Mutagenesis Of Recombinant Human Fibroblast Stromelysin C-Terminus Reveals the Autodegradation Pathway

Stromelysin as a member of the matrix metalloproteinases (MMP) family participates in connective tissue remodeling and diseases. Understanding MMPs' catalytic mechanism and designing specific inhibitors to control their activity is of great interest. A clone for mature truncated stromelysin (SL), F⁸³ to T²⁶⁰, without the C-terminal hemopexin-like domain was constructed a d expressed in E. coli. The purified, recombinant protein was active and had a specific activity and Ki</sun> values comparable to that of the native enzyme. However, protein degradation was observed which was attributed to autolysis. A specific autolytic site at S²⁴⁴-L²⁴⁵ was identified by N-terminal sequencing. A series of SL muteins and new truncations were engineered to evaluate their effects on autolysis. The replacement of L²⁴⁵ with an R prevents the autodegradation of SL at the S²⁴⁴-L²⁴⁵ autolytic site. This substitution reveals the primary site initiating SL autolysis (self-catalyzed cleavage) and locates it at Vl63-L₁₆₄.