TWEEDIE, E.P.*; SHAFER, T.H.: Purification and characterization of mucin-like glycoproteins from the cuticle of the blue crab Callinectes sapidus.
Two molecular species previously identified as electrophoretic bands at 66 and 79 kD in extracts from dorsal carpace cuticle disappear within a few hours after the molt in the blue crab, Callinectes sapidus. Disappearance of these bands occurs coincidentally with the onset of initial mineral deposition in the new cuticle exposed by molting. Identified as heavily glycosylated, mucin-like proteins, these two molecular species have been purified to homogeneity by jacalin lectin affinity and gel filtration chromatography columns and SDS-PAGE. The species contain both N- and O-glycosidic linkages, as indicated by PNGase electrophoretic band shifts, as well as affinity for jacalin and concanavalin A lectins. Polyclonal antibodies raised against the purified glycoproteins were used in Western blot and immunohistochemical analyses of early postmolt cuticle. Western blots revealed similar post-ecdysial cuticle alteration (PECA) changes as those recorded in previous research; namely, a dramatic electrophoretic band shift at one to two hours postmolt. Although antibodies demonstrated an affinity for epicuticular material in fluorescence-assisted histochemistry, they were not specific enough to distinguish or localize the mucin-like proteins in cuticle sections. Amino acid sequencing is being undertaken in order to raise more specific antibodies against epitopes in the core polypeptide.