Molecular characterization of a whirlin-like protein with biomineralization-related functions from the shell of Mytilus coruscus

Abstract

Mollusc shells are produced from calcified skeletons and have excellent mechanical properties. Shell matrix proteins (SMPs) have important functions in shell formation. A 16.6 kDa whirlin-like protein (WLP) with a PDZ domain was identified in the shell of Mytilus coruscus as a novel SMP. In this study, the expression, function, and location of WLP were analysed. The WLP gene was highly expressed and specifically located in the adductor muscle and mantle. The expression of recombinant WLP (rWLP) was associated with morphological change, polymorphic change, binding ability, and crystallization rate inhibition of the calcium carbonate crystals in vitro. In addition, an anti-rWLP antibody was prepared, and the results from immunohistochemistry and immunofluorescence analyses revealed the specific location of the WLP in the mantle, adductor muscle, and myostracum layer of the shell, suggesting multiple functions for WLP in biomineralization, muscle-shell attachment, and muscle attraction. Furthermore, results from a pull-down analysis revealed 10 protein partners of WLP in the shell matrices and a possible network of interacting WLPs in the shell. In addition, in this study, one of the WLP partners, actin, was confirmed to have the ability to bind WLP. These results expand the understanding of the functions of PDZ-domain-containing proteins in biomineralization and provide clues for determining the mechanisms of myostracum formation and muscle-shell attachment. OPEN ACCESS Citation: Jiang Y, Sun Q, Fan M, Zhang X, Shen W, Xu H, et al. (2020) Molecular characterization of a whirlin-like protein with biomineralization-related functions from the shell of Mytilus coruscus. PLoS ONE 15(4): e0231414. https://doi.org/10.

Recombinant transgelin‐like protein 1 fromMytilusshell induces formation of CaCO3polymorphic crystalsin vitro

“…According to the protocol of a previous study [33], quantitative real-time PCR (qRT-PCR) was used to evaluate the expression level of TLP-1 in various tissues. Briefly, total RNA was extracted from various tissues (mantle, adductor muscle, foot, gill, blood and gonad) by TRIzol reagent.…”

“…The localization of TLP-1 mRNA expressed in the mantle and adductor muscle was determined by in situ hybridization using a previously reported protocol [33,34]. The fixed Fig.…”

“…The effects of rTLP-1 on the morphology of CaCO 3 crystals (calcite and aragonite) were tested by in vitro crystal growth experiments [33,45]. Briefly, rTLP-1 was incubated with a freshly prepared saturated solution of calcium carbonate [46] on a siliconized cover glass with MgCl 2 (for the preparation of aragonite) or without MgCl 2 (for the preparation of calcite).…”

“…The morphologies and polymorphs of the CaCO 3 crystals were observed by a Nova Nano 450 (FEI) scanning electron microscope (SEM) and FTIR spectroscopy (Nicolet Nexus 670, Thermo Nicolet Corporation, Madison, WI, USA), respectively. The interaction between rTLP-1 and CaCO 3 crystals was tested by SDS/PAGE according to a previously reported protocol [33,46]. Briefly, 1 mgÁmL À1 rTLP-1 was dissolved in water (sample I) and then incubated with calcite or aragonite crystals at room temperature for 2 h. After centrifugation (10 000 g for 15 min), the supernatants were collected as sample II.…”

“…The inhibition calcium carbonate precipitation by rTLP-1 was tested using a previously reported protocol [33,45]. Briefly, 10 mM CaCl 2 containing rTLP-1 at various concentrations (10,30, and 50 lgÁmL À1 ) was dropped onto a 96well plate, and the plate was placed in a closed desiccator.…”