Takagaki K, Munakata H, Majima M, Kakizaki I, Endo M
Department of Biochemistry, Hirosaki University School of Medicine, Zaifu-cho, Hirosaki 036-8562, Japan.
J Biochem. 2000 Apr;127(4):695-702. doi: 10.1093/oxfordjournals.jbchem.a022659.
A method was developed for the reconstruction of glycosaminoglycan (GAG) oligosaccharides using the transglycosylation reaction of an endo-beta-N-acetylhexosaminidase, testicular hyaluronidase, under optimal conditions. Repetition of the transglycosylation using suitable combinations of various GAGs as acceptors and donors made it possible to custom-synthesize GAG oligosaccharides. 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 chondroitin 4-sulfate), GlcA-GalNAc6S (from chondroitin 6-sulfate), IdoA-GalNAc (from desulfated dermatan sulfate), and GlcA-GalNAc4,6-diS (from chondroitin sulfate E). The specificity of the hyaluronidase from Streptococcus dysgalactiae (hyaluronidase SD) was then investigated using these chimeric GAG oligosaccharides as model substrates. The results indicate that the specificity of hyaluronidase SD is determined by the following restrictions at the nonreducing terminal side of the cleavage site: (i) at least one disaccharide unit (GlcA-GlcNAc) is necessary for the enzymatic action of hyaluronidase 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 sulfated residues on the N-acetylgalactosamines in the disaccharide units were found to have no influence on the cleavage. Additionally, we found that hyaluronidase SD can specifically and endolytically cleave the internal unsulfated regions of chondroitin sulfate chains. This demonstration indicates that custom-synthesized GAG oligosaccharides will open a new avenue in GAG glycotechnology.
开发了一种利用内切β-N-乙酰己糖胺酶（睾丸透明质酸酶）在最佳条件下的转糖基化反应来重建糖胺聚糖（GAG）寡糖的方法。使用各种GAG作为受体和供体的合适组合重复转糖基化反应，使得定制合成GAG寡糖成为可能。因此，我们制备了一个嵌合GAG寡糖文库，其具有由二糖单元组成的杂合结构，如GlcA-GlcNAc（来自透明质酸）、GlcA-GalNAc（来自软骨素）、GlcA-GalNAc4S（来自硫酸软骨素4-硫酸酯）、GlcA-GalNAc6S（来自硫酸软骨素6-硫酸酯）、IdoA-GalNAc（来自脱硫酸皮肤素）和GlcA-GalNAc4,6-二硫酸酯（来自硫酸软骨素E）。然后使用这些嵌合GAG寡糖作为模型底物研究了来自无乳链球菌的透明质酸酶（透明质酸酶SD）的特异性。结果表明，透明质酸酶SD的特异性由切割位点非还原末端侧的以下限制决定：（i）透明质酸酶SD的酶促作用至少需要一个二糖单元（GlcA-GlcNAc）；（ii）N-乙酰半乳糖胺的硫酸化抑制切割；（iii）透明质酸酶SD释放GlcA-GalNAc和IdoA-GalNAc单元以及GlcA-GlcNAc。在切割位点的还原末端侧，发现二糖单元中N-乙酰半乳糖胺上的硫酸化残基对切割没有影响。此外，我们发现透明质酸酶SD可以特异性地和内切地切割硫酸软骨素链的内部未硫酸化区域。这一证明表明，定制合成的GAG寡糖将为GAG糖技术开辟一条新途径。
