In a cell-based assay for novel inhibitors, we have discovered that two
glycosides of 5-thiomannose, each containing an interglycosidic
nitrogen atom, prevented the correct
zymogen processing of the prohormone proopiomelanocortinin (
POMC) and the
transcription factor sterol-regulatory
element-binding protein-2 (SREBP-2) in mouse pituitary cells and Chinese hamster ovary (CHO) cells, respectively. In the case of SREBP-2, these effects were correlated with the altered N-linked glycosylation of
subtilisin/kexin-like isozyme-1 (SKI-1), the
protease responsible for SREBP-2 processing under
sterol-limiting conditions. Further examination of the effects of these compounds in CHO cells showed that they cause extensive
protein hypoglycosylation in a manner similar to type I
congenital disorders of glycosylation (CDGs) since the remaining N-
glycans in treated cells were complete (normal) structures. The under-glycosylation of
glycoproteins in 5-thiomannoside-treated cells is now shown to be caused by the compromised biosynthesis of the
dolichol-linked
oligosaccharide (DLO) N-glycosylation donor, although the
nucleotide sugars required for the synthesis of DLOs were neither reduced under these conditions, nor were their effects reversed upon the addition of exogenous
mannose. Analysis of DLO intermediates by fluorophore-assisted
carbohydrate electrophoresis demonstrated that 5-thiomannose-containing
glycosides block DLO biosynthesis most likely at a stage prior to the
GlcNAc(2) Man(3) intermediate, on the cytosolic face of the endoplasmic reticulum.