Specificity of a Vibrio vulnificus aminopeptidase toward kinins and other peptidyl substrates.
Recently, phosphoglucose isomerase with a lysyl aminopeptidase (PGI-LysAP) activity was identified in Vibrio vulnificus. In this paper, we demonstrate the proteolytic cleavage of human-derived peptides by PGI-LysAP of V. vulnificus using three approaches: (i) a quantitative fluorescent ninhydrin assay for free lysine, (ii) matrix-assisted laser desorption ionization-two-stage time of flight mass spectrometry (MALDI-TOF-TOF), and (iii) Tricine gel electrophoresis. PGI-LysAP hydrolyzed bradykinin, Lys-bradykinin, Lys-(des-Arg9)-bradykinin, neurokinin A, Met-Lys-bradykinin, histatin 8, and a myosin light chain fragment. We detected the proteolytic release of free L-lysine from peptide digests using a rapid, simple, sensitive, and quantitative fluorescent ninhydrin assay, and results were confirmed by MALDI-TOF-TOF. The use of the fluorescent ninhydrin assay to quantitatively detect free lysine hydrolyzed from peptides is the first application of its kind and serves as a paradigm for future studies. The visualization of peptide hydrolysis was accomplished by Tricine gel electrophoresis. Proteolytic processing of kinins alters their affinities toward specific cellular receptors and initiates signal transduction mechanisms responsible for inflammation, vasodilation, and enhanced vascular permeability. By applying novel approaches to determine the proteolytic potential of bacterial enzymes, we demonstrate that PGI-LysAP has broad exopeptidase activity which may enhance V. vulnificus invasiveness by altering peptides involved in signal transduction pathways.
Richards
Gary P
GP
USDA, ARS, Microbial Food Safety Research Unit, Delaware State University, 1200 N. DuPont Hwy., James W. W. Baker Center, Dover, DE 19901, USA. grichards@errc.ars.usda.gov
Nuñez
Alberto
A
D000626:Aminopeptidases / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D020689:Exopeptidases / Q000378:metabolism; D005453:Fluorescence; D005956:Glucose-6-Phosphate Isomerase / Q000378:metabolism*; D007202:Indicators and Reagents / Q000494:pharmacology; D008239:Lysine / Q000032:analysis; D009555:Ninhydrin / Q000494:pharmacology; D010455:Peptides / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D041261:Vibrio vulnificus / Q000201:enzymology*
D007202:Indicators and Reagents; D010455:Peptides; D020689:Exopeptidases; D000626:Aminopeptidases; C056860:lysyl aminopeptidase; D005956:Glucose-6-Phosphate Isomerase; D009555:Ninhydrin; D008239:Lysine