The dolichol cycle involves synthesis of the lipid-linked oligosaccharide (LLO) Glc3Man9GlcNAc2- P-P-dolichol (G3M9Gn2-P-P-Dol), transfer of G3M9Gn2 to asparaginyl residues of nascent endoplasmic reticulum (ER) polypeptides by oligosaccharyltransferase (OT), and recycling of the resultant Dol-P-P to Dol-P for new rounds of LLO synthesis. The importance of the dolichol cycle in secretory and membrane protein biosynthesis, ER function, and human genetic disease is now widely accepted. Elucidation of the fundamental properties of the dolichol cycle in intact cells was achieved through the use of radioactive sugar precursors, typically [3H]-labeled or [14C]-labeled D-mannose, D-galactose, or D-glucosamine. However, difficulties were encountered with cells or tissues not amenable to metabolic labeling, or in experiments influenced by isotope dilution, variable rates of LLO turnover, or special culture conditions required for the use of radioactive sugars. This article will review recently developed alternatives for LLO analysis that do not rely upon metabolic labeling with radioactive precursors, and thereby circumvent these problems. New information revealed by these methods with regard to regulation, genetic disorders, and evolution of the dolichol cycle, as well as caveats of radiolabeling techniques, will be discussed.
- Fluorophore-assisted carbohydrate electrophoresis
- Lipid-linked oligosaccharide
- N-linked glycosylation
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