Repnik Urska, Starr Amanda E, Overall Christopher M, Turk Boris
From the Department of Biochemistry and Molecular and Structural Biology, Jozef Stefan Institute, SI-1000 Ljubljana, Slovenia.
the Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
J Biol Chem. 2015 May 29;290(22):13800-11. doi: 10.1074/jbc.M115.638395. Epub 2015 Apr 1.
Cysteine cathepsins are primarily lysosomal proteases involved in general protein turnover, but they also have specific proteolytic functions in antigen presentation and bone remodeling. Cathepsins are most stable at acidic pH, although growing evidence indicates that they have physiologically relevant activity also at neutral pH. Post-translational proteolytic processing of mature chemokines is a key, yet underappreciated, level of chemokine regulation. Although the role of selected serine proteases and matrix metalloproteases in chemokine processing has long been known, little has been reported about the role of cysteine cathepsins. Here we evaluated cleavage of CXC ELR (CXCL1, -2, -3, -5, and -8) and non-ELR (CXCL9-12) chemokines by cysteine cathepsins B, K, L, and S at neutral pH by high resolution Tris-Tricine SDS-PAGE and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Whereas cathepsin B cleaved chemokines especially in the C-terminal region, cathepsins K, L, and S cleaved chemokines at the N terminus with glycosaminoglycans modulating cathepsin processing of chemokines. The functional consequences of the cleavages were determined by Ca(2+) mobilization and chemotaxis assays. We show that cysteine cathepsins inactivate and in some cases degrade non-ELR CXC chemokines CXCL9-12. In contrast, cathepsins specifically process ELR CXC chemokines CXCL1, -2, -3, -5, and -8 N-terminally to the ELR motif, thereby generating agonist forms. This study suggests that cysteine cathepsins regulate chemokine activity and thereby leukocyte recruitment during protective or pathological inflammation.
半胱氨酸组织蛋白酶主要是参与一般蛋白质周转的溶酶体蛋白酶，但它们在抗原呈递和骨重塑中也具有特定的蛋白水解功能。组织蛋白酶在酸性pH值下最稳定，尽管越来越多的证据表明它们在中性pH值下也具有生理相关活性。成熟趋化因子的翻译后蛋白水解加工是趋化因子调节的一个关键但未得到充分重视的层面。虽然早就知道某些丝氨酸蛋白酶和基质金属蛋白酶在趋化因子加工中的作用，但关于半胱氨酸组织蛋白酶的作用报道很少。在这里，我们通过高分辨率Tris-三羟甲基氨基甲烷SDS-聚丙烯酰胺凝胶电泳和基质辅助激光解吸电离飞行时间质谱法，评估了半胱氨酸组织蛋白酶B、K、L和S在中性pH值下对CXC ELR（CXCL1、-2、-3、-5和-8）和非ELR（CXCL9 - 12）趋化因子的切割作用。组织蛋白酶B尤其在C末端区域切割趋化因子，而组织蛋白酶K、L和S在N末端切割趋化因子，糖胺聚糖可调节组织蛋白酶对趋化因子的加工。通过钙离子动员和趋化性测定确定了切割的功能后果。我们表明，半胱氨酸组织蛋白酶使非ELR CXC趋化因子CXCL9 - 12失活，在某些情况下还会使其降解。相反，组织蛋白酶特异性地在ELR基序的N末端加工ELR CXC趋化因子CXCL1、-2、-3、-5和-8，从而产生激动剂形式。这项研究表明，半胱氨酸组织蛋白酶在保护性或病理性炎症过程中调节趋化因子活性，进而调节白细胞募集。
