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
1999-04-23
2001-12-11
Clark, Deborah J. R. (Department: 1633)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C536S023100, C536S023500, C536S023200, C435S069100, C435S320100, C435S325000, C435S243000, C435S455000
Reexamination Certificate
active
06329170
ABSTRACT:
1. FIELD OF THE INVENTION
The present invention relates to a rat ganglioside GM
1
-specific &agr;1→2fucosyltransferase. The invention provides novel nucleotide sequences of a rat &agr;1→2fucosyltransferase specific for a carbohydrate moiety found in ganglioside GM
1
, more particularly, specific for a terminal galactose &bgr;1→3N-acetylgalactosamine (Gal&bgr;1→3GalNAc) saccharide, amino acid sequences of its encoded protein (including peptide or polypeptide), and derivatives and analogs thereof. Merely for the ease of description, the enzyme is herein referred to as “GM
1
-specific” or “ganglioside GM
1
-specific”. The invention also relates to fragments (and derivatives and analogs thereof) which comprise a domain of rat ganglioside GM
1
-specific &agr;1→2fucosyltransferase with catalytic activity. Methods of production of rat ganglioside GM
1
-specific &agr;1→2fucosyltransferase and derivatives and analogs thereof (e.g. by recombinant means) are provided. In addition, the invention relates to methods of inhibiting the function of rat ganglioside GM
1
-specific &agr;1→2fucosyltransferase (e.g. by means of antisense RNA). The invention further relates to use of rat ganglioside GM
1
-specific &agr;1→2fucosyltransferase in the preparative production of fucosyl-GM
1
. Applications of fucosyl-GM
1
, for example as an immunotherapeutic for cancer, are disclosed.
2. BACKGROUND OF THE INVENTION
Citation of a reference herein shall not be construed as an admission that such reference is prior art to the present invention.
2.1. Fucosyltransferases
Fucosyltransferases are enzymes that catalyze the addition of a fucose residue to a terminal galactose acceptor of saccharide precursors. Fucosyltransferase activity is involved in the production of oligosaccharides, glycolipids or glycoproteins. There are four known classes of fucosyltransferases, namely those that catalyze the addition of fucose in &agr;1→2, &agr;1→3, &agr;1→4 and &agr;1→6 linkages.
Fucosyltransferases are best known for their roles in the synthesis of the oligosaccharide moieties that comprise blood group antigenic determinants. For example, the fucosyltransferase encoded by the H gene catalyzes the transfer of fucose in an &agr;1-2 linkage to the terminal galactose of Gal(&bgr;1-4)GlcNAc(&bgr;1-3)Gal-R to produce ‘H substance’ on the surface of erythrocytes. Further addition of N-acetylgalactosamine or galactose leads to the formation of the type A or type B blood group substances respectively. An analogous enzyme encoded by the Se locus catalyzes the formation of ‘H substance’ in epithelial tissues for secretion rather than presentation at the cell surface (Rosen et al., 1989, Dictionary of Immunology, Stockton Press, New York, pp. 1-3).
Previous experiments with H35 hepatoma cell extracts demonstrated that transfer of fucose to neolacto-series acceptors occurred at a rate only 2% of that found for GM
1
(Holmes., E. H., et al, 1983. J. Biol. Chem, 258:3706-3713). This substrate specificity is more restricted compared to other cloned &agr;1→2fucosyltransferases but is most closely related to secretor-type enzymes (Larsen, R. D., et al., 1990, Proc. Natl. Acad. Sci. USA 87:6674-6678; Kelly, R. J., et al., 1995, J. Biol. Chem. 270:4640-4649; Hitoshi, S. et al., 1995, J. Biol. Chem. 270:8844-8850; Hitoshi, S., et al., 1996, J. Biol. Chem. 271:16975-16981).
2.2. Structure of &agr;1→2Fucosyltransferases
To date, a number of genes encoding H-type and Se-type &agr;1→2fucosyltransferases have been cloned from several species of organisms. Three human &agr;1→2fucosyltransferases (Larsen et al., 1990, Biochemistry 87:6674-6678; Koda et al., 1997, Eur. J. Biochem. 246:750-755: Kelly et al., 1995, J. Biol. Chem. 270:4640-4649), three rabbit &agr;1→2fucosyltransferases (known as RFT-I, RFT-II and RFT-III) (Hitoshi et al., 1995, J. Biol. Chem. 270:8844-8850; Hitoshi et al., 1996, J. Biol. Chem. 271:16975-19681). and two mouse &agr;1→2fucosyltransferases (Tsuji, 1996, GenBank accession no. Y09882; Lin et al., 1998, GenBank accession no. AF064792) have been described. Piau et al. (1994, Eur. J. Biochem. 300:623-626) disclose fragments, designated FTA and FTB, of two rat &agr;1→2fucosyltransferases isolated from rat PROb colon adenocarcinoma cells. Piau et al. showed that antisense expression of the FTA or FTB nucleic acid fragments inhibited the endogenous &agr;1→2fucosyltransferase activity of PROb cells with respect to the synthetic fucose acceptor phenyl &bgr;-
D
-galactopyranoside; however the FTB fragment was not shown to be sufficient for &agr;1→2fucosyltransferase catalytic activity, nor was the substrate specificity of the PROb &agr;1→2fucosyltransferase activity determined.
H-type &agr;1→2fucosyltransferases are membrane localized whereas Se-type &agr;1→2fucosyltransferases are localized to the Golgi apparatus. Amino acid sequence alignment of membrane bound H-type &agr;1→2fucosyltransferases reveals that, like other glycosyltransferases, there exists a homologous domain structure comprising a short intracellular N-terminal domain, a transmembrane domain, an extracellular stem region not required for enzymatic activity, and finally, the catalytic domain at the C-terminus. Generally, there is little sequence homology outside the catalytic domain.
2.3. Ganglioside GM
1
and its Fucosylated Derivative Fucosyl-GM
1
Gangliosides are cell surface constituents comprising glycosphingolipids (produced by the linking of ceramides to oligosaccharides) with sialic acid residues. Depending on the number of sialic acid residues they possess, gangliosides are known as mono-, di-, tri- or polysialogangliosides. GM
1
stands for ganglioside mono(sialic acid)
1
.
Fucosyl-GM
1
, detected by monoclonal antibodies, is found largely in the nervous system, and in particular on a subpopulation of neurons in the dorsal root ganglia and dorsal horn of the spinal cord, as well as on surrounding satellite cells surrounding the fucosyl-GM
1
positive neurons (Kusunoki et al., 1989, Brain Res. 494:391-395; Kusonoki et al., 1992, Neurosci. Res. 15: 74-80).
Gangliosides have long been implicated in diseased states. They are often prominent cell surface constituents of transformed cells (see Section 2.5, infra) and alterations in their metabolism give rise to diseases of the nervous system. For example, several fatal hereditary diseases are caused by lysosomal storage of gangliosides wherein the absence or deficiency of lysosomal enzymes results in the deleterious accumulation of gangliosides. The most well known of these diseases is the neurodegenerative Tay-Sachs disease, which is characterized by the accumulation of ganglioside GM
2
. Accumulation of GM
1
results in GM
1
Gangliosidosis.
2.4. Regulation of Fucosyltransferase Expression
‘H substance’, the fucosylated precursor of blood group determinants, is strictly regulated temporally and spatially during vertebrate development (Fenderson et al., 1986, Dev. Biol. 1 14:12-21).
Dramatic changes in the expression of cell surface glycolipids are found with oncogenesis (Hakomori, 1989, Adv. Cancer Res. 52:257-331; Alhadeff, 1989, CRC Crit. Rev. Oncol./Hematol. 9:37-107). These changes frequently are oncofetal in nature in that a particular carbohydrate structure may be expressed during normal fetal development, disappear in adult tissues, and reappear in association with oncogenesis giving rise to a premalignant or malignant marker. One such example is expression of the ganglio-B determinant (II
3
NeuAcIV
3
&agr;GalIV
2
FucGg
4
) during early stages of chemical carcinogenesis in rat liver with N-2-acetylaminofluorene (AAF) (Holmes and Hakomori, 1982, J. Biol. Chem. 257:7698-7703; Scribncr et al., 1983, Environ. Health Perspect. 49:81-89). Expression of this determinant has been shown to be a property of liver parenchymal cells resulting from a carcinogenic stimulus but not hepatotoxicity (Holmes, 1990, Carcinogenesis 11:89-94). This determinant has also been shown to be developmental
Holmes Eric H.
Sherwood Anne L.
Clark Deborah J. R.
Kaushal Sumesh
Northwest Hospital
Townsend and Townsend / and Crew LLP
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