Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2002-05-22
2004-05-25
Lilling, Herbert J. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S072000, C435S084000, C435S097000, C435S193000, C435S208000, C435S212000
Reexamination Certificate
active
06740509
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to novel methods for the production of a mucin-type glycopeptide by utilizing a transglycosylation of endo-&agr;-N-acetylgalactosaminidase.
In particular, the present invention relates to the methods for the production of a mucin-type sugar chain adduct of peptide which comprises transferring a liberated sugar chain to a serine residue or a threonine residue of the peptide or protein as a sugar acceptor in the presence of the sugar acceptor of high concentration by an endo-&agr;-N-acetylgalactosaminidase, instead of the original use thereof to hydrolyze the sugar chain.
BACKGROUND OF THE INVENTION
It has been found that a sugar chain combined with a glycopeptide or a glycoprotein functions in interactions among cells and enhances a resistance of the glycopeptide or the glycoprotein to a protease. Therefore various technologies of combining a sugar chain with a peptide or a protein have been developed in order to develop, for example, peptidic medicaments having high effects.
For example, there has been known a method for combining a complex-type sugar chain or a mannose-rich sugar chain with a peptide or a protein comprising, by utilizing a transfer reaction of endo-&bgr;-D-N-acetylglucosaminidase, cleaving the complex-type sugar chain or the mannose-rich sugar chain between GlcNAc-GlcNAc of the three sugar base core; and then transferring the sugar chain of nonreducing end upward of the cleaving site to GlcNAc residue of the peptide or the protein.
However there has not been found an endo-type enzyme which can be used in the production of a mucin-type sugar chain adduct of peptide or protein. Although there has been known in the art an endo-&agr;-D-N-acetylgalactosaminidase capable of liberating a mucin-type sugar chain from the mucin-type sugar chain adduct of peptide or protein, a transfer reaction that the sugar chain is transferred to the peptide has not been reported. For example, R. M. Bardales and V. P. Bhavanandan reported that an endo-&agr;-D-N-acetylgalactosaminidase from
Diplococcus pneumoniae
transfers sugar chains of glycopeptides obtained by decomposing a protein to glycerol, serine or threonine, but did not report an activity of transferring the sugar chains to a peptide or a protein (J. Biol. Chem. Vol. 264, 19893-19897 (1989)).
Accordingly, in order to synthesize a mucin-type glycopeptide, there was a need to use a chemical synthesis method by C. M. Taylor (Tetrahedron, 54, 11317-11382 (1998)) or a chemoenzymatic method by K. Ajisaka and M. Miyasato (Biosci. Biotechnol. Biochem., 64, 1743-1746 (2000)). However these methods are inappropriate to apply in an industrial manufacturing. The chemical method needs many steps for the protection and deprotection of hydroxyl groups in the carbohydrate moiety and amino- or carboxyl groups in the peptide moiety. The chemoenzymatic method recently developed also includes synthesizing at first a GalNAc-linked peptide by a peptide synthesizer using peracetylated GalNAc-linked Fmoc serine derivative. The GalNAc-linked serine residue was very labile for recemization, especially during the deacetylation reaction with sodium methoxide in dry methanol. Moreover, the yield of the following enzymatic galactosylation or sialylation for the extension of sugar chain depends on the solubility of the glycopeptide used as an acceptor of transglycosylation. Therefore an industrially advantageous method for the production of mucin-type glycopeptides was not known in the art.
In view of industrially manufacturing the mucin-type glycopeptides, a method for the production thereof by combining a whole sugar chain with a peptide utilizing an endo-type enzyme is most appropriate among various methods for the synthesis of glycopeptides. An object of the present invention is to utilize an endo-&agr;-N-acetylgalactosaminidase having activities to liberate a mucin-type sugar chain in the endo-type form and to transfer the liberated sugar chain to a hydroxyl group of serine residue or threonine residue of another peptide or protein to accomplish a method for the production of glycopeptide.
It had been reported that endo-&agr;-N-acetylgalactosaminidases had an activity to hydrolyze the mucin-type sugar chain (Y. Tanaka, Y. Takahashi, M. Shinose, S. Omura, I. I. -Karakasa, H. Iwase and K. Hotta, J. Fermentation and bioengineering, Vol. 85, 381-387 (1998)). The inventors has further found out in their study that some endo-&agr;-N-acetylgalactosaminidases from microorganisms in particular, those belonging to the genus, Streptomyces, have an ability to transfer a mucin-type sugar chain with a glycosidic linkage to a peptide or a protein, in addition to the activity to hydrolyze the mucin-type sugar chain. The inventors have accomplished the present invention based on this finding.
SUMMARY OF THE INVENTION
The present invention provides a method for the production of mucin-type glycopeptide comprising a transglycosylation using a sugar donor and a sugar acceptor with an endo-&agr;-N-acetylgalactosaminidase.
More particularly, the present invention provides a method for the production of a mucin-type sugar chain adduct of peptide comprising the steps of providing a glycoside compound having a mucin-type sugar chain as a sugar donor and a peptide containing threonine residue(s) or serine residue(s) as a sugar acceptor; and applying an endo-&agr;-N-acetylgalactosaminidase to the glycoside compound and the peptide, transferring the mucin-type sugar chain of the glycoside compound to the serine residue or the threonine residue of the peptide to produce the mucin-type sugar chain adduct of peptide.
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Ajisaka, et al. “Efficient synthesis of O-linked glycopeptide by a transglycosylation using endo &agr;-N-acetylgalactosaminidase fromstreptomycessp.”,Biosci. Biotechnol. Biochem., (2001) vol. 65(5): 1240-1243.
Ishii-Karakasa, et al. “Partial purification and characterization of an endo &agr;-N-acetylgalactosaminidase from the culture medium ofstreptomycessp. OH-11242”,Biochem. J., (1992) vol. 288: 475-482.
Iwase, et al. “Release of oligosaccharides possessing reducing endo &agr;-N-acetylgalactosaminidase from mucus glycoprotein instreptomycessp. OH-11242 culture medium through action of endo-type glycosidase”,Biochem. Biophys. Res. Com., (1988) vol. 151(1): 422-428.
Ishii-Karakasa, et al. “Structural determination of the O-linked sialyl oligosaccharides liberated from fetuin with endo &agr;-N-acetylgalactosaminidase-S by HPLC analysis and 600-MHz1H-NMR spectroscopy”,Eur. J. Biochem., (1997) vol. 247: 709-715.
Tanaka, et al. “Screening and fermentation of endo &agr;-N-acetylgalactosaminidase S, a mucin-hydrolyzing enzyme fromstreptomycesacting on the GalNAc-O-Ser (Thr) linkage”,J. of Fermentation and Bioengineering, (1998) vol. 85(4): 381-387.
Ajisaka Katsumi
Karakasa Ikuko Ishii
Bozicevic Field & Francis LLP
Francis Carol L.
Ikuko Ishii Karakasa
Lilling Herbert J.
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