Characterization of the streptococcal C5a peptidase using a C5a-green fluorescent protein fusion protein substrate

A glutathione-S-transferase (GST)-C5a-green fluorescent protein (GFP) fusio n protein was designed for use as a substrate for the streptococcal C5a pep tidase (SCPA), The substrate was immobilized on a glutathione-Sepharose aff inity matrix and used to measure wild-type SCPA activity in the range of 0. 8 to 800 nM, The results of the assay demonstrated that SCPA is highly heat stable and has optimal activity on the synthetic substrate at or above pH 8.0. SCPA activity was unaffected by 0.1 to 10 mM Ca2+, Mg2+, and Mn2+ but was inhibited by the same concentrations of Zn2+, The assay shows high sens itivity to ionic strength; NaCl inhibits SCPA cleavage of GST-C5a-GFP in a dose-dependent manner. Based on previously published computer homology mode ling, four substitutions were introduced into the putative active site of S CPA: Asp(130)-Ala, His(193)-Ala, Asn(295)-Ala, and Ser(512)-Ala. All four m utant proteins had over 1,000-fold less proteolytic activity on C5a in vitr o, as determined both by the GFP assay described here and by a polymorphonu clear cell adherence assay. In addition, recombinant SCPA1 and SCPA49, from two distinct lineages of Streptococcus pyogenes (group A streptococci), an d recombinant SCPB, from Streptococcus agalactiae (group B streptococci), w ere compared in the GFP assay. The three enzymes had similar activities, al l cleaving approximately 6 mol of C5a mmol of SCP-1 liter(-1) min(-1).