Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1999-09-01
2001-04-03
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S193000, C536S023200
Reexamination Certificate
active
06210933
ABSTRACT:
BACKGROUND OF THE INVENTION
Sialyltransferases are a group of glycosyltransferases that transfer sialic acid from an activated sugar nucleotide to acceptor oligosaccharides found on glycoproteins, glycolipids or polysaccharides. Sialylated oligosaccharides play important roles in cell-cell recognition, cell differentiation and various receptor-ligand interactions in mammalian systems. The large number of sialylated oligosaccharide structures has lead to the characterization of many different sialyltransferases involved in the synthesis of these structures. Based on the linkage and acceptor specificity of the sialyltransferases studied so far, it has been determined that at least 13 distinct sialyltransferase genes are present in mammalian systems (Tsuji, S. et al. (1996)
Glycobiology
6:v-vii).
Sialylated glycoconjugates are also found in bacteria (Preston, A. et al. (1996)
Crit. Rev. Microbiol.
22:139-180; Reuter, G. et al. (1996)
Biol. Chem. Hoppe
-
Seyler
377:325-342) are thought to mimic oligosaccharides found in mammalian glycolipids to evade the host immune response (Moran, A. P. et al. (1996)
FEMS Immunol. Med. Microbiol.
16:105-115). The importance of sialylated lipooligosaccahride (LOS) in the pathogenesis of
Neisseria gonorrhoeae
has been established (Smith et al., (1992)
FEMS Microbiol Lett.
100:287-292) while for
N. meningitidis
both the polysialic acid capsule and the sialylated LOS were found to be important for pathogenicity (Vogel, U. et al. (1996)
Med. Microbiol. Immunol.
186:81-87).
Despite their importance as proven or potential virulence factors, few bacterial sialyltransferases have been cloned (Weisgerber, C. et al. (1991)
Glycobiol.
1:357-365; Frosch, M. et al. (1991)
Mol. Microbiol.
5:1251-1263; Gilbert, M. et al. (1996)
J. Biol. Chem.
271:28271-28276) or purified (Yamamoto, T. et al. (1996)
J. Biochem.
120:104-110). The &agr;-2,8-sialyltransferases involved in the synthesis of the polysialic acid capsules have been cloned and expressed from both
Escherichia coli
(Weisgerber, C. et al. (1991)
Glycobiol.
1:357-365) and
N. meningitidis
(Frosch, M. et al. (1991)
Mol. Microbiol.
5:1251-1263). Glycosyltransferases from
N. gonorrhoeae
involved in the synthesis of lipooligosaccharide (LOS) have been cloned (U.S. Pat. No. 5,545,553).
Because of biological activity of their products, mammalian sialyltransferases generally act in specific tissues, cell compartments and/or developmental stages to create precise sialyloglycans. Bacterial sialyltransferases are not subject to the same constraints and can use a wider range of acceptors than that of the mammalian sialyltransferases. For instance, the &agr;-2,6-sialyltransferase from
Photobacterium damsela
has been shown to transfer sialic acid to terminal galactose residues which are fucosylated or sialylated at the 2 or 3 position, respectively (Kajihara, Y. et al. (1996)
J. Org. Chem.
61:8632-8635). Such an acceptor specificity has not been reported so far for mammalian sialyltransferases.
Bacterial glycosyltransferases are useful in a number of applications, such as the synthesis of desired oligosaccharides with biological activity. Identification and characterization of new bacterial glycosyltransferases is thus useful in the development of these technologies. The present invention provides these and other advantages.
SUMMARY OF THE INVENTION
The present invention provides isolated nucleic acid molecules comprising a polynucleotide sequence which encodes an &agr;2,3-sialyltransferase polypeptide and which hybridizes to SEQ. ID. NOS: 1 or 3 under stringent conditions. Typically, the polynucleotide sequence encodes a &agr;2,3-sialyltransferase polypeptide having a molecular weight of about 40 kD, for instance as shown in SEQ. ID. NOS: 2 or 4. Exemplified polynucleotide sequences are shown in SEQ. ID. NOS: 1 and 3. The nucleic acid molecule may be isolated from
Neisseria meningitidis
or
N. gonorrhoeae.
If expression of the enzyme is desired, the nucleic acid molecules of the invention may further comprise an expression containing a promoter sequence operably linked to the polynucleotide sequence. In some embodiments, the promoter is active in prokaryotic cells, such as
E. Coli.
Also provided are cells (e.g.,
E. coli
) comprising the recombinant expression cassette of the invention.
The invention further provides methods of adding a sialic acid residue to an acceptor molecule comprising a terminal galactose residue. The methods comprise contacting the acceptor molecule with an activated sialic acid molecule and an &agr;2,3-sialyltransferase of the invention. The terminal galactose residue may linked through an &agr; or a &bgr; linkage to a second residue in the acceptor molecule. Exemplary linkages include &bgr;1,4 and &bgr;1,3 linkages. The activated sialic acid is typically CMP-Neu5Ac.
Definitions
The sialyltransferases of the invention are useful for transferring a monosaccharide from a donor substrate to an acceptor molecule. The addition generally takes place at the non-reducing end of an oligosaccharide or carbohydrate moiety on a biomolecule. Biomolecules as defined here include but are not limited to biologically significant molecules such as carbohydrates, proteins (e.g., glycoproteins), and lipids (e.g., glycolipids, phospholipids, sphingolipids and gangliosides).
The following abbreviations are used herein:
Ara=arabinosyl;
Fru=fructosyl;
Fuc=fucosyl;
Gal=galactosyl;
GalNAc=N-acetylgalacto;
Glc=glucosyl;
GlcNAc=N-acetylglico;
Man=mannosyl; and
NeuAc=sialyl (N-acetylneuraminyl).
The sialyltransferases of the invention can be used to add sialic acid residues of different forms to acceptor molecules. Typically, the sialic acid is 5-N-acetylneuraminic acid, (NeuAc) or 5-N-glcolylneuraminic acid (NeuGc). Other sialic acids may be used in their place, however. For a review of different forms of sialic acid suitable in the present invention see, Schauer,
Methods in Enzymology,
50: 64-89 (1987), and Schaur,
Advances in Carbohydrate Chemistry and Biochemistry,
40: 131-234.
Donor substrates for glycosyltransferases are activated nucleotide sugars. Such activated sugars generally consist of uridine, guanosine, and cytidine diphosphate derivatives of the sugars in which the nucleoside diphosphate serves as a leaving group. The donor substrate for the sialyltransferases of the invention are activated sugar nucleotides comprising the desired sialic acid. For instance, in the case of NeuAc, the activated sugar is CMP-NeuAc.
Oligosaccharides are considered to have a reducing end and a non-reducing end, whether or not the saccharide at the reducing end is in fact a reducing sugar. In accordance with accepted nomenclature, oligosaccharides are depicted herein with the non-reducing end on the left and the reducing end on the right.
All oligosaccharides described herein are described with the name or abbreviation for the non-reducing saccharide (e.g., Gal), followed by the configuration of the glycosidic bond (&agr; or &bgr;), the ring bond, the ring position of the reducing saccharide involved in the bond, and then the name or abbreviation of the reducing saccharide (e.g., GlcNAc). The linkage between two sugars may be expressed, for example, as 2,3, 2→3, or (2,3). Each saccharide is a pyranose or furanose.
Much of the nomenclature and general laboratory procedures required in this application can be found in Sambrook, et al.,
Molecular Cloning: A Laboratory Manual
(2nd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989. The manual is hereinafter referred to as “Sambrook et al.”
The term “nucleic acid” refers to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogues of natural nucleotides that hybridize to nucleic acids in manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence includes the complementary sequence thereof.
A “sialyltra
Gilbert Michel
Jennings Michael P.
Moxon Edward Richard
Wakarchuk Warren W.
Young N. Martin
National Research Council of Canada
Prouty Rebecca E.
Townsend and Townsend / and Crew LLP
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