Novel Adhesive Proteins from Freshwater Zebra Mussels

Hanifi, Arash (Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON, Canada.)
Manion, Joseph G (Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON, Canada.)
Rees, David J (Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON, Canada.)
Sone, Eli D (Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON, Canada.)

Introduction

For the past 30 years several types of bioadhesive materials have been used in clinical applications for tissue healing and regeneration purposes [1]. However, there have been complications with their poor adhesion, toxicity of biodegradation side products, and mechanical properties mismatch with the surrounding tissue. The freshwater zebra mussel (Dreissena polymorpha) presents underwater adhesion properties that can be used to overcome the current challenges of synthetic adhesives in aqueous environment. The mussel anchors itself to the substrate by means of a proteinaceous structure called byssus, which is secreted by foot organ and composed of series of threads connected to an adhesive plaque. Although the marine mussel adhesion has been correlated to 3,4 dihydroxyphenylalanine (DOPA)-rich proteins, the adhesion mechanism of freshwater mussel is not completely understood. Our previous studies identified eight new byssal proteins [2, 3]. We also discovered that the mussel attachment is mediated by a uniform layer on the plaque footprint. MALDI mass spectroscopic spatial analysis on byssus-substrate interface revealed the presence of low-molecular weight proteins at the mussel footprint that are not contained in the plaque bulk [4]. Therefore, the aim of this study was to identify the highly specialized low-molecular weight byssal proteins that play a key role in underwater adhesion of zebra mussel to the substrate.

Materials and Methods

Twenty freshly induced threads/plaques were extracted in a basic extraction buffer using an improved extraction and purification protocol based on the protocol described previously by Gantayet et al. [2, 3]. Samples were homogenized on ice, sonicated, and centrifuged. The supernatant (soluble extract) was then washed, concentrated, and separated by gel electrophoresis (10 threads/plaques per gel - total of two gels). The gels were then silver and blue stained to visualize portion bands. Blue stained gel bands at 6, 7, and 14 KDa were trypsin digested and analyzed by LC-MS/MS. Mass spectrometry spectra were searched against a zebra mussel cDNA library using Peaks to determine expressed sequence tags (ESTs). ESTs were then evaluated for; hydroxylation of tyrosine to locate possible DOPA sites, signal peptides, and sequence alignments. The theoretical mass, pI, and amino acid composition of virtual EST matches were also calculated.

Results

In addition to known protein bands, novel low molecular protein bands were appeared in both silver and blue stained gels showing the presence of zebra mussel proteins at 6, 7, and 14 KDa. These gel bands were subjected to trypsin digestion and LC-MS/MS analysis. Mass spectra were searched against zebra mussel cDNA library and ESTs with the -logP greater than 100 and peptide coverage greater than 20% were selected for following analysis; including 21, 19, and 12 ESTs for 6, 7, and 14 KDa gel bands, respectively. Among protein sequences a total of 17 unique EST families were obtained. Amino acid sequence of each of EST family was aligned against known zebra mussel proteins. Of these, six represented completely novel proteins and eight represented new variant of known proteins.

Discussion and Conclusion

Novel low-molecular byssal proteins were identified on stained gels, which led to identification of 14 new EST families including six families representing completely novel proteins. According to MALDI mass spectrometry experiments, these low molecular weight novel proteins are the most likely candidates responsible for the zebra mussel adhesion to substrates. Specific parts of EST families will be used to synthesize bioadhesive peptides, which will be tested by atomic force microscopy to evaluate their adhesive properties. The results of this work will serve as the groundwork for freshwater mussel inspired synthetic adhesive peptides.

Acknowledgements

This work was supported by an NCERC Discovery grant to EDS. AH is grateful for the support from the NSERC CREATE program in regenerative medicine.

References

1- B.P. Lee, P.B. Messersmith, J.N. Israelachvili, J.H. Waite, Mussel-Inspired adhesives and coatings. Annu Rev Mater Res, 2011; 41: 99-132. 2- A Gantayet, L. Ohana, E. D. Sone, Byssal proteins of the freshwater zebra mussel, Dreissena polymorpha, Biofouling. 2013; 29(1):77-85. 3- A. Gantayet, D. J. Rees, E. D. Sone, Novel Proteins Identified in the Insoluble Byssal Matrix of the Freshwater Zebra Mussel, Mar Biotechnol. 2014; 16(2): 144-55. 4- T. Gilbert, E. D. Sone, The byssus of the zebra mussel (Dreissena polymorpha): Spatial variations in protein composition. Biofouling 2010; 26: 829-836.

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