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).
Differential regulation of protein O-mannosylation in metazoans. O-mannosylation is predicted to be controlled by at least three distinct enzyme families; α-dystroglycan O-mannosylation is initiated by the POMT1/POMT2 family, the cadherin superfamily is glycosylated by the TMTC1-4 family and the plexin family (IPT/TIG domains) is O-mannosylated by unknown enzyme(s).
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.
O-linked mannose glycosylation on nucleocytoplasmic and secreted proteins in yeast and metazoans. Phosphorylation and O-GlcNAcylation work together and orchestrate signaling processes in metazoans; yeast appears to have a similar system (based on O-linked mannose) operating in nucleocytoplasmic compartments; however, the functions and enzyme(s) in yeast remain unknown.