défense publique de dissertation de doctorat de Madame Emilie Duthoo

Titre de la dissertation: « Morphological and molecular characterization of the adhesive system in the mussel Mytilus edulis and the tubeworm Sabellaria alveolata with a special emphasis on the enzymes involved in the maturation of adhesive proteins ».

Promoteurs de thèse: Monsieur Patrick Flammang et Monsieur Matthew J.Harrington

Résumé de la dissertation: The intertidal zone is a unique environment where both chemical and physical conditions can change considerably. Many marine organisms have adapted to this harsh environment by developing adhesive systems, notably the blue mussel (Mytilus edulis) and the honeycomb worm (Sabellaria alveolata). The mussel secretes numerous proteinaceous threads, collectively called byssus, each ending in a plaque that facilitates adhesion to the substrate. On the other hand, the honeycomb worm lives in a tube it constructs by cementing sand grains together, then lining it internally with an organic sheath. The adhesion mechanisms of mussels and tubeworms have been studied for several decades. They involve adhesive proteins rich in post-translationally modified amino acids. These modifications result in functional groups that are crucial for adhesion and their production is often facilitated by specific enzymes that are poorly investigated.

Both model organisms have a DOPA-based adhesive system. DOPA (3,4-dihydroxy-L-phenylalanine) is produced by the post-translational hydroxylation of tyrosine residues within the adhesive proteins catalyzed by tyrosinase enzymes. The first part of the thesis focuses on these tyrosinase enzymes. Through proteotranscriptomic analyses, we identified tyrosinase enzymes potentially involved in DOPA production and/or oxidation in the adhesive proteins of both organisms. Their roles were subsequently confirmed through localization via in situ hybridization, showing with a high gland-specificity in mussels but not in tubeworms. Phylogenetic analyses were also conducted to address a long-lasting question regarding potential similarities in the adhesion mechanisms of these organisms. These analyses support the hypothesis of independent expansions and parallel evolution of tyrosinases involved in adhesive protein maturation in both lineages, supporting the convergent evolution of their DOPA-based adhesion.

One of the tyrosinase candidates of the blue mussel, involved in byssal plaque formation, was then selected for further investigation. To gain deeper insights into its enzymatic function, this tyrosinase was recombinantly produced in bacteria with the aim of assessing its enzymatic activity by measuring DOPA production using different substrates and conditions. However, although the enzyme was successfully produced, it could not be purified in a soluble form.

Another crucial enzyme involved in marine adhesion is kinase. The honeycomb worm adhesive comprises two serine-rich proteins in which most serine residues are converted to phosphoserines. We hypothesized that this phosphorylation of serine residues is catalyzed by FAM20C kinase enzymes. We identified these enzymes through in silico analysis and confirmed their expression in the adhesive glands via in situ hybridization. Additionally, a better characterization of the honeycomb worm adhesive system was provided through a proteomic analysis of granules extracted from the cement glands, as well as morphological and elemental composition studies.

Finally, the tube lining, another important secreted system found in the honeycomb worm, has been studied. We examined the composition and ultrastructure of this complex fibrous sheath using electron microscopy, along with proteomic and molecular analyses. The morphology of the glands secreting this tube lining has also been investigated. The tube lining is a fibrous structure consisting of multiple layers of parallelly organised fibres embedded in a matrix. Three main types of secretory cells secrete this sheath: collagen-secreting, acidic mucopolysaccharide-secreting, and catechol-secreting cells. We identified eight main proteins within the tube lining. Interestingly, most of these proteins do not contain any known conserved functional domains nor share homology with any proteins from public databases. One of them, however, contains a collagen domain which shows some similarities with bacterial collagen triple helix repeat proteins.

Together, the works presented in this thesis significantly contribute to the understanding of marine adhesive systems, particularly the permanent adhesion of mussels and tubeworms. This knowledge may advance the design of new adhesive biomimetic materials, notably DOPA-based adhesive materials.