Complex interactions of vaginal microorganisms with the genital tract epithelium shape mucosal innate immunity, which holds the key to sexual and reproductive health. Bacterial vaginosis (BV), a microbiome-disturbance syndrome prevalent in reproductive-age women, occurs commonly in concert with trichomoniasis, and both are associated with increased risk of adverse reproductive outcomes and viral infections, largely attributable to inflammation. To investigate the causative relationships among inflammation, BV and trichomoniasis, we established a model of human cervicovaginal epithelial cells colonised by vaginal Lactobacillus isolates, dominant in healthy women, and common BV species (Atopobium vaginae, Gardnerella vaginalis and Prevotella bivia).
Colonised epithelia were infected with Trichomonas vaginalis (TV) or exposed to purified TV virulence factors (membrane lipophosphoglycan (LPG), its ceramide-phosphoinositol-glycan core (CPI-GC) or the endosymbiont Trichomonas vaginalis virus (TVV)), followed by assessment of bacterial colony-forming units, the mucosal anti-inflammatory microbicide secretory leucocyte protease inhibitor (SLPI), and chemokines that drive pro-inflammatory, antigen-presenting and T cells.
TV reduced colonisation by Lactobacillus but not by BV species, which were found inside epithelial cells. TV increased interleukin (IL)-8 and suppressed SLPI, likely via LPG/CPI-GC, and upregulated IL-8 and RANTES, likely via TVV as suggested by use of purified pathogenic determinants. BV species A vaginae and G vaginalis induced IL-8 and RANTES, and also amplified the pro-inflammatory responses to both LPG/CPI-GC and TVV, whereas P bivia suppressed the TV/TVV-induced chemokines.
These molecular host–parasite–endosymbiont–bacteria interactions explain epidemiological associations and suggest a revised paradigm for restoring vaginal immunity and preventing BV/TV-attributable inflammatory sequelae in women.
TRICHOMONAS; VAGINAL MICROBIOLOGY; IMMUNOLOGY; BACTERIAL VAGINOSIS; WOMEN
The extracellular human pathogen Trichomonas vaginalis is covered by a dense glycocalyx thought to play a role in host-parasite interactions. The main component of the glycocalyx is lipophosphoglycan (LPG), a polysaccharide anchored in the plasma membrane by inositol phosphoceramide. To study the role of LPG in trichomonads, we produced T. vaginalis LPG mutants by chemical mutagenesis and lectin selection and characterized them using morphological, biochemical, and functional assays. Two independently selected LPG mutants, with growth rates comparable to that of the wild-type (parent) strain, lost the ability to bind the lectins Ricinnus comunis agglutinin I (RCA120) and wheat germ agglutinin, indicating alterations in surface galactose and glucosamine residues. LPG isolated from mutants migrated faster than parent strain LPG on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting the mutants had shorter LPG molecules. Dionex high-performance anion exchange chromatography with pulsed amperometric detection analyses revealed galactosamine, glucosamine, galactose, glucose, mannose/xylose, and rhamnose as the main monosaccharides of T. vaginalis parent strain LPG. LPG from both mutants showed a reduction of galactose and glucosamine, corresponding with the reduced size of their LPG molecules and inability to bind the lectins RCA120 and wheat germ agglutinin. Mutant parasites were defective in attachment to plastic, a characteristic associated with avirulent strains of T. vaginalis. Moreover, the mutants were less adherent and less cytotoxic to human vaginal ectocervical cells in vitro than the parental strain. Finally, while parent strain LPG could inhibit the attachment of parent strain parasites to vaginal cells, LPG from either mutant could not inhibit attachment. These combined results demonstrate that T. vaginalis adherence to host cells is LPG mediated and that an altered LPG leads to reduced adherence and cytotoxicity of this parasite.
Trichomonad species are widespread unicellular flagellated parasites of vertebrates which interact with their hosts through carbohydrate-lectin interactions. In the past, some data has been accumulated regarding their lipo(phospho)glycans, a major glycoconjugate on their cell surfaces; on the other hand, other than biosynthetic aspects, few details about their N-linked oligosaccharides are known. In this study, we present both mass spectrometric and HPLC data about the N-glycans of different strains of Trichomonas vaginalis, a parasite of the human reproductive tract. The major structure in all strains examined is a truncated oligomannose form (Man5GlcNAc2) with α1,2-mannose residues, compatible with a previous bioinformatic examination of the glycogenomic potential of T. vaginalis. In addition, dependent on the strain, N-glycans modified by pentose residues, phosphate or phosphoethanolamine and terminal N-acetyllactosamine (Galβ1,4GlcNAc) units were found. The modification of N-glycans by N-acetyllactosamine in at least some strains is shared with the lipo(phospho)glycan and may represent a further interaction partner for host galectins, thereby playing a role in binding of the parasite to host epithelia. On the other hand, the variation in glycosylation between strains may be the result of genetic diversity within this species.
trichomonads; N-glycan; mass spectrometry; pentose; phosphoethanolamine
Protozoan parasites of the genus Leishmania synthesize lipophosphoglycans (LPGs), phosphoglycans and proteophosphoglycans that contain phosphosaccharide repeat units of [−6)Gal(β1-4)Man(α1-OPO3H−]. The repeat structures are assembled by sequential addition of Manα1-OPO3H and β-Gal. In this study, an UDP-Gal-dependent activity was detected in L. donovani and L. major membranes using synthetic phospho-oligosaccharide fragments of lipophosphoglycan as acceptor substrates. Incubation of a microsomal preparation from L. donovani or L. major parasites with synthetic substrates and UDP-[6-3H]Gal resulted in incorporation of radiolabel into these exogenous acceptors. The [3H]galactose-labeled products were characterized by degradation into radioactive, low molecular mass fragments upon hydrolysis with mild acid and treatment with β-galactosidases. We showed that the activity detected with L. donovani membranes is the elongating β-d-galactosyltransferase associated with LPG phosphosaccharide backbone biosynthesis (eGalT). The eGalT activity showed a requirement for the presence of at least one phosphodiester group in the substrate and it was enhanced dramatically when two or three phosphodiester groups were present. Using the same substrates we detected two types of galactosyltransferase activity in L. major membranes: the elongating β-d-galactosyltransferase and a branching β-d-galactosyltransferase (bGalT). Both L. major enzymes required a minimum of one phosphodiester group present in the substrate, but acceptors with two or three phosphodiester groups were found to be superior.
Trichomonas vaginalis is one of the most common nonviral sexually transmitted human infections and, worldwide, has been linked to increased incidence of human immunodeficiency virus type 1 transmission, preterm delivery, low birth weight, cervical cancer, and vaginitis. The molecular pathways that are important in initiating host inflammatory and immune responses to T. vaginalis are poorly understood. Here we report interactions of human cervicovaginal epithelial cells with the most abundant cell surface glycoconjugate of the parasite, the T. vaginalis lipophosphoglycan (LPG). Purified LPG mediated the adhesion of parasites to human vaginal epithelial cells in a dose-dependent manner. Furthermore, T. vaginalis LPG (but not LPG from Tritrichomonas foetus, the causative agent of bovine trichomoniasis) induced a selective upregulation of chemotactic cytokines by human endocervical, ectocervical, and vaginal epithelial cells, which do not express Toll-like receptor 4/MD2. The T. vaginalis LPG triggered interleukin 8 (IL-8), which promotes the adhesion and transmigration of neutrophils across the endothelium, and macrophage inflammatory protein 3α, which is a chemoattractant for immune cells and is essential for dendritic cell maturation. These effects were dose dependent and sustained in the absence of cytotoxicity and IL-1β release and utilized, at least in part, a signaling pathway independent from the Toll-like/IL-1 receptor adaptor protein MyD88.
Background: Trichomonas vaginalis lipoglycan (TvLG) mediates interactions between the parasite and human host.
Results: TvLG is composed of a polyrhamnose backbone with branches of poly-N-acetyllactosamine that are involved in attachment to host epithelium.
Conclusion: TvLG has a unique structure among solved parasite glycans.
Significance: This work provides a template to analyze TvLG from T. vaginalis with different binding properties.
The extracellular parasite Trichomonas vaginalis contains a surface glycoconjugate that appears to mediate parasite-host cell interaction via binding to human galectin-1. This glycoconjugate also elicits cytokine production from human vaginal epithelial cells, implicating its role in modulation of host immune responses. We have analyzed the structure of this glycoconjugate, previously described to contain the sugars rhamnose (Rha), N-acetylglucosamine (GlcNAc), galactose (Gal), xylose (Xyl), N-acetylgalactosamine (GalNAc), and glucose (Glc), using gas chromatograph mass spectrometry (GC-MS), matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF), electrospray MS/MS, and nuclear magnetic resonance (NMR), combined with chemical and enzymatic digestions. Our data reveal a complex structure, named T. vaginalis lipoglycan (TvLG), that differs markedly from Leishmania lipophosphoglycan and Entamoeba lipopeptidophosphoglycan and is devoid of phosphosaccharide repeats. TvLG is composed of an α1–3 linked polyrhamnose core, where Rha residues are substituted at the 2-position with either β-Xyl or chains of, on average, five N-acetyllactosamine (-3Galβ1–4GlcNAcβ1-) (LacNAc) units and occasionally lacto-N-biose (-3Galβ1-3GlcNAcβ1-) (LNB). These chains are themselves periodically substituted at the Gal residues with Xyl-Rha. These structural analyses led us to test the role of the poly-LacNAc/LNB chains in parasite binding to host cells. We found that reduction of poly-LacNAc/LNB chains decreased the ability of TvLG to compete parasite binding to host cells. In summary, our data provide a new model for the structure of TvLG, composed of a polyrhamnose backbone with branches of Xyl and poly-LacNAc/LNB. Furthermore, the poly-LacNAc side chains are shown to be involved in parasite-host cell interaction.
Adhesion; Glycoconjugate; Glycolipid Structure; Host-Pathogen Interactions; Pathogenesis; Trichomonas; TvLG
Trichomonas vaginalis is the most common non-viral sexually transmitted pathogen. The infection is prevalent in reproductive age women and is associated with vaginitis, endometritis, adnexitis, pyosalpinx, infertility, preterm birth, low birth weight, bacterial vaginosis, and increased risk of cervical cancer, HPV, and HIV infection. In men, its complications include urethritis, prostatitis, epididymitis, and infertility through inflammatory damage or interference with the sperm function. The infection is often asymptomatic and recurrent despite the presence of specific antibodies, suggesting the importance of the innate immune defense. T. vaginalis adhesion proteins, cysteine proteases, and the major parasite lipophosphoglycan (LPG) play distinct roles in the pathogenesis and evasion of host immunity. LPG plays a key role in the parasite adherence and signaling to human vaginal and cervical epithelial cells, which is at least in part mediated by galectins. The epithelial cells respond to T. vaginalis infection and purified LPG by selective upregulation of proinflammatory mediators. At the same time, T. vaginalis triggers an immunosuppressive response in monocytes, macrophages, and dendritic cells. The molecular mechanisms underlying reproductive complications and epidemiologic risks associated with T. vaginalis infection remain to be elucidated.
Trichomonas vaginalis; lipophosphoglycan; cytokines; galectins; human vaginal epithelial cells
CD24 is a glycosyl-phosphatidyl-inositol linked glycoprotein expressed in a broad range of cell types and is heavily glycosylated. It has been found to be over expressed in cancers and tumors and is also a costimulatory molecule. Therefore, this study was carried out to define the structures of the carbohydrates associated with the CD24 recombinant protein. The CD24 glycoprotein’s oligosaccharides were released by chemical and enzymatic means prior to being analyzed by MALDI-TOF-MS. The results obtained showed that CD24 is both N- and O-glycosylated. The major oligosaccharides were found to be Neu5Acα-2,3/6Galβ-1,3GalNAc, NeuAc2Gal β-1,3GalNAc1 (O-glycans), GalNAc2GlcNAc2Man3Fuc1, Gal1GalNAc2GlcNAc2Man3Fuc1, and Gal2GalNAc2GlcNAc2Man3Fuc1 (N-glycans). The results showed that Neu5Acα-2,3/6Galβ-1,3GalNAc (sialyl-tumor antigen, sT), a cancer-associated carbohydrate, was the most abundant glycan associated with CD24. This result raised the intriguing possibility that CD24 may be a major carrier of the sialyl-T abundantly found in cancer cells.
CD24; Glycans; MALDI-TOF-MS; T antigen
An in vitro culture system of bovine vaginal epithelial cells (BVECs) was developed to study the cytopathogenic effects of Tritrichomonas foetus and the role of lipophosphoglycan (LPG)-like cell surface glycoconjugates in adhesion of parasites to host cells. Exposure of BVEC monolayers to T. foetus resulted in extensive damage of monolayers. Host cell disruption was measured quantitatively by a trypan blue exclusion assay and by release of 3H from [3H]thymidine-labeled host cells. Results indicated contact-dependent cytotoxicity of host cells by T. foetus. The cytopathogenic effect was a function of T. foetus density. Metronidazole- or periodate-treated T. foetus showed no damage to BVEC monolayers. A related human trichomonad, Trichomonas vaginalis, showed no cytotoxic effects, indicating species-specific host-parasite interactions. A direct binding assay was developed and used to investigate the role of a major cell surface LPG-like molecule in host-parasite adhesion. The results of competition experiments showed that the binding to BVECs was displaceable, was saturable, and yielded a typical binding curve, suggesting that specific receptor-ligand interactions mediate the attachment of T. foetus to BVECs. Progesterone-treated BVECs showed enhanced parasite binding. T. foetus LPG inhibited the binding of T. foetus to BVECs; the LPG from T. vaginalis and a variety of other glycoconjugates did not. These data imply specificity of LPG on host-parasite adhesion. Periodate-treated parasites showed no adherence to host cells, indicating the involvement of carbohydrate containing molecules in the adhesion process.
Trichomonas vaginalis, the protist that causes vaginal itching, has a huge genome with numerous gene duplications. Recently we found that Trichomonas has numerous genes encoding putative dolichyl-phosphate-glucose (Dol-P-Glc) synthases (encoded by ALG5 genes) despite the fact that Trichomonas lacks the glycosyltransferases (encoded by ALG6, ALG8, and ALG10 genes) that use Dol-P-Glc to glucosylate dolichyl-PP-linked glycans. In addition, Trichomonas does not have a canonical DPM1 gene, encoding a dolichyl-P-mannose (Dol-P-Man) synthase. Here we show Trichomonas membranes have roughly 300 times the Dol-P-Glc synthase activity of Saccharomyces cerevisiae membranes and about one-fifth the Dol-P-Man synthase activity of Saccharomyces membranes. Endogenous Dol-P-hexoses of Trichomonas are relatively abundant and contain 16 isoprene units. Five paralogous Trichomonas ALG5 gene products have Dol-P-Glc synthase activity when expressed as recombinant proteins, and these Trichomonas Alg5s correct a carboxypeptidase N glycosylation defect in a Saccharomyces alg5 mutant in vivo. A recombinant Trichomonas Dpm1, which is deeply divergent in its sequence, has Dol-P-Man synthase activity. When radiolabeled Dol-P-Glc is incubated with Trichomonas membranes, Glc is incorporated into reducing and nonreducing sugars of O-glycans of endogenous glycoproteins. To our knowledge, this is the first demonstration of Dol-P-Glc as a sugar donor for O-glycans on glycoproteins.
Research on the conjugates of synthetic polyelectrolytes with antigenic molecules, such as proteins, peptides, or carbohydrates, is an attractive area due to their highly immunogenic character in comparison to classical adjuvants. For example, polyacrylic acid (PAA) is a weak polyelectrolyte and has been used in several biomedical applications such as immunological studies, drug delivery, and enzyme immobilization. However, to our knowledge, there are no studies that document immune-stimulant properties of PAA in Leishmania infection. Therefore, we aimed to develop a potential vaccine candidate against leishmaniasis by covalently conjugating PAA with an immunologically vital molecule of lipophosphoglycan (LPG) found in Leishmania parasites. In the study, LPG and PAA were conjugated by a multi-step procedure, and final products were analyzed with GPC and MALDI-TOF MS techniques. In cytotoxicity experiments, LPG-PAA conjugates did not indicate toxic effects on L929 and J774 murine macrophage cells. We assume that LPG-PAA conjugate can be a potential vaccine candidate, and will be immunologically characterized in further studies to prove its potential.
Polymer; Vaccine; Delivery; Adjuvant; Leishmania; Conjugation
Promastigotes of the intracellular protozoan parasite Leishmania major invade mononuclear phagocytes by a direct interaction between the cell surface lipophosphoglycan found on all Leishmania species and macrophage receptors. This interaction is mediated by phosphoglycan repeats containing oligomers of beta (1-3)Gal residues specific to L. major. We show here that although amastigotes also use lipophosphoglycan to bind to both primary macrophages and a cell line, this interaction is independent of the beta (1-3)Gal residues employed by promastigotes. Binding of amastigotes to macrophages could be blocked by intact lipophosphoglycan from L. major amastigotes as well as by lipophosphoglycan from promastigotes of several other Leishmania species, suggesting involvement of a conserved domain. Binding of amastigotes to macrophages could be blocked significantly by the monoclonal antibody WIC 108.3, directed to the lipophosphoglycan backbone. The glycan core of lipophosphoglycan could also inhibit attachment of amastigotes, but to a considerably lesser extent. The glycan core structure is also present in the type 2 glycoinositolphospholipids which are expressed on the surface of amastigotes at 100-fold-higher levels than lipophosphoglycan. However, their inhibitory effect could not be increased even when they were used at a 300-fold-higher concentration than lipophosphoglycan, indicating that lipophosphoglycan is the major macrophage-binding molecule on amastigotes of L. major. In the presence of complement, the attachment of amastigotes to macrophages was not altered, suggesting that lipophosphoglycan interacts directly with macrophage receptors.
Insect-transmitted protozoan parasites of the order Kinetoplastida, suborder Trypanosomatina, include Trypanosoma brucei (aetiological agent of African sleeping sickness), Trypanosoma cruzi (aetiological agent of Chagas' disease in South and Central America) and Leishmania spp. (aetiological agents of a variety of diseases throughout the tropics and sub-tropics). The structures of the most abundant cell-surface molecules of these organisms is reviewed and correlated with the different modes of parasitism of the three groups of parasites. The major surface molecules are all glycosylphosphatidylinositol (GPI)-anchored glycoproteins, such as the variant surface glycoproteins of T. brucei and the surface mucins of T. cruzi, or complex glycophospholipids, such as the lipophosphoglycans and glycoinositolphospholipids of the leishmanias. Significantly, all of the aforementioned structures share a motif of Man alpha 1-4GlcN alpha 1-6-myo-inositol-1-HPO4-lipid and can therefore be considered to be members of a GPI superfamily.
The genes encoding the enzymes required for UDP-xylose and UDP-galactose synthesis in Trichomonas vaginalis have been identified and the products of the recombinant enzymes analysed.
► Xylose and galactose are components of Trichomonas vaginalis glycans. ► T. vaginalis UDP-xylose synthase and UDP-galactose epimerase genes identified. ► Enzymes were expressed in recombinant form, purified and assayed.
The presence of xylose and galactose residues in the structure of trichomonad lipoglycans was indicated by previous studies and the modification of any glycoconjugate with either monosaccharide requires the respective presence of the nucleotide sugars, UDP-xylose and UDP-galactose. Biosynthesis of UDP-xylose de novo is mediated by UDP-xylose synthase (UXS; UDP-glucuronic acid decarboxylase), which converts UDP-glucuronic acid to UDP-xylose, whereas UDP-galactose can be generated from UDP-glucose by UDP-galactose epimerases (GalE). Trichomonas vaginalis cDNAs, encoding proteins with homology to these enzymes from other eukaryotes, were isolated. The recombinant T. vaginalis UDP-xylose synthase and UDP-galactose epimerase were expressed in Escherichia coli and tested via high pressure liquid chromatography to demonstrate their enzymatic activities. Thereby, in this first report on enzymes involved in glycoconjugate biosynthesis in this organism, we demonstrate the existence of xylose and galactose synthesising pathways in T. vaginalis.
GalE, UDP-galactose-4′-epimerase; UDP-GlcA, UDP-glucuronic acid; UXS, UDP-xylose synthase; UDP-xylose; UDP-galactose; Trichomonas vaginalis
In the protozoan parasite Leishmania, abundant surface
and secreted molecules such as lipophosphoglycan (LPG)1 and proteophosphoglycans (PPGs) contain extensive
galactose residues in the form of phosphoglycans (PGs) containing
[Gal-Man-PO4] 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. Database mining revealed twelve
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
knockout studies established that two genes (LPG5A and
LPG5B) encoded UDP-Gal NSTs. While the single
lpg5B− mutants produced PGs, an
double mutant was completely deficient. PG synthesis was restored in the
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, as the
lpg5A− mutant showed a partial
defect in LPG but not PPG phosphoglycosylation, while 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.
Sulfated epitopes of α-glucosamine (GlcN sulfoforms) were prepared by solid-phase synthesis as models of internal glucosamines within heparan sulfate. An orthogonally protected 2’-hydroxyethyl GlcN derivative was immobilized on a trityl resin support and subjected to regioselective deprotection and sulfonation conditions, which were optimized with the aid of on-resin infrared or Raman analysis. The sulfoforms were cleaved from the resin under mild Lewis acid conditions without affecting the O- or N-sulfate groups, and purified by reverse-phase reverse-phase high performance liquid chromatography (HPLC). The α-GlcN sulfoforms and their 4-O-benzyl ethers were examined by electrospray ionization tandem mass spectrometry (ESI-MS/MS), with product ion spectra produced by collision-induced dissociation (CID). ESI-MS/MS revealed significant differences in parent ion stabilities and fragmentation rates as a function of sulfate position. Ion fragmentation by CID resulted in characteristic mass losses with strong correlation to the positions of both free hydroxyl groups and sulfate ions. Most of these fragmentation patterns are consonant with elimination pathways, and suggest possible strategies for elucidating the structures of glucosamine-derived sulfoforms with identical m/z ratios. In particular, fragmentation analysis can easily distinguish GlcN sulfoforms bearing the relatively rare 3-O-sulfate from isomers with the more common 6-O-sulfate.
Leishmania synthesize abundant phosphoglycan-containing molecules made up of [Gal-Man-PO4] repeating units, including the surface lipophosphoglycan (LPG), and the surface and secreted proteophosphoglycan (PPG). The vector competence of Phlebotomus duboscqi and Lutzomyia longipalpis sand flies was tested using L. major knockout mutants deficient in either total phosphoglycans (lpg2− or lpg5A−/5B−) or LPG alone (lpg1−) along with their respective gene add-back controls. Our results confirm that LPG, the major cell surface molecule of Leishmania promastigotes known to mediate attachment to the vector midgut, is necessary to prevent the loss of infection during excretion of the blood meal remnants from a natural vector, P. duboscqi, but not an unnatural vector, L. longipalpis. Midgut digestive enzymes induced by blood feeding pose another potential barrier to parasite survival. Our results show that 36–72 h after the infective feed, all parasites developed well except the lpg2− and lpg5A−/5B− mutants, which showed significantly reduced survival and growth. Protease inhibitors promoted the early survival and growth of lpg2− in the blood meal. PPG was shown to be the key molecule conferring resistance to midgut digestive enzymes, as it prevented killing of lpg2− promastigotes exposed to midgut lysates prepared from blood-fed flies. The protection was not associated with inhibition of enzyme activities, but with cell surface acquisition of the PPG, which appears to function similar to mammalian mucins to protect the surface of developing promastigotes against proteolytic damage.
Background: The enzyme β4Gal-T1 synthesizes the LacNAc moiety of glycans.
Results: The extended oligosaccharide moiety of β1–6-branched GlcNAc acceptors binds to a different region on the enzyme.
Conclusion: β4Gal-T1 has two different oligosaccharide binding regions for extended oligosaccharide moieties of different acceptor substrates.
Significance: Multiple carbohydrate acceptor binding regions are observed on a glycosyltransferase.
N-Acetyllactosamine is the most prevalent disaccharide moiety in the glycans on the surface of mammalian cells and often found as repeat units in the linear and branched polylactosamines, known as i- and I-antigen, respectively. The β1–4-galactosyltransferase-I (β4Gal-T1) enzyme is responsible for the synthesis of the N-acetyllactosamine moiety. To understand its oligosaccharide acceptor specificity, we have previously investigated the binding of tri- and pentasaccharides of N-glycan with a GlcNAc at their nonreducing end and found that the extended sugar moiety in these acceptor substrates binds to the crevice present at the acceptor substrate binding site of the β4Gal-T1 molecule. Here we report seven crystal structures of β4Gal-T1 in complex with an oligosaccharide acceptor with a nonreducing end GlcNAc that has a β1–6-glycosidic link and that are analogous to either N-glycan or i/I-antigen. In the crystal structure of the complex of β4Gal-T1 with I-antigen analog pentasaccharide, the β1–6-branched GlcNAc moiety is bound to the sugar acceptor binding site of the β4Gal-T1 molecule in a way similar to the crystal structures described previously; however, the extended linear tetrasaccharide moiety does not interact with the previously found extended sugar binding site on the β4Gal-T1 molecule. Instead, it interacts with the different hydrophobic surface of the protein molecule formed by the residues Tyr-276, Trp-310, and Phe-356. Results from the present and previous studies suggest that β4Gal-T1 molecule has two different oligosaccharide binding regions for the binding of the extended oligosaccharide moiety of the acceptor substrate.
Enzyme Kinetics; Enzyme Structure; Glycosyltransferases; Oligosaccharide; Structural Biology; Multiple Carbohydrate Binding Site; Beta-4Gal-T1; i/I-Antigen Synthesis
Neuropeptides are essential cell-to-cell signaling molecules that influence diverse regulatory and behavioral functions within biological systems. Differing in their amino acid sequences and posttranslational modifications, hundreds of neuropeptides are produced via a series of enzymatic processing steps, and their levels vary with location, time, and physiological condition. Due to their wide range of endogenous concentrations and inherent chemical complexity, using mass spectrometry (MS) to accurately quantify changes in peptide levels can be challenging. Here we evaluate three different MS systems for their ability to accurately measure neuropeptide levels: capillary liquid chromatography-electrospray ionization-ion trap (CapLC-ESI-IT) MS, ultraperformance liquid chromatography- electrospray ionization-quadrupole-time-of-flight (UPLC-LC-ESI-Q-TOF) MS, and matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) MS. Specifically, eight sample mixtures composed of five neuropeptide standards, with four technical replicates of each, were labeled with H4/D4-succinic anhydride, followed by relative peptide quantitation using the three MS platforms. For these samples, the CapLC-ESI-IT MS platform offered the most robust ability to accurately quantify peptides over a concentration range of 1200-fold, although it required larger sample sizes than the other two platforms. Both the UPLC-ESI-Q-TOF MS and the MALDI-TOF MS systems had lower limits of quantification, with the MALDI-TOF having the lowest. By implementing several data acquisition schemes and optimizing the data analysis approaches, we were able to accurately quantify peptides over a three orders of magnitude concentration range using either the UPLC or MALDI-TOF platforms. Overall these results increase our understanding of both the capabilities and limits of using MS-based approaches to measure peptides.
Wild-type Escherichia coli grows more slowly on glucosamine (GlcN) than on N-acetylglucosamine (GlcNAc) as a sole source of carbon. Both sugars are transported by the phosphotransferase system, and their 6-phospho derivatives are produced. The subsequent catabolism of the sugars requires the allosteric enzyme glucosamine-6-phosphate (GlcN6P) deaminase, which is encoded by nagB, and degradation of GlcNAc also requires the nagA-encoded enzyme, N-acetylglucosamine-6-phosphate (GlcNAc6P) deacetylase. We investigated various factors which could affect growth on GlcN and GlcNAc, including the rate of GlcN uptake, the level of induction of the nag operon, and differential allosteric activation of GlcN6P deaminase. We found that for strains carrying a wild-type deaminase (nagB) gene, increasing the level of the NagB protein or the rate of GlcN uptake increased the growth rate, which showed that both enzyme induction and sugar transport were limiting. A set of point mutations in nagB that are known to affect the allosteric behavior of GlcN6P deaminase in vitro were transferred to the nagB gene on the Escherichia coli chromosome, and their effects on the growth rates were measured. Mutants in which the substrate-induced positive cooperativity of NagB was reduced or abolished grew even more slowly on GlcN than on GlcNAc or did not grow at all on GlcN. Increasing the amount of the deaminase by using a nagC or nagA mutation to derepress the nag operon improved growth. For some mutants, a nagA mutation, which caused the accumulation of the allosteric activator GlcNAc6P and permitted allosteric activation, had a stronger effect than nagC. The effects of the mutations on growth in vivo are discussed in light of their in vitro kinetics.
A novel sensitive liquid chromatography/mass spectrometry-based assay was developed for the quantitation of aminosugars, including 2-amino-2-deoxyglucose (glucosamine, GlcN), 2-amino-2-deoxygalactose (galactosamine, GalN), and 4-amino-4-deoxyarabinose (aminoarabinose, AraN), and for ethanolamine (EtN), present in lipid A. This assay enables the identification and quantitation of all amino-containing moieties present in lipopolysaccharide or lipid A from a single sample. The method was applied to the analysis of lipid A (endotoxin) isolated from a variety of biosynthetic and regulatory mutants of Salmonella enterica serovar Typhimurium and Francisella tularensis subspecies novicida. Lipid A is treated with trifluoroacetic acid to liberate and deacetylate individual aminosugars and mass tagged with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, which reacts with primary and secondary amines. The derivatives are separated using reversed-phase chromatography and analyzed using a single quadrupole mass spectrometer to detect quantities as small as 20 fmol. GalN was detected only in Francisella and AraN only in Salmonella, while GlcN was detected in lipid A samples from both species of bacteria. Additionally, we found an approximately 10-fold increase in the level of AraN in lipid A isolated from Salmonella grown in magnesium-limited versus magnesium-replete conditions. Salmonella with defined mutations in lipid A synthesis and regulatory genes were used to further validate the assay. Salmonella with null mutations in the phoP, pmrE, and prmF genes were unable to add AraN to their lipid A, while Salmonella with constitutively active phoP and pmrA exhibited AraN modification of lipid A even in the normally repressive magnesium-replete growth condition. The described assay produces excellent repeatability and reproducibility for the detection of amino-containing moieties in lipid A from a variety of bacterial sources.
Phlebotomine sand flies that transmit the protozoan parasite Leishmania differ greatly in their ability to support different parasite species or strains in the laboratory: while some show considerable selectivity, others are more permissive. In “selective” sand flies, Leishmania binding and survival in the fly midgut typically depends upon the abundant promastigote surface adhesin lipophosphoglycan (LPG), which exhibits species- and strain-specific modifications of the dominant phosphoglycan (PG) repeat units. For the “selective” fly Phlebotomus papatasi PpapJ, side chain galactosyl-modifications (scGal) of PG repeats play key roles in parasite binding. We probed the specificity and properties of this scGal-LPG PAMP (Pathogen Associated Molecular Pattern) through studies of natural isolates exhibiting a wide range of galactosylation patterns, and of a panel of isogenic L. major engineered to express similar scGal-LPG diversity by transfection of SCG-encoded β1,3-galactosyltransferases with different activities. Surprisingly, both ‘poly-scGal’ and ‘null-scGal’ lines survived poorly relative to PpapJ-sympatric L. major FV1 and other ‘mono-scGal’ lines. However, survival of all lines was equivalent in P. duboscqi, which naturally transmit L. major strains bearing ‘null-scGal’-LPG PAMPs. We then asked whether scGal-LPG-mediated interactions were sufficient for PpapJ midgut survival by engineering Leishmania donovani, which normally express unsubstituted LPG, to express a ‘PpapJ-optimal’ scGal-LPG PAMP. Unexpectedly, these “L. major FV1-cloaked” L. donovani-SCG lines remained unable to survive within PpapJ flies. These studies establish that midgut survival of L. major in PpapJ flies is exquisitely sensitive to the scGal-LPG PAMP, requiring a specific ‘mono-scGal’ pattern. However, failure of ‘mono-scGal’ L. donovani-SCG lines to survive in selective PpapJ flies suggests a requirement for an additional, as yet unidentified L. major-specific parasite factor(s). The interplay of the LPG PAMP and additional factor(s) with sand fly midgut receptors may determine whether a given sand fly host is “selective” or “permissive”, with important consequences to both disease transmission and the natural co-evolution of sand flies and Leishmania.
Phlebotomine sand flies are tiny blood-feeding insects that transmit Leishmania protozoan parasites, which cause diseases afflicting millions of people. The world-wide distribution of Leishmania is determined by the availability of transmission-competent vectors. In the laboratory, some vectors support many different Leishmania, while others are highly restricted. This is best exemplified by P. papatasi, which transmit only L. major despite a wide distribution in regions endemic for many Leishmania species. P. papatasi “selectivity” can be reproduced experimentally, and has been attributed to β1,3-linked galactose side chains decorating the abundant L. major surface lipophosphoglycan (LPG) adhesin, which mediate parasite attachment to the P. papatasi midgut to prevent elimination when the digested blood meal is excreted. As geographically diverse L. major display very different LPG galactosylation patterns (n = 0 - 8 βGals/side chain), we explored the consequences of this pattern diversity to survival in P. papatasi. Using natural isolates and L. major lines engineered to express a wide range of LPG galactosylation patterns, we showed L. major survival in P. papatasi PpapJ flies was optimized by expression of highly modified ‘mono-galactosylated’ LPG and extremely sensitive to LPG side chain length. Surprisingly, L. donovani lines engineered to express a “PpapJ-optimal” LPG mono-galactosylation pattern did not survive in PpapJ flies, suggesting that additional interactions are required. These studies reveal the fine specificity of Leishmania - sand fly interactions, and the nature of species- and strain-specific parasite molecules that have co-evolved to take advantage of midgut receptors specific to available sand fly vectors.
The essential role of the lipophosphoglycan (LPG) of Leishmania in innate immune response has been extensively reported. However, information about the role of the LPG-related glycoinositolphospholipids (GIPLs) is limited, especially with respect to the New World species of Leishmania. GIPLs are low molecular weight molecules covering the parasite surface and are similar to LPG in sharing a common lipid backbone and a glycan motif containing up to 7 sugars. Critical aspects of their structure and functions are still obscure in the interaction with the vertebrate host. In this study, we evaluated the role of those molecules in two medically important South American species Leishmania infantum and L. braziliensis, causative agents of visceral (VL) and cutaneous Leishmaniasis (CL), respectively. GIPLs derived from both species did not induce NO or TNF-α production by non-primed murine macrophages. Additionally, primed macrophages from mice (BALB/c, C57BL/6, TLR2−/− and TLR4−/−) exposed to GIPLs from both species, with exception to TNF-α, did not produce any of the cytokines analyzed (IL1-β, IL-2, IL-4, IL-5, IL-10, IL-12p40, IFN-γ) or p38 activation. GIPLs induced the production of TNF-α and NO by C57BL/6 mice, primarily via TLR4. Pre incubation of macrophages with GIPLs reduced significantly the amount of NO and IL-12 in the presence of IFN-γ or lipopolysaccharide (LPS), which was more pronounced with L. braziliensis GIPLs. This inhibition was reversed after PI-specific phospholipase C treatment. A structural analysis of the GIPLs showed that L. infantum has manose rich GIPLs, suggestive of type I and Hybrid GIPLs while L. braziliensis has galactose rich GIPLs, suggestive of Type II GIPLs. In conclusion, there are major differences in the structure and composition of GIPLs from L. braziliensis and L. infantum. Also, GIPLs are important inhibitory molecules during the interaction with macrophages.
Leishmania infantum (syn. L. chagasi) and L. braziliensis are the causative agents of VL and CL, respectively, in the New World. A vital part of the parasite's life cycle involves the circumvention of the host immune system and the infection of macrophages. This work focused on an important class of surface glycoconjugates, the glycoinositolphospholipids (GIPLs), and their role in the interaction with murine macrophages. GIPLs are expressed on every stage of the parasite life cycle and are the most abundant molecules on its surface. Here we show that these molecules modulate many macrophage functions such as cytokine production, release of nitric oxide and differentially activate MAPK. Although the GIPLs of both New World species are capable of modulating the same mechanisms, they do so to different degrees requiring an examination of their glycan composition. We show that L. infantum synthesize mannose rich GIPLs whereas L. braziliensis express galactose rich GIPLs.
We showed recently that contact of human vaginal epithelial cells (VECs) by Trichomonas vaginalis and incubation with trichomonad proteins in conditioned medium induced expression of VEC genes. We performed 2-D SDS-PAGE followed by MALDI-TOF to identify the major secreted proteins. Based on protein abundance and separation of spots in 2-D gels, 32 major secreted proteins were examined, which gave 19 proteins with accession numbers. These proteins included known secreted cysteine proteinases. In addition, other secreted proteins were enzymes of carbohydrate metabolism, adhesin protein AP65, heat shock proteins, thioredoxin reductase and coronins. We confirmed that the secreted trichomonad proteins induced expression of VEC genes, including interleukin 8 (IL-8), COX-2 and fibronectin. Purified AP65 added to VECs had a pronounced effect only on IL-8 gene expression, which was inhibited in the presence of 12G4 monoclonal antibody to AP65. Moreover, AP65 expressed episomally within epithelial cells was found to enhance the expression of IL-8 and COX-2. This may be the first report of analysis of the secreted proteins of T. vaginalis and of the host epithelial cell response to these proteins and to the prominent adhesin AP65.
The human Golgi enzyme CMP-NeuAc:Gal(β1–4)GlcNAc-R α2,6-sialyltransferase (ST6N) was stably coexpressed with human erythropoietin (EPO) from a BHK-21A cell line. The cell line was characterized with respect to the expression and in vitro activity of the ST6N and the endogenous α2,3-sialyltransferase. Detailed structural analysis of the N-linked carbohydrates of the rhuEPO expressed from the new cell line was performed by HPAE-PAD-mapping, MALDI/TOF-MS and methylation analysis after purification of the recombinant protein by immunoaffinity chromatography. This is the first report describing that the human α2,6-sialyltransferase is capable of sialylating, apart from Gal(β1–4)GlcNAc-R, also GalNAc(β1–4)GlcNAc-R motifs in vivo, which is not the case for the endogenous BHK-cell α2,3-sialyltransferase.
BHK-21 cells; human α2; 6-sialyltransferase; N-glycosylation genetic engineering; recombinant glycosyltransferaseexpression; tissue-type glycosylation