Identification, subcellular localization, biochemical properties, and high-resolution crystal structure of Trypanosoma brucei UDP-glucose pyrophosphorylase

Abstract

The protozoan parasite Trypanosoma brucei is the causative agent of the cattle disease Nagana and human African sleeping sickness. Glycoproteins play key roles in the parasite's survival and infectivity, and the de novo biosyntheses of the sugar nucleotides UDP-galactose (UDP-Gal), UDP-N-acetylglucosamine, and GDP-fucose have been shown to be essential for their growth. The only route to UDP-Gal in T. brucei is through the epimerization of UDP-glucose (UDP-Glc) by UDP-Glc 4'-epimerase. UDP-Glc is also the glucosyl donor for the unfolded glycoprotein glucosyltransferase (UGGT) involved in glycoprotein quality control in the endoplasmic reticulum and is the presumed donor for the synthesis of base J (β-D-glucosylhydroxymethyluracil), a rare deoxynucleotide found in telomere-proximal DNA in the bloodstream form of T. brucei. Considering that UDP-Glc plays such a central role in carbohydrate metabolism, we decided to characterize UDP-Glc biosynthesis in T. brucei. We identified and characterized the parasite UDP-glucose pyrophosphorylase (TbUGP), responsible for the formation of UDP-Glc from glucose-1-phosphate and UTP, and localized the enzyme to the peroxisome-like glycosome organelles of the parasite. Recombinant TbUGP was shown to be enzymatically active and specific for glucose-1-phosphate. The high-resolution crystal structure was also solved, providing a framework for the design of potential inhibitors against the parasite enzyme.

Fig. 1

Alignment of UDP-Glc pyrophosphorylase predicted amino acid sequences. The sequences of T. brucei, T. cruzi, L. major, Rattus norvegicus, H. sapiens and Candida albicans were aligned using Clustal W (http://www.ebi.ac.uk/) and Jalview (http://www.jalview.org/). Residues participating in contacts with the nucleoside (black circles), phosphate (triangles), and glucose residue (square) are highlighted. The highly conserved nucleotide-binding (pyrophosphorylase motif) loop (dashed line) and substrate-binding loop (full line) are boxed

Fig. 2

Detection of TbUGP mRNA and TbUGP protein in T. brucei. (A) RT–PCR reaction using 8, 16, 32 and 40 ng of RNA from T. brucei bloodstream (BSF) or procyclic (PCF) form parasites. (B) TbUGP western blot. Lane 1, E. coli recombinant TbUGP (25 ng); lane 2, BSF total lysate (5 × 10⁶ cell equivalent) and lane 3, TbUGP immunoprecipitate (2 × 10⁸ cell equivalent). The top arrow on the right indicates the native TbUGP, which has a slightly lower molecular weight than the recombinant protein due to the His₆ tag, and the faint band underneath is the heavy chain of IgG used in the immunoprecipitation. The molecular weight standards (kDa) are shown on the left

Fig. 5

Subcellular localization of TbUGP. Wild-type bloodstream form T. brucei cells were stained with affinity-purified mouse anti-TbUGP and Alexa 488-conjugated anti-mouse antibody (green channel, panels A, E) and with rabbit anti-GAPDH and Alexa 594-conjugated anti-rabbit antibody (red channel, B) to mark the glycosomes, or rabbit anti-enolase and Alexa 594-conjugated anti-rabbit antibody (red channel, F) to mark the cytosol. Merged images are shown in panels (C, G) and corresponding phase contrast images are shown in panels (D, H)

Fig. 3

Purification and characterization of recombinant His₆-TbUGP. (A) Coomassie blue-stained SDS–PAGE gel of proteins in uninduced E. coli cells (lane 1) and in IPTG-induced E. coli cells (lanes 2–7); total protein (lane 2), pellet after lysis and centrifugation (lane 3), supernatant after lysis and centrifugation (lane 4), flow through after Ni-affinity chromatography (lane 5), His₆-TbUGP purified by Ni-affinity chromatography and elution with imidazole (lane 6). Lane 7, His₆-TbUGP further purified by gel filtration. (B) Analytical ultracentrifugation profile of recombinant TbUGP at a final concentration of 0.75 mg/mL

Fig. 4

pH optimum and substrate specificity of recombinant TbUGP. (A) The pH optimum of TbUGP was determined by comparing the yields of UDP-Glc using the HPLC assay. The error bars indicate the SD values (n = 3). UDP-Glc was quantified by comparison with a UDP-Glc calibration curve. (B) The specificity of TbUGP was analyzed using the HPLC assay and different sugar-1-phosphate substrates, as indicated. Only Glc-1-P produced a UV-absorbing peak in the sugar nucleotide region of the chromatogram

Fig. 6

Crystal structure of TbUGP in complex with UDP-Glc. (A) Representation of the dimer observed as an artifact of crystallization. (B) Illustration of the three classical UGP domains present in TbUGP: the N-terminal domain (green), the catalytic central domain (red), and the C-terminal domain (yellow). (C) Overall structure comparison of TbUGP (green) with the unbound (yellow) and bound (cyan) forms of LmUGP. Arrow 1 shows the additional 12 degree closure of TbUGP onto substrate compared to both open and closed conformations of LmUGP. The actual active site is aligned almost perfectly with the closed LmUGP structure. Arrow 2 shows the substrate-binding loop (SB loop) aligning well with the closed conformation. Arrow 3 shows the nucleotide-binding loop (NB loop), similar to the one in LmUGP. (D) Electrostatic potential of TbUGP showing the extensive basic patch covering the substrate-binding pocket. Images were generated by Pymol