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

Yuting Jiang,Huanzhi Xu,Meihua Fan,Zhi Liao,Wang Shen,Xiaolin Zhang,Qi Sun,Linsheng Song

doi:10.1371/journal.pone.0231414

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.

Highlights

Bivalves are a widely spread molluscan class with more than 10 000 species [1], and they are characterized by an ability to build shells of different sizes, forms and structures
Quantitative real-time PCR analyses were performed with three independent replicates using SYBR1 Premix Ex TaqTM (TaKaRa) on an MX3000P Real-Time PCR system (Stratagene, US). qRT-PCR was performed with specific primers derived from the whirlin-like protein (WLP) sequence, including WLP/F (TCCTTCCGTACA GTGGG) and WLP/R (CTGGTTTAGTTTGTGCTCC)
The secondary structure of the WLP is primarily composed of 11% α-helices and 23% β-sheets (Fig 1A), which form a predicted tertiary structure with characteristics similar to the standard conformation of the PDZ domain with five β-strands and two αhelices (Fig 1B)

Summary

Introduction

Bivalves are a widely spread molluscan class with more than 10 000 species [1], and they are characterized by an ability to build shells of different sizes, forms and structures. Bivalve shells are very durable compared with inorganic geological forms and have been important to research in the fields of bioengineering and bionics for dozens of years [2, 3]. Shells are formed by a biologically controlled process, viz., biomineralization, which results in a composite material that is composed of approximately 95% calcium carbonate (aragonite and calcite) and less than 5% organic components (shell matrices) [4]. Jiang et al characterization of a whirlin-like protein from Mytilus coruscus shell

Methods

Results

Conclusion