Postecdysial cuticle alteration in the blue crab, Callinectes sapidus: Synchronous changes in glycoproteins and mineral nucleation

Abstract

In order to investigate the control of the onset of postecdysial mineralization of the cuticle of the blue crab, Callinectes sapidus Rathbun, EDTA-soluble proteins and glycoproteins were analyzed at short intervals after ecdysis by SDS-PAGE. Gels were either stained with silver, blotted to polyvinylidene difluoride membranes and stained with periodic acidlschiff reagent (PAS), or blotted to nitrocellulose membranes and probed with a variety of lectins [particularly the mannose-binding lectins concanavalin A (Con A) and GaZanthus niualis agglutinin (GNAjI.Pieces of cuticle were also tested for their ability to nucleate calcite in vitro. A major change occurred in the biochemistry of the cuticle between 1 and 3 hr after ecdysis. This change, which we term the post-ecdysial cuticle alteration (PECA), involved 1) loss of a major 66 kDa glycoprotein band, 2) decrease in Con A binding by a 79 kDa protein band, 3) complete loss of PASreactivity of both 66 and 79 kDa proteins, 4) gain in Con A affinity of an existing band a t 73 kDa, and 5) an increase in the intrinsic ability of cell-free cuticle pieces to mineralize, as measured by the in vitro calcite crystal nucleation assay o 1995 WiIey-Liss, Inc.

Purification of a soluble glycoprotein from the uncalcified ecdysial cuticle of the blue crabCallinectes sapidusand its possible role in initial mineralization

“…Based on the data presented, we speculate that the two previously described PECA bands (Shafer et al, 1995) represent glycosylation variants of the same protein.…”

“…The C. sapidus glycoprotein material previously studied (Shafer et al, 1994(Shafer et al, , 1995Coblentz et al, 1998) was purified from total soluble protein extracts of cuticle from crabs killed at ecdysis. A jacalin lectin affinity column was used to separate glycoproteins with O-linked carbohydrates, which were then separated on the basis of size using a gel filtration column (Fig.·1).…”

“…Based on these results, mucin-type glycans are probably the predominant structural feature of this molecule. However, since the glycoproteins that lose PAS reactivity during the first few hours post-molt do bind Concanavalin A as well as jacalin on lectin blots (Shafer et al, 1995), they presumably contain N-linked as well as O-linked oligosaccharides. The resistance to PNGase-F may be due to the presence of N-linked glycans α1,3-fucosylated on the asparagine-linked N-acetylglucosamine, a configuration found in insect glycoproteins and known to be unaffected by PNGase-F (Altmann et al, 1995(Altmann et al, , 1999.…”

“…A set of modifications of extracellular substances in the dorsal cuticle of C. sapidus termed the post-ecdysial cuticle alteration, or PECA, occurs immediately following ecdysis, in synchrony with a measured increase in the potential for calcite nucleation (Shafer et al, 1994(Shafer et al, , 1995. Specific changes include the loss of major protein bands at 66·kDa and 79·kDa in the first few hours post-ecydsis.…”

“…As little as 1·h after ecdysis, the glycoprotein bands cease to appear on PAS-stained membranes. Probing electroblots with lectins also demonstrated the PECA, as the two glycoproteins stain intensely at ecdysis with concanavalin A and jacalin, but lose their affinity for those lectins postecdysis (Shafer et al, 1995). It is unclear whether proteolysis causes the disappearance of these proteins or if deglycosylation causes the loss of lectin and PAS affinity.…”