Oxidase Activity of the Barnacle Adhesive Interface Involves Peroxide-Dependent Catechol Oxidase and Lysyl Oxidase Enzymes

Abstract

Oxidases are found to play a growing role in providing functional chemistry to marine adhesives for the permanent attachment of macrofouling organisms. Here, we demonstrate active peroxidase and lysyl oxidase enzymes in the adhesive layer of adult Amphibalanus amphitrite barnacles through live staining, proteomic analysis, and competitive enzyme assays on isolated cement. A novel full-length peroxinectin (AaPxt-1) secreted by barnacles is largely responsible for oxidizing phenolic chemistries; AaPxt-1 is driven by native hydrogen peroxide in the adhesive and oxidizes phenolic substrates typically preferred by phenoloxidases (POX) such as laccase and tyrosinase. A major cement protein component AaCP43 is found to contain ketone/aldehyde modifications via 2,4-dinitrophenylhydrazine (DNPH) derivatization, also called Brady's reagent, of cement proteins and immunoblotting with an anti-DNPH antibody. Our work outlines the landscape of molt-related oxidative pathways exposed to barnacle cement proteins, where ketone- and aldehyde-forming oxidases use peroxide intermediates to modify major cement components such as AaCP43.

Acorn Barnacles Secrete Phase‐Separating Fluid to Clear Surfaces Ahead of Cement Deposition

“…As previously mentioned, barnacle cement does not contain DOPA;10, 11, 12 however, Gohad et al and Dickinson et al demonstrated that cyprid cement and the barnacle cementing apparatus are phosphorylated, respectively 14, 30. Likewise, we observe that the cyprid adhesive plaque ( Figure 4 a), cuticular tissues, and some surface deposits positively stain for phosphorylation (Figure 4b).…”

The Quantitative Proteome of the Cement and Adhesive Gland of the Pedunculate Barnacle, Pollicipes pollicipes

“…Elastin is insoluble, and rich in hydrophobic amino acids, such as glycine and proline, which form mobile hydrophobic regions bounded by crosslinks between lysine residues [26]. Thus, elastin has been associated with a cross-linking mechanism of bulk proteins in the cement [10].…”

“…This model has been updated in the last years [10][11][12], including the notion that surface coupling relies on catechol groups, through various adhesive and cohesive forces, and that catechol, quinone, and lysine-based cross-linking are also involved in protein nanofibers' curing and proper holdfast to the adhesion surface [10]. However, in barnacles, the catechol groups responsible for β-amyloid fibrils' cross-linking of bulk proteins do not have a peptidyl origin, differently from other marine organisms, e.g., mussels and sandcastle worms; non-peptidyl catechol precursors and lysine are incorporated among bulk cement protein (CP52k and CP100k) fibrils through enzymatic reactions that involve peroxidases and lysyl oxidase [10], providing structure and mechanical characteristics to the composite. Six barnacle-specific cement proteins (CPs) have been identified, four of which are thought to be interface proteins, CP19k, −20 k, −43 k, and −68 k, and two bulk proteins, CP52k and CP100k, [13].…”

Semi‐aquatic spider silks: transcripts, proteins, and silk fibres of the fishing spider, Dolomedes triton (Pisauridae)

“…Three predicted proteins identified in peptide sequences from D. triton egg sacs are homologous to peroxidases. Peroxidases belong to a family of heme-containing enzymes that are involved in diverse biological roles such as peroxidase-catalysed protein cross-linking in barnacle cement (So et al, 2016(So et al, , 2017 and mosquito egg chorion layer (Li et al, 1996;Li and Li, 2006). A peroxidase has also been identified in the major and minor ampullate silk glands of the orb-web weaver Nephila senegalensis; this enzyme (NsPox) is thought to contribute to the initiation of disulphide links in outer layer silk proteins during fibre processing (Vollrath and Knight, 1999;Pouchkina et al, 2003).…”