Figure 1.
Restricted and unrestricted LLO synthesis, and the effect of CHX. (A) Hypothetical high-pressure liquid chromatograms of LLO glycans illustrate efficient (unrestricted) synthesis of G3M9Gn2-P-P-Dol versus accumulation of M2-7Gn2-P-P-Dol intermediates under restricted conditions that simulate metabolic dysfunction. The dashed lines indicate the mannosyl residues that vary in the respective intermediates (Helenius and Aebi, 2004). (B) Dermal fibroblasts were treated with the indicated concentrations of CHX for 50 min. Medium with 0.5 mM glucose was used. For the final 20 min, 40 μCi/ml [3H]mannose was added. [3H] LLO glycans were isolated and characterized by HPLC. The positions of standards are shown. Note that addition of CHX converts the pattern of LLO glycans from restricted to unrestricted. For comparison, an experiment is shown with 2 mM DTT added for 30 min instead of CHX.