Karthikeyan Vyshali, Vickram A S, Manian Rameshpathy
Department of Industrial Biotechnology, Vellore Institute of Technology, Vellore, India.
Young Scientist DST-SERB, Department of Biotechnology, Saveetha School of Engineering, Saveetha Institute of Medical and Technical Sciences, Chennai, India.
Int J Biol Macromol. 2020 Dec 1;164:735-747. doi: 10.1016/j.ijbiomac.2020.07.159. Epub 2020 Jul 18.
One of the major causes of varicocele is nitrosative stress. Two genes namely arginase 2 (ARG2) and nitric oxide synthase 1 (NOS1) are important in the mechanism of nitric oxide (NO) in the body. Semen samples from three different categories were taken, fertile (n = 20), Infertile (n = 20), and unilateral varicocele (n = 15). Quantitative estimation of ARG2 and NOS1 was carried out through the ELISA kit method. t-Test and ANOVA were calculated using Graphpad Prism. For the in silico analysis, the sequence of the proteins obtained from dbSNP was used in online tools such as nsSNPAnalyzer, PolyPhen-2, Fathmm, I-Mutant 2.0, SNPs&GO, PhD-SNP, PANTHER, SNAP2, PROVEAN, SIFT, and SNPeffect for screening deleterious mutants. These mutants were further evaluated with Swiss PDB and PyMOL for recording energy minimization, Root mean square deviation (RMSD) value, TM score, Hydrogen bonding, and Comparative modeling. Further, ConSurf, Netsurf, and STRING tools were used for evaluating conserved regions, stability, and protein-protein interactions respectively. The results of ARG2 protein were, fertile = 0.168 ± 0.007 U/ml, infertile = 0.201 ± 0.004 U/ml and, varicocele = 0.092 ± 0.002 U/ml. Results of NOS1 protein were fertile = 32.61 ± 2.8 nM/mg, infertile = 19.33 ± 3.7 nM/mg and, varicocele = 54.74 ± 4.8 nM/mg. From statistical analysis, the parameters were highly significant. From the in silico retrieved data, there were 130 and 499 nsSNPs (non-synonymous SNP) in ARG2 and NOS1 respectively. After screening with online tools, 6 deleterious nsSNPs each of ARG2 and NOS1 were considered for analysis through Swiss PDB. FoldX result of A52P mutant (ConSurf score = 9) of ARG2 was found to severely affect protein stability and that of A363T mutant (ConSurf score = 9) of NOS1 revealed a structural change. A52P had a higher RMSD value and A363T of NOS1 developed a new H-bond with 580th position. In varicocele cases, the ARG2 protein is found in lower quantity. The A52P variant of this protein can cause dysfunction and induce nitrosative stress. However, in infertile cases, NOS1 protein is found in lower quantity and the A363T variant can result in a decrease in NO leading to other forms of infertility.
精索静脉曲张的主要原因之一是亚硝化应激。精氨酸酶2（ARG2）和一氧化氮合酶1（NOS1）这两个基因在体内一氧化氮（NO）的机制中很重要。采集了来自三个不同类别的精液样本，分别是可育组（n = 20）、不育组（n = 20）和单侧精索静脉曲张组（n = 15）。通过酶联免疫吸附测定（ELISA）试剂盒法对ARG2和NOS1进行定量评估。使用Graphpad Prism计算t检验和方差分析。对于计算机模拟分析，从单核苷酸多态性数据库（dbSNP）获得的蛋白质序列用于在线工具，如非编码单核苷酸多态性分析器（nsSNPAnalyzer）、多酚-2（PolyPhen-2）、Fathmm、I-突变体2.0（I-Mutant 2.0）、单核苷酸多态性与基因本体（SNPs&GO）、博士单核苷酸多态性（PhD-SNP）、蛋白质分析通过进化关系（PANTHER）、SNAP2、蛋白质变异效应分析（PROVEAN）、筛选不良突变（SIFT）和单核苷酸多态性效应（SNPeffect），以筛选有害突变体。这些突变体通过瑞士蛋白质数据库（Swiss PDB）和PyMOL进一步评估，以记录能量最小化、均方根偏差（RMSD）值、TM分数、氢键和比较建模。此外，分别使用保守性表面分析（ConSurf）、网络表面分析（Netsurf）和STRING工具评估保守区域、稳定性和蛋白质-蛋白质相互作用。ARG2蛋白的结果为，可育组 = 0.168±0.007 U/ml，不育组 = 0.201±0.004 U/ml，精索静脉曲张组 = 0.092±0.002 U/ml。NOS1蛋白的结果为，可育组 = 32.61±2.8 nM/mg，不育组 = 19.33±3.7 nM/mg，精索静脉曲张组 = 54.74±4.8 nM/mg。从统计分析来看，这些参数具有高度显著性。从计算机模拟检索的数据来看，ARG2和NOS1分别有130个和499个非同义单核苷酸多态性（nsSNPs）。在用在线工具筛选后，分别考虑了ARG2和NOS1各6个有害的nsSNPs通过瑞士蛋白质数据库进行分析。发现ARG2的A52P突变体（保守性表面分析分数 = 9）的FoldX结果严重影响蛋白质稳定性，而NOS1的A363T突变体（保守性表面分析分数 = 9）显示出结构变化。A52P具有较高的RMSD值，NOS1的A363T在第580位形成了一个新的氢键。在精索静脉曲张病例中，发现ARG2蛋白的量较低。该蛋白的A52P变体可导致功能障碍并诱导亚硝化应激。然而，在不育病例中，发现NOS1蛋白的量较低，A363T变体可导致NO减少，从而导致其他形式的不育。
