Titre de la dissertation: « New insights into marine temporary adhesion: Characterization of the adhesive and de-adhesive proteins involved in the duo gland adhesive system of the sea star Asterias rubens ».

Promoteur: Monsieur Patrick Flammang

Co-promoteur: Monsieur Ruddy Wattiez

Résumé de la dissertation:Since several decades, our understanding of underwater adhesion has been constantly evolving. With the aim of mimicking the surrounding nature, scientists have studied many model organisms able to attach to various surfaces and focused on the characterization of biological adhesives. Sessile marine organisms, in particular, have been intensively studied due to their involvement in biofouling, the permanent adhesion organisms to man-made immersed surfaces which causes many problems. Another type of adhesion, temporary adhesion, is also interesting because organisms using it can attach strongly but reversibly in order to perform attachment and detachment cycles. Recently, the temporary adhesion of the common sea star Asterias rubens has been extensively investigated and a first adhesive protein, the sea star footprint protein 1 (Sfp1), has been characterized.

In this thesis, the duo-gland adhesive system used by A. rubens for temporary adhesion was investigated with a particular emphasis on the proteins involved in attachment and detachment. First, the evolutionary conservation of Sfp1 was studied by identification of Sfp1-like sequences in 17 sea star species, representative of 10 families from four out of the five sea star orders. The expression site of 34 other putative adhesive proteins was visualized by in situ hybridization, revealing that only 22 of them were expressed exclusively in the secretory cells of the adhesive epidermis. Then, we analysed all these candidates to retrieve their full-length sequences and to gain insights in their potential function. Overall, after grouping sequences, we identified 16 sea star footprint-specific proteins (Sfps) and mapped their expression to adhesive cell type 1 and/or 2, or to de-adhesive cells by double fluorescent in situ hybridization. Based on their expression and their conserved functional domains, we propose that the identified Sfps serve different functions during attachment and detachment. Fifteen Sfps form a complex adhesive material comprising structural cohesive proteins (Sfp1-6), surface-coupling adhesive proteins (Sfp7-8), and bulk binder proteins (Sfp9-15). The de-adhesive protein of this temporary adhesion system, Astacin-like Sfp, was also characterized. This putative protease presumably works enzymatically to jettison the adhesive footprint, allowing detachment. To investigate its function in detail, a recombinant Astacin-like Sfp was produced and purified. Despite diverse production strategies, however, the recombinant enzyme could not be activated in order to observe its proteolytic activity on a zymogram gel.

This thesis therefore contributes to the understanding of the temporary adhesion mechanism in the sea star Asterias rubens by the characterization of the full set of adhesive proteins as well as of the de-adhesive protein, Astacin-like Sfp. For the first time, the production of a recombinant de-adhesive protein was attempted.