Trypanosoma brucei UDP-Glucose:Glycoprotein Glucosyltransferase Has Unusual Substrate Specificity and Protects the Parasite from Stress

Abstract

In this paper, we describe the range of N-linked glycan structures produced by wild-type and glucosidase II null mutant bloodstream form Trypanosoma brucei parasites and the creation and characterization of a bloodstream form Trypanosoma brucei UDP-glucose:glycoprotein glucosyltransferase null mutant. These analyses highlight peculiarities of the Trypanosoma brucei UDP-glucose:glycoprotein glucosyltransferase, including an unusually wide substrate specificity, ranging from Man 5 GlcNAc 2 to Man 9 GlcNAc 2 glycans, and an unusually high efficiency in vivo, quantitatively glucosylating the Asn263 N-glycan of variant surface glycoprotein (VSG) 221 and 75% of all non-VSG N glycosylation sites. We also show that although Trypanosoma brucei UDP-glucose:glycoprotein glucosyltransferase is not essential for parasite growth at 37°C, it is essential for parasite growth and survival at 40°C. The null mutant was also shown to be hypersensitive to the effects of the N glycosylation inhibitor tunicamycin. Further analysis of bloodstream form Trypanosoma brucei under normal conditions and stress conditions suggests that it does not have a classical unfolded protein response triggered by sensing unfolded proteins in the endoplasmic reticulum. Rather, judging by its uniform Grp78/BiP levels, it appears to have an unregulated and constitutively active endoplasmic reticulum protein folding system. We suggest that the latter may be particularly appropriate for this organism, which has an extremely high flux of glycoproteins through its secretory pathway.

Unfolded Protein Response Pathways in Bloodstream-Form Trypanosoma brucei?

“…Tunicamycin does inhibit the forward trafficking of other highly glycosylated secretory cargo, such as the lysosomal membrane protein p67 (C. Tiengwe and J. D. Bangs, unpublished observations), but based on our current results, this is apparently not enough to induce a UPR-like response in BSF trypanosomes. Our findings with tunicamycin are consistent with those of Koumandou et al (12) and Izquierdo et al (38), both of whom found no induction of BiP. The latter authors also found that neither heat shock, nor knocking out a component of the ER folding machinery (UDPglucose:glycoprotein glycosyltransferase), affected BiP expression.…”

The disruption of GDP-fucose de novo biosynthesis suggests the presence of a novel fucose-containing glycoconjugate in Plasmodium asexual blood stages

“…Glycosyltransferase activity and sugar nucleotides donors have long been linked to protein folding and thermotolerance in many organisms, including different parasites353637. It has been suggested that Protein O -fucosyltransferase 2 (PoFUT2), of which a homolog is conserved and expressed in the P. falciparum genome18, may be involved in a novel quality control mechanism for the proper folding of TSR-domain containing proteins in the endoplasmic reticulum (ER)3738.…”

A Quaternary Mechanism Enables the Complex Biological Functions of Octameric Human UDP-glucose Pyrophosphorylase, a Key Enzyme in Cell Metabolism

“…In trypanosomatid parasites such as Leishmania and Trypanosoma , sugar-activating enzymes - including UGP - represent attractive drug targets since many vital processes within the parasites utilize UDP-Glc and UDP-Gal38394041. However, drugs targeting structurally conserved elements (i.e.…”