Chimeric glycosaminoglycan oligosaccharides synthesized by enzymatic reconstruction and their use in substrate specificity determination of Streptococcus hyaluronidase

A method was developed for the reconstruction of glycosaminoglycan (GAG) ol igosaccharides using the transglycosylation reaction of an endo-beta-N-acet ylhexosaminidase, testicular hyaluronidase, under optimal conditions. Repet ition of the transglycosylation using suitable combinations of various GAGs as accepters and donors made it possible to custom-synthesize GAG oligosac charides. Thus we prepared a library of chimeric GAG; oligosaccharides with hybrid structures composed of disaccharide units such as GlcA-GlcNAc (from hyaluronic acid), GlcA-GalNAc (from chondroitin), GlcA-GalNAc4S (from chon droitin 4-sulfate), GlcA-GalNAc6S (from chondroitin g-sulfate), IdoA-GalNAc (from desulfated dermatan sulfate), and GlcA-GalNAc4,6-diS (from chondroit in sulfate E). The specificity of the hyaluronidase from Streptococcus dysg alactiae (hyaluronidase SD) was then investigated using these chimeric GAG oligosaccharides as model substrates. The results indicate that the specifi city of hyaluronidase SD is determined by the following restrictions at the nonreducing terminal side of the cleavage site: (i) at least one disacchar ide unit (GlcA-GlcNAc) is necessary for the enzymatic action of hyaluronida se SD; (ii) cleavage is inhibited by sulfation of the N-acetylgalactosamine ; (iii) hyaluronidase SD releases GlcA-GalNAc and IdoA-GalNAc units as well as GlcA-GlcNAc, At the reducing terminal side of the cleavage site, the su lfated residues on the N-acetylgalactosamines in the disaccharide units wer e found to have no influence on the cleavage. Additionally, we found that h yaluronidase SD can specifically and endolytically cleave the internal unsu lfated regions of chondroitin sulfate chains. This demonstration indicates that custom-synthesized GAG; oligosaccharides will open a new avenue in GAG ; glycotechnology.