Two Be or Not Two Be: The Nuclear Autoantigen La/SS-B Is Able to Form Dimers and Oligomers in a Redox Dependent Manner

Abstract

According to the literature, the autoantigen La is involved in Cap-independent translation. It was proposed that one prerequisite for this function is the formation of a protein dimer. However, structural analyses argue against La protein dimers. Noteworthy to mention, these structural analyses were performed under reducing conditions. Here we describe that La protein can undergo redox-dependent structural changes. The oxidized form of La protein can form dimers, oligomers and even polymers stabilized by disulfide bridges. The primary sequence of La protein contains three cysteine residues. Only after mutation of all three cysteine residues to alanine La protein becomes insensitive to oxidation, indicating that all three cysteines are involved in redox-dependent structural changes. Biophysical analyses of the secondary structure of La protein support the redox-dependent conformational changes. Moreover, we identified monoclonal anti-La antibodies (anti-La mAbs) that react with either the reduced or oxidized form of La protein. Differential reactivities to the reduced and oxidized form of La protein were also found in anti-La sera of autoimmune patients.

Keywords: La/SS-B autoantigen; anti-La/SS-B antibodies; autoimmunity; primary Sjögren’s syndrome; systemic lupus erythematosus.

Figure 1

The La protein sequence and structure. (A) La protein is conserved during evolution [41,42]. For a detailed review of the sequence and structural comparisons, see also [17]. La proteins contain an N-terminal La motif (blue bar) followed by an RNA recognition motif (RRM1, red bar). The La motif contains an evolutionarily conserved cysteine residue (Cys18). La motif and RRM1 together form the La module (LaM) [17]. In previous studies, the LaM was termed LaN [41,42,43,44]. The sequence between the La motif and the RRM1 is unstructured but forms a helical structure in the case La protein binds to RNA [17]. The monoclonal anti-La antibody (anti-La mAb) 5B9 recognizes this linker (aa311-328 (KPLPEVTDEY, red box)) between the La motif and the RRM1 domain. Recently, mAbs were described by us being directed to conformational epitopes in the La motif (312B, 2F9, 32A, 27E) and RRM1 (22A, 24G7) [45]). Downstream of RRM1 La protein contains a second RRM (RRM2). The RRM2, together with the downstream sequence, represents the previously termed C-terminal La fragment LaC [41,42,43,44]. The RRM2 contains a dimerization domain (Dim, gray bar) [14]. Within this region, a nuclear retention element (NRE) [15] and a nucleolar localization signal (NoLS) [16] were identified. The epitope sequence (EKEALKKIIEDQQESLNK (aa311-328)) recognized by the anti-La mAb 7B6 is also part of this region (blue box, see also below). The data presented below show that aa304-344 is sufficient for dimerization (black line in the gray bar). The RRM2 domain contains the two cysteine residues (Cys232 and Cys245), playing a central role in the ALARM NMR assay [21,22,23]. (B) The structure of La protein is partially solved [17,18,19]. Structural data are available for the La motif, the RRM1 and RRM2 domain. The La motif contains two “wings”. The interdomain sequence, which contains the epitope recognized by the anti-La mAb 5B9, starts downstream of the second wing. The epitope of anti-La mAb 7B6 includes most of the α3-helix in the RRM2 domain.

Figure 2

Accessibility of the epitope recognized by the anti-La mAb 7B6. Influence of fixation. (A) Cells were stained with the anti-La mAb 7B6. (B) The cells in (A) were fixed with methanol that was stored in a plastic bottle. (C) Cells were stained by the anti-La mAb 7B6 (lower panel). DAPI staining was performed to visualize the presence of cells (upper panel). Cells in (C) were fixed with methanol of analytical grade that was stored in a dark brown glass bottle (D). A single drop of hydrogen peroxide was added to the PBS in the washing chamber (E), and the staining procedure of the cells in (C) was repeated (F).

Figure 3

The epitope recognized by the anti-La mAb 7B6 is part of the predicted dimerization domain (Dim), nuclear retention element (NRE) and nucleolar localization signal (NoLS). (A) A series of deletion mutants, which were truncated from either the N- or the C-terminus or from both sites, were cloned as His-tagged fusion proteins, expressed in E. coli and isolated by nickel affinity chromatography. (B) The deletion mutants were tested against either anti-His Abs (upper panel) or the anti-La mAb 7B6 (lower panel). Results for selected deletion mutants are shown, including the identified epitope sequence aa311-328 (EKEALKKIIEDQQESLNK). (C) The epitope sequence consists of most of the α3-helix in the RRM2 plus 3 aa (LNK) of the unstructured region located C-terminally of the α3-helix (see also Figure 1). (D) Besides the deletion mutants shown in (A,B), a series of further fragments related to the epitope sequence recognized by the anti-La mAb 7B6 were prepared and tested, which finally helped us to locate the epitope recognized by the anti-La mAb 7B6 to the aa311-328.

Figure 4

Posttranslational modifications in the epitope sequence of anti-La mAb 7B6. (A) The human (hLa) and the corresponding mouse (mLa) 7B6 epitope sequence differ with respect to the aa highlighted in red. The two aa IE in the human sequence is replaced by aa residues TD. Epitope (E) fusion proteins with an enhanced green fluorescent protein (EGFP) with (E-EGFP-NLS) or without a nuclear localization signal (NLS) (E-EGFP) were constructed. In addition, mutants were constructed in which either the aa isoleucine was replaced by threonine (TE) or the glutamate was replaced by aspartate (ID) (see B). The human epitope sequence was reported to undergo phosphorylation in serine 325 [48]. Therefore, we replaced serine 325 with alanine (S > A) or aspartate (S > D). After transfection, total extracts were prepared and analyzed by SDS–PAGE/immunoblotting (B). Alternatively, cells were fixed and analyzed by IF microscopy (C,D). (B) Cellular extracts were tested for their reactivity to anti-EGFP Abs ((B) anti-GFP, upper panel) or the anti-La mAb 7B6 ((B) anti-La 7B6, lower panel). Cells were manipulated to express either the (C) E-EGFP-NLS or (D) E-EGFP fusion protein and were analyzed by IF microscopy. Transfected cells were identified by the green fluorescence of EGFP (C,D). The reactivity of the anti-La mAb 7B6 was detected in the red channel (C,D). Non-transfected cells were identified besides transfected cells via DNA staining with DAPI (blue channel, C,D). * Due to a slightly different cloning procedure the linker upstream of the His-tag is smaller which explains the lower molecular weight of these fusion proteins.

Figure 5

Dimerization of La protein. Craig et al. reported [14] that La protein contains a dimerization domain (Dim). Within the same region, an NRE [15] as well as a NoLS [16] was identified in independent studies. Structural data argue against a dimerization of La protein [15]. The epitope region recognized by the anti-La mAb 7B6 is also part of the predicted Dim domain and consists mainly of the α3-helical domain in the RRM2. (A) La fragments truncated either from the N- or C-terminus or from both sites, including the epitope region recognized by the anti-La mAb 7B6 (aa311-328), were expressed in E. coli as His-tagged proteins and purified by nickel affinity chromatography. (B) Far-Western blotting analysis was performed. All La-related fragments were separated by SDS–PAGE, and their presence was verified by immunoblotting using an anti-His Ab (B, anti-His). None of the fragments, but full-length wild-type La protein (B, 1–408) reacted with the anti-La mAb SW5 (B, anti-La SW5), which requires N- and C-terminal portions of the RRM1 domain for reactivity [49,50]. Incubation of the blotted La fragments with full-length La protein leads to a stable protein–protein interaction, which could be detected with the anti-La mAb SW5 (La +anti-La SW5). Using this modified Far-Western blotting assay, we identified the fragment aa303-334 as the smallest fragment, which is still able to interact with full-length La protein. (C) The LaN fragment (aa1-192) lacks the Dim domain. Still, after Far-Western blotting, binding of LaN to LaC was detected by both the anti-La mAb SW5 and 5B9. (D) (I) According to these data, besides the proposed head–head/tail-tail dimers [14] also head to tail dimers (II) should be possible and also a mixture of oligomers and even polymers may be formed (III).

Figure 6

SPR studies to confirm La protein–protein interactions. Wildtype La protein was covalently linked to a sensor chip. Binding of full-length La protein (La 1-408) or N- or C-terminally truncated deletion mutants containing (highlighted in green) or lacking (highlighted in red) the identified Dim domain were tested for binding. As negative control served a sensor chip to which bovine serum albumin (BSA) was coupled.

Figure 7

La protein forms dimers and higher oligomers in a redox-dependent manner. (A) As schematically drawn in (1), La protein contains three cysteine residues. One cysteine residue Cys18 (C18) in the La motif and two cysteine residues Cys232 (C232) and Cys245 (C245) in the RRM2 domain. When recombinant La protein is stored in the fridge or at room temperature for several hours, it tends to precipitate ((A, 1), left tube). Mutating all the three cysteine residues (C18, C232, C245) to alanine residues (A18, A232, A245) abolishes this precipitation ((A, 2), right tube). We assumed that the cysteine residues in La protein form disulfide bridges within or between La molecules, finally leading to insoluble oligomers/polymers. (B) In order to differentiate which of the cysteine residue(s) contribute to this oxidation-dependent polymerization, additional mutants were prepared in which (3) either the cysteine residue Cys18 (C18) in the La motif was mutated to alanine (A18, mono cysteine mutant) or (4) both cysteine residues Cys232 and Cys245 (C232, C245) were mutated to alanine (A232, A245, double cysteine mutant). (C) SDS–PAGE/immunoblotting data of untreated, oxidized and reduced samples of wild-type La protein (lanes 1), triple cysteine mutant (lanes 2), mono cysteine mutant (lanes 3), and double cysteine mutant (lanes 4). As “untreated” samples, we used the respective La proteins as isolated by nickel affinity chromatography. Prior to SDS–PAGE, the proteins were prepared in a sample buffer lacking reducing agents. Oxidized samples were obtained by incubation of the respective proteins in the presence of CuSO₄. As for untreated samples, the proteins were prepared for SDS–PAGE in sample buffer lacking reducing agents. After separation by SDS–PAGE and transfer to blotting membranes, the respective samples were analyzed using either the anti-La mAb SW5 or anti-La mAb 7B6.

Figure 8

The α3-helix and the cysteine residues in the RRM2 domain. (A) The anti-La mAb 7B6 is directed to the α3-helix of the RRM2 domain. The accessibility of the epitope is sensitive to oxidation. The RRM2 contains two cysteine residues Cys232 and Cys245 (see arrows). (B) The cysteine residue Cys232 locates upstream of the ß-sheet1, while the cysteine residue Cys245 locates downstream of the ß-sheet in an unstructured region. In the RRM2 domain, the two cysteine residues are located at opposite sites (see arrows in (A,B)). In order to form a disulfide bridge, both cysteine residues must be positioned in close vicinity, which would require a refolding of the RRM2 domain.

Figure 9

Identification of anti-La mAbs to the reduced form of La protein. (A) The reactivities of the anti-La mAbs 2F9, 312B, 32A, and 27E, which are directed against the La motif and the anti-La mAbs 22A, 24G7, which are directed against the RRM1 domain [45] were tested by ELISA against wild-type La (black bars) and the triple cysteine mutant (gray bars). In parallel, the reactivities were determined for the anti-La mAbs 5B9, 7B6, SW5. (B) SDS–PAGE/immunoblotting using the anti-La mAb 2F9. A recombinantly expressed wild-type La protein sample was treated prior to electrophoresis under reducing conditions (sample reduced). The same protein sample was not reduced prior to electrophoresis (sample not reduced, membrane not reduced). The latter blot was cut into two halves. One-half was rinsed in PBS under reducing conditions and tested again against the anti-La mAb 2F9 (sample not reduced, membrane reduced). The position of wild-type La and dimers of La are indicated. (C) SDS–PAGE/immunoblotting using the anti-La mAb 312B. Wild-type La protein (lanes 1), the triple cysteine mutant (lanes 2), the mono cysteine mutant (lanes 3) and the double cysteine mutant (lanes 4) were analyzed. La protein samples were used as obtained after nickel affinity chromatography (untreated). Alternatively, the protein samples were treated with CuSO₄ prior to electrophoresis (oxidized). In both cases, the protein samples were not reduced prior to electrophoresis. As a third alternative, the protein samples were reduced prior to electrophoresis (reduced). (D) Redox sensitivity of the La motif. An ELISA was performed similarly to (A) using instead of wild-type La protein the C-terminally truncated La fragment LaN (gray bars). The LaN sequence contains only the cysteine residue Cys18. In parallel, we tested a mutant LaN fragment in which the cysteine residue Cys18 was mutated to alanine (black bars). The used La fragments are schematically shown in (I). The LaN fragments protein samples were used as obtained after nickel affinity chromatography (II, untreated) or treated with CuSO₄ during coating of the ELISA plates (III) or reduced during coating (IV, reduced).

Figure 10

Comparison of apparent K_D values of anti-La mAbs to the reduced and oxidized form of La protein. Wildtype La protein (wt) and the triple cysteine mutant (La_C₁₈A_C₂₃₂A_C₂₄₅A) were treated with CuSO_4, and the K_D values were determined by ELISA for the anti-La mAbs SW5, 5B9, 7B6, and 312B.

Figure 11

Circular dichroism (CD) analysis. (A) CD spectra were prepared for wild-type La protein (La wild-type), the mono (La_C₁₈A), double (La_C₂₃₂A_C₂₄₅A), and triple cysteine (La_C₁₈A_C₂₃₂A_C₂₄₅A) mutant, including under oxidative conditions. (B) Estimation of the melting temperature of La wild-type and the triple cysteine mutant (La_C₁₈A_C₂₃₂A_C₂₄₅A) in the absence or presence of 90 μM CuSO₄. The protein concentration of each sample was 0.2 mg/mL. CD spectra were collected in the temperature range between 10 °C and 80 °C. Each step represents a difference of 2.5 °C.

Figure 12

Comparison of the reactivity of patient’s anti-La sera against oxidized versus reduced La protein. Wild-type La protein was coated to ELISA plates. Prior to ELISA, La protein was either treated with H₂O₂ (black bars) or DTT (gray bars). Oxidoreduction was verified by the anti-La mAb 2F9. (A) Tested sera were grouped into three categories: (I) sera, which preferentially react with the reduced form of La protein, (II) sera, which preferentially react with the oxidized form of La protein, (III) sera, which do not show major differences in dependence on oxidoreduction. (B) Until now, 64 anti-La positive patient sera were tested for a differential reactivity to either the oxidized or reduced form of La protein. The sera were sorted for their preferential reactivity against either the oxidized (red bar) or reduced form of La protein (green bar). Each black bar represents one serum.