Vibrio cholerae HlyA hemolysin is processed by proteolysis

Abstract

The leukocidal activity of the Vibrio cholerae hemolysin (HlyA) was utilized to detect, enrich, and clone hybridoma cells expressing neutralizing monoclonal antibody in a new survivor selection protocol. A bank of 550 hybridoma clones was obtained from a mouse immunized with hemolysin by using standard techniques. The hybridoma bank was treated with a dose of HlyA hemolysin lethal to nonimmune clones. Five surviving hybridoma clones (X1 through X5) which possessed anti-HlyA activity were obtained. Western immunoblot analysis of V. cholerae culture supernatants with monoclonal antibody from clone X1 identified proteins with M(r)s of 83,200, 71,600, and 60,300. Amino-terminal sequence analysis of the 71,600-M(r) and 60,300-M(r) forms showed homology with the published predicted sequence of HlyA. Our data indicate that proteolytic cleavage occurs between residues 120 and 121 (Glu-Leu) of the 83,200-M(r) form, producing the 71,600-M(r) form with the terminus NH2-L-L-F-T-P-F-D-Q-A-E-E-. Cleavage between residues 150 and 151 (Gly-Phe) releases the 60,300-M(r) form with the terminus NH2-F-A-S-P-A-P-A-N-S-E-. Calculations based on the DNA sequence and the N termini indicated that the actual molecular masses of the 83,200-, 71,600-, and 60,300-M(r) forms were, respectively, 79.4 kilodaltons (kDa) 68.6 kDa, and 65.3 kDa. Survivor selection and amino-terminal microsequencing offer powerful tools for the analysis of leukotoxic agents.