Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1999-09-03
2002-10-08
Priebe, Scott D. (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S320100, C435S325000, C435S252300, C435S455000, C435S471000, C435S006120, C536S023100, C536S023200, C536S023500, C530S350000, C530S412000
Reexamination Certificate
active
06461835
ABSTRACT:
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
Not applicable.
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention is biological in nature and relates to the synthesis, structure and biological activities of novel &agr;-1,2 and &agr;-1,3 fucosyltransferases from
Caenorhabditis elegans
(“
C. elegans
”). The present invention also contemplates a transgenic non-human eukaryotic mammal whose germ cells and somatic cells incorporate nucleic acid sequence(s) encoding one or more of the novel &agr;-1,2 and &agr;-1,3 fucosyltransferases from
C. elegans
. The nucleic acid sequence(s) may be introduced into the non-human eukaryotic mammal, or an ancestor of the non-human eukaryotic mammal, at an embyonic stage.
2. Brief Description of Related Art
Glycoconjugates are a class of molecules having a carbohydrate component. Typical glycoconjugates include oligosaccharides; polysaccharides, glycoproteins, glycolipids, glycosaminoglycans, and carbohydrates covalently modifying other molecules, such as steroids, nucleic acids, and xenobiotics. Glycoconjugates having a-linked fucose residues are known in the art to have bioactivity in animals and are required for many cellular functions.
In addition to being important in many biological processes, glycoconjugates are major antigens recognized by the immune systems in higher animals. The pharmaceutical industry has exploited these antigenic properties in order to generate agents that are useful for treating and diagnosing diseases. In particular, pharmaceutical companies have developed recombinant glycoproteins, such as erythropoietin and tissue plasminogen activator, as well as therapeutic oligosaccharides and glycoconjugates, such as Cylexin and glycosylated antibiotics. Glycoconjugates are also important in the nutrition industry for use as food additives and supplements, such as lactose and its derivatives.
A major obstacle in the development, synthesis, and use of glycoconjugates for commercial use is that many glycoconjugates are difficult to synthesize on a commercial level by purely chemical means. Thus, in recent years, there has been increased emphasis on the identification of enzymes, e.g. glycosyltransferases, that can catalyze the formation of specific glycoside attachments to acceptor or substrate molecules both in vitro (e.g. enzyme catalyzed synthesis in the laboratory or production facility), and in vivo (e.g. transgenic animals).
In recent years there has also been an increase in awareness of the fact that glycoconjugates in higher animals and humans which contain the &agr;-fucosylated structures with Fuc&agr;1→3GlcNAc-R linkages (where R=H, OH, or an organic molecule) are important in the processes involving cellular adhesion of animal and bacterial cells. Examples of glycans with these linkages are shown in Table I. Some of these &agr;-1,3-fucosylated glycans are found on human leukocytes and are especially important in regulating cellular adhesion events in the early steps of the inflammatory response. Also, &agr;-1-3-fucosylated glycans, such as some of those shown in Table I, are synthesized by
Helicobacter pylori
and other pathogens and may be important in host-parasite interactions.
TABLE I
Glycans with fucosyl linkages
Lewis x antigen
Gal&bgr;1 → 4(Fuc&agr;1 → 3)GlcNAc-R (wherein
R is defined herein as a carbohydrate, a
glycoprotein, or a protein)
Lewis y antigen
Fuc&agr;1 → 2Gal&bgr;1 → 4(Fuc&agr;1 → 3)GlcNAc-R
Sialyl Lewis x antigen
NeuAc&agr;2 → 3Gal&bgr;1 → 4(Fuc&agr;1 → 3)
GlcNAc-R
Fucosylated lacdiNAc
GalNAc&bgr;1 → 4(Fuc&agr;1 → 3)GlcNAc-R
antigen
Fucosylated
Gal&bgr;1 → 4(Fuc&agr;1 → 3)GlcNAc&bgr;1 →
polylactosamine
[3Gal&bgr;1 → 4(Fuc&agr;1 → 3)GlcNAc_&bgr;1 → ]
n
-R,
where n = 1 to 1,000
Sialylated and fucosyl-
NeuAc&agr;2 → 3(6)Gal&bgr;1 → 4(Fuc&agr;1 → 3)
ated polylactosamine
GlcNAc&bgr;1 → [3Gal&bgr;1 → 4(Fuc&agr;1 → 3)
GlcNAc_&bgr;1 → ]
n
-R, where n = 1 to 1,000
O(H)-Blood group
Fuc&agr;1 → 2Gal&bgr;1 → 3(4)GlcNAc-R
antigen
Lewis a antigen
Gal&bgr;1 → 3(Fuc&agr;1 → 4)GlcNAc-R
Sialyl Lewis a antigen
NeuAc&agr;2 → 3Gal&bgr;1 → 3(Fuc&agr;1 → 4)
GlcNAc-R
Lewis b antigen
Fuc&agr;1 → 2Gal&bgr;1 → 3(Fuc&agr;1 → 4)GlcNAc-R
A-Blood group antigen
GalNAc&agr;1 → 3(Fuc&agr;1 → 2)Gal&bgr;1 → 3(4)
GlcNAc-R
B-Blood group antigen
Gal&agr;1 → 3(Fuc&agr;1 → 2)Gal&bgr;1 → 3(4)GlcNAc-R
In order to commercially exploit glycoconjugates and their unique functional abilities, it has been necessary to scale up the synthesis of glycans containing &agr;-1,3-, and &agr;-1,2-fucosylated glycans. These synthesized glycans can then be used as inhibitors of cell adhesion mediated by glycoconjugates containing these linkages. For example, the commercial production of &agr;-1,3-fucosylated glycans is ongoing in some pharmaceutical companies throughout the developed world and is expected to be a major pharmaceutical contribution in the coming years.
Because of the difficulty and costs in purely chemical synthetic methods for carbohydrate production, commercial production of &agr;-1,3-, &agr;1,4- and &agr;-1,2-fucosylated glycans has historically been dependent upon recombinant enzymes or &agr;-fucosyltransferases, which exhibit specific abilities to catalyze the appropriate linkages. These types of reactions are shown generally in Table II. Each of the &agr;-fucosyltransferases thus far identified in the art have limitations in the types of acceptors utilized, the rate of product formation, as well as potential undesirable side effects. For example, a human &agr;-fucosyltransferase termed Fuc-T-IV acts with the best efficiency toward non-sialylated acceptors and acts relatively poorly with sialylated acceptors. Also, another enzyme, termed Fuc-TIII, can catalyze both &agr;-1,3- and &agr;-1,4-linkages to GlcNAc residues in the two different acceptors.
Therefore, it is an object of the present invention to disclose and claim fucosyltransferases isolated and purified from
C. elegans
nematode, and in particular &agr;-1,3-, and &agr;-1,2-fucosyltransferases isolated and purified from
C. elegans.
The present invention further contemplates homologous variants of said &agr;-1,3 and &agr;-1,2 fucosyltransferases and nucleic acids which encode said fucosyltransferases.
It is also an object of the present invention to disclose and claim a transgenic mammal, such as a cow or goat, incorporating the genes or cDNA encoding the &agr;-1,3- and &agr;-1,2-fucosyltransferases isolated and purified from
C. elegans
and said homologous versions thereof.
These and other objects of the present invention will become apparent in light of the present specification, drawings, and claims.
It is still another object of the present invention to provide for the production of recombinant proteins from fucosyltransferases or products requiring action by fucosyltransferases in a non-human eukaryotic mammal's milk.
These and other features and advantages of the invention will be apparent given the present specification, figures, embodiments and claims.
SUMMARY OF THE INVENTION
Novel and previously unidentified &agr;-fucosyltransferases have been identified, isolated and purified from the
C. elegans
nematode. These novel
C. elegans
fucosyltransferases, described further herein, have been used to generate &agr;-fucosylated glycoconjugates. In particular, the
C. elegans
nematode contains fucosyltransferases which catalyze the reactions shown in Table III. The amino acid and nucleotide sequences for the novel &agr;1,3- and &agr;1,2-fucosyltransferases found in
C. elegans
can be found in SEQ ID NO:1 and SEQ ID NO:2, respectively.
The present invention also includes a transgenic non-human eukaryotic mammal whose germ cells and somatic cells incorporate nucleic acid sequence(s) encoding one or more of the novel &agr;1,3 and &agr;1,2-fucosyltransferases from
C. elegans
.
REFERENCES:
N
Cummings Richard D.
DeBose-Boyd Russell A.
Nyame A. Kwame
Becker Cheryl L.
Kaushal Sumesh
Priebe Scott D.
The University of Oklahoma
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