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

Abstract

We employed a genetic approach to study protein glycosylation in the procyclic form of the parasite Trypanosoma brucei. Two different mutant parasites, ConA 1-1 and ConA 4-1, were isolated from mutagenized cultures by selecting cells which resisted killing or agglutination by concanavalin A. Both mutant cells show reduced concanavalin A binding. However, the mutants have different phenotypes, as indicated by the fact that ConA 1-1 binds to wheat germ agglutinin but ConA 4-1 and wild type do not. A blot probed with concanavalin A revealed that many proteins in both mutants lost the ability to bind this lectin, and the blots resembled one of wild type membrane proteins treated with PNGase F. This finding suggested that the mutants had altered asparaginelinked glycosylation. This conclusion was confirmed by studies on a flagellar protein (Fla1) and procyclic acidic repetitive protein (PARP). Structural analysis indicated that the N-glycan of wild type PARP is exclusively Man 5 GlcNAc 2 whereas that in both mutants is predominantly a hybrid type with a terminal N-acetyllactosamine. The occupancy of the PARP glycosylation site in ConA 4-1 was much lower than that in ConA 1-1. These mutants will be useful for studying trypanosome glycosylation mechanisms and function.

Transposon Mutagenesis of Trypanosoma brucei Identifies Glycosylation Mutants Resistant to Concanavalin A

“…Finally, analysis of the C-terminal fragments after mild trifluoroacetic acid hydrolysis, which cleaves the EP procyclins at their mild acid-labile Asp-Pro bonds (7), showed the same characteristic C-terminal ions from EP1-1, EP1-2, and EP3 (m/z 7,001, 5,870, and 5,191) in all samples (data not shown). Taken together, these analyses suggest that, in contrast to wild-type The presence of peptides carrying a Hex 5 HexNAc 3 glycan (denoted as species B) suggests that one of the terminal ␣Man residues is capped with an N-acetyllactosamine (␤Gal-␤Glc-NAc) repeat that is similar to the modification found in the T. brucei ConA R clone ConA 1-1 (29,38). To confirm this interpretation, we incubated aqueous HF-treated ALG12 Ϫ/Ϫ procyclins with a mixture of bovine testis ␤-galactosidase and jack bean ␤-hexosaminidase.…”

“…T. brucei mutants with reduced ConA binding had been isolated after chemical mutagenesis (29,36) but, although these mutants were biochemically characterized, the genetic defect responsible for the altered phenotype could not be identified.…”

“…8, A and B). The implications of such a defect, with regard to the structure of mature glycans linked to surface glycoproteins, have been discussed extensively (29,36,38,39). One issue that has been clarified, from our study of ALG12 Ϫ/Ϫ cells, is that upon transfer of the truncated (Man 7 GlcNAc 2 ) OSL onto nascent proteins, the Man 4 GlcNAc 2 glycan that results from the trimming of all ␣1-2Man residues can also be modified by the addition of a terminal N-acetyllactosamine repeat.…”

“…In T. brucei Lister 427, the EP-procyclin isoforms are encoded by the EP1, EP2, and EP3 genes (4,8). Only the products of EP1 and EP3 contain a glycosylation site (Asn 29 ), which is modified exclusively by a Man 5 GlcNAc 2 glycan (7,9). Procyclins are important for survival in the tsetse (10 -12) but can be dispensed with in culture, after a period of adaptation that involves increased expression of free GPIs on the parasite surface (13).…”

“…ConA has been used to isolate glycosylation mutants of T. brucei after chemical mutagenesis (29), but the lack of methods for genetic mapping prevented the mutated genes from being identified.…”