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A novel dimer of a C‐type lectin‐like heterodimer from the venom of Calloselasma rhodostoma (Malayan pit viper)

Runhua Wang,C. Kong,P. Kolatkar,Maxey C M Chung

Published 2001 in FEBS Letters

ABSTRACT

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 αβ‐heterodimer, we propose that rhodoaggretin exists as a (αβ)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 αβ‐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 rs of 18 (α‐subunit) and 15 (β‐subunit) kDa. (d) Mass spectrometric analyses showed that purified rhodoaggretin had a M r of 30 155.39±3.25 Da while its s‐pyridylethylated α‐ and β‐subunits had M rs of 16 535.62±2.98 and 15 209.89±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 (αβ)2 complex. (e) Homology structural modeling studies showed that the extra cysteinyl residue can indeed form a disulfide bond that covalently links the two αβ‐heterodimers as proposed above.

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