钩端螺旋体M23超家族金属内肽酶生物学活性及致病相关性的研究

摘要

目的 了解并确定问号钩端螺旋体黄疸出血型赖株LA2582 和LA2901 基因产物金属内肽酶活性及其致病相关性.方法 采用生物信息学软件分析LA2582 和LA2901 基因结构与功能.构建LA2582 和LA2901 基因胞外区原核表达系统,Ni-NTA 亲和层析法提纯目的重组表达产物rLA2582 和rLA2901.采用分光光度法检测rLA2582 和rLA2901 水解偶氮酪蛋白底物的活性.采用荧光分光光度法检测rLA2582 和rLA2901 水解Dabsyl-Leu-Gly-Gly-Gly-Ala-Edans 荧光标记五肽底物的活性并测定其Km 和Kcat 值.采用SDS-PAGE 和分光光度法分别检测rLA2582 和rLA2901 水解细胞外基质(ECM)分子Ⅰ型胶原蛋白(COL1)、纤维连接蛋白(FN)和刚果红标记弹性蛋白(ELN)的活性.采用实时荧光定量RT-PCR 和Western blot 法分别检测问号钩端螺旋体赖株感染人脐静脉内皮细胞(HUVEC)后LA2582 和LA2901 基因mRNA 和蛋白质表达水平.结果 LA2582 和LA2901 基因产物均为含信号肽和HXH 基质金属蛋白酶基序的锌离子依赖M23 家族Gly-Gly 金属内肽酶.rLA2582 和rLA2901 不能水解偶氮酪蛋白,但能水解荧光五肽底物,其Km 和Kcat 值分别为126. 54μmol/ L 和4. 67/ s 、190. 25 μmol/ L 和4. 86/ s .rLA2582 和rLA2901 具有水解COL1、FN 和ELN 的活性.问号钩体赖株感染HUVEC 后,LA2582 和LA2901 基因mRNA、蛋白质表达水平均显著升高(P<0. 05).结论 问号钩体赖株LA2582 和LA2901 基因产物是具有水解ECM 分子活性的锌离子依赖M23 金属内肽酶,与问号钩体侵袭力密切相关.%Objective To understand and determine the biological activity and pathogenicity of metalloendopeptidases encoded by LA2582 and LA2901 genes of Leptospira interrogans(L.interrogans) sero-group Icterohaemorrhagiaeserovar Lai strain Lai. Methods Structures and functions of LA2582 and LA2901 genes were analyzed by using bioinformatic software. Prokaryotic expression systems for expressing the extra-cellular regions of LA2582 and LA2901 genes were generated. The target recombinant expression products, rLA2582 and rLA2901,were extracted by Ni-NTA affinity chromatography. The Azo-casein-hydrolyzingactiv-ity of rLA2582 and rLA2901 was detected by spectrophotometry. Activities of rLA2582 and rLA2901 in the hydrolysis of Dabsyl-Leu-Gly-Gly-Gly-Ala-Edans, a fluorescence-labeling pentapeptide substrate, were de-tected by fluorospectrophotometry,and then the Km and Kcat values were determined. SDS-PAGE and spec-trophotometry were performed to detect the activities of rLA2582 and rLA2901 in hydrolyzing extracellular matrix molecules such as collagen type-Ⅰ (COL1), fibronectin (FN) and Congo red-labeling elastin (ELN). Real-time fluorescent quantitative RT-PCR(qRT-PCR) and Western blot were respectively used to measure the expression of LA2582 and LA2901 genes at mRNA and protein levels after infecting human um-bilical vein endothelial cells(HUVEC) with L. interrogans strain Lai. Results The gene products of LA2582 and LA2901 genes were identified as the signal peptide and matrix metalloproteinase motif HXH-containing Zn2+-dependent Gly-Gly metalloendopeptidases belonging to the M23 superfamily. rLA2582 and rLA2901 did not hydrolyze Azo-casein (Km=126.54 μmol/L, Kcat=4.67/s), but could hydrolyze the pentapeptide substrate (Km=190. 25 μmol/L, Kcat 4. 86/s). rLA2582 and rLA2901 could hydrolyze COL1, FN and ELN. Expression of LA2582 and LA2901 genes at both mRNA and protein levels was signifi-cantly increased after infection of HUVEC with L.interrogans strain Lai(P<0.05). Conclusion The prod-ucts of LA2582 and LA2901 genes of L.interrogans strain Lai are Zn2+-dependent M23 metalloendopeptidas-es, which can hydrolyze multiple ECM molecules and are closely associated with the leptospiral invasiveness.