U.S. patent application number 09/839136 was filed with the patent office on 2002-06-27 for alpha 1-6 fucosyltransferase.
Invention is credited to Shiba, Tetsuo, Taniguchi, Naoyuki, Uozumi, Naofumi, Yanagidani, Shusaku.
Application Number | 20020081694 09/839136 |
Document ID | / |
Family ID | 27455380 |
Filed Date | 2002-06-27 |
United States Patent
Application |
20020081694 |
Kind Code |
A1 |
Taniguchi, Naoyuki ; et
al. |
June 27, 2002 |
Alpha 1-6 fucosyltransferase
Abstract
Alpha-1-6 fucosyltransferases with a human or swine origin which
have the following function; genes encoding these enzymes;
expression vectors containing these genes; transformants prepared
by using these expression vectors; and a process for producing a
recombinant .+-.1-6 fucosyltransferase by incubating such a
transformant. Function transferring fucose from guanosine
diphosphate to the hydroxyl group at the 6-position of GluNAc
closest to R in the receptor
(GluNAc.sup.21-2Man.+-.1-6)-(GlcNAc.sup.21-2Man.+-.1-3)Man.sup.21-4GlcNAc-
.sup.21-4GlucNac-R wherein R represents an asparagine residue or a
peptide chain carrying this residue to thereby form
(GlcNAc.sup.21-2Man.+-.1-6)(G-
lcNAc.sup.21-2Man.+-.1-3)Man.sup.21-4GlcNAc.sup.21-4(Fuc.+-.1-6)GlucNac-R
Inventors: |
Taniguchi, Naoyuki;
(Toyonaka-shi, JP) ; Uozumi, Naofumi; (Kobe-shi,
JP) ; Shiba, Tetsuo; (Toyonaka-shi, JP) ;
Yanagidani, Shusaku; (Ohtsu-shi, JP) |
Correspondence
Address: |
KENYON & KENYON
1500 K STREET, N.W., SUITE 700
WASHINGTON
DC
20005
US
|
Family ID: |
27455380 |
Appl. No.: |
09/839136 |
Filed: |
April 23, 2001 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09839136 |
Apr 23, 2001 |
|
|
|
09442629 |
Nov 18, 1999 |
|
|
|
Current U.S.
Class: |
435/193 ;
435/101; 435/320.1; 435/325; 435/69.1 |
Current CPC
Class: |
C12N 9/1051
20130101 |
Class at
Publication: |
435/193 ;
435/69.1; 435/325; 435/320.1; 435/101 |
International
Class: |
C12P 019/04; C12N
009/10; C12P 021/02; C12N 005/06 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 24, 1996 |
JP |
10365/1996 |
Jun 21, 1996 |
JP |
161648/1996 |
Jun 24, 1996 |
JP |
162813/1996 |
Jul 22, 1996 |
JP |
192260/1996 |
Claims
What is claimed is:
1. A porcine-derived .alpha.1-6 fucosyltransferase having the
following physico-chemical properties: (1) action: transferring
fucose from guanosine diphosphate-fucose to a hydroxy group at
6-position of GluNAc closest to R of a receptor
(GlcNAc.beta.1-2Man.alpha.1-6)
(GlcNAc.beta.1-2Man.alpha.1-3)Man.beta.1-4GlcNAc.alpha.1-4GlucNAc-R
wherein R is an asparagine residue or a peptide chain carrying said
residue, whereby to form
(GlcNAc.beta.1-2Man.alpha.1-6)-(GlcNAc.beta.1-2M-
an.alpha.1-3)Man.beta.1-4GlcNAc.beta.1-4(Fuc.alpha.1-6)GlucNAc-R
(2) optimum pH: about 7.0 (3) pH stability: stable in the pH range
of 4.0-10.0 by treatment at 4.degree. C. for 5 hours (4) optimum
temperature: about 30-37.degree. C. (5) inhibition or activation:
no requirement for divalent metal for expression of activity; no
inhibition of activity in the presence of 5 mM EDTA (6) molecular
weight: about 60,000 by SDS-polyacrylamide gel electrophoresis.
2. The porcine-derived .alpha.1-6 fucosyltransferase of claim 1,
which is purified from porcine brain.
3. A gene encoding porcine-derived .alpha.1-6
fucosyltransferase.
4. The gene of claim 3, comprising a gene encoding an amino acid
sequence as depicted in Sequence Listing, SEQ ID NO:2.
5. The gene of claim 3, comprising a nucleotide sequence as
depicted in Sequence Listing, SEQ ID NO:1.
6. The gene of claim 3, comprising a gene encoding an amino acid
sequence resulting from substitution, insertion, deletion or
addition with respect to at least one amino acid of amino acid
sequence depicted in Sequence Listing, SEQ ID NO:2.
7. The gene of claim 3, comprising a nucleotide sequence resulting
from substitution, insertion, deletion or addition with respect to
at least one nucleotide of nucleotide sequence depicted in Sequence
Listing, SEQ ID NO:1.
8. A gene which hybridizes to at least a part of a gene encoding
.alpha.1-6 fucosyltransferase and comprising nucleotide sequence as
depicted in Sequence Listing, SEQ ID NO:1.
9. An expression vector comprising a gene of any one of claims 3 to
8 which encodes .alpha.1-6 fucosyltransferase.
10. A transformant cell obtained by transforming a host cell with
the expression vector of claim 9.
11. A method for producing a recombinant .alpha.1-6
fucosyltransferase, comprising culturing the transformant cell of
claim 10, and harvesting the .alpha.1-6 fucosyltransferase from a
culture thereof.
12. A recombinant .alpha.1-6 fucosyltransferase produced according
to the method of claim 11.
13. An .alpha.1-6 fucosyltransferase derived from human, having the
following physico-chemical properties: (1) action: transferring
fucose from guanosine diphosphate-fucose to a hydroxy group at
6-position of GluNAc closest to R of a receptor
(GlcNAc.beta.1-2Man.alpha.1-6)(GlcNAc.b-
eta.1-2Man.alpha.1-3)Man.beta.1-4GlcNAc.beta.1-4GlucNAc-R wherein R
is an asparagine residue or a peptide chain carrying said residue,
whereby to form
(GlcNAc.beta.1-2Man.alpha.1-6)-(GlcNAc.beta.1-2Man.alpha.1-3)Man.bet-
a.1l-4GlcNAc.beta.1-4(Fuc.alpha.1-6)GlucNAc-R (2) optimum pH: about
7.5 (3) pH stability: stable in the pH range of 4.0-10.0 by
treatment at 4.degree. C. for 5 hours (4) optimum temperature:
about 30-37.degree. C. (5) inhibition or activation: no requirement
for divalent metal for expression of activity; no inhibition of
activity in the presence of 5 mM EDTA (6) molecular weight: about
60,000 by SDS-polyacrylamide gel electrophoresis.
14. The .alpha.1-6 fucosyltransferase of claim 13, which is
purified from a human cell culture medium.
15. The .alpha.1-6 fucosyltransferase of claim 14, wherein the
human cell culture medium is a human gastric cancer cell serum-free
medium.
16. A gene encoding .alpha.1-6 fucosyltransferase derived from
human.
17. The gene of claim 16, comprising a gene encoding amino acid
sequence as depicted in Sequence Listing, SEQ ID NO:10.
18. The gene of claim 16, comprising a nucleotide sequence as
depicted in Sequence Listing, SEQ ID NO:9.
19. The gene of claim 16, comprising a nucleotide sequence from
198th adenine to 1919th guanine as depicted in Sequence Listing,
SEQ ID NO:9.
20. The gene of claim 16, comprising a gene encoding an amino acid
sequence resulting from substitution, insertion, deletion or
addition with respect to at least one amino acid of amino acid
sequence as depicted in Sequence Listing, SEQ ID NO:10.
21. The gene of claim 16, comprising a nucleotide sequence
resulting from substitution, insertion, deletion or addition with
respect to at least one nucleotide of nucleotide sequence as
depicted in Sequence Listing, SEQ ID NO:9.
22. A gene which hybridizes to at least a part of a gene encoding
.alpha.1-6 fucosyltransferase and comprising nucleotide sequence as
depicted in Sequence Listing, SEQ ID NO:9.
23. The expression vector of any one of claims 16 to 22, which
comprises a gene encoding human .alpha.1-6 fucosyltransferase.
24. A transformant cell obtained by transforming a host cell with
the expression vector of claim 23.
25. A method for producing a recombinant .alpha.1-6
fucosyltransferase, comprising culturing the transformant cell of
claim 24, and harvesting the .alpha.1-6 fucosyltransferase from a
culture thereof.
26. A recombinant .alpha.1-6 fucosyltransferase produced according
to the method of claim 25.
Description
TECHNICAL FIELD
[0001] The present invention relates to an .alpha.1-6
fucosyltransferase derived from pig or human. More particularly,
the present invention relates to a novel .alpha.1-6
fucosyltransferase derived from human, which is an enzyme that
transfers fucose from guanosine diphosphate (GDP)-fucose by
.alpha.1.fwdarw.6 linkage to N-acetylglucosamine (GlcNAc) bound to
Asn at the stem of asparagine type sugar chain (Asn type sugar
chain) and which is useful in the field of glyco-technology for
modification and synthesis of sugar chain and/or for the diagnosis
of diseases such as malignant tumor, and to a gene encoding said
enzyme.
BACKGROUND ART
[0002] The structure and function of sugar chain moiety of complex
carbohydrates, such as glycoprotein and glycolipid, derived from
higher organisms have been drawing much attention in recent years,
and many studies are under way. While a sugar chain is formed by
the action of glycohydrolase and glycosyltransferase,
glycosyltransferase contributes greatly to its formation.
[0003] Using a sugar nucleotide as a sugar donor,
glycosyltransferase transfers a sugar to a receptor sugar chain,
thereby to elongate the sugar chain. The specificity for the
structure of receptor sugar chain is stringent, such that one
glycoside linkage is formed by the corresponding one transferase.
Hence, glycosyltransferases are used for structural studies of
sugar moiety of complex carbohydrate, for facilitated synthesis of
a particular sugar chain structure, and for modification of native
sugar chain structure.
[0004] Besides, glycosyltransferases are expected to be usable for
the modification of the nature of complex carbohydrate and cells,
by means of artificial alteration of sugar chain. For this end, the
development of various glycosyltransferases having identified
substrate specificity has been awaited.
[0005] An .alpha.1-6 fucosyltransferase is an important enzyme
found in Golgi appratus of organelle, which is considered to be one
of the enzymes that control processing of asparagine-linked sugar
chain. Therefore, the enzyme will be useful for the elucidation of
control mechanism and control of formation of sugar chain
structure, once acted on an asparagine-linked sugar chain.
[0006] In addition, the activity of .alpha.1-6 fucosyltransferase
and the proportion of reaction products of this enzyme are known to
increase in certain diseases such as liver cancer and cystic
fibrosis. Therefore, a rapid development of the method for
diagnosis of these diseases has been desired, which involves
determination of the activity of this enzyme, Northern blot using a
cDNA encoding .alpha.1-6 fucosyltransferase, or RT-PCR assay of
mRNA amount transcribed and expressed in the living body.
[0007] The activity of .alpha.1-6 fucosyltransferases has been
detected in body fluids or organs of various animals and culture
cells thereof, and there has been known, as a purified enzyme
product, an enzyme derived from human cystic fibrosis cell
homogenates [Journal of Biological Chemistry, vol. 266, pp.
21572-21577 (1991)]. According to this report, however, the enzyme
is associated with drawbacks in that (1) its optimum pH is 5.6
which is different from physiological pH, (2) it has relatively low
molecular weights (34,000 and 39,000) by SDS-polyacrylamide gel
electrophoresis, (3) its large scale and stable supply is
practically unattainable due to its being derived from human cell,
and others.
[0008] This enzyme is obtained as a membrane-bound enzyme, and
requires bovine serum for culturing the cells, which in turn
results in difficult purification of the enzyme and a huge amount
of money necessary for culture of the cells to be a starting
material. Consequently, stable supply of this enzyme preparation is
all but impractical.
[0009] While a chemical synthesis is often employed for
synthesizing a sugar chain, the synthesis of oligosaccharides
requires many steps that have been necessitated by its complicated
synthesis route and specificity of the reaction, so that it
involves various practical problems. Particularly, binding of
fucose to GlcNAc bound to Asn of asparagine-linked sugar chain by
.alpha.1-6 linkage is extremely difficult due to the instability of
fucose.
DISCLOSURE OF THE INVENTION
[0010] It is therefore an object of the present invention to stably
provide an .alpha.1-6 fucosyltransferase in large amounts, which is
useful as a reagent for structural analysis of sugar chain or
glyco-technology, or as diagnostics.
[0011] Another object of the present invention is to provide a
method of producing .alpha.1-6 fucosyltransferase in large amounts
by the use of a human- or porcine-derived .alpha.1-6
fucosyltransferase gene. It is aimed to use such specific genes so
as to enable development of a method for diagnosis of diseases by
Northern blot using a DNA encoding said enzyme, or by RT-PCR assay
of mRNA amount transcribed and expressed in the living body.
[0012] In an attempt to achieve the above-mentioned objects, the
present inventors started the study of an enzyme capable of linking
fucose to GlcNAc linked to Asn of asparagine type sugar chain by
.alpha.1.fwdarw.6 linkage, using a fluorescence-labeled substrate
analogous to an asparagine type sugar chain which is a receptor of
this enzyme. As a result, they have found the activity of this
enzyme in the extract fractions of porcine brain which is readily
available as a starting material to be purified, and they have
purified said enzyme from said fractions and elucidated the
enzymatic and physico-chemical properties, which resulted in the
completion of the invention.
[0013] Accordingly, the present invention relates to a
porcine-derived .alpha.1-6 fucosyltransferase having the following
physico-chemical properties (hereinafter this enzyme is referred to
as porcine .alpha.1-6 fucosyltransferase).
[0014] (1) Action: transferring fucose from guanosine
diphosphate-fucose to the hydroxy group at 6-position of GluNAc
closest to R of a receptor (GlcNAc.beta.1-2Man.alpha.1-6)
(GlcNAc.beta.1-2Man.alpha.1-3)Man.beta.1-4-
GlcNAc.beta.1-4GlucNAc-R wherein R is an asparagine residue or a
peptide chain carrying said residue, whereby to form
(GlcNAc.beta.1-2Man.alpha.1--
6)-(GlcNAc.beta.1-2Man.alpha.1-3)Man.beta.1-4GlcNAc.beta.1-4(Fuc.alpha.1-6-
)GlucNAc-R.
[0015] In the above formula, asparagine residue at R is a residue
wherein the acid amide group at the side chain of asparagine is
bound to the hydroxy group at the anomer position of the reducing
terminal of sugar chain, and a peptide chain having said residue is
a peptide chain having said residue in the peptide to which two or
more amino acids are bound, which is preferably a peptide chain
having --Asn--(X)--Ser/Thr--.
[0016] (2) optimum pH: about 7.0
[0017] (3) pH stability: stable in the pH range of 4.0-10.0 by
treatment at 4.degree. C. for 5 hours
[0018] (4) optimum temperature: about 30-37.degree. C.
[0019] (5) inhibition or activation: no requirement for divalent
metal ion for expression of activity; no inhibition of activity
even in the presence of 5 mM EDTA
[0020] (6) molecular weight : about 60,000 by SDS-polyacrylamide
gel electrophoresis.
[0021] The present inventors have purified .alpha.1-6
fucosyltransferase alone from porcine brain, analyzed the amino
acid sequence of this protein and cloned a gene based on the
partial amino acid sequence to accomplish the present
invention.
[0022] That is, the present invention provides a gene encoding
porcine .alpha.1-6 fucosyltransferase.
[0023] The present invention also provides an expression vector
containing a gene encoding porcine .alpha.1-6
fucosyltransferase.
[0024] The present invention further provides a transformant cell
obtained by transforming a host cell with an expression vector
containing a gene encoding porcine .alpha.1-6
fucosyltransferase.
[0025] The present invention yet provides a method for producing a
recombinant .alpha.1-6 fucosyltransferase, comprising culturing a
transformant cell obtained by transforming a host cell with an
expression vector containing a gene encoding porcine .alpha.1-6
fucosyltransferase, and harvesting the .alpha.1-6
fucosyltransferase from the culture thereof.
[0026] The present inventors have reached the present invention by
purifying protein having an .alpha.1-6 fucosyltransferase activity
from human cell culture broth and elucidating its enzymatic
property.
[0027] Accordingly, the present invention relates to an .alpha.1-6
fucosyltransferase derived from human, having the following
physico-chemical property (hereinafter this enzyme is to be
referred to as human .alpha.1-6 fucosyltransferase).
[0028] (1) Action: transferring fucose from guanosine
diphosphate-fucose to the hydroxy group at 6-position of GluNAc
closest to R of a receptor
(GlcNAc.beta.1-2Man.alpha.1-6)(GlcNAc.beta.1-2Man.alpha.1-3)Man.beta.1-4G-
lcNAc.beta.1-4GlucNAc-R wherein R is an asparagine residue or a
peptide chain carrying said residue, whereby to form
(GlcNAc.beta.1-2Man.alpha.1--
6)-(GlcNAc.beta.1-2Man.alpha.1-3)Man.beta.1-4GlcNAc.beta.1-4(Fuc.alpha.1-6-
)GlucNAc-R.
[0029] In the above formula, asparagine residue at R is a residue
wherein the acid amide group at the side chain of asparagine is
bound to the reducing terminal hydroxy group of sugar chain, and a
peptide chain having said residue is a peptide chain having said
residue in the peptide to which two or more amino acids are bound,
which is preferably a peptide chain having
--Asn--(X)--Ser/Thr--.
[0030] (2) optimum pH: about 7.5
[0031] (3) pH stability: stable in the pH range of 4.0-10.0 by
treatment at 4.degree. C. for 5 hours
[0032] (4) optimum temperature: about 30-37.degree. C.
[0033] (5) inhibition or activation: no requirement for divalent
metal ion for expression of activity; no inhibition of activity
even in the presence of 5 mM EDTA
[0034] (6) molecular weight: about 60,000 by SDS-polyacrylamide gel
electrophoresis.
[0035] The present inventors have purified .alpha.1-6
fucosyltransferase alone from human culture cell, analyzed the
amino acid sequence of this protein and cloned a gene based on the
partial amino acid sequence to accomplish the present
invention.
[0036] That is, the present invention provides a gene encoding
human .alpha.1-6 fucosyltransferase.
[0037] The present invention also provides an expression vector
containing a gene encoding human .alpha.1-6 fucosyltransferase.
[0038] The present invention further provides a transformant cell
obtained by transforming a host cell with an expression vector
containing a gene encoding human .alpha.1-6 fucosyltransferase.
[0039] The present invention yet provides a method for producing a
recombinant .alpha.1-6 fucosyltransferase, comprising culturing a
transformant cell obtained by transforming a host cell with an
expression vector containing a gene encoding human .alpha.1-6
fucosyltransferase, and harvesting the .alpha.1-6
fucosyltransferase from the culture thereof.
[0040] The starting material for the purification of the enzyme of
the present invention is, for example, the organ and body fluid of
pig having .alpha.1-6 fucosyltransferase activity. Examples of the
organ include brain, spermary, pancreas, lung, kidney and the like.
The body fluid of pig such as blood and sera can be also used.
[0041] The porcine .alpha.1-6 fucosyltransferase of the present
invention can be obtained by preparing a crude extract containing
the enzyme from, for example, homogenates of porcine brain and
separating the enzyme from this extract. In this case, since
.alpha.1-6 fucosyltransferase in the porcine brain is a
membrane-bound enzyme, a crude extract solution containing the
enzyme is generally obtained from brain lysate using a suitable
surfactant. This extract undergoes various known purification steps
to give a purified enzyme product. The purification may include,
for example, concentration using an ultrafiltration membrane,
desalting, affinity column chromatography wherein a substrate
analog is immobilized, ion exchange column chromatography,
hydrophobic column chromatography and the like in suitable
combination to give a substantially homogeneous enzyme product
which is free of contaminant proteins such as other transferases.
For example, porcine brain is disrupted in a Waring blender in a
phosphate buffer and membrane fractions are collected by
ultracentrifugation. The objective enzyme is extracted with a
phosphate buffer containing a surfactant (Triton X-100), and the
supernatants are collected by ultracentrifugation to give a crude
extract containing the enzyme. By applying affinity column
chromatography using a guanosine diphosphate
(GDP)-hexanolamine-sepharose, a GlcNAc.beta.1-2Man.alpha.1-6(-
GlcNAc.beta.1-2Man.alpha.1-3)Man.beta.1-4GlcNAc.beta.1-4GlcNAc-asparagine--
sepharose and the like, the fractions showing fucosyltransferase
activity are collected and purified.
[0042] The physico-chemical property of .alpha.1-6
fucosyltransferase derived from porcine brain, which is one aspect
of the present invention, is as follows.
[0043] (1) Action: transferring fucose from guanosine
diphosphate-fucose to the hydroxy group at 6-position of GluNAc
closest to R of a receptor
(GlcNAc.beta.1-2Man.alpha.1-6)(GlcNAc.beta.1-2Man.alpha.1-3)Man.beta.1-4G-
lcNAc.beta.1-4GlucNAc-R wherein R is an asparagine residue or a
peptide chain carrying said residue, whereby to form
(GlcNAc.beta.1-2Man.alpha.1--
6)-(GlcNAc.beta.1-2Man.alpha.1-3)Man.beta.1-4GlcNAc.beta.1-4(Fuc.alpha.1-6-
)GlucNAc-R.
[0044] (2) Determination of activity:
[0045] The activity of the porcine .alpha.1-6 fucosyltransferase
was determined as follows. That is, a compound of the following
formula, wherein the sugar chain end asparagine was
fluorescence-labeled with 4-(2-pyridylamino)butylamine [PABA:
--NH.sub.2(CH.sub.2).sub.4-NH-pyridin- e] was used as a substrate
for determination of enzyme activity: 1
[0046] wherein PA means pyridylamino. By the use of this substrate,
the product from the enzyme reaction, wherein fucose has been
transferred by .alpha.1.fwdarw.6 linkage, can be assayed by
detecting fluorescence by high performance liquid
chromatography.
[0047] Specifically, the determination includes the following
steps. A sample solution (10 .mu.l) and 1.25% Triton X-100 are
added to a 250 mM MES buffer containing 62.5 .mu.M of
fluorescence-labeled receptor substrate of the above formula and
625 .mu.M of a donor substrate (GDP-fucose), pH 7.0, 40 .mu.l, and
mixed. The mixture is reacted at 37.degree. C. for one hour, and
boiled for 5 minutes to stop the reaction. The reaction mixture is
subjected to high performance liquid chromatography and the peak of
the reaction product is assayed with a fluorescence detector. One
unit of the enzyme amount corresponds to the amount capable of
forming 1 pmole of GlcNAc .beta.1-2Man.alpha.1-6(GlcNAc-
.beta.1-2Man.alpha.1-3)Man.beta.1-4GlcNAc.beta.1-4(Fuc.alpha.1-6)GlcNAc-R
(wherein R is Asn--NH--(CH.sub.2).sub.4--NH--pyridine) in one
minute under these conditions.
[0048] (3) Optimum pH:
[0049] The .alpha.1-6 fucosyltransferase derived from porcine brain
(hereinafter this enzyme is referred to as porcine brain .alpha.1-6
fucosyltransferase) shows a high activity at nearly pH 7.0-7.5.
[0050] (4) pH Stability:
[0051] The porcine brain .alpha.1-6 fucosyltransferase is
relatively stable at pH 4-10, and more stable at pH 5-9.
[0052] (5) Optimum temperature:
[0053] The porcine brain .alpha.1-6 fucosyltransferase has an
optimum temperature at nearly 37.degree. C. and retains sufficient
activity at 20-40.degree. C.
[0054] (6) Divalent metal ion requirement:
[0055] The porcine brain .alpha.1-6 fucosyltransferase shows
sufficient activity even in the absence of divalent metal ion, such
as magnesium, manganese and the like. It also shows sufficient
activity even in the presence of 5 mM EDTA, which is a chelating
agent.
[0056] (7) Molecular weight:
[0057] A purified product of the porcine brain .alpha.1-6
fucosyltransferase shows a single band at a molecular weight of
about 60,000 by SDS-polyacrylamide gel electrophoresis.
[0058] Judging from the above properties, the porcine brain
.alpha.1-6 fucosyltransferase is a novel enzyme apparently
different from conventionally known .alpha.1-6 fucosyltransferase
derived from human cystic fibrosis cells (optimum pH 5.6, molecular
weights 34,000 and 39,000), in terms of optimum pH, metal ion
requirement and molecular weight.
[0059] The inventive porcine .alpha.1-6 fucosyltransferase is
expected to be extremely useful for (1) synthesis of sugar chain
compounds wherein sugar chain compounds having 1-6 fucose are
synthesized using the enzyme of the present invention, (2)
modification of sugar chain structure and functional analysis
wherein a fucose is newly introduced into asparagine type sugar
chain to artificially modify the sugar chain structure, whereby
changes in cell function and control mechanism of the processing of
complex carbohydrate, as well as the role of sugar chain, can be
elucidated, (3) diagnosis of lesions based on enzyme activity
wherein diseases such as cancer can be diagnosed by determining the
activity of the enzyme of the present invention which reflects
various lesions caused by tumorigenic transformation, (4) diagnosis
of various diseases wherein a specific antibody against the enzyme
of the present invention is prepared and used for the diagnosis,
and the like.
[0060] Another aspect of the present invention is a gene encoding
porcine .alpha.1-6 fucosyltransferase, which includes a gene
encoding .alpha.1-6 fucosyltransferase and including a gene
encoding amino acid sequence depicted in Sequence Listing, SEQ ID
NO:2. A different embodiment thereof is a gene encoding .alpha.1-6
fucosyltransferase and including nucleotide sequence depicted in
Sequence Listing, SEQ ID NO:1.
[0061] One aspect of the present invention is a gene encoding
porcine .alpha.1-6 fucosyltransferase and including a gene encoding
an amino acid sequence resulting from substitution, insertion,
deletion or addition with respect to at least one amino acid of the
amino acid sequence depicted in Sequence Listing, SEQ ID NO:2.
[0062] Another aspect of the present invention is a gene encoding
porcine .alpha.1-6 fucosyltransferase and including a nucleotide
sequence resulting from substitution, insertion, deletion or
addition with respect to at least one nucleotide of the nucleotide
sequence depicted in Sequence Listing, SEQ ID NO:1.
[0063] The present invention also includes, as one aspect thereof,
a gene that hybridizes to at least a part of a gene encoding
porcine .alpha.1-6 fucosyltransferase and including nucleotide
sequence depicted in Sequence Listing, SEQ ID NO:1.
[0064] The expression vector of the present invention contains a
gene encoding the above-mentioned porcine .alpha.1-6
fucosyltransferase.
[0065] The transformant host cell of the present invention has been
transformed with the above-mentioned expression vector.
[0066] The host cell is exemplified by microorganisms, such as
Escherichia coli, yeast, bacterial cells and the like. It also
includes animal cells such as insect cells, COS-1 cells, CHO cells
and the like, and plant cells, such as tobacco cells, Arabidopsis
cells and the like.
[0067] The vector may be any which is selected according to the
host to be transformed. In the case of Escherichia coli, for
example, pUC19 may be used; in the case of yeast, pYEUra3.TM. may
be used; in the case of insect cells, pBLUE Bac4 may be used; in
the case of COS-1 cells, pSVK3 may be used; and in the case of
tobacco cells and Arabidopsis cells, pBI may be used.
[0068] The method for preparing the inventive recombinant
.alpha.1-6 fucosyltransferase includes culturing the
above-mentioned transformant cells and harvesting .alpha.1-6
fucosyltransferase from the culture.
[0069] According to the present invention, .alpha.1-6
fucosyltransferase alone is purified from porcine brain, and
subjected to amino acid analysis of this protein. Its partial amino
acid sequence is determined and a primer for PCR is prepared based
on the amino acid sequence. Using this primer, PCR is performed
using cDNAs derived from porcine brain as a template to amplify a
gene encoding .alpha.1-6 fucosyltransferase to give a probe. This
probe is used to screen clones containing cDNA encoding .alpha.1-6
fucosyltransferase, from the cDNA library derived from porcine
brain. The cDNA encoding .alpha.1-6 fucosyltransferase is isolated
and used to express .alpha.1-6 fucosyltransferase.
[0070] To be specific, the purified porcine .alpha.1-6
fucosyltransferase is used to analyze amino acid sequences. For
example, SDS-polyacrylamide gel electrophoresis is applied, after
which the protein is transferred to PVDF membrane by
electroblotting, and the PVDF membrane containing ca. 60 kDa band
is cut out and subjected to sequencing using a protein sequencer.
As a result, the amino acid sequence of the amino terminal of
.alpha.1-6 fucosyltransferase depicted in Sequence Listing, SEQ ID
NO:3 is obtained.
[0071] Separately, purified .alpha.1-6 fucosyltransferase is
subjected to SDS-polyacrylamide gel electrophoresis and the peptide
fragments separated by electrophoresis are transferred to PVDF
membrane by electroblotting. Then, the PVDF membrane containing 60
kDa band is cut out and lysed on said PVDF membrane, using, for
example, a protease such as lysylendopeptidase. The lysate is
extracted from the sections of said PVDF membrane, and the extract
is subjected to reversed phase high performance liquid
chromatography to separate the lysate.
[0072] Then, using the amino acid sequences, a mixed primer for PCR
is prepared. For example, a mixed primer having a nucleotide
sequence depicted in SEQ ID NO:7 is synthesized from the amino acid
sequence depicted in SEQ ID NO:3, and a mixed primer having a
nucleotide sequence depicted in SEQ ID NO:8 is synthesized from the
amino acid sequence depicted in SEQ ID NO:4, respectively using a
DNA synthesizer, and used for the screening of cDNA of .alpha.1-6
fucosyltransferase.
[0073] For example, 25 cycles of PCR are performed to amplify DNA
fragments of ca. 1.45 kbp, using cDNA from porcine brain as a
template and mixed primers of SEQ ID NO:7 and SEQ ID NO:8, wherein
PCR at 94.degree. C. (1 min), 55.degree. C. (2 min) and 72.degree.
C. (3 min) is one cycle.
[0074] Then, using the amplified DNA fragments as a probe, clones
containing cDNA encoding .alpha.1-6 fucosyltransferase are screened
from the cDNA library derived from porcine brain by a plaque
hybridization method. The cDNA encoding .alpha.1-6
fucosyltransferase can be isolated from the obtained clones. The
nucleotide sequence of the obtained cDNA and the amino acid
sequence deduced from said nucleotide sequence are shown in SEQ ID
NO:1 and SEQ ID NO:2.
[0075] Said cDNA is subcloned into an expression vector such as
pSVK3. The host cells, such as COS-1 cells, transformed with said
subcloned plasmid, are cultured and .alpha.1-6 fucosyltransferase
is obtained from the culture.
[0076] In the present invention, the above-mentioned transformant
cells are cultured and .alpha.1-6 fucosyltransferase is harvested
from the culture, whereby recombinant .alpha.1-6 fucosyltransferase
is obtained. The method for harvesting the enzyme from the culture
is a conventional one.
[0077] The gene encoding the porcine .alpha.1-6 fucosyltransferase
of the present invention and DNA fragments (which are the lysates
thereof) may be used for the detection of the expression of
.alpha.1-6 fucosyltransferase in the living body, and thus are
useful for the genetic diagnosis of certain diseases such as liver
cancer and cystic fibrosis.
[0078] In addition, the polypeptide that is encoded by these genes
can be used to immunologically prepare various antibodies which are
useful for diagnosis and purification of .alpha.1-6
fucosyltransferase.
[0079] The starting material for the purification of the enzyme in
this invention may be any as long as it is a human cell culture
medium exhibiting .alpha.1-6 fucosyltransferase activity. For
example, human pancreatic cancer cells, human gastric cancer cells,
human myeloma tumor cells and the like may be used as the cells
having .alpha.1-6 fucosyltransferase activity.
[0080] While the human .alpha.1-6 fucosyltransferase is present in
the cell membrane as a membrane-bound enzyme, it is cleaved by
protease at a site unaffecting the enzyme activity and released
into the culture medium as a soluble enzyme. Thus, the culture
medium can be used as a crude enzyme solution, without complicated
steps such as disruption of cells and solubilizing of the enzyme.
Besides, the use of cells capable of growth in serum-free media
enables economical production of a crude enzyme solution having a
high purity. The culture medium is concentrated and desalted, and
subjected to ion exchange chromatography, affinity chromatography
and the like to give a purified enzyme product free of contaminant
transferases and glycosidase activity.
[0081] .alpha.1-6 Fucosyltransferase is purified from human gastric
cancer cells by, for example, culturing human gastric cancer cell
MKN45 without serum and purifying the enzyme from the obtained
culture medium. In this case, .alpha.1-6 fucosyltransferase of
human gastric cancer cell MKN45 is cleaved by protease in the cells
at a site unaffecting the enzyme activity and released into culture
medium as a soluble .alpha.1-6 fucosyltransferase. Therefore, the
culture medium can be used as a crude enzyme solution, without
complicated steps such as disruption of cells and solubilizing of
the enzyme with a surfactant. The crude enzyme solution is
subjected to known purification steps to give a purified enzyme
product.
[0082] In the present invention, a serum-free culture medium of
human gastric cancer cell MKN45 is concentrated by filtration
through an ultrafiltration membrane, and then the buffer is changed
to a Tris-HCl buffer containing 5 mM 2-mercaptoethanol and 0.1%
CHAPS [3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate],
pH 7.5, to give a crude enzyme solution.
[0083] This enzyme solution is subjected to column chromatography
using Q-sepharose, GDP-hexanolamine-sepharose,
(GlcNAc.beta.1-2Man.alpha.1-6)(G-
lcNAc.beta.1-2Man.alpha.1-3)Man.beta.1-4GlucNAc.beta.1-4GlucNAc-asparagine-
-sepharose and the like to collect active fractions, from which the
fucosyltransferase of the present invention can be purified.
[0084] The physico-chemical property of human .alpha.1-6
fucosyltransferase of the present invention is as follows.
[0085] (1) Action: transferring fucose from guanosine
diphosphate-fucose to the hydroxy group at 6-position of GluNAc
closest to R of a receptor
(GlcNAc.beta.1-2Man.alpha.1-6)(GlcNAc.beta.1-2Man.alpha.1-3)Man.beta.1-4G-
lcNAc.beta.1-4GlucNAc-R wherein R is an asparagine residue or a
peptide chain carrying said residue, whereby to form
(GlcNAc.beta.1-2Man.alpha.1--
6)-(GlcNAc.beta.1-2Man.alpha.1-3)Man.beta.1-4GlcNAc.beta.1-4(Fuc.alpha.1-6-
)GlucNAc-R.
[0086] (2) Determination of enzyme activity:
[0087] The activity of the human .alpha.1-6 fucosyltransferase was
determined as follows. That is, a compound of the above-mentioned
formula, wherein the asparagine on the end of sugar chain was
fluorescence-labeled with 4-(2-pyridylamino)butylamine [PABA:
--NH.sub.2 (CH.sub.2).sub.4--NH--pyridine], was used as a substrate
for determination of enzyme activity. By the use of this substrate,
the product from the enzyme reaction, wherein fucose is transferred
by .alpha.1-6 linkage, can be assayed by detecting fluorescence by
high performance liquid chromatography.
[0088] Specifically, the determination included the following
steps. An enzyme solution (10 .mu.l) was added to a 250 mM MES
buffer containing 62.5 .mu.M of fluorescence-labeled receptor
substrate of the above formula and 625 .mu.M of a donor substrate
(GDP-fucose), pH 7.0, 40 .mu.l, and mixed. The mixture was reacted
at 37.degree. C. for one hour, and boiled for 5 minutes to stop the
reaction. The reaction mixture is subjected to high performance
liquid chromatography and the peak of the reaction product is
assayed with a fluorescence detector.
[0089] One unit of the enzyme amount corresponded to the amount
capable of producing 1 pmole of
GlcNAc.beta.1-2Man.alpha.1-6(GlcNAc.beta.1-2Man.alph-
a.1-3)-Man.beta.1-4GlcNAc.beta.1-4(Fuc.alpha.1-6)GlcNAc-R (wherein
R is Asn--NH--(CH.sub.2).sub.4--NH--Pyridine) in one minute under
these conditions.
[0090] (3) Optimum pH:
[0091] The human .alpha.1-6 fucosyltransferase shows high activity
at nearly pH 7.0-7.5, as shown by a curve in FIG. 1. In FIG. 1, the
determination was performed using 500 mM MES buffer (black circle)
at pH 4.5-7.5 and 100 mM Tris-HCl buffer (white circle) at pH
7.0-9.0.
[0092] (4) pH Stability:
[0093] The human .alpha.1-6 fucosyltransferase is stable at about
pH 4-10, particularly at pH 5-9, as shown in FIG. 2. The buffers
used for the determination were 50 mM acetate buffer (black
triangle) at pH 3.5-5.5, 50 mM MES buffer (black circle) at pH
5.5-7.5, 50 mM Tris-HCl buffer (white circle) at pH 7.5-9.0, and
sodium hydrogencarbonate buffer (white triangle) at pH 9.0-11.5.
The enzyme of the present invention was treated in each buffer at
each pH at 4.degree. C. for 5 hours, and the residual activity was
determined. FIG. 1 is a graph showing the relationship between pH
(axis of abscissa) and relative activity (%, axis of ordinate) of
the human .alpha.1-6 fucosyltransferase obtained by the present
invention, and FIG. 2 is a graph showing pH (axis of abscissa) and
residual activity (%, axis of ordinate).
[0094] (5) Optimum temperature:
[0095] The human .alpha.1-6 fucosyltransferase has an optimum
temperature at nearly 37.degree. C. as shown in FIG. 3 and is
usable at 20-40.degree. C. A frozen product thereof is stable at
-20.degree. C. for at least several months.
[0096] (6) Divalent metal ion requirement:
[0097] Many glycosyltransferases require divalent metal ion for
their activity, such as magnesium, manganese and the like. This
human .alpha.1-6 fucosyltransferase shows sufficient activity in
the absence of divalent metal ion or in the presence of EDTA, which
is a chelating agent, and does not require divalent metal ion.
[0098] (7) Molecular weight:
[0099] A purified product of the human .alpha.1-6
fucosyltransferase of the present invention shows a single band at
a molecular weight of about 60,000 by SDS-polyacrylamide gel
electrophoresis.
[0100] (8) Morphology:
[0101] While the human .alpha.1-6 fucosyltransferase is
intrinsically present in cell membrane as a membrane-bound enzyme,
it is cleaved by protease in the cultured cell at a site
unaffecting the enzyme activity and released into a culture medium
as a soluble enzyme permitting easy handling, unlike
porcine-derived .alpha.1-6 fucosyltransferase and .alpha.1-6
fucosyltransferase derived from human cystic fibrosis cells
heretofore reported.
[0102] Judging from the above properties, the human .alpha.1-6
fucosyltransferase is a novel enzyme apparently different from
conventionally known .alpha.1-6 fucosyltransferase derived from
human cystic fibrosis cells (optimum pH 6.5, molecular weights
34,000 and 39,000), in terms of optimum pH, metal requirement and
molecular weight.
[0103] The human .alpha.1-6 fucosyltransferase is used for the
following purposes.
[0104] (1) Artificial modification of sugar chain structure by
introducing fucose anew into the asparagine-linked sugar chain,
whereby cell apparatus and control mechanism of processing of sugar
chain of complex carbohydrate, as well as the role of sugar chain,
can be elucidated.
[0105] (2) Diagnosis of various diseases based on the activity of
the inventive enzyme.
[0106] (3) Diagnosis of various diseases wherein a specific
antibody against the enzyme of the present invention is prepared
and used for the diagnosis.
[0107] The present invention is a gene encoding human .alpha.1-6
fucosyltransferase, which includes, as one embodiment, a gene
encoding .alpha.1-6 fucosyltransferase and including a gene
encoding an amino acid sequence depicted in Sequence Listing, SEQ
ID NO:10. A different embodiment thereof is a gene encoding
.alpha.1-6 fucosyltransferase inclusive of nucleotide sequence
depicted in Sequence Listing, SEQ ID NO:9. A further aspect of the
present invention is a gene encoding .alpha.1-6 fucosyltransferase
and including a nucleotide sequence from 198th adenine to 1919th
guanine as depicted in Sequence Listing, SEQ ID NO:9.
[0108] One aspect of the present invention is a gene encoding
.alpha.1-6 fucosyltransferase and including a gene encoding an
amino acid sequence resulting from substitution, insertion,
deletion or addition with respect to at least one amino acid of the
amino acid sequence depicted in Sequence Listing, SEQ ID NO:10.
[0109] Another aspect of the present invention is a gene encoding
.alpha.1-6 fucosyltransferase and including a nucleotide sequence
resulting from substitution, insertion, deletion or addition with
respect to at least one nucleotide of the nucleotide sequence
depicted in Sequence Listing, SEQ ID NO:9.
[0110] The present invention also includes, as one embodiment, a
gene which hybridizes to at least a part of gene encoding
.alpha.1-6 fucosyltransferase and including nucleotide sequence
depicted in Sequence Listing, SEQ ID NO:9.
[0111] The expression vector of the present invention contains a
gene encoding the above-mentioned .alpha.1-6
fucosyltransferase.
[0112] The transformant host cell of the present invention has been
transformed with the above-mentioned expression vector.
[0113] The host cell is exemplified by microorganisms, such as
Escherichia coli, yeast, bacterial cells and the like. It also
includes animal cells such as insect cells, COS-1 cells, CHO cells
and the like, and plant cells, such as tobacco cells, Arabidopsis
cells and the like.
[0114] The vector may be any which is selected according to the
host to be transformed. In the case of Escherichia coli, for
example, pUC19 may be used; in the case of yeast, pYEUra3.TM. may
be used; in the case of insect cells, pBLUE Bac4 may be used; in
the case of COS-1 cells, pSVK3 may be used; and in the case of
tobacco cells and Arabidopsis cells, pBI may be used.
[0115] The method for preparing the recombinant .alpha.1-6
fucosyltransferase includes culturing the above-mentioned
transformant cells and harvesting .alpha.1-6 fucosyltransferase
from the culture.
[0116] According to the present invention, .alpha.1-6
fucosyltransferase alone is purified from human gastric cancer
cells, and subjected to amino acid analysis of this protein. Its
partial amino acid sequence is determined and a primer for PCR is
prepared based on the amino acid sequence. Using this primer, PCR
is performed using cDNAs derived from human gastric cancer cells as
a template to amplify a gene encoding .alpha.1-6 fucosyltransferase
to give a probe. This probe is used to screen clones containing
cDNA encoding .alpha.1-6 fucosyltransferase, from the cDNA library
derived from human gastric cancer cells. The cDNA encoding
.alpha.1-6 fucosyltransferase is isolated and used to express
.alpha.1-6 fucosyltransferase.
[0117] To be specific, the purified .alpha.1-6 fucosyltransferase
is used to analyze amino acid sequence. For example, it is
subjected to SDS-polyacrylamide gel electrophoresis, after which
the protein is transferred to PVDF membrane by electroblotting, and
the PVDF membrane containing ca. 60 kDa band is cut out and
subjected to sequencing by a protein sequencer. As a result, the
amino acid sequence of the amino terminal of .alpha.1-6
fucosyltransferase depicted in Sequence Listing, SEQ ID NO:11 is
obtained.
[0118] Separately, purified .alpha.1-6 fucosyltransferase is
subjected to SDS-polyacrylamide gel electrophoresis, along with a
protease such as lysylendopeptidase, and the peptide fragments
separated by electrophoresis are transferred to PVDF membrane by
electroblotting. Then, the band containing the peptide fragments is
cut out and subjected to sequencing with a protein sequencer. Thus,
partial amino acid sequences of .alpha.1-6 fucosyltransferase as
depicted in Sequence Listing, SEQ ID NO:12 and SEQ ID NO:13 are
obtained. Then, using these amino acid sequences, a mixed primer
for PCR is prepared. For example, a mixed primer having a
nucleotide sequence depicted in SEQ ID NO:14 is synthesized from
the amino acid sequence depicted in SEQ ID NO:12, and a mixed
primer having a nucleotide sequence depicted in SEQ ID NO:15 is
synthesized from the amino acid sequence depicted in SEQ ID NO:13,
respectively using a DNA synthesizer, and used for the screening of
cDNA of .alpha.1-6 fucosyltransferase.
[0119] For example, 36 cycles of PCR are performed to amplify the
DNA fragments of ca. 200 bp, using cDNA from human gastric cancer
cells as a template and mixed primers of SEQ ID NO:14 and SEQ ID
NO:15, wherein PCR at 94.degree. C. (30 sec), 46.degree. C. (30
sec) and 72.degree. C. (1.5 min) is one cycle.
[0120] Then, using the amplified DNA fragments as a probe, clones
containing cDNA encoding .alpha.1-6 fucosyltransferase are screened
from the cDNA library derived from human gastric cancer cells by a
plaque hybridization method. The cDNA encoding .alpha.1-6
fucosyltransferase can be isolated from the obtained clones. The
nucleotide sequence of the obtained cDNA and the amino acid
sequences deduced from said nucleotide sequence are shown in SEQ ID
NO:9 and SEQ ID NO:10.
[0121] Said cDNA is subcloned into an expression vector such as
pSVK3. The host cells such as COS-1 cells transformed with said
subcloned plasmid are cultured and .alpha.1-6 fucosyltransferase is
obtained from the culture.
[0122] In the present invention, the above-mentioned transformant
cells are cultured and .alpha.1-6 fucosyltransferase is harvested
from the culture, whereby a recombinant .alpha.1-6
fucosyltransferase is obtained.
[0123] The method for harvesting the enzyme from the culture is a
conventional one.
[0124] The gene encoding the human .alpha.1-6 fucosyltransferase of
the present invention and DNA fragments (which are the lysates
thereof) may be used for the determination of the expression of
.alpha.1-6 fucosyltransferase in the living body and thus are
useful for genetic diagnosis of certain diseases such as liver
cancer and cystic fibrosis.
[0125] In addition, the polypeptide that is encoded by these genes
can be used to immunologically prepare various antibodies which are
useful for diagnosis and purification of .alpha.1-6
fucosyltransferase.
BRIEF DESCRIPTION OF THE DRAWINGS
[0126] FIG. 1 shows optimum pH of the porcine brain .alpha.1-6
fucosyltransferase of the present invention.
[0127] FIG. 2 shows pH stability of the porcine brain .alpha.1-6
fucosyltransferase of the present invention.
[0128] FIG. 3 shows optimum temperature of the porcine brain
.alpha.1-6 fucosyltransferase of the present invention.
[0129] FIG. 4 shows optimum pH of the human .alpha.1-6
fucosyltransferase of the present invention.
[0130] FIG. 5 shows pH stability of the human .alpha.1-6
fucosyltransferase of the present invention.
[0131] FIG. 6 shows optimum temperature of the human .alpha.1-6
fucosyltransferase of the present invention.
EMBODIMENT OF THE INVENTION
[0132] The present invention is described in more detail by way of
Examples.
[0133] In the present invention, the enzyme activity is determined
as follows.
[0134] A compound of the following formula, wherein the asparagine
on the end of sugar chain had been fluorescence-labeled with
4-(2-pyridyl-amino)butylamine [PABA:
--NH(CH.sub.2).sub.4--NH-pyridine] was used as a substrate for the
determination of enzyme activity. By the use of this substrate, the
product from the enzyme reaction wherein fucose has been
transferred by .alpha.1.fwdarw.6 linkage can be assayed by
detecting the fluorescence by high performance liquid
chromatography.
[0135] Specifically, the determination includes the following
steps. A sample solution (10 .mu.l) and 1.25% Triton X-100 are
added to a 250 mM MES buffer containing 62.5 .mu.M of
fluorescence-labeled receptor substrate of the above formula and
625 .mu.M of a donor substrate (GDP-fucose), pH 7.0, 40 .mu.l, and
mixed. The mixture is reacted at 37.degree. C. for one hour, and
boiled for 5 minutes to stop the reaction. The reaction mixture is
subjected to high performance liquid chromatography and the peak of
the reaction product is assayed with a fluorescence detector. One
unit of the enzyme amount corresponds to the amount capable of
producing 1 pmole of GlcNAc.beta.1-2Man.alpha.1-6(GlcNA-
c.beta.1-2Man.alpha.1-3)-Man.beta.1-4GlcNAc.beta.1-4(Fuc.alpha.1-6)GlcNAc--
R (wherein R is Asn--NH--(CH.sub.2).sub.4--NH--pyridine) in one
minute under these conditions.
EXAMPLE 1
[0136] (1) Preparation of Porcine Brain Lysate and Crude Extract
Solution
[0137] Porcine brain (100 g) was disrupted in a Waring blender in a
20 mM potassium phosphate buffer (pH 7.0) and membrane fractions
were collected by ultracentrifugation. The membrane fractions were
extracted with the same buffer containing Triton X-100
(concentration 0.5%) to extract the enzyme. After the extraction,
the supernatants were collected by centrifugation to give an
extract containing a crude enzyme.
[0138] (2) Purification of Enzyme from Crude Extract Solution
[0139] A
GlcNAc.beta.1-2Man.alpha.1-6(GlcNAc.beta.1l-2Man.alpha.1-3)Man.be-
ta.1-4GlcNAc.beta.1-4GlcNAc-asparagine-sepharose column (column of
asialoagalactoglycopeptide derived from transferrin) was
equilibrated with a 20 mM potassium phosphate buffer (pH 7.0)
containing 0.05% Triton X-100 and 50 mM KCl, and the crude extract
solution prepared in (1) above was applied. The column was washed
with said buffer until the protein was not detected in the
unadsorbed fractions. The active fractions were eluted with the
same buffer containing 1M KCl. Then, the active fractions of the
enzyme were concentrated using an ultrafiltration membrane and
desalted, and applied to a GDP-hexanolamine-sepharose column
equilibrated with the same buffer. The elution was performed using
the same buffer containing 100 mM GDP. Then, the active fractions
were collected and concentrated using an ultrafiltration membrane,
and desalted to give porcine brain .alpha.1-6 fucosyltransferase.
The porcine brain .alpha.1-6 fucosyltransferase thus obtained
showed a single band at a molecular weight of about 60,000 by
SDS-polyacrylamide gel electrophoresis. No other bands ascribed to
impurities were found and the enzyme was free of other transferase
activities, thus indicating that the enzyme obtained was highly
purified.
[0140] The optimum pH (determined by changing the pH of buffer) of
the enzyme of the present invention is shown in FIG. 1. The enzyme
showed high activity at around pH 7.0-7.5. The buffer used was 200
mM MES buffer (black circle). In this graph, the axis of abscissa
shows pH of .alpha.1-6 fucosyltransferase obtained in the present
invention and the axis of ordinate shows relative activity (%).
[0141] The pH stability of the enzyme of the present invention was
examined in the same manner. FIG. 2 shows residual activity after
treating the enzyme in each buffer at each pH, 4.degree. C. for 5
hours. The enzyme was comparatively stable at about pH 4-10, and
particularly stable at pH 5-9. The buffers used were 50 mM acetate
buffer (black triangle) at pH 3.5-5.5, 50 mM MES buffer (black
circle) at pH 5.5-7.5, 50 mM Tris-HCl buffer (white circle) at pH
7.5-9.0, and sodium hydrogencarbonate buffer (white triangle) at pH
9.0-11.5. The axis of abscissa of the graph shows pH of .alpha.1-6
fucosyltransferase obtained in the present invention and the axis
of ordinate shows residual activity (%).
[0142] As shown in FIG. 3, the optimum temperature of the enzyme of
the present invention was found to be at about 37.degree. C. and
the enzyme was considered to retain sufficient activity in the
range of 20-40.degree. C. A frozen product thereof was stable at
-20.degree. C. for at least several months. The buffer used was 200
mM MES buffer (black circle), pH 7.0. The axis of abscissa of the
graph shows treatment temperature (.degree. C.) and the axis of
ordinate shows relative activity (%) of the .alpha.1-6
fucosyltransferase obtained in the present invention.
[0143] While many glycosyltransferases require divalent metal ion
for their activity, such as magnesium, manganese and the like, the
enzyme showed sufficient activity in the absence of such divalent
metal ion. Inasmuch as it showed sufficient activity even in the
presence of 5 mM EDTA, which is a chelating agent, it is concluded
that the enzyme does not require a divalent metal ion.
EXAMPLE 2
Determination of Amino Terminal Amino Acid Sequence of Porcine
Brain .alpha.1-6 Fucosyltransferase
[0144] Purified porcine brain .alpha.1-6 fucosyltransferase (5
.mu.g) was subjected to SDS-polyacrylamide gel electrophoresis,
after which the protein was transferred to PVDF membrane
(Millipore) by electroblotting. The PVDF membrane was stained with
Coomassie Brilliant Blue G250, and a single band of porcine brain
.alpha.1-6 fucosyltransferase was detected at 60 kDa.
[0145] Then, the PVDF membrane containing said band was cut out,
and, after destaining with 50% methanol, subjected to Biosystem
473A protein sequencer (Applied Biosystems) to determine amino
terminal amino acid sequence of .alpha.1-6 fucosyltransferase. The
amino acid sequence determined is depicted in Sequence Listing, SEQ
ID NO:3.
EXAMPLE 3
Determination of Partial Amino Acid Sequence of Porcine Brain
.alpha.1-6 Fucosyltransferase
[0146] Purified porcine brain .alpha.1-6 fucosyltransferase (13
.mu.g) was subjected to SDS-polyacrylamide gel electrophoresis,
after which the protein was transferred to PVDF membrane
(Millipore) by electroblotting. The PVDF membrane was stained with
Coomassie Brilliant Blue G250, and a single band of porcine brain
.alpha.1-6 fucosyltransferase was detected at 60 kDa.
[0147] Then, the PVDF membrane containing said band was cut out and
destained with 50% methanol. Said PVDF membrane section was treated
in 100 mM Tris-HCl buffer-5% acetonitrile (pH 8.2) containing 1
.mu.g of lysylendopeptidase, at 37.degree. C. for 12 hours for
proteolysis. The PVDF membrane section which underwent proteolysis
was ultrasonicated to extract the proteolysis product. The
proteolysis product thus obtained was separated by a reversed phase
high performance liquid chromatography using a C-18 column to give
3 peptide fragments. The substance containing said peptide
fragments, which was separated by the reversed phase high
performance liquid chromatography, was applied to polybrene-coated
precycled glass fiber filter activated with trifluoroacetate and
dried, and then subjected to Biosystem 473A protein sequencer
(Applied Biosystems) to determine partial amino acid sequence of
porcine brain .alpha.1-6 fucosyltransferase. The determined amino
acid sequence is depicted in Sequence Listing, SEQ ID NOs:4-6.
EXAMPLE 4
Preparation of Probe DNA by PCR
[0148] Mixed primers shown in SEQ ID NO:7 and SEQ ID NO:8 were
synthesized from the amino acid sequences obtained in Examples 2
and 3. The mixed primer shown in SEQ ID NO:7 was used as a sense
primer, and the mixed primer shown in SEQ ID NO:8 was used as an
antisense primer for PCR. To be specific, 25 cycles of PCR were
performed wherein PCR at 94.degree. C. (1 min), 55.degree. C. (2
min) and 72.degree. C. (3 min) using 2 .mu.g of porcine
brain-derived cDNA, 25 pmole of sense primer (mixed primer shown in
SEQ ID NO: 7), 25 pmole of antisense primer (mixed primer shown in
SEQ ID NO:8) and a reaction mixture (50 .mu.l) of 50 mM potassium
chloride-10 mM Tris-HCl buffer (pH 8.3)-1.5 mM magnesium
chloride-0.001% gelatin-200 .mu.M dNTP, containing 2.5 units of Taq
DNA polymerase was one cycle.
[0149] The reaction mixture (10 .mu.l) after PCR was subjected to
0.7% agarose gel electrophoresis to confirm the PCR reaction
product DNA fragments. As a result of PCR performed using a mixed
primer shown in SEQ ID NO:7 and a mixed primer shown in SEQ ID NO:8
in combination, a 1.45 kbp DNA fragment was confirmed by agarose
gel electrophoresis.
[0150] This DNA fragment was subcloned into plasmid pT7BLUEt-Vector
(Novagen) and nucleotide sequence was confirmed. As a result, a DNA
corresponding to the amino acid sequence depicted in Sequence
Listing, SEQ ID Nos:3-6 was detected, whereby the DNA fragment was
confirmed to be a part of .alpha.1-6 fucosyltransferase gene.
EXAMPLE 5
Isolation of Porcine Brain .alpha.1-6 Fucosyltransferase Gene
[0151] The DNA fragments obtained in Example 4 were labeled with
.alpha..sup.-32P dCTP (3000 Ci/mmol, Amersham) and used as a probe
to screen clones containing cDNA encoding .alpha.1-6
fucosyltransferase, from porcine brain-derived .lambda.gt11 cDNA
library (Clonetech) by plaque hybridization method.
[0152] As a result of screening of about 400,000 plaques, 5
positive clones c1, c2, c3, c4 and c5 were obtained. Said clones c1
and c2 were postulated to contain a full length .alpha.1-6
fucosyltransferase gene in view of their length. Thus, the
nucleotide sequences of c1 and c2 were determined, and a nucleotide
sequence depicted in SEQ ID NO:1 was obtained.
EXAMPLE 6
Expression of Porcine Brain .alpha.1-6 Fucosyltransferase Gene
[0153] The coding region of .alpha.1-6 fucosyltransferase gene was
subcloned into expression vector pSVK3 from clones containing cDNA
encoding porcine brain .alpha.1-6 fucosyltransferase obtained in
Example 5. The expression vector containing said .alpha.1-6
fucosyltransferase gene was introduced into COS-1 cells. After 48
hours of incubation, culture cells were collected and the cells
were disrupted. The enzyme activity of .alpha.1-6
fucosyltransferase in the obtained lysate was determined.
[0154] As a control, the enzyme activity of .alpha.1-6
fucosyltransferase in the lysate of COS-1 cells, into which mock
pSVK3 had been introduced, was determined. As a result, the control
hardly showed activity, whereas COS-cells into which the expression
vector containing said .alpha.1-6 fucosyltransferase gene had been
introduced, showed a high activity of 2360 nmole/h/mg protein.
EXAMPLE 7
[0155] (1) Preparation of crude enzyme solution from serum-free
culture medium of human gastric cancer cell MKN45
[0156] Human gastric cancer cell MKN45 was cultured in a serum-free
medium (RPMI1640 medium:Ham's F-12 medium=1:1) containing sodium
selenite and canamycin, at 37.degree. C. in 5% CO.sub.2. The
resulting serum-free culture medium (100 l) was concentrated to 2 l
by ultrafiltration. The buffer was changed to a Tris-HCl buffer
containing 5 mM 2-mercaptoethanol and 0.1% CHAPS
[3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate- ], pH
7.5, to give a crude enzyme solution. This crude enzyme solution
was subjected to column chromatography using Q-sepharose,
GDP-hexanolamine-sepharose,
(GlcNAc.beta.1-2Man.alpha.1-6)(GlcNAc.beta.1--
2Man.alpha.1-3)Man.beta.1-4GlcNAc.beta.1-4GlcNAc-asparagine-sepharose
and the like to collect active fractions, from which the human
.alpha.1-6 fucosyltransferase of the present invention could be
purified.
[0157] (2) Preparation of enzyme
[0158] The crude enzyme solution obtained in (1) above was
subjected to the following purification steps. That is, the
solution was applied to a Q-sepharose column equilibrated with
Tris-HCl buffer containing 5 mM 2-mercaptoethanol and 0.1% CHAPS,
pH 7.5. The column was washed with a 5-fold amount of the same
buffer and the active fractions eluted with the same buffer
containing 0.1 M NaCl were collected. The active fractions were
concentrated using an ultrafiltration membrane and the buffer was
changed to Tris-HCl buffer containing 5 mM 2-mercaptoethanol and
0.7% CHAPS, pH 7.5, after which the fractions were applied to
GDP-hexanolamine-sepharose column equilibrated with the same
buffer. The elution was performed by the linear gradient of NaCl
from 0 M to 0.5 M.
[0159] The active fractions from 0.15 M to 0.3 M were collected and
concentrated using an ultrafiltration membrane. After desalting,
the fractions were applied to a
(GlcNAc.beta.1-2Man.alpha.1-6)(GlcNAc.beta.1--
2Man.alpha.1-3)Man.beta.1-4GlcNAc.beta.1-4GlcNAc-asparagine-sepharose
column equilibrated with Tris-HCl buffer containing 5 mM
2-mercaptoethanol and 0.7% CHAPS, pH 7.5. The elution was performed
by the linear gradient of NaCl from 0 M to 0.5 M.
[0160] The active fractions from 0.2 M to 0.5 M were collected and
concentrated using an ultrafiltration membrane. Desalting gave
human .alpha.1-6 fucosyltransferase.
[0161] The thus-obtained human .alpha.1-6 fucosyltransferase
fractions showed a single band at a molecular weight of about
60,000 by SDS-polyacrylamide gel electrophoresis. No other
activities, such as those of transferase and glycosidase, were
found and this purified enzyme was sufficiently usable as a reagent
for sugar chain studies.
[0162] The optimum pH (determined by changing the pH of buffer) of
the enzyme of the present invention is shown in FIG. 4. The enzyme
showed high activity at around pH 7.0-7.5. In this graph, the black
circle shows the case when MES buffer was used and white circle
shows the case when Tris-HCl buffer was used.
[0163] The pH stability of the enzyme of the present invention was
examined in the same manner. FIG. 5 shows residual activity after
treating the enzyme in each buffer at each pH, 4.degree. C. for 5
hours. The enzyme was comparatively stable at about pH 4-10, and
particularly stable at pH 5-9. In this graph, the black triangle
shows the case when acetate buffer was used, the black circle shows
the case when MES buffer was used, the white circle shows the case
when Tris-HCl buffer was used, and the white triangle shows the
case when sodium hydrogencarbonate buffer was used.
[0164] As shown in FIG. 6, the optimum temperature of the enzyme of
the present invention was found to be at about 37.degree. C. and
the enzyme was considered to retain sufficient activity in the
range of 20-40.degree. C. The frozen product was stable at
-20.degree. C. for at least several months.
[0165] The enzyme showed sufficient activity in the absence of
divalent metal ion. Inasmuch as it showed sufficient activity even
in the presence of 5 mM EDTA, which is a chelating agent, it is
concluded that the enzyme does not require a divalent metal
ion.
EXAMPLE 8
Determination of Amino Acid Sequence of Human .alpha.1-6
Fucosyltransferase
[0166] Purified human .alpha.1-6 fucosyltransferase (1 .mu.g) was
subjected to SDS-polyacrylamide gel electrophoresis, after which
the protein was transferred to PVDF membrane (Millipore) by
electroblotting. The PVDF membrane was stained with Coomassie
Brilliant Blue G250, and a single band of .alpha.1-6
fucosyltransferase was detected at about 60 kDa. Then, the PVDF
membrane containing said band was cut out, and, after destaining
with 50% methanol, subjected to Biosystem 473A protein sequencer
(Applied Biosystems) to determine amino terminal amino acid
sequence of human .alpha.1-6 fucosyltransferase. The amino acid
sequence determined is depicted in Sequence Listing, SEQ ID
NO:11.
EXAMPLE 9
Determination of Partial Amino Acid Sequence of Human .alpha.1-6
Fucosyltransferase
[0167] Purified human .alpha.1-6 fucosyltransferase (5 .mu.g) was
mixed with lysine endopeptidase and subjected to SDS-polyacrylamide
gel electrophoresis, after which the peptide fragments were
transferred to PVDF membrane (Millipore) by electroblotting. The
PVDF membrane was stained with Coomassie Brilliant Blue G250, and
several bands containing peptide fragments, inclusive of two main
bands, were detected. Then, the PVDF membrane containing each main
band was cut out and destained with 50% methanol. Said membrane was
subjected to Biosystem 473A protein sequencer (Applied Biosystems)
to determine the internal partial amino acid sequence of human
.alpha.1-6 fucosyltransferase. The determined amino acid sequences
are depicted in Sequence Listing, SEQ ID NO:12 and SEQ ID
NO:13.
EXAMPLE 10
Preparation of Probe DNA by PCR
[0168] Mixed primers shown by SEQ ID NO:14 and SEQ ID NO:15 were
synthesized from the amino acid sequences obtained in Example 9.
The mixed primer shown in SEQ ID NO:14 was used as a sense primer,
and the mixed primer shown in SEQ ID NO:15 was used as an antisense
primer for PCR. To be specific, 36 cycles of PCR were performed
wherein PCR at 94.degree. C. (30 sec), 46.degree. C. (30 sec) and
72.degree. C. (1.5 min) using 2 .mu.g of human-derived cDNA, 25
pmole of sense primer (mixed primer shown in SEQ ID NO: 14), 25
pmole of antisense primer (mixed primer shown in SEQ ID NO:15) and
a reaction mixture (50 .mu.l) of 50 mM potassium chloride-10 mM
Tris-HCl buffer (pH 8.3)-1.5 mM magnesium chloride-0.001%
gelatin-200 .mu.M dNTP, containing 2.5 units of Taq DNA polymerase,
was one cycle.
[0169] The reaction mixture (10 .mu.l) after PCR was subjected to
2.0% agarose gel electrophoresis to confirm the PCR reaction
product DNA fragments. As a result, about 200 bp DNA fragment was
confirmed by agarose gel electrophoresis.
[0170] This DNA fragment was subcloned into plasmid pT7BLUEt-Vector
(Novagen) and the nucleotide sequence was confirmed. As a result,
the DNA fragment was found to encode the amino acid sequence
depicted in Sequence Listing, SEQ ID NO:12 and SEQ ID NO:13,
whereby the DNA fragment was confirmed to be a part of .alpha.1-6
fucosyltransferase gene.
EXAMPLE 11
Isolation of Human .alpha.1-6 Fucosyltransferase Gene
[0171] The DNA fragment obtained in Example 10 was labeled with
[.alpha.-.sup.32P]dCTP (3000 Ci/mmol, Amersham) and used as a probe
to screen clones containing cDNA encoding human .alpha.1-6
fucosyltransferase, from human gastric cancer cell MKN45-derived
.lambda. ZAP cDNA library by plaque hybridization method. As a
result of screening of about 2,000,000 plaques, 8 positive clones
c1 to c8 were obtained. Said clones c1 to c7 were postulated to
contain a full length .alpha.1-6 fucosyltransferase gene in view of
the restriction enzyme cleavage site and their length. The
nucleotide sequences of c1 and c2 were determined, as a result of
which a nucleotide sequence depicted in SEQ ID NO:9 was
obtained.
EXAMPLE 12
Expression of Human .alpha.1-6 Fucosyltransferase
[0172] The coding region of human .alpha.1-6 fucosyltransferase
gene was subcloned into expression vector pSVK3 from clones
containing cDNA encoding the human .alpha.1-6 fucosyltransferase
obtained in Example 11. An expression vector containing said
.alpha.1-6 fucosyltransferase gene was introduced into COS-1 cells.
After 48 hours of incubation, culture cells were collected and
disrupted. The enzyme activity of .alpha.1-6 fucosyltransferase in
the obtained lysate was determined. As a control, the enzyme
activity of .alpha.1-6 fucosyltransferase in the lysate of COS-1
cells, into which mock pSVK3 had been introduced, was determined.
As a result, the control hardly showed activity, whereas COS-cells,
into which the expression vector containing said .alpha.1-6
fucosyltransferase gene had been introduced, showed a high activity
of 2130 nmole/h/mg protein.
Industrial Applicability
[0173] The porcine .alpha.1-6 fucosyltransferase of the present
invention differs significantly from known human .alpha.1-6
fucosyltransferase in physico-chemical properties, and shows
activity under optimum reaction conditions which are closer to the
physiological conditions.
[0174] The .alpha.1-6 fucosyltransferase derived from human also
shows physicochemical properties markedly different from those of
known human .alpha.1-6 fucosyltransferase, showing activity under
optimum reaction conditions which are closer to the physiological
conditions. Hence, the present invention enables development of
glyco-technology inclusive of modification and synthesis of sugar
chain, and of a method for diagnosis of diseases, such as cancer,
which includes the use of an antibody specific for the enzyme of
the present invention or the gene thereof.
Sequence CWU 1
1
15 1 1728 DNA Pig CDS (1)...(1728) 1 atg cgg cca tgg act ggt tcg
tgg cgt tgg att atg ctc att ctt ttt 48 Met Arg Pro Trp Thr Gly Ser
Trp Arg Trp Ile Met Leu Ile Leu Phe 1 5 10 15 gcc tgg ggg acc ttg
cta ttt tac ata ggt ggt cac ttg gta cga gat 96 Ala Trp Gly Thr Leu
Leu Phe Tyr Ile Gly Gly His Leu Val Arg Asp 20 25 30 aat gac cac
tct gat cac tct agc cga gaa ctg tcc aag att ttg gca 144 Asn Asp His
Ser Asp His Ser Ser Arg Glu Leu Ser Lys Ile Leu Ala 35 40 45 aag
ctg gaa cgc tta aaa caa caa aat gaa gac ttg agg aga atg gct 192 Lys
Leu Glu Arg Leu Lys Gln Gln Asn Glu Asp Leu Arg Arg Met Ala 50 55
60 gga tct ctc cga ata cca gaa ggc ccc att gat cag ggg cca gct tca
240 Gly Ser Leu Arg Ile Pro Glu Gly Pro Ile Asp Gln Gly Pro Ala Ser
65 70 75 80 gga aga gtt cgt gct tta gaa gag caa ttt atg aag gcc aaa
gaa cag 288 Gly Arg Val Arg Ala Leu Glu Glu Gln Phe Met Lys Ala Lys
Glu Gln 85 90 95 att gaa aat tat aag aaa caa act aaa aat ggt cca
ggg aag gat cat 336 Ile Glu Asn Tyr Lys Lys Gln Thr Lys Asn Gly Pro
Gly Lys Asp His 100 105 110 gaa atc cta agg agg agg att gaa aat gga
gct aaa gag ctc tgg ttt 384 Glu Ile Leu Arg Arg Arg Ile Glu Asn Gly
Ala Lys Glu Leu Trp Phe 115 120 125 ttt cta caa agt gag ttg aag aaa
tta aag aat tta gaa gga aat gaa 432 Phe Leu Gln Ser Glu Leu Lys Lys
Leu Lys Asn Leu Glu Gly Asn Glu 130 135 140 ctc caa aga cat gca gat
gaa ttt cta tca gat ttg gga cat cat gaa 480 Leu Gln Arg His Ala Asp
Glu Phe Leu Ser Asp Leu Gly His His Glu 145 150 155 160 agg tct ata
atg acg gat cta tac tac ctc agt caa aca gat ggg gca 528 Arg Ser Ile
Met Thr Asp Leu Tyr Tyr Leu Ser Gln Thr Asp Gly Ala 165 170 175 ggt
gat tgg cgt gaa aag gag gcc aaa gat ctg aca gag ctg gtc cag 576 Gly
Asp Trp Arg Glu Lys Glu Ala Lys Asp Leu Thr Glu Leu Val Gln 180 185
190 cgg aga ata aca tat ctt cag aat ccc aag gac tgc agc aaa gcc aag
624 Arg Arg Ile Thr Tyr Leu Gln Asn Pro Lys Asp Cys Ser Lys Ala Lys
195 200 205 aag cta gtg tgt aat atc aac aaa ggc tgt ggc tat ggc tgt
cag ctc 672 Lys Leu Val Cys Asn Ile Asn Lys Gly Cys Gly Tyr Gly Cys
Gln Leu 210 215 220 cat cat gta gtg tac tgc ttt atg att gca tat ggc
acc cag cga aca 720 His His Val Val Tyr Cys Phe Met Ile Ala Tyr Gly
Thr Gln Arg Thr 225 230 235 240 ctc gcc ttg gaa tct cac aat tgg cgc
tac gct act ggg gga tgg gaa 768 Leu Ala Leu Glu Ser His Asn Trp Arg
Tyr Ala Thr Gly Gly Trp Glu 245 250 255 act gtg ttt aga cct gta agt
gag acg tgc aca gac aga tct ggc agc 816 Thr Val Phe Arg Pro Val Ser
Glu Thr Cys Thr Asp Arg Ser Gly Ser 260 265 270 tcc act gga cat tgg
tca ggt gaa gta aag gac aaa aat gtt cag gtg 864 Ser Thr Gly His Trp
Ser Gly Glu Val Lys Asp Lys Asn Val Gln Val 275 280 285 gtt gag ctc
ccc att gta gac agt gtt cat cct cgt cct cca tat tta 912 Val Glu Leu
Pro Ile Val Asp Ser Val His Pro Arg Pro Pro Tyr Leu 290 295 300 ccc
ctg gct gtc cca gaa gac ctt gca gat cga ctt gta cga gtc cat 960 Pro
Leu Ala Val Pro Glu Asp Leu Ala Asp Arg Leu Val Arg Val His 305 310
315 320 ggt gat cct gca gtg tgg tgg gta tcc cag ttt gtc aag tac ttg
att 1008 Gly Asp Pro Ala Val Trp Trp Val Ser Gln Phe Val Lys Tyr
Leu Ile 325 330 335 cgc cca caa ccc tgg ctg gaa aag gaa ata gaa gag
gcc acc aag aag 1056 Arg Pro Gln Pro Trp Leu Glu Lys Glu Ile Glu
Glu Ala Thr Lys Lys 340 345 350 cta ggc ttc aaa cat cca gtt att gga
gtc cat gtt aga cgc aca gac 1104 Leu Gly Phe Lys His Pro Val Ile
Gly Val His Val Arg Arg Thr Asp 355 360 365 aaa gtg gga gcg gaa gca
gcc ttc cat ccc att gag gaa tac acg gtg 1152 Lys Val Gly Ala Glu
Ala Ala Phe His Pro Ile Glu Glu Tyr Thr Val 370 375 380 cac gtt gaa
gaa gac ttt cag ctt ctt gct cgc aga atg caa gtg gat 1200 His Val
Glu Glu Asp Phe Gln Leu Leu Ala Arg Arg Met Gln Val Asp 385 390 395
400 aaa aaa agg gtg tat ttg gcc aca gat gac cct gct ttg tta aaa gag
1248 Lys Lys Arg Val Tyr Leu Ala Thr Asp Asp Pro Ala Leu Leu Lys
Glu 405 410 415 gca aaa aca aag tac ccc agt tat gaa ttt att agt gat
aac tct atc 1296 Ala Lys Thr Lys Tyr Pro Ser Tyr Glu Phe Ile Ser
Asp Asn Ser Ile 420 425 430 tct tgg tca gct gga cta cat aat cga tat
aca gaa aat tca ctt cgg 1344 Ser Trp Ser Ala Gly Leu His Asn Arg
Tyr Thr Glu Asn Ser Leu Arg 435 440 445 ggt gtg atc ctg gat ata cac
ttt ctc tcc cag gca gac ttc cta gtg 1392 Gly Val Ile Leu Asp Ile
His Phe Leu Ser Gln Ala Asp Phe Leu Val 450 455 460 tgt act ttt tca
tcg cag gtc tgt aga gtt gct tat gaa atc atg caa 1440 Cys Thr Phe
Ser Ser Gln Val Cys Arg Val Ala Tyr Glu Ile Met Gln 465 470 475 480
gcg ctg cat cct gat gcc tct gcg aac ttc cgt tct ttg gat gac atc
1488 Ala Leu His Pro Asp Ala Ser Ala Asn Phe Arg Ser Leu Asp Asp
Ile 485 490 495 tac tat ttt gga ggc cca aat gcc cac aac caa att gcc
att tat cct 1536 Tyr Tyr Phe Gly Gly Pro Asn Ala His Asn Gln Ile
Ala Ile Tyr Pro 500 505 510 cac caa cct cga act gaa gga gaa atc ccc
atg gaa cct gga gat att 1584 His Gln Pro Arg Thr Glu Gly Glu Ile
Pro Met Glu Pro Gly Asp Ile 515 520 525 att ggt gtg gct gga aat cac
tgg gat ggc tat cct aaa ggt gtt aac 1632 Ile Gly Val Ala Gly Asn
His Trp Asp Gly Tyr Pro Lys Gly Val Asn 530 535 540 aga aaa ctg gga
agg acg ggc cta tat ccc tcc tac aaa gtt cga gag 1680 Arg Lys Leu
Gly Arg Thr Gly Leu Tyr Pro Ser Tyr Lys Val Arg Glu 545 550 555 560
aag ata gaa aca gtc aag tac ccc aca tat ccc gag gct gac aag taa
1728 Lys Ile Glu Thr Val Lys Tyr Pro Thr Tyr Pro Glu Ala Asp Lys *
565 570 575 2 575 PRT Pig 2 Met Arg Pro Trp Thr Gly Ser Trp Arg Trp
Ile Met Leu Ile Leu Phe 1 5 10 15 Ala Trp Gly Thr Leu Leu Phe Tyr
Ile Gly Gly His Leu Val Arg Asp 20 25 30 Asn Asp His Ser Asp His
Ser Ser Arg Glu Leu Ser Lys Ile Leu Ala 35 40 45 Lys Leu Glu Arg
Leu Lys Gln Gln Asn Glu Asp Leu Arg Arg Met Ala 50 55 60 Gly Ser
Leu Arg Ile Pro Glu Gly Pro Ile Asp Gln Gly Pro Ala Ser 65 70 75 80
Gly Arg Val Arg Ala Leu Glu Glu Gln Phe Met Lys Ala Lys Glu Gln 85
90 95 Ile Glu Asn Tyr Lys Lys Gln Thr Lys Asn Gly Pro Gly Lys Asp
His 100 105 110 Glu Ile Leu Arg Arg Arg Ile Glu Asn Gly Ala Lys Glu
Leu Trp Phe 115 120 125 Phe Leu Gln Ser Glu Leu Lys Lys Leu Lys Asn
Leu Glu Gly Asn Glu 130 135 140 Leu Gln Arg His Ala Asp Glu Phe Leu
Ser Asp Leu Gly His His Glu 145 150 155 160 Arg Ser Ile Met Thr Asp
Leu Tyr Tyr Leu Ser Gln Thr Asp Gly Ala 165 170 175 Gly Asp Trp Arg
Glu Lys Glu Ala Lys Asp Leu Thr Glu Leu Val Gln 180 185 190 Arg Arg
Ile Thr Tyr Leu Gln Asn Pro Lys Asp Cys Ser Lys Ala Lys 195 200 205
Lys Leu Val Cys Asn Ile Asn Lys Gly Cys Gly Tyr Gly Cys Gln Leu 210
215 220 His His Val Val Tyr Cys Phe Met Ile Ala Tyr Gly Thr Gln Arg
Thr 225 230 235 240 Leu Ala Leu Glu Ser His Asn Trp Arg Tyr Ala Thr
Gly Gly Trp Glu 245 250 255 Thr Val Phe Arg Pro Val Ser Glu Thr Cys
Thr Asp Arg Ser Gly Ser 260 265 270 Ser Thr Gly His Trp Ser Gly Glu
Val Lys Asp Lys Asn Val Gln Val 275 280 285 Val Glu Leu Pro Ile Val
Asp Ser Val His Pro Arg Pro Pro Tyr Leu 290 295 300 Pro Leu Ala Val
Pro Glu Asp Leu Ala Asp Arg Leu Val Arg Val His 305 310 315 320 Gly
Asp Pro Ala Val Trp Trp Val Ser Gln Phe Val Lys Tyr Leu Ile 325 330
335 Arg Pro Gln Pro Trp Leu Glu Lys Glu Ile Glu Glu Ala Thr Lys Lys
340 345 350 Leu Gly Phe Lys His Pro Val Ile Gly Val His Val Arg Arg
Thr Asp 355 360 365 Lys Val Gly Ala Glu Ala Ala Phe His Pro Ile Glu
Glu Tyr Thr Val 370 375 380 His Val Glu Glu Asp Phe Gln Leu Leu Ala
Arg Arg Met Gln Val Asp 385 390 395 400 Lys Lys Arg Val Tyr Leu Ala
Thr Asp Asp Pro Ala Leu Leu Lys Glu 405 410 415 Ala Lys Thr Lys Tyr
Pro Ser Tyr Glu Phe Ile Ser Asp Asn Ser Ile 420 425 430 Ser Trp Ser
Ala Gly Leu His Asn Arg Tyr Thr Glu Asn Ser Leu Arg 435 440 445 Gly
Val Ile Leu Asp Ile His Phe Leu Ser Gln Ala Asp Phe Leu Val 450 455
460 Cys Thr Phe Ser Ser Gln Val Cys Arg Val Ala Tyr Glu Ile Met Gln
465 470 475 480 Ala Leu His Pro Asp Ala Ser Ala Asn Phe Arg Ser Leu
Asp Asp Ile 485 490 495 Tyr Tyr Phe Gly Gly Pro Asn Ala His Asn Gln
Ile Ala Ile Tyr Pro 500 505 510 His Gln Pro Arg Thr Glu Gly Glu Ile
Pro Met Glu Pro Gly Asp Ile 515 520 525 Ile Gly Val Ala Gly Asn His
Trp Asp Gly Tyr Pro Lys Gly Val Asn 530 535 540 Arg Lys Leu Gly Arg
Thr Gly Leu Tyr Pro Ser Tyr Lys Val Arg Glu 545 550 555 560 Lys Ile
Glu Thr Val Lys Tyr Pro Thr Tyr Pro Glu Ala Asp Lys 565 570 575 3
26 PRT Artificial Sequence Primer 3 Lys Gln Thr Lys Asn Gly Pro Gly
Lys Asp His Glu Ile Leu Arg Arg 1 5 10 15 Arg Ile Glu Asn Gly Ala
Lys Glu Leu Gln 20 25 4 10 PRT Artificial Sequence Primer 4 Lys Tyr
Pro Thr Tyr Pro Glu Ala Asp Lys 1 5 10 5 12 PRT Artificial Sequence
Primer 5 Lys Tyr Leu Ile Arg Pro Gln Pro Trp Leu Glu Lys 1 5 10 6
14 PRT Artificial Sequence Primer 6 Lys Arg Val Tyr Leu Ala Thr Asp
Asp Pro Ala Leu Leu Lys 1 5 10 7 20 DNA Artificial Sequence Primer
7 aarsaracna araayggncc 20 8 20 DNA Artificial Sequence Primer 8
tcnggrtang tnggrtaytt 20 9 2100 DNA Homo sapiens CDS (198)...(1925)
9 aagcttccta cacatatcac caggaggatc tctttgaaag attcactgca ggactaccag
60 agagaataat ttgtctgaag catcatgtgt tgaaacaaca gaagtctatt
cacctgtgca 120 ctaactagaa acagagttac aatgttttca attctttgag
ctccaggact ccagggaagt 180 gagttgaaaa tctgaaa atg cgg cca tgg act
ggt tcc tgg cgt tgg att 230 Met Arg Pro Trp Thr Gly Ser Trp Arg Trp
Ile 1 5 10 atg ctc att ctt ttt gcc tgg ggg acc ttg ctg ttt tat ata
ggt ggt 278 Met Leu Ile Leu Phe Ala Trp Gly Thr Leu Leu Phe Tyr Ile
Gly Gly 15 20 25 cac ttg gta cga gat aat gac cat cct gat cac tct
agc cga gaa ctg 326 His Leu Val Arg Asp Asn Asp His Pro Asp His Ser
Ser Arg Glu Leu 30 35 40 tcc aag att ctg gca aag ctt gaa cgc tta
aaa cag cag aat gaa gac 374 Ser Lys Ile Leu Ala Lys Leu Glu Arg Leu
Lys Gln Gln Asn Glu Asp 45 50 55 ttg agg cga atg gcc gaa tct ctc
cgg ata cca gaa ggc cct att gat 422 Leu Arg Arg Met Ala Glu Ser Leu
Arg Ile Pro Glu Gly Pro Ile Asp 60 65 70 75 cag ggg cca gct ata gga
aga gta cgc gtt tta gaa gag cag ctt gtt 470 Gln Gly Pro Ala Ile Gly
Arg Val Arg Val Leu Glu Glu Gln Leu Val 80 85 90 aag gcc aaa gaa
cag att gaa aat tac aag aaa cag acc aga aat ggt 518 Lys Ala Lys Glu
Gln Ile Glu Asn Tyr Lys Lys Gln Thr Arg Asn Gly 95 100 105 ctg ggg
aag gat cat gaa atc ctg agg agg agg att gaa aat gga gct 566 Leu Gly
Lys Asp His Glu Ile Leu Arg Arg Arg Ile Glu Asn Gly Ala 110 115 120
aaa gag ctc tgg ttt ttc cta cag agt gaa ttg aag aaa tta aag aac 614
Lys Glu Leu Trp Phe Phe Leu Gln Ser Glu Leu Lys Lys Leu Lys Asn 125
130 135 tta gaa gga aat gaa ctc caa aga cat gca gat gaa ttt ctt ttg
gat 662 Leu Glu Gly Asn Glu Leu Gln Arg His Ala Asp Glu Phe Leu Leu
Asp 140 145 150 155 tta gga cat cat gaa agg tct ata atg acg gat cta
tac tac ctc agt 710 Leu Gly His His Glu Arg Ser Ile Met Thr Asp Leu
Tyr Tyr Leu Ser 160 165 170 cag aca gat gga gca ggt gat tgg cgg gaa
aaa gag gcc aaa gat ctg 758 Gln Thr Asp Gly Ala Gly Asp Trp Arg Glu
Lys Glu Ala Lys Asp Leu 175 180 185 aca gaa ctg gtt cag cgg aga ata
aca tat ctt cag aat ccc aag gac 806 Thr Glu Leu Val Gln Arg Arg Ile
Thr Tyr Leu Gln Asn Pro Lys Asp 190 195 200 tgc agc aaa gcc aaa aag
ctg gtg tgt aat atc aac aaa ggc tgt ggc 854 Cys Ser Lys Ala Lys Lys
Leu Val Cys Asn Ile Asn Lys Gly Cys Gly 205 210 215 tat ggc tgt cag
ctc cat cat gtg gtc tac tgc ttc atg att gca tat 902 Tyr Gly Cys Gln
Leu His His Val Val Tyr Cys Phe Met Ile Ala Tyr 220 225 230 235 ggc
acc cag cga aca ctc atc ttg gaa tct cag aat tgg cgc tat gct 950 Gly
Thr Gln Arg Thr Leu Ile Leu Glu Ser Gln Asn Trp Arg Tyr Ala 240 245
250 act ggt gga tgg gag act gta ttt agg cct gta agt gag aca tgc aca
998 Thr Gly Gly Trp Glu Thr Val Phe Arg Pro Val Ser Glu Thr Cys Thr
255 260 265 gac aga tct ggc atc tcc act gga cac tgg tca ggt gaa gtg
aag gac 1046 Asp Arg Ser Gly Ile Ser Thr Gly His Trp Ser Gly Glu
Val Lys Asp 270 275 280 aaa aat gtt caa gtg gtc gag ctt ccc att gta
gac agt ctt cat ccc 1094 Lys Asn Val Gln Val Val Glu Leu Pro Ile
Val Asp Ser Leu His Pro 285 290 295 cgt cct cca tat tta ccc ttg gct
gta cca gaa gac ctc gca gat cga 1142 Arg Pro Pro Tyr Leu Pro Leu
Ala Val Pro Glu Asp Leu Ala Asp Arg 300 305 310 315 ctt gta cga gtg
cat ggt gac cct gca gtg tgg tgg gtg tct cag ttt 1190 Leu Val Arg
Val His Gly Asp Pro Ala Val Trp Trp Val Ser Gln Phe 320 325 330 gtc
aaa tac ttg atc cgc cca cag cct tgg cta gaa aaa gaa ata gaa 1238
Val Lys Tyr Leu Ile Arg Pro Gln Pro Trp Leu Glu Lys Glu Ile Glu 335
340 345 gaa gcc acc aag aag ctt ggc ttc aaa cat cca gtt att gga gtc
cat 1286 Glu Ala Thr Lys Lys Leu Gly Phe Lys His Pro Val Ile Gly
Val His 350 355 360 gtc aga cgc aca gac aaa gtg gga aca gaa gct gcc
ttc cat ccc att 1334 Val Arg Arg Thr Asp Lys Val Gly Thr Glu Ala
Ala Phe His Pro Ile 365 370 375 gaa gag tac atg gtg cat gtt gaa gaa
cat ttt cag ctt ctt gca cgc 1382 Glu Glu Tyr Met Val His Val Glu
Glu His Phe Gln Leu Leu Ala Arg 380 385 390 395 aga atg caa gtg gac
aaa aaa aga gtg tat ttg gcc aca gat gac cct 1430 Arg Met Gln Val
Asp Lys Lys Arg Val Tyr Leu Ala Thr Asp Asp Pro 400 405 410 tct tta
tta aag gag gca aaa aca aag tac ccc aat tat gaa ttt att 1478 Ser
Leu Leu Lys Glu Ala Lys Thr Lys Tyr Pro Asn Tyr Glu Phe Ile 415 420
425 agt gat aac tct att tcc tgg tca gct gga ctg cac aat cga tac aca
1526 Ser Asp Asn Ser Ile Ser Trp Ser Ala Gly Leu His Asn Arg Tyr
Thr 430 435 440 gaa aat tca ctt cgt gga gtg atc ctg gat ata cat ttt
ctc tct cag 1574 Glu Asn Ser Leu Arg Gly Val Ile Leu Asp Ile His
Phe Leu Ser Gln 445 450 455 gca gac ttc cta gtg tgt act ttt tca tcc
cag gtc tgt cga gtt act 1622 Ala Asp Phe Leu Val Cys Thr Phe Ser
Ser Gln Val Cys Arg Val Thr 460 465 470 475 tat gaa att atg caa aca
cta cat cct gat gcc tct gca aac ttc cat 1670 Tyr Glu Ile Met Gln
Thr Leu His Pro Asp Ala Ser Ala Asn Phe His
480 485 490 tct tta gat gac atc tac tat ttt ggg ggc cag aat gcc cac
aat caa 1718 Ser Leu Asp Asp Ile Tyr Tyr Phe Gly Gly Gln Asn Ala
His Asn Gln 495 500 505 att gcc att tat gct cac caa ccc cga act gca
gat gaa att ccc atg 1766 Ile Ala Ile Tyr Ala His Gln Pro Arg Thr
Ala Asp Glu Ile Pro Met 510 515 520 gaa cct gga gat atc att ggt gtg
gct gga aat cat tgg gat ggc tat 1814 Glu Pro Gly Asp Ile Ile Gly
Val Ala Gly Asn His Trp Asp Gly Tyr 525 530 535 tct aaa ggt gtc aac
agg aaa ttg gga agg acg ggc cta tat ccc tcc 1862 Ser Lys Gly Val
Asn Arg Lys Leu Gly Arg Thr Gly Leu Tyr Pro Ser 540 545 550 555 tac
aaa gtt cca gag aag ata gaa acg gtc aag tac ccc aca tat cct 1910
Tyr Lys Val Pro Glu Lys Ile Glu Thr Val Lys Tyr Pro Thr Tyr Pro 560
565 570 gag gct gag aaa taa agctcacatg gaagagataa acgaccaaac
tcagttcgac 1965 Glu Ala Glu Lys * 575 caaactcagt tcaaaccatt
tcagccaaac tgtagatgaa gagggctctg atctaacaaa 2025 ataaggttat
atgagtagat actctcagca ccaagagcag ctgggaactg acataggctt 2085
caattggtgg aattc 2100 10 575 PRT Homo sapiens 10 Met Arg Pro Trp
Thr Gly Ser Trp Arg Trp Ile Met Leu Ile Leu Phe 1 5 10 15 Ala Trp
Gly Thr Leu Leu Phe Tyr Ile Gly Gly His Leu Val Arg Asp 20 25 30
Asn Asp His Pro Asp His Ser Ser Arg Glu Leu Ser Lys Ile Leu Ala 35
40 45 Lys Leu Glu Arg Leu Lys Gln Gln Asn Glu Asp Leu Arg Arg Met
Ala 50 55 60 Glu Ser Leu Arg Ile Pro Glu Gly Pro Ile Asp Gln Gly
Pro Ala Ile 65 70 75 80 Gly Arg Val Arg Val Leu Glu Glu Gln Leu Val
Lys Ala Lys Glu Gln 85 90 95 Ile Glu Asn Tyr Lys Lys Gln Thr Arg
Asn Gly Leu Gly Lys Asp His 100 105 110 Glu Ile Leu Arg Arg Arg Ile
Glu Asn Gly Ala Lys Glu Leu Trp Phe 115 120 125 Phe Leu Gln Ser Glu
Leu Lys Lys Leu Lys Asn Leu Glu Gly Asn Glu 130 135 140 Leu Gln Arg
His Ala Asp Glu Phe Leu Leu Asp Leu Gly His His Glu 145 150 155 160
Arg Ser Ile Met Thr Asp Leu Tyr Tyr Leu Ser Gln Thr Asp Gly Ala 165
170 175 Gly Asp Trp Arg Glu Lys Glu Ala Lys Asp Leu Thr Glu Leu Val
Gln 180 185 190 Arg Arg Ile Thr Tyr Leu Gln Asn Pro Lys Asp Cys Ser
Lys Ala Lys 195 200 205 Lys Leu Val Cys Asn Ile Asn Lys Gly Cys Gly
Tyr Gly Cys Gln Leu 210 215 220 His His Val Val Tyr Cys Phe Met Ile
Ala Tyr Gly Thr Gln Arg Thr 225 230 235 240 Leu Ile Leu Glu Ser Gln
Asn Trp Arg Tyr Ala Thr Gly Gly Trp Glu 245 250 255 Thr Val Phe Arg
Pro Val Ser Glu Thr Cys Thr Asp Arg Ser Gly Ile 260 265 270 Ser Thr
Gly His Trp Ser Gly Glu Val Lys Asp Lys Asn Val Gln Val 275 280 285
Val Glu Leu Pro Ile Val Asp Ser Leu His Pro Arg Pro Pro Tyr Leu 290
295 300 Pro Leu Ala Val Pro Glu Asp Leu Ala Asp Arg Leu Val Arg Val
His 305 310 315 320 Gly Asp Pro Ala Val Trp Trp Val Ser Gln Phe Val
Lys Tyr Leu Ile 325 330 335 Arg Pro Gln Pro Trp Leu Glu Lys Glu Ile
Glu Glu Ala Thr Lys Lys 340 345 350 Leu Gly Phe Lys His Pro Val Ile
Gly Val His Val Arg Arg Thr Asp 355 360 365 Lys Val Gly Thr Glu Ala
Ala Phe His Pro Ile Glu Glu Tyr Met Val 370 375 380 His Val Glu Glu
His Phe Gln Leu Leu Ala Arg Arg Met Gln Val Asp 385 390 395 400 Lys
Lys Arg Val Tyr Leu Ala Thr Asp Asp Pro Ser Leu Leu Lys Glu 405 410
415 Ala Lys Thr Lys Tyr Pro Asn Tyr Glu Phe Ile Ser Asp Asn Ser Ile
420 425 430 Ser Trp Ser Ala Gly Leu His Asn Arg Tyr Thr Glu Asn Ser
Leu Arg 435 440 445 Gly Val Ile Leu Asp Ile His Phe Leu Ser Gln Ala
Asp Phe Leu Val 450 455 460 Cys Thr Phe Ser Ser Gln Val Cys Arg Val
Thr Tyr Glu Ile Met Gln 465 470 475 480 Thr Leu His Pro Asp Ala Ser
Ala Asn Phe His Ser Leu Asp Asp Ile 485 490 495 Tyr Tyr Phe Gly Gly
Gln Asn Ala His Asn Gln Ile Ala Ile Tyr Ala 500 505 510 His Gln Pro
Arg Thr Ala Asp Glu Ile Pro Met Glu Pro Gly Asp Ile 515 520 525 Ile
Gly Val Ala Gly Asn His Trp Asp Gly Tyr Ser Lys Gly Val Asn 530 535
540 Arg Lys Leu Gly Arg Thr Gly Leu Tyr Pro Ser Tyr Lys Val Pro Glu
545 550 555 560 Lys Ile Glu Thr Val Lys Tyr Pro Thr Tyr Pro Glu Ala
Glu Lys 565 570 575 11 14 PRT Artificial Sequence Primer 11 Arg Ile
Pro Glu Gly Pro Ile Asp Gln Gly Pro Ala Ile Gly 1 5 10 12 25 PRT
Artificial Sequence Primer 12 Lys Leu Gly Phe Lys His Pro Val Ile
Gly Val His Val Arg Arg Thr 1 5 10 15 Asp Lys Val Gly Thr Cys Ala
Ala Phe 20 25 13 13 PRT Artificial Sequence Primer 13 Thr Lys Tyr
Pro Asn Tyr Glu Phe Ile Ser Asp Asn Ser 1 5 10 14 20 DNA Artificial
Sequence Primer 14 ttyaarcayc chgtbatygg 20 15 20 DNA Artificial
Sequence Primer 15 gwrttrtcrg wratraaytc 20
* * * * *