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First published online June 16, 2004
Journal of Experimental Biology 207, 2589-2598 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.01070
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Purification of a soluble glycoprotein from the uncalcified ecdysial cuticle of the blue crab Callinectes sapidus and its possible role in initial mineralization

Elizabeth P. Tweedie*, Francie E. Coblentz and Thomas H. Shafer{dagger}

Department of Biological Sciences, University of North Carolina at Wilmington, Wilmington, NC, USA



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  		Fig. 1. Chromatography of 0 h protein extract on a jacalin lectin affinity column (A) and subsequent chromatography of the jacalin-bound fraction on a Sephacryl S-200 gel filtration column (B). Arrows indicate peaks containing the glycoprotein of interest, as confirmed by reducing SDS-PAGE.

 


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  		Fig. 2. Two glycoprotein purifications (A,B and C,D) from 0 h cuticle extracts performed identically yet yielded different results. The purified material retains one major Coomassie Blue-stained band but either one (A,B) or two (C,D) broad carbohydrate bands detectable by PAS staining. A and C are SDS-PAGE gels stained with Coomassie Blue for protein. B and D are PVDF membrane blots of similar gels stained with PAS for carbohydrate. In all cases, the first lane contains molecular mass standards (MWS), lane 1 is unfractionated 0 h cuticle protein extract, lane 2 is the Jacalin-positive fraction (Jac+) following lectin affinity chromatography, and lane 3 is the final purified product after gel filtration chromatography of the Jac+ fraction.

 


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  		Fig. 3. Enzyme-treated purified glycoprotein subjected to SDS-PAGE and visualized by PAS staining of electroblotted membranes. (A) Lane 1, untreated glycoprotein; lane 2, chondroitinase-ABC treatment; lane 3, keratanase treatment; lane 4, heparinase II treatment. (B) Lane 1, untreated glycoprotein, lane 2, PNGase-F treatment. See Materials and methods for details.

 


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  		Fig. 4. An immunoblot experiment on unfractionated proteins from 0-h cuticle. Coomassie Blue staining of molecular mass standards (MWS) and cuticle extract (lane 1) are compared with immunoblots of the same extract probed with either anti-glycoprotein (lane 2) or anti-peptide (lane 3) antibodies. Lane 4 is an immunoblot of the purified glycoprotein probed with the anti-peptide antibody. Detection of the chemiluminescent product required 2 min exposure to film for the anti-glycoprotein blot (lane 2) and 30 s exposure for the anti-peptide (lanes 3 and 4).

 


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  		Fig. 5. Coomassie Blue-stained gel (A) and corresponding immunoblot (B) of soluble proteins extracted at ecdysis and at various times immediately thereafter. The immunoblot was probed with antibodies raised against the Edman degradation-derived N-terminal amino acid sequence (anti-peptide). In both A and B, lane 1 contains 0 h extract, lane 2 contains 1 hextract, lane 3 contains 2 h extract, and lane 4 contains 3 h extract. MWS, molecular mass standards (kDa).

 


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  		Fig. 6. Sections of 0 h (A–C), 1 h (D–F), 2 h (G–I) and 3 h (J–L) post-molt cuticle probed with antibodies raised against either the purified glycoprotein (A,D,G,J) or a 15-amino-acid peptide determined by Edman degradation of the purified material (B,E,H,K). Detection was by fluorescent-labeled secondary antibodies. Controls (C,F,I,L) lacking primary antibody show some autofluorescence in the epicuticle (arrowhead) and the hypodermal cells but none in the exocuticle (bracket). All photos were taken at the same light intensity and exposure time and were not digitally altered. Arrows indicate the interprismatic septa (IPS) staining less intensely at the later time periods. Scale bars, 50 µm.

 


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  		Fig. 7. Comparison of staining techniques visualizing the IPS (arrows) in 3 h post-molt exocuticle. (A) Immunohistochemistry using the antibody against the Edman degradation-derived amino acid sequence (anti-peptide). This image has been digitally enhanced to increase the contrast between the `prisms' and the IPS. (B) Toluidine Blue staining showing that the IPS may actually have a higher concentration of total proteins than the prisms, especially near the epicuticle boundary.

 




© The Company of Biologists Ltd 2004