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J. Biol. Chem., Vol. 282, Issue 19, 14006-14017, May 11, 2007
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1
From the
Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110 and the Department of Biochemistry, University of Kentucky Medical Center, Lexington, Kentucky 40536
Received for publication, November 27, 2006 , and in revised form, February 22, 2007.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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1,4)-Man(
1)PO4 (referred to hereafter as the PG repeat unit) (5). PG repeats frequently contain additional sugar substitutions in different species and strains, most commonly on the Gal residue, although modifications of the Man also occur (Fig. 1) (6).
Leishmania PGs can be linked to the cell surface via glycosylphosphatidylinositol anchor attachment, directly as in LPG or through attachment to the protein backbone in PPGs (Fig. 1). The basic LPG structure has four domains: a 1-alkyl-2-lyso-PI anchor, a heptasaccharide core consisting of Gal(1,6)-Gal(1,3)-Galf(1,3)-[Glc(1-PO4)6]-Man(1,3)-Man(1,4)-GlcN(1,6), the poly-PG "backbone" consisting of (Gal(1,4)-Man(1)P) PG repeat units (average n 
15–30), and a terminating oligosaccharide cap (Fig. 1). In Leishmania major, LPG plays several roles in parasite survival, including control of parasite binding to the sand fly midgut wall, resistance to lysis by complement, protection from oxidative damage, and delaying phagolysosomal fusion (6–8). PPGs arise from a large gene family that encodes large proteins (>200 kDa) containing Ser/Thr-rich regions to which PG repeating units are attached (9, 10). PPGs occur in different forms, including membrane-bound PPG, filamentous PPG, and secreted PPG (9). These PG-containing molecules play roles in parasite transmission and virulence following sand fly biting, such as modulating macrophage immune functions (11–13). The role(s) of secreted acid phosphatases in parasite virulence are not well understood, since their levels are relatively low in L. major (14), and secreted acid phosphatase null mutants in Leishmania mexicana are virulent (15).
The assembly of Leishmania PG repeating units to form LPG and PPGs occurs in the Golgi apparatus (16–18). Nucleotide sugar transporters (NSTs) transport cytoplasmically synthesized nucleotide sugars into the endoplasmic reticulum (ER) or Golgi lumen, where they are consumed in glycosylation reactions (19–22). A large number of NSTs have been identified, and they now constitute a well characterized family of membrane proteins (20, 23). Previously, we showed that the Leishmania LPG2 gene encoded the Golgi GDP-Man transporter, one of the earliest NSTs identified, and the first multispecific NST, since it was additionally able to transport GDP-D-Ara and GDP-L-Fuc (17). lpg2– mutants in three species of Leishmania were completely devoid of PGs (24–26); however, the consequences to parasite virulence differed greatly. Mutant lpg2– L. major were unable to survive within macrophages or to cause pathology (8), whereas lpg2– L. mexicana were able to survive and induce disease (26).
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Since our interests concerned the role of galactosylation reactions occurring within secretory compartments, our strategy focused on inactivation of UDP-Gal transport. Thus, it was necessary first to identify the parasite's UDP-Gal transporters. Twelve candidate NST sequences were identified in the L. major genome, with five showing varying degrees of similarity to known UDP-Gal transporters, including two pseudogenes. Our data indicate that two candidates, LPG5A and LPG5B, both encode UDP-Gal transporters and are required for phosphoglycan synthesis in L. major, although their functions differ significantly. More limited studies of another candidate, HUT1L, suggest that it may be the functional homolog of the ER UDP-Glc NST, shown recently to be important to the protein folding glucosylation/reglucosylation process (29, 30).
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Leishmania cells were transfected by electroporation using either a low voltage (31) or high voltage protocol (34). Following transfection, cells were allowed to grow 16–24 h in M199 medium with 10% fetal calf serum and then plated on semisolid media containing 1% Noble agar (Fisher) and the appropriate selective drugs. Individual colonies were picked and grown in liquid medium. Clones were maintained in selective medium and then removed from selection for one passage prior to experiments. Hygromycin B was from Calbiochem (now under EMD Biosciences (San Diego, CA)), G418 was from BioWhittaker (now under Cambrex Bio Science (Walkersville, MD)), puromycin powder was from Sigma, blasticidin was from Invitrogen, nourseothricin was from Werner BioAgents (Jena, Germany), and phleomycin was from InvivoGen (San Diego, CA).
[-32P]dCTP was from PerkinElmer Life Sciences. UDP-[6-3H]Gal and GDP-[6-3H]Man were from American Radiolabeled Chemicals (St. Louis, MO). The protease inhibitors leupeptin, 4-(2-aminoethyl)-benzenesulfonyl fluoride, and E-64 were from Sigma. Other reagents not mentioned were from either Sigma or Fisher. PCR was done using Taq polymerase (Roche Applied Science) or Platinum Taq Hi Fidelity (Invitrogen). Oligonucleotide primers were purchased from IDT (Coralville, IA), and their sequences are described in the supplemental materials. Restriction enzymes were from New England Biolabs Inc. (Ipswich, MA). Standard methods for general molecular biological techniques including Southern blotting were used as described (35). L. major Constructs—Molecular manipulations are summarized in Table S1. Briefly, 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). For expression, oligonucleotide primers (Table S2) added an optimal translation initiation sequence (CCACC) upstream of the ORF start codon. PCR products were directly cloned into the TA vector pGEM-TEasyTM (Promega, Madison, WI), sequenced, and then liberated with BamHI and inserted into Leishmania expression vectors. Where indicated, ORFs were liberated from pXG vectors with BamHI and cloned into the unique BamHI site of pCDNA3 (B1974; Table S1). pXG(NEO)-hUGT1-cHA (B5541) and pMKIT-hUGT1-cHA (B5034) were gifts from H. Segawa.
LPG5A allelic replacement constructs were made by inserting ORFs encoding hygromycin B (HYG) or puromycin (PAC) resistance between 0.95-kb 5' and 3' LPG5A flanking regions present in pBSK-LPG5Aflanks (B4748; Table S1), making pBSK-LPG5A-HYGKO (B5019) and pBSK-LPG5A-PACKO (B5020). Disruption constructs for LPG5B were made by inserting a 0.92-kb XhoI fragment from pXG(BSD) (B4098), which contains the DHFR-TS splice acceptor site and the BSD resistance gene, into the unique BsrGI site of pBSK-LPG5B subclone (B5025) to make pBSK-LPG5B-BSDdisrupt (B5025; Table S1). A 1.9-kb AccI/XhoI fragment from pXG(NEO) (B1288), which contains the DHFR-TS splice acceptor site and the NEO resistance gene, was cloned into the BsrGI site of pBSK-LPG5Bsubclone to make pBSK-LPG5B-NEOdisrupt (B5026; Table S1).
Mapping of the LPG5A Splice Acceptor Site—Mapping of splice acceptor sites was accomplished using RT-PCR (36). FV1 RNA was prepared from exponentially growing parasites using Trizol (Roche Applied Science) and used as a template to generate randomly primed cDNAs using Superscript II (Stratagene, La Jolla, CA). 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). The single product was cloned into pGEM-TEasyTM (Promega, Madison, WI) and sequenced.
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. For single-gene mutants, two rounds of transfection were done to recover homozygous mutants, and correct targeting was confirmed using Southern analysis. The names below follow the formal nomenclature for Leishmania (37). The 
lpg5A/lpg5A clonal strain A1 was chosen for biochemical analysis and is referred to as lpg5A–. The ^lpg5B/^lpg5B clonal strain A3 was chosen for biochemical analysis and is referred to as lpg5B–. To produce a strain targeted in both LPG5A and LPG5B, LPG5B was disrupted in the lpg5A– mutant. Clonal strain 2A-2, whose genotype lpg5A/lpg5A/^lpg5B/^lpg5B was confirmed by Southern analysis, is referred to as lpg5A–/5B–. Further details are discussed in supplemental materials. Parasite strains were passed in vitro no more than six times (M1P6) before use.
Immunoblotting—Whole cell lysates from L. major promastigotes were prepared from exponential growth or stationary phase parasites in SDS sample buffer; 5 x106 cell equivalents/lane were separated by discontinuous SDS-PAGE on 4% stacking and 12.5% resolving gels and then electrotransferred to nitrocellulose membranes (Hybond-ECL; Amersham Biosciences). Protein loading was assessed by staining with 5% Ponceau S, 1% glacial acetic acid (w/v) prior to Western analysis. Monoclonal antibody WIC79.3 (38) was used at a 1:1000 dilution; CA7AE (39) was used at a 1:1000 dilution. Secondary antibodies conjugated to peroxidase were from Amersham Biosciences, and ECL reactions were conducted using Pierce or PerkinElmer Life Sciences chemiluminescence kits.
Heterologous Expression—WT Chinese hamster ovary (CHO) and Lec8 cells (40, 41) were from ATCC (Manassas, VA) and maintained in culture following ATCC recommendations. Lec8 cells were transformed using Lipofectamine2000 (Invitrogen) per manufacturer's instructions. Transfectants were selected in medium containing 640 µg/ml G418; 5,000–10,000 stable transfectants were pooled and maintained in 300 µg/ml G418. Lec8 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.
Fluorescent Lectin Binding—The binding of fluorescein isothiocynate-labeled Maackia amurensis agglutinin (EY Laboratories, San Mateo, CA) was assessed by fluorescence microscopy and fluorescence-activated cell sorting. Transfectants were removed from antibiotic selection for one passage prior to experiments. Cells grown on coverslips for IFA or scraped from flasks for fluorescence-activated cell sorting were washed once with phosphate-buffered saline and fixed for 1 min in 3.7% formaldehyde (v/v) in phosphate-buffered saline and incubated for 1 h at 25°C in 20 or 40 µg/ml M. amurensis fluorescein isothiocyanate in phosphate-buffered saline. Cells were washed three times with phosphate-buffered saline and visualized using an Olympus AX-70 microscope (Melville, NY) or a BD Biosciences FACSCalibur.
Purification and Analysis of LPG—LPG was prepared from exponential and stationary phase parasites using previously described methods (42). To assess side chain modification, phosphoglycan repeating units were obtained by depolymerization of LPG using mild acid hydrolysis, dephosphorylated, and covalently labeled with either 8-aminonaphthalene-1,3,6-trisulfate or 1-aminopyrene-3,6,8-trisulfonate and analyzed using fluorescence-assisted carbohydrate electrophoresis and capillary electrophoresis (43).
The number of PG repeating units per LPG molecule was determined using two methods. The first method used gas chromatography-mass spectrometry (44). Briefly, the lipid anchor was cleaved by nitrous acid deamination, and the anhydromannose residue at the reducing end was further reduced with NaBH4 to produce a single anhydromannitol residue for each LPG molecule. The LPG was hydrolyzed with 2 N trifluoroacetic acid to release neutral monosaccharides and phosphorylated monosaccharides (mostly galactose 6-phosphate). The phosphorylated monosaccharides were removed by anion exchange chromatography, and then the neutral monosaccharides were acetylated before gas chromatography-mass spectrometry. The ratio of mannose to anhydromannitol was calculated, which was adjusted to account for the mannose present in the LPG core and capping sugars, to determine the number of PG repeats present per LPG molecule. The second method utilized capillary electrophoresis (43). Purified LPG was treated with nitrous acid, NaBH4, and trifluoroacetic acid as in the gas chromatography-mass spectrometry method. The released monosaccharides were directly labeled with 1-aminopyrene-3,6,8-trisulfonate, omitting acetylation, and separated on a Beckman P/ACE 5000 (Fullerton, CA). As with the gas chromatography-mass spectrometry method described above, the ratio of Man to anhydromannitol was determined to estimate the number of PG repeating units per LPG molecule. WT LPG was used as a control in all experiments.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Two additional potential L. major NSTs were only detected using more sensitive PSI-BLAST comparisons (Table 1). LmjF07.0400 showed a relationship to members of solute carrier subfamily family 35F, whose functions have not been determined. LmjF36.0670 showed a relationship to members of solute carrier family 35A, including human SLC35A3, which mediates UDP-GlcNAc uptake (47, 48).
Phylogenetic analysis showed that the candidates LmjF24.0360 and LmjF18.0400 associated with other functionally characterized UDP-Gal transporters from S. pombe, plants, and mammals (Fig. 2 and Table S3; data not shown). Although encouraging, this cluster contained transporters with specificities for a variety of UDP-sugars as well as CMP-sialic acid transporters (23), reinforcing prior observations that phylogeny is of only limited usefulness in predicting nucleotide sugar specificity in the NST family (20, 21, 49). Nonetheless, we used evolutionary relationship as a guide to focus on the two candidates showing the best homology to hUGT1, which we named LPG5A and LPG5B (Table 1), and carried out limited studies on the HUT1-related gene LmjF22.1010, which we named HUT1L (HUT1-like).
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Properties of the LPG5A and LPG5B Open Reading Frames—Typical eukaryotic NSTs are about 320–400 amino acids in length; however, the predicted LPG5A and LPG5B proteins comprised 600 and 561 amino acids, respectively (Table 1). We mapped the 5' end of the LPG5A mRNA using reverse transcription-PCR and the fact that all Leishmania mRNAs bear a common 39-nucleotide 5' leader sequence added by trans-splicing. This placed the splice acceptor at a position located 384 nucleotides 3' of the annotated AUG for the 600-amino acid LPG5A ORF (Fig. 3A). The next AUG following this mapped splice acceptor occurred 63 nucleotides downstream (Fig. 3B), leading to a predicted LPG5A protein of 451 amino acids (Table 1). As shown below, the 451-amino acid LPG5A ORF was functional. Although efforts to similarly map the LPG5B 5' end were unsuccessful, current data suggest that the 561-amino acid ORF for LPG5B is correct (discussed below).
Several transmembrane (TM) prediction algorithms suggested the occurrence of 8–10 TM domains for both LPG5A and LPG5B, consistent with predictions for other NSTs (Table 1, Figs. 4 and S1). We incorporated previous studies of the murine CMP-sialic acid transporter, whose topology has been experimentally determined and whose sequence is closely related to UDP-Gal transporters (50). From this and sequence alignment criteria, we developed models predicting 10 transmembrane domains in the LPG5A and LPG5B proteins (Figs. 4 and S1; Fig. S2 presents an amino acid alignment with shaded predicted TM domains).
One striking feature in both LPG5A and LPG5B was the prediction of a large cytoplasmic loop (134 and 199 amino acids, respectively) between TM domains 2 and 3 (Figs. 4 and S1). For comparison, this loop is 25–26 amino acids in the mammalian UDP-Gal and CMP-Sia NSTs, and no other characterized NSTs to date bear a loop comparable with those of LPG5A or LPG5B. Other conserved features included several buried charged residues, including conserved Glu and Lys residues in the second predicted TM2 domains of LPG5A, LPG5B, murine CMP-Sia, and human UDP-Gal NSTs (Figs. 4, S1, and S2). Residues shown to be functionally important in the mammalian UDP-Gal and CMP-Sia (21) were conserved in LPG5A and LPG5B as well (Figs. 4, S1, and S2).
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2,3)-galactose-specific M. amurensis agglutinin (41, 51). When a functional UDP-Gal transporter is expressed in these cells, M. amurensis agglutinin binding is restored (52), a widely used assay to functionally identify and characterize UDP-Gal transporters from a variety of species (19, 21, 22). M. amurensis agglutinin binding was examined in stably transfected Lec8 cells bearing constructs expressing LPG5A, LPG5B, or HUT1L. By fluorescence microscopy (Fig. 5A) and flow cytometry (Fig. 5B), LPG5A and LPG5B transfectants showed strong M. amurensis agglutinin binding, similar to that of WT CHO cells or to Lec8 cells expressing the human UDP-Gal transporter hUGT1. A caveat to the negative HUT1L result is that a specific antiserum to HUT1L was not available, precluding verification of its correct expression and/or localization in the transfectants. These data suggested that LPG5A and LPG5B probably encoded proteins with UDP-Gal transporter activity.
Generation of Null LPG5A, LPG5B, and Double Mutants—To further study the candidate UDP-Gal transporters, we created null mutants. Leishmania are asexual and predominantly disomic, necessitating the use of two successive rounds of transfection to generate null mutants (53, 54). For LPG5A, we designed constructs that would precisely replace the 451-amino acid LPG5A ORF with drug resistance marker ORFs encoding hygromycin B (HYG) or puromycin (PAC) resistance (Fig. 6A). Starting with the WT LV39 strain of L. major, correct replacement of both alleles was confirmed by Southern analysis (Fig. 6C), and this mutant is termed lpg5A–.
For LPG5B, we were unable to determine the mRNA start site by RT-PCR using a variety of LPG5B-specific primers and amplification conditions (data not shown); the reasons for this are not evident to us. The assignment of the LPG5B ORF, however, was supported by functional assays (Fig. 5), sequence alignment (Fig. S2), and transfection assays showing that several shorter ORFs initiating at downstream AUGs were inactive (data not shown). Thus, for LPG5B an insertional inactivation approach was used where autonomous cassettes mediating resistance to blastocidin (BSD) and G418 (NEO) were inserted into the LPG5B ORF (Fig. 6B). Correct targeting was confirmed by Southern analysis (Fig. 6D), and this mutant was designated lpg5B–. To make the lpg5A–/5B– mutant, we inactivated LPG5B in the background of the lpg5A– mutant above. Successful alteration of both genes was confirmed by Southern analysis (Fig. 6, C and D). All mutants grew at normal rates and to normal densities in standard M199 culture media in vitro (data not shown).
LPG5A or LPG5B Is Sufficient for PG Synthesis, but the lpg5A–/5B– Mutant Lacks PGs—L. major PG repeats are often modified by side chain Gal residues, added in consecutive 1,3-linkages to the Gal residue within the PG repeating unit by specific Gal-transferases (Fig. 1) (55). Gal-modified PG repeats can be recognized by the monoclonal antibody WIC79.3 (56), whereas unmodified PG repeats can be recognized by monoclonal antibody CA7AE (39). We used these in immunoblotting to probe the effects of LPG5A and LPG5B mutations on PG synthesis; similar results were obtained by immunofluorescence microscopy (data not shown).
WT extracts showed strong reactivity to WIC79.3 in the 25–100-kDa region, corresponding to LPG, whereas a much fainter reactivity was observed within the stacking gel, where the large PPGs migrate under these conditions (Fig. 7A, lane 1) (7, 10). In the lpg5A– mutant, strong WIC79.3 reactivity remained, but it migrated faster with an apparent molecular mass of 20–40 kDa (Fig. 7A, lane 2). A slight increase in WIC79.3 reactivity in the stacking gel/PPG region was also observed (Fig. 7A, lane 2). In contrast, for the lpg5B– mutant, strong WIC79.3 reactivity in the 25–100-kDa "LPG region" remained, whereas greatly elevated reactivity was observed within the stacking gel/"PPG region" (Fig. 7A, lane 3). These data suggested that loss of LPG5A primarily affected PGs associated with LPG, whereas loss of LPG5B primarily affected PGs associated with PPG.
Both the lpg5A– and lpg5B– mutant showed a small increase in CA7AE reactivity in the stacking gel/PPG region (Fig. 7B, lanes 2 and 3 versus lane 1). This was consistent with the idea that loss of UDP-Gal NST activity leads to decreased levels of galactosylated PGs. The lpg5B– PPG showed slightly decreased mobility relative to that of lpg5A– (Fig. 7B, lanes 2 and 3). The significance of this observation is uncertain, since structural studies of the PPG were not undertaken; potentially, the CA7AE-reactive PPG population is a minor fraction of total PPGs.
In contrast to the single mutants, no reactivity with either monoclonal antibody was seen for the double lpg5A–/5B– mutant, in either the LPG or stacking gel/PPG regions (Fig. 7, A and B, lanes 5). This phenotype was identical to that seen in the lpg2– mutant (Fig. 7, A and B, lanes 6), which is completely deficient in PGs through loss of the GDP-Man transporter (17). Thus, lpg5A–/5B– had similarly completely lost PG synthesis. PG expression was restored by reintroduction of either LPG5A or LPG5B, singly (data not shown) or in combination (Fig. 7C).
Altered LPG Structure in lpg5A– but Not lpg5B– Mutants—The altered mobility of lpg5A– LPG (Fig. 7A, lane 2) suggested that it was structurally altered, presumably arising through decreased levels of galactosylation. This could arise through decreased numbers of PG side chain Gal residues or PG repeats or both (Fig. 1A). We thus purified LPG from WT and mutant strains and determined its structure (Table 2). Side-chain galactosylation was assessed by depolymerizing LPG with mild acid to release the PG repeating units, which were then separated by capillary electrophoresis and quantitated. To examine the number of PG repeats in the LPG backbone of the lpg5A– mutant, we measured the ratio of mannose to anhydromanitol, the former arising from the PG repeats and the latter from the LPG core, following removal of the lipid anchor by nitrous acid deamination and reduction with NaBH4 and hydrolysis to monosaccharides (see "Experimental Procedures").
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Thus, the immunoblotting results are explicable as follows. The much greater reactivity of PPGs with WIC79.3 in the lpg5B– mutant can be attributed to increased levels of monogalactosylated PG repeats, relative to WT PPGs that bear predominantly oligo-/polygalactosylated PG repeats. Thus, loss of LPG5B appears to result in a decrease in galactosylation, but specifically affecting PPGs and not LPG (Table 1), in contrast to loss of LPG5A, where the reverse is seen.
The lpg5A–/5B– Mutant Is Rescued by Expression of a Heterologous UDP-Gal Transporter—Attempts to demonstrate decreased UDP-Gal uptake with purified lpg5A–/B– microsomes were unsuccessful (supplemental materials). This raised the formal possibility that the PG deficiency of the lpg5A–/5B– mutant might arise through mechanisms other than decreased UDP-Gal uptake. If this idea were correct, we reasoned that expression of a distantly related heterologous UDP-Gal transporter would be unable to rescue PG synthesis. Thus, we expressed the human UDP-Gal transporter hUGT1 in the lpg5A–/5B– mutant. However, in these transfectants, WIC79.3-reactive LPG was restored fully (Fig. 7D, lane 3 versus lane 1). It is highly unlikely that hUGT1 expression would rescue PG synthesis by any mechanism other than restoration of UDP-Gal uptake, and thus we conclude that the PG deficiency of the lpg5A–/B– double mutant arises from a lack of UDP-Gal transport activity.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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From the structures of known Leishmania glycoconjugates, one could predict that in addition to GDP-Man and UDP-Gal, NSTs mediating uptake of UDP-Glc, GDP-D-arabinopyranose, and UDP-galactofuranose are required. Many NSTs are multispecific and able to transport more than one nucleotide sugar, such as LPG2, which can transport guanine diphosphoarabinose and guanine diphosphofucose in addition to GDP-Man (17, 23, 57). Since UDP-Glc epimerase is localized to the glycosome in trypanosomatids (58), potentially UDP-Gal transport from this compartment may depend upon genes in the NST family.
The LPG5A/5B-related group of Leishmania NSTs appears to be a dynamic area of evolutionary variation, with the occurrence of at least two pseudogenes in L. major (Table 1), both of which may encode a functional gene in L. infantum (LinJ15.0890 and LinJ20.1080) but apparently not in L. braziliensis. Moreover, the NST gene families of Trypanosoma brucei and Trypanosoma cruzi appear to be as complex and diverse as seen in Leishmania (available on the World Wide Web at www.genedb.org; data not shown). Of the remaining NSTs, several showed a distant relationship to the LPG2 GDP-Man transporter (LmjF19.1490/19.1510 and LmjF30.2680), and one showed a relationship to the YEA4 group of NSTs, which includes UDP-glucuronic acid/UDP-GalNAc transporters (LmjF15.0840).
Orthogologous genes related to LmF36.0670 were restricted to Leishmania species, whereas an ortholog of LmjF07.0400 was present in T. brucei but not T. cruzi, and ones related to Lmj19.1490/19.1510 occurred in the T. cruzi but not T. brucei genomes. Potentially these NSTs could be redundant, have specificities for nucleotide sugars required uniquely by each species, or act in distinct cellular compartments. One attractive role for NSTs shared by L. major and T. cruzi, but not T. brucei, involves UDP-galactofuranose, since UDP-galactopyranose mutase, the enzyme responsible for UDP-Galf synthesis, is absent in the T. brucei genome (59). Correspondingly, Leishmania and T. cruzi, but not T. brucei, synthesize a variety of Galf-containing glycoconjugates (see references cited in Ref. 59).
A Probable Role for Leishmania HUT1L as an ER UDP-Glc NST—Leishmania HUT1L was of interest, since its closest relatives mediate UDP-Gal uptake in other species (30). However, heterologous expression of HUT1L did not rescue the UDP-Gal transport defect of Lec8 cells (Fig. 5). In preliminary studies, we attempted to generate null mutants for Leishmania HUT1L with the same methods successful with LPG5A and LPG5B (Fig. 6; data not shown). Although heterozygous replacements were readily obtained, we were unable to obtain homozygous null mutants (data not shown). Instead, a variety of aneuploid and tetraploid cell lines were recovered bearing the second replacement as well as the WT gene (data not shown), a common finding in other studies in Leishmania attempting to knock out essential genes (60).
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LPG5A and LPG5B Show Functional Divergence—Although complete ablation of PG synthesis required inactivation of both LPG5A and LPG5B, both the single lpg5A– and lpg5B– mutants made abundant PGs, albeit with different phenotypes. For the lpg5A– mutant, a structurally altered LPG was made (Fig. 7A), containing less side chain galactosylation and fewer PG repeating units (Table 2), but alterations in PPG immunoreactivity were not detected. In contrast, the structure of LPG was unchanged in the lpg5B– mutant (Table 2), but alterations in PPG immunoreactivity were seen (Fig. 7A). These were attributed to decreased galactosylation, leading to an increase in WIC79.3-reactive monogalactosylated PGs at the expense of oligo- or polygalactosylated PGs linked to PPG (Figs. 7 and S3).
What underlies the functional differences between LPG5A and LPG5B? Some NSTs display specific interactions with glycosyltransferases, as in the case of the mammalian Golgi UDP-galactose transporter UGT1 with UDP-galactose:ceramide galactosyltransferase (65). Building upon this model, perhaps key Gal transferases responsible for LPG versus PPG PG synthesis preferentially associate with the LPG5A and LPG5B UDP-Gal NSTs, respectively. Another possibility invokes differential localization within the secretory pathway of LPG5A and LPG5B. Current data strongly indicate that PGs incorporated into both LPG and PPG are synthesized in the parasite Golgi apparatus (16–18, 26). Although it has been generally assumed that relevant NSTs and glycosyltransferases are colocalized in the same compartment, recent data also raise the possibility that luminal nucleotide sugars may traffic between compartments as well (66, 67). Moreover, considerable data point to the existence of functionally divergent subcompartments within and after the eukaryotic Golgi apparatus (68).
Either model above could explain why, despite some degree of functional separation, both LPG5A and LPG5B function must be lost in order to completely ablate PG synthesis. For the association model, the coupling could be leaky, and for the compartmentalization, anterograde and/or retrograde vesicular trafficking of luminal UDP-Gal could result in limited redistribution.
The structure of the LPG5A and LPG5B proteins themselves provide some opportunities for functional divergence. Although most features closely resemble those of other known NSTs, LPG5A and LPG5B are unique in possessing a large, divergent 134–199-amino acid insert between the second and third TM domains (Figs. 4 and S1). Moreover, the C-terminal domain of several NSTs has been implicated in targeting NSTs to the ER and/or Golgi apparatus, through dilysine retention and/or hydrophobic export motifs (66, 69, 70). Both LPG5A and LPG5B show potential motifs of this sort (Fig. S2). Thus, it is reasonable to propose that these or other domains contribute in some way toward divergent functions of these two NSTs. Preliminary data suggest that at least some portion of C-terminally tagged LPG5A and LPG5B proteins are found within the parasite Golgi apparatus, and efforts are under way to more finely map the cellular distribution of these two proteins and the functional roles of the unique peptide insert domains in LPG, PPG, and other secretory pathway glycoconjugates, such as the glycosylinositolphospholipids.4
In summary, we have presented data for the existence of multiple NST candidate sequences in L. major. Pursuant to our interests in UDP-Gal transport, we found two functionally divergent UDP-Gal transporters, LPG5A and LPG5B, which both contribute to PG synthesis in L. major. These two NSTs show novel structural features and properties, studies of which are likely to increase our knowledge of the detailed Leishmania PG synthetic and secretory pathway in the future. Given the evolutionary diversity of glycoconjugates in both Leishmania and trypanosomes and their key roles during their infectious cycles, our studies further emphasize the roles of the large and diverse NST family in these important pathogens.
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FOOTNOTES |
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The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables S1–S3 and Figs. S1–S4.
1 To whom correspondence should be addressed: Dept. of Molecular Microbiology, 660 S. Euclid Ave., Box 8230, St. Louis, MO 63110. E-mail: beverley{at}borcim.wustl.edu.
2 The abbreviations used are: PG, phosphoglycan; PPG, proteophosphoglycans; LPG, lipophosphoglycan; NST, nucleotide sugar transporter; Galf, galactofuranose; ER, endoplasmic reticulum; WT, wild type; ORF, open reading frame; RT, reverse transcription; CHO, Chinese hamster ovary.
3 T. Nicholson, A. Capul, and S. M. Beverley, unpublished observations.
4 A. Capul, T. Nicholson, F. Hsu, J. Turk, S. J. Turco, and S. M. Beverley, manuscript in preparation.
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