Synthetic peptides derived from the central loop of fasciculin: structural analysis and evaluation as inhibitors of acetylcholinesterase.

Fasciculins are peptides present in the venom of green and black mamba snakes, with potent inhibitory activity towards acetylcholinesterase. In order to determine the role of fasciculin loop II in the acetylcholinesterase inhibition, two fasciculin fragments were synthesized by the solid phase procedure using N-alpha-Boc protected amino acids. The two peptides, Fas-A and Fas-B, span the 26-32 and 22-35 sequences of fasciculin and a disulfide bridge links each peptide end, thus ensuring the formation of a looped structure. Both peptides were characterized chemically, structurally and functionally. Circular dichroism indicated the existence of 19.4 and 24.9% of beta-sheet for Fas-A and Fas-B, respectively; SDS-PAGE patterns and mass spectrometry disclosed the intramolecular disulfide formation in both peptides. An inhibitory effect on eel acetylcholinesterase was observed with the longer peptide (Ki = 15.1 microM), without reaching the affinity level of the parent native toxin (Ki = 0.3 nM). This study confirms that fasciculin central loop residues strongly contribute to toxin interaction with acetylcholinesterase.

Interaction of synthetic peptides from fasciculin with acetylcholinesterase.

Fasciculins (Fas) are three-looped polypeptides isolated from mamba venom which exert their toxic action by inhibiting noncompetitively acetylcholinesterase (AChE). A peptide (Fas-D) encompassing the first loop sequence was synthesized and characterized chemically, structurally, and functionally. Fas-D possesses an intramolecular disulfide bridge, present in the native toxin. Circular dichroism (CD) indicated the existence of 21.8% beta-sheet content and 24.2% beta-turn in this peptide, compatible with crystallographic data of the native toxin. The peptide showed only low partial AChE inhibition at submillimolar concentrations, much lower than that observed with Fas and a peptide (Fas-B) encompassing the second loop sequence. The simultaneous presence of Fas-D and Fas-B produced an additive inhibitory effect on AChE activity; calculated Ki and alphaKi values (7.3 +/-2.4 microM and 10.0 +/- 1.8 microM, respectively) were not significantly different, thus indicating noncompetitive inhibition. These results are consistent with site-directed mutagenesis studies and analysis of the crystal structure of the Fas-AChE complex, which indicate that residues from loops I and II contribute to Fas binding to the enzyme.

Conformational comparison in the snake toxin family.

A theoretical method was applied to consensus sequences of several members of the snake toxin family as a further approach to examining their conformational homology. Some secondary-structure predictions as well as hydropathy profiles were also examined. A comparison of long neurotoxins themselves reveals a high homology degree. However, their C-terminal fragments show poor homology and the N-terminal fragments appear as the region of maximum variability. Moreover, when the matrix includes the consensus sequence of the genus Laticauda (LNTX1), lacking the disulfide bridge 31-35, the method detects a lower conformational homology in a molecular region centered at position 31. Unlike long neurotoxins, the N-terminal segments of short neurotoxins show a high homology degree, but when comparing short with long neurotoxins, a poor correlation is found in this zone of the molecule. Cytotoxins studied exhibit an excellent conformational homology except when the consensus sequence of cytotoxin homologues CTXE is one of the proteins in the matrix. A comparison between cytotoxins and short neurotoxins reveals homology only in two segments belonging to a beta-sheet structure. A considerable degree of homology is found between the short neurotoxin group and calciseptin and fasciculin as well as between the long neurotoxin group and kappa-neurotoxins.