Trypsin activation of enterotoxin from Clostridium perfringens type A fragmentation and some physicochemical properties

Granum, Per Einar; Whitaker, John R.; Skjelkvåle, Reidar

doi:10.1016/0005-2795(81)90165-3

Cited by 60 publications

(38 citation statements)

References 22 publications

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“…Since C-terminal enterotoxin fragments are not cytotoxic (11,12,14), some N-terminal CPE sequences are important for cytotoxicity. However, extreme N-terminal CPE sequences are not involved in cytotoxicity, since (i) tryptic or chymotryptic treatment removes the 25 or 36 N-terminal CPE amino acids, respectively, increasing cytotoxic activity by two-to threefold (8,9,13) and (ii) a recombinant CPE (rCPE) fragment lacking the first 44 amino acids of the native enterotoxin also exhibits a two-to threefold increase in large complex formation and cytotoxicity (19). Further deletion of N-terminal rCPE sequences produces an rCPE fragment that maintains wildtype binding activity, but neither forms a large complex nor elicits a cytotoxic response (19).…”

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confidence: 99%

Fine Mapping of the N-Terminal Cytotoxicity Region of Clostridium perfringens Enterotoxin by Site-Directed Mutagenesis

Smedley

McClane

2004

Infect Immun

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Clostridium perfringens enterotoxin (CPE) has a unique mechanism of action that results in the formation of large, sodium dodecyl sulfate-resistant complexes involving tight junction proteins; those complexes then induce plasma membrane permeability alterations in host intestinal epithelial cells, leading to cell death and epithelial desquamation. Previous deletion and point mutational studies mapped CPE receptor binding activity to the toxin's extreme C terminus. Those earlier analyses also determined that an N-terminal CPE region between residues D45 and G53 is required for large complex formation and cytotoxicity. To more finely map this N-terminal cytotoxicity region, site-directed mutagenesis was performed with recombinant CPE (rCPE). Alanine-scanning mutagenesis produced one rCPE variant, D48A, that failed to form large complexes or induce cytotoxicity, despite having normal ability to bind and form the small complex. Two saturation variants, D48E and D48N, also had a phenotype resembling that of the D48A variant, indicating that both size and charge are important at CPE residue 48. Another alanine substitution rCPE variant, I51A, was highly attenuated for large complex formation and cytotoxicity, but rCPE saturation variants I51L and I51V displayed a normal large complex formation and cytotoxicity phenotype. Collectively, these mutagenesis results identify a core CPE sequence extending from residues G47 to I51 that directly participates in large complex formation and cytotoxicity.

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confidence: 99%

Fine Mapping of the N-Terminal Cytotoxicity Region of Clostridium perfringens Enterotoxin by Site-Directed Mutagenesis

Smedley

McClane

2004

Infect Immun

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“…Limited information is available about CPE structure-function relationships. Trypsin removal of the 34 NH3-terminal residues from CPE increases CPE action (7), indicating that the entire CPE molecule is not required for bioactivity. Recent studies (9,27) have suggested that a C-terminal CPE fragment (putative amino acids 177 to 309), derived chemically by 2-nitro-5-thiocyanobenzoic acid (NTCB) cleavage of intact CPE, may inhibit CPE specific binding.…”

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confidence: 99%

Molecular cloning of the 3' half of the Clostridium perfringens enterotoxin gene and demonstration that this region encodes receptor-binding activity

1989

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“…Calculations for helix, beta-sheet and turns are made using the method of Krijbaum and Knutton [49] toxin. However, they may bear a resemblence to the tryptic fragments described [52,53] where treatment of C. perfringens type A enterotoxin with trypsin in the presence of 0.05% SDS results in the generation of a fraction of tryptic peptides of M , 16000. These peptides inhibited cell-free protein synthesis but lost the ability to disrupt Vero cells.…”

Section: Discussionmentioning

confidence: 96%

Purification and properties of an enterotoxin from a coatless spore mutant of Clostridium perfringens type A

Lindsay¹,

Sleigh²,

Ghitgas³

et al. 1985

European Journal of Biochemistry

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A method is described for isolating an enterotoxin from a coatless spore mutant (8-6) of Clostridium perfringens type A. The characteristics of this enterotoxin only slightly resembled those of previously isolated enterotoxins of C. perfringens. The type A (8-6) enterotoxin was found to be composed of two subunits of M , 18000 with isoelectric points of 3.8 and 4.3. The LD5,, for mice was 39 pg/kg with 0.10 pg corresponding to one erythemal unit. The type A (8-6) enterotoxin was inactivated by heating for 10 min at 60°C. The amino acid composition data of type A (8-6) and delta toxins was similar, but type A (8-6) and type A enterotoxins showed less similarity. This lack of similarity between type A and type A (8-6) enterotoxins was confirmed by the failure of anti-sera to type A enterotoxin to neutralize the type A (8-6) enterotoxin, in both the mouse and erythemal tests.Certain strains of Clostridium perfringens type A produce an enterotoxin that is linked to a major cause of mild food poisoning in humans [l]. The enterotoxin is a sporulationspecific gene product, whose production is correlated with late stage I1 to stage I11 of sporulation [2 -51. Several lines of evidence indicate that enterotoxin is possibly excess spore coat protein not incorporated as a structural component of the spore coat, and released from the sporangia only on lysis [6].While a large amount of data is available on the mode of action of enterotoxin [7-91 and more recently on a binding protein [lo] and membrane receptors [ll], information on structure and composition is still subject to some conjecture.The type A enterotoxin isolated by Granum and Skjelkvale [12] is a single polypeptide chain of M , 34000, however, although this molecule has now been fully sequenced [ 13 -151, the question still arises, whether this is the only enterotoxin produced by C. perfringens type A. This concern stems from some unresolved discrepancies in the literature as to the number of subunits, the M , of these subunits, their behaviour under SDS gel electrophoresis and some slight variation in amino acid composition of the enterotoxin when produced under different conditions [16 -231.In our studies on the development of spore heat resistance we have used a strain of C. perfringens type A [24] which produces a spore without a coat, although it produces coat protein(s) and heat-stable spores. Our rationale for this study was that if enterotoxin production is linked to coat protein synthesis, a coatless spore may provide enterotoxin in larger quantities than would normally be obtainable from coated spores. In this paper we report the isolation and characterization of a different enterotoxin from the coatless mutant of C. perfringens type A designated 8-6.Correspondence to J. A. Lindsay, CSIRO Division of Food Research, P. 0. Box 52, North Ryde, Sydney, New South Wales, Australia 21 13Abbreviations. i.p., intraperitoneally ; buffer A, 0.05 M phosphate buffer pH 6.7; SDS, sodium dodecyl sulphate; PAGE, polyacrylamide gel electrophoresis; HPLC, high performanc...

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Trypsin activation of enterotoxin from Clostridium perfringens type A fragmentation and some physicochemical properties

Cited by 60 publications

References 22 publications

Fine Mapping of the N-Terminal Cytotoxicity Region of Clostridium perfringens Enterotoxin by Site-Directed Mutagenesis

Fine Mapping of the N-Terminal Cytotoxicity Region of Clostridium perfringens Enterotoxin by Site-Directed Mutagenesis

Molecular cloning of the 3' half of the Clostridium perfringens enterotoxin gene and demonstration that this region encodes receptor-binding activity

Purification and properties of an enterotoxin from a coatless spore mutant of Clostridium perfringens type A

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