Identification and characterisation of two N-acetylglucosaminyltransferases associated with Trypanosoma brucei microsomes

doi:10.1016/0166-6851(82)90019-6

Molecular and Biochemical Parasitology

Volume 5, Issue 3, March 1982, Pages 173-187

https://doi.org/10.1016/0166-6851(82)90019-6 Get rights and content

Abstract

Microsomes from Trypanosoma brucei contain glycosyltransferases able to incorporate $N- [^{14} C]-acetylglucosamine$ into two different types of acceptors. A first transferase catalyzes the transfer of $N- [^{14} C]acetylglucosamine$ 1-phosphate from uridine diphosphate $N- [^{14} C]acetylglucosamine$ into dolichol monophosphate. The enzymatic activity requires Mn²⁺, is time and temperature dependent, has an optimum pH of 7.4 and is completely inhibited by the antibiotic tunicamycin. Exogenous dolichol monophosphate enhances the glycosyltransferase activity. The kinetics of incorporation are characterized by a K_m of 2.6 μM for uridine diphosphate N-acetylglucosamine and 1.45 μM for dolichol monophosphate. The characteristics of the N-acetylglucosaminyltransferase are comparable to those reported for the first enzyme of the dolichol cycle described in several eukaryotes. N-Acetylglucosaminylpyrophosphoryl-dolichol is essentially the only product of the reaction. A second type of activity which is responsible for the direct transfer of $N- [^{14} C]acetylglucosamine$ from uridine diphosphate $N- [^{14} C]acetylglucosamine$ into several endogenous polypeptide acceptors, is also associated with T. brucei microsomes. The reaction, which might be due to more than one enzyme, is dependent on Mn²⁺, but differs from the other transferase in all other characteristics. Time course and optimal temperature are different, and the optimum pH is 6.5. The reaction is independent of the external addition of dolichol monophosphate and tunicamycin has no inhibitory effect on the enzymatic activity. AK_m of 1.6 μM was calculated for uridine diphosphate N-acetylglucosamine.

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Cited by (7)

Identification and functional characterization of a highly divergent N-acetylglucosaminyltransferase I (TbGnTI) in Trypanosoma brucei
2014, Journal of Biological Chemistry
Citation Excerpt :
Thus, the actions of these two enzymes are suggested to be independent of each other, which would imply that the GlcNAc transferases involved in complex N-glycan biosynthesis in T. brucei may be different from their metazoan counterparts (15–18). Indeed, no obvious GnTI or GnTII homologs have been identified in the parasite genome (19) and, so far, only GPI anchor (20, 21) and unspecified GlcNAc transferase activities (22, 23) have been detected using T. brucei cell-free systems. A minimum of 38 distinct glycosidic linkages have been identified in the T. brucei glycome (19), however, so far only six glycosyltransferases have been experimentally related to specific genes: UDP-Glc:glycoprotein α1-3-glycosyltransferase to TbUGGT (24), dolichol phosphate mannose synthase to TbDPMS (25), Dol-P-Man:Man5GlcNAc2 α1–3-mannosyltransferase to TbALG3 (16), Dol-P-Man:Man7GlcNAc2 α1–6-mannosyltransferase to TbALG12 (17, 18), Dol-P-Man:Man2GlcNPI α1-2-mannosyltransferase to TbGPI10 (26), and UDP-GlcNAc:β-d-Gal-GPI β1-3-GlcNAc transferase to TbGT8 (19).
Trypanosoma brucei expresses a diverse repertoire of N-glycans, ranging from oligomannose and paucimannose structures to exceptionally large complex N-glycans. Despite the presence of the latter, no obvious homologues of known β1–4-galactosyltransferase or β1–2- or β1–6-N-acetylglucosaminyltransferase genes have been found in the parasite genome. However, we previously reported a family of putative UDP-sugar-dependent glycosyltransferases with similarity to the mammalian β1–3-glycosyltransferase family. Here we characterize one of these genes, TbGT11, and show that it encodes a Golgi apparatus resident UDP-GlcNAc:α3-d-mannoside β1–2-N-acetylglucosaminyltransferase I activity (TbGnTI). The bloodstream-form TbGT11 null mutant exhibited significantly modified protein N-glycans but normal growth in vitro and infectivity to rodents. In contrast to multicellular organisms, where the GnTI reaction is essential for biosynthesis of both complex and hybrid N-glycans, T. brucei TbGT11 null mutants expressed atypical “pseudohybrid” glycans, indicating that TbGnTII activity is not dependent on prior TbGnTI action. Using a functional in vitro assay, we showed that TbGnTI transfers UDP-GlcNAc to biantennary Man₃GlcNAc₂, but not to triantennary Man₅GlcNAc₂, which is the preferred substrate for metazoan GnTIs. Sequence alignment reveals that the T. brucei enzyme is far removed from the metazoan GnTI family and suggests that the parasite has adapted the β3-glycosyltransferase family to catalyze β1–2 linkages.
Tunicamycin-resistant variants from five species of Leishmania contain amplified DNA in extrachromosomal circles of different sizes with a transcriptionally active homologous region
1991, Molecular and Biochemical Parasitology
Twelve independent variants were selected from five species of Leishmania for resistance to tunicamycin by exposure of cultured promastigotes to increasing concentrations of this antibiotic, an inhibitor of the microsomal N-acetylglucosamine-1-phosphate transferase in the dolichol pathway of N-glycosylation. All variants obtained from all species, as found previously with Leishmania amazonensis, contain amplified chromosomal DNA exclusively as extrachromosomal circles. These circular amplicons hybridize with amplified DNAs cloned previously from tunicamycin-resistant Leishmania amazonensis, but not with those from Leishmania resistant to other drugs. The amplicons from tunicamycin-resistant cells vary with different species in size from 30 to 70 kb, but all share a homologous region of 20 kb. Multiple independent transcripts are overexpressed from this region. Elevation of the microsomal glycosyltransferase activity is demonstrated in these variants from representative species. The results thus provide further evidence that this enzyme is overexpressed due to amplification of the gene in these cells. The consistent observation of this event in all cases studied also suggests that this is the predominant, if not the only mechanism of tunicamycin resistance in Leishmania.
N-Glycosylation as a biochemical basis for virulence in Leishmania mexicana amazonensis
1988, Molecular and Biochemical Parasitology
Promastigotes of Leishmania mexicana amazonensis grown in vitro under different conditions showed variable degrees of virulence, as determined quantitatively by the size of the lesions and the number of intracellular parasites produced in mice and in cultured macrophages, respectively. Promastigotes newly transformed from amastigotes gave the highest degree of virulence, which decreased progressively with periods of their continuous in vitro cultivation. This loss of virulence was prevented by making virulent wildtype promastigotes resistant to tunicamycin, an inhibitor of N-acetylglucosamine-1-phosphate transferase in the dolichol pathway of protein glycosylation. In the wildtype cells, the progressive loss of virulence during a period of two years was marked by a gradual decrease in the activity of the glycosyltransferase, incorporation of 2-d-[³H]mannose and the expression of a surface glycoprotein (gp63). Virulent and avirulent wildtype cells differed in these activities by 3–4 fold. In contrast, during equivalent periods of in vitro cultivation, tunicamycin-resistant cells were found to consistently maintain the biochemical phenotypes of the virulent wildtype, except for a disproportional elevation of the glycosyltransferase activity. Thus, only a portion of the over-produced enzyme is relevant to leishmanial virulence in the drug-resistant variants. The bulk of its activity in these cells serves to overcome the inhibitory effect of tunicamycin responsible for their resistance to this drug, as shown previously. A role of N-glycosylation in leishmanial virulence is now indicated by studying cells of this phenotype selected by two independent methods. It is inferred that leishmanial virulence may be regulated by the level of the glycosyltransferases for N-glycosylation of proteins, e.g. gp63 in relation to their expression as virulent determinants.
Trypanosoma brucei brucei, T. brucei gambiense, and T. brucei rhodesiense: Common glycoproteins and glycoprotein oligosaccharide heterogeneity identified by lectin affinity blotting and endoglycosidase H treatment
1987, Experimental Parasitology
Whole cell extracts of 10 clones of bloodstream forms of African trypanosomes representing two strains of Trypanosoma brucei gambiense, one strain of T. b. rhodesiense and one strain of T. b. brucei were fractionated on sodium dodecyl sulfate-polyacrylamide gels, electrophoretically transferred to nitrocellulose paper, and probed with horseradish peroxidase conjugated lectins to detect glycoproteins. Variant specific glycoproteins of all 10 clones bound peroxidase labeled concanavalin A, but peroxidase labeled wheat germ agglutinin bound to the variant specific glycoproteins of only 3 of the 10 clones examined. In addition, 22 other glycoproteins expressed in common by all clones bound peroxidase labeled concanavalin A; 19 common glycoproteins bound peroxidase labeled wheat germ agglutinin. Lectin binding to transferred glycoproteins was specifically inhibited by appropriate monosaccharides, α-methyl mannoside for concanavalin A and N-acetyl glucosamine for wheat germ agglutinin. Prior incubation of blots in endo-β-N-acetylglucosaminidase H eliminated binding of peroxidase-labeled concanavalin A to most of the 22 common glycoproteins. Two glycoproteins, designated Gp 81 and Gp 110, were the major Endoglycosidase H resistant components. Endoglycosidase H treatment also reduced binding of peroxidase labeled concanavalin A to the variant specific glycoproteins of 7 clones. The variant specific glycoproteins from the 3 clones that bound peroxidase labeled concanavalin A following enzyme treatment were those that bound peroxidase labeled wheat germ agglutinin. These results show that African trypanosomes express a greater number of glycoproteins than has been reported previously and that only a limited number of these glycoproteins bear Endoglycosidase H resistant oligosaccharides.
Recent studies on inhibitors of macromolecular synthesis and function in trypanosomes
1984, Pharmacology and Therapeutics
The N-glycans of Trichomonas vaginalis contain variable core and antennal modifications
2012, Glycobiology

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Molecular and Biochemical Parasitology

Abstract

References (26)

The role of lipid intermediates in the glycosylation of proteins in the eukaryotic cell

Biochim. Biophys. Acta

The chemical composition of trypanosomes

Glycopeptides from variant surface glycoproteins of Trypanosoma brucei

Tunicamycin inhibition of polyisoprenol N-acetylglucosaminyl pyrophosphate formation in calf liver microsomes

Biochem. Biophys. Res. Commun.

The specific site of tunicamycin inhibition in the formation of dolichol-bound N-acetylglucosamine derivatives

FEBS Lett.

Studies on the biosynthesis of the variant surface glycoprotein of Trypanosoma brucei

Antigenic analysis in the Trypanosoma brucei group using the agglutination reaction

Trans. R. Soc. Trop. Med. and Hyg.

Protein measurement with folin phenol reagents

J. Biol. Chem.

Formation of α-1,2-mannosyl-mannose by an enzyme preparation from rabbit liver

J. Biol. Chem.

Membrane glycoproteins

Tunicamycin inhibition of [³H]glucosamine incorporation into yeast glycoproteins: binding of tunicamycin and interaction with phospholipids

Arch. Biochem. Biophys.

Solubilization and properties of mannose and N-acetylglucosamine transferases involved in formation of polyprenyl-sugar intermediates

J. Biol. Chem.

Properties of a soluble polyprenyl phosphate

Cited by (7)

Identification and functional characterization of a highly divergent N-acetylglucosaminyltransferase I (TbGnTI) in Trypanosoma brucei

Tunicamycin-resistant variants from five species of Leishmania contain amplified DNA in extrachromosomal circles of different sizes with a transcriptionally active homologous region

N-Glycosylation as a biochemical basis for virulence in Leishmania mexicana amazonensis

Trypanosoma brucei brucei, T. brucei gambiense, and T. brucei rhodesiense: Common glycoproteins and glycoprotein oligosaccharide heterogeneity identified by lectin affinity blotting and endoglycosidase H treatment

Recent studies on inhibitors of macromolecular synthesis and function in trypanosomes

The N-glycans of Trichomonas vaginalis contain variable core and antennal modifications