Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Citrullination alters immunomodulatory function of LL-37 essential for prevention of endotoxin-induced sepsis

doi: 10.4049/jimmunol.1303062

Figure Lengend Snippet: Human MDMs (A) and mouse RAW 264.7 (B) macrophages were stimulated with 10 ng/mL LPS in the presence of native or citrullinated LL-37 at the indicated concentrations (0.1–10 μg/mL). Citrullinated LL-37 was obtained by treatment of the native peptide with human PAD2 or PAD4 at 23.3 U/mg peptide). The level of TNF-α (A) and NO (B) in the culture supernatants was determined using ELISA or the Griess assay at 6 or 20 h post-stimulation, respectively. Since neither the LL-37 nor the PAD enzymes alone induced the release of NO or TNF-α, for the sake of clarity these controls are not shown in the figure. Data represent the mean ± SD of three independent experiments. ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001.

Article Snippet: Recombinant human PAD2 and PAD4 were obtained from Modiquest (The Netherlands).

Techniques: Enzyme-linked Immunosorbent Assay, Griess Assay

Journal: Disease Models & Mechanisms

Article Title: Inhibition of peptidyl-arginine deiminases reverses protein-hypercitrullination and disease in mouse models of multiple sclerosis

doi: 10.1242/dmm.010520

Figure Lengend Snippet: PAD activity in NAWM of MS patients. (A) Quantification of PAD2 protein in white matter from normal and MS brain by immunoslot blot ( n =5, P <0.0001). (B) Citrullinated protein in white matter from normal and MS brains by immunoslot blot as pixel density ( n =4, P <0.01). (C) PAD enzyme activity in normal and MS tissue, with or without pre-incubation with 2CA ( n =5, P <0.05). Each dot represents one patient analysed three times. The means (horizontal bar) for all the MS patients were compared with the normal.

Article Snippet: Recombinant PAD2 (5 μg)was reacted with 2CA in the presence of Ca 2+ in HEPES buffer pH 7.6 (BioShop Canada, Burlington, ON) at 52°C for one hour, after which it was dialyzed and lyophilized.

Techniques: Activity Assay, Incubation

Journal: Disease Models & Mechanisms

Article Title: Inhibition of peptidyl-arginine deiminases reverses protein-hypercitrullination and disease in mouse models of multiple sclerosis

doi: 10.1242/dmm.010520

Figure Lengend Snippet: Interaction of PAD with 2CA. (A) PAD2 and PAD4 inhibition curves in the presence of increasing 2CA concentrations. Insert: PAD1-4 enzymes contain a common C-terminal active-site cysteine residue (Cys656) bound by 2CA, confirmed by ESI mass spectrometry of tryptic digests of PAD2-acetamidine adducts. (B) Schematic of the nucleophilic reaction between 2CA and the Cys656 residue in the active site of PAD2 . (C) Tabular summary of peptide fragment atomic masses for 2CA-modified and native PAD2.

Article Snippet: Recombinant PAD2 (5 μg)was reacted with 2CA in the presence of Ca 2+ in HEPES buffer pH 7.6 (BioShop Canada, Burlington, ON) at 52°C for one hour, after which it was dialyzed and lyophilized.

Techniques: Inhibition, Residue, Mass Spectrometry, Modification

Journal: Disease Models & Mechanisms

Article Title: Inhibition of peptidyl-arginine deiminases reverses protein-hypercitrullination and disease in mouse models of multiple sclerosis

doi: 10.1242/dmm.010520

Figure Lengend Snippet: mAb4E12 (anti-2CA adduct) immunogold labeled optic nerve cryosections from control and 2CA-treated PAD2 transgenic mice. Minimal labeling in untreated mice (black arrows). Numerous gold particles (white arrows) in nuclei (N) and cytoplasm of oligodendrocytes, myelin and axonoplasm (Ax) of 2CA-treated mice. Scale bars: 500 nm.

Article Snippet: Recombinant PAD2 (5 μg)was reacted with 2CA in the presence of Ca 2+ in HEPES buffer pH 7.6 (BioShop Canada, Burlington, ON) at 52°C for one hour, after which it was dialyzed and lyophilized.

Techniques: Labeling, Control, Transgenic Assay

Journal: Disease Models & Mechanisms

Article Title: Inhibition of peptidyl-arginine deiminases reverses protein-hypercitrullination and disease in mouse models of multiple sclerosis

doi: 10.1242/dmm.010520

Figure Lengend Snippet: 2CA attenuates demyelinating disease in ND4 mice. (A) ND4 mice treated with PBS, 2CA (5 mg/kg), or 2CA+B12 (5 mg/kg and 10 mg/kg) starting at 2 months before disease onset ( n =5, P <0.0001). (B) ND4 mice treated at disease onset ( n =4, P <0.0001). (C) Stopping 2CA, but continuing B12 at 3.5 months in ND4 mice ( n =5, P <0.0001) demonstrates that B12 alone does not attenuate disease. (D) PAD activity in brains from animals shown in ( n =5, P <0.05). The first four bars represent results from animals at 6 months of age, whereas the post-treatment animals were 8 months of age. (E) PAD2 RT PCR in white matter extracts from normal, PBS-, 2CA- and 2CA+B12-treated ND4 mice ( n =9, P <0.05). (F) LFB and hematoxylin stain of cerebella from normal, PBS-, 2CA- and 2CA+B12-treated ND4 mice (40×). WM, white matter; GM, grey matter.

Article Snippet: Recombinant PAD2 (5 μg)was reacted with 2CA in the presence of Ca 2+ in HEPES buffer pH 7.6 (BioShop Canada, Burlington, ON) at 52°C for one hour, after which it was dialyzed and lyophilized.

Techniques: Activity Assay, Reverse Transcription Polymerase Chain Reaction, Staining

Journal: Disease Models & Mechanisms

Article Title: Inhibition of peptidyl-arginine deiminases reverses protein-hypercitrullination and disease in mouse models of multiple sclerosis

doi: 10.1242/dmm.010520

Figure Lengend Snippet: 2CA attenuates PAD2 overexpressor. (A) Demyelinating disease in PAD2 transgenic mice treated with PBS, 2CA or 2CA+B12 starting at 6 months of age ( n =5, P <0.0001). (B) PAD activity in brain extracts of PAD2 transgenic mice treated with PBS, 2CA or 2CA+B12, and non-transgenic littermates ( n =4, P <0.05). PAD activity is reduced to normal levels by treatment.

Article Snippet: Recombinant PAD2 (5 μg)was reacted with 2CA in the presence of Ca 2+ in HEPES buffer pH 7.6 (BioShop Canada, Burlington, ON) at 52°C for one hour, after which it was dialyzed and lyophilized.

Techniques: Transgenic Assay, Activity Assay

Journal: Frontiers in Immunology

Article Title: Citrullination of C1-inhibitor as a mechanism of impaired complement regulation in rheumatoid arthritis

doi: 10.3389/fimmu.2023.1203506

Figure Lengend Snippet: Confirmation of C1-INH citrullination by PAD4 and PAD2 using the PG-biotin probe on ELISA and Western blotting. C1-INH was incubated with PAD2 or PAD4 under reducing conditions and in the presence of 8 mM of CaCl 2 . Citrullination of C1-INH was confirmed using the biotin-PG probe at low pH and visualized in ELISA (A) and Western blotting (B) .

Article Snippet: Recombinant human PAD2 and PAD4 and biotin-phenylglyoxal (PG) were from Cayman Chemicals (Ann Arbor, MI, USA) (#10785, #10500, and #17450).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Incubation

Journal: Frontiers in Immunology

Article Title: Citrullination of C1-inhibitor as a mechanism of impaired complement regulation in rheumatoid arthritis

doi: 10.3389/fimmu.2023.1203506

Figure Lengend Snippet: ELISA confirming ACPA reactivity to citrullinated C1-INH. (A, B) RA patient samples were added to microtiter plates coated with native and PAD2-citrullinated (A) as well as PAD4-citrullinated (B) C1-INH. Autoantibody binding was detected using HRP-conjugated rabbit anti-human IgG and compared between ACPA-negative and ACPA-positive samples for PAD2-citrullinated (C, E) and PAD4-citrullinated C1-INH (D, F) for all RA patient samples (C, D) as well as for merely RF-negative samples (E, F) . Data are presented as median plus interquartile range, and significance was calculated using Mann–Whitney U and Kruskal–Wallis rank-sum tests. Citrullination of C1-INH using 100 U of the PAD enzymes per 1 mg of protein was confirmed using the biotin-PG probe visualized by Western blotting **p < 0.01, ***p < 0.001, ****p < 0.0001 (G) . ACPA, anti-citrullinated protein antibody; RA, rheumatoid arthritis; HRP, horseradish peroxidase; RF, rheumatoid factor.

Article Snippet: Recombinant human PAD2 and PAD4 and biotin-phenylglyoxal (PG) were from Cayman Chemicals (Ann Arbor, MI, USA) (#10785, #10500, and #17450).

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, MANN-WHITNEY, Western Blot

Journal: bioRxiv

Article Title: Development of Motif-Specific Monoclonal Antibodies for Global Protein Citrullination Detection with Minimal Cross-Reactivity to Homocitrullination

doi: 10.1101/2025.03.27.645732

Figure Lengend Snippet: (A) Reactivity of motif-specific monoclonal antibodies 12A2 and 21F10 against in vitro citrullinated or homocitrullinated HeLa proteomes. (-), untreated HeLa lysate. (B) Reactivity of three commercially available anti-pan citrullination antibodies (07-377, ab100932, and MABN328 (F95)) against citrullinated and homocitrullinated HeLa lysates. (-), untreated HeLa lysate. (C) Reactivity of motif-specific monoclonal antibodies 12A2 and 21F10 toward HeLa proteomes treated with different PAD isozymes (PAD1, PAD2, PAD3, PAD4). (-), untreated HeLa lysate.

Article Snippet: For comparison of the citrullination patterns of different PAD isozymes, 200 μg of HeLa lysate (5 μg/μL) was diluted with 160 μL PAD buffer and incubated with recombinant PAD1 (Cay10784, Biomol), PAD3 (Cay10786, Biomol), PAD2, or PAD4 (both kindly provided by Prof. Hui-Chih Hung) enzyme at 1:100 enzyme:protein ratio overnight at 37 °C with shaking at 600 rpm.

Techniques: Bioprocessing, In Vitro