中国寄生虫学与寄生虫病杂志 ›› 2019, Vol. 37 ›› Issue (4): 399-405.doi: 10.12140/j.issn.1000-7423.2019.04.005

• 论著 • 上一篇    下一篇

云南省1例伯氨喹诱发溶血患者的G6PD基因编码区突变分析及酶蛋白空间结构预测

董莹1,*(), 刘淑萍1,2, 徐艳春1, 刘言1, 邓艳1, 陈梦妮1   

  1. 1 云南省寄生虫病防治所,云南省疟疾研究中心,云南省虫媒传染病防控研究重点实验室,普洱 665000
    2 大理大学基础学院,大理 671000
  • 收稿日期:2019-01-28 出版日期:2019-08-30 发布日期:2019-09-05
  • 通讯作者: 董莹
  • 作者简介:

    作者简介:董莹(1964-),女,本科,主任医师,从事人体寄生虫病原学及分子生物学研究。E-mail: luxidongying@126.com

  • 基金资助:
    1 国家自然科学基金(No. 81660559);2 云南省科技项目应用基础研究计划青年项目(No. 2017FD007,No. 2017FD190)

Mutations and predicted structure change of G6PD isolated from a patient with primaquine-induced hemolysis in Yunnan Province

Ying DONG1,*(), Shu-ping LIU1,2, Yan-chun XU1, Yan LIU1, Yan DENG1, Meng-ni CHEN1   

  1. 1 Yunnan Institute of Parasitic Diseases,Yunnan Center of Malaria Research,Yunnan Provincial Key Laboratory of Vector-borne Diseases Control and Research,Puer 665000,China
    2 School of Basic Medical Sciences,Dali University,Dali 671000,China
  • Received:2019-01-28 Online:2019-08-30 Published:2019-09-05
  • Contact: Ying DONG
  • Supported by:
    Supported by National Science Foundation(No. 81660559)and the Youth Project of Yunnan Province Basic Research Program (No. 2017FD007, No. 2017FD190)

摘要:

目的 分析伯氨喹诱发溶血患者的葡萄糖-6-磷酸脱氢酶(G6PD)基因突变对空间结构形成的影响。方法 于2018年5月18日采集1例因服用伯氨喹出现溶血反应、G6PD酶活性下降75%的间日疟病例血样。提取血样的人基因组DNA,通过PCR分别扩增含G6PD基因exon2、exon3~7、exon8~9和exon10~13等12个外显子的片段并测序。整理获得的DNA序列与G6PD基因野生型、突变型序列比对,以确认12个外显子分别的起止点并拼接成exon2~13外显子的cDNA序列。用MEGA5.04软件分析cDNA序列的错义突变、同义突变和进行氨基酸链转换。采用SWISS-MODEL预测氨基酸链空间结构(www.swissmodel.expasy.org/interactive),PyMOL 2.2.0软件修饰空间结构预测图。结果 间日疟患者血样基因组经4个PCR反应体系扩增,分别获得含G6PD基因exon2、exon3~7、exon8~9、exon10~13外显子的336、2 277、976和1 421 bp等4种目标产物。由测序结果整理获得12个外显子的cDNA链为1 545 bp,与野生型序列比对的同义突变、错义突变位点分别为c.1311T > C和c.1376G > T,导致437、459氨基酸呈Y/Y不变和R/L变异,空间位置均未在G6PD与NADP+、乙醇酸配体的结合区。515 aa氨基酸链的二聚体空间结构模型GMQE、QMEAN分别为0.97和0.66,建模质量高,与野生型模型(6e07.1.A)比对,459氨基酸均处于模型表面,与NADP+配体的结合区均包括238、357等16个残基;四聚体的建模质量略差,乙醇酸的配体结合区仅为47等6个残基,少于野生型的11个。结论 G6PD基因编码区c.1311T > C同义突变与c.1376G > T错义突变同时存在,可能不影响该患者G6PD二个亚基的聚合及其与NADP+配体的结合,但四聚体的形成受到干扰。

关键词: 云南省, 伯氨喹, 溶血, 葡萄糖-6-磷酸脱氢酶, 突变, 空间结构

Abstract:

Objective Glucose-6-phosphate dehydrogenase (G6PD) deficiency is related to primaquine-induced hemolysis. This study aims to determine the mutations of G6PD gene isolated from a patient with primaquine-induced hemolysis and the possible structure change related to the pathology. Methods Blood sample was collected from a primaquine-induced hemolysis patient infected with Plasmodium vivax. The G6PD enzyme activity was measured to reduce up to 75% in this patient. The genomic DNA was extracted from the blood sample, and G6PD gene fragments containing exon2, exon3-7, exon8-9 and exon10-13 were amplified by PCR using primers designed based on the G6PD coding sequence. The amplified PCR products were sequenced to obtain the full-length coding sequence of G6PD and then aligned with the wildtype (GenBank: NC_000023.11) and mutant (GenBank: X55448.1) G6PD sequences to identify the mutations using MEGA5.04 software. The 3D structure of G6PD was predicted based on the deduced amino acid sequences by SWSS-MODEL(www.swissmodel.expasy.org/interactive) and modified by PyMOL 2.2.0 software. Results The DNA fragments containing G6PD exon2, exon3-7, exon8-9, exon10-13 were amplified from genomic DNA isolated from the blood sample of a P. vivax infected patient with primaquine-induced hemolysis, with the length of 336 bp, 2 277 bp, 976 bp and 1 421 bp, respectively. The full-length DNA of 1 545 bp coding for G6PD of the patient was obtained by aligning with reference wild-type and mutant G6PDs deposited in GenBank. Alignment of patient’s G6PD sequence with wild-type and mutant reference sequences identified two mutations with c.1 311T > C and c.1 376G > T. The first mutation(c.1 311T > C) was synonymous without changing the coding amino acid(Y), the second mutation(c.1 376G > T) leads to the coding amino acid changed from Arg(R) to Leu(L) at position 459. However, the mutation at position 459 was far away from the binding site of G6PD with NADP+ and glycolic acid ligands. The structure of patient G6PD could be the dimer form or tetramer form with the dimer more stable according to the GMQE and QMEAN modeling systems. Compared with the wild-type model of the dimer G6PD (6e07.1.A), the patient G6PD mutant amino acid at 459 was on the surface of the structure, and the binding region with NADP+ ligands contained 16 residues including 238 and 357 positon amino acids, similar to the wild-type form. However, the 3D structure of patient’s G6PD tetramer form was not as stable as wild-type G6PD. The ligand binding regions of glycolic acid contained only 6 residues including 47 position amino acid which is less than 11 residues of wild-type model. Conclusion The sequence of G6PD isolated from a P. vivax infected patient with primaquine-induced hemolysis contained a synonymous mutation of c.1 311T > C and a nonsynonymous mutation of c.1 376G > T compared to the wild-type G6PD. The nonsynonymous mutation at c.1 376G > T leads to the coding amino acid change from Arg(R) to Leu(L) at postion 459 which may not change the dimer structure, but interfere with the formation of tetramer structure of G6PD.

Key words: Yunnan Province, Primaquine, Hemolysis, Glucose-6-phosphate dehydrogenase, Mutation, Spatial structure

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