ROER, R.D.*; SHAFER, T.H.; TOWLE, D.W.; POWELL, K.H.; Univ. of N.C. at Wilmington; Univ. of N.C. at Wilmington; Mount Desert Island Biological Laboratory; Univ. of N.C. at Wilmington: Localization and partial sequence of a postmolt cuticular glycosidase from Callinectes sapidus

We previously demonstrated that a glycosidase appears in the calcifying cuticle during the early postecdysial hours. The enzyme is a putative N-acetylhexosaminidase (HexNAcase) and has affinities for p-nitrophenyl derivatives of both N-acetylglucosamine and N-acetylgalactosamine. The possible role of the HexNAcase in mineralization was previously described. These data are supported by the observation that the enzyme activity in postmolt calcifying cuticle is twice that in non-calcifying arthrodial cuticle. The enzyme was partially purified by Sephacryl size-exclusion chromatography followed by Concanavalin A affinity chromatography, but attempts to obtain an amino acid sequence failed. Consequently, an antibody was produced using a consensus sequence from Penaeus japonicus cuticular chitinase (Pjchi-2; Watanabe & Kono, 1997). The antibody inhibited the HexNAcase activity in vitro, and bound to cuticular sections in a pattern similar to that seen in histochemical localization of enzyme activity. A degenerate primer was constructed using part of the same consensus sequence and used for 3′-RACE in an attempt to amplify the cDNA for the HexNAcase. One prominent band appeared on agarose electrophoresis of the PCR product. The resulting sequence showed some similarity to parts of the Penaeus chitinase.