Rattlesnake presynaptic neurotoxins: primary structure and evolutionary origin of the acidic subunit

Abstract

Crotoxin and homologous crotalid presynaptic neurotoxins consist of a toxic, basic subunit and a slightly smaller, nontoxic, acidic subunit. The latter, in turn, consists of three chains, interconnected by disulfide bonds. The complete sequences of two of the three acidic subunit chains of crotoxin, from the venom of the South American rattlesnake Crotalus durissus terrificus, have been determined. In addition, all but the ten amino-terminal residues of the third chain have been sequenced. Sequence comparison data suggest that the acidic subunit has been derived from a nontoxic, homodimeric, crotalid phospholipase A2. When compared with sequences of phospholipases A2, the acidic subunit lacks a 22-residue amino-terminal segment and two additional segments that are implicated in phospholipid substrate binding. However, it apparently retains an intact active site, the calcium binding loop, and segments involved in subunit binding in homodimeric phospholipases A2. The C chain of the acidic subunit shows strong homology with mammalian neurophysins, lending possible support to the hypothesis that the acidic subunit functions as a chaperone to prevent nonspecific binding of the toxic basic subunit. Crystals suitable for X-ray diffraction studies have recently been produced [Achari, A., Radvanyi, F. R., Scott, D., Bon, C., & Sigler, P. B. (1985) J. Biol. Chem. 260, 9385-9387]; thus with these data it should now be possible to determine the three-dimensional structure of the intact neurotoxin and dissociated subunits.

The origin of the diversity of crotoxin isoforms in the venom of Crotalus durissus terrificus

“…However, no systematic screening of the cDNA library was performed to examine if it contains another cDNA encoding pro-CA precursor. The sequence of CA established with a mixture of isoforms by Aird et al [6,71 differs from that of the pro-CA precursor [17] at position 58 (Asn instead of Asp) and at position 87 (a mixture of Gly/Glu instead of Glu; numbering from the first amino acid of pro-CA). These differences, however, can be attributed either to deamidations or to the expression of different mRNAs.…”

Crotoxin: Structural Studies, Mechanism of Action and Cloning of Its Gene

“…When basic subunit antisera was cross-reacted with acidic subunit and acidic subunit antisera was cross-reacted with basic subunit, both showed moderate levels of cross-reactivity (Table 1). The reduced heterologous reaction seen between basic subunit antisera and acidic subunit is not too surprising, since the acidic subunit lacks three peptides present in the basic subunit protein (2). We also observed cross-reaction between both subunit antisera and phospholipase A 2 from both C admantju and C. atrgx venoms.…”

“…At least one of these must have been between subunits because recovered complex could not be dissociated in 6M urea. Sequence analyses indicate the presence of 10 Lys residues (the most likely reactant) in the basic subunit and one each in the A-chain and B-chain of the acidic subunit (2). The DMS-crotoxin had comparable levels of phospholipase A 2 activity to that of unmodified crotoxin (21 4mol/min-mg).…”

“…This region has little homology with known sequences in non-toxic phospholipases. We have now completed the amino acid sequence of the basic subunit (9) and two of the three acidic subunit chains (2). We have all but ten amino-terminal residues of the third chain sequenced.…”

“…11), and located by a post-column fluorescamine detection system that detects free primary amines. C-chain, which lacks Lys, was undetectable if deblocking of its amino-terminal end with pyroglutamate aminopeptidase was omitted (2). It contains 14 residues (Fig.…”