HALBROOK, K.E.*; ROER, R.D.; SHAFER, T.H.; PIERCE, D.C.; BUTLER, K.D.: Partial purification of a postmolt cuticular glycosidase from Callinectes sapidus and its putative role in mineralization

We have previously demonstrated a marked change in sugar moieties of glycoproteins of the cuticle of the blue crab, Callinectes sapidus, between 0.5 and 3 h postecdysis. The present study has identified a glycosidase that appears in the cuticle during the early postecdysial hours. The enzyme has affinities for p-nitrophenyl derivatives of both N-acetylglucosamine and N-acetylgalactosamine. Both activities are competitively inhibited by chitobiose, suggesting that the enzyme could be a N-acetylhexosaminidase (HexNAcase). Atypical of HexNAcases described to date, this enzyme has a pH optimum of 7.0. The enzyme activity is high during the postecdysial period coincident with the changes in glycoprotein profiles observed in vivo. Partial purification of the enzyme has been accomplished by Sephacryl size-exclusion chromatography followed by Concanavalin A (ConA) affinity chromatography. To test if a HexNAcase might be the causative agent in the alteration of the glycans and initiation of calcification, newly molted crab cuticle was treated with exogenous HexNAcase. Treating cuticular extracts from crabs at 0 h postecdysis with exogenous HexNAcase mimicked those changes observed in vivo. Specifically, the enzyme decreased the ConA affinity of an 83 kDa glycoprotein that binds to calcite crystals in vitro. Treating pieces of 0 h postecdysial cuticle with HexNAcase rendered them capable of nucleating calcite in vitro (similar to 5 h postecdysial cuticle), while untreated, 0 h controls remained uncalcified. The data imply a role of the cuticular HexNAcase-like enzyme in the initiation of calcite nucleation in the newly formed exoskeleton.