Identification of agmatinase from Vibrio splendidus and its roles in modulating arginine metabolism of Apostichopus japonicus

Innate immunity is the first line of defense against invading pathogens. However, pathogenic bacteria have developed various mechanisms, such as direct competition for common substrates and transformation of metabolic regulations of the host, which allow them to escape from the immune surveillance system and enhance their survival. In this study, the Vibrio splendidus agmatinase (designated as Vsagmatinase) gene was cloned, and its biological activities in Apostichopus japonicus coelomocytes were analyzed. Vsagmatinase encoded an open reading frame of 930 bp, which translated into a predicted protein of 309 amino acid residues. SMART analysis and multiple sequence alignment indicated that Vsagmatinase displayed an arginase domain, and the conserved histidine (H151 and H163) and aspartic acid (D125, D149, D153, D231, and D233) residues for catalytic activity were all strictly conserved in this domain. Moreover, four conserved residues, namely, Asp153, His163, Thr245, and Glu275, that bonded with the guanidinium portion of agmatine were conserved in V. splendidus agmatinase. The recombinant protein was expressed in Escherichia coli (BL21), and the purified Vsagmatinase showed high agmatinase activity with agmatine substrate. By site-directed mutagenesis, the agmatinase activity of mutated Vsagmatinase was reduced to approximately 8.9%-9.4% of wild-type activity under different Vsagmatinase protein concentrations, indicating that the Glu275 residue was necessary for the guanidinium group of agmatine. Notably, injection of Vsagmatinase in coelomocytes could significantly increase both the mRNA expression levels and enzyme activities of Ajarginase and Ajagmatinase and obviously inhibited AjNOS transcript and NO content in A. japonicus. By contrast, mutated Vsagmatinase downregulated the activity of arginase and promoted NO production at 12 h after injection. Our findings suggested that Vsagmatinase served as the competitor that utilizes host agmatine and modulates arginine metabolism in A. japonicus.