TbGT8 is a bifunctional glycosyltransferase that elaborates<em> N</em>-linked glycans on a protein phosphatase AcP115 and a GPI-anchor modifying glycan in <em>Trypanosoma brucei</em>

AbstractThe procyclic form of Trypanosoma brucei expresses procyclin surface glycoproteins with unusual glycosylphosphatidylinositol-anchor side chain structures that contain branched N-acetyllactosamine and lacto-N-biose units. The glycosyltransferase TbGT8 is involved in the synthesis of the branched side chain through its UDP-GlcNAc: βGal β1-3N-acetylglucosaminyltransferase activity. Here, we explored the role of TbGT8 in the mammalian bloodstream form of the parasite with a tetracycline-inducible conditional null T. brucei mutant for TbGT8. Under non-permissive conditions, the mutant showed significantly reduced binding to tomato lectin, which recognizes poly-N-acetyllactosamine-containing glycans. Lectin pull-down assays revealed differences between the wild type and TbGT8 null-mutant T. brucei, notably the absence of a broad protein band with an approximate molecular weight of 110kDa in the mutant lysate. Proteomic analysis revealed that the band contained several glycoproteins, including the acidic ecto-protein phosphatase AcP115, a stage-specific glycoprotein in the bloodstream form of T. brucei. Western blotting with an anti-AcP115 antibody revealed that AcP115 was approximately 10kDa smaller in the mutant. Enzymatic de-N-glycosylation demonstrated that the underlying protein cores were the same, suggesting that the 10-kDa difference was due to differences in N-linked glycans. Immunofluorescence microscopy revealed the colocalization of hemagglutinin epitope-tagged TbGT8 and the Golgi-associated protein GRASP. These data suggest that TbGT8 is involved in the construction of complex poly-N-acetyllactosamine-containing type N-linked and GPI-linked glycans in the Golgi of the bloodstream and procyclic parasite forms, respectively

Golgi self-correction generates bioequivalent glycans to preserve cellular homeostasis

Essential biological systems employ self-correcting mechanisms to maintain cellular homeostasis. Mammalian cell function is dynamically regulated by the interaction of cell surface galectins with branched N-glycans. Here we report that N-glycan branching deficiency triggers the Golgi to generate bioequivalent N-glycans that preserve galectin-glycoprotein interactions and cellular homeostasis. Galectins bind N-acetyllactosamine (LacNAc) units within N-glycans initiated from UDP-GlcNAc by the medial-Golgi branching enzymes as well as the trans-Golgi poly-LacNAc extension enzyme β1,3-N-acetylglucosaminyltransferase (B3GNT). Marginally reducing LacNAc content by limiting N-glycans to three branches results in T-cell hyperactivity and autoimmunity; yet further restricting branching does not produce a more hyperactive state. Rather, new poly-LacNAc extension by B3GNT maintains galectin binding and immune homeostasis. Poly-LacNAc extension is triggered by redistribution of unused UDP-GlcNAc from the medial to trans-Golgi via inter-cisternal tubules. These data demonstrate the functional equivalency of structurally dissimilar N-glycans and suggest a self-correcting feature of the Golgi that sustains cellular homeostasis

PLoS One

Rotavirus (RV) P[11] is an unique genotype that infects neonates. The mechanism of such age-specific host restriction remains unknown. In this study, we explored host mucosal glycans as a potential age-specific factor for attachment of P[11] RVs. Using in vitro binding assays, we demonstrated that VP8* of a P[11] RV (N155) could bind saliva of infants (60.3%, N\ue2\u20ac\u160=\ue2\u20ac\u160151) but not of adults (0%, N\ue2\u20ac\u160=\ue2\u20ac\u16048), with a significantly negative correlation between binding of VP8* and ages of infants (P<0.01). Recognition to the infant saliva did not correlate with the ABO, secretor and Lewis histo-blood group antigens (HBGAs) but with the binding of the lectin Lycopersicon esculentum (LEA) that is known to recognize the oligomers of N-acetyllactosamine (LacNAc), a precursor of human HBGAs. Direct evidence of LacNAc involvement in P[11] binding was obtained from specific binding of VP8* with homopolymers of LacNAc in variable lengths through a glycan array analysis of 611 glycans. These results were confirmed by strong binding of VP8* to the Lec2 cell line that expresses LacNAc oligomers but not to the Lec8 cell line lacking the LacNAc. In addition, N155 VP8* and authentic P[11] RVs (human 116E and bovine B223) hemagglutinated human red blood cells that are known to express poly-LacNAc. The potential role of poly-LacNAc in host attachment and infection of RVs has been obtained by abrogation of 116E replication by the PAA-conjugated poly-LacNAc, human milk, and LEA positive infant saliva. Overall, our results suggested that the poly-LacNAc could serve as an age-specific receptor for P[11] RVs and well explained the epidemiology that P[11] RVs mainly infect neonates and young children.GM098791/GM/NIGMS NIH HHS/United StatesGM62116/GM/NIGMS NIH HHS/United StatesP01 HD13021/HD/NICHD NIH HHS/United StatesR01 AI055649/AI/NIAID NIH HHS/United StatesR01 AI089634/AI/NIAID NIH HHS/United StatesR01 AI37093/AI/NIAID NIH HHS/United StatesUL1 TR000077/TR/NCATS NIH HHS/United State

A gene of the β3-glycosyltransferase family encodes <i>N</i>-acetylglucosaminyltransferase II function in <i>Trypanosoma brucei</i>

The bloodstream form of the human pathogen Trypanosoma brucei expresses oligomannose, paucimannose and complex N-linked glycans, including some exceptionally large poly-N-acetyllactosamine-containing structures. Despite the presence of complex N-glycans in this organism, no homologues of the canonical N-acetylglucosaminyltransferase I or II genes can be found in the T. brucei genome. These genes encode the activities that initiate the elaboration of the Manalpha1-3 and Manalpha1-6 arms, respectively, of the conserved trimannosyl-N-acetylchitobiosyl core of N-linked glycans. Previously, we identified a highly divergent T. brucei N-acetylglucosaminyltransferase I (TbGnTI) among a set of putative T. brucei glycosyltransferase genes belonging to the beta3-glycosyltransferase superfamily (1). Here, we demonstrate that TbGT15, another member of the same beta3-glycosyltransferase family, encodes an equally divergent N-acetylglucosaminyltransferase II (TbGnTII) activity. In contrast to multicellular organisms, where GnTII activity is essential, TbGnTII null mutants of T. brucei grow in culture and are still infectious to animals. Characterization of the large poly-N-acetyllactosamine containing N-glycans of the TbGnTII null mutants by methylation linkage analysis suggests that, in wild-type parasites, the Manalpha1-6 arm of the conserved trimannosyl core may carry predominantly linear poly-N-acetyllactosamine chains whereas the Manalpha1-3 arm may carry predominantly branched poly-N-acetyllactosamine chains. These results provide further detail on the structure and biosynthesis of complex N-glycans in an important human pathogen and provide a second example of the adaptation by trypanosomes of beta3-glycosyltransferase family members to catalyze beta1-2 glycosidic linkages

Loss of Effector Function of Human Cytolytic T Lymphocytes Is Accompanied by Major Alterations in N- and O-Glycosylation

Most human tumors are not eliminated by the immune system, and therapeutic vaccination shows poor results, a fact that can be explained at least partially by an immunosuppressive tumor microenvironment that is abundant in galectin-3. On cytolytic T lymphocyte (CTL) clones, maintained in culture by regular stimulation, recently activated CTLs present low effector functions. However, these functions are restored after a short treatment with LacNAc. The latter, which is in agreement with the glycoprotein-galectin lattice concept involving reduced motility, poses the question why galectin-3 ligands improve effector functions. We employed ultrasensitive MALDI-TOF-MS on resting and recently activated CTL clones combined with various glycosidase digestions and GC-MS linkage analyses. Our results showed that compared with the resting CTLs, the N-glycans of the recently activated CTLs consisted of (i) larger LacNAc oligomers of which a significant portion was longer than four-units and (ii) more multi-antennary structures. Interestingly, our results showed that the poly-LacNAc appeared to be equally distributed on all available N-glycan branches and not selectively enriched on a specific branch. The above structural alterations in the recently activated CTLs are expected to increase the galectin-3-LacNAc lattices and multivalent interactions and, therefore, reduce the motility of surface glycoproteins, such as the T-cell receptor. These findings suggest that the loss of effector functions on CTLs may be linked to reduced motility of surface glycoproteins. In addition, our results showed that recently activated CTLs had a reduced abundance of NeuAcα2,6-linked N-glycans and an increased abundance of disialylated core 1 and monosialylated core 2 O-glycan structures

Selective C-13-Labels on Repeating Glycan Oligomers to Reveal Protein Binding Epitopes through NMR: Polylactosamine Binding to Galectins

A combined chemo-enzymatic synthesis/NMR-based methodology is presented to identify, in unambiguous manner, the distinctive binding epitope within repeating sugar oligomers when binding to protein receptors. The concept is based on the incorporation of C-13-labels at specific monosaccharide units, selected within a repeating glycan oligomeric structure. No new chemical tags are added, and thus the chemical entity remains the same, while the presence of the C-13-labeled monosaccharide breaks the NMR chemical shift degeneracy that occurs in the non-labeled compound and allows the unique identification of the different components of the oligomer. The approach is demonstrated by a proof-of-concept study dealing with the interaction of a polylactosamine hexasaccharide with five different galectins that display distinct preferences for these entities.This research was funded by European Research Council for financial support (ERC-2017-AdG, project number 788143-RECGLYCANMR). We also thank Agencia Estatal de Investigacion (Spain) for project RTI2018-094751-B-C21 and the Severo Ochoa Excellence Accreditation (SEV-2016-0644

Selective C-13-Labels on Repeating Glycan Oligomers to Reveal Protein Binding Epitopes through NMR: Polylactosamine Binding to Galectins

A combined chemo-enzymatic synthesis/NMR-based methodology is presented to identify, in unambiguous manner, the distinctive binding epitope within repeating sugar oligomers when binding to protein receptors. The concept is based on the incorporation of C-13-labels at specific monosaccharide units, selected within a repeating glycan oligomeric structure. No new chemical tags are added, and thus the chemical entity remains the same, while the presence of the C-13-labeled monosaccharide breaks the NMR chemical shift degeneracy that occurs in the non-labeled compound and allows the unique identification of the different components of the oligomer. The approach is demonstrated by a proof-of-concept study dealing with the interaction of a polylactosamine hexasaccharide with five different galectins that display distinct preferences for these entities.This research was funded by European Research Council for financial support (ERC-2017-AdG, project number 788143-RECGLYCANMR). We also thank Agencia Estatal de Investigacion (Spain) for project RTI2018-094751-B-C21 and the Severo Ochoa Excellence Accreditation (SEV-2016-0644

Revealing the Specificity of Human H1 Influenza A Viruses to Complex N-Glycans

Influenza virus infection remains a threat to human health since viral hemagglutinins are constantly drifting, escaping infection and vaccine-induced antibody responses. Viral hemag-glutinins from different viruses display variability in glycan recognition. In this context, recent H3N2 viruses have specificity for alpha 2,6 sialylated branched N-glycans with at least three N- acetyllactosamine units (tri-LacNAc). In this work, we combined glycan arrays and tissue binding analyses with nuclear magnetic resonance experiments to characterize the glycan specificity of a family of H1 variants, including the one responsible for the 2009 pandemic outbreak. We also analyzed one engineered H6N1 mutant to understand if the preference for tri-LacNAc motifs could be a general trend in human-type receptor-adapted viruses. In addition, we developed a new NMR approach to perform competition experiments between glycans with similar compositions and different lengths. Our results point out that pandemic H1 viruses differ from previous seasonal H1 viruses by a strict preference for a minimum of di-LacNAc structural motifs.R.P.d.V. is a recipient of an ERC Starting grant from the European Commission (802780) and a Beijerinck Premium of the Royal Dutch Academy of Sciences. The glycan array setup was supported by the Netherlands Organization for Scientific Research (NWO, TOP-PUNT 718.015.003 to G.-J.P.H.B.). Dr. Lin Liu (CCRC) and Dr. Margreet A . Wolfert (Utrecht University) developed, printed, and validated the glycan microarray. We would like to thank Nikoloz Nemanichvili for technical assistance. A.C. acknowledges funding from Agencia Estatal de Investigacion "Spanish Ministry of Science and Innovation" (MICINN) project PID2019-105237GB-I00. J.P.C. acknowledges funding by the Spanish MICINN, grant no. RTI2018-095588-B-I00 (co-funded by the European Regional Development Fund/European Social Fund, "Invest-ing in your future"). JJB also tha n k s funding by the European Research Council (RECGLYCANMR, Advanced grant no. 788143), the Agencia Estatal de Investigacion (Spain) for grants RTI2018-094751-B-C21 and C22 and PDI2021-1237810B-C21 and C22, and CIBERES, an initiative of the Instituto de Salud Carlos III (ISCIII), Madrid, Spain. The NMR spectra were acquired at the NMR service of CIBMargarita Salas and in the NMR faci l i t y of the UCM. We also acknowledge Prof. Robert Woods group for sending us the coordinates of a glycan-hemagglut i n i n model