title: Rhodocetin, a novel platelet aggregation inhibitor from the venom of Calloselasma rhodostoma (Malayan pit viper): synergistic and noncovalent interaction between its subunits.
abstract: A novel platelet aggregation inhibitor, rhodocetin, was purified from the crude venom of Calloselasma rhodostoma. It inhibited collagen-induced platelet aggregation in a dose-dependent manner, with an IC50 of 41 nM. Rhodocetin has a heterodimeric structure with alpha and beta subunits, which could be separated on a nonreducing denaturing gel or reverse-phase HPLC column. Individually neither subunit inhibited platelet aggregation even at 2.0 microM concentration. Titration and reconstitution experiments showed that, when these subunits are mixed to give a 1:1 complex, most of its biological activity was recovered. The reconstituted complex inhibited platelet aggregation with an IC50 of 112 nM, about 3-fold less effective than the native molecule. Circular dichroism analysis revealed that the reconstituted complex had a spectrum similar to that of the native protein. By using surface plasmon resonance studies, we established that the stoichiometry of binding between the two subunits is 1:1 and the subunits interact with a Kd of 0.14 +/- 0.04 microM. The complete amino acid sequences of the alpha (15956.16 Da, 133 residues) and beta (15185.10 Da, 129 residues) subunits show a high degree of homology with each other (49%) and with the Ca2+-dependent lectin-related proteins (CLPs) (typically 29-48%) isolated from other snake venoms. Unlike the other members of the family in which the subunits are held together by an interchain disulfide bond, rhodocetin subunits are held together only through noncovalent interactions. The cysteinyl residues forming the intersubunit disulfide bridge in all other known CLPs are replaced by Ser-79 and Arg-75 in the alpha and beta subunits of rhodocetin, respectively. These studies support the noncovalent and synergistic interactions between the two subunits of rhodocetin. This is the first reported CLP dimer with such a novel heterodimeric structure.
authors.affiliation: Department of Biochemistry, Bioprocessing Technology Centre, and Bioscience Centre, National University of Singapore, Singapore.
authors.forename: R
authors.initials: R
authors.lastname: Wang
authors.forename: R M
authors.initials: RM
authors.lastname: Kini
authors.forename: M C
authors.initials: MC
authors.lastname: Chung
mesh_terms: D000595:Amino Acid Sequence; D001777:Blood Coagulation / Q000187:drug effects; D003435:Crotalid Venoms / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D019281:Dimerization; D004357:Drug Synergism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism*; D010974:Platelet Aggregation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
chemical_list: D003435:Crotalid Venoms; D010446:Peptide Fragments; D010975:Platelet Aggregation Inhibitors; C119730:rhodocetin