Buforin I, a natural peptide, inhibits botulinum neurotoxin B activity in vitro

Botulinum neurotoxin B (BoNT/B) serotype specifically cleaves between the a mino acids glutamine and phenylalanine (Q and F bond) in position 76-77 of synaptobrevin (VAMP2), We evaluated peptides that contain the QF cleavage s ite but are not identical in primary structure to the VAMP2 sequence surrou nding the QF site for both inhibition of BoNT/B proteolytic activity and as substrates for BoNT/B, A reverse-phase high-performance liquid chromatogra phy (RP-HPLC) method was used to measure digested peptides, A dose as high as 600 mu M of substance P, and 11-amino acid peptide containing the QF bon d, was neither a substrate nor inhibitor of BoNT/B in our assay, suggesting that more than the QF bond is required to be recognized by BoNT/B, Buforin I(B-I, QF site 24-25) is 39 amino acids in length, and sequence comparison of B-I and VAMP2 indicated a similarity of 18% for conserved amino acids a round the QF site. Furthermore, computer-aided secondary structure computat ions predict alpha-helical structures flanking the QF site for VAMP2 and fo r the upstream sequence of B-I, Although predictions for the downstream seq uence give nearly equal tendencies for alpha-helical and beta-sheet structu res, Yi et al, showed that the downstream sequence is likely to be the alph a-helix based on their examination of buforin II (B-II, a 21-amino acid sub set of B-I (16-36)), which includes the QF site and the downstream sequence of B-I, Buforin I was found not to be a substrate for BoNT/B; however, BI dose dependently and competitively inhibited BoNT/B activity, yielding IC50 = 1 X 10(-6) M, In contrast, B-II was not a substrate for BoNT/B and exhib ited only 25% of the B-I inhibition of BoNT/B, Two additional B-I deletion peptides were tested for inhibition of BoNT/B proteolysis: peptide 36 (36 m er; containing B-I amino acids 1-36) and peptide 24 (24 mer; B-I amino acid s 16-39), Peptide 24 had a similar inhibitory effect to B-II (ca, 25% of B- I) but peptide 36 was almost 50% as potent as B-I. These findings suggest t hat the buforin tertiary structure is important for the inhibitory activity of these peptides for BoNT/B.