作用于种属差异性表位的细菌α-半乳糖苷酶(EmGalase)的鉴定和研究

对脑膜炎败血伊丽莎白金菌进行糖苷酶功能基因组分析发现,该菌存在一个GH27家族α-半乳糖苷酶基因。克隆该基因并表达蛋白后,利用人工合成的pNP底物研究其酶学性质,发现该酶具有α连接的半乳糖(α-galactose,α-Gal)底物特异性,酶反应pH为3.0~8.0,反应温度为4~45 ℃。用不同寡糖底物进一步确定,该酶能够酶切直链末端α-1,3、1,4、1,6Gal。在猪红细胞上进行的酶切实验显示,该酶能够高效清除存在于猪红细胞表面的末端Gal α 1-3Gal表位。末端α-半乳糖基化修饰在免疫与感染中发挥着重要的生物学作用,作用于种属差异性表位的细菌α-半乳糖苷酶的发现将为该领域的研究提供一个新的工具,为缓解异种输血中的超急性免疫排斥反应提供一种可能。

Identification and analysis of an alpha galactosidase(EmGalase) acting on species-differentiated epitopes

Through a functional genomic analysis for glycosidase, we identified a candidate alpha galactosidase in the genome of Elizabethkingia meningoseptica. The gene was cloned and expressed in Escherichia coli (E.coli). The purified protein was subjected to assays with synthetic p-nitrophenol (pNP) substrates for its enzymatic properties. The results indicated that it had alpha-linked galactose (α-Gal) substrate specificity. The optimum pH was between 3.0 and 8.0, and the optimum temperature was between 4 and 45 ℃. Further assays with oligosaccharide substrates and Mass Spectrometry analysis found that this enzyme could digest the terminal α-1，3/1，4/1，6Gal, respectively. In addition, we also demonstrated that the enzyme can efficiently remove the terminal Galα1-3Gal antigen presented on porcine erythrocytes to alleviate hyperacute immune rejection in heterologous blood transfusion. As terminal alpha galactose modification plays an important biological role in immunity and infection, the reported alpha galactosidase may serve as a new tool for research in this field.