Abstract
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.
Highlights
Protozoan parasites of the genus Leishmania must survive in two separate and harsh environments to complete their life cycle: extracellularly within the midgut of the sand fly and intracellularly within the phagolysosome of vertebrate macrophages
10 10 NAg 5–13 NA 8–9 8–10 8–10 9–10 7 7–10 8–10 a Sequence comparisons were done using the BLAST or PSI-BLAST algorithms. b Assigned by the Leishmania Genome Project. c TM domains determined by submitting amino acid sequence to three different topology programs. d NS, no significant similarity. e LmjF24.0360 was assigned a start codon by the Leishmania Genome Project leading to a predicted 600-amino acid ORF; our results show that the correct ORF is 451 amino acids. f LmjF15.0840 is a pseudogene containing two internal stop codons, identified by both our and the Leishmania Genome Project sequence
UDP-Gal transporters (29 –38% identity, Table S3), but no significant similarity to Schizosaccharomyces pombe Hut1, Arabidopsis thaliana AtUTR1, or LPG2 (Table 1 and S3). These two Leishmania proteins were nearly as divergent from each other as they were from other UDP-Gal Nucleotide sugar transporters (NSTs) (26% versus 29 –38% amino acid identity; Table S3)
Summary
Cell Culture, Reagents, and Transfection—Leishmania were grown at 26 °C in M199 medium Cells were allowed to grow 16 –24 h in M199 medium with 10% fetal calf serum and plated on semisolid media containing 1% Noble agar (Fisher) and the appropriate selective drugs. ORFs were amplified from L. major DNA and cloned into the unique BamHI site of the Leishmania expression vectors pXG(NEO) (B1288) or pXG(PHLEO) (B3324). A universal miniexon primer (SMB936) was used in conjunction with oligonucleotide SMB1581 (Table S2) to amplify portions of gene-specific cDNAs from the pool of randomly primed cDNAs described above, using Taq polymerase (Roche Applied Science). Generation of LPG5 Mutants and Add-back Lines—Targeting fragments for LPG5A and LPG5B were liberated from their respective vectors and purified prior to transfection into L. major LV39. Lec cells were transfected with constructs pCDNA3, pCDNA-LPG5A, pCDNA-LPG5B, pCDNA-HUT1L, and pMKIT-hUGT1-cHA to make the strains Lec8/pCDNA3 (hereafter referred to as Lec8/ empty), Lec8/LPG5A, Lec8/LPG5B, Lec8/HUT1L, and Lec8/ hUGT1, respectively. The LPG was hydrolyzed with 2 N trifluoroacetic acid to release neutral monosaccharides and phos-
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