Zhang Xiaoxiao, Liu Xinyue, Su Guowei, Li Miaomiao, Liu Jian, Wang Chunyu, Xu Ding
Department of Oral Biology, The University at Buffalo, Buffalo, NY, USA.
Department of Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, USA.
Commun Biol. 2021 Feb 12;4(1):198. doi: 10.1038/s42003-021-01737-7.
Cystatin C (Cst-3) is a potent inhibitor of cysteine proteases with diverse biological functions. As a secreted protein, the potential interaction between Cst-3 and extracellular matrix components has not been well studied. Here we investigated the interaction between Cst-3 and heparan sulfate (HS), a major component of extracellular matrix. We discovered that Cst-3 is a HS-binding protein only at acidic pH. By NMR and site-directed mutagenesis, we identified two HS binding regions in Cst-3: the highly dynamic N-terminal segment and a flexible region located between residue 70-94. The composition of the HS-binding site by two highly dynamic halves is unique in known HS-binding proteins. We further discovered that HS-binding severely impairs the inhibitory activity of Cst-3 towards papain, suggesting the interaction could actively regulate Cst-3 activity. Using murine bone tissues, we showed that Cst-3 interacts with bone matrix HS at low pH, again highlighting the physiological relevance of our discovery.
胱抑素C（Cst-3）是一种对多种生物功能具有重要作用的半胱氨酸蛋白酶抑制剂。作为一种分泌蛋白，Cst-3与细胞外基质成分之间的潜在相互作用尚未得到充分研究。在此，我们研究了Cst-3与细胞外基质的主要成分硫酸乙酰肝素（HS）之间的相互作用。我们发现，Cst-3仅在酸性pH条件下是一种HS结合蛋白。通过核磁共振（NMR）和定点诱变，我们在Cst-3中鉴定出两个HS结合区域：高度动态的N端片段和位于第70至94位残基之间的一个柔性区域。由两个高度动态的部分组成的HS结合位点在已知的HS结合蛋白中是独一无二的。我们进一步发现，HS结合严重损害了Cst-3对木瓜蛋白酶的抑制活性，这表明这种相互作用可能会积极调节Cst-3的活性。利用小鼠骨组织，我们发现Cst-3在低pH条件下与骨基质HS相互作用，再次凸显了我们这一发现的生理相关性。
