Schematic representation of SweetBac.

<p>A glyco-module, consisting of the open reading frames of the <i>C. elegans N</i>-acetylglucosaminyltransferase II (GnTII) and the bovine β4-galactosyltransferase I (GalT), was integrated in the loxP site of a MultiBac genome resulting in SweetBac. The generated viral backbone was subsequently used for the expression of 3D6 antibody heavy (HC) and light (LC) chain genes, by integrating them in the standard Tn7 site.</p

Expression of 3D6 antibody in lepidopteran insect cells.

<p>(A) <i>Tnao</i>38 and Hi5 cells were infected in triplicate with recombinant baculovirus expressing 3D6 antibody. Samples were taken every 24 h and the amount of secreted antibody was measured by ELISA. (B) <i>Tnao</i>38 and Hi5 cells were again infected in triplicate with recombinant baculovirus (Tnao38, Hi5) and SweetBac virus (<i>Tnao</i>38 glyco, Hi5 glyco) expressing 3D6 antibody. Samples were taken 72 and 96 hours post infection (hpi) and the amount of secreted antibody was measured by ELISA. Significance of the results was confirmed by Student's t-test (<i>p</i>-value<0,05).</p

Lectin Blot using <i>Ricinus communis</i> agglutinin I.

<p>Cellular supernatants were harvested 96 hpi and purified 3D6 antibodies were separated on SDS-PAGE and binding of biotinylated <i>Riccinus communis</i> agglutinin I to antibody heavy chains was tested. The lectin only bound SweetBac expressed antibodies (Hi5Glyco, Tn38Glyco), because of terminal galactose present on N-glycans. CHO and Mimic insect cell expressed antibodies were used as controls.</p

Binding of insect cell expressed 3D6 antibodies to 3D6 epitope and human FcγRI.

<p><i>Sf</i>9 cells presenting the 3D6 epitope were incubated with purified antibodies and target binding was analysed by FACS. Antibodies expressed in Hi5 and <i>Tnao</i>38 cells using standard baculoviral vectors (Hi5, <i>Tnao</i>38) as well as SweetBac (Hi5 Glyco, <i>Tnao</i>38 Glyco) show a highly specific binding. The broadening of the peaks can be explained by different amounts of 3D6 epitope displayed on <i>Sf</i>9 cells (A). Binding of antibodies to human Fc gamma receptor I was measured by incubating 3D6 antibodies with U937 cells. FACS analysis showed that all antibodies bound FcγRI present on the cellular surface, but the binding of SweetBac expressed variants (Hi5 Glyco, <i>Tnao</i>38 Glyco) was significantly increased.</p

MALDI-TOF-MS spectra of 3D6 IgG₁ N-glycans.

<p>N-glycan structures of an IgG₁ antibody expressed in <i>Tnao</i>38 (A) and Hi5 (C) cells (96 hpi) mostly consist of single and double fucosylated tri-mannose structures with maximally one terminal <i>N</i>-acetylglucosamine. The spectra of the mammalianised cells (B, D) show a shift of the dominant structures towards higher m/z values. Fucosylated biantennary N-glycan structures carrying two <i>N</i>-acetylglucosamine residues with one or two terminal galactose residues were identified as dominant N-glycan on IgG₁ recombinantly expressed in <i>Tnao</i>38 cells using SweetBac (B). The N-glycan structures identified from IgG₁ expressed in Hi5 cells using SweetBac (D) resemble the ones found in SweetBac infected <i>Tnao</i>38 cells with an even higher dominance of terminal galactose. Oligomannosidic glycans (Man_5–6GlcNAc₂) are indicated by asterisks. Graphical representations of glycans are consistent with the nomenclature of the Consortium for Functional Glycomics.</p

Fucosyltransferases as Synthetic Tools: Glycan Array Based Substrate Selection and Core Fucosylation of Synthetic <i>N</i>-Glycans

Two recombinant fucosyltransferases were employed as synthetic tools in the chemoenzymatic synthesis of core fucosylated N-glycan structures. Enzyme substrates were rapidly identified by incubating a microarray of synthetic N-glycans with the transferases and detecting the presence of core fucose with four lectins and one antibody. Selected substrates were then enzymatically fucosylated in solution on a preparative scale and characterized by NMR and MS. With this approach the chemoenzymatic synthesis of a series of α1,3-, α1,6-, and difucosylated structures was accomplished in very short time and with high yields, which otherwise would have required extensive additional synthetic effort and a complete redesign of existing synthetic routes. In addition, valuable information was gathered regarding the specificities of the lectins employed in this study

Mass Spectrometric Analysis of Neutral and Anionic N‑Glycans from a <i>Dictyostelium discoideum</i> Model for Human Congenital Disorder of Glycosylation CDG IL

The HL241 mutant strain of the cellular slime mold <i>Dictyostelium discoideum</i> is a potential model for human congenital disorder of glycosylation type IL (ALG9-CDG) and has been previously predicted to possess a lower degree of modification of its N-glycans with anionic moieties than the parental wild-type. In this study, we first showed that this strain has a premature stop codon in its <i>alg9</i> mannosyltransferase gene compatible with the occurrence of truncated N-glycans. These were subject to an optimized analytical workflow, considering that the mass spectrometry of acidic glycans often presents challenges due to neutral loss and suppression effects. Therefore, the protein-bound N-glycans were first fractionated, after serial enzymatic release, by solid phase extraction. Then primarily single glycan species were isolated by mixed hydrophilic-interaction/anion-exchange or reversed-phase HPLC and analyzed using chemical and enzymatic treatments and MS/MS. We show that protein-linked N-glycans of the mutant are of reduced size as compared to those of wild-type AX3, but still contain core α1,3-fucose, intersecting <i>N-</i>acetylglucosamine, bisecting <i>N-</i>acetylglucosamine, methylphosphate, phosphate, and sulfate residues. We observe that a single N-glycan can carry up to four of these six possible modifications. Due to the improved analytical procedures, we reveal fuller details regarding the N-glycomic potential of this fascinating model organism

Carbohydrate-binding specificity of CCL2.

<p>Fluorescently labeled CCL2 was analyzed for binding to the mammalian glycan array (V3.1) of the Consortium for Functional Glycomics (CFG). Results shown are averages of triplicate measurements of fluorescence intensity at a lectin concentration of 200 µg/ml. Error bars indicate the standard deviations of the mean. Glycan structures are depicted for those epitopes with highest relative fluorescence. The raw data and the entire list of glycans with the respective spacers can be found on the CFG homepage [<a href="http://functionalglycomics.org/" target="_blank">http://functionalglycomics.org/</a>] or in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002706#ppat.1002706.s015" target="_blank">Tables S2</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002706#ppat.1002706.s016" target="_blank">S3</a>. Binding of 6'sulfo-sialyllactose (glycan #45) is likely to be an artifact since it is also bound by fucose-binding lectin AAL [<a href="http://functionalglycomics.org/" target="_blank">http://functionalglycomics.org/</a>].</p

NMR solution structure of the CCL2 lectin in complex with fucosylated chitobiose (GlcNAcβ1,4[Fucα1,3]GlcNAc).

<p>(A) Intermolecular NOEs observed in a 3D ¹³C F1-edited F3-filtered HSQC-NOESY spectrum in a schematic presentation. (B) Structural ensemble of 20 structures of the protein backbone and the carbohydrate in cyan. The subunits α, β and γ are colored green, yellow and orange, respectively. The orientation is identical to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002706#ppat-1002706-g004" target="_blank">Figure 4</a>. (C) Ribbon presentation of the most representative structure. (D) Stereo view of the carbohydrate recognition site. Potential intermolecular hydrogen bonds are shown with dashed magenta lines. (E) Details of the interaction site illustrating how the trisaccharide is recognized by hydrogen bonds. (F) Summary of the interactions between the trisaccharide and CCL2. Potential H-bonds are indicated as dotted lines in magenta and hydrophobic interactions by green lines. (G) Crystal structure of the β-trefoil domain of the fungal lectin MOA in complex with the trisaccharide Galα1,3[Fucα1,2]Gal <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002706#ppat.1002706-Grahn1" target="_blank">[19]</a> showing all three occupied canonical binding sites (pdb∶3EF2). For better comparison, the same orientation and colors as in panel B and C were used.</p

The surfaces of parasites, such as <i>Trypanosoma brucei brucei</i>, are covered by glycoconjugates forming a protective glycocalyx against the host defense systems.

<p>False-color scanning electron microscopy (EM) of a <i>T</i>. <i>b</i>. <i>brucei</i> procyclic interacting with cell microvilli in the tsetse fly proventriculus (bottom panel). Transmission EM of ruthenium-red stained ultrathin sections showing the surface glycocalyx of <i>T</i>. <i>b</i>. <i>brucei</i> procyclic cells (middle panel). Scheme summarizing the main surface glycosylphosphatidylinositol (GPI)-anchored (EP- and GPEET-procyclins and trans-sialidases) and transmembrane (including polytopic) glycoproteins and glycolipids expressed by <i>T</i>. <i>b</i>. <i>brucei</i> procyclics (top panel) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005169#ppat.1005169.ref002" target="_blank">2</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005169#ppat.1005169.ref024" target="_blank">24</a>]. Open rectangles linked to GPI molecules represent side chains characteristic of surface glycoconjugates from procyclic <i>T</i>. <i>b</i>. <i>brucei</i>. GIPLs: glycoinositolphospholipids, or free GPIs. EM images obtained by C. Rose, A. Beckett, L. Tetley, I. Prior, and A. Acosta-Serrano.</p