Journal: Nature Communications

Article Title: IgA subclasses have different effector functions associated with distinct glycosylation profiles

doi: 10.1038/s41467-019-13992-8

Figure Lengend Snippet: a Schematic overview of glycosylation sites in IgA1 and IgA2. Bold letters indicate glycosylation sites present in both IgA subclasses. b Representative lectin blots of IgA1 and IgA2 isolated from sera of healthy donors using antibodies against IgA or lectins against the core structure of N -glycans (=lens culinaris agglutinin), terminal α2,6-linked sialic acid (=sambuccus nigra agglutinin), and terminal galactose (=erythrina christagalli lectin). c Quantification of the lectin blots; n = 6 donors. d – f Mass spectrometric quantification of sialyation, galactosylation, bisection, fucosylation, and the presence of noncomplex structures for the glycosylation sites N144/N131 d , N340/N327 e , N41 f , and N205 g ; n = 12 donors. Significances were tested with paired two-sided Student’s t -test c – g or paired one-way ANOVA followed by Bonferroni correction for selected pairs of columns d – g . * p < 0,05; ** p < 0,01; and *** p < 0,001. Data are presented as scatter plots with mean ± s.e.m. c or box plots with medians and inter-quartile ranges + whiskers ranging from min to max d – g . Source data are provided as a Source Data file.

Article Snippet: After blocking with 3% deglycosylated gelatin (Sigma), blots were incubated with horseradish peroxidase (HRP)-labeled goat anti-human IgA (1:10,000; #2050-05; Southern Biotech), biotinylated lens culinaris agglutinin (5 µg/ml; #B-1045) for the detection of the core glycan, biotinylated erythrina cristagalli lectin (5 µg/ml; #B-1145) for galactose detection, or biotinylated sambuccus nigra lectin (2 µg/ml; #B-1305; all vector laboratories) for sialic acid detection, followed by incubation with HRP-labeled streptavidin (1:500; # DY998; R&D).

Techniques: Isolation

Journal: Cancers

Article Title: Decoding Single Cell Morphology in Osteotropic Breast Cancer Cells for Dissecting Their Migratory, Molecular and Biophysical Heterogeneity

doi: 10.3390/cancers14030603

Figure Lengend Snippet: Differential binding of lectins to breast cancer cell lines. ( A , B ) The parental cells MB-231 and its bone-seeking derivatives, MET and BONE, were cell-surface labeled with one of a panel of distinct FITC-conjugated lectins prior to cytochemistry (( A ), left panels) and flow cytometry (( A ), right panels, ( B )) analyses. See also . Percentages of positive cells are indicated in the histograms ( A ) and the median fluorescence intensity is presented for each lectin ( B ). Data from representative experiments acquired under uniform instruments setting for all cell lines are displayed. The lectins used were Concanavalin A (ConA), Datura Stramonium Lectin (DSL), Dolichos Biflorus Agglutinin (DBA), Erythrina Cristagalli Lectin (ECL), Griffonia Simplicifolia Lectin I (GSL-I), Griffonia Simplicifolia Lectin II (GSL-II), Lens Culinaris Agglutinin (LCA), Lycopersicon Esculentum Lectin (LEL), Peanut Agglutinin (PNA), Phaseolus Vulgaris Lectin E (PHA-E), Phaseolus Vulgaris Lectin L (PHA-L), Pisum Sativum Agglutinin (PSA), Ricinus Communis Agglutinin I (RCA), Sambucus Nigra Lectin (SNA), Solanum Tuberosum Lectin (STL), Soybean Agglutinin (SBA), Ulex Europaeus Agglutinin I (UEA-I), Vicia Villosa Lectin (VVL) and Wheat Germ Agglutinin (WGA, succinylated (succ)-WGA). Scale bar, 25 μm.

Article Snippet: After inactivation of trypsin, 2 washing steps with PBS and centrifugation (5 min at 300× g ), cells were resuspended in PBS or Ca/Mg-PBS (for lectin labeling), containing 1% BSA and 100 μL-cell suspension aliquots were incubated with unconjugated or fluorochrome-conjugated primary antibodies ( ) or FITC- or biotin-conjugated lectins (see above, Lectin kit I, Biotinylated (BK-1000) and SNA, Biotinylated (B-1305-2), both from Vector Laboratories) for 30 min at 4 °C.

Techniques: Binding Assay, Labeling, Flow Cytometry, Fluorescence

Journal: Frontiers in Microbiology

Article Title: Capsular Sialyltransferase Specificity Mediates Different Phenotypes in Streptococcus suis and Group B Streptococcus

doi: 10.3389/fmicb.2018.00545

Figure Lengend Snippet: Capsular polysaccharide (CPS) expression levels and sialic acid linkage in S. suis serotype 2 and 14 mutants carrying exogenous α-2,3-sialyltransferase. (A) Hydrophobicity (%) of the wild-type S. suis serotype 2 (SS2) and 14 (SS14) strains, the SS2sia2,3 (Δ cps2N / cpsK ) and SS14sia2,3 (Δ cps14N / cpsK ) mutants carrying the GBS α-2,3-sialyltransferase ( cpsK ). The non-encapsulated mutants SS2Δ cps and SS14Δ cps were used as control strains. (B,C) Whole-bacterial cell enzyme-linked lectin assay (ELLA) was performed to detect α-2,3 or α-2,6 capsular sialic acid linkage in SS2sia2,3 and SS14sia2,3 mutant strains. Whole bacteria were incubated with Sambucus nigra agglutinin (SNA-I) specific for Neu5Ac α-2,6 linkages, or Maackia amurensis leukoagglutinin (MAL-I) specific for Neu5Ac α-2,3 linkages. The non-encapsulated mutants SS2Δ cps and SS14Δ cps were used as negative controls. SS2 was used as positive control for SNA-I and wild-type GBS type III as positive control for MAL-I. Data in (A–C) are expressed as mean ± SEM of at least three independent experiments. Student's t -test analyses reported significant differences between “ a” and “ b” ( P < 0.05).

Article Snippet: In order to investigate the specific linkage of sialic acid in the sialyltransferase substitution mutants, a whole-bacterial cell ELLA was carried out with the biotinylated Sambucus nigra agglutinin (SNA-I, Vector Labs Canada, Burlington, ON, Canada) and the biotinylated Maackia amurensis leukoagglutinin (MAL-I, Vector Labs) which specifically recognize sialic acid as Neu5Acα-2,6-Gal p /Gal p NAc or as Neu5Acα-2,3-Galβ-1,4-GlcNAc, respectively (Shibuya et al., ; Geisler and Jarvis, ).

Techniques: Expressing, Mutagenesis, Incubation, Positive Control

Journal: Frontiers in Microbiology

Article Title: Capsular Sialyltransferase Specificity Mediates Different Phenotypes in Streptococcus suis and Group B Streptococcus

doi: 10.3389/fmicb.2018.00545

Figure Lengend Snippet: Capsular polysaccharide (CPS) expression levels and recognition of specific CPS sialic acid linkage in GBS type V isogenic mutants. (A) Hydrophobicity (%) of the wild-type GBS serotype V strain (GBS V), and the sialic acid synthesis GBSVΔsynth (Δ neu5B ) and sialyltransferase GBSVΔsiaT (Δ cps5K ) deficient mutants. The non-encapsulated strain (GBSVΔ cps ) was used as control. (B) Whole-bacterial cell enzyme-linked lectin assay (ELLA) was performed to detect α-2,3 or α-2,6 capsular sialic acid linkage in these mutant strains. Whole bacteria were incubated with Sambucus nigra agglutinin (SNA-I) specific for Neu5Ac α-2,6 linkages, or Maackia amurensis leukoagglutinin (MAL-I) specific for Neu5Ac α-2,3 linkages. The non-encapsulated mutant GBSVΔ cps was used as negative control. S. suis serotype 2 (SS2) was used as positive control for SNA-I and wild-type GBS type V as positive control for MAL-I. Data in (A,B) are expressed as mean ± SEM of at least three independent experiments. Student's t -test analyses reported significant differences between “ a” and “ b” ( P < 0.05).

Article Snippet: In order to investigate the specific linkage of sialic acid in the sialyltransferase substitution mutants, a whole-bacterial cell ELLA was carried out with the biotinylated Sambucus nigra agglutinin (SNA-I, Vector Labs Canada, Burlington, ON, Canada) and the biotinylated Maackia amurensis leukoagglutinin (MAL-I, Vector Labs) which specifically recognize sialic acid as Neu5Acα-2,6-Gal p /Gal p NAc or as Neu5Acα-2,3-Galβ-1,4-GlcNAc, respectively (Shibuya et al., ; Geisler and Jarvis, ).

Techniques: Expressing, Mutagenesis, Incubation, Negative Control, Positive Control

Journal: Frontiers in Microbiology

Article Title: Capsular Sialyltransferase Specificity Mediates Different Phenotypes in Streptococcus suis and Group B Streptococcus

doi: 10.3389/fmicb.2018.00545

Figure Lengend Snippet: Capsular polysaccharide (CPS) expression levels and sialic acid linkage in GBS type III and V mutants carrying exogenous α-2,6-sialyltransferase. (A) Hydrophobicity (%) of GBS type III and V wild-type strains and the mutants GBSIIIsia2,6 (Δ cps3K / cps2N ) and GBSVsia2,6 (Δ cps5K / cps2N ) carrying the S. suis α-2,6-sialyltransferase. The non-encapsulated mutant strains (GBSIIIΔ cps and GBSVΔ cps ) were used as controls. (B,C) Whole-bacterial cell enzyme-linked lectin assay (ELLA) was performed to detect α-2,3 or α-2,6 capsular sialic acid linkage in these mutant strains. Whole bacteria were incubated with Sambucus nigra agglutinin (SNA-I) specific for Neu5Ac α-2,6 linkages, or Maackia amurensis leukoagglutinin (MAL-I) specific for Neu5Ac α-2,3 linkages. The non-encapsulated mutants were used as negative controls. Data in (A–C) are expressed as mean ± SEM of at least three independent experiments. Student's t -test analyses reported significant differences between “ a” and “ b” , between “ a” and “ c” , and between “ b” and “ c” ( P < 0.05).

Article Snippet: In order to investigate the specific linkage of sialic acid in the sialyltransferase substitution mutants, a whole-bacterial cell ELLA was carried out with the biotinylated Sambucus nigra agglutinin (SNA-I, Vector Labs Canada, Burlington, ON, Canada) and the biotinylated Maackia amurensis leukoagglutinin (MAL-I, Vector Labs) which specifically recognize sialic acid as Neu5Acα-2,6-Gal p /Gal p NAc or as Neu5Acα-2,3-Galβ-1,4-GlcNAc, respectively (Shibuya et al., ; Geisler and Jarvis, ).

Techniques: Expressing, Mutagenesis, Incubation