Glycosylation is an essential cellular process that involves the covalent attachment of glycans (carbohydrates/sugars) to proteins; glycans play important roles in maintaining normal cellular functions and dysregulation of processes related to protein glycosylations are known to cause various diseases, e.g. cancer and developmental disorders. Our group is interested in the biosynthesis, regulation and biochemistry of O-linked glycosylations with a special focus on protein O-mannosylations (see our OA article in Cell). We use a combination of methods, including advanced mass spectrometry, to study structures, map site-specific locations and quantify changes of protein O-mannosylations on a proteome-wide scale. In addition, we use CRISPR/Cas9 gene editing for KO/KI of glycogenes in our efforts to study and understand enzymatic pathways involved in protein O-mannosylation.
Current Project Areas
Cadherin-specific O-mannosylation by TMTC1-4
O-mannosylation was for a long time believed to be a rare type of protein modification in mammals. Until recently, α-dystroglycan (αDG), a component of the dystrophin complex, remained the only well-characterized protein with respect to O-mannosylation sites and structures. Building on the SimpleCell technology developed at Copenhagen Center for Glycomics (CCG), we established a glycoproteomic workflow for studying protein O-mannosylations on a proteome-wide scale. This approach allowed us to greatly expand the human O-mannose glycoproteome and led to the discovery of cadherins and protocadherins as major carriers of O-mannosylations. Using the SimpleCell technology, we recently uncovered that O-mannosylation of the cadherin superfamily is not mediated by the classical POMT1/POMT2 enzymes and identified the glycosyltransferase family (GT105), composed of TMTC1-4, that initiates the cadherin-specific O-mannosylation. Currently, we are characterizing the TMTC1-4 enzyme family, exploring the substrate specificities/interactomes of individual TMTC family members and studying the disease-causing mutations in this enzyme family. Our ambitions are to gain further understanding on cellular processes related to the function of cadherin-specific O-mannosylation in health and disease (e.g. Cobblestone lissencephaly).
Identification of new genes and pathways involved in O-mannosylation and cell-cell communication
In addition to the cadherin superfamily, we have identified plexins as a major class of cell-surface O-mannosylated proteins. The IPT/TIG domains of plexins are frequently O-mannosylated on specific β-strands but, intriguingly, this O-mannosylation doesn’t seem to be mediated by POMT1/POMT2 or the TMTC1-4 family. Our results thus indicate that other, as yet unknown glycosyltransferases are present in mammalian systems and are responsible for directing O-mannosylation specifically to proteins with IPT/TIG folds. Using the SimpleCell platform, we are currently screening candidate genes to test this hypothesis and to identify the novel glycosyltransferase enzymes responsible for O-mannosylation in mammalian cells.
Nucleocytoplasmic O-mannosylation in yeast
All eukaryotes except yeast (e.g. S. cerevisiae) utilize a signaling mechanism that involves dynamic cycling of a sugar molecule on nucleocytoplasmic proteins, known as O-GlcNAcylation and dysregulation of this process is implicated in many common human illnesses e.g. diabetes, cardiovascular diseases and cancer. O-GlcNAcylation crosstalk with phosphorylation orchestrates essential cellular processes in all eukaryotic cells but the apparent lack of a nucleocytoplasmic O-GlcNAcylation system in yeast has been a longstanding conundrum. We recently discovered that yeast have a unique O-mannosylation capacity only found in nucleocytoplasmic compartments. The central hypothesis of this project suggests that yeast cells utilize the nucleocytoplasmic O-mannosylation machinery to modulate cellular processes, mirroring the signaling- and regulatory functions of the O-GlcNAcylation system of higher eukaryotes. Currently, we are exploring the dynamics of this nucleocytoplasmic O-mannosylation system and pursuing the hunt for the enzyme(s) responsible for nucleocytoplasmic O-mannosylation in yeast.
- Larsen ISB, Narimatsu Y, Clausen H, Joshi HJ, Halim A. (2019) Multiple distinct O-Mannosylation pathways in eukaryotes. Curr Opin Struct Biol. 2019 Jun;56:171-178.
- Larsen, I. S. B., Narimatsu, Y., Joshi, H. J., Siukstaite, L., Harrison, O. J., Brasch, J., Goodman, K. ., Hansen, L., Shapiro, L., Honig, B., Vakhrushev, S. Y., Clausen, H., and Halim, A. (2017) Discovery of an O-mannosylation pathway selectively serving cadherins and protocadherins. PNAS 114, 11163-11168
- Larsen, I. S. B., Narimatsu, Y., Joshi, H. J., Yang, Z., Harrison, O. J., Brasch, J., Shapiro, L., Honig, B., Vakhrushev, S. Y., Clausen, H., and Halim, A. (2017) Mammalian O-mannosylation of cadherins and plexins is independent of protein O-mannosyltransferases 1 and 2. J Biol Chem 292, 11586-11598
- Halim, A., Larsen, I. S., Neubert, P., Joshi, H. J., Petersen, B. L., Vakhrushev, S. Y., Strahl, S., and Clausen, H. (2015) Discovery of a nucleocytoplasmic O-mannose glycoproteome in yeast. PNAS 112, 15648-15653
- Vester-Christensen, M. B., Halim, A., Joshi, H. J., Steentoft, C., Bennett, E. P., Levery, S. B., Vakhrushev, S. Y., and Clausen, H. (2013) Mining the O-mannose glycoproteome reveals cadherins as major O-mannosylated glycoproteins. PNAS 110, 21018-21023
- Michael P. Rout (The Rockefeller University, NY, USA)
- John A. Hanover (National Institutes of Health, MD, USA)
- Sabine Strahl (University of Heidelberg, Germany)
- Daniel Hebert (University of Massachusetts, MA, USA)