pH-dependent and dynamic interactions of cystatin C with heparan sulfate

• Published in: Communications biology 2021-02, Vol.4 (1), p.198-11, Article 198
• Main Authors: Zhang, Xiaoxiao, Liu, Xinyue, Su, Guowei, Li, Miaomiao, Liu, Jian, Wang, Chunyu, Xu, Ding
• Format: Article
• Language: English
• Subjects: 101/6; 13/1; 13/106; 13/31; 13/51; 14/63; 631/45/221; 631/535/878/1263; 64/60; 82/103; 82/16; 82/29; 82/80; 82/83; Animals; Binding Sites; Biology; Biomedical and Life Sciences; Bone matrix; Cell Line; Cystatin C; Cystatin C - genetics; Cystatin C - metabolism; Extracellular matrix; Extracellular Matrix - metabolism; Femur - metabolism; Heparan sulfate; Heparitin Sulfate - metabolism; Hydrogen-Ion Concentration; Life Sciences; Male; Mice; Mutagenesis, Site-Directed; Mutation; NMR; Nuclear magnetic resonance; Papain; Papain - metabolism; pH effects; Protein Binding; Proteins; Proton Magnetic Resonance Spectroscopy; Site-directed mutagenesis
• ISSN: 2399-3642; 2399-3642
• 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. Zhang et al. identify Cystatin C as a new heparan sulfate (HS) binding protein, however only at acidic pH (≤6.5). They further identify two isolated HS binding motifs on Cystatin C that are required to come together to form a complete HS-binding site, which is a unique property among known HS binding proteins.