Synthesis and Characterization of Leiurotoxin I Analogs Lacking One Disulfide Bridge:  Evidence That Disulfide Pairing 3−21 Is Not Required for Full Toxin Activity

Abstract

Leiurotoxin I (Lei-NH2), a toxin isolated from the venom of the scorpion Leiurus quinquestriatus hebraeus, is a blocker of the apamin-sensitive Ca(2+)-activated K+ channels. It is a 31-residue polypeptide cross-linked by three disulfide bridges which are presumably between Cys3-Cys21, Cys8-Cys26, and Cys12-Cys28. To investigate the role of these disulfides, analogs of Lei-NH2 lacking one disulfide bridge (i.e., [Abu3,21]Lei-NH2, [Abu8,26]Lei-NH2, and [Abu12,28]Lei-NH2) were chemically synthesized by selective replacement of each pair of half-cystines forming a bridge by two alpha-aminobutyrate (Abu) residues. The two disulfide pairings of the main folded form of the synthetic analogs were established by enzymatic proteolysis. They were as expected between Cys8-Cys26 and Cys12-Cys28 for [Abu3,21]Lei-NH2 but were unexpectedly between Cys3-Cys12 and Cys21-Cys28 for [Abu8,26]Lei-NH2 and between Cys3-Cys8 and Cys21-Cys26 for [Abu12,28]Lei-NH2. The synthetic peptides were tested in vitro for their capacity to compete with the binding of [125I]apamin to rat brain synaptosomes and in vivo for their neurotoxicity in mice. In both assays, [Abu3,21]Lei-NH2 exhibited full Lei-NH2-like activity whereas [Abu8,26]Lei-NH2 and [Abu12,28]-Lei-NH2 possessed only residual activities (< 2% native toxin activity). This suggests that disulfide bridge Cys3-Cys21 is not essential per se for high toxin activity. Circular dichroism (CD) spectroscopy of the three analogs showed that only [Abu3,21]Lei-NH2 exhibited a CD spectrum similar to that of Lei-NH2, suggesting they both adopt closely related conformations, in agreement with the pharmacological data. Structural models of the analogs were constructed on the basis of the disulfide pairing assignment and compared with that of Lei-NH2.

Chemical synthesis and characterization of maurocalcine, a scorpion toxin that activates Ca2+ release channel/ryanodine receptors

“…In the present work, we have chemically synthesized MCa by the Fmoc/t-butyl strategy [9], and carefully characterized the folded synthetic product sMCa for its physicochemical and electrophysiological properties. The disul¢de bridge organization was formerly established by enzyme-based cleavage of sMCa followed by analyses of the proteolytic peptide fragments using mass spectrometry, amino acid content determination, and Edman sequencing techniques, as described [10,11]. sMCa was tested in vivo for lethal activity to [12], and by electrophysiology in vitro for its putative action on Ca 2 £ux through RyR1 channels incorporated into planar bilayer lipid membranes.…”

Structure–function study of a chlorotoxin-chimer and its activity on Kv1.3 channels

“…But instead of producing a four-disulfide bridge folded ClTx chimer, six different fragments were formed with only six cysteine residues. Although the specific pattern of the disulfide bridge folding in these fragments has not been determined yet, it has been shown, by NMR studies, for other scorpion toxin analogs (like the analogs of leiurotoxin I) that the absence of the first disulfide bridge does not affect the three-dimensional folding of the toxin molecule, nor it affects the bioactive conformation of the toxin [18,19]. The analogs were fully active in vitro on ion channels and in vivo when tested for neurotoxicity in mice [20].…”

“…The toxin was collected and dried. Commercially available ClTx was checked for purity by reversed-phase C 18 HPCL, in similar conditions as described above. Based on the law of Lambert-Beer, the concentration of this sample could be calculated and compared with the indicated amount of the purchased ClTx.…”

“…(A) The restriction digests were applied on a reversed-phase C18 HPLC column and all different peaks were tested for activity. (B) The active peak indicated by the arrow in A was further purified on a reversed-phase C 2 /C 18 HPLC column.…”

Gene Expression, Mutation, and Structure−Function Relationship of Scorpion Toxin BmP05 Active on SKCa Channels

“…Among them, toxins specifically active on low-conductance, Ca 2+ -activated K + channels and sharing less structure homology with others have been widely studied, such as LeTx1 from Leiurus quinquestriatus hebraeus (6), P05 from Androctomus mauretanicus mauretanicus (7), and BmP05 from Buthus martensi Karch (1). The toxins LeTx1 and P05 have been studied thoroughly on their primary sequences (6,7), structure conformations (8,9), chemical syntheses (10,11), and structure-function relationships (11)(12)(13). They are peptides of 31 residues with three disulfide bridges, composed of an R-helix from residue 5 to residue 14 and two antiparallel -sheets (residues 17-22 and 25-29) joined by a -turn from residue 22 to residue 25 ( Figure 1) (8,9).…”