一例Bw亚型的糖基转移酶等位基因鉴定及蛋白结构分析

目的对1例Bw亚型个体进行分子遗传学及蛋白结构分析。方法采用血清学技术进行红细胞表面抗原和血清中抗体的测定,并用PCR-基于序列的分型(PCR-sequence-based typing,PCR-SBT)方法进行ABO基因的全编码区序列和第1内含子区红系特异性调控元件的分析。进一步对存在突变位点的第5~7外显子扩增片段进行T-A克隆分离单倍体和序列验证分析。应用Pymol软件对突变蛋白进行3D结构的模拟,分析氨基酸残基的变化对蛋白结构稳定性的影响。结果该样本血清学表现为B抗原减弱,血清中含有抗-B抗体。ABO基因全编码区测序初步确定其基因型为ABO*B.01/ABO*O.01.01伴有c.734C/T的杂合突变。第1内含子红系特异性调控元件区碱基序列无异常。通过单倍体克隆分离,确定突变位点c.734T位于ABO*B.01等位基因链,另一个等位基因为ABO*O.01.01。该突变将B糖基转移酶活性中心245位Thr置换为Ile。蛋白3D结构的模拟分析发现氨基酸置换后,与之结合的残基未发生改变而连接的氢键距离发生变化,同时增加了与远距离水分子之间的连接。结论B糖基转移酶基因c.734C>T突变导致酶活性中心氨基酸置换,影响了突变B糖基转移酶蛋白稳定性,引起酶活性减弱,从而产生了Bw变异型。

Identification of a glycosyltransferase allele associated with Bw subtype and analysis of the protein structure

Objective To explore the molecular basis for an individual with Bw subtype.Methods Routine serological reactions were used to determine the surface antigens of erythrocytes and antibodies in serum.PCR-sequence-based typing(PCR-SBT)was used to analyze the coding regions of the ABO gene and erythroid-specific regulatory element in its intron 1.Amplicon for exons 5 to 7 containing the variant site was cloned by TA cloning for the isolation of the haploid and verification of the sequence.The 3D structure of mutant protein was predicted with Pymol software.Changes of amino acid residues and structural stability were also analyzed.Results Serological assay showed that the individual had weakened B antigen and anti-B antibody in his serum.His genotype was determined as ABO*B.01/ABO*O.01.01.Sequencing of the entire coding region of the ABO gene identified an additional heterozygous c.734C/T variant.No variant was found in the erythroid-specific regulatory element of intron 1.Haploid cloning and isolation has obtained an ABO*O.01.01 allele and a ABO*B.01 allele containing a c.734T variant,which has led to substitution of Thr by Ile at position 245 in the functional center of glycosyltransferase.Based on the 3D structure of the protein,the residues binding with the mutation were unchanged,but the bonding distance between the hydrogens was changed with the amino acid substitution,meanwhile,the connections with water molecules were increased.Conclusion The c.734C>T variant of the GTB gene can result in an amino acid substitution in the functional center of the enzyme,which in turn may affect the stability of glycosyltransferase B protein and reduce its enzymatic activity.