The ability of peptide‐N4‐(N‐acetyl‐β‐glucosaminyl)asparagine amidase F (PNGase F) from Flavobacterium meningosepticum and PNGase A from sweet almonds to deglycosylate N‐glycopeptides and N‐glycoproteins from plants was compared. Bromelain glycopeptide and horseradish peroxidase‐C glycoprotein, which contain xylose linked β1 → 2 to β‐mannose and fucose linked α1 → 3 to the innermost N‐acetylglucosamine, were used as substrates. In contrast to PNGase A, the enzyme from F. meningosepticum did not act upon these substrates even at concentrations 100‐fold higher than required for complete deglycosylation of commonly used standard substrates. After removal of α1 → 3‐linked fucose from the plant glycopeptide and glycoprotein by mild acid hydrolysis, they were readily degraded by PNGase F at moderate enzyme concentrations. Hence we conclude that α1 → 3 fucosylation of the inner N‐acetylglucosamine impedes the enzymatic action of PNGase F. Knowledge of this limitation of the deglycosylation potential of PNGase F may turn it from a pitfall into a useful experimental tool.

The N-glycans from 27 "plant" foodstuffs, including one from a gymnospermic plant and one from a fungus, were prepared by a new procedure and examined by means of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). For several samples, glycan structures were additionally investigated by size-fractionation and reverse-phase high-performance liquid chromatography in conjunction with exoglycosidase digests and finally also (1)H-nuclear magnetic resonance spectroscopy. The glycans found ranged from the typical vacuolar "horseradish peroxidase" type and oligomannose to complex Le(a)-carrying structures. Though the common mushroom exclusively contained N-glycans of the oligomannosidic type, all plant foods contained mixtures of the above-mentioned types. Apple, asparagus, avocado, banana, carrot, celery, hazelnut, kiwi, onion, orange, pear, pignoli, strawberry, and walnut were particularly rich in Le(a)-carrying N-glycans. Although traces of Le(a)-containing structures were also present in almond, pistachio, potato, and tomato, no such glycans could be found in cauliflower. Coconut exhibited almost exclusively N-glycans containing only xylose but no fucose. Oligomannosidic N-glycans dominated in buckwheat and especially in the legume seeds mung bean, pea, peanut, and soybean. Papaya presented a unique set of hybrid type structures partially containing the Le(a) determinant. These results are not only compatible with the hypothesis that the carbohydrate structures are another potential source of immunological cross-reaction between different plant allergens, but they also demonstrate that the Le(a)-type structure is very widespread among plants.

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In late summer, pollen grains originating from Compositae weeds (e.g., mugwort, ragweed) are a major source of allergens worldwide. Here, we report the isolation of a cDNA clone coding for Art v 1, the major allergen of mugwort pollen. Sequence analysis showed that Art v 1 is a secreted allergen with an N-terminal cysteine-rich domain homologous to plant defensins and a C-terminal proline-rich region containing several (Ser/Ala)(Pro)2-4 repeats. Structural analysis showed that some of the proline residues in the C-terminal domain of Art v 1 are posttranslationally modified by hydroxylation and O-glycosylation. The O-glycans are composed of 3 galactoses and 9-16 arabinoses linked to a hydroxyproline and represent a new type of plant O-glycan. A 3-D structural model of Art v 1 was generated showing a characteristic "head and tail" structure. Evaluation of the antibody binding properties of natural and recombinant Art v 1 produced in Escherichia coli revealed the involvement of the defensin fold and posttranslational modifications in the formation of epitopes recognized by IgE antibodies from allergic patients. However, posttranslational modifications did not influence T-cell recognition. Thus, recombinant nonglycosylated Art v 1 is a good starting template for engineering hypoallergenic vaccines for weed-pollen therapy.

Polyspecific human IgG preparations are indicated for the treatment of primary immunodeficiency disorders associated with defects in humoral immunity. In addition, intraveneous IgG (IVIG) is used to treat patients with autoimmune and systemic inflammatory diseases. Lectin chromatography on Sambucus nigra agglutinin stood at the cradle of the hypothesis that the anti-inflammatory properties depend on sialylation of the N-glycans in the Fc region of IgG. A detailed analysis of fractions obtained by lectin chromatography revealed that binding of IVIG is essentially mediated by Fab glycosylation. Moreover, experiments with a monoclonal antibody from a human cell line and IVIG Fc fragments indicated that at least two sialic acids in the Fc region of an antibody are required for lectin binding. Such glycoforms contain either two monosialylated glycans or a disialylated glycan and constitute 1% or less of the total human IgG. Arguably this small proportion holds the entire anti-inflammatory potency. A new mass spectrometric quantification method of IgG subclass ratio revealed that the IVIG Fc preparation essentially consists of IgG1. This observation may be relevant when studying the effect of human Fc in murine models of inflammation because mouse IgG subclasses differ substantially in their interaction with receptors.

The levels of beta 1,2-N-acetylglucosaminyltransferase (GlcNAc-T) I and II activities in cultured cells from Bombyx mori (Bm-N), Mamestra brassicae (IZD-Mb-0503) and Spodoptera frugiperda (Sf-9 and Sf-21) were investigated. Apart from initial experiments with Man alpha-3(Man alpha 1-6)-Man beta 1-O(CH2)8COOH3 and 3H-labelled UDP-GlcNAc as substrates, GlcNAc-T I activity was measured with a non-radioactive HPLC method using pyridylaminated Man3-GlcNAc2 and Man5GlcNAc2 as acceptor oligosaccharides. It was shown by reversed-phase HPLC, exoglycosidase digestion and methylation analysis that the product obtained with Man3GlcNAc2 contained a terminal GlcNAc residue linked beta 1,2 to the alpha 1,3 arm of the acceptor. Compared to the enzyme from the human hepatoma cell line HepG2, insect cell GlcNAc-T I exhibited a much higher preference for the Man5 substrate. The GlcNAc-T I from Mb-0503 cells had apparent Km and Vmax values for pyridylaminated Man3- and Man5GlcNAc2 of 2.15 and 0.21 mM, and of 3.4 and 11.4 nmol/h/mg of cell protein, respectively. When Man5GlcNAc2 was used as the acceptor substrate, the levels of GlcNAc-T I activity in the four insect cell lines ranged between 7.5 and 14.7 nmol/h/mg of cell protein, and thus were comparable to that of HepG2 cells. Evidence is presented for the dependence of lepidopteran fucosyltransferase on the presence of terminal N-acetylglucosamine.(ABSTRACT TRUNCATED AT 250 WORDS)

(2015) Processing of complex N-glycans in IgG Fc-region is affected by core fucosylation, mAbs, 7:5, 863-870, DOI: 10.1080DOI: 10. /19420862.2015 To link to this article: https://doi.org/10. 1080/19420862.2015 Keywords: core fucosylation, sialylation, Nicotiana benthamiana, bisected glycans, glycan modelling, IgG, cetuximab Abbreviations: 3-FucT, Zea maize core a1,3-fucosyltransferase;3-FucT GnTIV, human a1,3-mannosyl-b1,4-N-acetyL-glucosaminyltransferase fused to the CTS region of the Arabidopsis thaliana core a1,3-fucosyltransferase (FUT11); 6-FucT, Mus musculus core a1,6-fucosyltransferase; CH2, constant domain of an IgG heavy chain; CTS, cytoplasmic tail, transmembrane domain and stem region; CxMab cetuximab (Erbitux Ò ); Fab, fragment, antigen-binding; Fc, Fragment crystallizable region of immunoglobulin G; GlcNAc, N-acetylglucosamine; IgG1, Immunoglobulin G subclass 1; LC-ESI-MS, Liquid chromatography-electrospray ionisationmass spectrometry; mAb, monoclonal antibody; SDS-PAGE, Sodium dodecyl sulfate polyacrylamide gel electrophoresis; ST GalT, b1,4-galactosyltransferase fused to the CTS region of the rat a2,6-sialyltransferase; 6-SiaT, a2,6-sialyltransferase;ST GnT-III, b1,4-mannosyl-b1,4-N-acetylglucosaminyltransferase fused to the CTS region of the rat a2,6-sialyltransferase; DXT/FT, Nicotiana benthamiana glycosylation mutants lacking plant specific core b1,2-xylose and a1,3-fucose residuesWe investigated N-glycan processing of immunoglobulin G1 using the monoclonal antibody cetuximab (CxMab), which has a glycosite in the Fab domain in addition to the conserved Fc glycosylation, as a reporter. Three GlcNAc (Gn) terminating bi-antennary glycoforms of CxMab differing in core fucosylation (a1,3-and a1,6-linkage) were generated in a plant-based expression platform. These GnGn, GnGnF 3 , and GnGnF 6 CxMab variants were subjected in vivo to further processing toward sialylation and GlcNAc diversification (bisected and branching structures). Mass spectrometry-based glycan analyses revealed efficient processing of Fab glycans toward envisaged structures. By contrast, Fc glycan processing largely depend on the presence of core fucose. A particularly strong support of glycan processing in the presence of plant-specific core a1,3-fucose was observed. Consistently, molecular modeling suggests changes in the interactions of the Fc carbohydrate chain depending on the presence of core fucose, possibly changing the accessibility. Here, we provide data that reveal molecular mechanisms of glycan processing of IgG antibodies, which may have implications for the generation of glycan-engineered therapeutic antibodies with improved efficacies.

New SARS‐CoV‐2 variants are continuously emerging with critical implications for therapies or vaccinations. The 22 N ‐glycan sites of Spike remain highly conserved among SARS‐CoV‐2 variants, opening an avenue for robust therapeutic intervention. Here we used a comprehensive library of mammalian carbohydrate‐binding proteins (lectins) to probe critical sugar residues on the full‐length trimeric Spike and the receptor binding domain (RBD) of SARS‐CoV‐2. Two lectins, Clec4g and CD209c, were identified to strongly bind to Spike. Clec4g and CD209c binding to Spike was dissected and visualized in real time and at single‐molecule resolution using atomic force microscopy. 3D modelling showed that both lectins can bind to a glycan within the RBD‐ACE2 interface and thus interferes with Spike binding to cell surfaces. Importantly, Clec4g and CD209c significantly reduced SARS‐CoV‐2 infections. These data report the first extensive map and 3D structural modelling of lectin‐Spike interactions and uncovers candidate receptors involved in Spike binding and SARS‐CoV‐2 infections. The capacity of CLEC4G and mCD209c lectins to block SARS‐CoV‐2 viral entry holds promise for pan‐variant therapeutic interventions.