Hydrolysis of glycine-containing elastin pentapeptides by LasA, a metalloelastase from Pseudomonas aeroginosa

Pseudomonas aeruginosa is an opportunistic pathogen that causes severe infe ctions in vulnerable hosts. It may produce various virulence factors includ ing proteases. Among them, LasA possesses both elastolytic and staphylolyti c (hydrolysis of pentaglycine cross-links in the cell wall peptidoglycan) a ctivities. To understand if its elastolytic activity results from a prefere nce for glycine-rich substrates, we studied its ability to hydrolyse the 65 pentapeptides of human tropoelastin containing at least three glycines. As demonstrated by capillary electrophoresis (CE), 22 of these peptides were hydrolysed by LasA, generally at a single peptide bond and the catalytic ra tio k(cat)/K-M was determined for most of them. The highest value was obtai ned for LGGGA, 59 +/- 9 min(-1).mmol(-1).L. The specificity of hydrolysis w as elucidated by CE, liquid secondary ion mass spectrometry and, in some ca ses, collision activated dissociation-mass analysis of ion kinetic energy. The preferred cleavage sites are GG and GA peptide bonds, the sequence GGIA being especially sensitive to hydrolysis. Both positions P-2 and P'(2) mus t be occupied for hydrolysis and the presence of an amino acid in P-3 (but not in P'(3)) significantly increases the catalytic ratio. Considering thes e results, about 30 GGX sequences (X: G, A or Y) of human tropoelastin coul d be susceptible to LasA elastolysis.