Defects in the N-Linked Oligosaccharide Biosynthetic Pathway in a Trypanosoma brucei Glycosylation Mutant

Abstract

Concanavalin A (ConA) kills the procyclic (insect) form of Trypanosoma brucei by binding to its major surface glycoprotein, procyclin. We previously isolated a mutant cell line, ConA 1-1, that is less agglutinated and more resistant to ConA killing than are wild-type (WT) cells. Subsequently we found that the ConA resistance phenotype in this mutant is due to the fact that the procyclin either has no N-glycan or has an N-glycan with an altered structure. Here we demonstrate that the alteration in procyclin N-glycosylation correlates with two defects in the N-linked oligosaccharide biosynthetic pathway. First, ConA 1-1 has a defect in activity of polyprenol reductase, an enzyme involved in synthesis of dolichol.

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

“…The same ladder of [ 3 H]Man-labeled glycans was observed from ALG12 Ϫ/Ϫ OSLs, except that the most hydrophilic component was smaller and co-migrated with the Man 7 GlcNAc 2 standard. Thus, as predicted, disruption of ALG12 in procyclic T. brucei produces a mature OSL that has a shorter glycan, containing a Man 7 GlcNAc 2 instead of Man 9 GlcNAc 2 , as in wild-type cells (39).…”

“…Under these conditions, incubation with UDP-GlcNAc and GDP-[ 3 H]Man results in labeling of both OSL and GPI precursors and their biosynthetic intermediates (35). Although the mature OSL (Man 9 GlcNAc 2 -PP-lipid) was synthesized in the wild-type cell-free system, as expected (39), and by ALG12 ϩ/Ϫ membranes (Fig. 8A, first and second lanes), it was absent from the ALG12 Ϫ/Ϫ and the two mariner mutant samples (lanes 3-5), where less polar species, designated "mutant OSL," were observed, migrating slightly faster than PP1.…”

“…Subsequent work, however, showed that the dolichol-linked glycan precursor in ConA 1-1 also contained a modified glycan that would be consistent with a concomitant mutation in ALG12, although this has not been confirmed. The possibility remains that the ConA 1-1 phenotype is due to disruption of one polyprenol reductase allele and that the consequent dolichol deficiency causes an alteration in the structure of the OSL (39).…”

“…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.…”

The Suppression of Galactose Metabolism in Procylic Form Trypanosoma brucei Causes Cessation of Cell Growth and Alters Procyclin Glycoprotein Structure and Copy Number

“…A similar example of haploid insufficiency with respect to a biochemical phenotype (rather than viability) was reported for the ConA 1-1 procyclic form mutant that lacks one functional allele of polyprenol reductase, an enzyme involved in synthesis of dolichol. This partial defect affected procyclin Nglycosylation and rendered the parasites resistant to killing by the lectin concanavalin A (52).…”