Arg-Tyr-Asp (RYD) and Arg-Cys-Asp (RCD) motifs in dendroaspin promote selective inhibition of β1 and β3 integrins

Abstract

Arg-Gly-Asp (RGD) is a unique minimal integrin-binding sequence that is found within several glycoprotein ligands. This sequence has also been found in snake-venom anti-platelet proteins, including the disintegrins and dendroaspin, a natural variant of short-chain neurotoxins isolated from the venom of Dendroaspis jamesonii. In the present study, the motifs RYD and RCD were introduced into the dendroaspin scaffold to replace RGD. Both motifs in dendroaspin caused inhibition of ADP-induced platelet aggregation with IC50 values of 200 and 300nM respectively, similar to that of the wild-type RGD motif (170nM). In comparison with wild-type dendroaspin, both RYD- and RCD-containing dendroaspins were more selective in the inhibition of the adhesion of K562 cells to laminin rather than to fibrinogen and fibronectin, even though they were 10–30-fold less potent at inhibiting K562 cell (containing α5β1 integrin) adhesion to laminin compared with wild-type. Interestingly, the RYD motif produced a similar IC50 value to the RGD motif at inhibiting A375-SM cell (β3 integrin) adhesion to collagen, whereas the RCD motif was approx. 2–6-fold less potent compared with either RGD or RYD. These findings show that the selectivity of dendroaspin binding to β1 and β3 integrins can be modulated by the introduction of alternative cell recognition sequences.

The Effect of the Single Substitution of Arginine within the RGD Tripeptide Motif of a Modified Neurotoxin Dendroaspin on Its Activity of Platelet Aggregation and Cell Adhesion

“…Indeed, the NMR structural studies of several inhibitors including wild-type dendroaspin (34,36) have demonstrated that the positioning of the RGD motif at the apex of a solvent exposed loop is a prerequisite for inhibitory activity. On the other hand, the most potent tripeptide motifs are not limited to only RGD and KGD as our previous studies of RYD and RCD motifs have demonstrated (37).…”

Endothelial-cell apoptosis induced by cleaved high-molecular-weight kininogen (HKa) is matrix dependent and requires the generation of reactive oxygen species

“…10 However, despite the ability of aggretin to induce phosphorylation of endothelial-cell ERK 1/2, confirming its ability to activate ␣2␤1-dependent signaling 26 (Figure 2), aggretin did not inhibit HKa-induced endothelial-cell apoptosis. Identical results were obtained using mAb JBS2, a specific ␣2␤1-activating monoclonal antibody, 44,45 and mAb TS2/16, a specific ␤1 integrinactivating antibody, either in solution or as a coating for microplates on which cells were cultured (not shown). Although these results do not exclude a role for integrin ␣2␤1 in mediating collagen-dependent endothelial-cell survival, they suggest that if such an interaction is important, a more complex ligand-receptor interaction is involved than the monovalent or bivalent interactions associated with aggretin or monoclonal antibodies, respectively.…”

“…[17][18][19]45,69 While our results are consistent with such a mechanism, we were not able to demonstrate protection from apoptosis by soluble collagen or by smaller ligands such as aggretin or monoclonal antibodies that activate ␣2␤1. 26,27,44 Thus, we hypothesize that the ability of immobilized collagen to prevent HKa-induced apoptosis may reflect a need for receptor aggregation, or at least engagement by a multivalent ligand, perhaps accompanied by activation of mechanochemical signaling responses not induced by smaller, soluble ligands. 46,47,70 A second hypothesis, that the ability of collagen to quench ROSs may Figure 6.…”

Dynamics and functional differences between dendroaspin and rhodostomin: Insights into protein scaffolds in integrin recognition

“…24 They were consistent with the results of different integrin function-blocking mAbs. 25,26 In addition, the adhesion of K562 cells to fibronectin (FN) in the presence of 500 lM Mn 2þ was shown to be predominantly a5b1-dependent. 27 Their inhibitory constants are summarized in Table I.…”