Effects of site-specific acetylation on .omega.-conotoxin GVIA binding and function

Abstract

Chemical modification of omega-conotoxin GVIA (omega-CgTXGVIA) was performed using nonsaturating concentrations of acetic anhydride to generate seven distinct derivatives. Following separation of these peptides using reverse-phase HPLC (RP-HPLC), their individual molecular weights were determined using fast bombardment mass spectrometry (FAB-MS). Three peptides contained a single acetylated amino group, three possessed two acetylated amino groups, and the last contained three acetylations. For each peptide, the specific site of acetylation was confirmed using a scheme of tryptic digestion, under nonreducing conditions, followed by RP-HPLC and FAB-MS. Biological profiles for each peptide were obtained by analyzing their capacity to displace native 125I-omega-CgTx GVIA binding to rat hippocampal membranes and to block K(+)-stimulated 45Ca2+ influx into chick brain synaptosomes. The data indicate that successive additions of acetyl moieties to omega-CgTx GVIA lead to a loss of both binding affinity and Ca2+ influx inhibitory potency. Within the monoacetylated series, acetylation of the amino terminal of Cys-1, as compared to the epsilon-amino group of either Lys-2 or Lys-24, leads to the greatest shift in potency. In summary, these results indicate that basic (i.e., primary amino) groups, which are brought into close proximity as a result of disulfide bridging, are important in the functional blockade of neuronal Ca2+ channels by omega-CgTx GVIA.

A Consensus Structure for ω-Conotoxins with Different Selectivities for Voltage-sensitive Calcium Channel Subtypes: Comparison of MVIIA, SVIB and SNX-202

“…Second, the 3D structures of MVIIA and SVIB were calculated from NOE-based distance restraints and torsion angle restraints. These structures aid in the interpretation of activity data Haack et al, 1993;Lampe et al, 1993;Sato et al, 1993;Kim et al, 1994Kim et al, , 1995, which indicate that subtle differences in side-chain nature and positioning are likely to affect VSCC specificity.…”

Solution structure of ω‐conotoxin MVIIA using 2D NMR spectroscopy

“…Among the og-conotoxins, there are only a few conserved residues [43], the cysteines, and G5, and two with conservative substitutions Lys/Arg at positions 2 and 24 (og-CmTxa numbering) preserving the positive charge at these positions. It has been demonstrated, with acetylation of each of the lysines in og-CgTx [44], that no single lysine is very important for binding. There is some loss of binding when acetylating the amino-terminus of the peptide, but acetylating the amino-terminus along with both of the other lysines, at the positions equivalent to residues 2 and 24 of og-CmTxa, decreases the binding affinity by two orders of magnitude compared to native co-CgTx.…”

Structure-Function Relationships of ω-Conotoxin GVIA

“…The role of the ammonium groups was studied by selective acetylation, with the N terminus being found to be important for binding to rat hippocampal membranes and chick brain synaptosomes (18). Studies of GVIA biotinylated at Lys 2 and/or Lys 24 demonstrated that Lys 2 was more important than Lys 24 for binding to rat brain synaptic membranes (19).…”