&agr;1,3-fucosyltransferase

Details &agr;1,3-fucosyltransferase &agr;1,3-fucosyltransferase

1998-06-05

2002-06-04

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

C435S072000, C435S074000

Reexamination Certificate

active

06399337

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates generally to the field of &agr;1,3-fucosyltransferases and, more specifically, to &agr;1,3-fucosyltransferase polypeptides which are transmembrane segment-free.
BACKGROUND OF THE INVENTION
Helicobacter pylori
is an important human pathogen which causes both gastric and duodenal ulcers and has also been associated with gastric cancer and lymphoma. This microorganism has been shown to express cell surface glycoconjugates including Lewis X, Lewis Y. and sialyl Lewis X. These bacterial oligosaccharides are structurally similar to tumor-associated carbohydrate antigens found in mammals.
The presence of
H. pylori
isolate has been associated with an increased risk for development of gastric cancer (Wirth, H.-P., Yang, M., Karita, M., and Blaser, M. J. (1996)
Infect. Immun
. 64 4598-4605). This pathogen is highly adapted to colonize human gastric mucosa and may remain in the stomach with or without causing symptoms for many years. Although
H. pylori
elicits local as well as systemic antibody responses, it escapes elimination by the host immune response due to its sequestered habitation within human gastric mucosa. Another mechanism by which
H. pylori
may protect itself from the action of the host immune response is the production of surface antigens mimicking those in the host.
In mammalian cells the enzyme &agr;(1,3/1, 4)-fucosyltransferase (namely FucT) catalyzes the last step in the synthesis of two carbohydrate structures, Gal&bgr; 1-4[Fuc&agr;1-3] GlcNAc (Lewis X, Le
x
for short) or NeuAc&agr;2-3-Gal&bgr; 1-4[Fuc&agr;1-3]GlcNAc (sialyl Lewis X, sLe
x
for short). (Lowe et al., 1990, Cell 57: 475-484; Kukowska-Latallo et al., 1990, Genes & Development 4:1288-1303.) Cell surface &agr;(1,3)- and &agr;(1,2)-fucosylated oligosaccharides, that is, Lewis X (Le
x
), sialyl Lewis X (sLe
x
) and Lewis Y (Le
y
), are present on both eukaryotic and microbial cell surfaces. In mammals, Le
x
is a stage-specific embryonic antigen, however, Le
x
, sLe
x
and Le
y
are also regarded as tumor-associated markers. The biological functions of these bacterial oligosaccharide structures are not fully understood. It has been suggested that such glycoconjugates produced by
H. pylori
, may mimic host cell antigens and could mask the bacterium from the host immune response. It is also possible that these bacterial Lewis antigens could down regulate the host T-cell response. Therefore, production of such antigens may contribute to colonization and long-term infection of the stomach by
H. pylori.
Presently, use of carbohydrates as potential therapeutic drugs has become popular in the field of medical chemistry. In addition, qualitative and quantitative carbohydrates including Le
x
, Le
y
and sLe
x
are also required as reagents for assaying the enzymes which are involved in the biosynthesis of glycoconjugates in cells. Le
x
, Le
y
and sLe
x
products which are commercially available are chemically synthesized. However, synthesis of these products gives rise to several limitations such as time-consuming, complicated procedures and low yields. Although several mammalian fucosyltransferases have been cloned and expressed, enzymatic synthesis of Le
x
, Le
y
and sLe
x
products for a commercial purpose has not been reported.
SUMMARY OF THE INVENTION
The present invention is based on the discovery of a novel &agr;1,3-fucosyltransferase polypeptide and gene which encodes the polypeptide. The present invention includes a novel nucleic acid sequence of &agr;1,3-fucosyltransferase polypeptide which is useful in the detection and synthesis of &agr;1,3-fucosyltransferase polypeptide.
In another embodiment, the invention provides a method of using the novel &agr;1,3-fucosyltransferase to synthesize oligosaccharides such as Le
x
, Le
y
and sLe
x
.
In another embodiment the invention provides the novel polypeptide of &agr;1,3-fucosyltransferase which is useful in the development of antibodies to &agr;1,3-fucosyltransferase.
In another embodiment, the novel polypeptide of &agr;1,3-fucosyltransferase has a carboxyl terminal ~100 amino acids in length having therein a heptad repeat of X
1
X
2
LRX
3
X
4
Y, wherein X
1
is D or N; X
2
is D or N; X
3
is I, V or A; X
4
is N or D. In another embodiment, the &agr;1,3-fucosyltransferase is a peptide selected from SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3. In another embodiment the &agr;1,3-fucosyltransferase may have a variable number of heptad repeats.
Further provided is a method for producing &agr;1,3-fucosyltransferase. The method involves the step of culturing a gene expression system which comprises a host cell which has been recombinantly modified with a polynucleotide encoding &agr;1,3-fucosyltransferase or a portion thereof and harvesting the &agr;1,3-fucosyltransferase. A preferred embodiment of the method is directed to the use of the claimed genetic expression system which produces &agr;1,3-fucosyltransferase.
These and many other features and attendant advantages of the present invention will become better understood by reference to the following detailed description of the invention when taken in conjunction with the Examples.
ABBREVIATIONS
The abbreviation used are: FucT, &agr;1,3-fucosyltransferase unless specified otherwise; Le
x
, Lewis X; sLe
x
, sialyl-Lewis X; Le
y
, Lewis Y; nt, nucleotide (s); kb, kilobase (s); aa, amino acid (s); PCR, polymerase chain reaction; ORF, open reading frame; RSB, a ribosomal binding site; LPS, lipopolysaccharides; HD-Zip, homeodomain-leucine zipper; bZip, basic region-zipper; LacNAc-R, Gal&bgr;1-4GlcNAc&bgr;-O—(CH
2
),COOMe; Gal&bgr;1-3GlcNAc-R, Gal&bgr;1-3GlcNacb-O—(CH
2
)
8
COOMe; LacNAc-TMR, Gal&bgr;1-4GlcNAc&bgr;-O—(CH
2
)
8
CO—NHCH
2
CH
2
NH-TMR; Phenyl-Gal, phenyl-&bgr;-galactoside.


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