Hydrolysis of glycine-containing elastin pentapeptides by LasA, a metalloelastase from Pseudomonas aeruginosa.
Pseudomonas aeruginosa is an opportunistic pathogen that causes severe infections in vulnerable hosts. It may produce various virulence factors including proteases. Among them, LasA possesses both elastolytic and staphylolytic (hydrolysis of pentaglycine cross-links in the cell wall peptidoglycan) activities. To understand if its elastolytic activity results from a preference 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 ratio kcat/KM was determined for most of them. The highest value was obtained for LGGGA, 59 +/- 9 min(-1) x mmol(-1) x L. The specificity of hydrolysis was elucidated by CE, liquid secondary ion mass spectrometry and, in some cases, collision activated dissociation-mass analysis of ion kinetic energy. The preferred cleavage sites are GG and GA peptide bonds, the sequence GG(cleavage site)A being especially sensitive to hydrolysis. Both positions P2 and P'2 must be occupied for hydrolysis and the presence of an amino acid in P3 (but not in P'3) significantly increases the catalytic ratio. Considering these results, about 30 GGX sequences (X: G, A or Y) of human tropoelastin could be susceptible to LasA elastolysis.
Vessillier
S
S
Laboratoire de Biochimie Analytique et Synthèse Bioorganique, Université Lyon 1, Villeurbanne, France; Service Central d'Analyse, CNRS, Vernaison, France.
Delolme
F
F
Bernillon
J
J
Saulnier
J
J
Wallach
J
J
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D019075:Electrophoresis, Capillary; D005998:Glycine / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008666:Metalloendopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism; D018629:Spectrometry, Mass, Secondary Ion; D014333:Tropoelastin / Q000737:chemistry / Q000378:metabolism*
D001426:Bacterial Proteins; D009842:Oligopeptides; D014333:Tropoelastin; D008666:Metalloendopeptidases; C066371:staphylolytic protease; D005998:Glycine