Mapping the glycome of choanoflagellates to evaluate the role of protein glycosylation on the origin of multicellularity

• Applicant: Dr. Ulla Gerling-Driessen
• Subject Area: Biological and Biomimetic Chemistry
• Term: from 2015 to 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 284615620

Complex glycans that are covalently attached to secreted proteins or at the cell surface are involved in many cellular processes, such as protein quality control, cell adhesion, and receptor-ligand interactions. Glycomics is the attempt to experimentally quantify the glycome of a cell, tissue, or even a whole organism; it remains challenging due to the extreme heterogeneity and diversity of glycans. Current approaches to glycan characterization are based upon enrichment of the glycoprotein subtype and reduction of the glycan structure to a uniform and predictable mass. Circumventing the need for enrichment and/or enzymatic digestion and retaining the glycomic information, a novel mass-independent glycoproteomic platform, Isotope Targeted Glycoproteomics (IsoTaG), was established in the group of Carolyn R. Bertozzi. IsoTaG enables the characterization of intact glycopeptides at the whole proteome scale. The proposed project aims to investigate the glycome of choanoflagellates by means of IsoTaG technology. Choanoflagellates are the closest known living unicellular relatives of animals. These free-living, single-celled eukaryotes are able to form colonies, making them interesting organisms to study from an evolutionary perspective. So far, little is known about the glycome of choanoflagellates; however, genome sequencing has indicated that glycans may play an important role in diverse cellular functions, as is the case in animals. Thus, identifying the glycome of choanoflagellates could give new insights into the role that glycosylation plays in the origin of multicellularity. Since glycan binding has not yet been proven for choanoflagellates, the first part of the project is to qualitatively identify the types of glycans that are present and the particular cellular regions (cell surface, center, body, flagellum, collar, etc.) in which they are enriched. Using the concept of metabolic labeling, chemically functionalized azide- or alkyne-functionalized sugars are introduced in the cells glycome. The bioorthogonal azide or alkyne functional group can then be conjugated to imaging or affinity probes, such as fluorescent dyes, by using Cu-catalyzed or Cu-free click chemistries. The second part of the project deals with an extensive analysis of the glycoproteins using the mass-independent IsoTaG strategy, which is based on chemical tagging of the identified glycans with a unique bromine-based isotopic signature. The unique patterns produced can be recognized by a computational pattern searching algorithm, termed isotopic signature transfer and mass pattern prediction (IsoStamp) enabling identification of dibromide-tagged peptides from complex mixtures. The great advantage of the IsoTaG platform is that it can be applied even if the nature of the glycan structure (N-glycans, O-glycans, mucin-type O-glycans) is unknown, as would be the case for the herein investigated choanoflagellates.

• DFG Programme: Research Fellowships
• International Connection: USA
• Host: Professorin Dr. Carolyn Ruth Bertozzi