Journal:

Article Title: Increased Levels of Galactose-Deficient Anti-Gal Immunoglobulin G in the Sera of Hepatitis C Virus-Infected Individuals with Fibrosis and Cirrhosis ▿

doi: 10.1128/JVI.01600-07

Figure Lengend Snippet: Evidence that anti-Gal IgG molecules have altered glycosylation in patients with cirrhosis. Briefly, either HSA (column 1) or synthetic alpha-Gal-linked HSA (columns 2 and 3) was plated onto 96-well plates and incubated with human sera from four healthy control individuals or from three individuals with cirrhosis. The captured IgG was detected by using either anti-human IgG-conjugated secondary antibody (columns 1 and 2) or the fucose-specific lectin AAL (column 3).

Article Snippet: Briefly, lectin extraction was performed using agarose-bound Aleuria aurantia lectin (AAL) (Vector Laboratories, Ventura, CA) and Affi Sep-AAL absorption buffer and elution buffers (GALAB, Germany).

Techniques: Incubation

Journal:

Article Title: Increased Levels of Galactose-Deficient Anti-Gal Immunoglobulin G in the Sera of Hepatitis C Virus-Infected Individuals with Fibrosis and Cirrhosis ▿

doi: 10.1128/JVI.01600-07

Figure Lengend Snippet: Evidence that only anti-Gal IgG is reactive with fucose binding lectins. Removal of heterophilic alpha-Gal antibodies prevents lectin reactivity of IgGs from cirrhotic patients. IgGs from either healthy individuals or individuals with cirrhosis were captured from sera by using a mouse anti-human IgG antibody. The amount of captured IgG (lane 1) or the level of AAL lectin reactivity (lane 2) was determined as for Fig. ​Fig.3.3. Samples were precleared either with HSA (lanes 1 and 2) or with alpha-Gal-HSA (lanes 3 and 4) before analysis. H, precleared with HSA; A, precleared with alpha-Gal-HSA.

Article Snippet: Briefly, lectin extraction was performed using agarose-bound Aleuria aurantia lectin (AAL) (Vector Laboratories, Ventura, CA) and Affi Sep-AAL absorption buffer and elution buffers (GALAB, Germany).

Techniques: Binding Assay

Journal:

Article Title: Increased Levels of Galactose-Deficient Anti-Gal Immunoglobulin G in the Sera of Hepatitis C Virus-Infected Individuals with Fibrosis and Cirrhosis ▿

doi: 10.1128/JVI.01600-07

Figure Lengend Snippet: The reactivity of human IgG to the fucose binding lectin AAL is dependent on the type of N-linked glycan attached. (A) IgG from healthy individuals (purchased from Sigma Chemicals) was digested overnight with sialidase (A. ureafaciens) and beta(1-4)-galactosidase (jack bean) to create IgG molecules with the degalactosylated glycans observed in patients with cirrhosis. As a control, a mock sample, treated identically but without enzyme, was used. While IgG purified from healthy individuals had low reactivity with the fucose binding lectin, IgG purified either from cirrhotic individuals or from healthy serum that has been treated with the sialidase and beta-galactosidase had much greater reactivity, indicating that the lectin reactivity is directly associated with the FcA2G0 glycan structure. (B) Glycan analysis of IgG from either mock-treated samples (top) or enzyme-treated samples (bottom) to confirm enzymatic digestion. The major peaks are indicated. For the abbreviations, see the legend to Fig. 5B and C. Additional structures not presented in Fig. ​Fig.5:5: A2G2S1, monosialylated biantennary glycan; FCA2G2S1, monosialylated core fucosylated biantennary N-glycan; FCA2BG2S1, monosialylated core fucosylated biantennary glycan with a bisecting GlcNac; A2G2S2, disialylated biantennary glycan; FCA2BG2S2, disialylated core fucosylated biantennary glycan; FcA2BG2S2, disialylated core fucosylated biantennary glycan with a bisecting GlcNac. (C) Quantification of results shown in panel B. The x axis is the glycan structure as detailed in panel B; the y axis is the relative contribution of each glycan structure to the total N-linked glycan profile. Structures are as in panel B.

Article Snippet: Briefly, lectin extraction was performed using agarose-bound Aleuria aurantia lectin (AAL) (Vector Laboratories, Ventura, CA) and Affi Sep-AAL absorption buffer and elution buffers (GALAB, Germany).

Techniques: Binding Assay, Purification

Journal: The Journal of biological chemistry

Article Title: Architecture of the yeast cell wall. Beta(1-->6)-glucan interconnects mannoprotein, beta(1-->)3-glucan, and chitin.

doi: 10.1074/jbc.272.28.17762

Figure Lengend Snippet: FIG. 2. Affinity chromatography of 14C-Gal-labeled ConA2 and 14C-Gal-labeled ConA1 on E. cristagalli lectin-agarose. a, a por- tion of [14C]Gal-labeled ConA1 fraction (52,000 cpm of tritium and 20,500 cpm of 14C) was diluted to 500 ml with a solution containing 50 mM Hepes, pH 8.0, 100 mM KCl, 5 mM MgCl2, and 2 mM b-mercapto- ethanol (binding buffer) and applied to a column of E. cristagalli lectin- agarose (1 3 20 cm). Elution was done with the loading buffer, and 0.45-ml fractions were collected. At fraction 55 (arrow) the eluent was changed to a solution with the same composition of the binding buffer plus 0.2 M lactose and 0.5 M GlcNAc to displace the bound material. Of each fraction, a 50-ml sample was counted in separate channels for tritium (G) and 14C (å). b, a portion of [14C]Gal-labeled ConA2 (3.6 3 105 cpm of tritium and 7.5 3 104 cpm of 14C) was separated under the same conditions as in a, except that 200 ml of each fraction was counted, and the elution buffer was changed at fraction 60 (arrow).

Article Snippet: Sodium [3H]borohydride (100 mCi/mmol) was obtained from ICN; [1-14C]glucose (50–60 mCi/mmol) was from American Radiolabeled Chemicals; and uridine diphospho-[U-14C]galactose (305 mCi/mmol) was from Amersham Corp. Bio-Gel P-2 (fine and extra fine) and Bio-Gel P-4 (extra fine) were from Bio-Rad; concanavalin A-Sepharose and Sephadex G-100 were from Pharmacia Biotech Inc.; Erythrina cristagalli lectin-agarose was from Vector; and Rezex RSO-Oligosaccharides HPLC1 column was from Phenomenex.

Techniques: Affinity Chromatography, Labeling, Binding Assay

Journal: The Journal of biological chemistry

Article Title: Architecture of the yeast cell wall. Beta(1-->6)-glucan interconnects mannoprotein, beta(1-->)3-glucan, and chitin.

doi: 10.1074/jbc.272.28.17762

Figure Lengend Snippet: FIG. 7. Separation of tritiated and 14C-labeled oligosaccha- rides released from 14C-Gal-labeled ConA1 fraction by endo- b(136)-glucanase. a, the low molecular weight peak, isolated from Sephadex G-100 (Fig. 6b) after glucanase digestion of [14C]Gal-labeled ConA1 fraction (2.8 3 105 cpm of tritium and 1.6 3 105 cpm of 14C), was fractionated on an E. cristagalli lectin-agarose column to separate galactose-containing (14C-labeled, B) from galactose-free (tritium-la- beled, A) oligosaccharides. Conditions were as in Fig. 2. Since the 14C-labeled material was retarded rather than bound, the elution buffer was not required. Samples were counted in channels for tritium (G) or 14C (å). b, fraction A was concentrated by evaporation to ;1 ml, applied to an extra fine Bio-Gel P-2 column, and eluted as in Fig. 1. In addition to radioactivity, total carbohydrate (see “Experimental Procedures”) was measured in fractions (E). Positions of standards are indicated with arrows. 1, [14C]glucose (internal standard); 2, hexitol; 3–8, diol to hep- taol obtained by reduction of a dextran ladder (Oxford Glycosystems). c, fraction B was concentrated and chromatographed as fraction A. Stand- ards were the same as in b, except that the internal standard was [3H]mannose. In all cases, 0.45-ml fractions were collected. Samples of 10 ml were used for counting, and samples of 40 ml were used for carbohydrate determinations.

Article Snippet: Sodium [3H]borohydride (100 mCi/mmol) was obtained from ICN; [1-14C]glucose (50–60 mCi/mmol) was from American Radiolabeled Chemicals; and uridine diphospho-[U-14C]galactose (305 mCi/mmol) was from Amersham Corp. Bio-Gel P-2 (fine and extra fine) and Bio-Gel P-4 (extra fine) were from Bio-Rad; concanavalin A-Sepharose and Sephadex G-100 were from Pharmacia Biotech Inc.; Erythrina cristagalli lectin-agarose was from Vector; and Rezex RSO-Oligosaccharides HPLC1 column was from Phenomenex.

Techniques: Labeling, Molecular Weight, Isolation, Evaporation, Radioactivity

Journal: The Journal of Biological Chemistry

Article Title: Early Murine T-lymphocyte Activation Is Accompanied by a Switch from N -Glycolyl- to N -Acetyl-neuraminic Acid and Generation of Ligands for Siglec-E *

doi: 10.1074/jbc.M111.243410

Figure Lengend Snippet: Activation of murine T-lymphocytes leads to up-regulation in the binding of siglec-E to cell surface proteins in a sialic acid-dependent manner. A , T-lymphocytes were activated with anti-CD3 and anti-CD28 for 24 h and then cultured for up to 4 days in the presence of IL-2. Cells harvested at each time point were incubated with fluorophore-conjugated CD4 ( left panel ) and CD8 antibodies ( right panel ) together with a pre-complex of FITC-conjugated goat anti-human Fc and siglec-Fc. Mean Fluorescence Intensity ( MFI ) of sialic acid-dependent binding by FACS is shown following subtraction of MFI values for sialidase-treated cells. B , for the detection of MAL and SNA binding, harvested cells corresponding to each time point were incubated with biotinylated lectin followed by a secondary incubation with FITC-conjugated streptavidin. C , splenocytes harvested at 24 h were untreated ( open histograms ) or pretreated with V. cholerae sialidase ( left panel ) or proteinase K ( right panel ) (shaded histograms) and labeled with siglec-E-Fc/anti-Fc-FITC complexes. Data are representative of at least three independent experiments.

Article Snippet: 24 h-activated lysates were alternatively incubated with MAL-agarose (EY Labs) or SNA-agarose (Vector Labs) beads.

Techniques: Activation Assay, Binding Assay, Cell Culture, Incubation, Fluorescence, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Early Murine T-lymphocyte Activation Is Accompanied by a Switch from N -Glycolyl- to N -Acetyl-neuraminic Acid and Generation of Ligands for Siglec-E *

doi: 10.1074/jbc.M111.243410

Figure Lengend Snippet: Siglec-E-Fc binds subsets of sialylated proteins from activated T-lymphocytes. A , purified T cells were activated for 24 h and either untreated or sialidase-treated and then surface-labeled with biotin. Cell lysates were passed over protein G-Sepharose coupled noncovalently to siglec-E-Fc/anti-Fc complexes and bound proteins eluted with SDS-PAGE sample buffer. Western blots were probed with streptavidin-HRP to detect surface biotinylated proteins. Arrows indicated proteins that bound to Siglec-E-Fc in a sialidase-sensitive manner ( lane 4 ). B , purified T cells were activated for 24 h and treated or not with sialidase and lysates passed over MAL or SNA affinity columns. Bound sialylated proteins were eluted with SDS-PAGE sample buffer, separated by SDS-PAGE and transferred to nitrocellulose. Blots were probed with siglec-E-Fc precomplexed to anti-Fc-HRP. Arrows indicate proteins that are recognized by siglec-E-Fc in a sialidase-sensitive manner. C , purified T cells were activated for 24 h, cultured for 4 days and treated or not with sialidase. Cell lysates were passed over protein G-Sepharose coupled noncovalently to siglec-E-Fc/anti-Fc complexes and bound proteins eluted with SDS-PAGE sample buffer. Western blots were probed with goat anti-CD45 followed by HRP-conjugated anti-goat secondary antibody. The arrow indicates CD45 that is recognized by siglec-E-Fc in a sialidase-sensitive manner. The other bands represent nonspecifically bound proteins.

Article Snippet: 24 h-activated lysates were alternatively incubated with MAL-agarose (EY Labs) or SNA-agarose (Vector Labs) beads.

Techniques: Purification, Labeling, SDS Page, Western Blot, Cell Culture

Journal: Glycobiology

Article Title: Circulating blood and platelets supply glycosyltransferases that enable extrinsic extracellular glycosylation

doi: 10.1093/glycob/cww108

Figure Lengend Snippet: Activity toward GlcNAc and mucin-type Core 1 acceptor in WT murine serum and plasma. Panels A–C: Glycosyltransferase activity toward LacNAc, mucin-type Core 1 and GlcNAc acceptors in WT murine plasma and serum. A comparison of glycosyltransferase activity, namely SiaT (A), FucT (B) and GalT (C), in WT murine plasma (filled circle) and serum (unfilled squares) was assessed by measuring the amount of radiolabeled nucleotide-sugar donor transferred various acceptors (n = 5). SiaT activity toward Type-II LacNAc, whether displaying α2,3- or α2,6- linkage, was further deconvoluted using agarose- bound SNA lectin. GalT activity toward GlcNAc and SiaT activity toward mucin-type Core 1 acceptor is increased in WT murine serum compared to plasma by 1.3- and 2.2-fold, respectively. SiaT activity toward type-II LacNAc is predominantly α2,3-linked, shown by lectin chromatography. Using MSn analysis, it was determined that FucT activity in murine plasma and serum toward Type-II LacNAc produces predominantly Lewisx (Lex) structures (Figure ​(Figure33).

Article Snippet: Separation of α2,3- and α2,6-sialic acid fractions of sialylated Type-II LacNAc was performed with agarose-bound SNA ( S. nigra agglutnin) -lectin (Vector Laboratories, CA) affinity chromatography ( Jones et al. 2010 ).

Techniques: Activity Assay, Chromatography