A cDNA Cloning of a Novel Alpha-Class Tyrosinase of Pinctada fucata: Its Expression Analysis and Characterization of the Expressed Protein.

Ryousuke Takgi,Tomoyuki Miyashita

doi:10.1155/2014/780549

Abstract

Tyrosinase plays an important role in the formation of the shell matrix and melanin synthesis in mollusks shells. A cDNA clone encoding a 47 kDa protein was isolated from the pearl oyster Pinctada fucata. The cDNA was 1,957 base pairs long and encodes a 417 residue protein that has extensive sequence identity with tyrosinase (polyphenol oxidase: EC 1.14.18.1). This tyrosinase-like protein, termed PfTy, contains an N-terminal signal sequence and the two copper-binding domain signatures (CuA and CuB), suggesting that PfTy belongs to the α-subclass of type-3 copper proteins. Enzyme activity of PfTy was examined by a spectrophotometric method using the translation product derived from an S30 T7 high-yield protein expression system. Tyrosinase activity was seen in this recombinant product. RT-PCR analysis showed that PfTy mRNA was expressed in the mantle pallial, but not in the mantle edge. Therefore, PfTy may participate in insoluble shell matrix formation of the nacreous layer. PfTy expression was also observed in the foot, liver, and adductor muscle, suggesting that PfTy participates in the synthesis of melanins, which are effective scavengers of free radicals formed in multiple intracellular oxidative processes. This is the first report of a novel α-class tyrosinase from the pearl oyster P. fucata.

Highlights

Copper-containing proteins play very important roles in the regulation of the metabolism of an organism [1]
Nacrein β-Actin adductor muscle, and gill. These results suggest that PfTy participates in the synthesis of melanin, which is an effective scavenger of free radicals formed in multiple intracellular oxidative processes [30, 31]
Tyrosinases play an important role in the formation of the shell matrix and melanin synthesis in mollusks shells

Summary

Introduction

Copper-containing proteins play very important roles in the regulation of the metabolism of an organism [1]. They can be classified into three groups, namely, types 1 to 3, by the geometric structure of the active site and spectroscopic properties [2]. It is well known that tyrosinase (TYR)/polyphenoloxidase (PPO) (EC 1.14.18.1) is an important bifunctional copperdependent enzyme. This enzyme catalyzes a reaction that converts L-tyrosine to L-DOPA by hydroxylation and to L-dopaquinone. As for the antipathogenic activity of the insoluble form of melanin, the synthesis of melanin is observed in the wounds of various invertebrate animals. These products can act as reactive electrophile cross-linking agents between cuticular proteins to form the exoskeleton of arthropods [8]

Methods

Results

Conclusion