Immunolocalization of gastrolith matrix protein (GAMP) in the gastroliths and exoskeleton of crayfish, Procambarus clarkii

Abstract

Gastrolith matrix protein (GAMP) is a novel protein purified from gastroliths of the crayfish, Procambarus clarkii, and has been suggested to be associated with calcium carbonate deposition. In the present study, a specific antibody against GAMP was raised and distribution of GAMP immunoreactivity was studied in crayfish gastrolith and exoskeleton. Localization of calcium carbonate in the exoskeleton, determined by silver nitrate staining and energy dispersive X-ray microanalysis, was compared with that of GAMP immunoreactivity. Crystalline forms of calcium carbonate were also determined. SDS-PAGE and Western blotting revealed that the gastrolith extract contained a major band which was GAMP-immunopositive and showed the same mobility as that of purified GAMP. The exoskeleton extract showed smear bands, but no immunoreaction was detected. By using immunohistochemistry, the anti-GAMP antiserum reacted almost uniformly with gastrolith matrix irrespective of gastrolith size. Epithelial cells of the gastrolith disc were also immunopositive. In the exoskeleton, exocuticle was strongly GAMP-immunopositive, whereas the endocuticle and membrane layer was slightly positive. The epicuticle was immunonegative. Calcium carbonate was detected in exocuticle, endocuticle and a part of the membrane layer, but not in the epicuticle. Thus, the distribution of GAMP immunoreactivity roughly corresponded with that of calcium carbonate. X-ray diffraction study showed that calcium carbonate in the gastrolith was amorphous, whereas that in the exoskeleton consisted of calcite crystals. These data indicate that a GAMP-immunoreactive substance is commonly distributed in the mineralized tissues of the crayfish, but may exist in a chemically different form in other tissues.