Partial characterization of a 36-kDa protein from Clostridium botulinum type E that inhibits trypsin and chymotrypsin.

Abstract

Botulinum neurotoxin serotypes (A to G) are produced as ϳ150-kDa single-chain proteins by certain strains of Clostridium botulinum, C. butyricum, and C. baratii that are grouped as proteolytic, also called phenotypic group I, and nonproteolytic, or phenotypic group II (Collins and East, 1998;Hatheway, 1993;Minton, 1995). Protease(s) endogenous to the bacteria cleave (nick) peptide bond(s) between the two conserved Cys residues of the single-chain neurotoxin. The proteolytically processed (nicked) neurotoxin, a dichain protein, is more toxic than its precursor singlechain protein. The neurotoxin serotype A harvested after sufficient incubation (e.g., 96 h) of the proteolytic culture is found as the dichain protein, and earlier harvests yield only single-chain or a mixture of single and dichain molecules (Dekleva and DasGupta, 1989), and the neurotoxin serotype E remains in the singlechain form because the culture lacks the endogenous protease(s) (DasGupta and Rasmussen, 1983;DasGupta and Sugiyama, 1972a, b). The type B neurotoxin, produced by the proteolytic strain, harvested even after 96 h is a mixture of Ն90% single-chain and Յ10% dichain molecules (DasGupta and Sugiyama, 1976;Kozaki and Sakaguchi, 1975). One could speculate that a protease inhibitor produced by the organism inhibits the protease(s) responsible for nicking of type B neurotoxin and thus limits the endogenous single to dichain conversion (DasGupta and Sugiyama, 1976). Indeed, Höyem and Skulberg (1962) had reported the presence of a trypsin-inhibiting activity in the supernatants of cultures of C. botulinum that produced neurotoxin serotypes A, B, and E and noted maximum inhibitory activity in the serotype B culture. They partially characterized the inhibitor only from type E culture; it was easily dialyzable and heat stable (activity was not lost after 1-2 h at 100°C or 15 min autoclaving at 120°C). No other report on this trypsin inhibitor is apparent since its first report (Höyem and Skulberg, 1962).We report purification to homogeneity of a trypsin inhibitor formed in C. botulinum type E culture and Nterminal amino acid sequence of the ϳ36-kDa inhibitory protein. The protein inhibited both trypsin (EC 3.4.21.4) and chymotrypsin (EC 3.4.21.1). To our knowledge, this novel polypeptide has not been reported in any protein data bank and is the first polypeptide protease inhibitor of this large range isolated from a bacterial culture. We find it interesting that the culture phenotypically nonproteolytic produces this protease inhibitor. Materials and MethodsStock culture of C. botulinum type E (strain Alaska E-43 stored at Ϫ80°C) was grown (day 1) in cooked meat medium (Difco, Detroit, MI), at 30°C (Ϯ1°C). On day 2, 13-L glass carboys (Pyrex), each containing 9,500 ml of media (pH 7.3), were autoclaved for 1 h; then to each was added glucose (100 g in 500 ml water) separately autoclaved for 30 min. The final composition of the culture medium was yeast extract