Cloning, characterization and functional analysis of an Alveoline-like protein in the shell of Pinctada fucata

Jingjing Kong,Tianpeng Wang,Guilan Zheng,Liping Xie,Rongqing Zhang,Yi Yan,Yan Chen,Dong Yang,Jingliang Huang,Yangjia Liu,Chuang Liu

doi:10.1038/s41598-018-29743-6

Abstract

Shell matrix proteins (SMPs) have important functions in biomineralization. In the past decades, the roles of SMPs were gradually revealed. In 2015, our group identified 72 unique SMPs in Pinctada fucata, among which Alveoline-like (Alv) protein was reported to have homologous genes in Pinctada maxima and Pinctada margaritifera. In this study, the full-length cDNA sequence of Alv and the functional analysis of Alv protein during shell formation were explored. The deduced protein (Alv), which has a molecular mass of 24.9 kDa and an isoelectric point of 11.34, was characterized, and the functional analyses was explored in vivo and in vitro. The Alv gene has high expression in mantle and could response to notching damage. The functional inhibition of Alv protein in vivo by injecting recombinant Alv (rAlv) antibodies destroyed prism structure but accelerated nacre growth. Western blot and immunofluorescence staining showed that native Alv exists in the EDTA-insoluble matrix of both prismatic and nacreous layers and has different distribution patterns in the inner or outer prismatic layer. Taken together, the characterization and functional analyses of matrix protein Alv could expand our understanding of basic matrix proteins and their functions during shell formation.

Highlights

Biomineralization is a process during which minerals are regulated in an organized way by living organisms
We found that Alv mRNA was located in the outer epithelial cells of the outer fold (OF) and outer epithelial cells of the middle fold (MF); no hybridization signals were found in the inner fold (IF) and mantle pallial (MP) (Fig. 4a,b)
The mantle was reported to be important for biomineralization of which the mantle pallial is responsible for the nacreous layer formation and the mantle edge contributes to the prismatic layer formation[6]

Summary

Introduction

Biomineralization is a process during which minerals are regulated in an organized way by living organisms. Only few proteins, that is, the Shematrin family[21], tyrosinase-like protein 224, KRMPs17,18, Prisilkin-3919 and PfN2325, have all been demonstrated to play important roles in the process of shell formation. Alv was proven to have opposite functions in the prismatic and nacreous layers, which is a unique matrix protein with dual roles. Prisilkin-3919, Aspein[29] and MSI3130 play roles in the prismatic layer formation, while Nacrein[31], Pif9732, PfN4420, and N4033 only have important functions in the nacreous layer. Modified immunofluorescence staining has been applied and brought an interesting discovery These results offer insights into the functions of Alv during shell formation and shed light on the formation mechanism of prismatic layer

Methods

Results

Conclusion