Fibrinogen cleavage by the Streptococcus pyogenes extracellular cysteine protease and generation of antibodies that inhibit enzyme proteolytic activity

The extracellular cysteine protease from Streptococcus pyogenes is a virule nce factor that plays a significant role in host-pathogen interaction, Stre ptococcal protease is expressed as an inactive 40-kDa precursor that is aut ocatalytically converted into a 28-kDa mature (active) enzyme. Replacement of the single cysteine residue involved in formation of the enzyme active s ite with serine (C192S mutation) abolished detectable proteolytic activity and eliminated autocatalytic processing of zymogen to the mature form. In t he present study, we investigated activity of the wild-type (wt) streptococ cal protease toward human fibrinogen and bovine casein, The former is invol ved in blood coagulation, wound healing, and other aspects of hemostasis, T reatment with streptococcal protease resulted in degradation of the COOH-te rminal region of fibrinogen a chain, indicating that fibrinogen may serve a s an important substrate for this enzyme during the course of human infecti on. Polyclonal antibodies generated against recombinant 40- and 28-kDa (r40 - and r28-kDa) forms of the C192S streptococcal protease mutant exhibited h igh enzyme-linked immunosorbent assay titers but demonstrated different inh ibition activities toward proteolytic action of the wt enzyme. Activity of the wt protease was readily inhibited when the reaction was carried out in the presence of antibodies generated against r28-kDa C192S mutant, Antibodi es produced against r40-kDa C192S mutant had no significant effect on prote olysis. These data suggest that the presence of the NH2-terminal prosegment prevents generation of functionally active antibodies and indicate that in hibition activity of antibodies most likely depends on their ability to bin d the active-site region epitope(s) of the protein.