In vitro proteolytic processing and activation of the recombinant precursor of El Tor cytolysin/hemolysin (pro-HlyA) of Vibrio cholerae by soluble hemagglutinin/protease of V. cholerae, trypsin, and other proteases.
Vibrio cholerae produces a cytolytic toxin named El Tor cytolysin/hemolysin which is encoded by the hlyA gene. This cytolysin is produced as a 79-kDa precursor form (pro-HlyA) into the culture supernatant after cleavage of the signal peptide of the hlyA product (prepro-HlyA). The pro-HlyA is then processed to a 65-kDa mature cytolysin (mature HlyA) after cleavage of the 15-kDa N-terminal peptide (pro region) of the 79-kDa precursor, usually at the bond between Ala-157 and Asn-158. We investigated whether proteases could process the recombinant 79-kDa pro-HlyA to the 65-kDa mature HlyA. We observed that the soluble hemagglutinin/ protease (HA/protease; a major protease of V. cholerae), trypsin, alpha-chymotrypsin, subtilisin BPN', papain, and thermolysin all processed the pro-HlyA to the 65-kDa mature form of the protein. Along with this, the protease-processed HlyA showed drastically increased hemolytic activity. The N-terminal amino acid of the mature form of cytolysin generated by HA/protease was Phe-151, and that due to trypsin was Ser-149. Other proteases also cleaved the pro-HlyA at a nearby site, between Leu-146 and Ser-153, and all the processed cytolysins showed increased hemolytic activity. These data suggest that the active El Tor cytolysin of V. cholerae could be derived from the C-terminal region of a pro-HlyA following proteolytic cleavage of the bonds in the vicinity of Leu-146 to Asn-158 by any of a wide variety of proteases.
Department of Bacterial Infections, Research Institute for Microbial Diseases, Osaka University, Suita, Japan.
K
K
Nagamune
K
K
Yamamoto
A
A
Naka
J
J
Matsuyama
T
T
Miwatani
T
T
Honda
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D002918:Chymotrypsin / Q000378:metabolism; D003603:Cytotoxins / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism*; D006388:Hemagglutinins; D006460:Hemolysin Proteins / Q000378:metabolism*; D006461:Hemolysis; D008666:Metalloendopeptidases / Q000378:metabolism*; D008969:Molecular Sequence Data; D010206:Papain / Q000378:metabolism; D011498:Protein Precursors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D013381:Subtilisins / Q000378:metabolism; D013820:Thermolysin / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism; D014734:Vibrio cholerae / Q000201:enzymology
D001426:Bacterial Proteins; D003603:Cytotoxins; D006388:Hemagglutinins; D006460:Hemolysin Proteins; D011498:Protein Precursors; C084287:hlyA protein, Vibrio cholerae; D010450:Endopeptidases; D013381:Subtilisins; D002918:Chymotrypsin; D014357:Trypsin; D010206:Papain; D008666:Metalloendopeptidases; C039814:hemagglutinin-protease; D013820:Thermolysin