Protein N-glycosylation occurs by the transfer of an oligosaccharide from a lipid linked precursor (Glc3Man9GlcNAc2-PP-dolichol, DLO) onto newly synthesized proteins in the lumen of the endoplasmic reticulum (ER). This process is essential for life and mutations in genes required for biosynthesis of DLO, or the transfer of its oligosaccharide onto protein, underlie several of the rare Type I Congenital Disorders of Glycosylation (CDG-I). Although genetic lesions in the DLO biosynthetic pathway cause glycoprotein hypoglycosylation and accumulation of truncated DLO intermediates, for most of these multisystemic, often severe, diseases the link between genotype and phenotype is poorly understood. For the majority of CDG-I, there are no etiological treatments, in part, because the regulation of DLO biosynthesis in normal and disease situations is poorly understood. DLO diphosphatases (DLODPs), orphan enzymes whose genes have not been identified, cleave DLO intermediates seen in certain pathophysiological situations like CDG-I to generate oligosaccharyl phosphates (OSP), but the roles of these activities in normal and disease cells remain to be determined.

We hypothesise that DLODPs destroy toxic truncated DLO intermediates, which are generated under pathological/stress conditions like CDG-I, that would otherwise cause cell pathology. We have identified two OSP generating systems in mammalian cells. One OSP-generating process is associated with a Co²⁺-stimulated DLODP activity that co-localises with markers of the Golgi apparatus during subcellular fractionation and becomes apparent when cells are treated with brefeldin A, which causes elements of the Golgi apparatus to fuse with the ER. Under these conditions a population of OSP are generated from mature DLO (Glc3Man9GlcNAc2-PP-dolichol) within the ER/Golgi fusion compartment. In the second process, OSP are generated on the cytoplasmic face of the ER from truncated DLO intermediates by an as yet uncharacterised enzyme activity. This second process occurs in cells from CDG patients and is not affected by brefeldin A. Presently, we are identifying DLODP genes so that we can understand the roles of these enzymes in protein N-glycosylation. If the N-glycosylation pathway is to be understood in such a way that its behaviour can be predicted in the face of pharmacological or genetic perturbation, then the molecular identity, partners, regulation and biochemical characteristics of all the players must be described. Therefore, identification of DLDOP proteins and genes will lead to both fundamental insights into DLO and protein N-glycosylation homeostasis and the potential development of CDG-I treatment strategies.

Title

Middle east journal of medical genetics

Publisher

Wolters Kluwer - Medknow

Publisher Country

India

Publication Type

Online only

Volume

8

Year

2019

Pages

1-27