CSTX-9, a toxic peptide from the spider Cupiennius salei: amino acid sequence, disulphide bridge pattern and comparison with other spider toxins containing the cystine knot structure

CSTX-9 (68 residues, 7530.9 Da) is one of the most abundant toxic polypepti des in the venom of the wandering spider Cupiennius salei. The amino acid s equence was determined by Edman degradation using reduced and alkylated CST X-9 and peptides generated by cleavages with endoproteinase Asp-N and tryps in, respectively. Sequence comparison with CSTX-1, the most abundant and th e most toxic polypeptide in the crude spider venom, revealed a high degree of similarity (53 % identity). By means of limited proteolysis with immobil ised trypsin and RP-HPLC, the cystine-containing peptides of CSTX-9 were is olated and the disulphide bridges were assigned by amino acid analysis, Edm an degradation and nanospray tandem mass spectrometry. The four disulphide bonds present in CSTX-9 are arranged in the following pattern: 1-4, 2-5, 3- 8 and 6-7 (Cys(6)-Cys(21), Cys(13)-Cys(30), Cys(20)-Cys(48), Cys(32)-Cys(46 ))Sequence comparison of CSTX-1 with CSTX-9 clearly indicates the same disu lphide bridge pattern, which is also found in other spider polypeptide toxi ns, e.g. aga-toxins (omega -AGA-IVA, omega -AGA-IVB, mu -AGA-I and p-AGA-VI ) from Agelenopsis aperta, SNX-325 from Segestria florentina and curtatoxin s (CT-I, CT-II and CT-III) from Hololena curta. CSTX-1/CSTX-9 belong to the family of ion channel toxins containing the inhibitor cystine knot structu ral motif. CSTX-9, lacking the lysine-rich C-terminal tail of CSTX-1, exhib its a ninefold lower toxicity to Drosophila melanogaster than CSTX-1. This is in accordance with previous observations of CSTX-2a and CSTX-2b, two tru ncated forms of CSTX-1 which, like CSTX-9, also lack the C-terminal lysine- rich tail.