Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease
Reexamination Certificate
1998-10-30
2002-07-16
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Transferase other than ribonuclease
C435S004000, C435S183000, C435S252300, C435S264000, C435S325000, C435S410000, C435S419000, C800S008000, C800S295000
Reexamination Certificate
active
06420149
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a polypeptide having poly-N-acetyllactosamine sugar chains synthesis-related activity, a process for producing the polypeptide, DNA coding for the polypeptide, a process for producing the DNA, a recombinant vector having the DNA integrated therein, a transformant carrying the recombinant vector, an antibody recognizing the polypeptide, a process for producing poly-N-acetyllactosamine sugar chains by use of the polypeptide, and a process for producing poly-N-acetyllactosamine sugar chains by use of the transformant carrying the recombinant vector.
BACKGROUND OF THE INVENTION
It is estimated that sugar chains are involved not only in life phenomena such as development, differentiation and cell recognition but also in occurrence and progress of inflammations, cancers, infections, auto-immune diseases and a number of other diseases [A. Kobata, S. Hakomori and K. Nagai: Glycobiology Series (1) to (6), Kodansha (1993), Glycobiology, 3, 97 (1993)].
Sugar chains exsist not only as glycoproteins, proteoglycans or glycolipids, in which they are added to proteins or lipids, but also as oligosaccharides.
The poly-N-acetyllactosamine sugar chain as the subject of the present invention is a sugar chain with the structure having N-acetyllactosamine as the repeating unit bound via &bgr;1, 3 linkage [(Gal &bgr;1-4GlcNAc &bgr;1-3)n where n is 2 or more] , and it exists not only in N-glycoside linked sugar chains and O-glycoside linked sugar chains on glycoproteins but also in sugar chains of glycolipids and in oligosaccharides.
The poly-N-acetyllactosamine sugar chain is synthesized by alternately reaction of &bgr;1, 4-galactosyltransferases and &bgr;1, 3-N-acetylglucosaminyltransferases. The gene coding for the former enzyme &bgr;1,4-galactosyltransferase has already been cloned, but the gene coding for the latter enzyme &bgr;1,3-N-acetylglucosaminyltransferase is still not cloned. With respect to &bgr;1, 3-N-acetylglucosaminyltransferases having poly-N-acetyllactosamine synthesis-related activity, there are only reports on their partial purification resulting in no information of their amino acid sequences [J. Biol. Chem., 268, 27118 (1993), J. Biol. Chem., 267, 2994 (1992), J. Biol. Chem., 263, 12461 (1988), Jpn. J. Med. Sci. Biol., 42, 77 (1989)].
In some galactose residues in poly-N-acetyllactosamine sugar chains, an N-acetylglucosamine is bound via &bgr;1,6-linkage to synthesize poly-N-acetyllactosamine sugar chains having branched chains such as Gal&bgr;1-4GlcNAc&bgr;1-3(Gal&bgr;1-4GlcNAc&bgr;1-6) Gal&bgr;1-4GlcNAc. A glycosyltransferase transferring such branched &bgr;1,6-bound N-acetylglucosamine is &bgr;1,6-N-acetylglucosaminyltransferase (I-branching enzyme). A gene for this enzyme has also been cloned. The linear poly-N-acetyllactosamine sugar chain (i-antigen) is recognized by anti-i antibody and the branched poly-N-acetyllactosamine sugar chain (I-antigen) is recognized by anti-I antibody [J.
Biol. Chem., 254, 3221 (1979)].
Saccharides such as fucose, sialic acid, N-acetylgalactosamine, galactose etc. or sulfate groups etc are attached to linear or branched poly-N-acetyllactosamine sugar chains, and a wide variety of cell-specific or period-specific sugar chains (e.g. functional sugar chains, blood type sugar chains, cancer-related sugar chains) are formed [A. Kobata, S. Hakomori, K. Nagai: Glycobiology Series (1) to (6), Kodansha (1993), Japan].
It is known that poly-N-acetyllactosamine sugar chains having a sialyl-Lewis x sugar chain structure at their termini are present on granulocytes, monocytes or activated T cells, and these sugar chains function as ligands for E-selectin or P-selectin as adhesive molecules and are involved in accumulation of leukocytes into inflammation sites [A. Kobata, S. Hakomori, K. Nagai: Glycobiology Series (1) to (6), Kodansha (1993), Japan].
It is known that Poly-N-acetyllactosamine sugar chains having a sialyl-Lewis x sugar chain structure at their termini are present on cancer cells such as colon cancer cells, and it is suggested that these sugar chains also function as ligands for E-selectin or P-selectin and are involved in metastasis of cancer cells [A. Kobata, S. Hakomori, K. Nagai: Glycobiology Series (1) to (6), Kodansha (1993)].
It is known that the structure of poly-N-acetyllactosamine sugar chain is changed in the process of embryonic development, cell differentiation or cell transformation [A. Kobata, S. Hakomori, K. Nagai: Glycobiology Series (1) to (6), Kodansha (1993)]. While linear poly-N-acetyllactosamine sugar chains are expressed in human embryonic erythrocytes, branched poly-N-acetyllactosamine sugar chains are expressed in human adult erythrocytes [A. Kobata, S. Hakomori, K. Nagai: Glycobiology Series (1) “World of Various Sugar Chains” Kodansha (1993)]. ABO-type blood group antigens are expressed at the termini of poly-N-acetyllactosamine sugar chains in these erythrocytes. Expression of blood group antigens at each terminus of branched poly-N-acetyllactosamine sugar chain results in multivalent antigens to increase the ability of the antigens to bind to antibodies against blood group sugar chains more than 10
3
-fold compared with that of the linear antigens.
It is known that a series of sugar-chain antigens are expressed in a regulated manner during developmental process of mouse early embryo. SSEA-1 (stage specific embryonic antigen-1) is a Lewis x sugar chain [Gal&bgr;1-4(Fuc &agr;1-3)GlcNAc] which appears at the termini of poly-N-acetyllactosamine sugar chains, and expression of this antigen is initiated at the 8-cell stage, peaks at the morula stage, and disappears after the blastocyst stage [A. Kobata., S. Hakomori, K. Nagai: Glycobiology Series {circle around (
3
)} “Glycobiology of Cellular Society”, Kodansha (1993)]. The morula stage corresponds to the stage at which embryonic cells having increased merely numerically by repeated proliferation through cell division enter the blastocyst stage at which the cells come to have a differentiated “form”. Morula cells adhere to each other just before forming a blastocyst to cause cell compaction. If an oligosaccharide having SSEA-1 antigen is added, this cell compaction is inhibited and normal development thereafter is also inhibited [J. Exp. Med., 160, 1591 (1984)]. It is also known that adhesion of mouse teratocarcinoma cells is inhibited by anti-SSEA-l antibody [A. Kobata, S. Hakomori, K. Nagai: Glycobiology Series {circle around (
3
)} “Glycobiology of Cellular Society”, Kodansha (1993)]. The foregoing indicates that the SSEA-1 antigen acts as an adhesive molecule or a sugar chain signal to play an important role in development of early embryos.
It is known that poly-N-acetyllactosamine sugar chains are expressed at higher levels in cancer cells than in their corresponding normal cells [J.Biol. Chem., 259, 10834 (1984), J. Biol. Chem., 261, 10772 (1986), J. Biol. Chem., 266, 1772 (1991), J. Biol. Chem., 267, 5700 (1992)]. It is known that if N-ras protooncogene is expressed in NIH3T3 cells, the molecular weight of N-linked sugar chain on cells is increased, and the cells attain invasive activity, and at the same time, the amount of poly-N-acetyllactosamine sugar chains in the N-linked sugar chains is increased and simultaneously &bgr;1,4-galactosyltransferase and &bgr;1,3-N-acetylglucosaminyltransferase activities involved in synthesizing poly-N-acetyllactosamine sugar chains are increased [J. Biol. Chem., 266, 21674 (1991)].
Galectins are a family of lectins with affinity for &bgr;-galactoside, and are involved in cell adhesion and signal transduction, and their relation with diseases such as cancers is also suggested [Trends in Glycoscience and Glycotechnology, 9, 9 (1997)]. Ten types of Galectins have been found in mammals. Out of them, galectin-1 and galectin-3 are known to bind with high affinity to linear poly-N-acetyllactosamine sugar chains, and spec
Fukuda Minoru
Miura Kazumi
Nakagawa Satoshi
Nishi Tatsunari
Sasaki Katsutoshi
Achutamurthy Ponnathapu
Kyowa Hakko Kogyo Co. Ltd.
Rao Manjunath N.
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