A shell matrix protein of Pinctada mazatlanica produces nacre platelets in vitro

Crisalejandra Rivera-Perez,Josafat Jehu Ojeda Ramírez De Areyano,Norma Y Hernández-Saavedra,Iliana Alejandra Flores-Sánchez,Delia Irene Rojas Posadas

doi:10.1038/s41598-020-77320-7

Crisalejandra Rivera-Perez, Josafat Jehu Ojeda Ramírez De Areyano + Show 3 more

Open Access

https://doi.org/10.1038/s41598-020-77320-7

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Abstract

Nacre is the main component of the pearl oyster shells and it is synthesized by specialized soluble and insoluble shell matrix proteins. Insoluble proteins from the decalcification of the shell are the less studied proteins due to the technical problems to isolate them from the organic matrix. In this study, an insoluble shell matrix protein from Pinctada mazatlanica, pearlin (Pmaz-pearlin), was successfully cloned from the mantle tissue, and the native protein isolated from the shell was functionally characterized. The full coding sequence of Pmaz-pearlin mRNA consists of 423 base pairs, which encode to a 16.3 kDa pearlin. Analysis of the deduced amino acid sequence revealed that Pmaz-pearlin contained four acidic regions, an NG repeat domain, and Cys conserved residues, the latter potentially forms four disulfide bridges which might stabilize the protein structure. The isolated protein from the shell is a glycoprotein of ~ 16.74 kDa which can produce aragonite and calcite crystals in vitro. Our results show that Pmaz-pearlin is a well-conserved protein involved in nacre layer growth, which produces calcite crystals in the presence of CaCl2, aragonite crystal polymorphs with a hexagonal structure in the presence of MgCl2, and needle-like crystal structure polymorphs in the presence of CaCO3 The identity of the crystals was confirmed using RAMAN analyses.

Highlights

Nacre is the main component of the pearl oyster shells and it is synthesized by specialized soluble and insoluble shell matrix proteins
These structures are made by specialized proteins known as shell matrix proteins (SMPs), which are synthesized by the epithelial cells from the mantle of mollusks[1] and released into the extrapallial space, between the mantle and the shell, where they perform the crystal nucleation, crystal growth and crystal r egulation[2]
The SMPs have been classified according to their solubility after shell decalcification with EDTA or acetic acid solutions as soluble acidic matrix (Asp-rich proteins) and insoluble framework matrix proteins (Gly and Ala-rich proteins), the latter is mostly composed by chitin and s ilk[3,4]

Summary

Results

Sequence analysis of cDNA pearlin from the mantle of P. mazatlanica. The full-length Pmazpearlin was composed of a 726-bp including 5′-UTR and 3′-UTR (292 bp), and an open reading frame (ORF) of 423 bp encoding 140 amino acids (Fig. 1). Analysis of the Pmaz-pearlin sequence revealed the presence of several characteristic domains of the low molecular weight protein family from the shell matrix, including four acidic rich regions (AR1, residues 41–50; AR2, residues 68–73; AR3, residues 89–91 and AR4, residues 132–139), ten conserved cysteine residues (Cys18, Cys30, Cys36, Cys54, Cys55, Cys57, Cys66, Cys77, Cys[85], and Cys86) and an NG domain (N92-G111) (Fig. 2A). All the protein sequences contained a signal peptide, four aspartic residues (AR1-4), ten conserved Cys residues and the NG domain, the length differ among them, being pearlin from P. mazatlanica, P. margaritifera and N14 from P. maxima the longest from all the sequences analyzed, with 20 amino acid residues (Fig. 2A). Aragonite displays a Raman spectrum at 206.1 cm−1, crystal, contains four CaCO3 units, for 702.595 cm−1, and 1085.16 cm−1 with a total of twenty atoms, space group D21h6(Pnma) and cell parameters of 5.008, 8.029, and 5.861 Å for the a, b, and c axes, respectively[33]

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