&agr;1,2 fucosyltransferase

Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical

Reexamination Certificate

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C435S193000

Reexamination Certificate

active

06238894

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates generally to the field of &agr;1,2-fucosyltransferases and, more specifically, to &agr;1,2-fucosyltransferase polypeptides.
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 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.
The whole genome sequence of
H. pylori
26695 had been published, which will undoubtedly facilitate the genetic studies of
H. pylori. H. pylori
genome sequence revealed the existence of two copies of &agr;(1,3) fucT gene, whereas no putative &agr;(1,2) fucT gene had been annotated.
SUMMARY OF THE INVENTION
The present invention is based on the discovery of a &agr;1,2-fucosyltransferase polypeptide and gene which encodes the polypeptide. The gene was expressed in vitro and a mutagenesis study demonstrated that this gene is involved in Le
y
synthesis. The present invention includes a polynucleotide sequence encoding &agr;1,2-fucosyltransferase polypeptide which is useful in the detection and synthesis of &agr;1,2-fucosyltransferase polypeptide, and an &agr;1,2-fucosyltransferase that is able to synthesize Le
Y
, Le
B
and H type 1 structures.
Helicobacter pylori
lipopolysaccharide (LPS) express human oncofetal antigens Lewis X and Lewis Y. The synthesis of Lewis Y involves the actions of &agr;(1,3) and &agr;(1,2) fucosyltransferases (FucTs). Disclosed herein are the molecular cloning and characterization of genes encoding
H. pylori
&agr;(1,2) FucT (HpfucT2) from various
H. pylori
strains. Also provided are constructed Hp fucT2 knock-out mutants that demonstrate the loss of Lewis Y production in these mutants by ELISA and immunoelectron microscopy. The &agr;1,2fucT2 gene contains a hypermutable sequence (poly C and TAA repeats) which provides a possibility of frequent shifting into and out of coding frame by a polymerase slippage mechanism. Thus, &agr;1,2fucT2 gene displays two major genotypes: either encoding a single full-length open reading frame (ORF, as in the strain UA802), or truncated ORFs (as in the strain 26695). In vitro expression of Hp fucT2 genes demonstrated that both types of the gene have a potential to produce the full-length protein. The production of the full-length protein by the 26695 fucT2 gene could be attributed to translational −1 frameshifting, since a perfect translation frameshift cassette resembling that of
Escherichia coli
dnaX gene is present. The examination of the strain UA 1174 revealed that its fucT2 gene has a frameshifted ORF at the DNA level which cannot be compensated by translation frameshifting, accounting for its Lewis Y -off phenotype. In another strain, UA1218, the fucT2 gene is turned off apparently due to the loss of its promoter. Based on these data, we proposed a model for the variable expression of Lewis Y by
H. pylori
, in which the regulation at the level of replication, transcription, and translation of the fucT2 gene may all be involved.
In another embodiment, the invention provides a method of using the novel &agr;1,2-fucosyltransferase to synthesize oligosaccharides such as Le
x
, Le
y
, sLe
x
, Le
A
, Le
B
, H type 1 and H type 2.
In another embodiment the invention provides the novel polypeptide of &agr;1,2-fucosyltransferase which is useful in the development of antibodies to &agr;1,2-fucosyltransferase.
In another embodiment, a polypeptide of &agr;1,2-fucosyltransferase having a frameshift variant resulting from a “slippery” heptanucleic acid sequence X XXY YYZ, wherein X=C or A, Y=T or A and Z=A or G (e.g., A AAA AAG) is provided. In another embodiment, the &agr;1,2-fucosyltransferase is a polypeptide which has a sequence of SEQ ID NO:2. In another embodiment the polynucleotide sequence encoding &agr;1,2-fucosyltransferase has a variable number of poly-cytosine repeats and TAA repeats in different
H. pylori
strains.
Further provided is a method for producing &agr;1,2-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;1,2-fucosyltransferase or a portion thereof and harvesting the &agr;1,2-fucosyltransferase. A preferred embodiment of the method is directed to the use of the claimed genetic expression system which produces &agr;1,2-fucosyltransferase.
Further provided is a method to measure the enzymatic activity and acceptor specificity of &agr;1,2-fucosyltransferase. The method involves the use of a structurally defined oligosaccharide substrate (acceptor) in a radioactive labeled assay system and identification of the reaction products by capillary electrophoresis. In another embodiment, an &agr;1,2-fucosyltransferase has a substrate specificity that is distinct from the conventional &agr;1,2-fucosyltransferase of mammalian origin and uses a different pathway to synthesize Lewis antigens.
Also provided are knockout organisms in which expression of &agr;1,2-fucosyltransferase has been prevented or in which the &agr;1,2-fucosyltransferase expression results in a polypeptide lacking wild type biological activity.
These and

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