A mussel polyphenol oxidase-like protein shows thiol-mediated antioxidant activity

Highlights

• The full-length sequence of polyphenol oxidase- like protein (PPOL) from Mytilus galloprovincialis was identified.

• Recombinant PPOL (rPPOL) was successfully produced in Escherichia coli.

• rPPOL exhibits thiol-dependent antioxidant activity suppressing DOPA oxidation.

Abstract

Marine mussels adhere underwater to a variety of substrates using adhesive proteins with post-translationally modified amino acids, such as 3,4-dihydroxyphenylalanine (DOPA) residues, as a key chemical signature. DOPA can auto-oxidize easily in seawater reducing the adhesion strength, but contributing to subsequent cohesion (cross-linking) of the underlying proteins. To maintain both reduced and oxidized forms of DOPA with corresponding adhesion and cohesion properties, strict redox regulation is necessary for mussel underwater adhesion. In this study, a full-length polyphenol oxidase-like protein (PPOL) from Mytilus galloprovincialis was identified after screening of a mussel foot cDNA library using different degenerated PCR primers. The recombinant PPOL (rPPOL) was successfully produced in Escherichia coli. The rPPOL exhibits thiol-dependent antioxidant activity suppressing DOPA oxidation. This finding provides insights into how DOPA chemistry could be regulated and presumably inspires future applications of DOPA-mediated adhesion materials.

Introduction

In nature, marine mussels use a holdfast system containing a bundle of so-called byssal threads with a disk-like adhesive plaque at each tip, to adhere to various substrates underwater [1], [2]. The adhesive plaque comprises a variety of mussel foot proteins (mfps), which contain a catecholic amino acid (3,4-dihydroxyphenylalanine, DOPA) mediating strong underwater adhesion [3], [4]. DOPA residues are generated upon post-translational modification of tyrosine residues catalyzed by the tyrosine hydroxylase (cresolase) activity of polyphenol oxidase (PPO) [3], [5], [6]. In O₂ saturated and alkaline seawater (pH ∼ 8.0), DOPA exhibits high auto-oxidation tendency to Dopaquinone (DQ), which shows poor adhesion but good cohesion [3], [7], [8]. However, marine mussels are able to achieve strong adhesion forces, and obviously a tight and complex regulation of DOPA chemistry exists.

The incipient acidic (pH ∼ 3.0) secretion of mfps [9] as well as the strong reducing environment [8], [10] therein protect DOPA from auto-oxidation to begin with [3]. Previously, two reducing pathways were detected mediated by thiol-rich mfp-6 [8], [10] as well as DQ tautomerization [11], [12]. Along with mfp-3 and mfp-5, mfp-6 is secreted at the substratum’s interface during plaque formation [13], [14]. mfp-6 contains a great number of tyrosine residues (∼20 mol %) with inefficient post-translational modification into DOPA (<5 mol %), as well as 11 cysteine residues, 9 of which possessing presumably free thiols and 2 of which being disulfide bonded [13]. mfp-6 itself demonstrates poor adhesion properties, whereas it can effectively rescue the adhesive properties of other mfps, such as mfp-3 [8], [10], [15]. Approximately 9 free thiols and 4 DOPA residues with ∼17 electrons per molecule of mfp-6 devote to the reservoir of reducing electrons, reducing ∼8 DQ to DOPA per mfp-6 molecule (DQ + 2 Cys-SH → Cys-S-S-Cys + DOPA) [10]. Further, DQ reduces to α,β-dehydro-DOPA (Δ-DOPA) upon tautomerization at neutral to alkaline pH [11], [12]. Presumably, the Δ-DOPA bestows electrons on the reducing reservoir to extend the lifetime of DOPA as well [3].

Here, a full-length PPO-like cDNA sequence of Mytilus galloprovincialis (M. galloprovincialis, PPOL) has been identified in a mussel foot cDNA library [5], [16]. The PPOL comprises 15 cysteine residues, ∼2 of which are disulfide bonded leaving ∼13 endowed with free thiols. The recombinant PPOL (rPPOL) was produced in E. coli and exhibited antioxidant activity in its reduced state restraining DOPA oxidation into DQ.