Use of Giardia, which appears to have a single nucleotide-sugar transporter for UDP-GlcNAc, to identify the UDP-Glc transporter of Entamoeba

Abstract

Nucleotide-sugar transporters (NSTs) transport activated sugars (e.g. UDP-GlcNAc) from the cytosol to the lumen of the endoplasmic reticulum or Golgi apparatus where they are used to make glycoproteins and glycolipids. UDP-Glc is an important component of the N-glycan-dependent quality control (QC) system for protein folding. Because Entamoeba has this QC system while Giardia does not, we hypothesized that transfected Giardia might be used to identify the UDP-Glc transporter of Entamoeba. Here we show Giardia membranes transport UDP-GlcNAc and have apyrases, which hydrolyze nucleoside-diphosphates to make the antiporter nucleoside-monophosphate. The only NST of Giardia (GlNst), which we could identify, transports UDP-GlcNAc in transfected Saccharomyces and is present in perinuclear and peripheral vesicles and increases in expression during encystation. Entamoeba membranes transport three nucleotide-sugars (UDP-Gal, UDP-GlcNAc, and UDP-Glc), and Entamoeba has three NSTs, one of which has been shown previously to transport UDP-Gal (EhNst1). Here we show recombinant EhNst2 transports UDP-Glc in transfected Giardia, while recombinant EhNst3 transports UDP-GlcNAc in transfected Saccharomyces. In summary, all three NSTs of Entamoeba and the single NST of Giardia have been molecularly characterized, and transfected Giardia provides a new system for testing heterologous UDP-Glc transporters.

Fig. 1

Transport of UDP-GlcNAc by Giardia vesicles. A. Transport and transfer of various nucleotide sugars by Giardia membranes. Shaded bars indicate sealed vesicles, while unshaded bars indicate vesicles permeabilized with 0.1% Triton X-100 (negative control). B. Nucleotide-sugar transport by Giardia membranes with changing concentrations of UDP-GlcNAc. The K_m of transport is 1.9 μM.

Fig. 2

NDPase/apyrase activity of Giardia vesicles. Shaded bars indicate sealed vesicles, while unshaded bars indicate vesicles permeabilized with 0.1% Triton X-100. While nucleotide-sugar transport and transfer is increased in closed vesicles (Fig. 1A), NDPase/apyrase activity, which is normally present in the lumen of vesicles or on the cell surface, is increased in open vesicles.

Fig. 3

Myc-tagged GlNst is present in the perinuclear region and peripheral vesicles of transfected Giardia. A. GlNst is visualized with FITC-anti-myc antibodies (green) in permeabilized Giardia, the surface of which is labeled red with Alexafluor and the nucleus of which is labeled blue with DAPI. B. Negative control, which is transfected with an empty vector shows no binding with the anti-myc antibody.

Fig. 4

Quantitative RT-PCR, where Ct is the concentration of double stranded DNA produced by samples run in parallel, shows that mRNAs of GlNst are increased during encystation of Giardia. Trophozoites were induced to encyst for 6 h or 24 h and mRNAs for GlNst (putative UDP-GlcNAc transporter), GlOST (putative cytosolic O-GlcNAc transferase [27]), GNA (glucose-6-phosphate N-acetyltransferase, which is increased during encystation [26]), and tubulin were measured quantitatively.

Fig. 5

Phylogenetic tree of protist, fungal, and metazoan NSTs constructed by maximum likelihood method (21, 22). Protists include Giardia lamblia (Gl), Entamoeba histolytica (Eh), Trichomonas vaginalis (Tv), Trypanosoma cruzi (Tc), Trypanosoma brucei (Tb), Leishmania major (Lm), Plasmodium falciparum (Pf), Toxoplasma gondii (Tg), Cryptosporidium parvum (CP), and Dictyostelium discoideum (Dd). Fungi include Saccharomyces cerevisiae (Sc), Schizosaccharomyces pombe (Sp), and Encephalitizoon cuniculi (Ec), while metazoans include Homo sapiens (Hs), Caenorhabditis elegans (Ce), and Drosophila melanogaster (Dm). Arabidopsis thaliana (At) is the only plant. NCBI gene id numbers for each NST are listed in Supplemental Table 1. Giardia and Entamoeba NSTs are circled. Lengths of branches are proportional to differences between sequences, while numbers at nodes refer to bootstrap values for 100 trees. These bootstraps strongly support three distinct families of NSTs (3, 4).

Fig. 6

Expression of Giardia Nst in Saccharomyces. A. Western blot with anti-His antibodies of Saccharomyces transfected with GlNst with a C-terminal polyHis tag (lane 1) and Saccharomyces transfected with an empty vector (lane 2). B. Transport activity of closed vesicles of Saccharomyces transfected with a vector containing GlNst (black bars) or an empty vector (white bars). While transport of GDP-Man and UDP-Glc were the same, UDP-GlcNAc transport was dramatically increased in fungi transfected with GlNst. C. Plot of nucleotide-sugar transport by Saccharomyces transfected with GlNst with changing concentrations of UDP-GlcNAc (solid line). The K_m of UDP-GlcNAc transport is 2.7 μM. In contrast, there was minimal or no transport of UDP-GlcNAc in Saccharomyces transfected with an empty vector (dotted line).

Fig. 7

Expression of Entamoeba Nst3 in Saccharomyces. A. Transport and transfer of various nucleotide sugars by Entamoeba membranes. Shaded bars indicate sealed vesicles, while unshaded bars indicate vesicles permeabilized with 0.1% Triton X-100 (negative control). Transport and transfer of UDP-Glc and UDP-Gal by Entamoeba membranes were previously shown in ref. , while transport and transfer of UDP-GlcNAc is new. B. Transport of UDP-GlcNAc by closed vesicles of Saccharomyces transfected with a vector containing EhNst3 (black bars) was dramatically increased versus transport by control vesicles from yeast transfected with an empty vector (white bars). No UDP-GlcNAc transport was observed in open vesicles (negative control).

Fig. 8

Expression of Entamoeba Nst2 in Giardia. A. Western blot with anti-FLAG antibodies to proteins of Giardia transfected with a vector expressing Entamoeba Nst2 with a C-terminal FLAG tag (lane 1) or Giardia transfected with an empty vector (lane 2). B. Transport activities of closed vesicles of Giardia transfected with Entamoeba Nst2 (black bars) or Giardia transfected with an empty vector (white bars). While transport of UDP-GlcNAc and UDP-Gal were the same, UDP-Glc transport was dramatically increased in Giardia transfected with EhNst2. C. Plot of nucleotide-sugar transport by Giardia transfected with EhNst2 with changing concentrations of UDP-Glc (solid line). The calculated K_m of transport was 1.6 μM. In contrast, there was minimal or no transport of UDP-Glc in Giardia transfected with an empty vector (dotted line).