Journal: Life Science Alliance

Article Title: FAT10 inhibits TRIM21 to down-regulate antiviral type-I interferon secretion

doi: 10.26508/lsa.202402786

Figure Lengend Snippet: (A) HEK293T cells were transiently transfected with expression constructs for HA-FAT10 and Myc-DDK-TRIM21. After 24 h, cells were harvested and lysed. Cleared lysate was subjected to immunoprecipitation (IP) using FLAG M2 affinity gel, which specifically recognizes the DDK (FLAG) tag. Proteins were visualized by Western blot analysis under reducing conditions (4% 2-ME) using antibodies reactive to HA or FLAG (DDK). GAPDH was used as a loading control. (B) HEK293T cells were transiently co-transfected with expression constructs for HA-FAT10, its conjugation incompetent variant HA–FAT10AV and Myc-DDK-TRIM21. After 24 h, cells were harvested and lysed. Cleared lysate was subjected to immunoprecipitation (IP) using FLAG M2 affinity gel. Proteins were visualized by Western blot analysis under reducing conditions (4% 2-ME) using antibodies reactive to HA or FLAG. GAPDH was used as loading control. (C) In vitro FAT10ylation assay was performed using recombinant proteins. FLAG–UBA6 was incubated with tagless recombinant FAT10 or FAT10AV and C–Myc-DDK-TRIM21 for 30 min at 37°C. Reaction was stopped by adding 4x sample buffer and boiling. SDS–PAGE and subsequent Western blotting was performed using the indicated antibodies under reducing conditions (4% 2-ME). Asterisks indicate TRIM21–FAT10 conjugates. (D) Schematic representation of full-length and truncation mutants of human TRIM21 generated and used in this study. The truncations were constructed using site directed mutagenesis. (E) HEK293T cells were transiently transfected with expression plasmids for HA–FAT10 or HA–FAT10AV and full length or truncation mutants of Myc-DDK-tagged TRIM21. After 24 h, cells were collected and lysed. Cleared cell lysate was subjected to immunoprecipitation using FLAG–M2 affinity gel. SDS–PAGE and Western blot analysis was performed under reducing conditions (4% 2-ME) using antibodies reactive to HA or FLAG (DDK). GAPDH was used as loading control. Shown is one representative experiment out of three independent experiments with similar outcomes for all experiments. Source data are available for this figure.

Article Snippet: Recombinant FLAG-UBA6 was purchased from Enzo Life Sciences and human C-Myc-DDK-TRIM21 was purchased from Origene (#TP302088).

Techniques: Transfection, Expressing, Construct, Immunoprecipitation, FLAG-tag, Western Blot, Control, Conjugation Assay, Variant Assay, In Vitro, Recombinant, Incubation, SDS Page, Generated, Mutagenesis

Journal: Life Science Alliance

Article Title: FAT10 inhibits TRIM21 to down-regulate antiviral type-I interferon secretion

doi: 10.26508/lsa.202402786

Figure Lengend Snippet: (A) A549 FLAG-FAT10 cells were infected with IAV (MOI: 1) as indicated and uninfected A549 cells were used as control. After 24 h, cells were harvested and lysed. Precleared cell lysates were subjected to Ni–IDA pull-down assay and incubated overnight at 4°C. Beads were washed four times with lysis buffer. SDS–PAGE under reducing conditions (4% 2-ME) followed by Western blotting was performed using the indicated antibodies. GAPDH was used as the loading control. Asterisk marks nonspecific binding of TRIM21 to the beads. (B) A549 WT and FLAG–FAT10 cells were infected with IAV (MOI: 1), as indicated. After 24 h, cells were harvested and lysed. Cleared cell lysates were subjected to SDS–PAGE under reducing conditions (4% 2-ME) followed by Western blotting using the indicated antibodies. GAPDH was used as the loading control. (C) Densitometric quantification of the fluorescent signal obtained in (B). The total TRIM21 fluorescent signal was normalized to GAPDH. The signal intensity of other samples was calculated relative to uninfected A549 WT cells in which the normalized TRIM21 signal was set to 100%. (D) A549 WT and A549 FLAG-FAT10 cells were infected with IAV (MOI: 1), as indicated. After 24 h, cells were harvested and lysed. Cleared cell lysates were subjected to immunoprecipitation with an antibody reactive to TRIM21. SDS–PAGE under reducing conditions (4% 2-ME) followed by Western blotting was performed using the indicated antibodies. GAPDH was used as loading control. Shown is one representative experiment out of three independent experiments with similar outcomes for each experiment. Error bars in (C) indicate SD (n = 3). * P < 0.05 (one-way anova), ns means not significant. Source data are available for this figure.

Article Snippet: Recombinant FLAG-UBA6 was purchased from Enzo Life Sciences and human C-Myc-DDK-TRIM21 was purchased from Origene (#TP302088).

Techniques: Infection, Control, Pull Down Assay, Incubation, Lysis, SDS Page, Western Blot, Binding Assay, Immunoprecipitation

Journal: Life Science Alliance

Article Title: FAT10 inhibits TRIM21 to down-regulate antiviral type-I interferon secretion

doi: 10.26508/lsa.202402786

Figure Lengend Snippet: (A) A549 WT, FLAG–FAT10, TRIM21 KO, TRIM21 KO/FLAG–FAT10 cells were infected with IAV (MOI: 1). After 24 h, supernatants and cell pellets were collected. Cell pellets were lysed and cleared cell lysates were subject to SDS–PAGE under reducing conditions (4% 2-ME) followed by Western blot analysis with the indicated antibodies. GAPDH was used as the loading control. (B) IFNβ ELISA was performed with the cell culture supernatants from (A). (C) A549 WT, FLAG–FAT10, mCherry-TRIM21, mCherry-TRIM21/FLAG–FAT10 cells were infected with IAV (MOI: 1). After 24 h, supernatants and cell pellets were collected. Cell pellets were lysed and cleared cell lysate was subjected to SDS–PAGE under reducing conditions (4% 2-ME) followed by Western blot analysis with the indicated antibodies. GAPDH was used as the loading control. (D) IFNβ ELISA was performed with the cell culture supernatants from (C). (E) A549 WT, TRIM21 KO, mCherry–TRIM21, and FAT10 KO cells were treated with TNF/IFNγ for 24 h followed by IAV infection (MOI: 0.5). After 12 h, supernatant and cell pellets were collected. IFNβ ELISA was performed with the supernatants. Shown is one representative experiment out of at least three independent experiments with similar outcomes for each experiment. Error bars in (B, D, E) indicate SD (n = 3), * P < 0.05 (One-way Anova (B, D), student’s t test (E)), ns means not significant. Source data are available for this figure.

Article Snippet: Recombinant FLAG-UBA6 was purchased from Enzo Life Sciences and human C-Myc-DDK-TRIM21 was purchased from Origene (#TP302088).

Techniques: Infection, SDS Page, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Life Science Alliance

Article Title: FAT10 inhibits TRIM21 to down-regulate antiviral type-I interferon secretion

doi: 10.26508/lsa.202402786

Figure Lengend Snippet: (A) HEK293 WT, or where indicated UBA6 KO and USE1 KO cells, were transiently transfected with HA–FAT10 or HA–FAT10AV and Myc–DDK–TRIM21 expression plasmids. After 24 h, cells were collected and lysed. Cleared cell lysate was subjected to SDS–PAGE and subsequent Western blotting under reducing conditions (4% 2-ME) using antibodies reactive to HA or FLAG (DDK). GAPDH was used as loading control. (B) HEK293 WT, or UBA6 KO and USE1 KO cells were transiently transfected with expression constructs for HA–FAT10 and Myc-DDK-TRIM21. Where indicated, HA-UBA6 expression plasmid was transiently transfected in HEK293 UBA6 KO cells, and HIS–USE1 or HIS-USE1 C188A expression plasmids were transiently transfected in HEK293 USE1 KO cells. After 24 h, cells were collected and lysed. Cleared cell lysates were subjected to SDS–PAGE and subsequent Western blotting was performed under reducing conditions (4% 2-ME) using antibodies reactive to HA or FLAG (DDK). GAPDH was used as loading control. In case of UBA6 KO cells, a 4x times amount of the cell lysate was used for immunoprecipitation because overexpression of proteins is always low in this cell line. (C) HEK293 WT, UBA6 KO and USE1 KO cells were transiently transfected with expression constructs for HA–FAT10 and Myc–DDK–TRIM21. Where indicated, cells were treated with 600 U/ml TNF. After 24 h, cells were collected, lysed, and cleared cell lysate was subjected to immunoprecipitation using FLAG M2 affinity gel, which specifically recognizes the DDK (FLAG) tag. SDS–PAGE and Western blotting was performed under reducing conditions (4% 2-ME) using antibodies reactive to HA or FLAG. GAPDH was used as loading control. Shown is one representative experiment out of three independent experiments with similar outcomes for each experiment. Source data are available for this figure.

Article Snippet: Recombinant FLAG-UBA6 was purchased from Enzo Life Sciences and human C-Myc-DDK-TRIM21 was purchased from Origene (#TP302088).

Techniques: Transfection, Expressing, SDS Page, Western Blot, Control, Construct, Plasmid Preparation, Immunoprecipitation, Over Expression, FLAG-tag

Journal: Life Science Alliance

Article Title: FAT10 inhibits TRIM21 to down-regulate antiviral type-I interferon secretion

doi: 10.26508/lsa.202402786

Figure Lengend Snippet: (A) HEK293T cells were transiently transfected with expression constructs for HA–FAT10 and Myc–DDK–TRIM21. After 24 h, cells were treated with cycloheximide and/or MG132 for the indicated time points. Cleared cell lysates were subjected to immunoprecipitation using FLAG M2 affinity gel, which is specific for the DDK-tag. SDS–PAGE and Western blot analysis was performed under reducing conditions (4% 2-ME) using antibodies reactive to HA or FLAG (DDK). GAPDH was used as loading control. Shown is one representative experiment out of three independent experiments with similar outcomes. (B) Densitometric quantification of the FAT10-TRIM21 conjugate fluorescent signal, normalized to the respective GAPDH fluorescent signal. The value of untreated sample was set to 100%. Shown is the mean three independent experiments with similar outcomes. Source data are available for this figure.

Article Snippet: Recombinant FLAG-UBA6 was purchased from Enzo Life Sciences and human C-Myc-DDK-TRIM21 was purchased from Origene (#TP302088).

Techniques: Transfection, Expressing, Construct, Immunoprecipitation, SDS Page, Western Blot, Control

Journal: Life Science Alliance

Article Title: FAT10 inhibits TRIM21 to down-regulate antiviral type-I interferon secretion

doi: 10.26508/lsa.202402786

Figure Lengend Snippet: (A) A549 cells were treated with TNF/IFNγ for 24 h followed by influenza A virus (IAV) infection (MOI: 1), as indicated. Additional TNF/IFNγ treatment was performed for 24 h. After 24 h, cells were harvested, and cleared cell lysates were subjected to immunoprecipitation with antibodies reactive to TRIM21 or a nonspecific IgG control antibody. Proteins were separated on SDS–PAGE followed by Western blot analysis with the indicated antibodies. GAPDH was used as loading control. Shown is one representative experiment out of three independent experiments with similar outcomes. (B) A549 cells were treated and harvested as indicated in (A). Cleared cell lysates from the lysed cells were immunoprecipitated with antibodies reactive to FAT10 or with a nonspecific IgG control antibody. Proteins were separated on SDS–PAGE followed by Western blot analysis using the indicated antibodies. GAPDH was used as loading control. Shown is one representative experiment out of three independent experiments with similar outcomes. (C) A549 WT and stable FLAG-FAT10 expressing A549 cells were infected with influenza A virus (MOI: 1), as indicated. After 24 h, cells were harvested and lysed. Cleared cell lysates were immunoprecipitated using antibodies reactive to TRIM21 or by using an unspecific IgG control antibody. SDS–PAGE followed by Western blotting was performed using the indicated antibodies. GAPDH was used as loading control. Shown is one representative experiment out of three independent experiments with similar outcomes for each experiment. Source data are available for this figure.

Article Snippet: Recombinant FLAG-UBA6 was purchased from Enzo Life Sciences and human C-Myc-DDK-TRIM21 was purchased from Origene (#TP302088).

Techniques: Virus, Infection, Immunoprecipitation, Control, SDS Page, Western Blot, Expressing

Journal: Life Science Alliance

Article Title: FAT10 inhibits TRIM21 to down-regulate antiviral type-I interferon secretion

doi: 10.26508/lsa.202402786

Figure Lengend Snippet: (A) Densitometric quantification of fluorescence signal intensities from . TRIM21 signal intensities were normalized to GAPDH and normalized to the WT + influenza A virus (IAV). Shown is the mean of three independent experiments with similar outcomes. (B) IFNβ ELISA was performed with the cell culture supernatants from uninfected A549 WT, FLAG–FAT10, TRIM21 KO, TRIM21 KO/FLAG–FAT10 cells. (C) A549 WT, FLAG–FAT10, TRIM21 KO, TRIM21 KO/FLAG–FAT10 cells were infected with IAV (MOI: 1). After 24 h, supernatants and cell pellets were collected. Supernatants were used for plaque assay to determine IAV titers. (D) mRNA was isolated from cell pellets obtained in (C) and subjected to real-time PCR analysis. (E) Densitometric quantification of fluorescence signal intensities from . TRIM21 signal intensities were normalized to GAPDH and normalized to the WT + IAV. Shown is the mean of three independent experiments with similar outcomes. (F) IFNβ ELISA was performed with the cell culture supernatants from uninfected A549 WT, FLAG-FAT10, mCherry-TRIM21, mCherry-TRIM21/FLAG–FAT10 cells. (G) A549 WT, FLAG–FAT10, mCherry-TRIM21, mCherry-TRIM21/FLAG–FAT10 cells were infected with IAV (MOI: 1). After 24 h, supernatants and cell pellets were collected. Supernatants were used for plaque assay to determine IAV titers. (H) mRNA was isolated from cell pellets obtained in (E) and subjected to real-time PCR analysis. Error bars in (A, E) indicate SD (n = 3). * P < 0.05 ( t test), ns means not significant. Error bars in (D, H) indicate SD (n = 3). * P < 0.05 (multiple t test).

Article Snippet: Recombinant FLAG-UBA6 was purchased from Enzo Life Sciences and human C-Myc-DDK-TRIM21 was purchased from Origene (#TP302088).

Techniques: Fluorescence, Virus, Enzyme-linked Immunosorbent Assay, Cell Culture, Infection, Plaque Assay, Isolation, Real-time Polymerase Chain Reaction

Journal: Life Science Alliance

Article Title: FAT10 inhibits TRIM21 to down-regulate antiviral type-I interferon secretion

doi: 10.26508/lsa.202402786

Figure Lengend Snippet: (A) A549 cells were treated with TNF/IFNγ for 24 h followed by influenza A virus (IAV) infection (MOI: 1), as indicated. Additional TNF/IFNγ treatment was performed for 24 h. Cells were immunostained with antibodies reactive to TRIM21 and FAT10 and visualized by confocal microscopy. Scale bars, 20 μm. (B) Cells from (A) were immunostained with antibodies reactive to M1 and with DAPI. Scale bars, 20 μm. (C) A549 mCherry-TRIM21/FLAG–FAT10 cells were infected with influenza A virus (MOI: 1). Cells were visualized by confocal microscopy. Scale bars, 20 μm. Shown here is one representative experiment out of three independent experiments with similar outcomes.

Article Snippet: Recombinant FLAG-UBA6 was purchased from Enzo Life Sciences and human C-Myc-DDK-TRIM21 was purchased from Origene (#TP302088).

Techniques: Virus, Infection, Confocal Microscopy

Journal: Life Science Alliance

Article Title: FAT10 inhibits TRIM21 to down-regulate antiviral type-I interferon secretion

doi: 10.26508/lsa.202402786

Figure Lengend Snippet: (A) Cleared cell lysates from were subjected to SDS–PAGE followed by Western blot analysis using the indicated antibodies. (B) A549 WT, TRIM21 KO, mCherry-TRIM21, and FAT10 KO cells were treated with TNF/IFNγ for 24 h followed by influenza A virus infection (MOI: 0.5). Cells were harvested and cleared cell lysates were subjected to immunoprecipitation using an antibody reactive to FAT10 (4F1, ). SDS–PAGE followed by Western blot analysis was performed using the indicated antibodies. GAPDH was used as the loading control. Shown is one representative experiment out of three independent experiments with similar outcomes. Source data are available for this figure.

Article Snippet: Recombinant FLAG-UBA6 was purchased from Enzo Life Sciences and human C-Myc-DDK-TRIM21 was purchased from Origene (#TP302088).

Techniques: SDS Page, Western Blot, Virus, Infection, Immunoprecipitation, Control

Journal: Life Science Alliance

Article Title: FAT10 inhibits TRIM21 to down-regulate antiviral type-I interferon secretion

doi: 10.26508/lsa.202402786

Figure Lengend Snippet: (A) C57BL/6 WT or FAT10KO mice were infected with LCMV (200 pfu), as indicated. Relative FAT10 mRNA expression in mice livers was determined by quantitative real-time PCR. (B) C57BL/6 WT or FAT10KO mice were infected with LCMV (200 pfu), where indicated. Cleared tissue lysates from the livers of the mice were prepared day three post-infection. 40 μg of protein was separated on a SDS–PAGE followed by Western blotting using the indicated antibodies. GAPDH was used as the loading control. (C) Densitometric quantification of fluorescence signal intensities from . TRIM21 signal intensities were normalized to GAPDH. (D) TRIM21 levels in spleen of LCMV infected mice as described in (B). (E) Densitometric quantification of fluorescence signal intensities from . TRIM21 signal intensities were normalized to GAPDH. Shown is one representative experiment out of three independent experiments with similar outcomes. Error bars in (A, C, E) indicate SD (n = 3). * P < 0.05 (one-way Anova). Source data are available for this figure.

Article Snippet: Recombinant FLAG-UBA6 was purchased from Enzo Life Sciences and human C-Myc-DDK-TRIM21 was purchased from Origene (#TP302088).

Techniques: Infection, Expressing, Real-time Polymerase Chain Reaction, SDS Page, Western Blot, Control, Fluorescence

Journal: Life Science Alliance

Article Title: FAT10 inhibits TRIM21 to down-regulate antiviral type-I interferon secretion

doi: 10.26508/lsa.202402786

Figure Lengend Snippet: TNF/IFNγ induces the expression of FAT10 (blue arrow). FAT10-mediated down-regulation of type-I IFN happens in two ways: (I) influenza A virus infection upregulates TRIM21 expression (blue arrow). TRIM21 positively regulates the antiviral type-I IFN production through a positive feedback loop (blue arrow). FAT10 inhibits TRIM21 by directly binding to its PRYSPRY domain, causing either degradation of TRIM21 by the 26S proteasome, and/or inhibiting TRIM21 auto-ubiquitination, thus down-regulating the production of type-I IFN. (II) FAT10 gets phosphorylated upon influenza A virus infection. Phosphorylated FAT10 stabilizes and activates OTUB1, which inhibits type-I IFN production .

Article Snippet: Recombinant FLAG-UBA6 was purchased from Enzo Life Sciences and human C-Myc-DDK-TRIM21 was purchased from Origene (#TP302088).

Techniques: Expressing, Virus, Infection, Binding Assay

Journal: Frontiers in Immunology

Article Title: TRIM21 and Fc-engineered antibodies: decoding its complex antibody binding mode with implications for viral neutralization

doi: 10.3389/fimmu.2024.1401471

Figure Lengend Snippet: SPR assay orientations to characterize the interaction between TRIM21 and an antibody. Symmetrical and asymmetrical antibody Fc variants are investigated. In case of an asymmetrical Fc part, one Fc heavy chain contains a AAA mutation (schematically shown by red star), that completely abolishes TRIM21 binding. The used Fc variants and assay setups allow determining how Fc mutations influence the avidity-binding mode and dissecting avidity from affinity. (A) Antibody Fc variants are captured on the biosensor surface via an anti-Fab nanobody (vhh), Fc-only variants are coupled using standard amine coupling chemistry and cytokine Fc-Fusions are captured via anti-PGLALA F(ab’)2 fragment , while TRIM21 PRYSPRY domain is the analyte in solution (see Materials and Methods). Configuration (B) schematically shows the inverse to (A) while the PRYSPRY domain is captured via monovalent streptavidin. (C) To analyze the dimeric TRIM21 engagement of both IgG heavy chains, the antibody is captured via its Fab fragment, cytokine Fc-Fusions are captured via anti-PGLALA F(ab’)2 fragment (identical capture setup as in (A) and TRIM21-coiled-coil-PYRSPRY (TRIM21-CC-PS) is injected. Illustrations are created with BioRender.com .

Article Snippet: Recombinant human TRIM21 proteins, including the PRYSPRY domain variant and the PRYSPRY-coiled-coil (TRIM21-CC-PS) variant, were produced and purified by Proteros Biostructures GmbH (Planegg, Germany).

Techniques: SPR Assay, Mutagenesis, Binding Assay, Injection

Journal: Frontiers in Immunology

Article Title: TRIM21 and Fc-engineered antibodies: decoding its complex antibody binding mode with implications for viral neutralization

doi: 10.3389/fimmu.2024.1401471

Figure Lengend Snippet: Interaction analysis of human IgG1 (mAb1) Fc variants and TRIM21 PRYSPRY domain. (A–C) showing sensorgrams (SPR data) where PRYSPRY was injected in five different concentrations as two-fold dilution series to immobilized mAb1 Fc variants (capture level approx. 60 RU). Each plot shows the measured raw data (colored gradient) and the global fit analysis as solid black lines. For immobilized mAb1 WT (A) and mAb1 WT-AAA (B) PRYSPRY was injected at 500 nM highest concentration and for mAb1 AAA (C) at 2000 nM. The sensorgrams show the affinity binding mode applying a mono-exponential fit model (Langmuir 1:1). The determined kinetic parameters are described in (D) . The k ON , k OFF and K D values are results from a global fit analysis ± fitting error. (E, F) show the complementary mass photometry (MP) data displaying a 2:1 binding stoichiometry confirming the SPR data. For the PRYSPRY - mAb1 WT complex, the data reveals a double bound state and for mAb1 WT-AAA a single bound state, while the control mAb1 AAA shows no binding at all. A Gaussian distribution model was used to analyze the MP data. For individual masses of the molecules, see SI Info Supplementary Figure S1 .

Article Snippet: Recombinant human TRIM21 proteins, including the PRYSPRY domain variant and the PRYSPRY-coiled-coil (TRIM21-CC-PS) variant, were produced and purified by Proteros Biostructures GmbH (Planegg, Germany).

Techniques: Injection, Concentration Assay, Binding Assay, Control

Journal: Frontiers in Immunology

Article Title: TRIM21 and Fc-engineered antibodies: decoding its complex antibody binding mode with implications for viral neutralization

doi: 10.3389/fimmu.2024.1401471

Figure Lengend Snippet: Kinetic characterization of TRIM21 PRYSPRY binding to immobilized antibody Fc variants and cytokine-Fc Fusion constructs, and Fc only variant (Raw data SI Info Supplementary Figure S2 ). Detailed SPR assay setup is described in materials and methods. (A) The Affinity Rate Scale Plot enables the kinetic comparison of several binding experiments at one glance. The association rate (k ON ) and corresponding dissociation rate (k OFF ) are juxtaposed in opposition, connected via a vertical line, representing the binding strength (affinity). The further apart both parameters (k ON and k OFF ) the stronger the interaction is. Compared to mAb1 Fc WT, the Fc variants YTE (M252Y, S254T, T256E), HH (T307H, N434H) and Y436A show decreased PRYSPRY affinity. The YTE affinity is 1.7-fold, HH 2.4-fold and Y436A 180-fold decreased. As shown in (B) the altered binding strength is mostly off rate driven, which becomes apparent in the overlay of normalized dissociation. The start of dissociation is normalized to 100%.

Article Snippet: Recombinant human TRIM21 proteins, including the PRYSPRY domain variant and the PRYSPRY-coiled-coil (TRIM21-CC-PS) variant, were produced and purified by Proteros Biostructures GmbH (Planegg, Germany).

Techniques: Binding Assay, Construct, Variant Assay, SPR Assay, Comparison

Journal: Frontiers in Immunology

Article Title: TRIM21 and Fc-engineered antibodies: decoding its complex antibody binding mode with implications for viral neutralization

doi: 10.3389/fimmu.2024.1401471

Figure Lengend Snippet: Affinity rate scale plot for captured TRIM21 PRYSPRY domain (ligand) and antibody (human IgG1) variable domain variants or antigen fusion constructs in solution (analyte). The injected constructs have different Fab regions but share the same Fc region. This allows the investigation of a potential Fab contribution to the PRYSPRY binding. All constructs were analyzed by applying a simple 1:1 Langmuir fit. The analyzed antibody variants do not show any Fab contribution. Notably, there is a faster on-rate (2x) for all constructs when compared to the reverse assay setup up (PRYSPRY as analyte). Raw data is shown in SI Info Supplementary Figure S3 .

Article Snippet: Recombinant human TRIM21 proteins, including the PRYSPRY domain variant and the PRYSPRY-coiled-coil (TRIM21-CC-PS) variant, were produced and purified by Proteros Biostructures GmbH (Planegg, Germany).

Techniques: Construct, Injection, Binding Assay

Journal: Frontiers in Immunology

Article Title: TRIM21 and Fc-engineered antibodies: decoding its complex antibody binding mode with implications for viral neutralization

doi: 10.3389/fimmu.2024.1401471

Figure Lengend Snippet: Characterization of the TRIM21 dimeric nature (TRIM21-CC-PS) applying different technologies. (A) SEC-MALS data reveals 94% TRIM21-CC-PS dimer (90 kDa). (B) Mass photometry technology shows 99% TRIM21-CC-PS with 82 kDa. (C) Selection of EM 2D classes confirming TRIM21-CC-PS dimers. The coiled coil domains facilitate dimerization whereas the C-terminal PRYSPRY domains are placed at the opposite end of each coiled-coil domain.

Article Snippet: Recombinant human TRIM21 proteins, including the PRYSPRY domain variant and the PRYSPRY-coiled-coil (TRIM21-CC-PS) variant, were produced and purified by Proteros Biostructures GmbH (Planegg, Germany).

Techniques: Selection

Journal: Frontiers in Immunology

Article Title: TRIM21 and Fc-engineered antibodies: decoding its complex antibody binding mode with implications for viral neutralization

doi: 10.3389/fimmu.2024.1401471

Figure Lengend Snippet: Characterizing the interaction of TRIM21-CC-PS with three different Antibody Fc variants. (A–D) Applying MP, dashed lines indicate the main peak of the respective species over all measurements. The applied 3-dimensional Gaussian Fit Distribution is shown in black lines. (A) MP of TRIM21-CC-PS with mAb 1 WT, (B) MP of TRIM21-CC-PS with mAb 1 WT-AAA (C) MP of TRIM21-CC-PS with mAb 1 AAA. Only mAb 1 WT shows binding to TRIM21-CC-PS at low nM concentration in accordance with its low nM binding strength. (D) The amount (%) of TRIM21-CC-PS - mAb1 WT complex increases with excess of TRIM21-CC-PS, while TRIM21-CC-PS - mAb1 WT-AAA shows single events of complexed species for the applied concentrations, that could not be fitted robustly. (E–H) selected 2D averages of EM data, resolving TRIM21-CC-PS with Fc WT (E) , TRIM21-CC-PS with Fc WT-AAA (F) , TRIM21-CC-PS with mAb1 WT (G) and mAb1 WT alone (H) .

Article Snippet: Recombinant human TRIM21 proteins, including the PRYSPRY domain variant and the PRYSPRY-coiled-coil (TRIM21-CC-PS) variant, were produced and purified by Proteros Biostructures GmbH (Planegg, Germany).

Techniques: Binding Assay, Concentration Assay

Journal: Frontiers in Immunology

Article Title: TRIM21 and Fc-engineered antibodies: decoding its complex antibody binding mode with implications for viral neutralization

doi: 10.3389/fimmu.2024.1401471

Figure Lengend Snippet: Characterization of TRIM21-CC-PS with Antibody Fc variants. (A) shows the sensorgram (SPR) of mAb1 WT (ligand, approx. 8–10 RU) and TRIM21-CC-PS (analyte) where TRIM21-CC-PS was injected in seven different concentration, each for 180 sec as two-fold dilution series with 100 nM as highest concentration. Applied fit model is a simple 1:1 interaction reflecting 100% avid bound, 1:1 antibody - TRIM21-CC-PS species. (B) Variation in association time (10 -300 sec) injecting a constant concentration of 25 nM TRIM21-CC-PS to captured mAb1 WT reveals a biphasic binding kinetic, which can be described by applying a two state model providing fast and slow kinetic rates. (C) Rate-scale-plot comparing affinity and avidity measurements of Fc variants towards PRYSPRY or TRIM21-CC-PS. (D) The altered binding strength from affinity to avidity is mostly off rate driven, which becomes apparent in the overlay of normalized dissociation phases (k OFF,AVIDITY ) but can also occur as combination of both kinetic rate parameters, namely on and off rate.

Article Snippet: Recombinant human TRIM21 proteins, including the PRYSPRY domain variant and the PRYSPRY-coiled-coil (TRIM21-CC-PS) variant, were produced and purified by Proteros Biostructures GmbH (Planegg, Germany).

Techniques: Injection, Concentration Assay, Binding Assay

Journal: Frontiers in Immunology

Article Title: TRIM21 and Fc-engineered antibodies: decoding its complex antibody binding mode with implications for viral neutralization

doi: 10.3389/fimmu.2024.1401471

Figure Lengend Snippet: TRIM21-CC-PS-Antibody-AAV2 Characterization. SPR Assay data is shown in (A, B) . (A1, A2) Schematic SPR assay configuration to analyze the affinity to avidity interplay of TRIM21-CC-PS, anti-capsid antibody variants A20 and rAAVv-2. Biotinylated TRIM21-CC-PS is captured via monovalent streptavidin achieving a captured level of 190 RU ( A1 , high density) and 35 RU ( A2 , low density). Subsequent, anti-AAV2 capsid antibody variants (bivalent A20 Fc WT, one-armed A20 Fc WT and Fc WT-AAA) are injected to saturate the TRIM21-CC-PS surface, followed by the injection of rAAV-2. (B) Overlay of the normalized dissociation phases (Start of Dissociation: 100%) after the injection of 3.32 nM rAAVv-2 over low and high TRIM21-CC-PS-A20 densities. At higher antibody densities, more avid complexation occurs and a higher degree of rAAVv-2 surface decoration is possible. This allows less complex to dissociate over time to due to simultaneous engagement of both, TRIM21-CC-PS and AAV2, mediated via the A20 antibody variants. (C) Electron microscopy images of rAAVv-2 interactions with antibodies alone (left column) and TRIM21 additionally (right column). The scale bars represent 50 nm.

Article Snippet: Recombinant human TRIM21 proteins, including the PRYSPRY domain variant and the PRYSPRY-coiled-coil (TRIM21-CC-PS) variant, were produced and purified by Proteros Biostructures GmbH (Planegg, Germany).

Techniques: SPR Assay, Injection, Electron Microscopy

Journal: Frontiers in Immunology

Article Title: TRIM21 and Fc-engineered antibodies: decoding its complex antibody binding mode with implications for viral neutralization

doi: 10.3389/fimmu.2024.1401471

Figure Lengend Snippet: Schematic model suggesting how one TRIM21 dimer engages both sites of the Fc region in a two-step process. Upon Fc binding, the PRYSPRY detaches from the coiled-coil domain. The linker domain allows enough freedom of movement to allow engagement of the second PRYSPRY domain. Only after initial binding bivalent engagement of both Fc heavy chains is possible.

Article Snippet: Recombinant human TRIM21 proteins, including the PRYSPRY domain variant and the PRYSPRY-coiled-coil (TRIM21-CC-PS) variant, were produced and purified by Proteros Biostructures GmbH (Planegg, Germany).

Techniques: Binding Assay