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) fusion protein was designed for use as a substrate for the streptococcal C5a peptidase (SCPA). The substrate was immobilized on a glutathione-Sepharose affinity 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 Ca(2+), Mg(2+), and Mn(2+) but was inhibited by the same concentrations of Zn(2+). The assay shows high sensitivity to ionic strength; NaCl inhibits SCPA cleavage of GST-C5a-GFP in a dose-dependent manner. Based on previously published computer homology modeling, four substitutions were introduced into the putative active site of SCPA: Asp(130)-Ala, His(193)-Ala, Asn(295)-Ala, and Ser(512)-Ala. All four mutant proteins had over 1,000-fold less proteolytic activity on C5a in vitro, as determined both by the GFP assay described here and by a polymorphonuclear cell adherence assay. In addition, recombinant SCPA1 and SCPA49, from two distinct lineages of Streptococcus pyogenes (group A streptococci), and recombinant SCPB, from Streptococcus agalactiae (group B streptococci), were compared in the GFP assay. The three enzymes had similar activities, all cleaving approximately 6 mol of C5a mmol of SCP(-1) liter(-1) min(-1).
Stafslien
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DK
Department of Microbiology, University of Minnesota, Minneapolis 55455, USA.
Cleary
P P
PP
D018829:Adhesins, Bacterial; D015936:Complement C5a / Q000235:genetics / Q000378:metabolism*; D003169:Complement Inactivator Proteins / Q000032:analysis*; D010450:Endopeptidases / Q000032:analysis*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism*
D018829:Adhesins, Bacterial; D003169:Complement Inactivator Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D015936:Complement C5a; D010450:Endopeptidases; C053088:C5a peptidase