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

In the protozoan parasite Leishmania, abundant surface and secreted molecules, such as lipophosphoglycan (LPG) and proteophosphoglycans (PPGs), contain extensive galactose in the form of phosphoglycans (PGs) based on (Gal-Man-PO 4 ) repeating units. PGs are synthesized in the parasite Golgi apparatus and require transport of cytoplasmic nucleotide sugar precursors to the Golgi lumen by nucleotide sugar transporters (NSTs). GDP-Man transport is mediated by the LPG2 gene product, and here we focused on transporters for UDP-Gal. Data base mining revealed 12 candidate NST genes in the L. major genome, including LPG2 as well as a candidate endoplasmic reticulum UDP-glucose transporter (HUT1L) and several pseudogenes. Gene knock-out studies established that two genes (LPG5A and LPG5B) encoded UDP-Gal NSTs. Although the single lpg5A ؊ and lpg5B ؊ mutants produced PGs, an lpg5A ؊ /5B ؊ double mutant was completely deficient. PG synthesis was restored in the lpg5A ؊ /5B ؊ mutant by heterologous expression of the human UDP-Gal transporter, and heterologous expression of LPG5A and LPG5B rescued the glycosylation defects of the mammalian Lec8 mutant, which is deficient in UDP-Gal uptake. Interestingly, the LPG5A and LPG5B functions overlap but are not equivalent, since the lpg5A ؊ mutant showed a partial defect in LPG but not PPG phosphoglycosylation, whereas the lpg5B ؊ mutant showed a partial defect in PPG but not LPG phosphoglycosylation. Identification of these key NSTs in Leishmania will facilitate the dissection of glycoconjugate synthesis and its role(s) in the parasite life cycle and further our understanding of NSTs generally.

Section: Discussionmentioning

confidence: 99%

Two Functionally Divergent UDP-Gal Nucleotide Sugar Transporters Participate in Phosphoglycan Synthesis in Leishmania major

Capul

Barron

Dobson

et al. 2007

“…L. major UGM was localized in the cytoplasm as predicted from the lack of polypeptide sequences typical of peroxisomal targeting, membrane association, or anchorage. Interestingly, the UDPglucose 4Ј-epimerase of Trypanosoma brucei has been localized in the glycosome (42) and several other trypanosomatid enzymes involved in UDP-Gal production are predicted to be in this microbody (46). The cytoplasmic localization of UGM implies thus the existence of a UDP-Gal transporter exporting UDP-Gal from the glycosome into the cytoplasm and of a Golgi-localized UDP-Gal f transporter because the galactofuranosyltransferase LPG1 is situated in this organelle (37).…”

Section: Discussionmentioning

confidence: 99%

Targeted Gene Deletion of Leishmania major UDP-galactopyranose Mutase Leads to Attenuated Virulence

Kleczka

Lamerz

Zandbergen

et al. 2007

Considering the high incidence of galactofuranose (Gal f ) in pathogens and its absence from higher eukaryotes, the enzymes involved in the biosynthesis of this unusual monosaccharide appear as attractive drug targets. However, although the importance of Gal f in bacterial survival or pathogenesis is established, its role in eukaryotic pathogens is still undefined. Recently, we reported the identification and characterization of the first eukaryotic UDP-galactopyranose mutases. This enzyme holds a central role in Gal f metabolism by providing UDP-Gal f to all galactofuranosyltransferases. In this work, the therapeutical potential of Gal f metabolism in Leishmania major was hence evaluated by targeted replacement of the GLF gene encoding UDP-galactopyranose mutase. In L. major, Gal f is present in the membrane anchor of the lipophosphoglycan (LPG) and in glycoinositolphospholipids. Accordingly, the generated glf ؊ mutant is deficient in LPG backbone and expresses truncated glycoinositolphospholipids. These structural changes do not influence the in vitro growth of the parasite but lead to an attenuation of virulence comparable with that observed with a mutant exclusively deficient in LPG.

“…1). Because the trypanosomatid parasites Trypanosoma brucei and Trypanosoma cruzi are unable to take up galactose from the environment (9,10), the UDP-Glc 4-epimerase is indispensable for biosynthesis of UDP-Gal and derived glycoconjugates in these organisms and is essential for their survival (11)(12)(13)(14). In contrast, a salvage pathway for UDP-Gal synthesis is known to occur in Leishmania because radiolabeled Gal is taken up by promastigotes and incorporated into surface molecules (15).…”

mentioning

confidence: 99%

Leishmania UDP-sugar Pyrophosphorylase

Damerow

Lamerz

Haselhorst

et al. 2010

The Leishmania parasite glycocalyx is rich in galactosecontaining glycoconjugates that are synthesized by specific glycosyltransferases that use UDP-galactose as a glycosyl donor. UDP-galactose biosynthesis is thought to be predominantly a de novo process involving epimerization of the abundant nucleotide sugar UDP-glucose by the UDP-glucose 4-epimerase, although galactose salvage from the environment has been demonstrated for Leishmania major. Here, we present the characterization of an L. major UDP-sugar pyrophosphorylase able to reversibly activate galactose 1-phosphate into UDP-galactose thus proving the existence of the Isselbacher salvage pathway in this parasite. The ordered bisubstrate mechanism and high affinity of the enzyme for UTP seem to favor the synthesis of nucleotide sugar rather than their pyrophosphorolysis. Although L. major UDP-sugar pyrophosphorylase preferentially activates galactose 1-phosphate and glucose 1-phosphate, the enzyme is able to act on a variety of hexose 1-phosphates as well as pentose 1-phosphates but not hexosamine 1-phosphates and hence presents a broad in vitro specificity. The newly identified enzyme exhibits a low but significant homology with UDP-glucose pyrophosphorylases and conserved in particular is the pyrophosphorylase consensus sequence and residues involved in nucleotide and phosphate binding. Saturation transfer difference NMR spectroscopy experiments confirm the importance of these moieties for substrate binding. The described leishmanial enzyme is closely related to plant UDP-sugar pyrophosphorylases and presents a similar substrate specificity suggesting their common origin.