Comparative Analysis of the Adhesive Proteins of the Adult Stalked Goose Barnacle Pollicipes pollicipes (Cirripedia: Pedunculata)

Abstract

Adhesion in barnacles is still poorly understood. The cement gland secretes an insoluble multi-protein complex, which adheres very strongly to a variety of substrates in the presence of water. This adhesion mechanism is bioinspiring for the engineering of new adhesive materials, but to replicate this adhesive system, the genes coding for the cement constitutive proteins must be identified and elucidated, and their products characterised. Here, the complete sequences of three cement protein (CP) genes (CP-100K, CP-52K, and CP-19K) isolated from the cement gland of the stalked barnacle Pollicipes pollicipes (order Scalpelliformes) were obtained using RACE PCR. The three genes were compared to the 23 other acorn barnacle CP genes so far sequenced (order Sessilia) to determine common and differential patterns and molecular properties, since the adhesives of both orders have visibly different characteristics. A shotgun proteomic analysis was performed on the cement, excreted at the membranous base of specimens, where the products of the three genes sequenced in the gland were identified, validating their function as CPs. A principal component analysis (PCA) was performed, to cluster CPs into groups with similar amino acid composition. This analysis uncovered three CP groups, each characterised by similar residue composition, features in secondary structure, and some biochemical properties, including isoelectric point and residue accessibility to solvents. The similarity among proteins in each defined group was low despite comparable amino acid composition. PCA can identify putative adhesive proteins from NGS transcriptomic data regardless of their low homology. This analysis did not highlight significant differences in residue composition between homologous acorn and stalked barnacle CPs. The characteristics responsible for the structural differences between the cement of stalked and acorn barnacles are described, and the presence of nanostructures, such as repetitive homologous domains and low complexity regions, and repetitive β-sheets are discussed relatively to self-assembly and adhesion.

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References

  1. Almeida JR, Correia-da-Silva M, Sousa E, Antunes J, Pinto M, Vasconcelos V, Cunha I (2017) Antifouling potential of nature-inspired sulfated compounds. Sci Rep 7:42424

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Anderson DT (1994) Reproductive system, mating and oviposition. In: Anderson DT (ed) Barnacles. Structure, function, development and evolution, 1st edn. Chapman & Hall, London, pp 127–170

    Google Scholar 

  3. Blobel G, Dobberstein B (1975) Transfer of proteins across membranes: I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma. J Cell Biol 67:835–851

    Article  CAS  PubMed  Google Scholar 

  4. Boëtius J (1952) Some notes on the relation to the substratum of Lepas anatifera L. and Lepas. Oikos 4:112–117

    Article  Google Scholar 

  5. Cejas MA, Kinney WA, Chen C, Vinter JG, Almond HR, Balss KM, Maryanoff CA, Schmidt U, Breslav M, Mahan A, Lacy E, Maryanoff BE (2008) Thrombogenic collagen-mimetic peptides: self-assembly of triple helix-based fibrils driven by hydrophobic interactions. Proc Natl Acad Sci U S A 105:8513–8518

    Article  PubMed  PubMed Central  Google Scholar 

  6. Clancy SK, Sodano A, Cunningham DJ, Huang SS, Zalicki PJ, Shin S, Ahn BK (2016) Marine bioinspired underwater contact adhesion. Biomacromolecules 17:1869–1874

    Article  CAS  PubMed  Google Scholar 

  7. Cruz T, Castro JJ, Hawkins SJ (2010) Recruitment, growth and population size structure of Pollicipes pollicipes in SW Portugal. J Exp Mar Biol Ecol 392(1–2):200–209

    Article  Google Scholar 

  8. Cui M, Ren S, Wei S, Sun C, Zhong C (2017) Natural and bio-inspired underwater adhesives. Curr Prog New Persp 5(11):116102

    Google Scholar 

  9. Delaney JS (2004) ESOL: estimating aqueous solubility directly from molecular structure. J Chem Inf Comput Sci 44:1000–1005

    Article  CAS  PubMed  Google Scholar 

  10. Dickinson GH, Vega IE, Wahl KJ, Orihuela B, Beyley V, Rodriguez EN, Everett RK, Bonaventura J, Rittschof D (2009) Barnacle cement: a polymerization model based on evolutionary concepts. J Exp Biol 212:3499–3510

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Frey S, Richter RP, Gorlich D (2006) FG-rich repeats of nuclear pore proteins form a three-dimensional meshwork with hydrogel-like properties. Science 314:815–817

    Article  CAS  PubMed  Google Scholar 

  12. Fyhn UE, Costlow JD (1976) A histological study of cement secretion during the intermolt cycle in barnacles. Biol Bull 150:47–56

    Article  CAS  PubMed  Google Scholar 

  13. Gohad NV, Aldred N, Hartshorn CM, Jong Lee Y, Cicerone MT, Orihuela B, Clare AS, Rittschof D, Mount AS (2014) Synergistic roles for lipids and proteins in the permanent adhesive of barnacle larvae. Nat Commun 5:1–9

    Article  CAS  Google Scholar 

  14. Guruprasad K, Reddy BVB, Pandit MW (1990) Correlation between stability of a protein and its dipeptide composition: a novel approach for predicting in vivo stability of a protein from its primary sequence. Prot Eng 4:155–161

    Article  CAS  Google Scholar 

  15. He LS, Zhang G, Qian PY (2013) FG-rich repeats of nuclear pore proteins form a three-dimensional meshwork with hydrogel-like properties. PLoS One 8:1–9

    CAS  Google Scholar 

  16. Hennebert E, Maldonado B, Ladurner P, Flammang P, Santos R (2014) Experimental strategies for the identification and characterization of adhesive proteins in animals: a review. Interface Focus 5:20140064

    Article  Google Scholar 

  17. Hoffman DL (1984) Size-frequency distribution patterns of the juvenile stages of the pedunculate barnacle, Pollicipes polymerus Sowerby, 1833 (Cirripedia, Lepadomorpha). Crustaceana 46:295–299

    Article  Google Scholar 

  18. Hughes CS, Foehr S, Garfield DA, Furlong EE, Steinmetz LM, Krijgsveld J (2014) Ultrasensitive proteome analysis using paramagnetic bead technology. Mol Syst Biol 10:757

    Article  CAS  PubMed  Google Scholar 

  19. Ikai AJ (1980) Thermostability and aliphatic index of globular proteins. J Biochem 88:1895–1898

    CAS  Google Scholar 

  20. Jonker J-L, von Byern J, Flammang P, Klepal W, Power AM (2012) Unusual adhesive production system in the barnacle Lepas anatifera: an ultrastructural and histochemical investigation. J Morphol 273:1377–1391

    Article  PubMed  Google Scholar 

  21. Jonker JL, Abram F, Pires E, Varela Coelho A, Grunwald I, Power AM (2014) Adhesive proteins of stalked and acorn barnacles display homology with low sequence similarities. PLoS One 9:e108902

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Jonker J-L, Morrison L, Lynch EP, Grunwald I, von Byern J, Power AM (2015) The chemistry of stalked barnacle adhesive (Lepas anatifera). Interface Focus 5:20140062

    Article  PubMed  PubMed Central  Google Scholar 

  23. Kamino K (2006) Barnacle underwater attachment. In: Biological adhesives. Springer Berlin Heidelberg, Heidelberg, pp 145–166

    Google Scholar 

  24. Kamino K (2008) Underwater adhesive of marine organisms as the vital link between biological science and material science. Mar Biotechnol 10:111–121

    Article  CAS  PubMed  Google Scholar 

  25. Kamino K (2010) Molecular design of barnacle cement in comparison with those of mussel and tubeworm. J Adhes 86:96–110

    Article  CAS  Google Scholar 

  26. Kamino K (2013) Mini-review: barnacle adhesives and adhesion. Biofouling 29:735–749

    Article  CAS  PubMed  Google Scholar 

  27. Kamino K (2016) Barnacle underwater attachment. In: Biological adhesives. Springer, Cham, pp 153–176

    Google Scholar 

  28. Kamino K, Odo S, Maruyama T (1996) Cement proteins of the acorn barnacle, Megabalanus rosa. Biol Bull 190:403–409

    Article  CAS  PubMed  Google Scholar 

  29. Kamino K, Inoue K, Maruyama T, Takamatsu N, Harayama S, Shizuri Y (2000) Barnacle cement proteins. Importance of disulfide bonds in their insolubility. J Biol Chem 275:27360–27365

    CAS  PubMed  Google Scholar 

  30. Kamino K, Nakano M, Kanai S (2012) Significance of the conformation of building blocks in curing of barnacle underwater adhesive. FEBS J 279:1750–1760

    Article  CAS  PubMed  Google Scholar 

  31. Kugele M, Yule AB (2000) Active relocation in lepadomorph barnacles. J Mar Biol Assoc UK 80:103–111

    Article  Google Scholar 

  32. Kyte J, Doolittle RF (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol 57:105–132

    Article  Google Scholar 

  33. Lee H, Dellatore SH, Miller WM, Messersmith PB (2007) Mussel-Inspired Surface Chemistry for Multifunctional Coatings. Science 318(5849):426

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Lin HC, Wong YH, Tsang LM, Chu KH, Qian PY, Chan BKK (2014) First study on gene expression of cement proteins and potential adhesion-related genes of a membranous-based barnacle as revealed from next-generation sequencing technology. Biofouling 30:169–181

    Article  CAS  PubMed  Google Scholar 

  35. Lobo-da-Cunha A, Alves Ï, Oliveira E, Cunha I (2017) The cement apparatus of the stalked barnacle Pollicipes pollicipes. Mar Biol 164:11

    Article  CAS  Google Scholar 

  36. Meusemann K, Von Reumont BM, Simon S et al (2010) A phylogenomic approach to resolve the arthropod tree of life. Mol Biol Evol 27:2451–2464

    Article  CAS  PubMed  Google Scholar 

  37. Minchin D (1996) Tar pellets and plastics as attachment surfaces for lepadid cirripedes in the North Atlantic Ocean. Mar Pollut Bull 32:855–859

    Article  CAS  Google Scholar 

  38. Molliex A, Temirov J, Lee J, Coughlin M, Kanagaraj AP, Kim HJ, Mittag T, Taylor JP (2015) Phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization. Cell 163:123–133

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Mori Y, Urushida Y, Nakano M, Uchiyama S, Kamino K (2007) Calcite-specific coupling protein in barnacle underwater cement. FEBS J 274:6436–6446

    Article  CAS  PubMed  Google Scholar 

  40. Moriarty JE, Sachs JA, Jones K (2008) Directional locomotion in a turtle barnacle, Chelonibia testudinaria, on green turtles, Chelonia mydas. Mar Turt Newsl 119:1–4

    Google Scholar 

  41. Murr MM, Morse DE (2005) Fractal intermediates in the self-assembly of silicatein filaments. Proc Natl Acad Sci 102:11657–11662

    Article  CAS  PubMed  Google Scholar 

  42. Nakano M, Kamino K (2015) Amyloid-like conformation and interaction for the self-assembly in barnacle underwater cement. Biochemistry 54:826–835

    Article  CAS  PubMed  Google Scholar 

  43. Naldrett MJ (1993) The importance of sulphur cross-links and hydrophobic interactions in the polymerization of barnacle cement. J Mar Biol Assoc UK 73:689–702

    Article  CAS  Google Scholar 

  44. Naldrett MJ, Kaplan DL (1997) Characterization of barnacle (Balanus eburneus and B. cenatus) adhesive proteins. Mar Biol 127:629–635

    Article  CAS  Google Scholar 

  45. Nishida J, Higaki Y, Takahara A (2015) Synthesis and characterization of barnacle adhesive mimetic towards underwater adhesion. Chem Lett 44:1047–1049

    Article  CAS  Google Scholar 

  46. Osório H, de Almeida AM, Campos A (2018) Sample preparation for 2DE using samples of animal origin. In: Proteomics in domestic animals: from farm to systems biology. Springer International Publishing, Cham, pp 37–53

    Google Scholar 

  47. OSPAR (2008) OSPAR list of threatened and/or declining species and habitats (agreement 2008–6). OSPAR Comm

  48. Park KH, Seong KY, Yang SY, Seo S (2017) Advances in medical adhesives inspired by aquatic organisms' adhesion. Biomater Res 21:16

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Pérez-Losada M, Harp M, Høeg JT, Achituv Y, Jones D, Watanabe H, Crandall KA (2008) The tempo and mode of barnacle evolution. Mol Phylogenet Evol 46:328–346

    Article  PubMed  Google Scholar 

  50. Petersen TN, Brunak S, Von Heijne G, Nielsen H (2011) SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods 8:785–786

    Article  CAS  Google Scholar 

  51. Pochai A, Kingtong S, Sukparangsi W, Khachonpisitsak S (2017) The diversity of acorn barnacles (Cirripedia, Balanomorpha) across Thailand’s coasts: the Andaman Sea and the Gulf of Thailand. Zoosystematics Evol 93:13–34

    Article  Google Scholar 

  52. Power AM, Klepal W, Zheden V, Jonker J, Mcevilly P, von Byern J (2010) Mechanisms of Adhesion in Adult Barnacles. In: von Byern J, Grunwald I (eds) Biological Adhesive Systems. Springer, Vienna

    Google Scholar 

  53. Raman S, Kumar R (2011a) Construction and nanomechanical properties of the exoskeleton of the barnacle, Amphibalanus reticulatus. J Struct Biol 176:360–369

    Article  PubMed  Google Scholar 

  54. Raman S, Kumar R (2011b) Interfacial morphology and nanomechanics of cement of the barnacle, amphibalanus reticulatus on metallic and non-metallic substrata. Biofouling 27:569–577

    Article  CAS  PubMed  Google Scholar 

  55. Raman S, Malms L, Utzig T, Shrestha BR, Stock P, Krishnan S, Valtiner M (2017) Adhesive barnacle peptides exhibit a steric-driven design rule to enhance adhesion between asymmetric surfaces. Colloids Surf B Biointerfaces 152:42–48

    Article  CAS  PubMed  Google Scholar 

  56. Saroyan JR, Lindner E, Dooley CA (1970) Repair and reattachment in the balanidae as related to their cementing mechanism. Biol Bull 139:333–350

    Article  CAS  PubMed  Google Scholar 

  57. Schultz MP, Bendick JA, Holm ER, Hertel WM (2010) Economic impact of biofouling on a naval surface ship. Biofouling 27(1):87–98

    Article  Google Scholar 

  58. So CR, Fears KP, Leary DH, Scancella JM, Wang Z, Liu JL, Orihuela B, Rittschof D, Spillmann CM, Wahl KJ (2016) Sequence basis of barnacle cement nanostructure is defined by proteins with silk homology. Sci Rep 6:36219

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Stewart RJ (2011) Protein-based underwater adhesives and the prospects for their biotechnological production. Appl Microbiol Biotechnol 89:27–33

    Article  CAS  PubMed  Google Scholar 

  60. Suh J, Hutter H (2012) A survey of putative secreted and transmembrane proteins encoded in the C. elegans genome. BMC Genomics 13:333

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Waite JH (1987) Nature’s underwater adhesive specialist. Int J Adhes Adhes 7:9–14

    Article  CAS  Google Scholar 

  62. Walker G (1970) The histology, histochemistry and ultrastructure of the cement apparatus of three adult sessile barnacles, Elminius modestus, Balanus balanoides and Balanus hameri. Mar Biol 7:239–248

    Article  Google Scholar 

  63. Walker G, Youngson A (1975) The biochemical composition of Lepas Anatifera (L.) cement (Crustacea: Cirripedia). J Mar Biol Assoc UK 55:703–707

    Article  CAS  Google Scholar 

  64. Whitehead TO, Biccard A, Griffiths CL (2011) South African pelagic goose barnacles (Cirripedia, Thoracica): substratum preferences and influence of plastic debris on abundance and distribution. Crustaceana 84:635–649

    Article  Google Scholar 

  65. Wei W, Petrone L, Tan Y, Cai H, Israelachvili JN, Miserez A, Waite JH (2016) An Underwater Surface-Drying Peptide Inspired by a Mussel Adhesive Protein. Adv Funct Mat 26(20):3496–3507

    Article  CAS  Google Scholar 

  66. Wiegemann M, Watermann B (2004) The impact of desiccation on the adhesion of barnacles attached to non-stick coatings. Biofouling 20:147–153

    Article  PubMed  Google Scholar 

  67. Yule AB, Walker G (1987) Adhesion in barnacles. In: Crustacean Issues: Barnacle Biology, vol 5. Balkema, pp 389–402

  68. Zhao H, Sun C, Stewart RJ, Waite JH (2005) Cement proteins of the tube building polychaete Phragmatopoma californica. J Biol Chem 280:42938–42944

    Article  CAS  PubMed  Google Scholar 

  69. Zheden V, Von Byern J, Kerbl A et al (2012) Morphology of the cement apparatus and the cement of the buoy barnacle Dosima fascicularis (Crustacea, Cirripedia, Thoracica, Lepadidae). Biol Bull 223:192–204

    Article  PubMed  Google Scholar 

  70. Zheden V, Klepal W, Gorb SN, Kovalev A (2014a) Mechanical properties of the cement of the stalked barnacle dosima fascicularis (cirripedia, crustacea). Interface Focus 5:1–9

    Google Scholar 

  71. Zheden V, Klepal W, von Byern J, Bogner FR, Thiel K, Kowalik T, Grunwald I (2014b) Biochemical analyses of the cement float of the goose barnacle Dosima fascicularis – a preliminary study. Biofouling 30:949–963

    Article  CAS  PubMed  Google Scholar 

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Funding

This research was partially supported by the Structured Program of R&D&I INNOVMAR – Innovation and Sustainability in the Management and Exploitation of Marine Resources (reference NORTE-01-0145-FEDER-000035, Research Line NOVELMAR), funded by the Northern Regional Operational Programme (NORTE2020) through the European Regional Development Fund (ERDF). The research was also funded by the Portuguese Foundation for Science and Technology (FCT) through the strategic project “UID/Multi/04423/2013” and a postdoctoral fellowship to IC (SFRH/BPD/110020/2015) and AC (SFRH/BPD/103683/2014).

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Correspondence to Filipe Pereira or Isabel Cunha.

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All authors have disclosed any actual or potential competing interests (financial or non-financial) regarding the content of this article. All experiments were conducted in accordance with ethical guidelines of the European Union Council (Directives 86/609/EEC and 2010/63/EU) and the Portuguese Agricultural Ministry (Portaria nr.1005/92 of 23 October 2010) for the protection of animals used for experimental and other scientific purposes.

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Miguel Rocha and Paulo Antas are Joint First authors

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Rocha, M., Antas, P., Castro, L.F.C. et al. Comparative Analysis of the Adhesive Proteins of the Adult Stalked Goose Barnacle Pollicipes pollicipes (Cirripedia: Pedunculata). Mar Biotechnol 21, 38–51 (2019). https://doi.org/10.1007/s10126-018-9856-y

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Keywords

  • Adhesive proteins
  • Cement proteins
  • Biased residue composition
  • Adult barnacle
  • Interface surface adhesion
  • Underwater adhesion