Biochemical and biological properties of a bifunctional GH27 enzyme from rice

Glycobiology is the study of carbohydrates, their synthesis, modification, transport and binding partners. Within the glycobiology field, lectins occupy a prominent place. Lectins are remarkable proteins equipped with carbohydrate-binding lectin domains and exhibit reversible binding towards sugars, polysaccharides and glycans. Lectins are implied in a wide range of physiological processes including: cell-cell adhesion, intra and intercellular communication, receptor functionality in defense responses. Within the plant kingdom, lectin domains often occur in combination with non-lectin domains, including enzymatically-active domains such as glycoside hydrolases (GH). The combination of a GH27 domain and a ricin-B-like lectin domain was observed in OsAPSE, an enzyme from Japanese rice, which is the subject to this doctoral dissertation. The aim of this doctoral dissertation was to study the biochemical properties and to elucidate the biological relevance of OsAPSE for the rice plant. Findings: 1- The research pointed out that the presence of a ricin-B-like lectin domain is a distinctive and discriminating trait within the GH27 family of enzymes from land plants. Furthermore, the ricin-B-like lectin domain occurring in OsAPSE and its APSE homologs does not resemble the closely-related CBM13/ricin-B domains. Unfortunately, we were not able to demonstrate the carbohydrate-binding properties of the recombinantly produced ricin-B-like domain. 2- The GH27 domain of OsAPSE was produced recombinantly in a cell-free expression system and allowed to determine some of the biochemical and enzymatic parameters. OsAPSE is a GH27 enzyme with thermal optimum at 25°C and alkaline optimum at pH = 7.5-8, and displays activity towards cell wall polysaccharides but also alternative substrates such as raffinose family oligosaccharides. 3- Both in silico predictions and microscopic subcellular localization experiments indicate that OsAPSE is located at the cell wall and displays enzymatic activity on arabinogalactan protein O-glycans and pectic rhamnogalacturonan I, thereby modulating the non-covalent interactions between aforementioned carbohydrate structures. RT-qPCR analyses on transgenic rice plants overexpressing OsAPSE and mutant (CRISPR/Cas9) OsAPSE-knock out plants demonstrated that OsAPSE is particularly relevant during the seed germination process. It was shown that the germination capacity of transgenic and mutant rice plants were significantly affected compared to wild type seedlings, indicating that well-dosed transcription of OsAPSE is essential to ensure normal rice seed germination. We hypothesize that the activity of OsAPSE contributes to providing cell wall flexibility, which is typically required during fast-paced developmental processes such as rice seed germination. The findings reported in this doctoral dissertation are of particular importance for the plant glycobiology research field, as the relationship between the structure of arabinogalactan protein O-glycans and their biological function remains largely understudied and not well understood. Furthermore, important insights were gathered regarding the success of recombinant protein production of eukaryotic proteins of interest, providing a learning ground for peer researchers. It is recommended for future research to focus on the recombinant expression of other APSE homologs, and to further explore the biology of arabinogalactan protein O-glycan modifying enzymes.