Relationship between hemagglutinin and sialidase from Clostridium perfringens CN3870: chromatographic characterization of the biologically active proteins

Abstract

Biochemical characterization of hemagglutinin and sialidase activities from Clostridium perfringens strain CN3870 revealed that this strain produced three sialidase enzymes that were separable by gel filtration, ion exchange chromatography, and polyacrylamide gel electrophoresis. The molecular weights of MATERIALS AND METHODS Bacteria. The strains used in these studies were C. perfringens strains CN3870 and ATCC 10543. The growth and maintenance of these strains and preparation of culture media were as described previously (17). Cooked meat medium cultures were used as stock cultures of strain ATCC 10543. The growth media used were fructose-Trypticase soy broth (16) and MacLennan broth. Materials. Reagents and materials were from sources previously described (17). In addition, human immunoglobulin was from Commonwealth Serum Laboratories, Parkville, Australia; acrylamide, N,N'-methylenebisacrylamide, and N,N,N', N'-tetramethylethylenediamine from Eastman Kodak Co., Rochester, N. Y.; aldolase (rabbit muscle, lot 102C-1120) and xanthine oxidase (buttermilk, lot 124C-01371) from Sigma Chemical Co., St. Louis, 831 on August 5, 2020 by guest http://jb.asm.org/ Downloaded from 832 ROOD AND WILKINSON Mo.; apoferritin (horse spleen, lot 801863), ovalbumin (lot 901688), and dithiothreitol (DTT) from Calbiochem, Los Angeles, Calif.; blue dextran 2000, DEAE-Sephadex A-25 (40 to 120 ,um), and Sephadex G-200 (40 to 120 ,um) from Pharmacia, Uppsala, Sweden; Coomassie brilliant blue R250 from Colab Laboratories Inc., Chicago Heights, Ill.; dimethyldichlorosilane from Applied Science Laboratories Inc., State College, Pa.; Sarkosyl NL 97 from Geigy Industrial Chemicals, Ardsley, N. Y.; tris(hydroxymethyl)aminomethane (Tris) from E. Merck, Darmstadt, West Germany; and Triton X-100 from Packard Instrument Co., Downer's Grove, Ill.

Relationship between hemagglutinin and sialidase from Clostridium perfringens CN3870: gel filtration of mutant and reverant activities

“…MacLennan broth (MB) was used instead of fructose-Trypticase soy broth for reasons previously discussed (3). All reagents and materials were from sources previously described (3,4).…”

“…In the preceding publication (4) it is shown that the wild-type Clostridium perfringens, strain CN3870, produced three mucopolysaccharide N-acetylneuraminylhydrolase (EC 3.2.1.18) (sialidase or neuraminidase) enzymes designated as sialidases I, II, and III in order of decreasing molecular weight. Sialidase I and II activities were associated with hemagglutinin I and II activities, respectively.…”

Molecular genetics and pathogenesis of Clostridium perfringens.

“…All C. perfringens types produce a neuraminidase, or sialidase, which cleaves acylneuraminic acid residues from glycoproteins, glycolipids, and oligosaccharides and may play a role in disease by destroying receptor molecules on cell surfaces or by disrupting connective tissue (125). In early studies, three distinct forms of the enzyme (64, 105, and 310 kDa) were found by gel filtration (170). The larger two of these enzyme forms appeared to correspond to different multimers and appeared to be associated both physically and genetically with hemagglutinins (169,171).…”

Carbohydrate Recognition by a Large Sialidase Toxin from Clostridium perfringens

“…In addition to the major toxins, C. perfringens produces a battery of toxins that are classified as minor due to their inability to cause lethal effects in mice. Among these minor toxins are the sialidases ( − ). These enzymes remove terminal sialic acids from glycans, which has two important outcomes during the infection of humans.…”