Characterization of two different endo-α-N-acetylgalactosaminidases from probiotic and pathogenic enterobacteria, Bifidobacterium longum and Clostridium perfringens
Characterization of two different endo-α-N-acetylgalactosaminidases from probiotic and pathogenic enterobacteria, Bifidobacterium longum and Clostridium perfringens
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DOI：
10.1093/glycob/cwn053
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发表时间：
2008-09-01
期刊：
影响因子：
4.3
通讯作者：
Yamamoto, Kenji
中科院分区：
文献类型：
--
作者：
Ashida, Hisashi;Maki, Riichi;Yamamoto, Kenji
内切-α-N-乙酰半乳糖胺酶(Endo-α-GalNAc-ase)催化粘蛋白糖链还原末端的α-GalNAc与蛋白质的丝氨酸/苏氨酸之间的O-糖苷键的水解，从而释放寡糖。此前，我们从长双歧杆菌中克隆了编码Endo-α-GalNAc-ase的基因EN-GBF，该基因特异性地释放二糖半乳糖β1-3GalNAc(Fujita K，Oura F，Nagamamine N，Katayama T，Hiratake J，Sakata K，Kumagai H，Yamamoto K.2005)。长双歧杆菌核心1型O-糖特异性内切酶糖苷水解酶家族的鉴定和分子克隆。J生物化学。280：37415-37422)。在此，我们从致病肠杆菌产气荚膜梭菌中克隆了一个类似的基因engCP，并对其基因产物EngCP进行了鉴定。用二叔丁基硅烯直接法合成的一系列对硝基苯基-α-糖苷对EngCP和EngBF的底物特异性进行了详细的分析，结果表明这两种酶都释放了Hex/HexNAcβ1-3GalNAc(Hex=Gal或GLC)。EngCP还能释放核心2三糖半乳糖β1-3(GlcNAcβ1-6)GalNAc、核心8二糖半乳糖α1-3GalNAc和单糖GalNAc。我们的结果表明，EngCP比EngBF具有更广泛的底物特异性。研究了这两种酶对天然糖蛋白和细胞表面糖蛋白的作用。
Endo-alpha-N-acetylgalactosaminidase (endo-alpha-GalNAc-ase) catalyzes the hydrolysis of the O-glycosidic bond between alpha-GalNAc at the reducing end of mucin-type sugar chains and serine/threonine of proteins to release oligosaccharides. Previously, we identified the gene en-gBF encoding endo-alpha-GalNAc-ase from Bifidobacterium longum, which specifically released the disaccharide Gal beta 1-3GalNAc (Fujita K, Oura F, Nagamine N, Katayama T, Hiratake J, Sakata K, Kumagai H, Yamamoto K. 2005. Identification and molecular cloning of a novel glycoside hydrolase family of core 1 type O-glycan-specific endo-alpha-N-acetylgalactosaminidase from Bifidobacterium longum. J Biol Chem. 280:37415-37422). Here we cloned a similar gene named engCP from Clostridium perfringens, a pathogenic enterobacterium, and characterized the gene product EngCP. Detailed analyses on substrate specificities of EngCP and EngBF using a series of p-nitrophenyl-alpha-glycosides chemically synthesized by the di-tert-butylsilyiene-directed method revealed that both enzymes released Hex/HexNAc beta 1-3GalNAc (Hex = Gal or Glc). EngCP could also release the core 2 trisaccharide Gal beta 1-3(GlcNAc beta 1-6)GalNAc, core 8 disaccharide Gal alpha 1-3GalNAc, and monosaccharide GalNAc. Our results suggest that EngCP possesses broader substrate specificity than EngBF. Actions of the two enzymes on native glycoproteins and cell surface glycoproteins were also investigated.