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. Metabolic incorporation of [3H]mevalonate showed that ConA 1-1 synthesizes equal amounts of dolichol and polyprenol, whereas WT cells make predominantly dolichol. Second, we found that ConA 1-1 synthesizes and accumulates an oligosaccharide lipid (OSL) precursor that is smaller in size than that from WT cells. The glycan of OSL in WT cells is apparently Man9GlcNAc2, whereas that from ConA 1-1 is Man7GlcNAc2. The smaller OSL glycan in the ConA 1-1 explains how some procyclin polypeptides bear a Man4GlcNAc2 modified with a terminal N-acetyllactosamine group, which is poorly recognized by ConA.

FIG. 1.

Structure of procyclin N-glycans from WT and mutant trypanosomes. (A) WT; (B) ConA 1-1 (1, 11, 12). “+/−” indicates heterogeneity in ConA 1-1 N-glycan.

FIG. 2.

Identification of the prenol in WT T. brucei and ConA 1-1. (A) Synthesis of dolichol from polyprenol by polyprenol reductase. (B) Lipids, which were labeled during a 36-h incubation of growing cells with [³H]mevalonolactone, were extracted, saponified, and dephosphorylated. Prenols were then separated from sterols, and the prenols were analyzed on a silica column by normal-phase HPLC. The elution positions of coenzyme Q₉ (CoQ9), polyprenol-11 (P11), and dolichol-11 (D11) were determined by absorbance (A₂₁₀) of internal standards. The elution positions and the amounts of the radioactive products were determined by counting the entire fraction. The polar metabolites eluting at the breakthrough have not been characterized.

FIG. 3.

Cell-free synthesis of OSL. Glycolipids were synthesized in a cell-free system containing washed trypanosome membranes. The membranes were incubated with GDP-[³H]Man and UDP-GlcNAc and then chased with nonradioactive GDP-Man. In both cases, glycolipids were extracted with organic solvents, fractionated by TLC, and detected by autoradiography. (A) Synthesis of glycolipids in WT membranes in the absence or presence of 1.6 μg of tunicamycin/ml (Tunic). (B) Synthesis of glycolipids in WT and ConA 1-1 membranes under the same conditions as shown in panel A, except that tunicamycin was omitted. Dol-P-M, dolichol phosphoryl-Man; PP1 and P3, GPI precursors; Man1, Man2, and Man3, GPI biosynthetic intermediates (Ints) with the indicated number of Man residues; X, unknown species.

FIG. 4.

Bio-Gel P4 analyses of WT and ConA 1-1 OSL glycans. [³H]Man-labeled glycans (labeled in the cell-free system with GDP-[³H]Man and isolated from hydrolysates of TLC-purified OSL) from WT and ConA 1-1 cells were mixed with unlabeled dextran Glc oligomers and analyzed by P4 gel filtration. Arrowheads indicate elution positions of Glc oligomers, starting with Glc₁ on the right.

FIG. 5.

Structural characterization of OSL oligosaccharides. [³H]Man-labeled glycans were analyzed by TLC and autoradiography. (A) Lane 1, ³H-labeled Glc₃Man₉GlcNAc₂ from WT CHO cells (K12); lane 2, ³H-labeled Man₉GlcNAc₂ from MI8-5 mutant CHO cells; lane 3, ³H-labeled OSL glycan from WT T. brucei. (B) ³H-labeled OSL glycans from MI8-5 CHO cells (lane 1), WT T. brucei (lane 2), C. fasciculata (lanes 3 and 5), and ConA 1-1 (lanes 4 and 6). Glycans in lanes 5 and 6 were treated with A. saitoi α-mannosidase (ASAM), and those in lanes 3 and 4 were mock treated. Positions of glycan standards (2 μg; Sigma) visualized by orcinol-H₂SO₄ staining are indicated on the right. Cf, C. fasciculata glycan.

FIG. 6.

Proposed structure and processing of WT and ConA 1-1 OSLs. After formation of WT OSL (containing Man₉GlcNAc₂) (A) and ConA 1-1 OSL (containing Man₇GlcNAc₂) (B), glycans are transferred to EP-procyclins. Whereas the WT glycan is efficiently transferred to procyclins, those from mutants are only partially transferred, forming underglycosylated proteins. Subsequently, all the α1-2Man residues are removed from glycans by specific mannosidases, forming procyclins bearing Man₅GlcNAc₂ (WT) or Man₄GlcNAc₂ (ConA 1-1). Whereas WT glycans are not further processed, most glycans in ConA 1-1 EP-procyclins are modified to hybrid types by the addition of an N-acetyllactosamine group to the terminal α1-3Man residue. *, Man residue in WT whose transfer is defective in ConA 1-1. “(+/−)” indicates that some procyclin glycosylation sites are unmodified in ConA 1-1 cells.