A novel dimer of a C-type lectin-like heterodimer from the venom of Calloselasma rhodostoma (Malayan pit viper).
We have isolated a potent platelet aggregation inducer from the crude venom of Calloselasma rhodostoma (Malayan pit viper), termed rhodoaggretin, with a novel oligomeric structure consisting of a dimer of C-type lectin-like heterodimers. On the basis of its native molecular mass of 66 kDa, and a M(r) of 30 kDa for its disulfide-linked alphabeta-heterodimer, we propose that rhodoaggretin exists as a (alphabeta)2 complex in the native state. We postulate that the di-dimer is stabilized by non-covalent interactions as well as by an intersubunit disulfide bridge between the two alphabeta-heterodimers. This conclusion is based on the following observations: (a) sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the non-reduced rhodoaggretin gave a major 28 and a minor 52 kDa band. (b) Prior treatment of rhodoaggretin with a limited amount of 2-mercaptoethanol (2-ME; 0.1%) resulted in the complete abolishment of the 52 kDa band in SDS-PAGE. (c) Two-dimensional SDS-PAGE in the presence of 3% 2-ME showed that both the 28 and 52 kDa bands gave two bands each with M(r)s of 18 (alpha-subunit) and 15 (beta-subunit) kDa. (d) Mass spectrometric analyses showed that purified rhodoaggretin had a M(r) of 30155.39+/-3.25 Da while its s-pyridylethylated alpha- and beta-subunits had M(r)s of 16535.62+/-2.98 and 15209.89+/-1.61 Da respectively. These molecular weight data suggested the presence of 15 cysteinyl residues in rhodoaggretin as compared to the 14 that are reported for the heterodimeric C-type lectin-like proteins. This extra cysteinyl residue is a candidate for the formation of the intersubunit disulfide bond in the (alphabeta)2 complex. (e) Homology structural modeling studies showed that the extra cysteinyl residue can indeed form a disulfide bond that covalently links the two alphabeta-heterodimers as proposed above.
Wang
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Department of Biochemistry, National University of Singapore, Singapore.
Kong
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C
Kolatkar
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P
Chung
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MC
D000818:Animals; D003435:Crotalid Venoms / Q000737:chemistry* / Q000302:isolation & purification*; D003545:Cysteine / Q000737:chemistry; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007526:Isoelectric Point; D037102:Lectins / Q000737:chemistry; D008623:Mercaptoethanol / Q000494:pharmacology; D008958:Models, Molecular; D008970:Molecular Weight; D010449:Peptide Mapping; D010974:Platelet Aggregation / Q000187:drug effects; D011487:Protein Conformation; D020836:Protein Structure, Quaternary; D021122:Protein Subunits; D020539:Sequence Analysis, Protein; D014757:Viper Venoms / Q000737:chemistry* / Q000302:isolation & purification* / Q000494:pharmacology; D017819:Viperidae
D003435:Crotalid Venoms; D004220:Disulfides; D037102:Lectins; D021122:Protein Subunits; D014757:Viper Venoms; C442235:rhodoaggretin; D008623:Mercaptoethanol; D003545:Cysteine