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. 2011 Oct 21;286(42):36478-91.
doi: 10.1074/jbc.M111.241786. Epub 2011 Aug 23.

Anti-Ro52 autoantibodies from patients with Sjögren's syndrome inhibit the Ro52 E3 ligase activity by blocking the E3/E2 interface

Alexander Espinosa  1 , Janosch Hennig, Aurélie Ambrosi, Madhanagopal Anandapadmanaban, Martina Sandberg Abelius, Yi Sheng, Filippa Nyberg, Cheryl H Arrowsmith, Maria Sunnerhagen, Marie Wahren-Herlenius
Affiliations

Anti-Ro52 autoantibodies from patients with Sjögren's syndrome inhibit the Ro52 E3 ligase activity by blocking the E3/E2 interface

Alexander Espinosa et al. J Biol Chem. .

Abstract

Ro52 (TRIM21) is an E3 ligase of the tripartite motif family that negatively regulates proinflammatory cytokine production by ubiquitinating transcription factors of the interferon regulatory factor family. Autoantibodies to Ro52 are present in patients with lupus and Sjögren's syndrome, but it is not known if these autoantibodies affect the function of Ro52. To address this question, the requirements for Ro52 E3 ligase activity were first analyzed in detail. Scanning a panel of E2 ubiquitin-conjugating enzymes, we found that UBE2D1-4 and UBE2E1-2 supported the E3 ligase activity of Ro52 and that the E3 ligase activity of Ro52 was dependent on its RING domain. We also found that the N-terminal extensions in the class III E2 enzymes affected their interaction with Ro52. Although the N-terminal extension in UBE2E3 made this E2 enzyme unable to function together with Ro52, the N-terminal extensions in UBE2E1 and UBE2E2 allowed for a functional interaction with Ro52. Anti-Ro52-positive patient sera and affinity-purified anti-RING domain autoantibodies inhibited the E3 activity of Ro52 in ubiquitination assays. Using NMR, limited proteolysis, ELISA, and Ro52 mutants, we mapped the interactions between Ro52, UBE2E1, and anti-Ro52 autoantibodies. We found that anti-Ro52 autoantibodies inhibited the E3 ligase activity of Ro52 by sterically blocking the E2/E3 interaction between Ro52 and UBE2E1. Our data suggest that anti-Ro52 autoantibodies binding the RING domain of Ro52 may be actively involved in the pathogenesis of rheumatic autoimmune disease by inhibiting Ro52-mediated ubiquitination.

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Figures

FIGURE 1.
FIGURE 1.
MaBP and His-tagged Ro52 mutants. Ro52-1 is the fusion of MaBP to full-length human Ro52 (residues 1–475). Ro52-2 lacks the B30.2 domain; Ro52-3 consists of the coiled-coil domain only, whereas Ro52-4 and Ro52-5 comprise the zinc binding and B30.2 domains, respectively. The deletion constructs Ro52β and Ro52βΔRING lack the leucine zipper or the RING and the leucine zipper, respectively. MaBP fused mouse Ro52, mouse Ro52ΔRING, and mouse Ro52 with point mutations (indicated by asterisks), and mRo52mut1 (C20A, C23A, and mRo52mut2 (C55A and C58A) were also used as well as His6-tagged RING and B-box constructs.
FIGURE 2.
FIGURE 2.
Ro52 is a RING domain-dependent E3 that can form polyubiquitin chains both through Lys-48 and Lys-63. A, Ro52 was used in autoubiquitination reactions together with E1, ubiquitin, reaction buffer, and different E2 enzymes. Ro52 is autoubiquitinated in reactions with UBE2D1–4, UBE2E1, UBE2E2, and CDC34. The monoclonal antibody 7.8C7 was used for anti-Ro52 immunoblotting. NEG, reaction without any E2. B, in vitro ubiquitination assays with different Ro52 mutants demonstrating that only mutants containing the RING domain have E3 activity. C, Ro52 without the RING domain or with point mutation in the RING domain lack E3 activity. mRo52ΔRING, mRo52mut1 (C20A,C23A), and mRo52mut2 (C55A,C58A) were used for in vitro ubiquitination assays with UBE2E1. D, two isoforms Ro52 and Ro52β (lacking exon 4) both had E3 activity, whereas Ro52β lacking the RING domain had no E3 activity. E, 293T cells were transfected with His6-ubiquitin and Myc-Ro52, Myc-Ro52β, or empty Myc plasmid. After purifying ubiquitinated proteins with Ni-NTA resin, immunoblotting was made against the Myc epitope. Both Ro52α and Ro52β are ubiquitinated in vivo. F, ubiquitination assays using Ro52 with UBE2E1 and two ubiquitin mutants containing a single lysine residue only, Lys-48-only or Lys-63-only. Ro52 is autoubiquitinated with both Lys-48-only and Lys-63-only ubiquitin mutants.
FIGURE 3.
FIGURE 3.
E2 class III N-terminal extensions mediate specificity to E3s. A, ClustalW (49) sequence alignment of UBE2 class I (UBE2D1–4) and class III (UBE2E1–3) E2-conjugating enzymes. The figure was generated using ESPript (50). Black background indicates 100% similarity, and framed residues indicate amino acids of similar chemical character. B, in vitro ubiquitination assays with class III E2 ubiquitin-conjugating enzymes, with and without N-terminal extensions. UBE2E1 and UBE2E2 support Ro52 activity, whereas full-length UBE2E3 does not support the activity of Ro52; in contrast, UBE2E3ΔN (lacking the N terminus) is active and generates polyubiquitin chains together with Ro52.
FIGURE 4.
FIGURE 4.
Antibodies toward the RING domain are common in sera from patients with Sjögren's syndrome. A, number of Ro/SSA-positive patients with Sjögren's syndrome or SLE that had Ro52 autoantibodies in their sera; B, percentage of patients positive for Ro52 full-length zinc binding region and RING and B-box domains, respectively. Antibody levels to the full-length Ro52 (Ro52-1), zinc binding region (Ro52-4), RING, and B-box domains in patients with Sjögren's syndrome (C) and SLE (D).
FIGURE 5.
FIGURE 5.
Patient sera reactive to the Ro52 RING domain inhibit the E3 activity of Ro52. A, Ro52 was used with UBE2E1 for in vitro ubiquitination reactions. Serum P1, not containing anti-Ro52 antibodies, did not inhibit the E3 activity of Ro52. In contrast, serum P2 containing anti-RING antibodies markedly inhibited the ubiquitination reaction. B, Ig fractions of the sera P2, P3, and P6 all inhibited ubiquitination, whereas Ig fractions of the anti-RING-negative sera P1, P4, and P5 did not inhibit ubiquitination. A monoclonal antibody toward the linker peptide of the zinc binding domain (7.12E11) did not inhibit Ro52 E3 activity. An anti-FLAG monoclonal antibody was used as a negative control. HC, heavy chain. C, immunoblotting of Ig-purified sera and affinity-purified serum P2 to detect the IgG content. The affinity-purified serum is shown in the 3rd lane and is denoted by P2 aff. D, Ro52-binding activity in patient sera and corresponding Ig fractions was analyzed by ELISA using Ro52. E, Ig fraction and affinity-purified antibodies of patient serum P2 were tested in ELISA for retained activity to the RING domain using the RING-RBL construct in ELISA. F, no antibodies were detected against ubiquitin, UBE2E1, UBE2E2, or E1 using patient sera P1–P6 in ELISA. (Pos, positive control antibodies.)
FIGURE 6.
FIGURE 6.
Binding interface mapping of the RING-RBL/UBE2E1 and RING-RBL/P6 interaction. A, overlay view of a series of 1H,15N-HSQC spectra of the RING-RBL at pH 8 with decreasing RING-RBL/UBE2E1 ratios from 1:0 (dark blue), 1:0.25 (light blue), 1: 0.5 (purple), 1: 0.8 (pink), and 1:1.4 (orange). The red cross-bars indicate residues that exhibited the strongest CSPs. A zoom of each of those residues is shown to the right of the full HSQC spectrum with the same color code. B, upper panel shows a schematic representation of the RING-RBL region, indicating the position of loop 1 and loop 2 (L1 and L2), and the secondary structure elements. The lower panel shows the chemical shift perturbation per residue for the UBE2E1 titration. Only assigned residues are listed on the x axis. The dashed line indicates the significance level (see “Experimental Procedures”) above which residues are considered to be involved in the E2/E3 interaction. Among these residues, Ile-18, Val-24, Ile-29, Cys-39, Ile-40, Cys-50, Val-53, and Cys-54 as well as residues Glu-25 and Leu-59 below the CSP cutoff exhibit significant line broadening (>40% larger line widths compared with the signals in the absence of E2). Other residues exhibit line broadening below 5%. C, three-dimensional view visualization of combined V8 and trypsin proteolysis of the RING-RBL (upper panel) and the RING-RBL-UBE2E1 complex (lower panel). Plotted are the relative cleavage propensities (z axis), versus sequence (x axis), versus time (y axis). Only those residues that exhibit relative cleavage propensities are labeled. Because residues Glu-12, Glu-13, Asp-21, Glu-25, Glu-30, Glu-38, Lys-45, and Arg-55 do not show any relative cleavage propensities in the absence or presence of UBE2E1, the region comprising residues 12–57 is omitted to increase clarity. Cleavage sites for RING-RBL are as follows: Arg-6, Glu-12, Glu-13, Asp-21, Glu-25, Glu-30, Glu-38, Lys-45, Arg-55, Arg-57, Lys-61, Arg-64, Arg-67, Lys-77, Glu-78, Glu-82, Arg-84, Glu-85, Glu-90, and Arg-91.
FIGURE 7.
FIGURE 7.
Mutational analysis of Ro52/autoantibody interaction in the RING domain. A, 35 anti-Ro52 RING-RBL antibodies containing human patient sera were tested in ELISA to assess binding to the RING-RBL, as well as a mutant designed to disrupt the L2 epitope (RING-RBL V50D,L59D) and a mutant designed to disrupt the RBL epitope (RING-RBL A83D,R84A). B, RING-RBL binding monoclonal 7.12E11, which does not affect ubiquitination, was tested in ELISA with the same mutants as in A. C, E3 ligase activity of the mutants in A assessed by in vitro ubiquitination. All lanes were derived from the same blot with the same exposure but were nonadjacent in the original image. w/o, without.
FIGURE 8.
FIGURE 8.
A structural model for antibody interference with Ro52-mediated ubiquitination. A, model of the RING domain generated from previous homology modeling and mass spectrometry (27), with residues of the E2 binding interface as determined by NMR spectroscopy highlighted in cobalt blue. Loops L1 and L2 continue N- and C-terminally but are not part of this model. The third region found to potentially contribute to the Ro52 E3 interaction with E2 UbcH6 (Cys-39–Gln-42) is highlighted in light blue, and the remainder of the Ro52 RING-RBL backbone amino acid chain is colored gray. Residue Val-50, which interacts with both the E2 and patient autoantibodies, is highlighted in purple. This figure was generated with PyMOL. B, schematic illustration of how patient Ro52 autoantibodies targeting the RING domain obstruct interaction of the E2 with the E3 Ro52 and thus inhibit the ubiquitination process. C, antibodies specific for the RBL-linker domain (monoclonal 12E11 and a subset of patient autoantibodies) do not interfere with the E2/E3 interaction, and the ubiquitination process is therefore not disrupted.
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References

    1. Strandberg L., Ambrosi A., Espinosa A., Ottosson L., Eloranta M. L., Zhou W., Elfving A., Greenfield E., Kuchroo V. K., Wahren-Herlenius M. (2008) J. Clin. Immunol. 28, 220–231 - PubMed
    1. Wahren M., Tengnér P., Gunnarsson I., Lundberg I., Hedfors E., Ringertz N. R., Pettersson I. (1998) J. Autoimmun. 11, 29–38 - PubMed
    1. Reymond A., Meroni G., Fantozzi A., Merla G., Cairo S., Luzi L., Riganelli D., Zanaria E., Messali S., Cainarca S., Guffanti A., Minucci S., Pelicci P. G., Ballabio A. (2001) EMBO J. 20, 2140–2151 - PMC - PubMed
    1. Borden K. L., Freemont P. S. (1996) Curr. Opin. Struct. Biol. 6, 395–401 - PubMed
    1. Borden K. L., Martin S. R., O'Reilly N. J., Lally J. M., Reddy B. A., Etkin L. D., Freemont P. S. (1993) FEBS Lett. 335, 255–260 - PubMed

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