Adaptation of Caddisfly Larval Silks to Aquatic Habitats by

Mar 2, 2010 - (1) They occupy freshwater habitats ranging from cold fast moving mountain streams to still marshes, often with several species dividing...
0 downloads 0 Views 2MB Size
Download PDF
Biomacromolecules 2010, 11, 969–974

969

Adaptation of Caddisfly Larval Silks to Aquatic Habitats by Phosphorylation of H-Fibroin Serines Russell J. Stewart* and Ching Shuen Wang Department of Bioengineering, University of Utah, Salt Lake City, Utah 84112 Received December 14, 2009; Revised Manuscript Received February 10, 2010

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 Ca2+ in the caddisfly larval silk proteins suggest phosphorylated serines could contribute to silk fiber periodic substructure through Ca2+ crossbridging.

Introduction Caddisflies (order Trichoptera) are a large group of aquatic insects familiar to fly fishermen around the world.1 They occupy freshwater habitats ranging from cold fast moving mountain streams to still marshes, often with several species dividing resources within each habitat.2 The larval stages feed, mature, and pupate underwater. The pupae “hatch” into short-lived winged adults that leave the water to mate. The caddisflies’ successful penetration into diverse aquatic habitats is largely due to the inventive use by their larva of underwater silk to build elaborate structures for protection and food gathering. Ancestral caddisflies diverged into three suborders distinguished by their larval silk constructions.2 The larvae of retreat-makers (suborder Annulipalpia) live in stationary composite structures assembled with fragments of leaves, sticks, or stones bound together with underwater silk. These elaborate structures are often equipped with silk nets for capturing food from water channeled through the retreat. Spicipalpia larvae, the cocoonmakers, construct closed, rigid silk cases for pupation. The casemaker larvae (Integripalpia) build composite tubular structures from silk-bonded leaves, sticks, or stones (Figure 1A). The case makers are mobile foragers that drag their portable cases with them for camouflage as well as physical protection from trout and other predators. Caddisflies are closely related to silk spinning terrestrial moths and butterflies, an order (Lepidoptera) that includes the domesticated silkworm moth (Bombyx mori) from which textile silk has been gathered for millennia. Moth and butterfly caterpillars spin dry silken cocoons and chrysalises in which to pupate. The close relation of these orders raises the question “What molecular adaptations of the dry silks of their terrestrial ancestors were necessary for caddisflies to move into aquatic habitats and for their silks to bond diverse substrates, organic and inorganic, underwater?”. Commercially important silkworm silk is comparatively well studied. The core of the fibers is comprised of heavy chain fibroin (H-fibroin, 250-500 kDa), light chain fibroin (L-fibroin, ∼25 kDa), and the glycoprotein P25 (∼30 * To whom correspondence should be addressed. E-mail: rstewart@ eng.utah.edu.

Figure 1. (A) Brachycentrus echo larva in a case partially constructed with glass beads in a laboratory aquarium. (B-D) SEMs of the inside of the glass case at increasing magnification (scale bars: 500, 250, 100 µm, respectively). (E) SEM of the region in F analyzed by EDS: blue ) phosphorus, purple ) silicon (scale bars: 10 µm).

kDa). These proteins are produced in posterior silk gland cells, assembled into elementary secretory units in a 6:6:1 molar ratio, and secreted into the silk gland lumen.3 The heavy and light chain fibroins are covalently linked through a single intermolecular disulfide bond.4 On the way to being drawn-out of labial

10.1021/bm901426d  2010 American Chemical Society Published on Web 03/02/2010

970

Biomacromolecules, Vol. 11, No. 4, 2010

spinnerets as an insoluble filament, the concentrated fibroin suspension is coated with a heterogeneous mixture of sticky sericins, aligned into microfibrils, and possibly dehydrated as it passes through the middle and anterior regions of the silk gland.5,6 The final spun-out silk consists of two filaments from the paired silk glands fused into a single fiber coated with adhesive sericins. Caddisfly silk is produced in a pair of silk glands functionally partitioned into fibroin producing posterior and middle regions7 and anterior regions that narrow into a common duct connected to the labial spinneret.6,8 Sequences homologous to moth Hand L-fibroins have been identified in species representing all three caddisfly suborders by sequencing random silk glandderived cDNAs.7,9,10 The caddisfly H-fibroins share several structural design features with moth H-fibroins: nonrepetitive N- and C-termini flanking a long central region of conserved motifs arranged in repeating blocks, regularly alternating hydrophobic and hydrophilic regions in the central core,11 and conserved positions and spacing of cysteine residues that covalently cross-link H- and L-fibroins. At the amino acid level, the commonalities include a preponderance of simple motifs like GX, GGX, GPGXX, and SXSXSX, which is reflected in the high levels of G and S in both caddisfly and moth H-fibroins (Table 2). A conspicuous difference in amino acid composition is the comparatively low incidence of alanine in caddisfly, which in moth and spider H-fibroins occurs in runs of poly(A) and poly(GA) that confer β-crystallinity and mechanical strength to their silk fibers.12,13 Another striking difference is the high concentration (around 15 mol %) of positively charged basic residues, especially arginine, which are comparatively scarce in moth silks. Neither a cDNA nor protein homologue of P25 could be identified in three caddisfly species.9,10 The important role of P25 in moth silk filament assembly and secretion suggests this may be another important distinction in the processing and assembly of dry versus wet silks.

Materials and Methods Sample Preparation. Brachycentrus echo (family Brachycentridae) caddisfly larvae were collected from the lower Provo River, Utah. Larvae with natural cases were maintained in an aquarium with circulating water at 12 °C. The natural stone cases were either partially or completely removed with fine forceps. The larva were then placed on a bed of prewashed 0.5 mm glass beads in a clean glass vial. After 1-2 days partial glass built onto the end of natural cases or completely rebuilt glass cases were taken away from the larva who would start over rebuilding their case. The harvested glass cases were frozen at -80 °C, lyophilized, and then mounted on conductive carbon tape for SEM and EDS analysis (FEI Company, Quanta 600 FEG). Amino Acid and Elemental Analysis. Glass beads from reconstructed cases were carefully examined for contaminating sand or minerals. After lyophilization, weighed samples of beads with silk and without silk collected from the same vial were digested in 500 µL of 5.7 N HCl with 0.1% phenol in vacuo for 24 h at 110 °C. An aliquot of the hydrolysate was analyzed for amino acids (Beckman 6300) and a second aliquot from the same hydrolysate was analyzed for elements by ICP-OES (PerkinElmer, Optima 3100XL) after dilution in 40% nitric acid. Elements were quantified by comparison to standard curves prepared with commercial mixed metal standards (PerkinElmer). Gel Electrophoresis and Western Blot Analysis. Dissected silk glands were transferred to a clean eppendorf tube containing DI water at 4 °C. Silk proteins were released from the gland within 20 min. To collect soluble fraction of silk proteins, samples were spun at 13000 rpm for 5 min at room temperature. The supernatant was transferred to a new tube for protein concentration determination (Bio-Rad). A total of 40 mg of soluble silk proteins were subjected to SDS-PAGE

Stewart and Wang on 15% gels. For Western blot analysis, the separated proteins were transferred to PVDF membranes that were then blocked with 2 mg/ mL BSA in phosphate buffered saline (pH 7.4) at room temperature for at least 2 h. The blots were probed with an antiphosphoserine mouse antibody (Abcam, #PSR45, 1:1000) at 4° overnight. After incubation with Horseradish peroxidase goat antimouse-HRP (1:5000) secondary antibody (Jackson Immuno Research, #115-035-166) at room temperature for 1 h signals were developed with ECL (Pierce, #32109). Silk Gland Immunostaining. Larvae were killed with 7% ethanol in DI water before the paired silk glands were removed still attached to the head. The glands were fixed with 4% paraformaldehyde in PBS at room temperature for 30 min before permeabilization with proteinase digestion buffer (2 µg/mL proteinase K, 1% SDS, 0.1% Triton X-100 in PBS) at room temperature for 15 min. The silk gland was blocked with 2 mg/mL BSA in PBS at room temperature for at least 2 h and then incubated with anti-pS antibody (Abcam, #PSR45, 1:1000) at room temperature for 1 h. The primary antibody was labeled with a goat antimouse alkaline phosphatase conjugated secondary antibody (Abcam, #6729, 1:5000) at RT for another 1 h. The blue signal was developed with NBT/BCIP (3:1 molar ratio) in AP buffer (150 mM NaCl, 100 mM Tris (pH 8.8), 5 mM MgCl2 and 0.05% Tween-20) until blue color appeared. Glands were then dehydrated with serial dilution of ethanol (100, 70, and 50% and TBS) to remove nonspecific staining and followed by serial hydration for photoimagining. Tandem Mass Spectrometry. Silk proteins were isolated from dissected B. echo silk glands in 25 mM ammonium bicarbonate. The silk proteins were heat denatured at 100 °C, quickly cooled on ice to limit renaturation, and digested with trypsin at an ∼1:25 ratio of enzyme to silk protein for 2 h at 37 °C. Phosphopeptides from the silk protein digests were enriched by immobilized metal affinity chromatography (IMAC) using a SwellGel Gallium Disc (Pierce) according to the manufacturers instructions for phosphopeptide enrichment. The IMAC enriched peptides were analyzed by LC/MS/MS using a LTQ-FT hybrid mass spectrometer (ThermoElectron Corp). Peptides were introduced into the spectrometer by nanoLC (Eksigent, Inc.) using a C18 nanobore column and nanoelectrospray ionization (ThermoElectron Corp). Peptides were eluted with a 50 min linear gradient of 5-60% acetonitrile with 0.1% formic acid. Primary peptide molecular masses were determined by FT-ICR and peptide sequences by collision-induced dissociation in the linear ion trap of the LTQ-FT hybrid mass spectrometer. Peptides were identified by MS/MS search using the Mascot search engine (ver. 2.2.1, Matrix Science). Possible phosphorylation on S, T, and Y were included in the search. Mascot thresholds were primary mass errors of 20, and expect values 200 kDa (consistent with H-fibroin), at ∼50, at ∼30 (the approximate MW expected for L-fibroin), and at 17 kDa and below (Figure 2A, lanes 1,2). The band pattern depended on the extraction method; sodium dodecyl sulfate (SDS) solubilized caddisfly H-fibroin while 8 M urea did not. Proteins extracted from the silk gland of B. mori with either urea or SDS and probed with R-pS did not have bands corresponding to H-fibroin but did have weak immunoreactive bands at ∼30 kDa and below 17 kDa (Figure 2A, lanes 3,4). Further confirmation of phosphorylated silk proteins was obtained by immunostaining isolated caddisfly larval silk glands with anti-pS. The posterior region of the paired glands stained for pS (Figure 2C). Silk fibers on glass beads retrieved from cases were also strongly labeled with the anti-pS antibody (Figure 2E). Caddisfly silk proteins isolated from dissected silk glands were heat denatured, rapidly cooled, and digested with trypsin. Tryptic peptides enriched for phosphopeptides were isolated by immobilized metal affinity chromatography (IMAC) and analyzed by tandem mass spectrometry. Experimental peptide masses were compared using the Mascot search engine14 against peptide masses calculated from translated caddisfly fibroin sequences deposited in GenBank. Genbank contains partial H-fibroin sequences for Hydropsyche augustipennis (Annulipalpia), Limnephilus decipiens (Integripalpia), Rhyacophila obliterata (Spicipalpia), and Stenopsyche marmorata (Annulipalpia) and complete L-fibroin sequences for all four caddisfly species. A total of 18 unique peptides were identified, 16 of which were in most instances multiply phosphorylated (Table 1). The central regions of caddisfly H-fibroins are repeating sets of unique repeats that have been assigned letters A-F.9,10 All four species share a conserved D repeat that is shown in Table 1. Together, the identified peptides spanned an entire D repeat taken at random from the L. dicipiens H-fibroin sequence. Identification of peptides in B. echo silk with the same sequence as L. decipiens H-fibroin demonstrates these species are closely related. Conservation of the position in all four species of two

Biomacromolecules, Vol. 11, No. 4, 2010

971

(SX)4 motifs suggests the B. echo phosphorylation pattern is likely conserved as well. The larger repeating motif of two phosphorylated blocks flanking a hydrophobic region with a central proline must be an important structural element of caddisfly silks. The L. decipiens F repeats contain (SX)3-5 motifs in 15-18 residue tryptic peptides but corresponding peptides or phosphopeptides were not identified in the B. echo peptide mass analysis. The peptides may not be exactly conserved in B. echo, or the site(s) may not be accessible to trypsin. Likewise, no phosphoproteins from L-fibroin were identified. Manual searches were not performed. Beads recovered from glass cases constructed by B. echo in a laboratory aquarium were lyophilized and subjected to amino acid analysis after hydrolysis in 50% HCl. The amino acid composition of the acid digested silk fibers was comparable to amino acid compositions of three caddisfly species deduced from their partial H-fibroin sequences in GenBank. The alanine content was higher but this is likely due to the comparison of whole silk fibers to H-fibroin only. The L. decipiens L-fibroin, for example, contains 14 mol % alanine. A similar mol % alanine in the B. echo L-fibroin and a 1:1 ratio of H- to L-fibroin would bring the composition in line with the other caddisflies. To estimate the ratio of phosphate to serine residues, aliquots from two of the acid hydrolysates were also analyzed by inductively coupled plasma-optical emission spectroscopy. There was no appreciable serine or P in background measurements made with unglued beads collected from the same area of the aquarium at the same time as the glass cases. The caddisfly silk contained 114 nmol of P corresponding to 166 nmol of serine in the first hydrolysate and 164 nmol P to 256 nmol serine in the second hydrolysate for ratios of 0.69 and 0.64, respectively. These estimates seem reasonable given the ratio of phosphorylated serines found by mass spectrometry. The second most abundant element in the silk proteins was Ca2+ at ratios to P of 0.5 and 0.7 (Table 3).

Discussion Phosphorylated serines have been identified previously in three phylogenetically disparate marine underwater bioadhesives. The mussel (mollusca) foot protein Mefp-5 at the interface of the adhesive plaque and substrate contains several pS residues which may promote interfacial adhesion to calcareous minerals.15 Evidence for pS in the adhesive secreted by Cuvierian tubules of sea cucumbers (echinodermata) was shown with an anti-pS antibody.16 The marine sandcastle worm, P. californica (annelida), that builds reef-like structures by gluing together sand grains has the most pronounced content of pS in its adhesive proteins; Pc3B contains repeating blocks of 4-13 consecutive serines running for more than 300 residues17 of which at least 95% are phosphorylated.18 The P. californica polyphosphoproteins may promote interfacial adhesion to mineral substrates but probably play an important role in cohesion and triggered setting of the glue as well. The adhesive contains divalent Mg2+ and Ca2+ at combined concentrations roughly equal to phosphate (unpublished). The solubility of Mg2+/Ca2+ and polyphosphoproteins is higher at pHs < pKa2 than at pHs > pKa2 of phosphate, which could trigger the adhesive to solidify as it is released from secretory granules (pH ∼ 5) into seawater (pH 8.2). These diverse examples of pS in natural adhesives now include a fourth phylum (arthropoda) and a freshwater insect order (trichoptera). What is the role of pS in wet caddisfly silk? Polymeric phosphates are well-known adhesion promoters in the coatings

972

Biomacromolecules, Vol. 11, No. 4, 2010

Stewart and Wang

Table 1. Phosphorylated Peptides Identified by Tandem Mass Spectrometrya

a The top four peptide rows are the conserved D repeats from H. augustipennis (Ha), S. marmorata (Sm), R. obliterata (Ro), and L. dicipiens (Ld). Conserved serines are shaded. Phosphorylated residues are underlined. b Experimental relative molecular mass (Mr(expt)) errors: E ) (Mexpt - Mcalc)/Mcalc are presented in parts per million (ppm). The MS/MS ion score is -10(LogP) where P is the probability the observed peptide is a random match.14

industry and in dentistry as examples. Addition of phosphate groups to water-borne latex paints greatly increases their wet interfacial adhesion and scrub resistance.19 Likewise, priming teeth with polymerizable phosphates greatly increases interfacial bond strengths with restorative dental materials.20 Analogously, the caddisfly silk phosphates may function to promote adhesion to wet substrates in an aquatic environment. Labeling of untreated silk fibers with an anti-pS antibody (Figure 2E) was consistent with the phosphate groups being surface exposed and may also account for the positive reaction of acidophilic stains with the peripheral layer covering the fiber core of caddisfly silks.8 Dense phosphorylation of clustered serine residues could also have a profound effect on the structure of the silk fibers and consequently on the mechanism of fiber assembly. There is broad consensus that silks from both lepidopteran and spiders behave as semicrystalline elastomers, with alanine-rich regions forming ordered β-sheet structures in a disordered glycine-rich matrix. Mechanical properties like tensile strength and extensibility of silks correlate with the fraction of ordered to disordered regions.21 The nearly complete absence of alanine suggests caddisfly silks departed from this consistent design feature of terrestrial silks. Yonemura et al.9 suggested the serinerich motifs (SX)n that occur at least once in most of the repeating sequence blocks of caddisfly H-fibroins can form β-sheets to

compensate for the lack of alanine. However, the multiple phosphate groups on the serines likely preclude formation of typical β-sheets by the (SX)n motifs due to the bulkiness of the phosphate groups and repulsion between like charges on the surface of the sheet. Destabilization of β-sheets by charged side chains has been experimentally observed.22,23 High extensibility and tensile strength on the lower end of terrestrial silks may be consistent with lower content of ordered regions in caddisfly silk.24 A critical structural role and direct promotion of interfacial adhesion are not mutually exclusive functions of the extensive caddisfly silk phosphorylation. The B. echo silk proteins contain a 2- to 3-fold excess of negative relative to positive charges (assuming 60% of the serines are phosphorylated) that must be balanced by small counterions (Tables 2 and 3). Association of the observed silk fiber Ca2+ with the phosphate side chains could create intraand intermolecular cross-bridging of the (pSX)n motifs into rigid domains analogous to the β-crystalline regions of spider and silkworm silks (Figure 3). Indeed, X-ray diffraction studies of several caddisfly silks provided evidence of a repeating threesheet ordered structure despite the absence of alanine.25 Formation of Ca2+ crossbridged phosphoserine domains would also contribute to dehydration of the predominantly hydrophilic silk proteins while submerged in water because the solubility of polyphosphates and Ca2+ is low at neutral pH.26 This role would

Caddisfly Larval Silks

Biomacromolecules, Vol. 11, No. 4, 2010

973

Table 2. Amino Acid Composition of Four Caddisfly and Two Moth Species residue

B. echoa (mol % ( s.d.)

L. decipiens (mol %)

R. obliterata (mol %)

H. augustipennis (mol %)

B. mori (mol %)

G. mellonella (mol %)

Gly Ala Ser Thr Ile Leu Val Tyr Phe Pro Asx Glx Arg His Lys

20.1 ( 0.4 6.3 ( 1.1 15.4 ( 1.8 3.2 ( 0.5 3.8 ( 0.4 6.0 ( 0.6 4.1 ( 0.5 4.1 ( 0.8 1.2 ( 0.06 4.0 ( 0.3 11.7 ( 1.6 3.5 ( 0.4 8.8 ( 0.5 0.7 ( 0.2 4.2 ( 0.4

24.6 0.4 17.2 2.3 4.3 5.0 12.2 2.7 0.8 4.8 2.6 3.5 14.1 0.2 2.3

24.9 1.9 14.7 1.9 9.1 9.5 5.9 1.8 0.2 3.3 3.8 4.0 7.7 6.2 3.3

19.4 4.5 12.5 2.5 6.6 5.4 9.4 6.2 0.1 9.6 3.4 3.8 9.6 2.7 2.1

45.9 30.3 12.1 0.9 0.2 0.1 1.8 5.3 0.6 0.3 0.9 0.8 0.3 0.1 0.2

28.6 21.3 17.0 3.2 4.2 6.6 6.2 0.5 0.4 3.8 2.8 2.5 1.6 0.1 0.3

a Experimental amino acid composition from four independent analyses of B. echo silk. The amino acid compositions of the other species were deduced from H-fibroin sequences available in GenBank.

Table 3. Elements in B. echo Silk Proteina element

sample 1 (nmol ( sd)

sample 2 (nmol ( sd)

Ca Fe Mg Mn Zn S P

55.7 ( 3.39 14.4 ( 0.07 24.0 ( 0.09 1.0 ( 0.0 3.1 ( 0.02 38.5 ( 0.35 114.2 ( 0.48

123.0 ( 3.16 28.7 ( 0.06 15.4 ( 0.11 0.90 ( 0.0 3.5 ( 0.01 55.4 ( 0.23 164.2 ( 0.14

a The amounts are nmols per 70 mg of glass beads from caddisfly cases after background subtraction. Backgrounds were determined with an equivalent mass of non-bonded glass beads collected from the same aquarium.

phase separation as complex coacervates. This hypothesis is analogous in some regards to the complex coacervation model suggested for formation of the underwater adhesive of P. californica from polycationic proteins, divalent cations, and highly phosphorylated proteins.18 Complex coacervation occurs when oppositely charged polyelectrolytes associate in aqueous solution through mutual charge neutralization. When the solution is near net charge neutrality a dense concentrated polymer aqueous phase separates from a polymer depleted aqueous phase driven in part by entropic gains from the release of small counterions and water.29 In subsequent steps in the fiber formation process, stress-induced elongation and reorganization of the coacervated protein phase could lead to nanofibril formation, additional charge neutralization, and dehydration of the fiber during extrusion. Perfect registry of the oppositely charged segments could cause the proteins to precipitate, while some imperfections in charge alignments would result in retained counterions and water to provide localized plasticity.

Conclusions

Figure 3. Schematic diagram of hypothetical repeating domain structure formed by phosphoserine and Ca2+.

be analogous to water exclusion by extensive β-sheet formation in dry silks.21 Dehydration of water-borne proteins through electrostatic charge neutralization may be an under appreciated phenomenon driving the formation of insoluble biological materials from water-soluble components in the presence of water. At a longer length scale, phase separation of alternating hydrophilic and hydrophobic blocks is a major aspect of silk fiber assembly models for both spiders and silkworms. Their amphiphilic structures may lead first to liquid crystal27 or micelle formation28 in the posterior silk gland, then fibril formation as staggered amphiphilic blocks associate laterally during stressinduced elongation of silk proteins during fiber extrusion.11,12 Aquatic caddisflies may use a mechanism with broad similarities but key variations. Rather than alternating hydrophilic and hydrophobic blocks, staggered electrostatic association of alternating blocks with opposite charge may drive liquid-liquid

In conclusion, caddisfly silks share many design features common to spider and moth silks. Features that include blocky and highly repetitive sequences in H-fibroin cores, nonrepetitive terminal domains, conserved terminal domain cysteine residues that may covalently link H- and L-fibroin into a 1:1 complex, and alternating blocks of dissimilar amino acids: hydrophilic versus hydrophobic in spiders and moths, and positive versus negatively charged in caddisflies. Important molecular adaptations for underwater silks may be the accumulation of basic residues in their sequences and multiple phosphorylation of repeating serine-rich motifs. It appears that a fairly simple biochemical change, phosphorylation of serines that are also present in their moth and butterfly terrestrial cousins, may be largely responsible for adapting a dry ancestral silk to the underwater environment of caddisfly larva. The phosphorylated underwater caddisfly silks add a fascinating variation to the already rich diversity of silk materials that have remarkable but so far unreplicable mechanical properties. Acknowledgment. R.J.S. thanks Dr. Christy Geraci at the Smithsonian Institute for the introduction to the wonderful diversity of caddisflies. The authors thank Drs. Dennis Winge and Hyung Kim for amino acid and elemental analysis, Drs. Krishna Parsawar and Chad Nelson for expert help with peptide mass spectrometry, Nic Ashton for collecting caddisfly lar-

974

Biomacromolecules, Vol. 11, No. 4, 2010

vae, and Fred Hayes for photography. This work was supported by a grant (#0906014) from the NSF Division of Materials Research.

References and Notes (1) LaFontaine, G., Caddisflies; The Lyons Press: Guilford, CT, 1981. (2) Wiggins, G. B.; Currie, D. C., Trichoptera Families. In An Introduction to the Aquatic Insects of North America; Merritt, R. W., Cummins, K. W., Berg, M. B., Eds.; Kendall/Hunt Publishing Company: Dubuque, IA, 2008. (3) Inoue, S.; Tanaka, K.; Arisaka, F.; Kimura, S.; Ohtomo, K.; Mizuno, S. Silk fibroin of Bombyx mori is secreted, assembling a high molecular mass elementary unit consisting of H-chain, L-chain, and P25, with a 6:6:1 molar ratio. J. Biol. Chem. 2000, 275 (51), 40517– 28. (4) Tanaka, K.; Kajiyama, N.; Ishikura, K.; Waga, S.; Kikuchi, A.; Ohtomo, K.; Takagi, T.; Mizuno, S. Determination of the site of disulfide linkage between heavy and light chains of silk fibroin produced by Bombyx mori. Biochim. Biophys. Acta 1999, 1432 (1), 92–103. (5) Sehnal, F.; Sutherland, T. Silks produced by insect labial glands. Prion 2008, 2 (4), 145–153. (6) Sehnal, F.; Akai, H. Insect silk glands: their types, development, and function, and effects of environmental factors and morphogenetic hormones on them. Int. J. Insect Morphol. Embryol. 1990, 19 (2), 79–132. (7) Wang, Y.; Sanai, K.; Wen, H.; Zhao, T.; Nakagaki, M. Characterization of unique heavy chain fibroin filaments spun underwater by the caddisfly Stenopsyche marmorata (Trichoptera; Stenopsychidae). Mol. Biol. Rep. 2009. (8) Engster, M. Studies on silk secretion in the Trichoptera (F. Limnephilidae). I. Histology, histochemistry, and ultrastructure of the silk glands. J. Morph. 1976, 150, 183–212. (9) Yonemura, N.; Sehnal, F.; Mita, K.; Tamura, T. Protein composition of silk filaments spun under water by caddisfly larvae. Biomacromolecules 2006, 7 (12), 3370–8. (10) Yonemura, N.; Mita, K.; Tamura, T.; Sehnal, F. Conservation of silk genes in Trichoptera and Lepidoptera. J. Mol. EVol. 2009, 68 (6), 641– 53. (11) Bini, E.; Knight, D. P.; Kaplan, D. L. Mapping domain structures in silks from insects and spiders related to protein assembly. J. Mol. Biol. 2004, 335 (1), 27–40. (12) Sehnal, F.; Zurovec, M. Construction of silk fiber core in lepidoptera. Biomacromolecules 2004, 5 (3), 666–74.

Stewart and Wang (13) Simmons, A. H.; Michal, C. A.; Jelinski, L. W. Molecular orientation and two-component nature of the crystalline fraction of spider dragline silk. Science 1996, 271 (5245), 84–7. (14) Perkins, D. N.; Pappin, D. J.; Creasy, D. M.; Cottrell, J. S. Probabilitybased protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 1999, 20 (18), 3551–67. (15) Waite, J. H.; Qin, X. Polyphosphoprotein from the adhesive pads of Mytilus edulis. Biochemistry 2001, 40 (9), 2887–93. (16) Flammang, P.; Lambert, A.; Bailly, P.; Hennebert, E. Polyphosphoprotein-containing marine adhesives. J. Adhes. 2009, 85, 447–464. (17) Zhao, H.; Sun, C.; Stewart, R. J.; Waite, J. H. Cement proteins of the tube-building polychaete Phragmatopoma californica. J. Biol. Chem. 2005, 280 (52), 42938–44. (18) Stewart, R. J.; Weaver, J. C.; Morse, D. E.; Waite, J. H. The tube cement of Phragmatopoma californica: a solid foam. J. Exp. Biol. 2004, 207 (Pt 26), 4727–34. (19) Hui, S. Y.; Herve, A.; Kiplinger, J. Phosphate polymerizable adhesion promoters. JCT Coat. Technol. 2005, 2 (13), 44–49. (20) Janda, R.; Homburg, B.; Eppinger, B. Photopolymerizable phosphatecontaining adhesion promoting dental composition. U.S. Patent 4,640,936, 1987. (21) Porter, D.; Vollrath, F. Silk as a biomimetic ideal for structural polymers. AdV. Mater. 2009, 21, 487–492. (22) Lassila, K. S.; Datta, D.; Mayo, S. L. Evaluation of the energetic contribution of an ionic network to β-sheet stability. Protein Sci. 2002, 11 (3), 688–90. (23) Winkler, S.; Wilson, D.; Kaplan, D. L. Controlling β-sheet assembly in genetically engineered silk by enzymatic phosphorylation/dephosphorylation. Biochemistry 2000, 39 (41), 12739–46. (24) Brown, S. A.; Ruxton, G. D.; Humphries, S. Physical properties of Hydropsyche siltalai (Trichoptera) net silk. J. North Am. Benthol. Soc. 2004, 23 (4), 771–779. (25) Engster, M. Studies on silk secretion in the Trichoptera (F. limnephilidae). Cell Tissue Res. 1976, 169, 77–92. (26) Shao, H.; Stewart, R. J. Biomimetic underwater adhesives with environmentally triggered setting mechanisms. AdV. Mater. 2009, 21. (27) Vollrath, F.; Knight, D. P. Liquid crystalline spinning of spider silk. Nature 2001, 410, 541–548. (28) Jin, H. J.; Kaplan, D. L. Mechanism of silk processing in insects and spiders. Nature 2003, 424 (6952), 1057–61. (29) Bungenberg de Jong, H. G. Morphology of coacervates. In Colloid Science; Kruyt, H. R., Ed.; Elsevier Publishing Company, Inc.: New York, 1949; Vol. II, pp 431-482.

BM901426D