cisAB03亚型分子生物学鉴定及分子对接

Molecular biological identification and molecular docking of cisAB03 subtype

目的:通过1例罕见的cisAB03亚型的研究,探讨关键氨基酸突变后糖基转移酶影响糖供体和受体结合的分子机制。方法:采用血清学方法鉴定血型表型,直接测序方法鉴定基因型,转移酶同源建模后进行UDP和H糖链的对接,分析突变后糖基转移酶结构差异对底物结合的影响,最后利用细胞体外表达实验间接验证突变后糖基转移酶合成产物的效率。结果:血清学鉴定表型为cisAB,基因测序显示患者基因型是ABO*cisAB.03/O.01.01,同源建模显示与野生型相比,cisAB03糖基转移酶p.Pro234Ser突变导致Met266结构朝向改变,分子对接后表面显示呈现催化活性中心结合凹槽结构改变,更有利于UDP-GalNAc加成到H抗原。细胞体外表达实验证实cisAB03合成的A抗原强于B抗原。结论:p.Pro234Ser突变是形成cisAB03糖基转移酶双功能活性的关键位点。

Aim:To investigate the molecular basis of glycosyltransferase affecting the binding of sugar donor with acceptor after key amino acid mutations through a study of a rare cisAB03 subtype.Methods:Serological methods were used to detect the blood type.The genotype was identified by direct sequencing.The homologous modeling of cisAB03 glycosyltransferase was used to dock the UDP and H sugar chains into catalytic active center,and the effects of structural changes of cisAB03 glycosyltransferase on substrates binding were evaluated.Finally,the efficiency of the glycosyltransferase synthesizing product after mutation was indirectly verified by in vitro expression assay.Results:The patient′s serological phenotype was cisAB.Gene sequencing showed that the patient′s genotype was ABO*cisAB.03/O.01.01.Homology modeling showed that compared with wild type,cisAB03 glycosyltransferase p.Pro234Ser mutation resulted in the orientation of the Met266 structure changes and surface representation of molecular docking exhibited alteration in the binding groove of catalytic active center,which was more favorable for the addition of UDP-GalNAc to the H antigen.In vitro cell expression assays confirmed that the A antigen synthesized by cisAB03 was stronger than the B antigen.Conclusion:The p.Pro234Ser mutation is a key site for the bifunctional activity of cisAB03 glycosyltransferase.