Angela Mehlert

Hydrophilic-interaction chromatography of complex carbohydrates

Complex carbohydrates can frequently be separated using hydrophilic-interaction chromatography (HILIC). The mechanism was investigated using small oligosaccharides and a new column, PolyGLYCOPLEX. Some carbohydrates exhibited anomer separation, which made it possible to determine the orientation of the reducing end relative to the stationary phase. Amide sugars were consistently good contact regions. Relative to amide sugars, sialic acids and neutral hexoses were better contact regions at lower levels of organic solvents than at higher levels. HILIC readily resolved carbohydrates differing in residue composition and position of linkage. Complex carbohydrate mixtures could be resolved using volatile mobile phases. This was evaluated with native glycans and with glycans derivatized with 2-aminopyridine or a nitrobenzene derivative. Both asialo- and sialylated glycans could be resolved using the same set of conditions. With derivatized carbohydrates, detection was possible at the picomole level by UV detection or on-line electrospray mass spectrometry. Selectivity compared favorably with that of other modes of HPLC. HILIC is promising for a variety of analytical and preparative applications.

The Lipid Structure of the Glycosylphosphatidylinositol-anchored Mucin-like Sialic Acid Acceptors of Trypanosoma cruzi Changes during Parasite Differentiation from Epimastigotes to Infective Metacyclic Trypomastigote Forms

The major acceptors of sialic acid on the surface of metacyclic trypomastigotes, which are the infective forms of Trypanosoma cruzi found in the insect vector, are mucin-like glycoproteins linked to the parasite membrane via glycosylphosphatidylinositol anchors. Here we have compared the lipid and the carbohydrate structure of the glycosylphosphatidylinositol anchors and the O-linked oligosaccharides of the mucins isolated from metacyclic trypomastigotes and noninfective epimastigote forms obtained in culture. The single difference found was in the lipid structure. While the phosphatidylinositol moiety of the epimastigote mucins contains mainly 1-O-hexadecyl-2-O-hexadecanoylphosphatidylinositol, the phosphatidylinositol moiety of the metacyclic trypomastigote mucins contains mostly (70%) inositol phosphoceramides, consisting of a C sphinganine long chain base and mainly C and C fatty acids. The remaining 30% of the metacyclic phosphatidylinositol moieties are the same alkylacylphosphatidylinositol species found in epimastigotes. In contrast, the glycosylphosphatidylinositol glycan cores of both molecules are very similar, mainly Manα1-2Manα1-2Manα1-6Manα1-4GlcN. The glycans are substituted at the GlcN residue and at the third αMan distal to the GlcN residue by ethanolamine phosphate or 2-aminoethylphosphonate groups. The structures of the desialylated O-linked oligosaccharides of the metacyclic trypomastigote mucin-like molecules, released by β-elimination with concomitant reduction, are identical to the structures reported for the epimastigote mucins (Previato, J. O., Jones, C., Gon¸alves, L. P. B., Wait, R., Travassos, L. R., and Mendo¸a-Previato, L.(1994) Biochem. J. 301, 151-159). In addition, a significant amount of nonsubstituted N-acetylglucosaminitol was released from the mucins of both forms of the parasite. Taken together, these results indicate that when epimastigotes transform into infective metacyclic trypomastigotes, the phosphatidylinositol moiety of the glycosylphosphatidylinositol anchor of the major acceptor of sialic acid is modified, while the glycosylphosphatidylinositol anchor and O-linked sugar chains remain essentially unchanged.

The Phosphoproteome of Bloodstream Form Trypanosoma brucei, Causative Agent of African Sleeping Sickness

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 eukaryote.

The glycosylation of the variant surface glycoproteins and procyclic acidic repetitive proteins of Trypanosoma brucei.

Trypanosoma brucei, in common with the other African trypanosomes, exhibits unusual cell-surface molecular architecture. The bloodstream form of the parasite is coated with a continuous layer of approximately five million variant surface glycoprotein (VSG) dimers that provide the parasite with a macromolecular diffusion barrier to guard against lysis by the alternative complement pathway. The procyclic form of the parasite has a more diffuse cell-surface coat made up of approximately 2.5 million copies of procyclic acidic repetitive protein (PARP). Within the VSG and PARP coats exist lower-abundance surface glycoproteins such as receptors and nutrient transporters. Both the VSG molecules and the PARP molecules are attached to the membrane via glycosylphosphatidylinositol (GPI) membrane anchors and the VSGs and one form of PARP are N-glycosylated. In this article, the structures of the N-glycans and the GPI anchors of T. brucei VSGs and PARPs are reviewed and simple models of the surfaces of bloodstream and procyclic trypomastigotes are presented.

Glycosyl-phosphatidylinositol molecules of the parasite and the host.

The glycosyl-phosphatidylinositol (GPI) protein-membrane anchors are ubiquitous among the eukaryotes. However, while mammalian cells typically express in the order of 100 thousand copies of GPI-anchor per cell, the parasitic protozoa, particularly the kinetoplastids, express up to 10-20 million copies of GPI-anchor and/or GPI-related glycolipids per cell. Thus GPI-family members dominate the cell surface molecular architecture of these organisms. In several cases, GPI-anchored proteins, such as the variant surface glycoprotein (VSG) of the African trypanosomes, or GPI-related glycolipids, such as the lipophosphoglycan (LPG) of the Leishmania, are known to be essential for parasite survival and infectivity. The highly elevated levels and specialised nature of GPI metabolism in the kinetoplastid parasites suggest that the GPI biosynthetic pathways might be good targets for the development of chemotherapeutic agents. This article introduces the range of GPI structures found in protozoan parasites, and their mammalian hosts, and discusses some aspects of GPI biosynthesis.

The Procyclin Repertoire of Trypanosoma brucei IDENTIFICATION AND STRUCTURAL CHARACTERIZATION OF THE GLU-PRO-RICH POLYPEPTIDES

The surface of the insect stages of the protozoan parasite Trypanosoma brucei is covered by abundant glycosyl phosphatidylinositol (GPI)-anchored glycoproteins known as procyclins. One type of procyclin, the EP isoform, is predicted to have 22–30 Glu-Pro (EP) repeats in its C-terminal domain and is encoded by multiple genes. Because of the similarity of the EP isoform sequences and the heterogeneity of their GPI anchors, it has been impossible to separate and characterize these polypeptides by standard protein fractionation techniques. To facilitate their structural and functional characterization, we used a combination of matrix-assisted laser desorption ionization and electrospray mass spectrometry to analyze the entire procyclin repertoire expressed on the trypanosome cell. This analysis, which required removal of the GPI anchors by aqueous hydrofluoric acid treatment and cleavage at aspartate-proline bonds by mild acid hydrolysis, provided precise information about the glycosylation state and the number of Glu-Pro repeats in these proteins. Using this methodology we detected in a T. bruceiclone the glycosylated products of the EP3 gene and two different products of the EP1 gene (EP1-1 and EP1-2). Furthermore, only low amounts of the nonglycosylated products of theGPEET and EP2 genes were detected. Because all procyclin genes are transcribed polycistronically, the latter finding indicates that the expression of the GPEET andEP2 genes is post-transcriptionaly regulated. This is the first time that the whole procyclin repertoire from procyclic trypanosomes has been characterized at the protein level.

The glycan core of GPI-anchored proteins modulates aerolysin binding but is not sufficient: the polypeptide moiety is required for the toxin-receptor interaction.

Sensitivity of mammalian cells to the bacterial toxin aerolysin is due to the presence at their surface of glycosylphosphatidyl inositol (GPI)‐anchored proteins which act as receptors. Using a panel of mutants that are affected in the GPI biosynthetic pathway and Trypanosoma brucei variant surface glycoproteins, we show that addition of an ethanolamine phosphate residue on the first mannose of the glycan core does not affect binding. In contrast, the addition of a side chain of up to four galactose residues at position 3 of this same mannose leads to an increase in binding. However, protein free GPIs, which accumulate in mutant cells deficient in the transamidase that transfers the protein to the pre‐formed GPI‐anchor, were unable to bind the toxin indicating a requirement for the polypeptide moiety, the nature and size of which seem of little importance although two exceptions have been identified.

Modeling of the N-Glycosylated Transferrin Receptor Suggests How Transferrin Binding Can Occur within the Surface Coat of Trypanosoma brucei

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.

Chemical structure of Trichomonas vaginalis surface lipoglycan: a role for short galactose (β1-4/3) N-acetylglucosamine repeats in host cell interaction

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.

Identification of a glycosylphosphatidylinositol anchor‐modifying β1‐3 N‐acetylglucosaminyl transferase in Trypanosoma brucei

Trypanosoma brucei expresses complex glycoproteins throughout its life cycle. A review of its repertoire of glycosidic linkages suggests a minimum of 38 glycosyltransferase activities. Of these, five have been experimentally related to specific genes and a further nine can be associated with candidate genes. The remaining linkages have no obvious candidate glycosyltransferase genes; however, the T. brucei genome contains a family of 21 putative UDP sugar‐dependent glycosyltransferases of unknown function. One representative, TbGT8, was used to establish a functional characterization workflow. Bloodstream and procyclic‐form TbGT8 null mutants were created and both exhibited normal growth. The major surface glycoprotein of the procyclic form, the procyclin, exhibited a marked reduction in molecular weight due to changes in the procyclin glycosylphosphatidylinositol (GPI) anchor side‐chains. Structural analysis of the mutant procyclin GPI anchors indicated that TbGT8 encodes a UDP‐GlcNAc: β‐Gal‐GPI β1‐3 GlcNAc transferase. This is only the second GPI‐modifying glycosyltransferase to have been identified from any organism. The glycosylation of the major glycoprotein of bloodstream‐form T. brucei, the variant surface glycoprotein, was unaffected in the TbGT8 mutant. However, changes in the lectin binding of other glycoproteins suggest that TbGT8 influences the processing of the poly N‐acetyllactosamine‐containing asparagine‐linked glycans of this life cycle stage.