Cloning and Characterization of Prisilkin-39, a Novel Matrix Protein Serving a Dual Role in the Prismatic Layer Formation from the Oyster Pinctada fucata

Abstract

Molluscs form their shells out of CaCO 3 and a matrix of biomacromolecules. Understanding the role of matrices may shed some light on the mechanism of biomineralization. Here, a 1401-bp full-length cDNA sequence encoding a novel matrix protein was cloned from the mantle of the bivalve oyster, Pinctada fucata. The deduced protein (Prisilkin-39), which has a molecular mass of 39.3 kDa and an isoelectric point of 8.83, was fully characterized, and its role in biomineralization was demonstrated using both in vivo and in vitro crystal growth assays. Prisilkin-39 is a highly repetitive protein with an unusual composition of Gly, Tyr, and Ser residues. Expression of Prisilkin-39 was localized to columnar epithelial cells of the mantle edge, corresponding to the calcitic prismatic layer formation. Immunostaining in situ and immunodetection in vitro revealed the presence of a characteristic pattern of Prisilkin-39 in the organic sheet and in sheaths around the prisms. Prisilkin-39 binds tightly with chitin, an insoluble polysaccharide that forms the highly structured framework of the shell. Antibody injection in vivo resulted in dramatic morphological deformities in the inner shell surface structure, where large amounts of CaCO 3 were deposited in an uncontrolled manner. Moreover, Prisilkin-39 strictly prohibited the precipitation of aragonite in vitro. Taken together, Prisilkin-39 is the first protein shown to have dual function, involved both in the chitinous framework building and in crystal growth regulation during the prismatic layer mineralization. These observations may extend our view on the rare group of basic matrices and their functions during elaboration of the molluscan shell.

α2β1 integrin-mediated mechanical signals during osteodifferentiation of stem cells from the Wharton’s jelly of the umbilical cord

“…Subsequently, the membrane was washed with TBST and incubated with www.fhc.viamedica.pl a secondary antibody coupled to horseradish peroxidase (DAKO, Glostrup, Denmark), diluted in TBST as per the manufacturer's instructions. The signal of the secondary antibody was detected by chemiluminescence (using the ECL detection kit, Amersham), followed by autoradiography [14]. Films were quantified using IMAGE-Quant version 5.2 (Molecular Dynamic, Sunnyvale, CA, USA).…”

Novel Molluskan Biomineralization Proteins Retrieved from Proteomics: A Case Study with Upsalin

“…Unlike other biomineralization transcripts that are exclusively expressed in the mantle, [13,36] the expression of Upsalin is particularly high in the mantle and adductor muscles and it is also faintly detectable in the gills and foot. A similar situation has been observed previously with some members of the shematrin family and with the methionine-rich protein MRNP-34, [25] which are thought to provide frameworks for calcification.…”

Erosion of the prismatic layer by the organic matrix during the formation of the nacre-prism transition layer in the shell of Pinctada fucata (Bivalvia, Mollusca)

“…Pif, a nacre-specific protein, had been shown to exist in the nacre-prism transition layer [18]. A prismatic layer-specific protein, Prisilikin-39, also exists in the transition layer [19]. A study by Dauphin et al of the structure and composition of the nacre-prism transition layer also indicated that these layers are not sharply defined from each other [20].…”