关节软骨 Kunitz 蛋白酶抑制蛋白的回顾性分析表明，这些蛋白是内 α-胰蛋白酶抑制剂超家族的成员，具有保护关节表面的潜在作用。
A Retrospective Analysis of the Cartilage Kunitz Protease Inhibitory Proteins Identifies These as Members of the Inter-α-Trypsin Inhibitor Superfamily with Potential Roles in the Protection of the Articulatory Surface.
机构信息
Raymond Purves Bone and Joint Research Laboratory, Kolling Institute, Northern Sydney Local Health District, St. Leonards, NSW 2065, Australia.
Graduate School of Biomedical Engineering, University of New South Wales, Sydney, NSW 2052, Australia.
出版信息
Int J Mol Sci. 2019 Jan 24;20(3):497. doi: 10.3390/ijms20030497.
AIM
The aim of this study was to assess if the ovine articular cartilage serine proteinase inhibitors (SPIs) were related to the Kunitz inter-α-trypsin inhibitor (ITI) family.
METHODS
Ovine articular cartilage was finely diced and extracted in 6 M urea and SPIs isolated by sequential anion exchange, HA affinity and Sephadex G100 gel permeation chromatography. Selected samples were also subjected to chymotrypsin and concanavalin-A affinity chromatography. Eluant fractions from these isolation steps were monitored for protein and trypsin inhibitory activity. Inhibitory fractions were assessed by affinity blotting using biotinylated trypsin to detect SPIs and by Western blotting using antibodies to α1-microglobulin, bikunin, TSG-6 and 2-B-6 (+) CS epitope generated by chondroitinase-ABC digestion.
RESULTS
2-B-6 (+) positive 250, 220,120, 58 and 36 kDa SPIs were detected. The 58 kDa SPI contained α1-microglobulin, bikunin and chondroitin-4-sulfate stub epitope consistent with an identity of α1-microglobulin-bikunin (AMBP) precursor and was also isolated by concanavalin-A lectin affinity chromatography indicating it had -glycosylation. Kunitz protease inhibitor (KPI) species of 36, 26, 12 and 6 kDa were autolytically generated by prolonged storage of the 120 and 58 kDa SPIs; chymotrypsin affinity chromatography generated the 6 kDa SPI. KPI domain 1 and 2 SPIs were separated by concanavalin lectin affinity chromatography, domain 1 displayed affinity for this lectin indicating it had -glycosylation. KPI 1 and 2 displayed potent inhibitory activity against trypsin, chymotrypsin, kallikrein, leucocyte elastase and cathepsin G. Localisation of versican, lubricin and hyaluronan (HA) in the surface regions of articular cartilage represented probable binding sites for the ITI serine proteinase inhibitors (SPIs) which may preserve articulatory properties and joint function.
DISCUSSION/CONCLUSIONS: The Kunitz SPI proteins synthesised by articular chondrocytes are members of the ITI superfamily. By analogy with other tissues in which these proteins occur we deduce that the cartilage Kunitz SPIs may be multifunctional proteins. Binding of the cartilage Kunitz SPIs to HA may protect this polymer from depolymerisation by free radical damage and may also protect other components in the cartilage surface from proteolytic degradation preserving joint function.
目的
本研究旨在评估绵羊关节软骨丝氨酸蛋白酶抑制剂（SPIs）是否与 Kunitz 型胰蛋白酶抑制剂（ITI）家族有关。
方法
将绵羊关节软骨切成细块，在 6 M 尿素中提取，通过顺序阴离子交换、HA 亲和和 Sephadex G100 凝胶渗透层析分离 SPIs。选择的样品还进行胰凝乳蛋白酶和伴刀豆球蛋白 A 亲和层析。从这些分离步骤的洗脱液中监测蛋白质和胰蛋白酶抑制活性。使用生物素化胰蛋白酶检测 SPIs 的亲和印迹法和针对 α1-微球蛋白、 bikunin、TSG-6 和软骨素酶 ABC 消化产生的 2-B-6（+）CS 表位的抗体进行 Western 印迹法评估抑制性馏分。
结果
检测到 2-B-6（+）阳性 250、220、120、58 和 36 kDa 的 SPI。58 kDa SPI 含有 α1-微球蛋白、 bikunin 和软骨素-4-硫酸盐短链表位，与 α1-微球蛋白- bikunin（AMBP）前体的同一性一致，并且也通过伴刀豆球蛋白 A 凝集素亲和层析分离出来，表明它具有 -糖基化。通过长时间储存 120 和 58 kDa SPI，自动产生 36、26、12 和 6 kDa 的 Kunitz 蛋白酶抑制剂（KPI）物种；胰凝乳蛋白酶亲和层析生成 6 kDa SPI。通过伴刀豆球蛋白 A 凝集素亲和层析分离 KPI 结构域 1 和 2 SPI，结构域 1 对该凝集素显示出亲和力，表明其具有 -糖基化。KPI 1 和 2 对胰蛋白酶、糜蛋白酶、激肽释放酶、白细胞弹性蛋白酶和组织蛋白酶 G 具有强烈的抑制活性。在关节软骨表面区域中发现 versican、lubricin 和透明质酸（HA）的定位可能是 ITI 丝氨酸蛋白酶抑制剂（SPIs）的可能结合位点，这可能有助于保持关节的运动特性和功能。
讨论/结论：关节软骨细胞合成的 Kunitz SPI 蛋白是 ITI 超家族的成员。通过与其他组织中出现这些蛋白质的类比，我们推断软骨中的 Kunitz SPIs 可能是多功能蛋白。软骨中的 Kunitz SPIs 与 HA 的结合可以防止聚合物因自由基损伤而解聚，还可以保护软骨表面的其他成分免受蛋白水解降解，从而保持关节功能。
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