Maria Lucia S. Güther

Bio: Maria Lucia S. Güther is an academic researcher from University of Dundee. The author has contributed to research in topics: Trypanosoma brucei & Glycolipid. The author has an hindex of 17, co-authored 29 publications receiving 1070 citations.

Galactose metabolism is essential for the African sleeping sickness parasite Trypanosoma brucei

Abstract: The tsetse fly-transmitted protozoan parasite Trypanosoma brucei is the causative agent of human African sleeping sickness and the cattle disease Nagana. The bloodstream form of the parasite uses a dense cell-surface coat of variant surface glycoprotein to escape the innate and adaptive immune responses of the mammalian host and a highly glycosylated transferrin receptor to take up host transferrin, an essential growth factor. These glycoproteins, as well as other flagellar pocket, endosomal, and lysosomal glycoproteins, are known to contain galactose. The parasite is unable to take up galactose, suggesting that it may depend on the action of UDP-glucose 4′-epimerase for the conversion of UDP-Glc to UDP-Gal and subsequent incorporation of galactose into glycoconjugates via UDP-Gal-dependent galactosyltransferases. In this paper, we describe the cloning of T. brucei galE, encoding T. brucei UDP-Glc-4′-epimerase, and functional characterization by complementation of a galE-deficient Escherichia coli mutant and enzymatic assay of recombinant protein. A tetracycline-inducible conditional galE null mutant of T. brucei was created using a transgenic parasite expressing the TETR tetracycline repressor protein gene. Withdrawal of tetracycline led to a cessation of cell division and substantial cell death, demonstrating that galactose metabolism in T. brucei proceeds via UDP-Glc-4′-epimerase and is essential for parasite growth. After several days without tetracycline, cultures spontaneously recovered. These cells were shown to have undergone a genetic rearrangement that deleted the TETR gene. The results show that enzymes and transporters involved in galactose metabolism may be considered as potential therapeutic targets against African trypanosomiasis.

Distinct donor and acceptor specificities of Trypanosoma brucei oligosaccharyltransferases

Abstract: 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.

The role of inositol acylation and inositol deacylation in GPI biosynthesis in Trypanosoma brucei.

Abstract: The compound diisopropylfluorophosphate (DFP) selectively inhibits an inositol deacylase activity in living trypanosomes that, together with the previously described phenylmethylsulfonyl fluoride (PMSF)-sensitive inositol acyltransferase, maintains a dynamic equilibrium between the glycosylphosphatidylinositol (GPI) anchor precursor, glycolipid A [NH2(CH2)2PO4-6Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcN alpha 1-6myo-inositol-1-PO4-sn-1,2-dimyristoylglycerol], and its inositol acylated form, glycolipid C. Experiments using DFP in living trypanosomes and a trypanosome cell-free system suggest that earlier GPI intermediates are also in equilibrium between their inositol acylated and nonacylated forms. However, unlike mammalian and yeast cells, bloodstream form trypanosomes do not appear to produce an inositol acylated form of glucosaminylphosphatidylinositol (GlcN-PI). A specific function of inositol acylation in trypanosomes may be to enhance the efficiency of ethanolamine phosphate addition to the Man3GlcN-(acyl)PI intermediate. Inositol deacylation appears to be a prerequisite for fatty acid remodelling of GPI intermediates that leads to the exclusive presence of myristic acid in glycolipid A and, ultimately, in the variant surface glycoprotein (VSG). In the presence of DFP, the de novo synthesis of GPI precursors cannot proceed beyond glycolipid C' (the unremodelled version of glycolipid C) and lyso-glycolipid C'. Under these conditions glycolipid C'-type GPI anchors appear on newly synthesized VSG molecules. However, the efficiencies of both anchor addition to VSG and N-glycosylation of VSG were significantly reduced. A modified model of the GPI biosynthetic pathway in bloodstream form African trypanosomes incorporating these findings is presented.

High-confidence glycosome proteome for procyclic form Trypanosoma brucei by epitope-tag organelle enrichment and SILAC proteomics

Abstract: The glycosome of the pathogenic African trypanosome Trypanosoma brucei is a specialized peroxisome that contains most of the enzymes of glycolysis and several other metabolic and catabolic pathways. The contents and transporters of this membrane-bounded organelle are of considerable interest as potential drug targets. Here we use epitope tagging, magnetic bead enrichment, and SILAC quantitative proteomics to determine a high-confidence glycosome proteome for the procyclic life cycle stage of the parasite using isotope ratios to discriminate glycosomal from mitochondrial and other contaminating proteins. The data confirm the presence of several previously demonstrated and suggested pathways in the organelle and identify previously unanticipated activities, such as protein phosphatases. The implications of the findings are discussed.

The Genome of the African Trypanosome Trypanosoma brucei

Abstract: African trypanosomes cause human sleeping sickness and livestock trypanosomiasis in sub-Saharan Africa. We present the sequence and analysis of the 11 megabase-sized chromosomes of Trypanosoma brucei. The 26-megabase genome contains 9068 predicted genes, including ∼900 pseudogenes and ∼1700 T. brucei–specific genes. Large subtelomeric arrays contain an archive of 806 variant surface glycoprotein (VSG) genes used by the parasite to evade the mammalian immune system. Most VSG genes are pseudogenes, which may be used to generate expressed mosaic genes by ectopic recombination. Comparisons of the cytoskeleton and endocytic trafficking systems with those of humans and other eukaryotic organisms reveal major differences. A comparison of metabolic pathways encoded by the genomes of T. brucei, T. cruzi, and Leishmania major reveals the least overall metabolic capability in T. brucei and the greatest in L. major. Horizontal transfer of genes of bacterial origin has contributed to some of the metabolic differences in these parasites, and a number of novel potential drug targets have been identified.

Protein glycosylation. Structural and functional aspects.

Abstract: During the last decade, there have been enormous advances in our knowledge of glycoproteins and the stage has been set for the biotechnological production of many of them for therapeutic use. These advances are reviewed, with special emphasis on the structure and function of the glycoproteins (excluding the proteoglycans). Current methods for structural analysis of glycoproteins are surveyed, as are novel carbohydrate-peptide linking groups, and mono- and oligo-saccharide constituents found in these macromolecules. The possible roles of the carbohydrate units in modulating the physicochemical and biological properties of the parent proteins are discussed, and evidence is presented on their roles as recognition determinants between molecules and cells, or cell and cells. Finally, examples are given of changes that occur in the carbohydrates of soluble and cell-surface glycoproteins during differentiation, growth and malignancy, which further highlight the important role of these substances in health and disease.

The structure, biosynthesis and functions of glycosylphosphatidylinositol anchors, and the contributions of trypanosome research

Abstract: The discovery of glycosylphosphatidylinositol (GPI) membrane anchors has had a significant impact on several areas of eukaryote cell biology. Studies of the African trypanosome, which expresses a dense surface coat of GPI-anchored variant surface glycoprotein, have played important roles in establishing the general structure of GPI membrane anchors and in delineating the pathway of GPI biosynthesis. The major cell-surface molecules of related parasites are also rich in GPI-anchored glycoproteins and/or GPI-related glycophospholipids, and differences in substrate specificity between enzymes of trypanosomal and mammalian GPI biosynthesis may have potential for the development of anti-parasite therapies. Apart from providing stable membrane anchorage, GPI anchors have been implicated in the sequestration of GPI-anchored proteins into specialised membrane microdomains, known as lipid rafts, and in signal transduction events.