Enzymatic attachment of glycosaminoglycan chain to peptide using the sugar chain transfer reaction with endo-beta-xylosidase.
Endo-beta-xylosidase from the mid-gut gland of the molluscus Patinopecten is an endo-type glycosidase that hydrolyzes the xylosyl serine linkage between a core protein and a glycosaminoglycan (GAG) chain, releasing the intact GAG chain from proteoglycan. In this study, we investigated GAG chain transfer activity of this enzyme, in order to develop a method for attaching GAG chains to peptide. Peptidochondroitin sulfate (molecular mass of sugar chain, 30 kDa) from bovine tracheal cartilage as a donor and butyloxycarbonyl-leucyl-seryl-threonyl-arginine-(4-methylcoumaryl-7-amide) as an acceptor were incubated with endo-beta-xylosidase. As a result, a reaction product with the same fluorescence as the acceptor peptide was observed. High pressure liquid chromatography analysis, cellulose acetate membrane electrophoresis, and enzymatic digestion showed that this reaction product had the chondroitin sulfate (ChS) from the donor. Furthermore, the acceptor peptide was released from this reaction product after hydrolysis by endo-beta-xylosidase. Therefore, it was confirmed that the ChS chain released from the donor was transferred to the acceptor peptide by the GAG chain transfer reaction of endo-beta-xylosidase. The optimal pH for hydrolysis by this enzyme was found to be about 4.0, whereas that for this reaction was about 3.0. Not only the ChS but also the dermatan sulfate and the heparan sulfate were transferred to the acceptor peptide by this reaction. By using this reaction, the GAG chain could be attached to the peptide in one step. The GAG chain transfer reaction of endo-beta-xylosidase should be a significant glycotechnological tool for the artificial synthesis of proteoglycan.
Ishido
Keinosuke
K
Department of Biochemistry and Second Department of Surgery, Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki 036-8562, Japan.
Takagaki
Keiichi
K
Iwafune
Mito
M
Yoshihara
Syuichi
S
Sasaki
Mutsuo
M
Endo
Masahiko
M
D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D006025:Glycosaminoglycans / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D013058:Mass Spectrometry; D008956:Models, Chemical; D008974:Mollusca; D009844:Oligosaccharides / Q000494:pharmacology; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013552:Swine; D013997:Time Factors; D014995:Xylosidases / Q000378:metabolism*
D006025:Glycosaminoglycans; D009844:Oligosaccharides; D010455:Peptides; D014995:Xylosidases; C056547:endo-beta-xylosidase