title: Proteolytic cleavage of a C-terminal prosequence, leading to autoprocessing at the N Terminus, activates leucine aminopeptidase from Pseudomonas aeruginosa.
abstract: At high bacterial cell density the gene expression program of Pseudomonas aeruginosa is regulated by quorum sensing. Among the gene products highly up-regulated by this system is an exoprotease, leucine aminopeptidase (PA-LAP), which is coexpressed with several known virulence factors and secreted as a proenzyme. We undertook a study of its activation by expressing the full-length proform of PA-LAP recombinantly in Escherichia coli (here termed, rLAP55) and characterizing individual steps in its conversion to an active enzyme. Activation is initiated with the proteolytic removal of a C-terminal prosequence. Removal of approximately 20 amino acids is accomplished by Pseudomonas elastase, which is also positively regulated by quorum sensing. Activation is also mediated by other proteases that cleave rLAP55 near its C terminus. The importance of the C terminus was confirmed by showing that C-terminal deletions of 1-24 amino acids produce a fully active enzyme. The removal of C-terminal prosequences either by proteolysis or deletion leads to an unusual autoprocessing event at the N terminus. Autoprocessing is apparently an intramolecular event, requires the active site of LAP, and results in the removal of 12 N-terminal amino acids. Furthermore, a detailed analysis of the C-terminal prosequence suggests that the proenzyme state is dependent on the presence of a basic side chain contributed by the last amino acid, lysine 536. Our data support a model whereby full-length PA-LAP is activated in a two-step process; proteolytic cleavage at the C terminus is followed by an intramolecular autocatalytic removal of a 12-amino acid propeptide at the N terminus.
authors.affiliation: Laboratory of Molecular Biology, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Robert
authors.initials: R
authors.lastname: Sarnovsky
authors.forename: Jennifer
authors.initials: J
authors.lastname: Rea
authors.forename: Matt
authors.initials: M
authors.lastname: Makowski
authors.forename: Ralf
authors.initials: R
authors.lastname: Hertle
authors.forename: Colleen
authors.initials: C
authors.lastname: Kelly
authors.forename: Antonella
authors.initials: A
authors.lastname: Antignani
authors.forename: Diana V
authors.initials: DV
authors.lastname: Pastrana
authors.forename: David J
authors.initials: DJ
authors.lastname: Fitzgerald
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002852:Chromatography, Ion Exchange; D004926:Escherichia coli / Q000378:metabolism; D007931:Leucyl Aminopeptidase / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D008969:Molecular Sequence Data; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000378:metabolism*; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid
chemical_list: D011994:Recombinant Proteins; D007931:Leucyl Aminopeptidase