg Lys Leu Ala Trp Glu Asp
Gln Arg 50 55 60Tyr Asn Asp Ile Ile Gln Gln Asp Phe Val Asp Ser Phe
Tyr Asn Leu65 70 75 80Thr Leu Lys Leu Leu Met Gln Phe Ser Trp Ala
Asn Thr Tyr Cys Pro 85 90 95His Ala Lys Phe Leu Met Thr Ala Asp Asp
Asp Ile Phe Ile His Met 100 105 110Pro Asn Leu Ile Glu Tyr Leu Gln
Ser Leu Glu Gln Ile Gly Val Gln 115 120 125Asp Phe Trp Ile Gly Arg
Val His Arg Gly Ala Pro Pro Ile Arg Asp 130 135 140Lys Ser Ser Lys
Tyr Tyr Val Ser Tyr Glu Met Tyr Gln Trp Pro Ala145 150 155 160Tyr
Pro Asp Tyr Thr Ala Gly Ala Ala Tyr Val Ile Ser Gly Asp Val 165 170
175Ala Ala Lys Val Tyr Glu Ala Ser Gln Thr Leu Asn Ser Ser Leu Tyr
180 185 190Ile Asp Asp Val Phe Met Gly Leu Cys Ala Asn Lys Ile Gly
Ile Val 195 200 205Pro Gln Asp His Val Phe Phe Ser Gly Glu Gly Lys
Thr Pro Tyr His 210 215 220Pro Cys Ile Tyr Glu Lys Met Met Thr Ser
His Gly His Leu Glu Asp225 230 235 240Leu Gln Asp Leu Trp Lys Asn
Ala Thr Asp Pro Lys Val Lys Thr Ile 245 250 255Ser Lys Gly Phe Phe
Gly Gln Ile Tyr Cys Arg Leu Met Lys Ile Ile 260 265 270Leu Leu Cys
Lys Ile Ser Tyr Val Asp Thr Tyr Pro Cys Arg Ala Ala 275 280 285Phe
Ile 290
* * * * *