Journal: Science immunology

Article Title: Enzymatic Synthesis of Core 2 O-Glycans Governs the Tissue-Trafficking Potential of Memory CD8 + T Cells

doi: 10.1126/sciimmunol.aan6049

Figure Lengend Snippet: (A) Expression of CD62L and KLRG1 on primary and tertiary memory P14 CD8+ T cells 90 days post-LCMV infection and (B) binding to E- and P-selectin. (C,D) Quantification of (B). (E) Expression of CD122 and CD132. (F,G) Quantification of (E). (H) Expression of total core 1 O-glycans (Jacalin) and unsialylated core 1 O-glycans (PNA). (I,J) Quantification of (H). (K) Primary and tertiary memory P14 CD8+ T cells were purified and stimulated with the indicated concentration of IL-15 for 15 minutes and phosphorylation of STAT5 (Y694) was analyzed by immunoblot. (L) Primary and tertiary memory CD8+ T cells were purified and cultured with IL-15 for 3 days. Binding of MAL II, PNA and the 1B11 antibody was analyzed. (M) Same as (L) except the glycosylation of PSGL-1 was analyzed by immunoblot. (N) Same as (L) except binding to E-selectin was analyzed by flow cytometry. (O) Quantification of (N) from 4 independent experiments. (P,Q) Same as (N,O) except for P-selectin.

Article Snippet: Lectin and Selectin Binding Fluorescein or biotin-conjugated Jacalin, Peanut Agglutinin (PNA), and MAL II (VectorLabs) were incubated with cells for 30 minutes in 1% FBS/PBS at room temperature.

Techniques: Expressing, Infection, Binding Assay, Purification, Concentration Assay, Western Blot, Cell Culture, Flow Cytometry

Journal: Cell reports

Article Title: Discovery of O-Linked Carbohydrate on HIV-1 Envelope and Its Role in Shielding against One Category of Broadly Neutralizing Antibodies

doi: 10.1016/j.celrep.2020.01.056

Figure Lengend Snippet: Equal amounts of monomeric gp120 were loaded into the wells of three 4%–12% SDS-PAGE gels. Recombinant proteins were quality checked by Coomassie (top rows) and gp120 antibody binding (middle rows). The presence of O-glycosylation was determined using jacalin-HRP (bottom rows). (A) Identification of three O-glycosylated HIV-1 Envelopes. (B) O-glycosylation of HIV-1 gp120 maps to the V1 domain. See also Figure S2.

Article Snippet: Artocarpus integrifolia (jacalin) HRP , United States Biological , Cat# A3590-11.

Techniques: SDS Page, Recombinant, Binding Assay, Glycoproteomics

Journal: Cell reports

Article Title: Discovery of O-Linked Carbohydrate on HIV-1 Envelope and Its Role in Shielding against One Category of Broadly Neutralizing Antibodies

doi: 10.1016/j.celrep.2020.01.056

Figure Lengend Snippet: (A) Matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectra of permethylated O-glycans isolated from HEK293T-produced HIV-1 GU329473 gp120. The sugar symbols in the represented glycan structures are those used by the Consortium for Functional Glycomics. Structural assignments are based on monosaccharide composition (obtained by MALDI-TOF mass spectrometry [MS]), fragmentation analyses (MALDI-TOF/TOF MS/MS), and knowledge of glycan biosynthetic pathways. All molecular ions are [M+Na]+. “M” and “m” designations indicate major and minor abundances, respectively. The asterisk on the ion at m/z 1,713 indicates that the hydroxyl of the carboxylic group of a NeuAc residue of the corresponding glycan structure is sodiated shifting the m/z value 8 units higher than the permethylated one. (B) Jacalin-binding epitopes. See also Figure S2.

Article Snippet: Artocarpus integrifolia (jacalin) HRP , United States Biological , Cat# A3590-11.

Techniques: Isolation, Produced, Glycoproteomics, Functional Assay, Mass Spectrometry, Tandem Mass Spectroscopy, Residue, Binding Assay

Journal: Cell reports

Article Title: Discovery of O-Linked Carbohydrate on HIV-1 Envelope and Its Role in Shielding against One Category of Broadly Neutralizing Antibodies

doi: 10.1016/j.celrep.2020.01.056

Figure Lengend Snippet: (A–F) Recombinant NL4–3 strains each containing a unique full-length gp160 were produced in either wild-type HEK293T or GaleGalk2 KO cells. TZM-bl cells were infected with each recombinant NL4–3 in the presence or absence of 250 μg/mL jacalin, a lectin that specifically binds O-linked glycans: (A) NL-AD8, (B) NL-VA40774, (C) NL-HM215335, (D) NL-KX693469, (E) NL-GU329473, and (F) SIVmac239. The black and blue curves represent the infectivity in the presence or absence of jacalin, respectively. Results are presented as mean ± SEM from three experiments. See also Figure S2 and S3.

Article Snippet: Artocarpus integrifolia (jacalin) HRP , United States Biological , Cat# A3590-11.

Techniques: Recombinant, Produced, Infection

Journal: Cell reports

Article Title: Discovery of O-Linked Carbohydrate on HIV-1 Envelope and Its Role in Shielding against One Category of Broadly Neutralizing Antibodies

doi: 10.1016/j.celrep.2020.01.056

Figure Lengend Snippet: The V1 domains of 32 HIV-1 Envelopes were screened using ELISA for their ability to elicit a jacalin-HRP signal in the context of the non-jacalin-reactive VAgp120 backbone. Samples 33–40 served as internal controls for the assay. The name and V1 domain characteristics of samples 1–37 are found in Table S1. Sample 38, GU329473 gp120; sample 39, GUgp120_(AD8)V1; sample 40, AD8 gp120. Results are presented as mean ± SEM from two experiments. See also Figures S2 and S5–S7 and Table S1 and S2.

Article Snippet: Artocarpus integrifolia (jacalin) HRP , United States Biological , Cat# A3590-11.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Cell reports

Article Title: Discovery of O-Linked Carbohydrate on HIV-1 Envelope and Its Role in Shielding against One Category of Broadly Neutralizing Antibodies

doi: 10.1016/j.celrep.2020.01.056

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Artocarpus integrifolia (jacalin) HRP , United States Biological , Cat# A3590-11.

Techniques: Virus, Recombinant, Plasmid Preparation, Reporter Gene Assay, Knock-Out, Expressing, Software, Western Blot

Journal: Frontiers in Cell and Developmental Biology

Article Title: Stimulation of Acanthamoeba castellanii excystment by enzyme treatment and consequences on trophozoite growth

doi: 10.3389/fcell.2022.982897

Figure Lengend Snippet: Influence of cellulase treatment on carbohydrate localization in A. castellanii cysts. After a treatment in the presence or absence (control) of 7.5 U/mL cellulase at 27°C for 48 h, carbohydrates were labeled by FITC-lectins (green). Phase contrast and merge images are shown on top and at the bottom of the FITC-lectin image, respectively. Nine lectins were used in this experiment: Con A, GSL II, Jacalin, LCA, LEL, SBA, SWGA, VVL and WGA. Scale bars = 10 µm.

Article Snippet: The FITC-lectins used in the present study were: Con A (Concanavalin A), GSL II ( Griffonia Simplicifolia Lectin II), Jacalin, LCA ( Lens Culinaris Agglutinin), LEL ( Lycopersicon Esculentum Lectin), SBA (SoyBean Agglutinin), VVL ( Vicia Villosa Lectin, also called VVA), WGA (Wheat Germ Agglutinin) or SWGA (Succinylated Wheat Germ Agglutinin; Eurobio, Les Ulis, France).

Techniques: Labeling

Journal: mBio

Article Title: Making Home Sweet and Sturdy: Toxoplasma gondii ppGalNAc-Ts Glycosylate in Hierarchical Order and Confer Cyst Wall Rigidity

doi: 10.1128/mBio.02048-16

Figure Lengend Snippet: Lectin profiling of ppGalNAc-T-knockout strains. Purified lysates from T. gondii strains were separated by SDS-PAGE and transferred onto a PVDF membrane which was then incubated with either biotin-conjugated jacalin (A) or VVA (B). The orange arrowheads indicate the bands missing in both ppGalNAc-Δ t2 and -Δ t3 strains. The blue arrowheads indicate the bands missing in ppGalNAc-Δ t2 but not in -Δ t3 . The gray arrowheads indicate the nonspecific endogenous biotinylated proteins (streptavidin was used for detection). This blot demonstrates that multiple T. gondii glycoproteins require ppGalNAc-T2 and -T3 glycosyltransferase activities. The duplicate blots on the right were subjected to β-elimination and demonstrate that the identified bands are due to O-linked glycosylation. This figure demonstrates T. gondii cultivated under tachyzoite (pH 7.1 with 5% CO 2 ) culture conditions, and Fig. S3 in the supplemental material demonstrates T. gondii cultivated under bradyzoite culture conditions (pH 8.1 with 0.1% CO 2 ). WT, wild type.

Article Snippet: The samples were centrifuged at 13,600 × g at 4°C for 16 min, and the collected supernatants were applied to either a jacalin- or a VVA-agarose bead affinity column (EY Laboratories).

Techniques: Knock-Out, Purification, SDS Page, Incubation

Journal: mBio

Article Title: Making Home Sweet and Sturdy: Toxoplasma gondii ppGalNAc-Ts Glycosylate in Hierarchical Order and Confer Cyst Wall Rigidity

doi: 10.1128/mBio.02048-16

Figure Lengend Snippet: Binding of lectins to the cyst wall of ppGalNAc-T-knockout strains. Human foreskin fibroblasts (HFF) infected with wild-type and ppGalNAc-T deletion mutant T. gondii were cultured in pH 8.1 medium, facilitating the formation of in vitro cysts. Infected HFF cells were stained with jacalin (red, upper row), VVA (red, middle row), or HPA (red, bottom row) as well as the green fluorescent protein (green) driven by bradyzoite-specific promoter LDH2 and DAPI (blue). The Δ t2 parasite lacks jacalin, VVA, and HPA cyst wall staining, while the Δt3 parasite lacks jacalin staining only. This suggests that the various lectin-binding glycoproteins on the cyst wall are posttranslationally modified by ppGalNAc-T2 and -T3 glycosyltransferases.

Article Snippet: The samples were centrifuged at 13,600 × g at 4°C for 16 min, and the collected supernatants were applied to either a jacalin- or a VVA-agarose bead affinity column (EY Laboratories).

Techniques: Binding Assay, Knock-Out, Infection, Mutagenesis, Cell Culture, In Vitro, Staining, Modification