Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

Article Title: L1210 Cells Overexpressing ABCB1 Drug Transporters Are Resistant to Inhibitors of the N- and O-glycosylation of Proteins

doi: 10.3390/molecules22071104

Figure Lengend Snippet: Detection of ConA and GNA binding to glycoproteins in S, R and T cells passaged three times in the absence or presence of either tunicamycin (0.1 μmol·dm −3 ) or GalNAc-α- O -benzyl (0.1 mmol·dm −3 ). Panel ( a ): Representative sample of flow cytometry measurement of FITC-ConA binding to S (upper plot), R (middle plot) and T (lower plot) cells. Black histogram – unlabeled S, R and T cells, red histogram – FITC-ConA-labeled S cells, blue histogram FITC-ConA-labeled R cells, and green histogram – FITC-ConA-labeled T cells. Parameter Δ (represents the difference between medians of labeled and unlabeled cells) was ascertained using this measurement. Similar measurements were obtained for S, R and T cells untreated and treated with tunicamycin and GalNAc-α- O -benzyl and documented in panels ( b ) (for ConA) and ( c ) (for GNA). Binding of lectin to untreated S cells was arbitrarily selected as 100%. White column—untreated cells; gray—column cell treated with GalNAc-α- O -benzyl and black column—cell treated with tunicamycin. Significance: * and +—value significantly differs from the corresponding value for S cells at p < 0.02 and p < 0.05, respectively. The data represent the means ± S.E.M. of five independent measurements. Panels ( d ) (for ConA) and ( e ) (for GNA) represent Eastern blot identification of glycoproteins in crude membrane fractions isolated from S, R and T cells untreated C, or treated with inhibitor of O-glycosylation (GalNAc-α- O -benzyl, O) or N-glycosylation (tunicamycin, N). Data are representative of three independent measurements. Red arrows indicate the P-gp form glycosylated with saccharides that are GNA ligands.

Article Snippet: For glycoprotein detection by Eastern blots, ConA and GNA conjugated with biotin (EY Laboratories Inc., San Mateo, CA, USA), and avidin conjugated with horseradish peroxidase (Sigma-Aldrich, San Diego, CA, USA) was used.

Techniques: Binding Assay, Flow Cytometry, Labeling, Isolation

Journal: International Journal of Molecular Sciences

Article Title: Overview of the Structure–Function Relationships of Mannose-Specific Lectins from Plants, Algae and Fungi

doi: 10.3390/ijms20020254

Figure Lengend Snippet: Overview of plant, algae and fungi lectins with a mannosyl-binding specificity (β-sandwich: βs, β-prism: βp, n.d.: not determined).

Article Snippet: A similar observation was previously reported in a series of mutant HIV-1 isolates resistant to GNA ( Galanthus nivalis ) and HHA ( Hippeastrum sp. hybrid) lectins, showing that the major amino acid mutations occur at several putative N -glycosylation sites NXT and NXS, and especially, at the ultimate T or S residues [ ].

Techniques: Algae, Antiviral Assay

Journal: International Journal of Molecular Sciences

Article Title: Overview of the Structure–Function Relationships of Mannose-Specific Lectins from Plants, Algae and Fungi

doi: 10.3390/ijms20020254

Figure Lengend Snippet: Structural diversity of the mannose-binding lectins. ( A ). Two-chain lectin protomer of Lathyrus ochrus (PDB code 1LOE ). Light chain and heavy chains are colored green and red, respectively. ( B ). Homodimeric organization of the L. ochrus isolectin-I (1LOE). The light and heavy chains of the dimer are colored differently. ( C ). Homotetrameric organization of Con A (PDB code 3CNA). The four single-chain protomers are shown in different colors. ( D ). The β-prism organization of the artocarpin protomer from Artocarpus integrifolia (PDB code 1J4S). The three bundles of β-strands forming the β-prism are colored green, red and orange, respectively. ( E ). Homotetrameric organization of artocarpin from A. integrifolia (1J4U). The β-prism protomers are colored differently. ( F ). Homooctameric organization of Heltuba from Helianthus tuberosus (1C3M) . The β-prism protomers are colored differently. ( G ). The β-prism II organization of the protomer of GNA from Galanthus nivalis (PDB code 1MSA). ( H ). Organization of the β-prism II protomers in the GNA tetramer (PDB code 1MSA). ( I ). Hexameric structure of the tarin lectin from Colocasia esculenta (PDB code 5T20). The six β-prism-folded protomers are colored differently.

Article Snippet: A similar observation was previously reported in a series of mutant HIV-1 isolates resistant to GNA ( Galanthus nivalis ) and HHA ( Hippeastrum sp. hybrid) lectins, showing that the major amino acid mutations occur at several putative N -glycosylation sites NXT and NXS, and especially, at the ultimate T or S residues [ ].

Techniques: Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Overview of the Structure–Function Relationships of Mannose-Specific Lectins from Plants, Algae and Fungi

doi: 10.3390/ijms20020254

Figure Lengend Snippet: PDB codes of lectins from plants and fungi, complexed with simple sugars ( m ), oligomannosides (o), and complex (c) mannose-containing glycans.

Article Snippet: A similar observation was previously reported in a series of mutant HIV-1 isolates resistant to GNA ( Galanthus nivalis ) and HHA ( Hippeastrum sp. hybrid) lectins, showing that the major amino acid mutations occur at several putative N -glycosylation sites NXT and NXS, and especially, at the ultimate T or S residues [ ].

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Overview of the Structure–Function Relationships of Mannose-Specific Lectins from Plants, Algae and Fungi

doi: 10.3390/ijms20020254

Figure Lengend Snippet: ( A , B ). ConA from Canavalia ensiformis in complex with α-methylmannoside (PDB code 5CNA). ( C , D ). Isolectin LoLI from Lathyrus ochrus in complex with Man (PDB code 1LOB). ( E , F ). Artocarpin from Artocarpus integrifolia in complex with α-mthylmannoside (PDB code 1J4U). ( G , H ). Heltuba from Helianthus tuberosus in complex with Manα1,3Man (PDB code 1C3M). ( I , J ). Third Man-binding site of GNA from Galanthus nivalis in complex with α-methylmannoside (PDB code 1MSA). Hydrogen bonds connecting the monosaccharides to the amino acid residues forming the monosaccharide-binding site are represented by black dashed lines. Aromatic residues participating in stacking interactions with the sugar rings are colored orange. The molecular surface of the lectins is colored dark grey and their extended oligosaccharide-binding areas are delineated by white dashed lines. The shallow depression corresponding to the monosaccharide-binding site that accommodates simple sugars is delineated by a green dashed line. The green and violet spheres correspond to the Ca 2+ and Mn 2+ ions, that have a stabilizing effect on the carbohydrate-binding site.

Article Snippet: A similar observation was previously reported in a series of mutant HIV-1 isolates resistant to GNA ( Galanthus nivalis ) and HHA ( Hippeastrum sp. hybrid) lectins, showing that the major amino acid mutations occur at several putative N -glycosylation sites NXT and NXS, and especially, at the ultimate T or S residues [ ].

Techniques: Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Overview of the Structure–Function Relationships of Mannose-Specific Lectins from Plants, Algae and Fungi

doi: 10.3390/ijms20020254

Figure Lengend Snippet: ( A , B ). ConA from Canavalia ensiformis in complex with β-D-GlcNAc-(1,2)-α-D-Man-(1,6)-[β-D-GlcNAc-(1,2)-α-D-Man-(1,6]-αD-Man (PDB code 1TEI) . ( C , D ). Isolectin LoLII from Lathyrus ochrus in complex with a biantennary octasaccharide of the N -acetyllactosamine type from lactotransferrin (PDB code 1LOF). ( E , F ). GNA from Galanthus nivalis in complex with three mannosyl residues from a mannopentaose (PDB code 1JPC). ( G , H ). PAL from Pterocarpus angolensis in complex with a mannotetraose (PDB code 2PHF). Hydrogen bonds connecting the oligosaccharides to the amino acid residues forming the extended carbohydrate-binding site are represented by black dashed lines. Aromatic residues participating in stacking interactions with the sugar rings are colored orange. The electrostatic potentials were calculated and mapped on the molecular surface of the lectins, using YASARA. The extended oligosaccharide-binding areas are delineated by white dashed lines. The shallow depression corresponding to the monosaccharide-binding site that accommodates simple sugars, is delineated by a green dashed line.

Article Snippet: A similar observation was previously reported in a series of mutant HIV-1 isolates resistant to GNA ( Galanthus nivalis ) and HHA ( Hippeastrum sp. hybrid) lectins, showing that the major amino acid mutations occur at several putative N -glycosylation sites NXT and NXS, and especially, at the ultimate T or S residues [ ].

Techniques: Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Overview of the Structure–Function Relationships of Mannose-Specific Lectins from Plants, Algae and Fungi

doi: 10.3390/ijms20020254

Figure Lengend Snippet: List of Man-specific lectins investigated for their toxicity towards aphids (aphid predator species are indicated with an asterisk *).

Article Snippet: A similar observation was previously reported in a series of mutant HIV-1 isolates resistant to GNA ( Galanthus nivalis ) and HHA ( Hippeastrum sp. hybrid) lectins, showing that the major amino acid mutations occur at several putative N -glycosylation sites NXT and NXS, and especially, at the ultimate T or S residues [ ].

Techniques: Multiple Displacement Amplification

Journal: International Journal of Molecular Sciences

Article Title: Overview of the Structure–Function Relationships of Mannose-Specific Lectins from Plants, Algae and Fungi

doi: 10.3390/ijms20020254

Figure Lengend Snippet: List of the mannose-specific lectins inhibiting HIV infection by binding to the viral gp120 envelope protein.

Article Snippet: A similar observation was previously reported in a series of mutant HIV-1 isolates resistant to GNA ( Galanthus nivalis ) and HHA ( Hippeastrum sp. hybrid) lectins, showing that the major amino acid mutations occur at several putative N -glycosylation sites NXT and NXS, and especially, at the ultimate T or S residues [ ].

Techniques: Infection, Binding Assay, Algae

Journal: Nature Communications

Article Title: Red blood cell mannoses as phagocytic ligands mediating both sickle cell anaemia and malaria resistance

doi: 10.1038/s41467-021-21814-z

Figure Lengend Snippet: Whole blood flow cytometry analysis of HbAA (normal hemoglobin) and HbSS (homozygous sickle cell hemoglobin) RBCs using fluorescently labeled plant lectins, detailed in “Methods” section. Vertical axes show normalized geometric mean fluorescence (gMFI). Symbols of terminal carbohydrates detected by plant lectins are indicated. Data shown as median +/− IQR, n = 7 per group for significant differences, 2 tailed Mann-Whitney p values shown, distinct samples measured once each, 3 separate experiments. Annotation uses conventional symbols for carbohydrates in accordance with http://www.functionalglycomics.org guidelines: purple diamond, sialic acids; yellow circle, galactose; blue square, N-acetyl glucosamine; green circle, mannose; red triangle, fucose. a Galanthus nivalis Agglutinin (GNA) lectin staining (red) of HbSS and HbAA RBCs, immunofluorescence, merged with the bright field. b MALDI-ToF mass spectra ( m / z versus relative intensity) for glycomic analysis of N-glycans from membrane ghosts from individual HbSS and HbAA donors. Red boxes indicate high mannose structures. Annotation uses conventional symbols for carbohydrates in accordance with http://www.functionalglycomics.org guidelines: purple diamond, sialic acids; yellow circle, galactose; blue square, N-acetyl glucosamine; green circle, mannose; red triangle, fucose. Only major structures are annotated for clarity. Full spectra from both HbSS and HbAA donors are shown in Supplementary Fig. c.

Article Snippet: GNA lectin/anti-spectrin stained RBCs were gravity sedimented onto poly-L-lysine treated chamber slides (LabTek).

Techniques: Flow Cytometry, Labeling, Fluorescence, MANN-WHITNEY, Staining, Immunofluorescence

Journal: Nature Communications

Article Title: Red blood cell mannoses as phagocytic ligands mediating both sickle cell anaemia and malaria resistance

doi: 10.1038/s41467-021-21814-z

Figure Lengend Snippet: a Normalized geometric mean fluorescence (gMFI) of Galanthus nivalis Agglutinin (GNA) lectin staining of RBCs from peripheral blood samples, comparing RBC from patients with sub-types of sickle cell disease (milder phenotypes: HbSC (compound heterozygotes for hemoglobin S with C) ( n = 34), HbS/beta-thal ( n = 8)(compound heterozygotes for hemoglobin S with β-thalassemia), HbSS microcytic ( n = 12))(compound heterozygotes for hemoglobin S homozygosity with α-thalassemia) and HbSS HPFH ( n = 3)) (compound heterozygotes for hemoglobin S homozygosity with hereditary persistence of fetal hemoglobin) versus healthy donors ( n = 45), sickle cell trait ( n = 57)(HbAS) and β-thalassemia ( n = 6). The dotted line indicates the 90th centile of GNA lectin binding within healthy samples. Data are shown as median +/− IQR, 2 tailed Mann–Whitney, p values shown, numerous (>20) experiments. 2 tailed p values relative to HbSS: HbAA, p = 3.9 × 10 −14 , HbAS, p = 3.9 × 10 −16 , HbSC, p = 1.8 × 10 −6 . Each data point derived from an independent RBC donor. No adjustments made for multiple comparisons. b Plot of RBC count against normalized GNA gMFI for sickle cell disease: HbS/B + and HbSC indicates compound heterozygosity for HbS with β-thalassemia and HbC respectively; Spearman’s rank correlation, p = 7.0 × 10 −23 , numerous (>20) experiments. Plots of: ( c ) RBC count versus serum lactate dehydrogenase (LDH), Spearman’s rank, d) RBC count vs GNA binding, Spearman’s rank, ( p = 5.2 × 10 −9 ) e LDH vs. GNA lectin binding; HbSS RBCs for which corresponding serum LDH values were available; Spearman’s rank correlation, numerous (>20) experiments. f Percentage phagocytosis of HbAA and HbSS RBCs by human monocyte-derived macrophages analyzed by microscopy. Mannan inhibition as shown. n = 4–7 independent RBC donors over two independent experiments. Data are shown as median +/− IQR, 2 tailed Mann–Whitney.

Article Snippet: GNA lectin/anti-spectrin stained RBCs were gravity sedimented onto poly-L-lysine treated chamber slides (LabTek).

Techniques: Fluorescence, Staining, Binding Assay, MANN-WHITNEY, Derivative Assay, Microscopy, Inhibition

Journal: Nature Communications

Article Title: Red blood cell mannoses as phagocytic ligands mediating both sickle cell anaemia and malaria resistance

doi: 10.1038/s41467-021-21814-z

Figure Lengend Snippet: a Peanut agglutinin (PNA) and Galanthus nivalis Agglutinin (GNA) lectin binding to normal hemoglobin (HbAA) RBCs with or without oxidation. Mannan blockade for GNA lectin binding shown in blue. 2 tailed Wilcoxon, paired data, PNA, n = 7; GNA, n = 8 biologically independent RBC donors over two independent experiments. b Immunofluorescence microscopy of GNA lectin/streptavidin (red) staining of healthy HbAA RBCs (above) and after the oxidative insult (below). c Normalized geometric mean fluorescence (gMFI) for binding analyzed by flow cytometry of murine Fc fusions with C-type lectins or sub-domains applied to oxidized versus undamaged RBCs. Mannan blockade of mannose receptor-carbohydrate recognition domain (MR-CRD) binding is shown in blue. MAH, macrophage antigen H. MR-CR, mannose receptor cysteine-rich domain. 2 tailed Wilcoxon, paired data, n = 7 biologically independent RBC donors over two independent experiments. d Immunofluorescence microscopy image of human monocyte-derived macrophages (HMDM) stained with DAPI (blue) and for mannose receptor (green) after incubation with oxidized HbAA RBCs, shown in magenta. e Percentage phagocytosis of oxidized RBCs by HMDM treated with human MR specific or scrambled siRNA. 2 tailed Mann–Whitney, n = 4 biologically independent RBC donors over two independent experiments. f Percentage phagocytosis of healthy or oxidized HbAA RBCs by HMDM with or without pre-blocking by mannan, chitin, or laminarin, 2 tailed Mann–Whitney, n = 4–8 biologically independent RBC donors over two independent experiments. No adjustments made for multiple comparisons. g As f but oxidized RBCs are blocked by MR-CRD blocking antibody 15.2 as indicated. 2 tailed Mann–Whitney, n = 6 biologically independent RBC donors over two independent experiments. h Percentage phagocytosis of sickle cell homozygote (HbSS) RBCs with or without pre-blocking by MR-CRD blocking antibody 15.2. 2 tailed Mann–Whitney, n = 5 biologically independent RBC donors over three independent experiments. i HbAA unblocked and HbSS unblocked or mannan and chitin blockade phagocytosis experiments as shown. 2 tailed Mann–Whitney, n = 4-7 biologically independent RBC donors. No adjustments made for multiple comparisons. All data are shown as median +/− IQR.

Article Snippet: GNA lectin/anti-spectrin stained RBCs were gravity sedimented onto poly-L-lysine treated chamber slides (LabTek).

Techniques: Binding Assay, Immunofluorescence, Microscopy, Staining, Fluorescence, Flow Cytometry, Derivative Assay, Incubation, MANN-WHITNEY, Blocking Assay

Journal: Nature Communications

Article Title: Red blood cell mannoses as phagocytic ligands mediating both sickle cell anaemia and malaria resistance

doi: 10.1038/s41467-021-21814-z

Figure Lengend Snippet: a Galanthus nivalis Agglutinin (GNA) lectin western blot from healthy (HbAA) and homozygote sickle hemoglobin (HbSS) ghosts. b Above are shown further GNA lectin western blots from HbAA and HBSS ghosts. The histogram below the blot shows the flow cytometrically measured surface GNA lectin staining values of the RBCs used to make the ghosts, with each bar corresponding to the cells used to make the western lane above. The r -value to the right of the 100 kDa size label is Spearman’s rank correlation coefficient between GNA lectin staining values and band intensities, both classified ordinally as high, medium, or low (post hoc analysis p = 1.3 × 10 −6 , n = 27 measurements from 22 individuals, no statistical adjustment made for other bands). c GNA lectin blot from HbSS ghosts: untreated (U), treated by PNGase (P) or Endo-H (E). d High exposure β-spectrin blot showing PNGase and partial/full (0.1×/1×) Endo-H digestion of two HbSS ghosts. e Lectin precipitation of healthy ghosts with GNA or Maackia amurensis Lectin II (MAL-II) lectins. No lectin control is also shown. Immunoblot with the β-spectrin specific antibody. f Super-resolution microscopy image of spectrin membrane skeleton (blue) from healthy (AA) and sickle cells (SS). Yellow clusters of GNA staining are overlaid. 3D image is sliced to reveal a single sheet of membrane skeleton network. g GNA lectin blot of spectrin released from HbSS ghosts after digestion with trypsin for one hour. Untreated (UT), Tx indicates the dilution factor of trypsin relative to spectrin material. h The amino acid sequence of α-spectrin showing peptide coverage (presence or absence of bar above the line) and intensity (proportional to the height of the bar) from proteomic analysis of F40 following chymotrypsin treatment. i GNA lectin blots showing Endo-H treatment of the 10 kDa concentrate from e under native or denaturing conditions (urea/SDS/2-mercaptoethanol) for 24 h. j 3D SIM super-resolution microscopy of surface GNA lectin binding and internal β-spectrin in HbSS RBC. Cells are first stained with GNA lectin (yellow), then permeabilized, and stained with anti-spectrin antibody (blue) ( k ).

Article Snippet: GNA lectin/anti-spectrin stained RBCs were gravity sedimented onto poly-L-lysine treated chamber slides (LabTek).

Techniques: Western Blot, Staining, Microscopy, Sequencing, Binding Assay

Journal: Nature Communications

Article Title: Red blood cell mannoses as phagocytic ligands mediating both sickle cell anaemia and malaria resistance

doi: 10.1038/s41467-021-21814-z

Figure Lengend Snippet: a Galanthus nivalis Agglutinin (GNA) lectin binding to healthy (HbAA) ( n = 18) versus sickle cell trait (HbAS) ( n = 21), compound heterozygotes for hemoglobins S and C (HbSC) ( n = 16), homozygotes for hemoglobin S (HbSS) ( n = 14) RBCs in response to oxidative stress, expressed as a ratio to mean of oxidized HbAA samples. Data are shown as median +/− IQR, 2 tailed Mann–Whitney. p values relative to HbAA: HbSC, p = 8.6 × 10 −6 , HbSS, p = 3.5 × 10 −5 . Each data point indicates an independent RBC donor over more than five independent experiments. b HbS percentage is plotted against elevation of GNA lectin binding in response to oxidative stress as in a . (**** p = 3.5 × 10 −7 ). HbAA, HbAS, and HbSC donor samples are shown as indicated. Spearman’s rank statistics shown. c) GNA binding to HbAA, HbAS, and HbSS RBCs in response to infection with P. falciparum . Values for rings, trophozoites, and schizonts are expressed as ratios relative to the uninfected gate. Linked one-way ANOVA analysis for HbAA ( p = 0.027), HbAS ( p = 9.5 × 10 5 ), HbSS (n.s. p = 0.192); Tukey’s multiple comparisons within each genotype. n = 27 independent RBC donors over five independent experiments. Data are shown as mean +/− SD. d Plot of relative GNA lectin binding for RBCs infected by schizonts against relative PfEMP1 expression. Hemoglobin phenotypes of donors as indicated, Spearman’s rank.

Article Snippet: GNA lectin/anti-spectrin stained RBCs were gravity sedimented onto poly-L-lysine treated chamber slides (LabTek).

Techniques: Binding Assay, MANN-WHITNEY, Infection, Expressing