Electroextraction of Insoluble Proteins from the Organic Matrix of the Nacreous Layer of the Japanese Pearl Oyster, Pinctada fucata.

Daisuke Funabara,Satoshi Kanoh,Kaoru Maeyama,Kiyohito Nagai,Natsumi Miyashita

doi:10.3390/mps2020037

Daisuke Funabara, Satoshi Kanoh + Show 3 more

Open Access

https://doi.org/10.3390/mps2020037

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Abstract

The nacreous layer of shells and pearls is composed of aragonite crystals arranged in an organic matrix. The organic matrix contains chitin and several proteins that regulate the formation of the nacreous layer. Owing to their strong interactions in the organic matrix, the current method for extraction of insoluble proteins from the pre-powdered nacreous layer involves heating to high temperatures in the presence of a detergent (e.g., sodium dodecyl sulfate, SDS) and reductant (e.g., dithiothreitol, DTT), which is likely to induce protein degradation. Therefore, we have developed an electroextraction method to isolate proteins from the organic matrix of a nacreous organic sheet, that was obtained following the decalcification of shells in their original shape. Our electroextraction method employs milder conditions without heating or detergent. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns of the electro-extracted proteins (EEPs) under non-reduced and reduced conditions revealed that this method yielded a greater number of different proteins compared with the conventional extraction method and the isolated EEPs retained their disulfide bonds. Our method is able to easily extract insoluble proteins from the nacreous layer under mild conditions and will undoubtedly aid future analyses into the functions of the nacreous layer proteins.

Highlights

The Japanese pearl oyster, Pinctada fucata, is one of the most important molluscan species in the pearl culture industry
Nacreous layers are repeatedly secreted on the surface of the nucleus that eventually results in the formation of a pearl [1]
Formation of the nacreous layer in P. fucata shells is thought to be regulated by several types of proteins that are secreted from the mantle tissue

Summary

Introduction

The Japanese pearl oyster, Pinctada fucata, is one of the most important molluscan species in the pearl culture industry These oysters form pearls using the same process that produces the nacreous inner layer of their shells: Mantle tissue and a nucleus implanted into an oyster body cause the formation of a pearl sac, which subsequently closes. Formation of the nacreous layer in P. fucata shells is thought to be regulated by several types of proteins that are secreted from the mantle tissue. The nacreous layer is composed of aragonite tablets arranged in an organic matrix comprised of chitin that forms compartments filled with silk-like proteins associated with acidic glycoproteins [3]. N16, an aragonite forming protein, was extracted from the nacreous matrix with an alkaline solution of NH4OH, following shell decalcification using 10% EDTA [9]. We developed a novel method to electro-extract nacreous layer proteins under milder conditions, which enabled the extraction of proteins that are cross-linked by intermolecular disulfide bonds

Decalcification of the Nacreous Layer

Heat Extraction of Proteins from the NOS

Electroextraction of Proteins from the NOS

Methods