Investigation of the glycosyltransferase enzymes involved in the initial stages of the N-linked protein glycosylation pathway in Aspergillus niger.

A protocol has been developed to produce functional microsomes from mycelium of Aspergillus niger. Using these preparations, radioactive biochemical assays have been designed and optimised to measure the in vitro activities of three of the enzymes of the N-linked dolichol phosphate (Dol-P) glycosylation pathway: UDP-N-GlcNAc:dolichol-P GlcNAc-1-P transferase (GPT), Dol-P mannose synthase (DPMS) and Dol-P glucose synthase (DPGS). Exogenous Dol-P and Triton X-100 are essential for in vitro activity. All three Dol-P:glycosyl transferases had alkaline pH optima and activity rapidly decreased below neutrality. Characterisation of the glycolipid products by anion-exchange chromatography and TLC showed that they were Dol-P-P-GlcNAc, Dol-P-Glc and Dol-P-Man for GPT, DPGS and DPMS, respectively. The antibiotic tunicamycin completely inhibited GPT activity at a concentration of 100-200 ng ml(-1) and an IC50 of 40 ng ml(-1), but had little effect on the other two enzymes. The ratio of the activity of the three enzymes to each other suggested that DPMS may be involved in other cellular activities and is probably under different control mechanisms than the other two.