纤维连接蛋白瓜氨酸化后对聚蛋白多糖酶-1活性的影响

Fibronectin decreases its inhibiting activity on disintegrin and metalloproteinase with thrombospondinmotifs-4 by citrullination

目的通过观察聚蛋白多糖酶-1（ADAMTS-4）与纤维连接蛋白（FN）以及瓜氨酸化的FN（cFN）的结合活性及结合后的酶解活性，探讨ADAMTS-4的活性调节机制。方法应用肽酰基精氨酸脱亚胺酶4（PADl4）对纤维连接蛋白（FN）进行瓜氨酸化并采用蛋白印迹法进行验证；应用酶联免疫吸附法测定FN和CFN与2种不同相对分子质量的ADAMTS-4的结合活性；使用聚蛋白多糖酶活性分析试剂盒测定2种ADAMTS-4的活性和其与FN、cFN结合后酶活性的变化。统计分析采用单因素方差分析，LSD-t检验或t检验。结果蛋白印迹法证实PADl4可以使FN瓜氨酸化为cFN；cFN与相对分子质量为93000的ADAMTS-4的结合活性明显低于FN（分别为0．624＋0．033，2．1824-0．042；t：50．522，P〈0．01），而两者与缺失碳端的相对分子质量为53000的ADAMTS．4的结合活性差异无统计学意义（分别为0．934＋0．012，0．971±0．024；t=2．388，P〉0．05）。相对分子质量为93000的ADAMTS．4能够酶解聚蛋白多糖产生大量酶解产物[ARGxx肽段浓度：（0．908±0．088）nmol／L]，与FN结合后降解聚蛋白多糖的能力明显下降[ARGxx肽段浓度：（0．5734-0．000）nmol／L，P〈0．05]，与cFN孵育后的ADAMTS-4降解聚蛋白多糖产生的ARGxx肽段浓度[（0．8304-0．020）nmol／L]明显高于与FN孵育后产生的ARGxx肽段浓度[（O．5734-0．000）nmol／L，P〈0．05]。相对分子质量为53000的ADAMTS-4的酶活性在与FN或eFN孵育前后酶活性无明显变化（P均〉0．05）。结论FN能结合ADAMTS-4并抑制它的活性，PADl4能将FN转化为cFN，cFN与ADAMTS-4的结合活性明显降低，从而对ADAMTS-4的活性抑制作用减弱。

Objective To observe the inhibiting activities of fibronectin （FN） and citrullinated fibre- nectin （cFN） on disintegrin and metalloproteinase with thrombospondin motifs-4 （ADAMTS-4）, and to explore the extracellular regulative mechanisms of ADAMTS-4. Methods FN was incubated with peptidylarginine deaminase type 4 （PADI4）. Western blotting analysis was used to verify the citrullination of FN. The binding activity of FN and cFN to ADAMTS-4 were investigated by enzyme-linked immunosorbent assay （ELISA）. The pretealytic ability of ADAMTS-4 after binding to FN and ：cFN were measured with the aggrecanase activity assay kit. One-way ANOVA, LSD-t test and t-test were used for statistical analysis. Results The immuno- signal of citrulline was detected in FN after incubated with PADI4, hut not in the absence of PADI4. A higher absorbance at 405 nm was detected when the full-length ADAMTS-4 protein was incubated with FN （2.182± 0.042） than cFN （0.624＋0.033; t=50.522, P〈0.01）. Additionally, the recombinant ADAMTS-4 protein with a truncation at the C-terminus displayed low absorbance at 405 nm when the enzyme was incubated with both FN（0.971±0.024） and cFN（0.934±0.012; t=2.388, P〉0.05）. Large amounts of ARGxx peptide were detected with full-length ADAMTS-4 in aggrecanase activity assay [ （0.908±0.088） nmol/L], but significantly less whenin the presence of FN and ADAMTS-4 [ （0.573±0.000） nmol/L, P〈0.05 ]. The production of this peptide was more when full-length ADAMTS-4 was incubated with cFN [ （0.830＋0.020） nmol/L, P〈0.05 ] than with FN. The reaction containing the truncated ADAMTS-4 without FN or cFN yielded the highest concentration of ARGxx peptide [（36.420±3.673） nmol/L], peptide production was not significantly altered when FN [（41.099±0.101） nmol/L] or cFN [（41.064±0.083） nmol/L] were added to the reaction. Conclusion FN could bind to ADAMTS-4 and inhibit its proteolytic activity. After citndlinated by PADI4, the binding activity of cFN is weakened and less inhibition to ADAMTS-4.