Ching Shuen Wang

Complex coacervates as a foundation for synthetic underwater adhesives

Complex coacervation was proposed to play a role in the formation of the underwater bioadhesive of the Sandcastle worm (Phragmatopoma californica) based on the polyacidic and polybasic nature of the glue proteins and the balance of opposite charges at physiological pH. Morphological studies of the secretory system suggested that the natural process does not involve complex coacervation as commonly defined. The distinction may not be important because electrostatic interactions likely play an important role in the formation of the sandcastle glue. Complex coacervation has also been invoked in the formation of adhesive underwater silk fibers of caddisfly larvae and the adhesive plaques of mussels. A process similar to complex coacervation, that is, condensation and dehydration of biopolyelectrolytes through electrostatic associations, seems plausible for the caddisfly silk. This much is clear, the sandcastle glue complex coacervation model provided a valuable blueprint for the synthesis of a biomimetic, water-borne, underwater adhesive with demonstrated potential for repair of wet tissue.

Adaptation of Caddisfly Larval Silks to Aquatic Habitats by Phosphorylation of H-Fibroin Serines

Aquatic caddisflies diverged from a silk-spinning ancestor shared with terrestrial moths and butterflies. Caddisfly larva spin adhesive silk underwater to construct protective shelters with adventitiously gathered materials. A repeating (SX)(n) motif conserved in the H-fibroin of several caddisfly species is densely phosphorylated. In total, more than half of the serines in caddisfly silk may be phosphorylated. Major molecular adaptations allowing underwater spinning of an ancestral dry silk appear to have been phosphorylation of serines and the accumulation of basic residues in the silk proteins. The amphoteric nature of the silk proteins could contribute to silk fiber assembly through electrostatic association of phosphorylated blocks with arginine-rich blocks. The presence of Ca(2+) in the caddisfly larval silk proteins suggest phosphorylated serines could contribute to silk fiber periodic substructure through Ca(2+) crossbridging.

Multipart Copolyelectrolyte Adhesive of the Sandcastle Worm, Phragmatopoma californica (Fewkes): Catechol Oxidase Catalyzed Curing through Peptidyl-DOPA

Tube-building sabellariid polychaetes have major impacts on the geology and ecology of shorelines worldwide. Sandcastle worms, Phragmatopoma californica (Fewkes), live along the western coast of North America. Individual sabellariid worms build tubular shells by gluing together mineral particles with a multipart polyelectrolytic adhesive. Distinct sets of oppositely charged components are packaged and stored in concentrated granules in separate cell types. Homogeneous granules contain sulfated macromolecules as counter-polyanion to polycationic Pc2 and Pc5 proteins, which become major components of the fully cured glue. Heterogeneous granules contain polyphosphoproteins, Pc3A/B, paired with divalent cations and polycationic Pc1 and Pc4 proteins. Both types of granules contain catechol oxidase that catalyzes oxidative cross-linking of L-DOPA. Co-secretion of catechol oxidase guarantees rapid and spatially homogeneous curing with limited mixing of the preassembled adhesive packets. Catechol oxidase remains active long after the glue is fully cured, perhaps providing an active cue for conspecific larval settlement.

Localization of the bioadhesive precursors of the sandcastle worm, Phragmatopoma californica (Fewkes)

SUMMARY The marine sandcastle worm bonds mineral particles together into underwater composite dwellings with a proteinaceous glue. The products of at least four distinct secretory cell types are co-secreted from the building organ to form the glue. Prominent hetereogeneous granules contain dense sub-granules of Mg and the (polyphospho)proteins Pc3A and B, as well as at least two polybasic proteins, Pc1 and Pc4, as revealed by immunolabeling with specific antibodies against synthetic peptides. Equally prominent homogeneous granules comprise at least two polybasic proteins, Pc2 and Pc5, localized by immunolabeling with anti-synthetic peptide antibodies. The components of the sub-micrometer granule types are unknown, though positive staining with a redox-sensitive dye suggests the contents include o-dihydroxy-phenylalanine (dopa). Quantitative PCR and in situ hybridization demonstrated that a tyrosinase-like enzyme with a signal peptide was highly expressed in both the heterogeneous and homogeneous granules. The contents of the granules are poorly mixed in the secreted mixture that forms the glue. Subsequent covalent cross-linking of the glue may be catalyzed by the co-secreted tyrosinase. The first three parapodia of the sandcastle worm also contain at least two distinct secretory tissues. The Pc4 protein was immunolocalized to the anterior secretory cells and the tryosinase-like gene was expressed in the posterior secretory cells, which suggests these proteins may have multiple roles.

Peroxinectin catalyzed dityrosine crosslinking in the adhesive underwater silk of a casemaker caddisfly larvae, Hysperophylax occidentalis.

Aquatic caddisfly larvae use sticky silk fibers as an adhesive tape to construct protective composite structures under water. Three new silk fiber components were identified by transcriptome and proteome analysis of the silk gland: a heme-peroxidase in the peroxinectin (Pxt) sub-family, a superoxide dismutase 3 (SOD3) that generates the H2O2 substrate of the silk fiber Pxt from environmental reactive oxygen species (eROS), and a novel structural component with sequence similarity to the elastic PEVK region of the muscle protein, titin. All three proteins are co-drawn with fibroins to form silk fibers. The Pxt and SOD3 enzymes retain activity in drawn fibers. In native fibers, Pxt activity and dityrosine crosslinks are co-localized at the boundary of a peripheral layer and the silk fiber core. To our knowledge, dityrosine crosslinks, heme peroxidase, and SOD3 activities have not been previously reported in an insect silk. The PEVK-like protein is homogeneously distributed throughout the fiber core. The results are consolidated into a model in which caddisfly silk Pxt-catalyzed dityrosine crosslinking occurs post-draw using H2O2 generated within the silk fibers by SOD3. The ROS substrate of caddisfly silk SOD3 occurs naturally in aquatic environments, from biotic and abiotic sources. The radially inhomogeneous dityrosine crosslinking and a potential titin-like PEVK protein network have important implications for the mechanical properties of caddifly silk fibers.

Morphology of the Adhesive System in the Sandcastle Worm, Phragmatopoma californica

The marine Sandcastle worm (P. californica) and related species live in composite mineralized tubes for shelter. They gather the mineral phase for free from the environment as sand grains and seashell bits with a crown of ciliated tentacles. The captured mineral particles are conveyed for inspection to the building organ — a pincer-shaped pair of dexterous palps in front of the mouth (Fig. 10.1). A dab of proteinaceous adhesive (Jensen and Morse, 1988) is secreted from the building organ onto suitable particles as they are pressed onto the end of the tube. The major protein components of the adhesive are a group of heterogeneous proteins, referred to as Pc3x, characterized by serial runs of 10–14 serine residues punctuated with single tyrosine residues (Zhao et al., 2005). Phosphorylation of more than 90% of the serines (Stewart et al., 2004) makes the Pc3 proteins polyacidic (pI<3). Other potential protein components identified biochemically (Waite et al., 1992) and by sequencing random cDNAs from an adhesive gland library (Endrizzi and Stewart, 2009) are generally polybasic with predicted pIs greater than 9. Amino acid analysis of secreted glue revealed that, in total, close to 50% of the adhesive protein residues are charged when serine phosphorylation is taken into account. The adhesive also contains Mg2+ and Ca2+ and a large fraction of the tyrosines are post-translationally hydroxylated to form 3,4-dihydroxyphenylalanine (DOPA), a residue shared with the adhesive plaque proteins of the mussel (Waite and Tanzer, 1981). Phosphates and o-dihydroxyphenols are well-known adhesion promoters.

The role of coacervation and phase transitions in the sandcastle worm adhesive system.

Sandcastle worms, Phragmatopoma californica (Fewkes), live along the western coast of North America. Individual worms build tubular shells under seawater by gluing together sandgrains and biomineral particles with a multipart, rapid-set, self-initiating adhesive. The glue comprises distinct sets of condensed, oppositely charged polyelectrolytic components-polyphosphates, polysulfates, and polyamines-that are separately granulated and stored at high concentration in distinct cell types. The pre-organized adhesive modules are secreted separately and intact, but rapidly fuse with minimal mixing and expand into a crack-penetrating complex fluid. Within 30s of secretion into seawater, the fluid adhesive transitions (sets) into a porous solid adhesive joint. The nano- and microporous structure of the foamy solid adhesive contributes to the strength and toughness of the adhesive joint through several mechanisms. A curing agent (catechol oxidase), co-packaged into both types of adhesive granules, covalently cross-links the adhesive and becomes a structural component of the final adhesive joint. The overall effectiveness of the granulated sandcastle glue is more a product of the cellular sorting and packaging mechanisms, the transition from fluid to solid following secretion, and its final biphasic porous structure as it is of its composition or any particular amino acid modification.

Peroxidase-catalysed interfacial adhesion of aquatic caddisworm silk

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.

The Adhesive Tape-Like Silk of Aquatic Caddisworms

Aquatic caddisfly larva spin a sticky silk tape used underwater to construct a protective composite stone case. Caddisworm silk fibers are drawn on-demand from fluid precursors stored in the posterior region of the silk gland. Fibers begin to form in the gland at a cuticular narrowing at the entrance into the short (2–3 mm) anterior conducting channel leading to the spinneret. The caddisworm silk comprises a thin adhesive peripheral coating on a tough viscoelastic core fiber. The thin adhesive layer contains glycoproteins and a heme-peroxidase in the peroxinectin subfamily (Pxt). Pxt catalyzes dityrosine cross-linking in the fiber periphery and may catalyze covalent adhesive cross-links to surface-active natural polyphenolic compounds. The major component of the silk core, H-fibroin, contains around 13 mol% phosphoserines (pS) in repeating (pSX) n motifs, wherein X is usually hydrophobic, and n is 4 or 5. The (pSX) n motifs form β-domains crossbridged and stabilized by multivalent metal ions, predominantly Ca2+ in natural fibers. During loading, the Ca2+/(pSX) n β-domains reversibly rupture to reveal hidden length and dissipate strain energy. The tough fibers can be strained to more than 100 % of their initial length before fracture. The work of extension to failure, ~17.3 ± 6.2 MJ/m3, is higher than articular cartilage. Silk fibers cycled to 20 % elongation completely recover their initial stiffness, strength, and hysteresis within 120 min as an elastic covalent network guides the post-yield recovery of the Ca2+/(pSX) n β-domains.