Proteolytic cleavage of staphylococcal exoproteins analyzed by two-dimensional gel electrophoresis.
Extracellular proteases of Staphylococcus aureus are emerging as potential virulence factors that are relevant to the pathogenicity of staphylococcal infections. These proteases may also be involved in the proteolytic cleavage of other exoproteins released from this organism. To define the target exoproteins and their sites of cleavage by proteases, high-resolution two-dimensional polyacrylamide gel electrophoresis followed by N-terminal amino acid sequencing of exoprotein spots was performed. Two to three hundred exoprotein spots were detected at the early-stationary phase of cultures of S. aureus NCTC8325, and then at the late-stationary stage most of these high molecular protein spots became invisible due to further proteolytic degradation. As the result of N-terminal analysis, lipase, triacylglycerol lipase, orf619 protein and orf388 protein were detected as multiple spots at the early-stationary phase. We found that these exoproteins were cleaved at 3, 7, 4 and 4 different sites, respectively, by proteases. According to the M.W. and pI of each peptide spot obtained from the gel and their matches with calculated values in addition to their N-terminal sequences, we showed that the positions of putative peptides resulted from proteolytic cleavage of these proteins.
Kawano
Y
Y
Department of Molecular Bacteriology, Nagoya University Graduate School of Medicine, Aichi, Japan.
Kawagishi
M
M
Nakano
M
M
Mase
K
K
Yamashino
T
T
Hasegawa
T
T
Ohta
M
M
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification*; D001665:Binding Sites; D015180:Electrophoresis, Gel, Two-Dimensional; D010450:Endopeptidases / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D008049:Lipase / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D013211:Staphylococcus aureus / Q000201:enzymology* / Q000235:genetics / Q000472:pathogenicity; D014774:Virulence
D001426:Bacterial Proteins; D008049:Lipase; D010450:Endopeptidases