Contribution to the characterization of MYORG, a putative transmembrane glycosidase

Abstract

Background.
MYORG (Myogenesis Regulatory Glycosidase) is a 714 amino acid transmembrane protein with a putative glycosidase activity. Its deficiency has been associated with familial brain calcification, and with impaired differentiation of myocytes. However, the intracellular function of MYORG remains unknown. The “Prolocate” database (http://prolocate.cabm.rutgers.edu) indicates that MYORG might be localized in lysosomes and microsomes in rat liver, whereas other teams reported it in the nuclear envelope.
Aim.
To uncover the role of MYORG in normal and pathological conditions, a first step is to elucidate its subcellular localization. Here, we will investigate its localization in several cell types. Then we will analyze its putative α-glycosidase activity.
Methods.
We engineered and expressed GFP or MYC-tagged MYORG (with the tag in N-ter or C-ter position) in HeLa (cervical), C2C12 (muscle), N1E-115 (neuronal) and Hep3B (hepatocytes) cells and studied MYORG localization by fluorescence microscopy. In HeLa cells, we also investigated whether MYORG is glycosylated, using endo-glycosidase F and H treatments. In addition, we tested the putative enzymatic activity of MYORG using three different α-linked fluorogenic substrates and a D463A MYORG mutant as control. Aspartate 463 is predicted as key amino acid of the catalytic site of MYORG.
Results.
Our results show that, in all cell types analyzed, MYORG is predominantly detected around the nucleus, in structures that exhibit a thick and filamentous appearance. We also detected MYORG in the endoplasmic reticulum, in accordance with the presence of high-mannose glycans on the protein. The presence of a fraction of MYORG proteins bearing complex-N-glycans, and a weak colocalization with a Golgi apparatus marker also suggested that MYORG proteins are, at least to some extent, able to reach this compartment. Enzyme assays conducted with HeLa cells overexpressing MYORG did not highlight any MYORG-dependent α-glycosidase activity (using three different α-linked fluorogenic substrates).
Conclusion.
MYORG is glycosylated, partly localizes to the ER and Golgi apparatus and likely to the nuclear envelope. His putative α-glycosidase activity remains hypothetical at this stage.

Date of Award	2022
Original language	English
Awarding Institution	University of Namur
Supervisor	Marielle Boonen (Supervisor)