Peroxidase-catalysed interfacial adhesion of aquatic caddisworm silk

Abstract

Casemaker caddisfly (Hesperophylax occidentalis) larvae use adhesive silk fibres to construct protective shelters under water. The silk comprises a distinct peripheral coating on a viscoelastic fibre core. Caddisworm silk peroxinectin (csPxt), a haem-peroxidase, was shown to be glycosylated by lectin affinity chromatography and tandem mass spectrometry. Using high-resolution H2O2 and peroxidase-dependent silver ion reduction and nanoparticle deposition, imaged by electron microscopy, csPxt activity was shown to be localized in the peripheral layer of drawn silk fibres. CsPxt catalyses dityrosine cross-linking within the adhesive peripheral layer post-draw, initiated perhaps by H2O2 generated by a silk gland-specific superoxide dismutase 3 (csSOD3) from environmental reactive oxygen species present in natural water. CsSOD3 was also shown to be a glycoprotein and is likely localized in the peripheral layer. Using a synthetic fluorescent phenolic copolymer and confocal microscopy, it was shown that csPxt catalyses oxidative cross-linking to external polyphenolic compounds capable of diffusive interpenetration into the fuzzy peripheral coating, including humic acid, a natural surface-active polyphenol. The results provide evidence of enzyme-mediated covalent cross-linking of a natural bioadhesive to polyphenol conditioned interfaces as a mechanism of permanent adhesion underwater.

Keywords: bioadhesive; caddisworms; dityrosine cross-linking; peroxinectin; silk.

Figure 1.

Synthesis of rhodamine-labelled, phenol-conjugated poly(HEMA-co-MAA). Numbers indicate the sidechain mol% in the final copolymer. Rho, rhodamine.

Figure 2.

Thin silk fibre cross sections stained for carbohydrates. (a) Unstained; (b) stained with PAS. CO, fibre core; PL, peripheral layer.

Figure 3.

SDS-PAGE of silk gland contents. Lane 1: general protein stain coomassie blue (CB); lane 2: carbohydrate stain (PAS) to identify glycoproteins; lane 3: phosphoprotein stain (ProQ Diamond).

Figure 4.

CsPxt localization by silver deposition. (a) SEM image of whole fibres with Ag⁺ and H₂O₂. (b) Whole fibres pre-treated with sodium azide. (c) SEM image of a thin silk fibre cross section treated with Ag⁺ and H₂O₂. (d) Thin cross section pre-treated with sodium azide before Ag⁺ and H₂O₂.

Figure 5.

Fibre ultrastructure and csPxt localization in the fibre peripheral layer. (a) TEM image of native fibre cross section. (b) Boxed region in (a). (c) Longitudinal section of fibres treated with Ag⁺ in the presence of H₂O₂. Electron dense metallic Ag nanoparticles are indicated by arrowhead. (d) Longitudinal section without H₂O₂. PL, peripheral layer; CO, fibre core.

Figure 6.

CsPxt-catalysed cross-linking of a rhodamine-labelled synthetic polyphenol into the silk peripheral layer. (a–d) Differential interference contrast (DIC) and fluorescent images of unsectioned silk fibres incubated with polyphenol with and without H₂O₂. (e–h) DIC and fluorescent images of cross-sectioned silk fibres incubated with polyphenol with and without H₂O₂. (i) Dityrosine autofluorescence of silk fibre in (e) and (f). (j) Overlay of fluorescent polyphenol, (f), and dityrosine autofluoresence, (i). (k,l) Higher magnification of boxed regions in (j). All scale bars represent 5 µm, except where indicated otherwise.

Figure 7.

Peroxidase inhibitors prevent fibre labelling with polyphenol. Silk fibres were incubated with rhodamine-labelled polyphenol, 10 µM H₂O₂, and either 100 µM sodium azide (a,b) or 100 µM l-tyrosine (c,d). Scale bars, 5 µm.

Figure 8.

CsPxt-catalysed cross-linking of humic acid into the silk peripheral layer. (a–d) Differential interference contrast (DIC) and fluorescent images of silk fibres incubated with humic acid with and without H₂O₂. (e–h) DIC and fluorescent images of cross-sectioned silk fibres incubated with humic acid with and without H₂O₂. (i) Humic acid autofluorescence of silk fibre in (e) and (f). (j) Overlay of humic acid fluorescence, (f), and dityrosine autofluoresence, (i). (k,l) Higher magnification of boxed regions in panel (j). All scale bars represent 5 µm, except were indicated otherwise.

Figure 9.

Schematic of csPxt-catalysed underwater interfacial adhesion. CsPxt embedded in the peripheral adhesive fibre coating catalyses covalent cross-linking between tyrosine sidechains in the peripheral coating and between tyrosine sidechains and natural polyphenolic primers on submerged surfaces. The csPxt H₂O₂ substrate may be generated, wholly or in part, by proximate csSOD3 acting on environmental ROS.