Protein glycosylation mutants of procyclic Trypanosoma brucei: defects in the asparagine-glycosylation pathway

We employed a genetic approach to study protein glycosylation in the procyc lic form of the parasite Trypanosoma brucei. Two different mutant parasites , ConA 1-1 and ConA;4-1, were isolated from mutagenized cultures by selecti ng cells which resisted killing or agglutination by concanavalin A. Both mu tant cells show reduced concanavalin A binding. However, the mutants have d ifferent phenotype, as indicated by the fact that ConA 1-1 binds to wheat g erm agglutinin but ConA 4-1 and wild type do not. A blot probed with concan avalin A revealed that many proteins in both mutants lost the ability to bi nd this lectin, and the blots resembled one of wild type membrane proteins treated with PNGase F. This finding suggested that the mutants had altered asparagine-linked glycosylation, This conclusion was confirmed by studies o n a flagellar protein (Fla1) and procyclic acidic repetitive protein (PARP) , Structural analysis indicated that the N-glycan of wild type PARP is excl usively Man(5)GlcNAc(2) whereas that in both mutants is predominantly a hyb rid type with a terminal N-acetyllactosamine. The occupancy of the PARP gly cosylation site in ConA 4-1 was much lower than that in ConA 1-1, These mut ants will be useful for studying trypanosome glycosylation mechanisms and f unction.