人来源α-1,3/1,6甘露糖转移酶Alg2的异源表达及活性测定

Heterologous Expression and Activity Assay of Humanα-1,3/1,6 Mannosyltransferase Alg2

在N-糖基化途径中,双功能型的甘露糖转移酶Alg2蛋白催化两个甘露糖分别以α-1,3/1,6连键组装到多萜醇寡糖Man1GlcNAc2-PP-Dol上,形成Man3GlcNAc2-PP-Dol。作者探究人来源的Alg2蛋白(hAlg2)在酿酒酵母中的体内功能和在大肠杆菌中表达并纯化后的体外活性。构建酿酒酵母w303a-GAL1pr-ALG2,并利用该菌株的生长被葡萄糖抑制的特点,发现过表达hALG2基因能够回补其生长缺陷,证实了hALG2和ScALG2的功能同源性。作者利用大肠杆菌表达系统,成功表达并纯化了TrxA-hAlg2,并以Man1GlcNAc2-PP-Phy(PPGn2M1)代替hAlg2的天然底物,测定了其体外活性。结合液质联用的方法,检测发现TrxA-hAlg2具有催化PPGn2M1生成PPGn2M2和PPGn2M3的甘露糖基转移酶活性,为建立基于大肠杆菌表达纯化的hAlg2体外反应体系奠定了基础。

In the N-glycosylation pathway,the bifunctional mannosyltransferase Alg2 protein catalyzes the assembly of two mannose onto dolichol linked oligosaccharide Man1GlcNAc2-PP-Dol byα-1,3/1,6 linkages to form Man3GlcNAc2-PP-Dol.In this study,the function of human Alg2 protein(hAlg2)in S.cerevisiae cells was explored and its activity in vitro after expressed and purified from E.coli was investigated.The growth of constructed yeast strain w303a-GAL1pr-ALG2 was suppressed by glucose,while the overexpression of hALG2 gene could rescue such growth defect,confirming the functional homology of hALG2 and ScALG2.In addition,TrxA-hAlg2 was successfully purified from E.coli and its activity in vitro was further tested using Man1GlcNAc2-PP-Phy(PPGn2M1),a natural substrate analogue of hAlg2.LC-MS analysis showed that the purified TrxA-hAlg2 was capable of producing PPGn2M2 and PPGn2M3 from PPGn2M1,providing the foundation for in vitro reaction system of hAlg2 purified from E.coli.