Thomas H. Shafer

Cuticular proteins from the blue crab alter in vitro calcium carbonate mineralization

Extracellular proteins have long been implicated in the control of mineralization. Cuticular proteins of the blue crab, Callinectes sapidus, undergo changes concurrent with mineralization after molting. A pH drift in vitro mineralization assay was used to assess the effects of cuticular extracts on the inhibition of crystal deposition and the morphology of the resulting crystals. Crystal nucleation was strongly delayed by material extracted into 2% acetic acid within the first 1.5 h post-ecdysis. This effect was not seen with other extracts. No consistent effects of the cuticular extracts on calcium carbonate crystal morphology were observed. Proteins associating with these crystals were extracted and subjected to electrophoresis. These crystal-associated proteins (CAPs) differed depending upon the extraction method and the time after ecdysis when the crab was sacrificed. The early acetic acid extracts produced a unique combination of CAP bands at 66, 42 and 24 kDa. These results are consistent with a model of mineralization in C. sapidus involving (a) certain proteins serving as nucleation sites and attached to the cuticle at all times by acid labile bonds, and (b) other proteins acting as inhibitors of crystal nucleation until being altered at approximately 1 h post-ecdysis.

Postecdysial changes in the protein and glycoprotein composition of the cuticle of the blue crab Callinectes sapidus

ABSTRACT Postecdysial modifications of the cuticle of Callinectes sapidus were evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of soluble protein extracts. Gels stained with Coomassie blue and with silver demonstrated that major changes in protein composition or mobility occurred immediately after ecdysis. Electroblots of SDS-PAGE gels were probed with various biotinylated lectins. The patterns of lectin binding were also markedly different between blots of pre- and postecdysial cuticle extracts, demonstrating a change in glycosylation or glycoprotein mobility in the early postecdysial period. The dramatic alteration in cuticular composition is concurrent with the onset of sclerotization and mineralization of the preexuvially deposited layers of the cuticle.

Purification of a soluble glycoprotein from the uncalcified ecdysial cuticle of the blue crab Callinectes sapidus and its possible role in initial mineralization.

SUMMARY A heavily glycosylated soluble protein was purified using a combination of lectin affinity and size exclusion chromatography from a soluble extract of uncalcified dorsal cuticle of blue crab Callinectes sapidus removed at ecdysis. Similarities in apparent molecular mass and carbohydrate composition suggest that this protein is the same species previously shown to disappear from soluble extracts coincidentally with the onset of mineral deposition in the newly exposed post-molt cuticle. The amino acid sequence of the N-terminal portion of the core polypeptide was determined and polyclonal antibodies were raised against both the purified glycoprotein and the peptide. Immunoblots of unfractionated soluble extracts taken at various times post-molt illustrated that the anti-peptide antibody recognized several polypeptides with electrophoretic mobilities that differ from the purified glycoprotein. These bands may be deglycosylation products which would not have been purified due to different lectin affinity or size. Immunohistochemical analysis indicated uniform protein distribution in the exocuticle at ecdysis, but decreased antibody binding at the interprismatic septa by 2 h post-molt. The location of the protein is therefore the negative image of the calcification pattern in the exocuticle and provides a spatial pattern to correlate with the previously reported temporal events. This strengthens the hypothesis that the glycoprotein under investigation is an inhibitor of calcite nucleation or of initial amorphous calcium carbonate accumulation.

Identifying exoskeleton proteins in the blue crab from an expressed sequence tag (EST) library

A blue crab (Callinectes sapidus) expressed sequence tag project was designed for multiple purposes including discovery of genes for cuticular (exoskeletal) proteins, some of which may regulate mineralization. One of the expression libraries sequenced was from the hypodermis (the epithelium depositing the cuticle). RNAs used for cDNA synthesis were pooled from arthrodial and mid-dorsal hypodermis at both pre-ecdysis and post-ecdysis. This ensured representation from both calcifying and non-calcifying regions and from layers of cuticle deposited both before and after ecdysis. The EST database was mined for cuticular protein sequences in three ways. First, we searched for sequences coding for known cuticle-specific motifs like the Rebers-Riddiford chitin-binding sequence and a motif known only from proteins extracted from mineralized exoskeletons of other decapods. Second, we checked the associated annotations in the EST project for similarity to known cuticular proteins, often from insects. Third, BLAST was used to search the EST data for significant homology to published cuticular protein sequences from other crustaceans. In all, the database contains at least 73 contigs or singlets representing transcripts of cuticular proteins. Forty-five of these distribute among ten clusters of very similar transcripts, possibly representing alternative splicing or recent gene duplications. The rest share less similarity. We have obtained complete sequences for 25 of the transcripts, have produced phylogenetics trees comparing them with similar proteins from insects and other crustaceans, and have determined expression patterns across the molt in calcifying versus non-calcifying cuticle. The combination of homology analysis and gene expression analysis allows us to infer putative functions in cuticle synthesis and calcification.

Glycosidase activity in the post-ecdysial cuticle of the blue crab, Callinectes sapidus

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 post-ecdysis. The present study has identified a glycosidase that appears in the cuticle during the early post-ecdysial 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 (EC 3.2.1.52). Atypical of N-acetylhexosaminidases described to date, this enzyme has a pH optimum of 7.0. The enzyme activity is high during the post-ecdysial 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 affinity chromatography.

Variation in adenylate energy charge and phosphoadenylate pool size in estuarine organisms after an oil spill

Adenylate energy charge (AEC) is the proportion of the total phosphoadenylate pool charged with high-energy bonds. AEC values vary between zero and one by definition. Since AEC can be measured in any organism, decreases might be a universal measure of sublethal environmental stress. In some organisms which maintain high AEC while withstanding natural or anthropogenic stress, the absolute concentration of ATP and the total phosphoadenylate pool (TPP) decrease proportionally. However, in certain organisms the TPP shows dramatic natural fluctuations unrelated to pollution or stress. On 28 June 1983, a tanker spilled approximately 42,000 gallons of number6 diesel oil in the Cape Fear River, North Carolina, USA. Oil covered the tidal marshes on the east side of the river and provided an opportunity to determine if either the AEC or TPP in a variety of organisms would respond to this stress. Five test species were examined as long as one year after the spill. AEC and TPP values of the organisms were compared between contaminated and uncontaminated sites at all seasons. This is the first investigation to monitor AEC in a number of taxonomically distinct estuarine species during an extended period after an oil spill.