Reactions involving removal and addition of glucose to N-glycans in the ER (endoplasmic reticulum) are performed in higher eukaryotes by glucosidases I and II and the UDP-glucose:glycoprotein glucosyltransferase respectively. Monoglucosylated N-glycan structures have been implicated in glycoprotein folding or ER quality control. Components of the system appear across a range of organisms; however, the precise combination differs between organisms. We have identified putative components of the system in the protozoal organism Trypanosoma brucei by local alignment searching. The function of one of these components, a glucosidase II α-subunit homologue, has been confirmed by phenotyping a null mutant, and an ectopic expression cell line. A combination of MS, methylation linkage analysis, exoglycosidase digestion and partial acetolysis have been used to characterize three novel N-glycan structures on the variant surface glycoprotein of the null mutant. On the basis of our results, we propose that two N-glycan precursors are available for transfer to variant surface glycoprotein (variant 221) in the ER of T. brucei; only one of these precursors is glucosylated after transfer.

In higher eukaryotes, removal of the final glucose residues from Asn-linked glycans in the ER (endoplasmic reticulum) is performed by glucosidase II. The reverse of this reaction is performed on N-linked Man9GlcNAc2 structures on unfolded glycoproteins by the UGGT (UDP-glucose:glycoprotein glucosyltransferase). Monoglucosylated Asn-linked glycans have been implicated in ER quality control, particularly in association with the lectin-like chaperones calnexin and calreticulin [1] (see Figure 1A).

Asn-linked glycans in the ER

Figure 1
Asn-linked glycans in the ER

(A) A model for higher eukaryotes based on information provided in Figures 2 and 3 from [1]. (B) A modified version of the model for T. brucei considering the results of our homology searching and characterization of sVSG221 purified from the Tb10.05.0080 null mutant T. brucei cells. CNX, calnexin; CRT, calreticulin.

Figure 1
Asn-linked glycans in the ER

(A) A model for higher eukaryotes based on information provided in Figures 2 and 3 from [1]. (B) A modified version of the model for T. brucei considering the results of our homology searching and characterization of sVSG221 purified from the Tb10.05.0080 null mutant T. brucei cells. CNX, calnexin; CRT, calreticulin.

In Schizosaccharomyces pombe mutants, where the Man9GlcNAc2-PP-Dol precursor cannot be glucosylated, UGGT and calnexin are essential for viability [2]. Furthermore, disruption of glucosidase II or UGGT leads to an induction of BiP RNA [3]. Induction of BiP in glucosidase II-defective mammalian cells has also been reported [4]. However, induction of the unfolded protein response is only observed in Saccharomyces cerevisiae glucosidase II-defective cells under conditions of ER stress [5]. S. cerevisiae also lacks a UGGT homologue [6], and the calnexin homologue (encoded by CNE1) differs significantly from its mammalian counterparts [7]. In both of these yeasts, only one calnexin/calreticulin homologue is found, and this is membrane-bound [7,8]. This homologue is essential in S. pombe but not in S. cerevisiae [8].

We analysed the available Trypanosoma brucei genomic databases for homologues of genes whose products are involved in N-glycan biosynthesis, transfer and interaction in the ER. The dolichol-P-mannose synthetase, the dolichol kinase, the various GlcNAc and Man transferases and RFT1 are found with varying strengths of similarity. The three Glc transferases on the luminal side of the ER and glucosidase I are noticeably absent. Glucosidase IIα shows the greatest homology in the system to both yeast and murine sequences. UGGT, calreticulin (but not calnexin), Erp57, EDEM, ER mannosidase I and ERGIC 53 all have identifiable homologues in T. brucei. These results led to a modification of the model proposed in [1] for the T. brucei system (see Figure 1B).

Two homologues of the ER glucosidase IIα subunit were found, one homologue (Tb10.05.0080) had the greatest similarity. The second homologue (Tb11.01.2140) showed greater similarity to lysosomal α glucosidase and was not studied further. The Tb10.05.0080 ORF (open reading frame) was cloned, sequenced and deleted from the bloodstream form of T. brucei by replacement with two antibiotic-resistant genes. A rescue cell line was generated by transforming the null mutant with an ectopic expression construct based on pLew82 [9]. The genotypes of cell lines were confirmed by Southern blotting.

VSG (variant surface glycoprotein, variant 221) was purified from 2E8 T. brucei cells by osmotic lysis followed by DE52 anion exchange as described previously [10]. The intact glycoprotein from the null mutant, wild-type and the ectopic rescue cell lines was analysed by ESI–MS (electrospray ionization mass spectrometry) (see Figure 2A). These results showed a mass increase corresponding to three hexose residues resulting from deletion of the Tb10.05.0080 ORF. This biochemical phenotype was mimicked by treatment of wild-type T. brucei with the potent glucosidase inhibitor 1-deoxynojirimicin and was reversed in the null mutant by expression of an ectopic copy of the glucosidase II gene. sVSG was digested with Pronase and the glycopeptides were analysed by ESI–MS and ESI–MS/MS; these results indicated that the high mannose glycan at the Asn-454 position along with the GPI anchor were unaffected by the deletion of the ORF. The predominant glycans at the Asn-289 position were altered from Hex3HexNAc2, Hex3HexNAc3 and Hex4HexNAc3 in the wild-type to Hex6HexNAc2 in the null mutant, and this phenotype was reversed by the expression of an ectopic copy of the Tb10.05.0080 gene.

Effect of glucosidase II deletion on sVSG221 glycan repertoire

Figure 2
Effect of glucosidase II deletion on sVSG221 glycan repertoire

(A) Deconvoluted ESI–MS spectra of intact sVSG221 isolated from T. brucei. sVSG221 is compared with wild-type cells treated with or without glucosidase inhibitor 1-deoxynojirimicin, glucosidase II null mutant cells and the ectopic rescue cell line. (B) The three novel Asn-glycan structures purified from the large-scale glycan preparation from sVSG on glucosidase II null mutant cells are shown.

Figure 2
Effect of glucosidase II deletion on sVSG221 glycan repertoire

(A) Deconvoluted ESI–MS spectra of intact sVSG221 isolated from T. brucei. sVSG221 is compared with wild-type cells treated with or without glucosidase inhibitor 1-deoxynojirimicin, glucosidase II null mutant cells and the ectopic rescue cell line. (B) The three novel Asn-glycan structures purified from the large-scale glycan preparation from sVSG on glucosidase II null mutant cells are shown.

A large-scale sVSG221 isolation was performed from the blood of rats infected with the null mutant (2E11 cells), and glycans were released from 25 mg of the resulting sVSG221 by peptide N-glycosidase F and partially purified by ethanol precipitation and S200 size-exclusion chromatography. The glycan pool was then separated by high-pH anion-exchange chromatography and three novel structures (Hex6HexNAc2, Hex6HexNAc3 and Hex7HexNAc3) from the Asn-289 site identified by ESI–MS. Digestion with glycosidase enzymes or sensitivity to partial acetolysis (selective for cleavage of Manα1-6Man glycosidic bonds) was assessed by high-performance TLC and fluorography of NaB3H4-reduced oligosaccharides.

The Hex6HexNAc2 structure was analysed by GC–MS compositional analysis, one- and two-dimensional 1H-NMR (correlated spectroscopy, total correlated spectroscopy and rotating-frame Overhauser enhancement spectroscopy) and, after permethylation, ESI–MS/MS and GC–MS methylation linkage analysis. The NMR analysis suggested the linear sequence Glcα1-3Manα1-2Manα1-2Manα1. These assignments were consistent with the ESI–MS/MS data, which show a linear Hex4 branch, indicated by the m/z 853 B-type and m/z 953 Y-type daughter ions. Linkage analysis showed the presence of a terminal Glc residue together with a terminal Man residue, a 3-O-substituted Man and 2-O-substituted and 3,6-di-O-substituted Man residues. Partial acetolysis resulted in the loss of one hexose residue consistent with a single αMan attached to the 6-arm and the Glcα1-3Manα1-2Manα1-2Man branch attached to the 3-arm.

Similar analyses of the Hex6HexNAc3 and Hex7HexNAc3 structures suggested an additional GlcNAc and Galβ1-4GlcNAc residues on the 6-arm of the structures respectively. These data provide support for the structures proposed in Figure 2(B). The Hex8HexNAc2 and Hex9HexNAc2 species from the Asn-454 glycosylation site were studied by positive ion ESI–MS/MS after permethylation and by digestion with jack bean α-mannosidase. The results were identical with those expected from conventional Man9GlcNAc2 and Man8GlcNAc2.

Our results, together with those of Bangs et al. [11], suggest that the Asn-289 glycosylation site of VSG221 may be added as a Man5GlcNAc2 structure (rather than as a conventional Man9GlcNAc2 structure) and that it is the Man5GlcNAc2 structure that takes part in the UGGT/calnexin protein-folding cycle. This is in contrast with other eukaryotes, where only Man9GlcNAc2 and Man8GlcNAc2 are thought to be substrates for UGGT.

Structure Related to Function: Molecules and Cells: A Focus Topic at BioScience2004, held at SECC Glasgow, U.K., 18–22 July 2004. Edited by D. Alessi (Dundee, U.K.), T. Cass (Imperial College London, U.K.), T. Corfield (Bristol, U.K.), M. Cousin (Edinburgh, U.K.), A. Entwistle (Ludwig Institute for Cancer Research, London, U.K.), I. Fearnley (Cambridge, U.K.), P. Haris (De Montfort, Leicester, U.K.), J. Mayer (Nottingham, U.K.) and M. Tuite (Canterbury, U.K.).

Abbreviations

     
  • ER

    endoplasmic reticulum

  •  
  • ESI–MS

    electrospray ionization mass spectrometry

  •  
  • ORF

    open reading frame

  •  
  • UGGT

    UDP-glucose:glycoprotein glucosyltransferase

  •  
  • VSG

    variant surface glycoprotein

D.J. thanks the MRC for a Ph.D. studentship. This work was supported by a Programme Grant from the Wellcome Trust to M.A.J.F.

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