Molecular characterization of Limulus polyphemus C-reactive protein. I. Subunit composition.

The molecular mass of whole native C-reactive protein (CRP) from Limulus polyphemus, the horseshoe crab, was precisely determined for the first time, by analytical ultracentrifugation after measurement of its absorption coefficient, A1cm, 1% at 280 nm (15.49) and its partial specific volume (0.72 +/- 0.003 ml/g). The apparent weight-average molecular mass, in the range over which it was independent of the concentration, was 300 kDa. Limulus CRP, isolated from an individual animal, was also analyzed by electrospray mass spectrometry after dissociation into its subunits, and nine components were detected in the mass range 24935-25617 Da. Four of these components corresponded precisely to one of the previously reported protein sequences of Limulus CRP subunits which is glycosylated by a single oligosaccharide chain of composition, Man2GlcNAc2, Man3GlcNAc2, Man4GlcNAc2 or Man5GlcNAc2, the structures of which are elucidated in the accompanying paper [Amatayakul-Chantler, S., Dwek, R. A., Tennent, G. A., Pepys, M. B. & Rademacher, T. W. Molecular characterization of Limulus polyphemus C-reactive protein. II. Asparagine-linked oligosaccharides (1993) Eur. J. Biochem. 214, 99-110]. All the remaining components, except one minor component, corresponded to the analogous glycoforms of peptides of molecular mass 24281 +/- 2 Da and 24523 +/- 1 Da, neither of which have previously been characterized. These results are consistent with the presence of 12 subunits in the Limulus CRP molecule, in contrast to the number of subunits in vertebrate members of the CRP family. The CRP family members in vertebrates are known as pentraxins because of the symmetrical pentameric arrangement of their subunits. The concentration of Limulus CRP in hemolymph from 20 individual animals, measured precisely by specific electroimmunoassay, covered the wide range 0.275-6.64 mg/ml, mean (SD) = 1.83 (2.06) mg/ml. It remains to be determined whether this protein is an acute-phase reactant, like its human and some other mammalian homologues.