Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1999-06-24
2002-04-30
Prats, Francisco (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S072000, C435S084000, C435S100000, C435S101000, C435S170000, C435S871000
Reexamination Certificate
active
06379933
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method of transferring at least two saccharide units with a polyglycosyltransferase, a polyglycosyltransferase and a gene encoding such a polyglycosyltransferase.
2. Discussion of the Background
Biosynthesis of Oligosaccharides
Oligosaccharides are polymers of varying number of residues, linkages, and subunits. The basic subunit is a carbohydrate monosaccharide or sugar, such as mannose, glucose, galactose, N-acetylglucosamine, N-acetylgalactosamine, and the like. The number of different possible stereoisomeric oligosaccharide chains is enormous.
Oligosaccharides and polysaccharides play an important role in protein function and activity, by serving as half-life modulators, and, in some instances, by providing structure. Oligosaccharides are critical to the antigenic variability, and hence immune evasion, of Neisseria, especially gonococcus.
Numerous classical techniques for the synthesis of carbohydrates have been developed, but these techniques suffer the difficulty of requiring selective protection and deprotection. Organic synthesis of oligosaccharides is further hampered by the lability of many glycosidic bonds, difficulties in achieving regioselective sugar coupling, and generally low synthetic yields. In short, unlike the experience with peptide synthesis, traditional synthetic organic chemistry cannot provide for quantitative, reliable synthesis of even fairly simple oligosaccharides.
Recent advances in oligosaccharide synthesis have occurred with the isolation of glycosyltransferases from natural sources. These enzymes can be used in vitro to prepare oligosaccharides and polysaccharides (see, e.g., Roth, U.S. Pat. No. 5,180,674). The advantage of biosynthesis with glycosyltransferases is that the glycosidic linkages formed by enzymes are highly stereo and regiospecific. However, each enzyme catalyzes linkage of specific sugar donor residues to other specific acceptor molecules, e.g., an oligosaccharide or lipid. Thus, synthesis of a desired oligosaccharide has required the use of a different glycosyltransferase for each different saccharide unit being transferred.
More specifically, such glycosyltransferases have only provided for the transfer of a single saccharide unit, specific for the glycosyltransferase. For example, a galactosyltransferase would transfer only galactose, a glucosyltransferase would transfer only glucose, an N-acetylglucosaminlytransferase would transfer only N-acetylglucosamine and a sialyl transferase would transfer only sialic acid.
However, the lack of generality of glycosyltransferases makes it necessary to use a different glycosyltransferase for every different sugar donor being transferred. As the usefulness of oligosaccharide compounds expands, the ability to transfer more than one sugar donor would provide a tremendous advantage, by decreasing the number of glycosyltransferases necessary to form necessary glycosidic bonds.
In addition, a glycosyltransferase which transferred at least two different sugar donors would be advantageous in synthesizing two glycosidic bonds of at least a trisaccharide, using the same glycosyltransferase.
A locus involved in the biosynthesis of gonococcal lipooligosaccharide (LOS) has been reported as being cloned from the gonococcal strain F62 (Gotschlich J. Exp. Med. (1994) 180, 2181-2190). Five genes lgtA, lgtB, lgtC, lgtD and lgtE are reported, and based on deletion experiments, activities are postulated, as encoding for glycosyltransferases. Due to the uncertainty caused by the nature of the deletion experiments, the exact activity of the proteins encoded by each of the genes was not ascertained and some of the genes are only suggested as being responsible for one or another activity, in the alternative. The gene lgtA is suggested as most likely to code for a GlcNAc transferase.
The transfer of more than one different saccharide moiety, by a polyglycosyltransferase has heretofore been unreported.
SUMMARY OF THE INVENTION
The present invention is directed to a method of transferring at least two saccharide units with a polyglycosyltransferase, a polyglycosyltransferase and nucleic acids encoding a polyglycosyltransferase.
Accordingly, in one aspect, the invention is directed to a method of transferring at least two saccharide units with a polyglycosyltransferase.
Accordingly, another aspect of the invention is directed to a method of transferring at least two saccharide units with a polyglycosyltransferase, which transfers both GlcNAc, and GalNAc, from the corresponding sugar nucleotides to a sugar acceptor.
According to another aspect of the invention, a polyglycosyltransferase is obtained from a bacteria of the genus Neisseria, Escherichia or Pseudomonas.
Another aspect of the invention, is directed to a method of making at least two oligosaccharide compounds, from the same acceptor, with a polyglycosyltransferase.
Another aspect of the invention is directed to a method of making at least two oligosaccharide compounds from the same acceptor with a polyglycosyltransferase, which transfers both GlcNAc and GalNAc from the corresponding sugar nucleotides to the sugar acceptor.
Another embodiment of the present invention is directed to a method of transferring an N-acetylgalactosamine using a glycosyltransferase of SEQ ID NO: 8.
In specific embodiments, the invention relates to a nucleic acid that has a nucleotide sequence which encodes for the polypeptide sequence shown in SEQ ID NO. 8.
The functionally active polyglycosyltransferase of the invention is characterized by catalyzing both the addition of GalNAc &bgr;1→3 to Gal and the addition of GlcNAc &bgr;1→3 to Gal.
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Andree and Berliner, 1978, “Glucosyl Transferase Activity of Bovine Galactosyl Transferase”, Biochim. Biophys. Acta 544:489-495.
Avigad et al., 1962, “The D-Galactose Oxidase ofPolyporus circinatus”, J. Biol. Chem 237:2736-2743.
Gotschlich, 1994, “Genetic Locus for the Biosynthesis of the Variable Portion ofNeisseria gonorrhoeaeLipooligosaccharide”, J. Exp. Med. 180:2181-2190.
Greenwell et al., 1986, “UDP-N-Acetyl-D-Galactosamine as a Donor Substrate for the Glycosyltransferase Encoded by the B Gene at the Human Blood Group ABO Locus”, Carbohydrate Res. 149:149-170.
Greenwell et al., 1979, Blood Group A Synthesising Activity of the Blood Group B Gene Specified &agr;-3-D-Galactosyl Transferase.
Palcic and Hindsgaul, 1991, “flexibility in the Donor Substrate Specificity of &bgr; 1,4-Galactosyltransferase: Application in the Synthesis of Complex Carbohydrates”, Glycobiol. 1:205-209.
Schram et al., 1977, “The Identity of &agr;-Galactosidase B from Human Liver”, Biochim. Biopys. Acta 482:138-144.
Takeya et al., 1993, “Biosynthesis of the Blood Group P Antigen-Like GalAc&bgr;1→3Gal&bgr;1→4GlcNAc/Glc Structure: Kinetic Evidence for the Responsibility of N-Acetylglucosaminyl-Transferase”, Jpn. J. Med. Sci. Biol. 46:1-15.
Buczala Stephanie L.
Johnson Karl F.
Roth Stephen
Morgan & Lewis & Bockius, LLP
Neose Technologies, Inc.
Prats Francisco
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