Hennebert E et al. (2015), Experimental strategies for the identification ...

ECB-ART-50037

Interface Focus 2015 Feb 06;51:20140064. doi: 10.1098/rsfs.2014.0064.

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Experimental strategies for the identification and characterization of adhesive proteins in animals: a review.

Hennebert E , Maldonado B , Ladurner P , Flammang P , Santos R .

Abstract
Adhesive secretions occur in both aquatic and terrestrial animals, in which they perform diverse functions. Biological adhesives can therefore be remarkably complex and involve a large range of components with different functions and interactions. However, being mainly protein based, biological adhesives can be characterized by classical molecular methods. This review compiles experimental strategies that were successfully used to identify, characterize and obtain the full-length sequence of adhesive proteins from nine biological models: echinoderms, barnacles, tubeworms, mussels, sticklebacks, slugs, velvet worms, spiders and ticks. A brief description and practical examples are given for a variety of tools used to study adhesive molecules at different levels from genes to secreted proteins. In most studies, proteins, extracted from secreted materials or from adhesive organs, are analysed for the presence of post-translational modifications and submitted to peptide sequencing. The peptide sequences are then used directly for a BLAST search in genomic or transcriptomic databases, or to design degenerate primers to perform RT-PCR, both allowing the recovery of the sequence of the cDNA coding for the investigated protein. These sequences can then be used for functional validation and recombinant production. In recent years, the dual proteomic and transcriptomic approach has emerged as the best way leading to the identification of novel adhesive proteins and retrieval of their complete sequences.

PubMed ID: 25657842
PMC ID: PMC4275877
Article link: Interface Focus

Article Images: [+] show captions

	Figure 1. Model animals used for the study of biological adhesives. (a) Sea star of the species Asterias rubens attached to a rock by its tube feet. (b) Group of barnacles of the species Elminius modestus attached on a rock (picture courtesy of N. Aldred, Newcastle University, UK). (c) Polychaete of the species Sabellaria alveolata extracted from its tube. (d) Mussel of the species Mytilus edulis attached to a Teflon surface by means of byssal threads. (e) Three-spine stickleback of the species Gasterosteus aculeatus assembling its nest (picture courtesy of I. Barber, University of Leicester, UK). (f) Slug from the species Arion fasciatus creeping on a rock on an adhesive mucus film (picture courtesy of A. Smith, Ithaca College, USA). (g) Velvet worm of the species Principapillatus hitoyensis ejecting sticky threads for defence or prey capture (picture courtesy of A. Bär, University of Leipzig, Germany). (h) Spider of the species Nephila pilipes on its web (picture courtesy of J. Delroisse, University of Mons, Belgium). (i) Tick of the genus Ixodes (picture courtesy of J. Delroisse, University of Mons, Belgium). (Online version in colour.)
	Figure 2. Schematic illustration of the synthesis and secretory pathway followed by adhesive proteins in a typical adhesive cell (not to scale). The names of the different molecules, cellular compartments and processes involved are indicated in black, green and blue, respectively. See text for a detailed description.
	Figure 3. Molecular tools used to characterize protein-based adhesives, illustrated by examples from sea stars. Nucleic acids (DNA and mRNA; blue pathway) are extracted from the adhesive organ(s) (here, tube feet). Both can be submitted to next-generation sequencing to obtain, respectively, the genome of the animal or the transcriptome of the adhesive organ(s). Proteins (yellow pathway) are extracted from the secreted material (here, adhesive footprints) or the adhesive organ(s). After purification, they can be analysed for the presence of post-translational modifications (PTMs; red pathway) and/or submitted to peptide sequencing by Edman degradation or tandem mass spectrometry (MS/MS). The peptide sequences can be directly used for a BLAST search in the genome or transcriptome, or to design degenerate primers to perform RT-PCR, both allowing the recovery of the sequence of the cDNA coding for the investigated protein. The sequence is then analysed in silico to extract information required to perform experiments allowing the validation of the adhesive function of the protein (green pathway).
	Figure 4. Consecutive steps for transcriptome sequencing (a) and experimental set-up for differential gene expression (b). See text for details. (a) mRNA (black); sequencing library (red), note adapters at the ends are not shown; paired-end reads (green–red–green), each sequenced stretch at both ends (green) of the DNA fragment (red) is about 100 bp assembled transcripts (purple lines). (b) Transcripts of transcriptome (purple lines) and adhesive transcript (blue line). Note that no reads can be mapped to the adhesive transcript when tissue without adhesive cells used.

References [+] :

Adams, Complementary DNA sequencing: expressed sequence tags and human genome project. 1991, Pubmed