The diagnosis of Human African Trypanosomiasis relies mainly on the Card Agglutination Test for Trypanosomiasis (CATT). While this test is successful, it is acknowledged that there may be room for improvement. Our aim was to develop a prototype lateral flow test based on the detection of antibodies to trypanosome antigens.
We took a non-biased approach to identify potential immunodiagnostic parasite protein antigens. The IgG fractions from the sera from Trypanosoma brucei gambiense infected and control patients were isolated using protein-G affinity chromatography and then immobilized on Sepharose beads. The IgG-beads were incubated with detergent lysates of trypanosomes and those proteins that bound were identified by mass spectrometry-based proteomic methods. This approach provided a list of twenty-four trypanosome proteins that selectively bound to the infection IgG fraction and that might, therefore, be considered as immunodiagnostic antigens. We selected four antigens from this list (ISG64, ISG65, ISG75 and GRESAG4) and performed protein expression trials in E. coli with twelve constructs. Seven soluble recombinant protein products (three for ISG64, two for ISG65 and one each for ISG75 and GRESAG4) were obtained and assessed for their immunodiagnostic potential by ELISA using individual and/or pooled patient sera. The ISG65 and ISG64 construct ELISAs performed well with respect to detecting T. b. gambiense infections, though less well for detecting T. b. rhodesiense infections, and the best performing ISG65 construct was used to develop a prototype lateral flow diagnostic device.
Using a panel of eighty randomized T. b. gambiense infection and control sera, the prototype showed reasonable sensitivity (88%) and specificity (93%) using visual readout in detecting T. b. gambiense infections. These results provide encouragement to further develop and optimize the lateral flow device for clinical use.
Human African Trypanosomiasis is caused by infection with Trypanosoma brucei gambiense or T. b. rhodesiense. Preliminary diagnosis of T. b. gambiense infection relies mainly on a Card Agglutination Test for Trypanosomiasis (CATT), which has acknowledged limitations. New approaches are needed, first to identify new diagnostic antigens and, second, to find a more suitable platform for field-based immunodiagnostic tests. We took an unbiased approach to identify candidate diagnostic antigens by asking which parasite proteins bind to the antibodies of infected patients and not to the antibodies of uninfected patients. From this list of twenty-four candidate antigens, we selected four and from these we selected the one that worked the best in conventional immunodiagnostic tests. This antigen, ISG65, was used to make lateral flow devices, where a small sample of patient serum is added to a pad and thirty minutes later infection can be inferred by simple optical read out. This simple prototype device works as well as the CATT test and may be developed and optimized for clinical use in the field.
The title compound, C14H17N5O, a 1,2,3-triazole derivative of benzoxadiazole (C14H17N5O), was synthesized via Cu-catalysed azide–alkyne cycloaddition (CuAAC) from the corresponding n-octyne and 4-azidobenzoxadiazole. The benzoxadiazole and triazole rings show a roughly planar orientation [dihedral angle between the ring planes = 12.18 (5)°]. The alkane chain adopts a zigzag conformation, which deviates from the central triazole ring by 20.89 (6)°. These two torsion angles result in an overall twist to the structure, with a dihedral angle of 32.86 (7)° between the benzoxadiazole group and the hexyl chain. The crystal structure features C—H⋯N hydrogen bonds leading to chains propagating along [2-10] and offset parallel stacking interactions of the triazole and benzoxadiazole rings. The centroid of the extended π-system formed by the benzoxadiazole and triazole rings (14 atoms total) was calculated; the centroid–centroid distance was 4.179 Å, interplanar separation was 3.243 Å, and the resulting offset was 2.636 Å.
The protozoan parasite Trypanosoma brucei has a complex digenetic lifecycle between a mammalian host and an insect vector, and adaption of its proteome between lifecycle stages is essential to its survival and virulence. We have optimized a procedure for growing Trypanosoma brucei procyclic form cells in conditions suitable for stable isotope labeling by amino acids in culture (SILAC) and report a comparative proteomic analysis of cultured procyclic form and bloodstream form T. brucei cells. In total we were able to identify 3959 proteins and quantify SILAC ratios for 3553 proteins with a false discovery rate of 0.01. A large number of proteins (10.6%) are differentially regulated by more the 5-fold between lifecycle stages, including those involved in the parasite surface coat, and in mitochondrial and glycosomal energy metabolism. Our proteomic data is broadly in agreement with transcriptomic studies, but with significantly larger fold changes observed at the protein level than at the mRNA level.
The transferrin receptor of bloodstream form Trypanosoma brucei is a heterodimer encoded by expression site associated genes 6 and 7. This low-abundance glycoprotein with a single glycosylphosphatidylinositol membrane anchor and eight potential N-glycosylation sites is located in the flagellar pocket. The receptor is essential for the parasite, providing its only source of iron by scavenging host transferrin from the bloodstream. Here, we demonstrate that both receptor subunits contain endoglycosidase H-sensitive and endoglycosidase H-resistant N-glycans. Lectin blotting of the purified receptor and structural analysis of the released N-glycans revealed oligomannose and paucimannose structures but, contrary to previous suggestions, no poly-N-acetyllactosamine structures were found. Overlay experiments suggest that the receptor can bind to other trypanosome glycoproteins, which may explain this discrepancy. Nevertheless, these data suggest that a current model, in which poly-N-acetyllactosamine glycans are directly involved in receptor-mediated endocytosis in bloodstream form Trypanosoma brucei, should be revised. Sequential endoglycosidase H and peptide-N-glycosidase F treatment, followed by tryptic peptide analysis, allowed the mapping of oligomannose and paucimannose structures to four of the receptor N-glycosylation sites. These results are discussed with respect to the current model for protein N-glycosylation in the parasite. Finally, the glycosylation data allowed the creation of a molecular model for the parasite transferrin receptor. This model, when placed in the context of a model for the dense variant surface glycoprotein coat in which it is embedded, suggests that receptor N-glycosylation may play an important role in providing sufficient space for the approach and binding of transferrin to the receptor, without significantly disrupting the continuity of the protective variant surface glycoprotein coat.
The tsetse fly transmitted parasite that causes human African trypanosomiasis, or sleeping sickness, scavenges iron from the bloodstream of the infected individual so that it can live, multiply and ultimately cause disease. To do this, it places a glycoprotein (a protein with carbohydrate chains attached) called the transferrin receptor on its surface to capture circulating human transferrin, an iron transport protein. It then internalizes transferrin receptor/transferrin complex and digests the transferrin part, releasing the iron for its own use. By analyzing the parasite transferrin receptor, we have been able to describe the carbohydrate chains of the transferrin receptor and thus complete a molecular model of this important glycoprotein. We have further built models of how we expect this low abundance glycoprotein will sit in the surface coat of the parasite, which is made of millions of copies of another glycoprotein. The results provide a ‘molecule's eye view’ of how the carbohydrate chains of the transferrin receptor provide the space necessary for the transferrin to bind to it without disrupting the protective coat.
This study evaluates the relationship between cytochrome P450 (CYP) 3A5 genotype and vincristine-induced peripheral neuropathy in children with precursor B cell acute lymphoblastic leukemia (preB ALL). We have shown in vitro that vincristine is metabolized significantly more efficiently by CYP3A5 than by CYP3A4. We also found that vincristine neurotoxicity is less common in African-Americans (70% express CYP3A5) than in Caucasians. We test the hypothesis that CYP3A5 expressers experience less vincristine neuropathy than do CYP3A5 non-expressers.
This study of pharmacogenetics of vincristine neuropathy in children with preB ALL was completed at Indiana University Simon Cancer Center. Whole blood for DNA extraction and genotyping was collected as well as plasma from a single time-point for analysis of vincristine and primary metabolite (M1) concentrations. Vincristine neuropathy was captured via chart review and graded per the National Cancer Institute Common Terminology Criteria for Adverse Events, version 3.0.
89% of CYP3A5 expressers experienced neurotoxicity versus 100% of non-expressers (p=0.03). The proportion of treatment months with neurotoxicity was significantly different between the expressers and non-expressers (16% vs. 27%, p=0.0007). Limited pharmacokinetic data suggest different rates of vincristine metabolism between CYP3A5 genotype groups with higher primary metabolite (M1) plasma concentrations (p=0.0004) and lower metabolic ratios ([vincristine]/[M1]) (p=0.036) in the CYP3A5 expressers compared to the CYP3A5 non-expressers. M1 concentration was also inversely related to severity of neuropathy (p=0.0316).
In children with preB ALL, CYP3A5 expressers experience less vincristine-induced peripheral neuropathy, produce more M1, and have lower metabolic ratios compared to CYP3A5 non-expressers.
vincristine; pharmacogenetics; acute lymphoblastic leukemia; peripheral neuropathy
N-Myristoyltransferase (NMT) represents
drug target for human African trypanosomiasis (HAT), which is caused
by the parasitic protozoa Trypanosoma brucei. We
report the optimization of a high throughput screening hit (1) to give a lead molecule DDD85646 (63), which
has potent activity against the enzyme (IC50 = 2 nM) and T. brucei (EC50 = 2 nM) in culture. The compound
has good oral pharmacokinetics and cures rodent models of peripheral
HAT infection. This compound provides an excellent tool for validation
of T. brucei NMT as a drug target for HAT as well
as a valuable lead for further optimization.
DYRK-family kinases employ an intramolecular mechanism to autophosphorylate a critical tyrosine residue in the activation loop. Once phosphorylated, DYRKs lose tyrosine kinase activity and function as serine/threonine kinases. DYRKs have been characterized in organisms from yeast to human; however, all entities belong to the Unikont supergroup, only one of five eukaryotic supergroups. To assess the evolutionary age and conservation of the DYRK intramolecular kinase-activation mechanism, we surveyed 21 genomes representing four of the five eukaryotic supergroups for the presence of DYRKs. We also analyzed the activation mechanism of the sole DYRK (class 2 DYRK) present in Trypanosoma brucei (TbDYRK2), a member of the excavate supergroup and separated from Drosophila by ∼850 million years. Bioinformatics showed the DYRKs clustering into five known subfamilies, class 1, class 2, Yaks, HIPKs and Prp4s. Only class 2 DYRKs were present in all four supergroups. These diverse class 2 DYRKs also exhibited conservation of N-terminal NAPA regions located outside of the kinase domain, and were shown to have an essential role in activation loop autophosphorylation of Drosophila DmDYRK2. Class 2 TbDYRK2 required the activation loop tyrosine conserved in other DYRKs, the NAPA regions were critical for this autophosphorylation event, and the NAPA-regions of Trypanosoma and human DYRK2 complemented autophosphorylation by the kinase domain of DmDYRK2 in trans. Finally, sequential deletion analysis was used to further define the minimal region required for trans-complementation. Our analysis provides strong evidence that class 2 DYRKs were present in the primordial or root eukaryote, and suggest this subgroup may be the oldest, founding member of the DYRK family. The conservation of activation loop autophosphorylation demonstrates that kinase self-activation mechanisms are also primitive.
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
A gene predicted to encode Trypanosoma brucei glucosamine 6-phosphate N-acetyltransferase (TbGNA1; EC 184.108.40.206) was cloned and expressed in Escherichia coli. The recombinant protein was enzymatically active, and its high-resolution crystal structure was obtained at 1.86 Å. Endogenous TbGNA1 protein was localized to the peroxisome-like microbody, the glycosome. A bloodstream-form T. brucei
GNA1 conditional null mutant was constructed and shown to be unable to sustain growth in vitro under nonpermissive conditions, demonstrating that there are no metabolic or nutritional routes to UDP-GlcNAc other than via GlcNAc-6-phosphate. Analysis of the protein glycosylation phenotype of the TbGNA1 mutant under nonpermissive conditions revealed that poly-N-acetyllactosamine structures were greatly reduced in the parasite and that the glycosylation profile of the principal parasite surface coat component, the variant surface glycoprotein (VSG), was modified. The significance of results and the potential of TbGNA1 as a novel drug target for African sleeping sickness are discussed.
We report the extension of the copper(II) tetrafluoroborate catalysed opening of epoxides with alcohols to include a wider variety of alcohols, a range of solvents and a method to purify the products from the reaction.
Epoxide; Copper(II) tetrafluoroborate; Lewis acid; Alcohols
Available studies indicate that both genetic background and aging influence collateral growth capacity, but it is not known how their combination affects collateral growth. We evaluated collateral growth induced by ileal artery ligation in Fischer 344 (F344), Brown Norway (BN), and the first generation hybrid of F344 × BN (F1) rats available for aging research from the National Institute on Aging. Collateral growth was determined by paired diameter measurements in anesthetized rats immediately and 7 days postligation. In 3-mo-old rats, significant collateral growth occurred only in BN (35% ± 11%, P < 0.001). The endothelial cell number in arterial cross sections was also determined, since this precedes shear-mediated luminal expansion. When compared with the same animal controls, the intimal cell number was increased only in BN rats (92% ± 21%, P < 0.001). The increase in intimal cell number and the degree of collateral luminal expansion in BN rats was not affected by age from 3 to 24 mo. Immunohistochemical studies demonstrated that intimal cell proliferation was much greater in the collaterals of BN than of F1 rats. The remarkable difference between these three strains of rats used in aging research and the lack of an age-related impairment in the BN rats are novel observations. These rat strains mimic clinical observations of interindividual variation in collateral growth capacity and the impact of age on arteriogenesis and should be useful models to investigate the molecular mechanisms responsible for such differences.
strain dependent; endothelial proliferation; macrophage recruitment
Sand flies are the exclusive vectors of the protozoan parasite Leishmania1, but the mechanism of transmission by fly bite has not been determined nor incorporated into experimental models of infection. In sand flies with mature Leishmania infections the anterior midgut is blocked by a gel of parasite origin, the promastigote secretory gel (PSG)2,3. Here, we analyse for the first time the inocula from Leishmania mexicana infected Lutzomyia longipalpis sand flies. This revealed the size of the infectious dose, the underlying mechanism of parasite delivery by regurgitation, and the novel contribution made to infection by filamentous proteophosphoglycan (fPPG), a component of PSG found to accompany the parasites during transmission. Collectively, these results have important implications for understanding the relationship between parasite and its vector, the pathology of cutaneous leishmaniasis in humans and also the development of effective vaccines and drugs. These findings emphasise that to fully understand transmission of vector-borne diseases the interaction between all three participants must be considered.
The Ptdlns-3-kinase (PI3-K) signaling pathway plays a vital role in cell survival, proliferation, apoptosis and differentiation in normal cells, as well as in diseases such as cancer and diabetes. Quantification of phospho-Akt is a standard way of assessing the activity of the PI3-K signaling pathway in both cells and tumors. This measurement is traditionally performed semiquantitatively using immunoassays such as Western blot. Here we report an LC-MS method to accurately measure the stoichiometry of Akt phosphorylation in biological samples. The procedure includes immunoprecipitation, gel electrophoresis, in-gel digestion, addition of isotopicaly labeled internal standards and LC-MS/MS. Two proteolytic enzymes, chymotrypsin and trypsin, were used to generate suitable peptide fragments for measuring Thr308 and Ser473 phosphorylation, respectively. The interday imprecision was estimated to be 3.8% and 2.3% for Thr308 and Ser473, respectively. This method has been tested on human T-cells grown in presence and absence of pervanadate and with or without a PI3-K inhibitor and on human glioblastoma cells (U-87 MG) grown in presence and absence of wortmannin (PI3-K inhibitor).The results of T cells suggest that the levels of Akt phosphorylation in untreated cells were below 1% for both phosphorylation sites. Pervanadate treatment provoked an 18-fold increase in phosphorylation of Thr308 and the PI3-K inhibitor partially reversed the increase. A comparison between LC-MS/MS and Western blotting suggests that the LC-MS based method is of comparable sensitivity and provides a more accurate phosphorylation stoichiometry, a wider dynamic range and more in-depth information. The application of the new method and its utility to providing predictive markers of response to targeted therapies is discussed.
The Ptdlns-3-kinase signaling pathway plays a vital role in normal physiology and pathophysiology. An LC-MS method using stable isotope peptides to accurately measure the stoichiometry of Akt phosphorylation, a biomarker of Ptdlns-3-kinase activation, in biological samples was developed. A comparison between LC-MS/MS and Western blotting suggests that the LC-MS based method is of comparable sensitivity and provides a more accurate phosphorylation stoichiometry, a wider dynamic range and more in-depth information.
Akt; PKB; phosphorylation; stoichiometry; LC-MS/MS; T cells; U-87 MG cells
Conventional drug design embraces the “one gene, one drug, one disease” philosophy. Polypharmacology, which focuses on multi-target drugs, has emerged as a new paradigm in drug discovery. The rational design of drugs that act via polypharmacological mechanisms can produce compounds that exhibit increased therapeutic potency and against which resistance is less likely to develop. Additionally, identifying multiple protein targets is also critical for side-effect prediction. One third of potential therapeutic compounds fail in clinical trials or are later removed from the market due to unacceptable side effects often caused by off-target binding. In the current work, we introduce a multidimensional strategy for the identification of secondary targets of known small-molecule inhibitors in the absence of global structural and sequence homology with the primary target protein. To demonstrate the utility of the strategy, we identify several targets of 4,5-dihydroxy-3-(1-naphthyldiazenyl)-2,7-naphthalenedisulfonic acid, a known micromolar inhibitor of Trypanosoma brucei RNA editing ligase 1. As it is capable of identifying potential secondary targets, the strategy described here may play a useful role in future efforts to reduce drug side effects and/or to increase polypharmacology.
Proteins play a critical role in human disease; bacteria, viruses, and parasites have unique proteins that can interfere with human health, and dysfunctional human proteins can likewise lead to illness. In order to find cures, scientists often try to identify small molecules (drugs) that can inhibit disease-causing proteins. The goal is to identify a molecule that can fit snugly into the pockets and grooves, or “active sites,” on the protein's surface. Unfortunately, drugs that inhibit a single disease-causing protein are problematic. A single protein can evolve to evade drug action. Additionally, when only one protein is targeted, drug potency is often diminished. Single drugs that simultaneously target multiple disease-causing proteins are much more effective. On the other hand, if scientists are not careful, the drugs they design might inhibit essential human proteins in addition to inhibiting their intended targets, leading to unexpected side effects. In our current work, we have developed a computer-based procedure that can be used to identify proteins with similar active sites. Once unexpected protein targets have been identified, scientists can modify drugs under development in order to increase the simultaneous inhibition of multiple disease-causing proteins while avoiding potential side effects by decreasing the inhibition of useful human proteins.
Glycosylphosphatidylinositol (GPI) anchored proteins are ubiquitous in eukaryotic cells. Earlier analysis methods required large amounts of purified protein to elucidate the structure of the GPI. This paper describes methods for analyzing GPIs on a ‘proteomic’ scale. Partially purified proteins may be run on sodium dodecyl sulphate polyacrylamide gel electrophoresis and then blotted onto a polyvinylidene difluoride (PVDF) membrane. Following identification of the protein the piece of PVDF may be subjected to various chemical treatments, which are specific for GPI structures. The first method uses gas chromatography–mass spectrometry and it enables the presence of a GPI anchor to be confirmed. The second method depends on the cleavage of phosphate bonds and permits the carbohydrate structure to be elucidated by electrospray or matrix assisted laser desorption ionization-time of flight mass spectrometry. The final method described uses deamination of the glucosamine residue to release the lipid moiety for analysis by mass spectrometry.
Glycosylphosphatidylinositol (GPI); Mass spectrometry; Proteomic techniques
A small zinc-binding group (ZBG) library of deoxy-2-C-branched-monosaccharides, for example, 1,5-anhydroglucitols, consisting of either monodentate ligand binding carboxylic acids or bidentate ligand binding hydroxamic acids, were prepared to assess the zinc affinity of the putative metalloenzyme 2-acetamido-2-deoxy-α-d-glucopyranosyl-(1→6)-phosphatidylinositol de-N-acetylase (EC 220.127.116.11) of glycosylphosphatidylinositol biosynthesis. The N-ureido thioglucoside was also synthesised and added to the ZBG library because a previous N-ureido analogue, synthesised by us, had inhibitory activity against the aforementioned de-N-acetylase, presumably via the N-ureido motif.
Glycosylphosphatidylinositol (GPI) biosynthesis; Zinc metalloenzyme inhibitor; Zinc-binding group; Branched monosaccharides, Phosphatidylinositol de-N-acetylase
Phosphorylation on tyrosine residues is a key signal transduction mechanism known to regulate intercellular and intracellular communication in multicellular organisms. Despite the lack of conventional tyrosine kinases in the genome of the single cell organism Trypanosoma brucei, phosphorylation on trypanosomal protein tyrosine residues has been reported for this parasite. However, the identities of most of the tyrosine-phosphorylated proteins and their precise site(s) of phosphorylation were unknown. Here, we have applied a phosphotyrosine-specific proteomics approach to identify 34 phosphotyrosine-containing proteins from whole-cell extracts of procyclic form T. brucei. A significant proportion of the phosphotyrosine-containing proteins identified in this study were protein kinases of the CMGC kinase group as well as some proteins of unknown function and proteins involved in energy metabolism, protein synthesis, and RNA metabolism. Interestingly, immunofluorescence microscopy using anti-phosphotyrosine antibodies suggests that there is a concentration of tyrosine-phosphorylated proteins associated with cytoskeletal structures (basal body and flagellum) and in the nucleolus of the parasite. This localization of tyrosine-phosphorylated proteins supports the idea that the function of signaling molecules is controlled by their precise location in T. brucei, a principle well known from higher eukaryotes.
Asparagine-linked glycosylation is catalysed by oligosaccharyltransferase (OTase). In Trypanosoma brucei OTase activity is catalysed by single-subunit enzymes encoded by three paralogous genes of which TbSTT3B and TbSTT3C can complement a yeast Δstt3 mutant. The two enzymes have overlapping but distinct peptide acceptor specificities, with TbSTT3C displaying an enhanced ability to glycosylate sites flanked by acidic residues. TbSTT3A and TbSTT3B, but not TbSTT3C, are transcribed in the bloodstream and procyclic life cycle stages of T. brucei. Selective knockdown and analysis of parasite protein N-glycosylation showed that TbSTT3A selectively transfers biantennary Man5GlcNAc2 to specific glycosylation sites whereas TbSTT3B selectively transfers triantennary Man9GlcNAc2 to others. Analysis of T. brucei glycosylation site occupancy showed that TbSTT3A and TbSTT3B glycosylate sites in acidic to neutral and neutral to basic regions of polypeptide, respectively. This embodiment of distinct specificities in single-subunit OTases may have implications for recombinant glycoprotein engineering. TbSTT3A and TbSTT3B could be knocked down individually, but not collectively, in tissue culture. However, both were independently essential for parasite growth in mice, suggesting that inhibiting protein N-glycosylation could have therapeutic potential against trypanosomiasis.
glycosylation; oligosaccharyltransferase; STT3;
In this paper, we describe the range of N-linked glycan structures produced by wild-type and glucosidase II null mutant bloodstream form Trypanosoma brucei parasites and the creation and characterization of a bloodstream form Trypanosoma brucei UDP-glucose:glycoprotein glucosyltransferase null mutant. These analyses highlight peculiarities of the Trypanosoma brucei UDP-glucose:glycoprotein glucosyltransferase, including an unusually wide substrate specificity, ranging from Man5GlcNAc2 to Man9GlcNAc2 glycans, and an unusually high efficiency in vivo, quantitatively glucosylating the Asn263 N-glycan of variant surface glycoprotein (VSG) 221 and 75% of all non-VSG N glycosylation sites. We also show that although Trypanosoma brucei UDP-glucose:glycoprotein glucosyltransferase is not essential for parasite growth at 37°C, it is essential for parasite growth and survival at 40°C. The null mutant was also shown to be hypersensitive to the effects of the N glycosylation inhibitor tunicamycin. Further analysis of bloodstream form Trypanosoma brucei under normal conditions and stress conditions suggests that it does not have a classical unfolded protein response triggered by sensing unfolded proteins in the endoplasmic reticulum. Rather, judging by its uniform Grp78/BiP levels, it appears to have an unregulated and constitutively active endoplasmic reticulum protein folding system. We suggest that the latter may be particularly appropriate for this organism, which has an extremely high flux of glycoproteins through its secretory pathway.
A Trypanosoma brucei TbGPI12 null mutant that is unable to express cell surface procyclins and free glycosylphosphatidylinositols (GPI) revealed that these are not the only surface coat molecules of the procyclic life cycle stage. Here, we show that non-GPI-anchored procyclins are N-glycosylated, accumulate in the lysosome, and appear as proteolytic fragments in the medium. We also show, using lectin agglutination and galactose oxidase-NaB3H4 labeling, that the cell surface of the TbGPI12 null parasites contains glycoconjugates that terminate in sialic acid linked to galactose. Following desialylation, a high-apparent-molecular-weight glycoconjugate fraction was purified by ricin affinity chromatography and gel filtration and shown to contain mannose, galactose, N-acetylglucosamine, and fucose. The latter has not been previously reported in T. brucei glycoproteins. A proteomic analysis of this fraction revealed a mixture of polytopic transmembrane proteins, including P-type ATPase and vacuolar proton-translocating pyrophosphatase. Immunolocalization studies showed that both could be labeled on the surfaces of wild-type and TbGPI12 null cells. Neither galactose oxidase-NaB3H4 labeling of the non-GPI-anchored surface glycoconjugates nor immunogold labeling of the P-type ATPase was affected by the presence of procyclins in the wild-type cells, suggesting that the procyclins do not, by themselves, form a macromolecular barrier.
In the title compound, [Yb(C24H40BN6)2]·C7H8, the Yb atom is coordinated by two tris(3-tert-butyl-5-methylpyrazol-1-yl)hydridoborate [TptBu,Me] ligands. One ligand binds in the κ3 mode, throuh three N atoms of the pyrazolyl rings, the other ligand coordinates through two N atoms of the pyrazolyl rings and the H atom attached to the central B via an agostic-type interaction through the B—H group of the second TptBu,Me ligand, giving an overall distorted octahedral geometry. One of the tert-butyl groups is disordered over two sites, with occupancies of 0.65 and 0.35.
The protozoan parasite Trypanosoma brucei is the causative agent
of human African sleeping sickness and related animal diseases, and it has over
170 predicted protein kinases. Protein phosphorylation is a key regulatory
mechanism for cellular function that, thus far, has been studied in
T.brucei principally through putative kinase mRNA knockdown
and observation of the resulting phenotype. However, despite the relatively
large kinome of this organism and the demonstrated essentiality of several
T. brucei kinases, very few specific phosphorylation sites
have been determined in this organism. Using a gel-free, phosphopeptide
enrichment-based proteomics approach we performed the first large scale
phosphorylation site analyses for T.brucei. Serine, threonine,
and tyrosine phosphorylation sites were determined for a cytosolic protein
fraction of the bloodstream form of the parasite, resulting in the
identification of 491 phosphoproteins based on the identification of 852 unique
phosphopeptides and 1204 phosphorylation sites. The phosphoproteins detected in
this study are predicted from their genome annotations to participate in a wide
variety of biological processes, including signal transduction, processing of
DNA and RNA, protein synthesis, and degradation and to a minor extent in
metabolic pathways. The analysis of phosphopeptides and phosphorylation sites
was facilitated by in-house developed software, and this automated approach was
validated by manual annotation of spectra of the kinase subset of proteins.
Analysis of the cytosolic bloodstream form T. brucei kinome
revealed the presence of 44 phosphorylated protein kinases in our data set that
could be classified into the major eukaryotic protein kinase groups by applying
a multilevel hidden Markov model library of the kinase catalytic domain.
Identification of the kinase phosphorylation sites showed conserved
phosphorylation sequence motifs in several kinase activation segments,
supporting the view that phosphorylation-based signaling is a general and
fundamental regulatory process that extends to this highly divergent lower
VSG MITat1.8 was characterized with respect to its N-glycosylation, GPI anchor structure and found to be a disulfide-linked homodimer.
Following a switch from variant surface glycoprotein MITat1.4 to variant surface glycoprotein MITat1.8 expression by Lister strain 427 Trypanosoma brucei brucei parasites, the latter uncharacterized variant surface glycoprotein was analysed. Variant surface glycoprotein MITat1.8 was found to be a disulphide-linked homodimer, containing a complex N-linked glycan at Asn58 and a glycosylphosphatidylinositol membrane anchor attached to Asp419. Mass spectrometric analyses demonstrated that the N-glycan is exclusively Galβ1-4GlcNAcβ1-2Manα1-3(Galβ1-4GlcNAcβ1-2Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAc and that the conserved Man3GlcN-myo-inositol glycosylphosphatidylinositol anchor glycan core is substituted with an average of 4 hexose, most likely galactose, residues. The presence of a complex N-glycan at Asn58 is consistent with the relatively acidic environment of the Asn58 N-glycosylation sequon, that predicts N-glycosylation by T. brucei oligosaccharyltransferase TbSTT3A with a Man5GlcNAc2 structure destined for processing to a paucimannose and/or complex N-glycan (Izquierdo L, Schulz B, Rodrigues JA et al. EMBO J 2009;28:2650–61 ).
Trypanosoma brucei; N-linked oligosaccharides; N-glycosylation; Glycosylphosphatidylinositol; GPI; Mass spectrometry
The title compound, a bis-5,5′-triazole, C38H28N6O4, was observed as a side-product from the Sharpless–Meldal click reaction of the corresponding coumarin alkyne and benzylazide. Although the compound was present as a minor component, it crystallized in preference to the major product. The two triazole rings are almost orthogonal to each other [dihedral angle = 83.8 (1)°]. However the 4 and 4′ coumarin systems are close to coplanar with their respective triazole rings [23.6 (1) and 15.1 (1)°]. Each of the benzene rings packs approximately face-to-face with the opposing coumarin ring systems, with interplanar angles of 7.7 (1) and 25.3 (1)° and distances of 3.567 (2) and 3.929 (2) Å between the respective centroids of the opposing rings.
A gene encoding Trypanosoma brucei
UDP-N-acetylglucosamine pyrophosphorylase was identified, and the
recombinant protein was shown to have enzymatic activity. The parasite enzyme
is unusual in having a strict substrate specificity for
N-acetylglucosamine 1-phosphate and in being located inside a
peroxisome-like microbody, the glycosome. A bloodstream form T.
brucei conditional null mutant was constructed and shown to be unable to
sustain growth in vitro or in vivo under nonpermissive
conditions, demonstrating that there are no alternative metabolic or
nutritional routes to UDP-N-acetylglucosamine and providing a genetic
validation for the enzyme as a potential drug target. The conditional null
mutant was also used to investigate the effects of
N-acetylglucosamine starvation in the parasite. After 48 h under
nonpermissive conditions, about 24 h before cell lysis, the status of parasite
glycoprotein glycosylation was assessed. Under these conditions,
UDP-N-acetylglucosamine levels were less than 5% of wild type. Lectin
blotting and fluorescence microscopy with tomato lectin revealed that
poly-N-acetyllactosamine structures were greatly reduced in the
parasite. The principal parasite surface coat component, the variant surface
glycoprotein, was also analyzed. Endoglycosidase digestions and mass
spectrometry showed that, under UDP-N-acetylglucosamine starvation,
the variant surface glycoprotein was specifically underglycosylated at its
C-terminal Asn-428 N-glycosylation site. The significance of this
finding, with respect to the hierarchy of site-specific
N-glycosylation in T. brucei, is discussed.