Substitutions of proline 42 to alanine and methionine 46 to asparagine around the RGD domain of the neurotoxin dendroaspin alter its preferential antagonism to that resembling the disintegrin elegantin.
Previous studies have shown that the neurotoxin dendroaspin and the disintegrin kistrin, which show little overall sequence homology but similar residues around RGD (PRGDMP), preferentially inhibited platelet adhesion to fibrinogen. In contrast, the elegantin which has different amino acids around RGD (ARGDNP) preferentially inhibited platelet adhesion to fibronectin. To investigate further the role of amino acids around RGD in disintegrins, we have constructed the genes of a wild-type and of two mutant dendroaspins with substitutions around the RGD, namely [Asn46]- and [Ala42,Asn46]-dendroaspins. Proteins were expressed in Escherichia coli as glutathione S-transferase fusion recombinants and purified to homogeneity by affinity chromatography and reversed phase high performance liquid chromatography. Platelet aggregation studies revealed that wild-type dendroaspin showed an IC50 value similar to that of native dendroaspin, with [Ala42,Asn46]-dendroaspin showing an IC50 value similar to that of elegantin. Interestingly, in platelet adhesion assays, the mutants showed a progressive shift in inhibitory preference, in particular, [Ala42,Asn46]dendroaspin showed nearly identical behavior as elegantin when fibronectin was the immobilized ligand (IC50 = 0.33 microM and 0.6 microM, respectively, compared with 20 microM for native dendroaspin). Native and recombinant wild-type dendroaspin bound to a single class of binding site exhibiting a Kd = 67 nM; [Asn46]- and [Ala42,Asn46]dendroaspins, however, both produced biphasic isotherms with Kd values = 87 nM and 361 nM for [Asn46]dendroaspin and 33 nM and 371 nM for [Ala42,Asn46]dendroaspin, which are close to those of elegantin (Kd values = 18 nM and 179 nM). These studies prove that the amino acids flanking RGD provide an extended locus that regulate the affinity and selectivity of RGD protein dendroaspin.
Lu
X
X
Platelet Section, Thrombosis Research Institute, London, United Kingdom.
Rahman
S
S
Kakkar
V V
VV
Authi
K S
KS
D000409:Alanine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001216:Asparagine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004546:Elapid Venoms / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D008715:Methionine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides; D010455:Peptides / Q000494:pharmacology*; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology*; D011392:Proline / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D004546:Elapid Venoms; D009842:Oligopeptides; D010455:Peptides; D010975:Platelet Aggregation Inhibitors; D011994:Recombinant Proteins; C064594:elegantin; C074772:mambin; D001216:Asparagine; C047981:arginyl-glycyl-aspartic acid; D011392:Proline; D008715:Methionine; D000409:Alanine