Journal: Nucleic Acids Research

Article Title: InterPro in 2022

doi: 10.1093/nar/gkac993

Figure Lengend Snippet: Release version and number of member database signatures integrated into InterPro version 90.0

Article Snippet: InterPro combines 13 protein signature databases into one central resource: CATH-Gene3D , the Conserved Domains Database (CDD) , HAMAP , PANTHER , Pfam , PIRSF , PRINTS , PROSITE Patterns , PROSITE Profiles , SMART , the Structure–Function Linkage Database (SFLD) , SUPERFAMILY ( ) and TIGRFAMs ( ).

Techniques:

Journal: Nature Communications

Article Title: Unraveling function and diversity of bacterial lectins in the human microbiome

doi: 10.1038/s41467-022-29949-3

Figure Lengend Snippet: a Cbeg5 functional domains (InterPro) and protein constructs created for bioactivity assays (box). b Cbeg4 and Cbeg5 protein constructs were generated containing only the CBM domain and a GFP tag. The Cbeg5 CBM protein (CBM5) and the Cbeg4 CBM protein (CBM4) were assessed in a binding assay against a panel of 609 N-linked and O-linked glycans (Functional Glycomics Consortium). CBM5 and CBM4 were assayed at 5 μg ml −1 and 50 μg ml −1 in 6 replicates and glycan binding quantified as relative fluorescent units (RFU). Dot plots of CBM4 and CBM5 glycan binding screens at a concentration of 5 μg ml −1 (mean ± s.e.m). c Plot of CBM4 vs CBM5 glycan binding normalized to the top glycan binder (set at RFU = 1). Protein concentration is 50 μg ml −1 . d The structure of the top bound glycan (red dot in b , c ) is pictured. Analysis of CBM4/CBM5 bound and unbound glycans identifies a conserved binding motif outlined in red dotted line (glycopattern). e A Glyconnect search of glycomics datasets for samples containing the conserved N-linked glycan motif was performed. Glycan structures containing the conserved binding motif were most commonly identified in human leukocyte datasets (11/12 glycan structures). Four glycan structures were shared by human leukocyte, urine and kidney datasets. One glycan structure was exclusive to a kidney dataset.

Article Snippet: Fig. 1 Cbeg4 and Cbeg5 glycomics. a Cbeg5 functional domains (InterPro) and protein constructs created for bioactivity assays (box). b Cbeg4 and Cbeg5 protein constructs were generated containing only the CBM domain and a GFP tag.

Techniques: Functional Assay, Construct, Generated, Binding Assay, Glycoproteomics, Concentration Assay, Protein Concentration

Journal: Nature Communications

Article Title: Unraveling function and diversity of bacterial lectins in the human microbiome

doi: 10.1038/s41467-022-29949-3

Figure Lengend Snippet: Fresh PBMC were isolated from healthy patients and exposed to Cbeg5, Fn5 and PBS for 6 h and analyzed by CyTOF with a panel of 16 cell surface and 10 cytokine markers. a Cell surface markers distinguish major immune cell populations (A–K) that correlate to groups identified by tSNE analysis (Supplementary Fig. ). b Fold induction of 10 cytokines across cell populations A–K from ( a ). Fold induction is calculated relative to PBS control. Cytokine responses > 3-fold to > 100-fold are marked with gray-shaded boxes. Proteins assayed at 100 nM or 1000 nM as indicated. (Data is a single experiment. PBS data is normalized to a replicate experiment with PBS). c Bar graph (mean ± s.e.m) of percent positive cells for each cytokine in specific cell populations in response to Cbeg5 or FN3 control at 500 nM concentration ( N = 5 from three independent experiments, unpaired, two-sided Mann–Whitney test comparing Cbeg5 to Fn3 control in each cell population) *** p < 0.001 ** p < 0.01 * p < 0.05.

Article Snippet: Fig. 1 Cbeg4 and Cbeg5 glycomics. a Cbeg5 functional domains (InterPro) and protein constructs created for bioactivity assays (box). b Cbeg4 and Cbeg5 protein constructs were generated containing only the CBM domain and a GFP tag.

Techniques: Isolation, Control, Concentration Assay, MANN-WHITNEY

Journal: Nature Communications

Article Title: Unraveling function and diversity of bacterial lectins in the human microbiome

doi: 10.1038/s41467-022-29949-3

Figure Lengend Snippet: Fresh colon tissue was obtained from patients at the time of surgery for isolation of lamina propria leukocytes (LPL). a LPL cells were exposed to Cbeg5 or Fn5, analyzed by CyTOF and the data evaluated by SPADE. A SPADE plot for Cbeg5 induced TNF-α production from intestinal leukocytes. Fold induction calculated relative to Fn5. Cbeg5 induces cytokine production in two myeloid cell clusters (CD19 − CD56 − CD66b − HLADR + ) that are CD14 hi or CD1c hi . Clusters are annotated by manual gating of indicated cell surface markers. b Cells from the CD14 cluster and CD1c cluster were analyzed to identify CD14 + CD4 + cells (monocyte derived macrophage markers) and CD14 + CD1c + cells (monocyte derived dendritic cell markers). Bar graphs depict Cbeg5 induced cytokine production in CD14 + CD4 + and CD14 + CD1c + cells (mean ± s.e.m, N = 2, two independent experiments). c Isolated blood CD14 + monocytes were differentiated in vitro into macrophages (MDM) and dendritic cells (moDC) and exposed to 500 nM concentrations of Cbeg5, Fn5 and PBS. Cytokines, chemokines, growth factors and adhesion molecules were analyzed by Luminex (Procarta Plex TM ). Heatmap of cytokine production with > 3-fold to > 100-fold induction shown with gray shaded boxes (induction relative to PBS control, data is mean of triplicate experiments for CD14 + monocytes and duplicate experiments for mMAC, mDC). d Bar graph of cytokine or chemokine and adhesion molecule production for cell populations in response to Cbeg5. Bar graph colors are derived from labels used in the heatmap ( c ).

Article Snippet: Fig. 1 Cbeg4 and Cbeg5 glycomics. a Cbeg5 functional domains (InterPro) and protein constructs created for bioactivity assays (box). b Cbeg4 and Cbeg5 protein constructs were generated containing only the CBM domain and a GFP tag.

Techniques: Isolation, Derivative Assay, In Vitro, Luminex, Control

Journal: Nature Communications

Article Title: Unraveling function and diversity of bacterial lectins in the human microbiome

doi: 10.1038/s41467-022-29949-3

Figure Lengend Snippet: Wild-type 8-week C57BL/6 mice were colonized with E. coli engineered to express Cbeg5 (Treatment, EC:Cbeg5) or E. coli with an empty vector (Control, EC:Con). After 1 week of colonization the colon was analyzed for inflammatory changes by histology ( a ) and for change in immune cell populations by flow cytometry ( b , c ). a No difference was observed in colon histologic inflammatory indices (one representative image, Supplementary Fig. ). b After gating for singlets and live cells (Aqua LIVE/DEAD) the gating schema is shown for identification of macrophages MHCII + CD11c + CD64 + CD11b + and monocytes MHCII - Lyc + CD11b + . c There was a significant increase in lamina propria MHCII + CD11c + CD64 + CD11b + macrophages and a decrease in MHCII − Lyc + CD11b + monocytes in the treatment group ( N = 16 (8 M/8 F) Treatment, N = 18 (9 M/9 F) Control. Data represents 4 independent experiments. Error bars are mean ± s.e.m. Control and treatment populations compared using an unpaired, two-sided Mann–Whitney test).

Article Snippet: Fig. 1 Cbeg4 and Cbeg5 glycomics. a Cbeg5 functional domains (InterPro) and protein constructs created for bioactivity assays (box). b Cbeg4 and Cbeg5 protein constructs were generated containing only the CBM domain and a GFP tag.

Techniques: Plasmid Preparation, Control, Flow Cytometry, MANN-WHITNEY