Sea stars adhere firmly but temporarily to various substrata as a result of underwater efficient adhesive secretions released by their tube feet. Previous studies showed that this material is mainly made up of proteins, which play a key role in its adhesiveness and cohesiveness. Recently, we solubilized the majority of these proteins and obtained 43 de novo-generated peptide sequences by tandem MS. Here, one of these sequences served to recover the full-length sequence of Sea star footprint protein 1 (Sfp1), by RT-PCR and tube foot transcriptome analysis. Sfp1, a large protein of 3,853 aa, is the second most abundant constituent of the secreted adhesive. By using MS and Western blot analyses, we showed that Sfp1 is translated from a single mRNA and then cleaved into four subunits linked together by disulphide bridges in tube foot adhesive cells. The four subunits display specific protein-, carbohydrate-, and metal-binding domains. Immunohistochemistry and immunocytochemistry located Sfp1 in granules stockpiled by one of the two types of adhesive cells responsible for the secretion of the adhesive material. We also demonstrated that Sfp1 makes up the structural scaffold of the adhesive footprint that remains on the substratum after tube foot detachment. Taken together, the results suggest that Sfp1 is a major structural protein involved in footprint cohesion and possibly in adhesive interactions with the tube foot surface. In recombinant form, it could be used for the design of novel sea starinspired biomaterials.echinoderms | Asterias rubens | marine adhesion

In sea stars, adhesion takes place at the level of a multitude of small appendages, the tube feet. It involves the secretion of an adhesive material which, after tube foot detachment, remains on the substratum as a footprint. It was previously reported that the two main organic components of this material are proteins and carbohydrates. The carbohydrate moiety of the adhesive secretion of Asterias rubens was investigated using a set of 16 lectins which were used on sections through tube feet, on footprints, and on the proteins extracted from these footprints. After gel electrophoresis, these proteins separate into eight protein bands which were named sea star footprint proteins (Sfps). Eleven lectins label the tube foot epidermis at the level of the adhesive cells, four react with footprints, and eight with two of the extracted footprint proteins, which are therefore classified as glycoproteins. Sfp-290 appears to bear mostly N-linked oligosaccharides and Sfp-210 principally O-linked oligosaccharides. The outer chains of both glycoproteins enclose galactose, N-acetylgalactosamine, fucose, and sialic acid residues. Another part of the carbohydrate fraction of the footprints would be in the form of larger molecules, such as sialylated proteoglycans. These two types of glycoconjugates are presumably key components of the sea star temporary adhesive providing both cohesive and adhesive contributions through electrostatic interactions by the polar and hydrogen-bonding functional groups of their glycan chains.

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.

Sea stars rely on epidermal secretions to cope with their benthic life. Their integument produces a mucus, which represents the first barrier against invaders; and their tube feet produce adhesive secretions to pry open mussels and attach strongly but temporarily to rocks. In this study, we combined high-throughput sequencing of expressed mRNA and mass-spectrometry-based identification of proteins to establish the first proteome of mucous and adhesive secretions from the sea star Asterias rubens. We show that the two secretions differ significantly, the major adhesive proteins being only present in trace amounts in the mucus secretion. Except for 41 proteins which were present in both secretions, a total of 34 and 244 proteins were identified as specific of adhesive secretions and mucus, respectively. We discuss the role of some of these proteins in the adhesion of sea stars as well as in their protection against oxygen reactive species and microorganisms. In addition, 58% of the proteins identified in adhesive secretions did not present significant similarity to other known proteins, revealing a list of potential novel sea star adhesive proteins uncharacterized so far. The panel of proteins identified in this study offers unprecedented opportunities for the development of sea star-inspired biomimetic materials.

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