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Infiltration of chitin by protein coacervates defines the squid beak mechanical gradient

Nature Chemical Biology volume 11, pages 488–495 (2015)Cite this article

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Abstract

The beak of the jumbo squid Dosidicus gigas is a fascinating example of how seamlessly nature builds with mechanically mismatched materials. A 200-fold stiffness gradient begins in the hydrated chitin of the soft beak base and gradually increases to maximum stiffness in the dehydrated distal rostrum. Here, we combined RNA-Seq and proteomics to show that the beak contains two protein families. One family consists of chitin-binding proteins (DgCBPs) that physically join chitin chains, whereas the other family comprises highly modular histidine-rich proteins (DgHBPs). We propose that DgHBPs play multiple key roles during beak bioprocessing, first by forming concentrated coacervate solutions that diffuse into the DgCBP-chitin scaffold, and second by inducing crosslinking via an abundant GHG sequence motif. These processes generate spatially controlled desolvation, resulting in the impressive biomechanical gradient. Our findings provide novel molecular-scale strategies for designing functional gradient materials.

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Figure 1: D. gigas beaks and protein extraction from a beak.
Figure 2: Schematic representations of DgCBP-1–DgCBP-4 and DgHBP-1–DgHBP-3.
Figure 3: Primary sequence and chemical characteristics of DgHBPs.
Figure 4: Coacervation and characterization of recDgHBP-1.
Figure 5: Self-coacervation and crosslinking of hydrophobic DgHBP-pep.
Figure 6: Proposed model for jumbo squid beak gradient formation.

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References

  1. Farren, L., Shayler, S. & Ennos, A.R. The fracture properties and mechanical design of human fingernails. J. Exp. Biol. 207, 735–741 (2004).

    Article  CAS  PubMed  Google Scholar 

  2. Benjamin, M. et al. Where tendons and ligaments meet bone: attachment sites ('entheses') in relation to exercise and/or mechanical load. J. Anat. 208, 471–490 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Lu, H.H. & Thomopoulos, S. Functional attachment of soft tissues to bone: development, healing, and tissue engineering. Annu. Rev. Biomed. Eng. 15, 201–226 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Zajac, F.E. Muscle and tendon: properties, models, scaling, and application to biomechanics and motor control. Crit. Rev. Biomed. Eng. 17, 359–411 (1989).

    CAS  PubMed  Google Scholar 

  5. Sun, C.J. & Waite, J.H. Mapping chemical gradients within and along a fibrous structural tissue, mussel byssal threads. J. Biol. Chem. 280, 39332–39336 (2005).

    Article  CAS  PubMed  Google Scholar 

  6. Miserez, A., Schneberk, T., Sun, C., Zok, F.W. & Waite, J.H. The transition from stiff to compliant materials in squid beaks. Science 319, 1816–1819 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Suresh, S. Graded materials for resistance to contact deformation and damage. Science 292, 2447–2451 (2001).

    Article  CAS  PubMed  Google Scholar 

  8. Waite, J.H., Lichtenegger, H.C., Stucky, G.D. & Hansma, P. Exploring molecular and mechanical gradients in structural bioscaffolds. Biochemistry 43, 7653–7662 (2004).

    Article  CAS  PubMed  Google Scholar 

  9. Mikos, A.G. et al. Engineering complex tissues. Tissue Eng. 12, 3307–3339 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Helton, K.L., Ratner, B.D. & Wisniewski, N.A. Biomechanics of the sensor-tissue interface-effects of motion, pressure, and design on sensor performance and the foreign body response-part I: theoretical framework. J. Diabetes Sci. Technol. 5, 632–646 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  11. Fleckman, P. & Olerud, J.E. Models for the histologic study of the skin interface with percutaneous biomaterials. Biomed. Mater. 3, 034006 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Fox, J.D., Capadona, J.R., Marasco, P.D. & Rowan, S.J. Bioinspired water-enhanced mechanical gradient nanocomposite films that mimic the architecture and properties of the squid beak. J. Am. Chem. Soc. 135, 5167–5174 (2013).

    Article  CAS  PubMed  Google Scholar 

  13. Zvarec, O., Purushotham, S., Masic, A., Ramanujan, R.V. & Miserez, A. Catechol-functionalized chitosan/iron oxide nanoparticle composite inspired by mussel thread coating and squid beak interfacial chemistry. Langmuir 29, 10899–10906 (2013).

    Article  CAS  PubMed  Google Scholar 

  14. Miserez, A., Rubin, D. & Waite, J.H. Cross-linking chemistry of squid beak. J. Biol. Chem. 285, 38115–38124 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Rubin, D.J., Miserez, A. & Waite, J.H. Diverse strategies of protein sclerotization in marine invertebrates: structure-property relationships in natural biomaterials. Adv. Insect Physiol. 38, 75–133 (2010).

    Article  Google Scholar 

  16. Dilly, P.N. & Nixon, M. The cells that secrete the beaks in octopods and squids (Mollusca, Cephalopoda). Cell Tissue Res. 167, 229–241 (1976).

    Article  CAS  PubMed  Google Scholar 

  17. Grabherr, M.G. et al. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat. Biotechnol. 29, 644–652 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. de Kruif, C.G., Weinbreck, F. & De Vries, R.K. Complex coacervation of proteins and anionic polysaccharides. Curr. Opin. Colloid Interface Sci. 9, 340–349 (2004).

    Article  CAS  Google Scholar 

  19. Smith, B.J. in The Protein Protocols Handbook (ed. Walker, J.M.) 507–510 (Humana Press Inc., 2002).

  20. Miserez, A., Li, Y., Waite, J.H. & Zok, F. Jumbo squid beaks: inspiration for design of robust organic composites. Acta Biomater. 3, 139–149 (2007).

    Article  CAS  PubMed  Google Scholar 

  21. Sahal, D. et al. Specific and instantaneous one-step chemodetection of histidine-rich proteins by Pauly's stain. Anal. Biochem. 308, 405–408 (2002).

    Article  CAS  PubMed  Google Scholar 

  22. de Castro, E. et al. ScanProsite: detection of PROSITE signature matches and ProRule-associated functional and structural residues in proteins. Nucleic Acids Res. 34, W362–W365 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Li, A.Q. et al. Chemical cleavage at aspartyl residues for protein identification. Anal. Chem. 73, 5395–5402 (2001).

    Article  CAS  PubMed  Google Scholar 

  24. Tan, Y., Yildiz, U.H., Wei, W., Waite, J.H. & Miserez, A. Layer-by-layer polyelectrolyte deposition: a mechanism for forming biocomposite materials. Biomacromolecules 14, 1715–1726 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Mithieux, S.M. & Weiss, A.S. in Fibrous Proteins: Coiled-Coils, Collagen and Elastomers (Advances in Protein Chemistry vol. 7) (eds. Parry, D.A.D. & Squire, J.M.) 437–461 (Elsevier Academic Press Inc., 2005).

  26. Yeo, G.C., Keeley, F.W. & Weiss, A.S. Coacervation of tropoelastin. Adv. Colloid Interface Sci. 167, 94–103 (2011).

    Article  CAS  PubMed  Google Scholar 

  27. Wei, W. et al. A mussel-derived one component adhesive coacervate. Acta Biomater. 10, 1663–1670 (2014).

    Article  CAS  PubMed  Google Scholar 

  28. Ortony, J.H., Hwang, D.S., Franck, J.M., Waite, J.H. & Han, S. Asymmetric collapse in biomimetic complex coacervates revealed by local polymer and water dynamics. Biomacromolecules 14, 1395–1402 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Kizilay, E., Kayitmazer, A.B. & Dubin, P.L. Complexation and coacervation of polyelectrolytes with oppositely charged colloids. Adv. Colloid Interface Sci. 167, 24–37 (2011).

    Article  CAS  PubMed  Google Scholar 

  30. Betre, H., Setton, L.A., Meyer, D.E. & Chilkoti, A. Characterization of a genetically engineered elastin-like polypeptide for cartilaginous tissue repair. Biomacromolecules 3, 910–916 (2002).

    Article  CAS  PubMed  Google Scholar 

  31. Spruijt, E., Sprakel, J., Cohen-Stuart, M.A. & van der Gucht, J. Interfacial tension between a complex coacervate phase and its coexisting aqueous phase. Soft Matter 6, 172–178 (2010).

    Article  CAS  Google Scholar 

  32. Weinbreck, F., de Vries, R., Schrooyen, P. & de Kruif, C.G. Complex coacervation of whey proteins and gum arabic. Biomacromolecules 4, 293–303 (2003).

    Article  CAS  PubMed  Google Scholar 

  33. Kaibara, K., Okazaki, T., Bohidar, H.B. & Dubin, P.L. pH-induced coacervation in complexes of bovine serum albumin and cationic polyelectrolytes. Biomacromolecules 1, 100–107 (2000).

    Article  CAS  PubMed  Google Scholar 

  34. Lin, J. & Brown, C.W. Spectroscopic measurement of NaCl and seawater salinity in the near-IR region of 680–1230 nm. Appl. Spectrosc. 47, 239–241 (1993).

    Article  CAS  Google Scholar 

  35. Liu, B., Burdine, L. & Kodadek, T. Chemistry of periodate-mediated cross-linking of 3,4-dihydroxylphenylalanine-containing molecules to proteins. J. Am. Chem. Soc. 128, 15228–15235 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Kerwin, J.L. et al. Mass spectrometric analysis of catechol-histidine adducts from insect cuticle. Anal. Biochem. 268, 229–237 (1999).

    Article  CAS  PubMed  Google Scholar 

  37. Guerette, P.A. et al. Accelerating the design of biomimetic materials by integrating RNA-seq with proteomics and materials science. Nat. Biotechnol. 31, 908–915 (2013).

    Article  CAS  PubMed  Google Scholar 

  38. Zhao, H., Sun, C., Stewart, R.J. & Waite, J.H. Cement proteins of the tube-building polychaete Phragmatopoma californica. J. Biol. Chem. 280, 42938–42944 (2005).

    Article  CAS  PubMed  Google Scholar 

  39. Oparin, A.I. & Synge, A. The Origin of Life on the Earth 3rd edn. (Academic Press, 1957).

  40. Hwang, D.S. et al. Viscosity and interfacial properties in a mussel-inspired adhesive coacervate. Soft Matter 6, 3232–3236 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Fournier, R.L. Basic Transport Phenomena in Biomedical Engineering 3rd edn. (CRC Press, Taylor & Francis Group, 2012).

  42. Vrhovski, B. & Weiss, A.S. Biochemistry of tropoelastin. Eur. J. Biochem. 258, 1–18 (1998).

    Article  CAS  PubMed  Google Scholar 

  43. Giesa, T. & Buehler, M.J. Nanoconfinement and the strength of biopolymers. Annu. Rev. Biophys. 42, 651–673 (2013).

    Article  CAS  PubMed  Google Scholar 

  44. Gosline, J.M., Guerette, P.A., Ortlepp, C.S. & Savage, K.N. The mechanical design of spider silks: from fibroin sequence to mechanical function. J. Exp. Biol. 202, 3295–3303 (1999).

    CAS  PubMed  Google Scholar 

  45. d'Orbigny, A.D. et al. Voyage dans l'Amérique méridionale: Tome Neuviéme (Pitois-Levrault, Paris, 1847).

  46. Schägger, H. Tricine-SDS-PAGE. Nat. Protoc. 1, 16–22 (2006).

    Article  PubMed  Google Scholar 

  47. Paz, M.A., Fluckiger, R., Boak, A., Kagan, H.M. & Gallop, P.M. Specific detection of quinoproteins by redox-cycling staining. J. Biol. Chem. 266, 689–692 (1991).

    CAS  PubMed  Google Scholar 

  48. Stajich, J.E. et al. The Bioperl toolkit: Perl modules for the life sciences. Genome Res. 12, 1611–1618 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Shevchenko, A., Tomas, H., Havlis, J., Olsen, J.V. & Mann, M. In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat. Protoc. 1, 2856–2860 (2006).

    Article  CAS  PubMed  Google Scholar 

  50. Edgar, R.C. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Waterhouse, A.M., Procter, J.B., Martin, D.M.A., Clamp, M. & Barton, G.J. Jalview Version 2-a multiple sequence alignment editor and analysis workbench. Bioinformatics 25, 1189–1191 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Larkin, M.A. et al. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948 (2007).

    CAS  PubMed  Google Scholar 

  53. Newman, A.M. & Cooper, J.B. XSTREAM: A practical algorithm for identification and architecture modeling of tandem repeats in protein sequences. BMC Bioinformatics 8, 382 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  54. Sievers, F. et al. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol. Syst. Biol. 7, 539 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  55. Rebers, J.E. & Willis, J.H. A conserved domain in arthropod cuticular proteins binds chitin. Insect Biochem. Mol. Biol. 31, 1083–1093 (2001).

    Article  CAS  PubMed  Google Scholar 

  56. Goodarzi, M.T., Fotinopoulou, A. & Turner, G.A.A. in The Protein Protocols Handbook Springer Protocols Handbooks (ed. Walker, J.M.) 1205–1214 (Humana Press, 2009).

  57. Hwang, D.S., Waite, J.H. & Tirrell, M. Promotion of osteoblast proliferation on complex coacervation-based hyaluronic acid—recombinant mussel adhesive protein coatings on titanium. Biomaterials 31, 1080–1084 (2010).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

The authors would like to thank J. Pavlovich of the UCSB Mass Spectrometry Facility for his help with obtaining nanoLC-MS/MS data. We thank D. DeMartini (UCSB) and the crew of the R/V New Horizon for obtaining squid beak samples from the Guayamas Basin (Mexico), and K.W. Kong and V. Gangu (Molecular Engineering Lab, Agency for Science, Technology and Research (A*STAR), Singapore) for technical assistance with the RNA-Seq analysis. Some of the work was conducted in the Biological NanoStructures Laboratory within the California NanoSystems Institute, supported by the University California, Santa Barbara, and the University of California, Office of the President. This work was supported in whole or in part by US National Institutes of Health Grant R01-DE018468 and the Materials Research Science & Engineering Centers (MRSEC) Program of the US National Science Foundation under Award No. DMR 1121053 (J.H.W.). A.M. was supported by the Singapore National Research Foundation (NRF) through a NRF Fellowship, and S.H. by the Biomedical Research Council, A*STAR, Singapore.

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Authors and Affiliations

  1. Biomolecular Science and Engineering Program, University of California, Santa Barbara, California, USA

    YerPeng Tan & J Herbert Waite

  2. Molecular Engineering Lab, Biomedical Sciences Institutes, Agency for Science, Technology and Research (A*STAR), Singapore

    Shawn Hoon

  3. School of Biological Sciences, Nanyang Technological University, Singapore

    Shawn Hoon & Ali Miserez

  4. Energy Research Institute at Nanyang Technological University (ERI@N), Nanyang Technological University, Singapore

    Paul A Guerette

  5. Biological & Biomimetic Material Laboratory, School of Materials Science & Engineering, Nanyang Technological University, Singapore

    Paul A Guerette, Ali Ghadban, Cai Hao & Ali Miserez

  6. Materials Research Laboratory, University of California, Santa Barbara, California, USA

    Wei Wei & J Herbert Waite

  7. Department of Molecular Cellular and Developmental Biology, University of California, Santa Barbara, California, USA

    J Herbert Waite

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Contributions

Y.T. carried out beak protein extraction and MS/MS experiments and prepared figures and tables. P.A.G. extracted and purified RNA samples, conducted 3′ RACE and designed DgHBP-1 cloning, expression and purification experiments. S.H. performed and analyzed RNA-Seq, did transcriptome analysis and RACE sequence confirmation and prepared figures. P.A.G., S.H., Y.T. and A.M. analyzed protein sequence data. C.H. expressed, purified and analyzed DgHBP-1. A.G. helped to purify DgHBP-1 and conducted rheological experiments. W.W. designed and carried out coacervation and infiltration experiments on recDgHBP-1. A.G. and C.H. conducted the characterization and infiltration of coacervates under the supervision of P.A.G. and A.M. Y.T. and W.W. carried out and analyzed coacervation and crosslinking experiments on DgHBP-pep. Y.T., J.H.W., P.A.G., S.H. and A.M. wrote the manuscript. J.H.W. and A.M. designed and supervised the research.

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Correspondence to Ali Miserez or J Herbert Waite.

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Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Tables 1–6 and Supplementary Figures 1–10. (PDF 15591 kb)

Coacervation of recDgHBP-1.

Part 1: Formation of a coacervate phase when a dilute solution of recDgHBP-1 is added to a 100 mM PBS buffer (pH 6.5), with a protein:buffer volume ratio of 1:9. Part 2: Dispersion of the coacervate phase by stirring. (MP4 14095 kb)

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Tan, Y., Hoon, S., Guerette, P. et al. Infiltration of chitin by protein coacervates defines the squid beak mechanical gradient. Nat Chem Biol 11, 488–495 (2015). https://doi.org/10.1038/nchembio.1833

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