Purification and characterization of a clostripain-like protease from a recombinant Clostridium perfringens culture

Abstract

Clostridium perfringens produces a homologue of clostripain (Clo), the arginine-specific endopeptidase of Clostridium histolyticum. To determine the biochemical and biological properties of the C. perfringens homologue (Clp), it was purified from the culture supernatant of a recombinant C. perfringens strain by cation-exchange chromatography and ultrafiltration. Analysis by SDS-PAGE, N-terminal amino acid sequencing and TOF mass spectrometry revealed that Clp consists of two polypeptides comprising heavy (38 kDa) and light (16 kDa or 15 kDa) chains, and that the two light chains differ in the N-terminal cleavage site. This difference in the light chain did not affect the enzymic activity toward N-benzoyl-l-arginine p-nitroanilide (Bz-l-arginine pNA), as demonstrated by assaying culture supernatants differing in the relative ratio of the two light chains. Although the purified Clp preferentially degraded Bz-dl-arginine pNA rather than Bz-dl-lysine pNA, it degraded the latter more efficiently than did Clo. Clp showed 2.3-fold higher caseinolytic activity than Clo, as expected from the difference in substrate specificity. Clp caused an increase in vascular permeability when injected intradermally into mice, implying a possible role of Clp in the pathogenesis of clostridial myonecrosis.