Crustacean cuticular structures are key features formed during a molt cycle. These structures are complex biomaterials comprising chitin and different mineral forms in distinct scaffold organizations. The formation of these complex biomaterials is controlled by the organic extracellular matrix including structural proteins. Since cuticular structures are formed de novo during each molt cycle, the spatial and temporal expression patterns of structural proteins are tightly linked to molt cycle events. As a model scenario, we demonstrate the molt-related pattern of expression of the gene encoding GAP65, a core structural protein involved in the formation of the cuticular structures of Cherax quadricarinatus. Based on this typical pattern of expression and using a binary-patterning approach, which is a specialized tool for the study of molt-related proteins, we revealed and characterized additional candidate proteins involved in the formation of crustacean cuticular structures. We propose that our approach be applied as a framework in the search for proteins involved in the formation of the crustacean cuticle. To stimulate research on this important aspect of structural biology, we put forward a schematic representation of the extracellular matrix and its proteins in three cuticular structures of C. quadricarinatus, the gastroliths, the mandibles, and the mineralized cuticle.
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Abehsera, S., L. Glazer, J. Tynyakov, I. Plaschkes, V. Chalifa-Caspi, I. Khalaila, E. D. Aflalo & A. Sagi, 2015. Binary gene expression patterning of the molt cycle: the case of chitin metabolism. PLoS ONE 10(4): e0122602.
Abehsera, S., S. Peles, J. Tynyakov, S. Bentov, E. D. Aflalo, S. Li, F. Li, J. Xiang & A. Sagi, 2017. MARS: a protein family involved in the formation of vertical skeletal elements. Journal of Structural Biology 198(2): 92–102.
Addadi, L. & S. Weiner, 1985. Interactions between acidic proteins and crystals – stereochemical requirements in biomineralization. Proceedings of the National academy of Sciences of the United States of America 82(12): 4110–4114.
Addadi, L., S. Raz & S. Weiner, 2003. Taking advantage of disorder: amorphous calcium carbonate and its roles in biomineralization. Advanced Materials 15(12): 959–970.
Akiva-Tal, A., S. Kababya, Y. S. Balazs, L. Glazer, A. Berman, A. Sagi & A. Schmidt, 2011. In situ molecular NMR picture of bioavailable calcium stabilized as amorphous CaCO3 biomineral in crayfish gastroliths. Proceedings of the National academy of Sciences of the United States of America 108(36): 14763–14768.
Al-Sawalmih, A., C. H. Li, S. Siegel, P. Fratzl & O. Paris, 2009. On the stability of amorphous minerals in lobster cuticle. Advanced Materials 21(40): 4011–4015.
Becker, A., A. Ziegler & M. Epple, 2005. The mineral phase in the cuticles of two species of Crustacea consists of magnesium calcite, amorphous calcium carbonate, and amorphous calcium phosphate. Dalton Transactions 10: 1814–1820.
Behr, M. & M. Hoch, 2005. Identification of the novel evolutionary conserved obstructor multigene family in invertebrates. FEBS Letters 579(30): 6827–6833.
Bentov, S., P. Zaslansky, A. Al-Sawalmih, A. Masic, P. Fratzl, A. Sagi, A. Berman & B. Aichmayer, 2012. Enamel-like apatite crown covering amorphous mineral in a crayfish mandible. Nature Communications 3: 839.
Bentov, S., E. D. Aflalo, J. Tynyakov, L. Glazer & A. Sagi, 2016. Calcium phosphate mineralization is widely applied in crustacean mandibles. Scientific Reports 6: 22118.
Buchholz, F., 1989. Molt cycle and seasonal activities of chitinolytic enzymes in the integument and digestive-tract of the antarctic krill, euphausia-superba. Polar Biology 9(5): 311–317.
Charles, J. P., H. Bouhin, B. Quennedey, A. Courrent & J. Delachambre, 1992. cDNA cloning and deduced amino acid sequence of a major, glycine-rich cuticular protein from the coleopteran Tenebrio molitor. The FEBS Journal 206(3): 813–819.
Cho, H. J., H. J. Cho & H. S. Kim, 2009. Osteopontin: a multifunctional protein at the crossroads of inflammation, atherosclerosis, and vascular calcification. Current Atherosclerosis Reports 11(3): 206–213.
Compere, P., M. F. Jaspar-Versali & G. Goffinet, 2002. Glycoproteins from the cuticle of the Atlantic shore crab Carcinus maenas: I. Electrophoresis and western-blot analysis by use of lectins. Biological Bulletin 202(1): 61–73.
Ding, D., P. A. Guerette, S. Hoon, K. W. Kong, T. Cornvik, M. Nilsson, A. Kumar, J. Lescar & A. Miserez, 2014. Biomimetic production of silk-like recombinant squid sucker ring teeth proteins. Biomacromolecules 15(9): 3278–3289.
Dosztanyi, Z., V. Csizmok, P. Tompa & I. Simon, 2005. IUPred: web server for the prediction of intrinsically unstructured regions of proteins based on estimated energy content. Bioinformatics 21(16): 3433–3434.
Dyson, H. J. & P. E. Wright, 2002. Coupling of folding and binding for unstructured proteins. Current Opinion in Structural Biology 12(1): 54–60.
Endo, H., P. Persson & T. Watanabe, 2000. Molecular cloning of the crustacean DD4 cDNA encoding a Ca2 + -binding protein. Biochemical and Biophysical Research Communications 276(1): 286–291.
Faircloth, L. M. & T. H. Shafer, 2007. Differential expression of eight transcripts and their roles in the cuticle of the blue crab, Callinectes sapidus. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology 146(3): 370–383.
Fisher, L. W., D. A. Torchia, B. Fohr, M. F. Young & N. S. Fedarko, 2001. Flexible structures of SIBLING proteins, bone sialoprotein, and osteopontin. Biochemical and Biophysical Research Communications 280(2): 460–465.
Gasteiger, E., C. Hoogland, A. Gattiker, S. E. Duvaud, M. R. Wilkins, R. D. Appel & A. Bairoch, 2005. Protein identification and analysis tools on the ExPASy server. Springer, New York.
Glazer, L. & A. Sagi, 2012. On the involvement of proteins in the assembly of the crayfish gastrolith extracellular matrix. Invertebrate Reproduction & Development 56(1): 57–65.
Glazer, L., A. Shechter, M. Tom, Y. Yudkovski, S. Weil, E. D. Aflalo, R. R. Pamuru, I. Khalaila, S. Bentov, A. Berman & A. Sagi, 2010. A protein involved in the assembly of an extracellular calcium storage matrix. The Journal of Biological Chemistry 285(17): 12831–12839.
Glazer, L., Z. Roth, S. Weil, E. D. Aflalo, I. Khalaila & A. Sagi, 2015. Proteomic analysis of the crayfish gastrolith chitinous extracellular matrix reveals putative protein complexes and a central role for GAP 65. Journal of Proteomics 128: 333–343.
Guerette, P. A., S. Hoon, D. Ding, S. Amini, A. Masic, V. Ravi, B. Venkatesh, J. C. Weaver & A. Miserez, 2014. Nanoconfined beta-sheets mechanically reinforce the supra-biomolecular network of robust squid Sucker Ring Teeth. ACS Nano 8(7): 7170–7179.
Habraken, W. J. E. M., A. Masic, L. Bertinetti, A. Al-Sawalmih, L. Glazer, S. Bentov, P. Fratzl, A. Sagi, B. Aichmayer & A. Berman, 2015. Layered growth of crayfish gastrolith: about the stability of amorphous calcium carbonate and role of additives. Journal of Structural Biology 189(1): 28–36.
Hild, S., O. Marti & A. Ziegler, 2008. Spatial distribution of calcite and amorphous calcium carbonate in the cuticle of the terrestrial crustaceans Porcellio scaber and Armadillidium vulgare. Journal of Structural Biology 163(1): 100–108.
Huber, J., H. O. Fabritius, E. Griesshaber & A. Ziegler, 2014. Function-related adaptations of ultrastructure, mineral phase distribution and mechanical properties in the incisive cuticle of mandibles of Porcellio scaber Latreille, 1804. Journal of Structural Biology 188(1): 1–15.
Iijima, M., T. Hashimoto, Y. Matsuda, T. Nagai, Y. Yamano, T. Ichi, T. Osaki & S. I. Kawabata, 2005. Comprehensive sequence analysis of horseshoe crab cuticular proteins and their involvement in transglutaminase-dependent cross-linking. The FEBS Journal 272(18): 4774–4786.
Inoue, H., N. Yuasa-Hashimoto, M. Suzuki & H. Nagasawa, 2008. Structural determination and functional analysis of a soluble matrix protein associated with calcification of the exoskeleton of the crayfish, Procambarus clarkii. Bioscience, Biotechnology, and Biochemistry 72(10): 2697–2707.
Ishii, K., T. Yanagisawa & H. Nagasawa, 1996. Characterization of a matrix protein in the gastroliths of the crayfish Procambarus clarkii. Bioscience, Biotechnology, and Biochemistry 60(9): 1479–1482.
Kragh, M., L. Molbak & S. O. Andersen, 1997. Cuticular proteins from the lobster, Homarus americanus. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology 118(1): 147–154.
Kuballa, A. V., D. J. Merritt & A. Elizur, 2007. Gene expression profiling of cuticular proteins across the moult cycle of the crab Portunus pelagicus. BMC Biology 5(1): 45.
Lowenstam, H. A. & S. Weiner, 1989. On biomineralization. Oxford University Press, Oxford.
Luquet, G., 2012. Biomineralizations: insights and prospects from crustaceans. Zookeys 176: 103–121.
Luquet, G., Y. Dauphin, A. Percot, M. Salome, A. Ziegler, M. S. Fernandez & J. L. Arias, 2016. Calcium deposits in the crayfish, Cherax quadricarinatus: microstructure versus elemental distribution. Microscopy and Microanalysis 22(1): 22–38.
Marin, F., G. Luquet, B. Marie & D. Medakovic, 2007. Molluscan shell proteins: primary structure, origin, and evolution. Current Topics in Developmental Biology 80: 209–276.
Marsh, J. J. & H. G. Lebherz, 1992. Fructose-bisphosphate aldolases: an evolutionary history. Trends in Biochemical Sciences 17(3): 110–113.
Merzendorfer, H. & L. Zimoch, 2003. Chitin metabolism in insects: structure, function and regulation of chitin synthases and chitinases. Journal of Experimental Biology 206(Pt 24): 4393–4412.
Patino, M. G., M. E. Neiders, S. Andreana, B. Noble & R. E. Cohen, 2002. Collagen: an overview. Implant Dent 11(3): 280–285.
Pesch, Y. Y., D. Riedel & M. Behr, 2015. Obstructor a organizes matrix assembly at the apical cell surface to promote enzymatic cuticle maturation in drosophila. Journal of Biological Chemistry 290(16): 10071–10082.
Petersen, T. N., S. Brunak, G. von Heijne & H. Nielsen, 2011. SignalP 4.0: discriminating signal peptides from transmembrane regions. Nature Methods 8(10): 785–786.
Rebers, J. E. & L. M. Riddiford, 1988. Structure and expression of a Manduca sexta larval cuticle gene homologous to Drosophila cuticle genes. Journal of Molecular Biology 203(2): 411–423.
Rebers, J. E. & J. H. Willis, 2001. A conserved domain in arthropod cuticular proteins binds chitin. Insect Biochemistry and Molecular Biology 31(11): 1083–1093.
Ringli, C., B. Keller & U. Ryser, 2001. Glycine-rich proteins as structural components of plant cell walls. Cellular and Molecular Life Sciences 58(10): 1430–1441.
Roer, R. & R. Dillaman, 1984. The structure and calcification of the crustacean cuticle. American Zoologist 24(4): 893–909.
Roer, R., S. Abehsera & A. Sagi, 2015. Exoskeletons across the Pancrustacea: comparative morphology, physiology, biochemistry and genetics. Integrative and Comparative Biology 55(5): 771–791.
Roth, Z., S. Parnes, S. Wiel, A. Sagi, N. Zmora, J. S. Chung & I. Khalaila, 2010. N-glycan moieties of the crustacean egg yolk protein and their glycosylation sites. Glycoconjugate Journal 27(1): 159–169.
Sato, A., S. Nagasaka, K. Furihata, S. Nagata, I. Arai, K. Saruwatari, T. Kogure, S. Sakuda & H. Nagasawa, 2011. Glycolytic intermediates induce amorphous calcium carbonate formation in crustaceans. Nature Chemical Biology 7(4): 197–199.
Shechter, A., M. Tom, Y. Yudkovski, S. Weil, S. A. Chang, E. S. Chang, V. Chalifa-Caspi, A. Berman & A. Sagi, 2007. Search for hepatopancreatic ecdysteroid-responsive genes during the crayfish molt cycle: from a single gene to multigenicity. Journal of Experimental Biology 210(Pt 20): 3525–3537.
Shechter, A., A. Berman, A. Singer, A. Freiman, M. Grinstein, J. Erez, E. D. Aflalo & A. Sagi, 2008a. Reciprocal changes in calcification of the gastrolith and cuticle during the molt cycle of the red claw crayfish Cherax quadricarinatus. The Biological Bulletin 214(2): 122–134.
Shechter, A., L. Glazer, S. Cheled, E. Mor, S. Weil, A. Berman, S. Bentov, E. D. Aflalo, I. Khalaila & A. Sagi, 2008b. A gastrolith protein serving a dual role in the formation of an amorphous mineral containing extracellular matrix. Proceedings of the National Academy of Sciences United States of America 105(20): 7129–7134.
Shechter, A., L. Glazer, S. Cheled, E. Mor, S. Weil, A. Berman, S. Bentov, E. D. Aflalo, I. Khalaila & A. Sagi, 2008c. A gastrolith protein serving a dual role in the formation of an amorphous mineral containing extracellular matrix. Proceedings of the National Academy of Sciences of the United States of America 105(20): 7129–7134.
Shibata, T., S. Ariki, N. Shinzawa, R. Miyaji, H. Suyama, M. Sako, N. Inomata, T. Koshiba, H. Kanuka & S. Kawabata, 2010. Protein crosslinking by transglutaminase controls cuticle morphogenesis in Drosophila. PLoS ONE 5(10): e13477.
Simkiss, K. & K. M. Wilbur, 2012. Biomineralization. Elsevier, Amsterdam.
Tellam, R. L., T. Vuocolo, S. E. Johnson, J. Jarmey & R. D. Pearson, 2000. Insect chitin synthase cDNA sequence, gene organization and expression. European Journal of Biochemistry 267(19): 6025–6043.
Travis, D. F., 1963. The deposition of skeletal structures in the crustacea. 2. The histochemical changes associated with the development of the nonmineralized skeletal components of the gastrolith discs of the crayfish, Orconectes virilis hagen. Acta Histochemica 15: 251–268.
Tweedie, E. P., F. E. Coblentz & T. H. Shafer, 2004. Purification of a soluble glycoprotein from the uncalcified ecdysial cuticle of the blue crab Callinectes sapidus and its possible role in initial mineralization. Journal of Experimental Biology 207(Pt 15): 2589–2598.
Tynyakov, J., S. Bentov, S. Abehsera, I. Khalaila, R. Manor, A. L. Katzir, S. Weil, E. Aflalo & A. Sagi, 2015a. A novel chitin binding crayfish molar tooth protein with elasticity properties. PLoS ONE 10(5): e0127871.
Tynyakov, J., S. Bentov, S. Abehsera, G. Yehezkel, Z. Roth, I. Khalaila, S. Weil, A. Berman, I. Plaschkes, M. Tom, E. D. Aflalo & A. Sagi, 2015b. A crayfish molar tooth protein with putative mineralized exoskeletal chitinous matrix properties. Journal of Experimental Biology 218(Pt 21): 3487–3498.
Tynyakov, J., S. Bentov, S. Abehsera, G. Yehezkel, Z. Roth, I. Khalaila, S. Weil, A. Berman, I. Plaschkes, M. Tom, E. D. Aflalo & A. Sagi, 2015c. A crayfish molar tooth protein with putative mineralized exoskeletal chitinous matrix properties. Journal of Experimental Biology 218(Pt 21): 3487–3498.
Ueno, M., 1980. Calcium transport in crayfish gastrolith disc: morphology of gastrolith disc and ultrahistochemical demonstration of calcium. Journal of Experimental Zoology 213(2): 161–171.
Ueno, M. & V. Mizuhira, 1984. Calcium transport mechanism in crayfish gastrolith epithelium correlated with the molting cycle. II. Cytochemical demonstration of Ca2+-ATPase and Mg2+ -ATPase. Histochemistry 80(3): 213–217.
Vatcher, H. E., R. D. Roer & R. M. Dillaman, 2015. Structure, molting, and mineralization of the dorsal ossicle complex in the gastric mill of the blue crab, Callinectes sapidus. Journal of Morphology 276(11): 1358–1367.
Vincent, K. & M. C. Durrant, 2013. A structural and functional model for human bone sialoprotein. Journal of Molecular Graphics and Modelling 39: 108–117.
Vuzman, D. & Y. Levy, 2012. Intrinsically disordered regions as affinity tuners in protein-DNA interactions. Molecular BioSystems 8(1): 47–57.
Weiner, S. & P. M. Dove, 2003. An overview of biomineralization processes and the problem of the vital effect. Reviews in Mineralogy and Geochemistry 54(1): 1–29.
Welinder, B. S., 1974. The crustacean cuticle – I. Studies on the composition of the cuticle. Comparative Biochemistry and Physiology Part A, Physiology 47(2): 779–787.
Wynn, A. & T. H. Shafer, 2005. Four differentially expressed cDNAs in Callinectes sapidus containing the Rebers-Riddiford consensus sequence. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology 141(3): 294–306.
Yan, Z., Z. Fang, Z. Ma, J. Deng, S. Li, L. Xie & R. Zhang, 2007. Biomineralization: functions of calmodulin-like protein in the shell formation of pearl oyster. Biochimica et Biophysica Acta 1770(9): 1338–1344.
Zhong, Y. S., K. Mita, T. Shimada & H. Kawasaki, 2006. Glycine-rich protein genes, which encode a major component of the cuticle, have different developmental profiles from other cuticle protein genes in Bombyx mori. Insect Biochemistry and Molecular Biology 36(2): 99–110.
This research was supported by the Israel Science Foundation (Grant No. 613/13) and the National Institute for Biotechnology in the Negev (NIBN).
Guest editors: Guiomar Rotllant, Ferran Palero, Peter Mather, Heather Bracken-Grissom & Begoña Santos / Crustacean Genomics
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Abehsera, S., Weil, S., Manor, R. et al. The search for proteins involved in the formation of crustacean cuticular structures. Hydrobiologia 825, 29–45 (2018). https://doi.org/10.1007/s10750-018-3684-y
- Chitinous scaffold
- Crustacean cuticle
- Extracellular matrix
- Molt cycle
- Structural proteins
- Transcript binary patterning