PROTEINS OF CRUSTACEAN EXOSKELETON .3. GLYCOPROTEINS IN THE BERMUDA LAND CRAB GECARCINUS-LATERALIS

Chitin and protein are major components of crustacean exoskeletons. In insect cuticles, glycosylation is one of the predominant post-transla tional modifications of the proteins. Investigations of glycosylation of crustacean exoskeletal proteins are much more limited. We have used lectins to follow changes in glycosylation of proteins in the exoskel etal layers of the Bermuda land crab Gecarcinus lateralis at selected stages of the intermolt cycle. Proteins extracted from the individual layers during anecdysis and late proecdysis as well as from the layers of exuviae were electrophoresed on sodium dodecyl sulfate (SDS)-polya crylamide gels, blotted to polyvinylidene difluoride membranes, and re acted with biotinylated lectins. Seven lectins were tested: concanaval in A (Con A), soybean agglutinin (SEA), wheat germ agglutinin (WGA), D olichos biflorus agglutinin (DBA), Ulex europaeus agglutinin (UEA I), Ricinus communis agglutinin (RCA(120)), and peanut agglutinin (PNA). C on A, which binds primarily to mannose residues, produced the stronges t signals. During anecdysis, most of the binding occurred with protein bands larger than 31 kDa, while in late proecdysis and in layers from exuviae, small as well as large proteins were bound. N-glycosidase F digestion of electrotransferred proteins and subsequent treatment with Con A indicated both N-linked and O-linked glycosylation. SEA, which binds to both alpha- and beta-linked N-acetylgalactosamine, was the se cond most reactive lectin, and the number of exoskeletal proteins to w hich it bound also increased in late proecdysis. Little or no binding of DBA, RCA(120), or PNA, which react with alpha-linked N-acetylgalact osamine, indicated that the N-acetylgalactosamine was beta-linked. The specificity of binding of Con A or SEA was demonstrated by inhibition with methyl-alpha-D-mannopyranoside and N-acetyl-D-galactosamine, res pectively. Binding of UEA I revealed the presence of fucose residues o n a few proteins. As the exoskeleton was degraded during proecdysis, p rotein bands that were not detected by lectin binding during anecdysis were modified in such a way that they became accessible to lectins. ( C) 1995 Wiley-Liss, Inc.