Formation Of Complex N-glycans Research Articles

In Planta Production of the Receptor-Binding Domain From SARS-CoV-2 With Human Blood Group A Glycan Structures.

Glycosylation of viral envelope proteins is important for infectivity and immune evasion. The SARS-CoV-2 spike protein is heavily glycosylated and host-derived glycan modifications contribute to the formation of specific immunogenic epitopes, enhance the virus-cell interaction or affect virus transmission. On recombinant viral antigens used as subunit vaccines or for serological assays, distinct glycan structures may enhance the immunogenicity and are recognized by naturally occurring antibodies in human sera. Here, we performed an in vivo glycoengineering approach to produce recombinant variants of the SARS-CoV-2 receptor-binding domain (RBD) with blood group antigens in Nicotiana benthamiana plants. SARS-CoV-2 RBD and human glycosyltransferases for the blood group ABH antigen formation were transiently co-expressed in N. benthamiana leaves. Recombinant RBD was purified and the formation of complex N-glycans carrying blood group A antigens was shown by immunoblotting and MS analysis. Binding to the cellular ACE2 receptor and the conformation-dependent CR3022 antibody showed that the RBD glycosylation variants carrying blood group antigens were functional. Analysis of sera from RBD-positive and RBD-negative individuals revealed further that non-infected RBD-negative blood group O individuals have antibodies that strongly bind to RBD modified with blood group A antigen structures. The binding of IgGs derived from sera of non-infected RBD-negative blood group O individuals to blood group A antigens on SARS-CoV-2 RBD suggests that these antibodies could provide some degree of protection from virus infection.

Complex-Type N-Glycans Influence the Root Hair Landscape of Arabidopsis Seedlings by Altering the Auxin Output.

Roots supply plants with nutrients and water, besides anchoring them in the soil. The primary root with its lateral roots constitutes the central skeleton of the root system. In particular, root hairs increase the root surface, which is critical for optimizing uptake efficiency. During root-cell growth and development, many proteins that are components of, e.g., the cell wall and plasma membrane are constitutively transported through the secretory system and become posttranslationally modified. Here, the best-studied posttranslational modification is protein N-glycosylation. While alterations in the attachment/modification of N-glycans within the ER lumen results in severe developmental defects, the impact of Golgi-localized complex N-glycan modification, particularly on root development, has not been studied in detail. We report that impairment of complex-type N-glycosylation results in a differential response to synthetic phytohormones with earlier and increased root-hair elongation. Application of either the cytokinin BAP, the auxin NAA, or the ethylene precursor ACC revealed an interaction of auxin with complex N-glycosylation during root-hair development. Especially in gntI mutant seedlings, the early block of complex N-glycan formation resulted in an increased auxin sensitivity. RNA-seq experiments suggest that gntI roots have permanently elevated nutrient-, hypoxia-, and defense-stress responses, which might be a consequence of the altered auxin responsiveness.

Substrate Engagement and Catalytic Mechanisms of N-Acetylglucosaminyltransferase V

α-Mannoside β-1,6-N-acetylglucosaminyltransferase V (MGAT5) is a mammalian glycosyltransferase involved in complex N-glycan formation, which strongly drives cancer when overexpressed. Despite intense interest, the catalytic mechanism of MGAT5 is not known in detail, precluding therapeutic exploitation. We solved structures of MGAT5 complexed to glycosyl donor and acceptor ligands, revealing an unforeseen role for donor-induced loop rearrangements in controlling acceptor substrate engagement. QM/MM metadynamics simulations of MGAT5 catalysis highlight the key assisting role of Glu297 and reveal considerable conformational distortions imposed upon the glycosyl donor during transfer. Detailed mechanistic characterization of MGAT5 will aid inhibitor development to correct cancer-associated N-glycosylation.

Identification of methylated GnTI-dependent N-glycans in Botryococcus brauni.

In contrast to mammals and vascular plants, microalgae show a high diversity in the N-glycan structures of complex N-glycoproteins. Although homologues for β1,2-N-acetylglucosaminyltransferase I (GnTI), a key enzyme in the formation of complex N-glycans, have been identified in several algal species, GnTI-dependent N-glycans have not been detected so far. We have performed an N-glycoproteomic analysis of the hydrocarbon oils accumulating green microalgae Botryococcus braunii. Thereby, the analysis of intact N-glycopeptides allowed the determination of N-glycan compositions. Furthermore, insights into the role of N-glycosylation in B.braunii were gained from functional annotation of the identified N-glycoproteins. In total, 517 unique N-glycosylated peptides have been identified, including intact N-glycopeptides that harbored N-acetylhexosamine (HexNAc) at the nonreducing end. Surprisingly, these GnTI-dependent N-glycans were also found to be modified with (di)methylated hexose. The identification of GnTI-dependent N-glycans in combination with N-glycan methylation in B.braunii revealed an uncommon type of N-glycan processing in this microalgae.

Influence of an ER-retention signal on the N-glycosylation of recombinant human α-l-iduronidase generated in seeds of Arabidopsis

Processes associated with late events of N-glycosylation within the plant Golgi complex are a major limitation to the use of plant-based systems to produce recombinant pharmaceutical proteins for parenteral administration. Specifically, sugars added to the N-glycans of a recombinant protein during glycan maturation to complex forms (e.g. β1,2 xylose and α1,3 fucose) can render the product immunogenic. In order to avoid these sugars, the human enzyme α-L-iduronidase (IDUA, EC 3.2.1.76), with a C-terminal ER-retention sequence SEKDEL, was expressed in seeds of complex-glycan-deficient (cgl) mutant and wild-type (Col-0) Arabidopsis thaliana, under the control of regulatory (5'-, signal-peptide-encoding-, and 3'-) sequences from the arcelin 5-I gene of Phaseolus vulgaris (cgl-IDUA-SEKDEL and Col-IDUA-SEKDEL, respectively). The SEKDEL motif had no adverse effect on the specific activity of the purified enzyme. Surprisingly, the majority of the N-glycans of Col-IDUA-SEKDEL were complex N-glycans (i.e. contained xylose and/or fucose) (88 %), whereas complex N-glycans comprised a much lower proportion of the N-glycans of cgl-IDUA-SEKDEL (26 %), in which high-mannose forms were predominant. In contrast to the non-chimeric IDUA of cgl seeds, which is mainly secreted into the extracellular spaces, the addition of the SEKDEL sequence to human recombinant IDUA expressed in the same background led to retention of the protein in ER-derived vesicles/compartments and its partial localization in protein storage vacuoles. Our data support the contention that the use of a C-terminal ER retention motif as an effective strategy to prevent or reduce complex N-glycan formation, is protein specific.

MHCII glycosylation modulates Bacteroides fragilis carbohydrate antigen presentation

N-linked glycans are thought to protect class II major histocompatibility complex (MHC) molecules (MHCII) from proteolytic cleavage and assist in arranging proteins within the immune synapse, but were not thought to directly participate in antigen presentation. Here, we report that antigen-presenting cells (APCs) lacking native complex N-glycans showed reduced MHCII binding and presentation of the T cell activating glycoantigen (GlyAg) polysaccharide A from Bacteroides fragilis but not conventional peptides. APCs lacking native N-glycans also failed to mediate GlyAg-driven T cell activation but activated T cells normally with protein antigen. Mice treated with the mannosidase inhibitor kifunensine to prevent the formation of complex N-glycans were unable to expand GlyAg-specific T cells in vivo upon immunization, yet adoptive transfer of normally glycosylated APCs into these animals overcame this defect. Our findings reveal that MHCII N-glycosylation directly impacts binding and presentation of at least one class of T cell-dependent antigen.

GDP-mannose pyrophosphorylase is essential in the bloodstream form of Trypanosoma brucei

A putative GDP-Man PP (guanidine diphosphomannose pyrophosphorylase) gene from Trypanosoma brucei (TbGDP-Man PP) was identified in the genome and subsequently cloned, sequenced and recombinantly expressed, and shown to be a catalytically active dimer. Kinetic analysis revealed a Vmax of 0.34 mumol/min per mg of protein and Km values of 67 muM and 12 muM for GTP and mannose 1-phosphate respectively. Further kinetic studies showed GDP-Man was a potent product feedback inhibitor. RNAi (RNA interference) of the cytosolic TbGDP-Man PP showed that mRNA levels were reduced to ~20% of wild-type levels, causing the cells to die after 3-4 days, demonstrating that TbGDP-Man PP is essential in the bloodstream form of T. brucei and thus a potential drug target. The RNAi-induced parasites have a greatly reduced capability to form GDP-Man, leading ultimately to a reduction in their ability to synthesize their essential GPI (glycosylphosphatidylinositol) anchors. The RNAi-induced parasites also showed aberrant N-glycosylation of their major cell-surface glycoprotein, variant surface glycoprotein, with loss of the high-mannose Man9GlcNAc2 N-glycosylation at Asn428 and formation of complex N-glycans at Asn263.

Molecular cloning and characterization of Arabidopsis thaliana Golgi α‐mannosidase II, a key enzyme in the formation of complex N‐glycans in plants

N-glycosylation is one of the major post-translational modifications of proteins in eukaryotes; however, the processing reactions of oligomannosidic N-glycan precursors leading to hybrid-type and finally complex-type N-glycans are not fully understood in plants. To investigate the role of Golgi alpha-mannosidase II (GMII) in the formation of complex N-glycans in plants, we identified a putative GMII from Arabidopsis thaliana (AtGMII; EC 3.2.1.114) and characterized the enzyme at a molecular level. The putative AtGMII cDNA was cloned, and its deduced amino acid sequence revealed a typical type II membrane protein of 1173 amino acids. A soluble recombinant form of the enzyme produced in insect cells was capable of processing different physiologically relevant hybrid N-glycans. Furthermore, a detailed N-glycan analysis of two AtGMII knockout mutants revealed the predominant presence of unprocessed hybrid N-glycans. These results provide evidence that AtGMII plays a central role in the formation of complex N-glycans in plants. Furthermore, conclusive evidence was obtained that alternative routes in the conversion of hybrid N-glycans to complex N-glycans exist in plants. Transient expression of N-terminal AtGMII fragments fused to a GFP reporter molecule demonstrated that the transmembrane domain and 10 amino acids from the cytoplasmic tail are sufficient to retain a reporter molecule in the Golgi apparatus and that lumenal sequences are not involved in the retention mechanism. A GFP fusion construct containing only the transmembrane domain was predominantly retained in the ER, a result that indicates the presence of a motif promoting ER export within the last 10 amino acids of the cytoplasmic tail of AtGMII.

Molecular basis of N-acetylglucosaminyltransferase I deficiency in Arabidopsis thaliana plants lacking complex N-glycans

GnTI (N-acetylglucosaminyltransferase I) is a Golgi-resident enzyme essential for the processing of high-mannose to hybrid and complex N-glycans. The Arabidopsis thaliana cgl mutant lacks GnTI activity and as a consequence accumulates oligomannosidic structures. Molecular cloning of cgl GnTI cDNA revealed a point mutation, which causes a critical amino acid substitution (Asp144-->Asn), thereby creating an additional N-glycosylation site. Heterologous expression of cgl GnTI in insect cells confirmed its lack of activity and the use of the N-glycosylation site. Remarkably, introduction of the Asp144-->Asn mutation into rabbit GnTI, which does not result in the formation of a new N-glycosylation site, led to a protein with strongly reduced, but still detectable enzymic activity. Expression of Asn144 rabbit GnTI in cgl plants could partially restore complex N-glycan formation. These results indicate that the complete deficiency of GnTI activity in cgl plants is mainly due to the additional N-glycan, which appears to interfere with the proper folding of the enzyme.

The synthesis of a series of modified mannotrisaccharides as probes of the enzymes involved in the early stages of mammalian complex N-glycan formation

A series of mannotrisaccharides were synthesized by two distinct chemical pathways as probes of the enzymes involved in the early stages of mammalian complex N-glycan formation. Methyl (α- d-mannopyranosyl)-(1→3)-[(α- d-mannopyranosyl)-(1→6)]-β- d-mannopyranoside ( 6) and methyl (2-deoxy-2-fluoro-α- d-mannopyranosyl)-(1→3)-[(2-deoxy-2-fluoro-α- d-mannopyranosyl)-(1→6)]-β- d-mannopyranoside ( 8) were rapidly synthesized from unprotected methyl β- d-mannopyranoside ( 12). Methyl (2-deoxy-2-fluoro-α- d-mannopyranosyl)-(1→3)-[(α- d-mannopyranosyl)-(1→6)]-β- d-mannopyranoside ( 7) and methyl (α- d-mannopyranosyl)-(1→3)-[(2-deoxy-2-fluoro-α- d-mannopyranosyl)-(1→6)]-β- d-mannopyranoside ( 9) were synthesized from the common orthogonally protected precursor methyl 2- O-acetyl-4,6- O-benzylidene-β- d-mannopyranoside ( 15). The 2-deoxy-2-fluoro substitution common to trisaccharides 7– 9 renders these analogues resistant to enzyme action in two distinct ways. Firstly the fluorine serves as a non-nucleophilic isostere for the acceptor hydroxyl in studies with glycosyl transferases GnT-I and GnT-II ( 7 and 9, respectively). Secondly it should render trisaccharide 8 stable to hydrolysis by the mannosidases Man-II and Man-III by inductive destabilization of their oxocarbenium ion-like transition states. These analogues should be useful for structural studies on these enzymes.

Unaltered complex N-glycan profiles in Nicotiana benthamiana despite drastic reduction of beta1,2- N -acetylglucosaminyltransferase I activity.

UDP-GlcNAc:alpha3-D-mannoside beta1,2- N -acetylglucosaminyltransferase I (GnTI; EC 2.4.1.101) is a Golgi-resident glycosyltransferase that is essential for the processing of oligomannose to hybrid and complex N-glycans in higher eukaryotes. The cDNA of Nicotiana tabacum GnTI has been cloned and characterised previously. To assess the influence of GnTI expression levels on the formation of complex N-glycans we used posttranscriptional gene silencing to knock down the expression of GnTI in the tobacco related species Nicotiana benthamiana. 143 independent transgenic plants containing GnTI constructs in either sense or antisense orientation were generated. 23 lines were selected for measurement of GnTI activity and 10 lines thereof showed a reduction of more than 85% in in vitro assays as compared to wildtype plants. GnTI reduction was stably inherited and did not interfere with the viability of the transformants. Noteworthy one line, 34S/2, exhibited a residual GnTI activity below the detection limit. beta1,2- N -acetylglucosaminyltransferase II (GnTII), an enzyme which acts further downstream in the N-glycosylation pathway, as well as other control enzymes (alpha-mannosidase, beta- N -acetylglucosaminidase) were not affected indicating the specific downregulation of GnTI. Remarkably, immunoblots and mass spectrometric N-glycan profiling revealed no significant changes of the total N-glycan comparable to wildtype plants.

Modulation of O-glycans and N-glycans on murine CD8 T cells fails to alter annexin V ligand induction by galectin 1.

Thymic negative selection and contraction of responding T cell oligoclones after infection represent important cell ablation processes required for maintaining T cell homeostasis. It has been proposed that galectin 1 contributes to these processes through interaction with lactosyl sequences principally on cell surface glycoproteins bearing core 2 (C2GnT1)-branched O-glycans. According to this model, specific T cell surface proteins cross-linked by galectin 1 induce signaling, ligand redistribution, and apoptosis in both immature thymocytes and activated T cells. The influence of lactosyl residues contained in branched O-glycans or complex N-glycans on galectin 1 binding and induction of annexin V ligand in murine CD8 T cells was assessed. Neither galectin binding nor galectin-induced expression of annexin V ligand was perturbed under conditions in which: 1) C2GnT1 activity was differentially induced by CD8 T cell activation/culture with IL-2 vs IL-4; 2) activated CD8(+) T cells lacked C2GnT1 expression; or 3) complex N-glycan formation was blocked by swainsonine. The maintenance of galectin 1 binding and induced annexin V expression under conditions that alter lactosamine abundance on O- or complex N-glycans suggest that galectin 1-mediated apoptosis is neither a simple function of fluctuating C2GnT1 activity nor a general C2GnT1-dependent mechanism underlying contraction of CD8 T cells subsequent to activation.