Biochemical activities of the calpain peptidase homologue from Actinomyces israelii

Abstract

Objectives: Calpains represent a large family of calcium ion-activated cysteine peptidases, which are widely distributed in eukaryotes, and have been well-studied. However, the thiol protease (Tpr) protein of Porphyromonas gingivalis represents the only bacterial calpain peptidase characterized to date. The aim of the study is to characterize the biochemical activities of the putative calpain-family peptidase (AiCP; 476aa) encoded by Actinomyces israelii DSM 6844. Methods: The AiCP protein was cloned, expressed and purified as an N-terminal glutathione S-transferase fusion protein, using standard techniques. Its self-digestion (autolytic) properties were determined by peptide mass fingerprinting (PMF) of self-digested protein fragments resolved on SDS-polyacrylamide gels. Peptidolytic activities were determined using spectrophotometric/fluorometric assays using commercially-available fluorogenic/chromogenic peptide/protein substrates (Spectramax multi-wavelength plate-reader) at 25°C, Tris pH 7.4, 5mM CaCl2. Results: Size exclusion chromatography analysis revealed that AiCP formed a trimeric arrangement in solution. It digested itself in a calcium ion-dependent manner at several distinct loci, creating two major bands with apparent molecular weights of 42 kDa and 36 kDa. It digested casein, but not gelatin. Z-Gly-Gly-Arg-AMC and H-Arg-Arg-AMC were digested with specific molar activities of 0.053±0.015 and 0.044±0.009 micromoles/min.micromoles protein, respectively. H-Gly-Arg-AMC, H-Arg-AMC, H-Ala-Phe-pNA (SAAPFNA), H-Ala-Phe-Lys-AMC, N-alpha-benzoyl-Arg-pNA (BAPNA), Suc-Leu-Leu-Val-Tyr-AMC, were not hydrolyzed to detectable levels. Further investigations are ongoing. Conclusions: These preliminary results indicate that the calpain homologue (AiCP) from A. israelii has certain ‘trypsin-like’ peptidolytic activities; similar to, but notably distinct from, those of the P. gingivalis Tpr protein. Whilst the biological roles of AiCP remain to be determined, our results suggest that it may play a role in peptide/amino acid acquisition and/or metabolism in this bacterium, which is a common component of oral biofilms.