title: Purification of a soluble glycoprotein from the uncalcified ecdysial cuticle of the blue crab Callinectes sapidus and its possible role in initial mineralization.
abstract: 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.
authors.affiliation: Department of Biological Sciences, University of North Carolina at Wilmington, Wilmington, NC, USA.
authors.forename: Elizabeth P
authors.initials: EP
authors.lastname: Tweedie
authors.forename: Francie E
authors.initials: FE
authors.lastname: Coblentz
authors.forename: Thomas H
authors.initials: TH
authors.lastname: Shafer
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003386:Brachyura / Q000378:metabolism* / Q000502:physiology; D002113:Calcification, Physiologic / Q000502:physiology; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000235:genetics / Q000302:isolation & purification*; D015151:Immunoblotting; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D018978:Molting / Q000502:physiology*; D009943:Organophosphorus Compounds; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D020539:Sequence Analysis, Protein
chemical_list: C043517:2-(4-isothiocyanatophenoxy)-1,3,2-dioxaphosphinene 2-oxide; D006023:Glycoproteins; D009943:Organophosphorus Compounds; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase