Purification and characterization of a novel hemagglutinin from Vibrio cholerae.
A lectin with strong hemagglutinating activity toward erythrocytes of several animal species was isolated from an 18-h culture supernatant of a diarrheagenic strain, V2, of non-O1 Vibrio cholerae. The hemagglutinin (HA) was purified free of lipopolysaccharide by salt fractionation followed by gel filtration, hydrophobic interaction chromatography, and, finally, gel filtration in the presence of urea and deoxycholate. The purification procedure resulted in an HA preparation with 80-fold enhancement of specific activity. The HA consisted of noncovalently bound subunits of Mr 62,000 and behaved essentially as a single component with pI 6.0. Nonpolar and acidic amino acids contributed 46 and 24%, respectively, to the total amino acid residues. Electron micrographs of the HA showed it to consist of large, nonstoichiometric aggregates' of disklike molecules of 10-nm diameter. Inhibition of the HA by the glycoproteins fetuin, asialofetuin, and mucin, but not by ovalbumin and simple sugars, suggested the specific requirement of complex carbohydrates for binding. Rabbit antisera to the purified HA inhibited the hemagglutinating activities of the crude cell-free HA preparations, but not cell-associated HA activities of the parent (V2) or of other O1 and non-O1 V. cholerae strains. This suggested that the released and cell-associated HA activities were mediated by antigenically distinct components. Immunoblotting experiments showed that the antisera recognized a polypeptide component of Mr 62,000 in the cell envelope preparations of the parent and several other V. cholerae O1 and non-O1 strains. These data suggested that the HA was a nonfimbrial lectin of somatic origin with no protease activity and was apparently distinct from V. cholerae HAs described so far.
Banerjee
K K
KK
Division of Immunology, National Institute of Cholera and Enteric Diseases, Calcutta, India.
Ghosh
A N
AN
Dutta-Roy
K
K
Pal
S C
SC
Ghose
A C
AC
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000494:pharmacology; D004912:Erythrocytes / Q000187:drug effects / Q000382:microbiology; D006388:Hemagglutinins / Q000032:analysis* / Q000737:chemistry / Q000494:pharmacology; D006358:Hot Temperature; D006801:Humans; D007525:Isoelectric Focusing; D011817:Rabbits; D014734:Vibrio cholerae / Q000032:analysis*
D000596:Amino Acids; D006388:Hemagglutinins; D010450:Endopeptidases