gp38  (Miltenyi Biotec)

Journal: JCI Insight

Article Title: Molecular control of PDPN hi macrophage subset induction by ADAP as a host defense in sepsis

doi: 10.1172/jci.insight.186456

Figure Lengend Snippet: ( A ) Gating strategy for FACS of CD11b + F4/80 + and CD11b + F4/80 – cells from the peritoneal cells of WT and Adap –/– mice 18 hours after E . coli injection (2 × 10 7 CFU, i.p.). RNA-Seq analysis identified DEGs (log 2 [fold change] ≤ −1/ ≥ 1, 5% FDR) in sorted cell populations. ( B ) Schematic of RNA-Seq analysis of WT and Adap –/– PMs exposed to LPS (100 ng/mL) for 12 hours in vitro (upper panel). Venn diagram of DEGs in WT versus Adap –/– PMs from E. coli –infected mice (209 DEGs) and WT versus Adap –/– PMs treated with LPS (166 DEGs), revealing 13 overlapping DEGs (lower panel). ( C ) Heatmap and hierarchical clustering of the 13 overlapping DEGs from B . ( D – F ) PDPN expression in WT and Adap –/– PMs with or without LPS stimulation (100 ng/mL) was analyzed by Western blotting ( D ), flow cytometry ( E ), and qPCR ( F , n = 3 each, 2-way ANOVA, Šidák’s multiple-comparison test). Relative mRNA levels were normalized to Hprt . ( G ) Immunoblot analysis of PDPN in WT or ADAP-knockdown (ADAP KD ) RAW264.7 cells stimulated with or without LPS (100 ng/mL) for the indicated time points. ( H ) Pdpn mRNA expression in WT and Adap –/– iBMMs with or without LPS stimulation (100 ng/mL, 12 hours), analyzed by qPCR ( n = 3 each, 2-way ANOVA, Šidák’s multiple-comparison test). Relative mRNA levels were normalized to Hprt . ( I ) ADAP KD RAW264.7 cells were reconstituted via lentiviral transduction with ADAP to induce overexpression (OE) or with an empty vector (EV) as a control, followed by mock treatment or stimulation with LPS (100 ng/mL) for 24 hours. Whole cell lysates were prepared and subjected to Western blot analysis of PDPN. α-Tubulin blots are derived from the same samples run contemporaneously in parallel gels.

Article Snippet: The antibodies used in this study are listed as follows: rabbit anti-ADAP (MilliporeSigma, 07-546), rabbit anti–α-tubulin (Abcam, ab4074), rat anti-PDPN (Abcam, ab256559), rabbit anti-Stat3 (Proteintech, 10253-2-AP), rabbit anti–p-Stat3 (tyr705) (CST, 9131), mouse anti–p-tyrosine (p-Tyr 100 ) (CST, 9411), HRP-conjugated goat anti-rat IgG (H+L) (ABclonal, AS028), HRP-conjugated goat anti–rabbit IgG (H+L) (CST, 7074), PE-Cy7–conjugated anti–mouse CD45 (BD Bioscience, 552848), FITC-conjugated anti–mouse CD11b (BD Bioscience, 553310), Alexa Fluor 647–conjugated anti–mouse F4/80 (BD Bioscience, 565853), PE anti–mouse PDPN (BD Bioscience, 566390), PE-conjugated anti–mouse Ly-6G (BioLegend, 127608), Alexa Fluor 647–conjugated anti–mouse PDPN (BioLegend, 156204), InVivoMAb polyclonal Syrian hamster IgG (BioXCell, BE0087), and InVivoMAb anti–mouse PDPN (gp38) (BioXCell, BE0236).

Techniques: Injection, RNA Sequencing, In Vitro, Infection, Expressing, Western Blot, Flow Cytometry, Comparison, Knockdown, Transduction, Over Expression, Plasmid Preparation, Control, Derivative Assay

Journal: JCI Insight

Article Title: Molecular control of PDPN hi macrophage subset induction by ADAP as a host defense in sepsis

doi: 10.1172/jci.insight.186456

Figure Lengend Snippet: ( A ) Vertical scatter plots summarizing the mRNA expression levels of Adap , Pdpn , and Il6 as measured by qPCR analysis of peritoneal cells from WT mice 18 hours after injection of saline or E . coli (2 × 10 7 CFU, i.p.) ( n = 4 each, unpaired t test). Relative mRNA levels were normalized to Hprt . ( B ) Vertical scatter plots showing the expression of Adap and Pdpn genes (expressed as RNA-Seq reads) extracted from RNA-Seq data of peritoneal cells from LPS-induced peritonitis in mice ( n = 6 each, unpaired t test). ( C ) The expression of Adap and Pdpn in the peritoneal cells of E . coli –infected mice was analyzed using a published database (GEO accession no. GSE34114, naive 2 hours, n = 2; naive 18 hours, n = 3; E . coli 1 hour, n = 2; E . coli 2 hours, n = 2; E . coli 18 hours, n = 3). Gene expression was analyzed in GEO2R. ( D and E ) Peritoneal exudate cells were isolated from WT, Adap –/– , and Skap1 –/– mice 18 hours after injection of E . coli (2 × 10 7 CFU, i.p.), and CD11b + F4/80 + PDPN hi macrophages were analyzed by flow cytometry. Left panels: Representative contour plots showing the frequency of PDPN hi macrophages in the peritoneal cavity of WT, Adap –/– , and Skap1 –/– mice at 18 hours after E . coli injection. Right panels: Bar graphs showing the percentage of PDPN hi macrophages ( D , n = 6 each; E , n = 3 each; unpaired t test).

Article Snippet: The antibodies used in this study are listed as follows: rabbit anti-ADAP (MilliporeSigma, 07-546), rabbit anti–α-tubulin (Abcam, ab4074), rat anti-PDPN (Abcam, ab256559), rabbit anti-Stat3 (Proteintech, 10253-2-AP), rabbit anti–p-Stat3 (tyr705) (CST, 9131), mouse anti–p-tyrosine (p-Tyr 100 ) (CST, 9411), HRP-conjugated goat anti-rat IgG (H+L) (ABclonal, AS028), HRP-conjugated goat anti–rabbit IgG (H+L) (CST, 7074), PE-Cy7–conjugated anti–mouse CD45 (BD Bioscience, 552848), FITC-conjugated anti–mouse CD11b (BD Bioscience, 553310), Alexa Fluor 647–conjugated anti–mouse F4/80 (BD Bioscience, 565853), PE anti–mouse PDPN (BD Bioscience, 566390), PE-conjugated anti–mouse Ly-6G (BioLegend, 127608), Alexa Fluor 647–conjugated anti–mouse PDPN (BioLegend, 156204), InVivoMAb polyclonal Syrian hamster IgG (BioXCell, BE0087), and InVivoMAb anti–mouse PDPN (gp38) (BioXCell, BE0236).

Techniques: Expressing, Injection, Saline, RNA Sequencing, Infection, Gene Expression, Isolation, Flow Cytometry

Journal: JCI Insight

Article Title: Molecular control of PDPN hi macrophage subset induction by ADAP as a host defense in sepsis

doi: 10.1172/jci.insight.186456

Figure Lengend Snippet: ( A ) RNA-Seq analysis of CD11b + F4/80 + PDPN hi and CD11b + F4/80 + PDPN lo PMs sorted from WT mice 18 hours after E . coli injection (2 × 10 7 CFU, i.p.), showing the numbers of upregulated and downregulated DEGs (log 2 [fold change] ≤ −1/ ≥ 1, 5% FDR). ( B ) Top 10 KEGG pathways identified from DEGs in A by KEGG enrichment analysis. ( C ) Heatmaps showing the DEGs of the chemokine-related genes in CD11b + F4/80 + PDPN hi PMs, compared with CD11b + F4/80 + PDPN lo , grouped by hierarchical clustering analysis of the RNA-Seq data. ( D and E ) qPCR analysis of M2-related chemokine DEGs ( D ) and polarization markers ( E ) in PDPN hi versus PDPN lo PMs from WT septic mice ( n = 4 each, unpaired t test). Relative mRNA levels were normalized to Hprt . ( F ) Flow cytometric analysis of E . coli –GFP uptake by CD11b + F4/80 + PDPN lo and CD11b + F4/80 + PDPN hi PMs in LPS-treated septic WT mice. GFP fluorescence (mean fluorescence intensity) was compared ( n = 3 each, unpaired t test). ( G ) GSEA histogram for the “phagosome” gene set in PDPN hi versus PDPN lo PMs. The normalized enrichment score (NES) and FDR q value are indicated. ( H ) The mRNA levels of Cd36 and Marco in PDPN hi and PDPN lo PMs from WT septic mice were determined using qPCR ( n = 4 each, unpaired t test). Relative mRNA levels were normalized to Hprt . ( I ) Kaplan-Meier survival analysis of WT and Adap –/– mice injected with anti-PDPN blocking antibodies (100 μg/mouse, i.v.) 2 hours after E . coli infection (2 × 10 7 CFU, i.p.) (WT E . coli , n = 15; Adap –/– E . coli , n = 10; WT E . coli –anti-PDPN, n = 16; Adap –/– E . coli –anti-PDPN, n = 11). Log-rank test was used to compare survival curve.

Article Snippet: The antibodies used in this study are listed as follows: rabbit anti-ADAP (MilliporeSigma, 07-546), rabbit anti–α-tubulin (Abcam, ab4074), rat anti-PDPN (Abcam, ab256559), rabbit anti-Stat3 (Proteintech, 10253-2-AP), rabbit anti–p-Stat3 (tyr705) (CST, 9131), mouse anti–p-tyrosine (p-Tyr 100 ) (CST, 9411), HRP-conjugated goat anti-rat IgG (H+L) (ABclonal, AS028), HRP-conjugated goat anti–rabbit IgG (H+L) (CST, 7074), PE-Cy7–conjugated anti–mouse CD45 (BD Bioscience, 552848), FITC-conjugated anti–mouse CD11b (BD Bioscience, 553310), Alexa Fluor 647–conjugated anti–mouse F4/80 (BD Bioscience, 565853), PE anti–mouse PDPN (BD Bioscience, 566390), PE-conjugated anti–mouse Ly-6G (BioLegend, 127608), Alexa Fluor 647–conjugated anti–mouse PDPN (BioLegend, 156204), InVivoMAb polyclonal Syrian hamster IgG (BioXCell, BE0087), and InVivoMAb anti–mouse PDPN (gp38) (BioXCell, BE0236).

Techniques: RNA Sequencing, Injection, Fluorescence, Blocking Assay, Infection

Journal: JCI Insight

Article Title: Molecular control of PDPN hi macrophage subset induction by ADAP as a host defense in sepsis

doi: 10.1172/jci.insight.186456

Figure Lengend Snippet: ( A – D ) WT PMs were pretreated with resatorvid (0.1, 0.5, and 1 μM), IKK-16 (0.1, 0.3, and 0.5 μM), Bay 11-7085 (0.5, 1, and 5 μM), SB203580 (1, 5, and 10 μM), or SP600125 (1, 10, and 20 μM) for 1 hour, followed by LPS stimulation (100 ng/mL) for 24 hours ( A , C , and D ) or 12 hours ( B ). ADAP and PDPN expression was analyzed by Western blotting and qPCR ( B , n = 3 each, 1-way ANOVA, Tukey’s multiple-comparison test). Relative mRNA levels were normalized to Hprt . α-Tubulin blots are derived from the same samples run contemporaneously in parallel gels ( D ). ( E ) RAW264.7 cells transduced with ADAP were treated with LPS (100 ng/mL, 24 hours) in the presence of IKK-16 (0.5 μM). ADAP and PDPN expression was analyzed by Western blotting. α-Tubulin blots are derived from the same samples run contemporaneously in parallel gels. ( F ) Schematic of kinase inhibitors screening and summary of inhibitors blocking LPS-induced PDPN upregulation. ( G ) WT PMs pretreated with ibrutinib (1, 5, and 10 μM) or dasatinib (0.01, 0.1, and 1 μM) for 1 hour were stimulated with LPS (100 ng/mL, 24 hours). PDPN and ADAP expression was assessed by Western blotting. ( H ) MS confirmed LPS-induced (100 ng/mL, 1 hour) ADAP phosphorylation at Y 571 in PMs. MS/MS spectra of the phosphorylated peptide (TTAVEIDYDSLKR) are shown. ( I ) ADAP KD RAW264.7 cells reconstituted with empty vector (EV), ADAP-WT, or ADAP (Y571F) were stimulated with LPS (100 ng/mL, 24 hours). PDPN and ADAP expression was analyzed by Western blotting. ( J ) CD11b + F4/80 + PDPN hi (C2) and CD11b + F4/80 + PDPN lo (C1) PMs were sorted from WT septic mice 18 hours after E . coli injection (2 × 10 7 CFU, i.p.). Lysates were immunoprecipitated with anti-ADAP and immunoblotted for p-Tyr 100 and ADAP.

Article Snippet: The antibodies used in this study are listed as follows: rabbit anti-ADAP (MilliporeSigma, 07-546), rabbit anti–α-tubulin (Abcam, ab4074), rat anti-PDPN (Abcam, ab256559), rabbit anti-Stat3 (Proteintech, 10253-2-AP), rabbit anti–p-Stat3 (tyr705) (CST, 9131), mouse anti–p-tyrosine (p-Tyr 100 ) (CST, 9411), HRP-conjugated goat anti-rat IgG (H+L) (ABclonal, AS028), HRP-conjugated goat anti–rabbit IgG (H+L) (CST, 7074), PE-Cy7–conjugated anti–mouse CD45 (BD Bioscience, 552848), FITC-conjugated anti–mouse CD11b (BD Bioscience, 553310), Alexa Fluor 647–conjugated anti–mouse F4/80 (BD Bioscience, 565853), PE anti–mouse PDPN (BD Bioscience, 566390), PE-conjugated anti–mouse Ly-6G (BioLegend, 127608), Alexa Fluor 647–conjugated anti–mouse PDPN (BioLegend, 156204), InVivoMAb polyclonal Syrian hamster IgG (BioXCell, BE0087), and InVivoMAb anti–mouse PDPN (gp38) (BioXCell, BE0236).

Techniques: Expressing, Western Blot, Comparison, Derivative Assay, Transduction, Blocking Assay, Tandem Mass Spectroscopy, Plasmid Preparation, Injection, Immunoprecipitation

Journal: JCI Insight

Article Title: Molecular control of PDPN hi macrophage subset induction by ADAP as a host defense in sepsis

doi: 10.1172/jci.insight.186456

Figure Lengend Snippet: ( A ) PDPN expression in PMs pretreated with BP-1-102 (0.5, 1, and 5 μM) or fludarabine (10, 50, and 100 μM) for 1 hour and stimulated with LPS (100 ng/mL, 24 hours), analyzed by Western blotting and densitometry normalized to α-tubulin ( n = 6 each, 1-way ANOVA, Tukey’s multiple-comparison test). ( B ) STAT3 binding motifs in the Pdpn promoter and CUT & RUN qPCR showing STAT3 enrichment at site 2 in untreated or LPS-treated PMs (100 ng/mL, 1 hour) ( n = 3 each, 2-way ANOVA, Tukey’s multiple-comparison test). ( C ) Luciferase activity of Pdpn -WT-Luc or Pdpn -Mut-Luc in WT or ADAP KD RAW264.7 cells following LPS stimulation (100 ng/mL, 6 hours) ( n = 3 each, 2-way ANOVA, Tukey’s multiple-comparison test). ( D ) EMSA showing STAT3-DNA binding in nuclear extracts of WT or ADAP KD RAW264.7 cells after LPS stimulation (100 ng/mL, 1 hour). ( E ) Flow cytometry of CD11b + F4/80 + PDPN hi PMs in WT mice treated with colivelin (1 mg/kg, i.p.) after E . coli infection (2 × 10 7 CFU, i.p.) ( n = 5 each, unpaired t test). ( F and G ) Western blot of PDPN, p-STAT3, and STAT3 in LPS-treated PMs (100 ng/mL) with or without mTOR inhibitors or rapamycin (1 μM). ( H ) LPS-stimulated PMs (100 ng/mL, 1 hour) pretreated with rapamycin (1 μM, 1 hour) were immunoprecipitated with anti-ADAP and immunoblotted for STAT3. ( I ) PDPN expression in WT and Adap –/– PMs treated with LPS (10 ng/mL) and MHY1485 (10 μM, 24 hours) was analyzed by Western blotting. ( J and K ) Kaplan-Meier survival curves and bacterial burden in WT mice treated with colivelin (1 mg/kg, i.p.) after E . coli infection (5 × 10 7 CFU, i.p.), showing survival ( n = 15 each, log-rank test) and bacterial load in peritoneal lavage fluid and blood at 18 hours ( n = 5 each; unpaired t test).

Article Snippet: The antibodies used in this study are listed as follows: rabbit anti-ADAP (MilliporeSigma, 07-546), rabbit anti–α-tubulin (Abcam, ab4074), rat anti-PDPN (Abcam, ab256559), rabbit anti-Stat3 (Proteintech, 10253-2-AP), rabbit anti–p-Stat3 (tyr705) (CST, 9131), mouse anti–p-tyrosine (p-Tyr 100 ) (CST, 9411), HRP-conjugated goat anti-rat IgG (H+L) (ABclonal, AS028), HRP-conjugated goat anti–rabbit IgG (H+L) (CST, 7074), PE-Cy7–conjugated anti–mouse CD45 (BD Bioscience, 552848), FITC-conjugated anti–mouse CD11b (BD Bioscience, 553310), Alexa Fluor 647–conjugated anti–mouse F4/80 (BD Bioscience, 565853), PE anti–mouse PDPN (BD Bioscience, 566390), PE-conjugated anti–mouse Ly-6G (BioLegend, 127608), Alexa Fluor 647–conjugated anti–mouse PDPN (BioLegend, 156204), InVivoMAb polyclonal Syrian hamster IgG (BioXCell, BE0087), and InVivoMAb anti–mouse PDPN (gp38) (BioXCell, BE0236).

Techniques: Expressing, Western Blot, Comparison, Binding Assay, Luciferase, Activity Assay, Flow Cytometry, Infection, Immunoprecipitation

Journal: JCI Insight

Article Title: Molecular control of PDPN hi macrophage subset induction by ADAP as a host defense in sepsis

doi: 10.1172/jci.insight.186456

Figure Lengend Snippet: ADAP is upregulated in sepsis and serves as an LPS stimulus–responsive protein that governs the upregulation of PDPN to form a distinct PDPN hi PM subset during sepsis, which displays a phenotype closely akin to M2 macrophages with enhanced phagocytic activity and provides enhanced protection against sepsis in the host. Mechanistically, while TLR4 signaling stimulates the upregulation of ADAP via the NF-κB pathway, TLR4-triggered phosphorylation of ADAP at Y 571 by BTK primes the subsequent direct phosphorylation of STAT3 by mTOR, which transactivates PDPN transcription.

Article Snippet: The antibodies used in this study are listed as follows: rabbit anti-ADAP (MilliporeSigma, 07-546), rabbit anti–α-tubulin (Abcam, ab4074), rat anti-PDPN (Abcam, ab256559), rabbit anti-Stat3 (Proteintech, 10253-2-AP), rabbit anti–p-Stat3 (tyr705) (CST, 9131), mouse anti–p-tyrosine (p-Tyr 100 ) (CST, 9411), HRP-conjugated goat anti-rat IgG (H+L) (ABclonal, AS028), HRP-conjugated goat anti–rabbit IgG (H+L) (CST, 7074), PE-Cy7–conjugated anti–mouse CD45 (BD Bioscience, 552848), FITC-conjugated anti–mouse CD11b (BD Bioscience, 553310), Alexa Fluor 647–conjugated anti–mouse F4/80 (BD Bioscience, 565853), PE anti–mouse PDPN (BD Bioscience, 566390), PE-conjugated anti–mouse Ly-6G (BioLegend, 127608), Alexa Fluor 647–conjugated anti–mouse PDPN (BioLegend, 156204), InVivoMAb polyclonal Syrian hamster IgG (BioXCell, BE0087), and InVivoMAb anti–mouse PDPN (gp38) (BioXCell, BE0236).

Techniques: Activity Assay

Journal: Cell reports

Article Title: Crimean-Congo hemorrhagic fever survivors elicit protective non-neutralizing antibodies that target 11 overlapping regions on glycoprotein GP38

doi: 10.1016/j.celrep.2024.114502

Figure Lengend Snippet: (A) Matrix of percent sequence identity of GP38 amino acid residues (AA) across six CCHFV isolates. (B) Single concentration BLI binding analysis of 188 antibodies to the six rGP38 proteins as a whole panel (top) and broken down by bin (bottom). Shades of green represent the number of rGP38 proteins bound by a single antibody (from 0 in gray to 6 in darkest green). Total number of mAbs is indicated in the circular diagram and total mAbs from each bin are indicated above the bar graph. (C) Carterra system HT-SPR binding analysis of six lead antibody candidates binding to six rGP38 proteins. The highest binding affinities are in dark green and the lowest binding affinities are in white. Calculated K D values appear in each rectangle of the heatmap; for samples that were off-rate limited, K D values are denoted as < the calculated K D . The one interaction for which a curve could not be fit is denoted as P.F.

Article Snippet: Cells that bound the non-competing anti-GP38 antibodies were sorted and plated on complete minimal media glucose agar plates minus tryptophan (Teknova).

Techniques: Sequencing, Concentration Assay, Binding Assay

Journal: Cell reports

Article Title: Crimean-Congo hemorrhagic fever survivors elicit protective non-neutralizing antibodies that target 11 overlapping regions on glycoprotein GP38

doi: 10.1016/j.celrep.2024.114502

Figure Lengend Snippet: (A) Yeast-based mapping strategy of select antibodies to identify critical binding residues on GP38. The percentage of antibody binding retained by each GP38 variant is colored according to the key. Critical residues are defined as mutations that led to a binding disruption of 75% or more and are colored by the assigned antigenic site. (B) Yeast-based critical residues mapped on the surface of GP38: bin I (blue, residues Val385, Pro388), bin II (green, residues Gly371, Leu374, Ile375, Lys404, Lys488, Leu499), bin III (yellow, residues Ser428-Ala429, Asp444-Asp446, Lys474-Leu475, Asp477), bin IV (orange, residues Ile253-Leu255, Leu257, Lys262, Gly266, Glu277, Glu281), bin V (red, residues Glu285, Arg289, Gly292). (C) Composite structure of GP38 bound with representative antibodies. GP38 is shown as a rainbow ribbon and Fabs as molecular surfaces. Heavy chains are colored to represent the five non-overlapping bins, and light chains are white. Black dashed lines highlight the vertical alignment of Fabs along one plane (left) and the opposing binding directions to another plane (right).

Article Snippet: Cells that bound the non-competing anti-GP38 antibodies were sorted and plated on complete minimal media glucose agar plates minus tryptophan (Teknova).

Techniques: Binding Assay, Variant Assay, Disruption

Journal: Cell reports

Article Title: Crimean-Congo hemorrhagic fever survivors elicit protective non-neutralizing antibodies that target 11 overlapping regions on glycoprotein GP38

doi: 10.1016/j.celrep.2024.114502

Figure Lengend Snippet: (A) Crystal structure of GP38 bound with ADI-46143 (bin I, blue) with heavy-chain interactions (top) and light-chain interactions (bottom). (B) Cryo-EM structure of GP38 bound with ADI-58048 (bin II, green, left) and ADI-46152 (bin IV+V, red, right). Heavy-chain interactions (top left, top right) and light-chain interactions (bottom left, bottom right) are shown in the insets. (C) Crystal structure of GP38 bound with c13G8 (bin IV+V, red) with heavy-chain interactions (top) and light-chain interactions (bottom). For all panels, heavy chains are colored, light chains are gray, polar interactions are indicated by black dashed lines, and GP38 residues are labeled in white text with a black outline.

Article Snippet: Cells that bound the non-competing anti-GP38 antibodies were sorted and plated on complete minimal media glucose agar plates minus tryptophan (Teknova).

Techniques: Cryo-EM Sample Prep, Labeling

Journal: Cell reports

Article Title: Crimean-Congo hemorrhagic fever survivors elicit protective non-neutralizing antibodies that target 11 overlapping regions on glycoprotein GP38

doi: 10.1016/j.celrep.2024.114502

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Cells that bound the non-competing anti-GP38 antibodies were sorted and plated on complete minimal media glucose agar plates minus tryptophan (Teknova).

Techniques: Derivative Assay, Virus, Recombinant, Protease Inhibitor, Reverse Transcription, Membrane, Cell Isolation, Mutagenesis, Expressing, Plasmid Preparation, Software, Affinity Column, Sequencing, Transfection, Electron Microscopy

Journal: Cell reports

Article Title: Crimean-Congo hemorrhagic fever survivors elicit protective non-neutralizing antibodies that target 11 overlapping regions on glycoprotein GP38

doi: 10.1016/j.celrep.2024.114502

Figure Lengend Snippet: (A) Matrix of percent sequence identity of GP38 amino acid residues (AA) across six CCHFV isolates. (B) Single concentration BLI binding analysis of 188 antibodies to the six rGP38 proteins as a whole panel (top) and broken down by bin (bottom). Shades of green represent the number of rGP38 proteins bound by a single antibody (from 0 in gray to 6 in darkest green). Total number of mAbs is indicated in the circular diagram and total mAbs from each bin are indicated above the bar graph. (C) Carterra system HT-SPR binding analysis of six lead antibody candidates binding to six rGP38 proteins. The highest binding affinities are in dark green and the lowest binding affinities are in white. Calculated K D values appear in each rectangle of the heatmap; for samples that were off-rate limited, K D values are denoted as < the calculated K D . The one interaction for which a curve could not be fit is denoted as P.F.

Article Snippet: Gene fragments (Integrated DNA Technologies) of each isolate’s MLD-GP38 sequence encoding for residues 1–515, as denoted by CCHFV IbAr10200 strain GPC numbering, were codon-optimized for human cell expression (GenScript Codon Optimization Tool).

Techniques: Sequencing, Concentration Assay, Binding Assay

Journal: Cell reports

Article Title: Crimean-Congo hemorrhagic fever survivors elicit protective non-neutralizing antibodies that target 11 overlapping regions on glycoprotein GP38

doi: 10.1016/j.celrep.2024.114502

Figure Lengend Snippet: (A) Yeast-based mapping strategy of select antibodies to identify critical binding residues on GP38. The percentage of antibody binding retained by each GP38 variant is colored according to the key. Critical residues are defined as mutations that led to a binding disruption of 75% or more and are colored by the assigned antigenic site. (B) Yeast-based critical residues mapped on the surface of GP38: bin I (blue, residues Val385, Pro388), bin II (green, residues Gly371, Leu374, Ile375, Lys404, Lys488, Leu499), bin III (yellow, residues Ser428-Ala429, Asp444-Asp446, Lys474-Leu475, Asp477), bin IV (orange, residues Ile253-Leu255, Leu257, Lys262, Gly266, Glu277, Glu281), bin V (red, residues Glu285, Arg289, Gly292). (C) Composite structure of GP38 bound with representative antibodies. GP38 is shown as a rainbow ribbon and Fabs as molecular surfaces. Heavy chains are colored to represent the five non-overlapping bins, and light chains are white. Black dashed lines highlight the vertical alignment of Fabs along one plane (left) and the opposing binding directions to another plane (right).

Article Snippet: Gene fragments (Integrated DNA Technologies) of each isolate’s MLD-GP38 sequence encoding for residues 1–515, as denoted by CCHFV IbAr10200 strain GPC numbering, were codon-optimized for human cell expression (GenScript Codon Optimization Tool).

Techniques: Binding Assay, Variant Assay, Disruption

Journal: Cell reports

Article Title: Crimean-Congo hemorrhagic fever survivors elicit protective non-neutralizing antibodies that target 11 overlapping regions on glycoprotein GP38

doi: 10.1016/j.celrep.2024.114502

Figure Lengend Snippet: (A) Crystal structure of GP38 bound with ADI-46143 (bin I, blue) with heavy-chain interactions (top) and light-chain interactions (bottom). (B) Cryo-EM structure of GP38 bound with ADI-58048 (bin II, green, left) and ADI-46152 (bin IV+V, red, right). Heavy-chain interactions (top left, top right) and light-chain interactions (bottom left, bottom right) are shown in the insets. (C) Crystal structure of GP38 bound with c13G8 (bin IV+V, red) with heavy-chain interactions (top) and light-chain interactions (bottom). For all panels, heavy chains are colored, light chains are gray, polar interactions are indicated by black dashed lines, and GP38 residues are labeled in white text with a black outline.

Article Snippet: Gene fragments (Integrated DNA Technologies) of each isolate’s MLD-GP38 sequence encoding for residues 1–515, as denoted by CCHFV IbAr10200 strain GPC numbering, were codon-optimized for human cell expression (GenScript Codon Optimization Tool).

Techniques: Cryo-EM Sample Prep, Labeling

Journal: Cell reports

Article Title: Crimean-Congo hemorrhagic fever survivors elicit protective non-neutralizing antibodies that target 11 overlapping regions on glycoprotein GP38

doi: 10.1016/j.celrep.2024.114502

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Gene fragments (Integrated DNA Technologies) of each isolate’s MLD-GP38 sequence encoding for residues 1–515, as denoted by CCHFV IbAr10200 strain GPC numbering, were codon-optimized for human cell expression (GenScript Codon Optimization Tool).

Techniques: Derivative Assay, Virus, Recombinant, Protease Inhibitor, Reverse Transcription, Membrane, Cell Isolation, Mutagenesis, Expressing, Plasmid Preparation, Software, Affinity Column, Sequencing, Transfection, Electron Microscopy

Journal: Cell reports

Article Title: Crimean-Congo hemorrhagic fever survivors elicit protective non-neutralizing antibodies that target 11 overlapping regions on glycoprotein GP38

doi: 10.1016/j.celrep.2024.114502

Figure Lengend Snippet: (A) Matrix of competition-binning experiments. For on-yeast competition experiments (top left quadrant), results are displayed with surface-presented IgGs on the y axis and competitive pre-complexed Fabs on the x axis. For BLI competition assays (the other three quadrants), binning was performed in an IgG vs. IgG format. (B) Binning analysis of on-yeast competition assays of all 188 antibodies; each color represents 1 of 11 overlapping bins and the Unknown/Weak Affinity mAbs are shown in gray . Distribution of overlapping bins of the antibody panel (left) and by each donor (right). Total number of mAbs is indicated in the circular diagram and total mAbs from each donor are indicated above each bar graph.

Article Snippet: Models of CCHFV GP38 bound to Fabs of GP38-specific mAbs have been deposited at Worldwide Protein DataBank (wwPDB) under accession codes 8VVK, 8VVL, and 8VVW.

Techniques:

Journal: Cell reports

Article Title: Crimean-Congo hemorrhagic fever survivors elicit protective non-neutralizing antibodies that target 11 overlapping regions on glycoprotein GP38

doi: 10.1016/j.celrep.2024.114502

Figure Lengend Snippet: (A) Matrix of percent sequence identity of GP38 amino acid residues (AA) across six CCHFV isolates. (B) Single concentration BLI binding analysis of 188 antibodies to the six rGP38 proteins as a whole panel (top) and broken down by bin (bottom). Shades of green represent the number of rGP38 proteins bound by a single antibody (from 0 in gray to 6 in darkest green). Total number of mAbs is indicated in the circular diagram and total mAbs from each bin are indicated above the bar graph. (C) Carterra system HT-SPR binding analysis of six lead antibody candidates binding to six rGP38 proteins. The highest binding affinities are in dark green and the lowest binding affinities are in white. Calculated K D values appear in each rectangle of the heatmap; for samples that were off-rate limited, K D values are denoted as < the calculated K D . The one interaction for which a curve could not be fit is denoted as P.F.

Article Snippet: Models of CCHFV GP38 bound to Fabs of GP38-specific mAbs have been deposited at Worldwide Protein DataBank (wwPDB) under accession codes 8VVK, 8VVL, and 8VVW.

Techniques: Sequencing, Concentration Assay, Binding Assay

Journal: Cell reports

Article Title: Crimean-Congo hemorrhagic fever survivors elicit protective non-neutralizing antibodies that target 11 overlapping regions on glycoprotein GP38

doi: 10.1016/j.celrep.2024.114502

Figure Lengend Snippet: (A) Neutralization curves for CCHFV IbAr10200 tecVLPs, as measured by the reduction in luciferase activity compared with no-antibody treatment on Vero cells. (B–E) Neutralization curves of the indicated mAbs against authentic (B) CCHFV IbAr10200, (C) CCHFV Afg09, (D) CCHFV Turkey2004, and (E) CCHFV Oman as measured by the reduction in infection compared with no-antibody treatment on SW-13 cells. The average of n = 3 replicates each from two independent experiments ( n = 6 total) is shown for all neutralization curves.

Article Snippet: Models of CCHFV GP38 bound to Fabs of GP38-specific mAbs have been deposited at Worldwide Protein DataBank (wwPDB) under accession codes 8VVK, 8VVL, and 8VVW.

Techniques: Neutralization, Luciferase, Activity Assay, Infection

Journal: Cell reports

Article Title: Crimean-Congo hemorrhagic fever survivors elicit protective non-neutralizing antibodies that target 11 overlapping regions on glycoprotein GP38

doi: 10.1016/j.celrep.2024.114502

Figure Lengend Snippet: (A–C) IFNAR1 −/− mice were treated with the indicated mAbs at 1 mg/mouse 1 and 4 days post-challenge (2 mg total; n = 10 mice per group) with IbAr10200. (A) Survival curves (vehicle vs. test mAb), (B) associated mean weight loss, and (C) clinical score data are shown. (D–L) STAT1 −/− mice were challenged with (D–F) CCHFV-Afg09, (G–I) CCHFV-Turkey2004, or (J–L) CCHFV-Oman and then treated with 0.2 mg/mouse of mAb or vehicle 30 min post-exposure (n = 5–6 mice per study; represented by 2 replicate studies). (D, G, and J) Survival curves. (E, H, and K) Associated mean weight loss. (F, I, and L) Clinical scores are defined as: 1 = decreased grooming and/or ruffled fur; 2 = subdued behavior when un-stimulated; 3 = lethargy, hunched posture, and/or subdued behavior even when stimulated; 4 = bleeding, unresponsiveness, severe weakness, or inability to walk. Mice scoring a 4 were considered moribund and were humanely euthanized based on IACUC-approved criteria (denoted as X over white).

Article Snippet: Models of CCHFV GP38 bound to Fabs of GP38-specific mAbs have been deposited at Worldwide Protein DataBank (wwPDB) under accession codes 8VVK, 8VVL, and 8VVW.

Techniques: