Autoantibodies to dsDNA cross-react with the arginineglycine-rich domain of heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2) and promote methylation of hnRNP A2
K.-H. Sun,
S.-J. Tang1,
Y.-S. Wang,
W.-J. Lin2 and
R.-I. You
Faculty of Medical Technology and Institute of Biotechnology in Medicine, National Yang-Ming University, Taipei,
1 Institute of Marine Biotechnology, National Taiwan Ocean University, Keelung and
2 Institute of Biopharmaceutical Science, National Yang-Ming University, Taiwan, ROC
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Abstract
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Objective. This study was designed to clarify the internalization of anti-DNA antibodies (anti-DNA) into living cells in the pathogenesis of systemic lupus erythematosus (SLE) using anti-DNA monoclonal antibodies (mAbs).
Methods. Anti-DNA mAbs 9D7, 9D7D2, 9A4, 5E3F5, 12B3H2 and 6E11E3 were prepared by a standard hybridoma procedure to determine the interaction of anti-DNA with proteins in different types of cells.
Results. The anti-DNA mAbs reacted with two protein antigens (35 and 50 kDa) in the cells. The 35-kDa antigen was shown to have 100% homology with hnRNP A2. The arginineglycine-rich domain in hnRNP A2 was found to be the reaction site, and the methylation of hnRNP A2 by PRMT1 (protein arginine methyltransferase 1) was increased by anti-DNA. Moreover, anti-DNA was demonstrated to bind and internalize into the cytoplasm and nucleus.
Conclusion. Nuclear localizing anti-DNA may cross-react with hnRNP A2 to modulate the inflammatory responses and polarize immune reactions associated with SLE.
KEY WORDS: Antigen, Autoantibody, Epitope, Systemic lupus erythematosus.
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Introduction
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Circulating autoantibodies to double-stranded DNA (dsDNA) are not only specific for systemic lupus erythematosus (SLE) but also play an important role in the pathogenesis and activity of this disease. Although deposition of circulating DNA-anti-DNA or nucleosome-anti-DNA immune complexes may lead to lupus nephritis [13], the receptor-mediated cellular entry of nuclear localizing anti-DNA antibodies (anti-DNA) has been documented to be a potential link for the functional perturbations at the cellular level [47]. It has been reported that a 30-amino acid peptide corresponding to the combination of the complementarity-determining regions 2 and 3 of the heavy-chain variable region of anti-DNA is able to penetrate into the cytoplasm and nucleus in several cell lines [8]. These findings suggest that there is a relationship between the binding and internalization of anti-DNA into different types of cells and the initiation of cellular events associated with SLE.
A number of membrane components have been reported to be the receptors for the internalization of anti-DNA [47, 916]. Although the membrane receptors for anti-DNA are highly heterogeneous, anti-DNA may interact with receptors on the membrane or penetrate into the cells to localize in the cytoplasm or enter the nucleus [17]. However, information on the mechanisms of the anti-DNA-mediated cellular dysfunction is not abundant [13, 18, 19]. It has been reported that anti-DNA may exert its injurious effects through direct interaction with kidney cells by C-mediated cytotoxicity and potential cell cycle dysfunctions [13]. Moreover, the binding of anti-DNA to DNase I inhibits the activity and leads to resistance to apoptotic stimuli [19].
In a previous study, we demonstrated that anti-DNA monoclonal antibodies (mAbs) derived from lupus-prone mice were able to internalize into human mononuclear cells and in turn stimulate the release of proinflammatory cytokines and interleukin (IL)-10 [18]. This specific effect of anti-DNA may participate in the pathogenesis of lupus by augmenting inflammatory reactions and autoantibody production. In the present study we attempted to identify the cognate antigens for anti-DNA in different cell lines and their interactions after internalization into the cells. The results indicate that heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2) may be a cognate antigen for anti-DNA. By interaction with the arginineglycine-rich domain (RGG) of this protein, anti-DNA may increase the methylation of hnRNP A2 by protein arginine methyltransferase 1 (PRMT1) to trigger the cellular events associated with SLE.
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Materials and methods
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Cell lines
A human acute T-cell leukaemia line (Jurkat), a human lung cancer line (H1299), a human colon cancer line (RKO) and a mouse monocytic leukaemia cell line (J774A.1) were obtained from the American Type Culture Collection (Rockville, MD, USA). The normal rat brain astrocyte line (RBA-1) was obtained from the cell bank at Veterans General Hospital, Taipei, Taiwan.
Preparation of anti-DNA mAbs
The anti-DNA mAbs 9D7, 9D7D2, 9A4, 5E3F5, 12B3H2 and 6E11E3 were prepared by the fusion of NS-1 myeloma cells with spleen cells of autoimmune MRL-lpr/lpr mice. These antibodies have been shown to belong to immunoglobulin (Ig) type G2b, with the exception of 12B3H2, which is an IgG2a [18]. The anti-DNA mAbs were purified from the culture supernatants by protein Aagarose (Sigma, St Louis, MO, USA) affinity chromatography. The purity of each preparation was assessed by the use of 13% SDS-PAGE (sodium dodecyl sulphatepolyacrylamide gel electrophoresis). Although bovine IgG from fetal calf serum could be co-purified, only the heavy chains and light chains of IgG were found in the affinity-purified antibodies. The concentration of mouse IgG was then determined with an enzyme-linked immunosorbent assay (ELISA; Boehringer Mannheim Biochemicals, Mannheim, Germany). Corresponding concentrations of commercially available normal mouse IgG2b (Sigma), WH27 anti-Der p7 (IgG2b) (donated by Horng-Der Shen) and 9B6-4 anti-ribosomal P (IgG1) (prepared by the authors) mAbs were used as non-specific isotype or subclass-matched antibody controls.
DNA binding assay
Polystyrene microtitre plates (Corning, New York, NY, USA) were prepared by precoating with 0.5 mg/ml protamine chloride (150 µl) (Sigma) per well before incubating for 2 h, and were washed with phosphate-buffered saline solution (PBS) (pH 7.2). Calf thymus DNA (Sigma) (0.2 µg in 100 µl/well) was incubated in the microwells at room temperature overnight. After washing with PBS, 2% bovine serum albumin (BSA) (200 µl) in PBS was added and the samples were incubated for 2 h. The plates were washed with PBS and 100 µl of sample was added. After incubating for 1 h, the plates were washed with PBS. Diluted horseradish peroxidase-conjugated goat anti-mouse IgG (Jackson Immunoresearch, West Grove, PA, USA) (100 µl) was then added and incubation was continued for another 1 h. The plates were washed with PBS and 100 µl of a freshly prepared substrate solution (1.37 mg/ml of 2,2'-azinodi-13-ethylbenzthiazoline sulphonic acid in 100 mM phosphate buffer, pH 4.2, containing 0.6 µl/ml 30% H2O2) was added to each well. Optical density was measured at 410 nm with a Dynatech enzyme immunoassay reader (Dynex Technologies, Chantilly, VA, USA) after incubating for 30 min. The anti-DNA antibodies of SLE sera were quantitated with an ELISA kit (BioHyTech, Ramat Gan, Israel).
Western blotting
Lysates from various cell lines and recombinant P proteins were prepared by a method described previously [5]. The cell lysates (5x105 per lane), recombinant P proteins (2 µg/lane) and DNase I (Boehringer Mannheim) were electrophoresed on 13% SDS-PAGE. After electrotransferring and immunoblotting with anti-DNA mAb (10 µg/ml), the antigenantibody complexes on the nitrocellulose paper were detected with peroxidase-coupled anti-mouse IgG and enhanced with chemiluminescence detection kits (NEN, Boston, MA, USA).
Analysis of cognate antigens for anti-DNA
Two-dimensional (2D) gel electrophoresis of Jurkat cell lysate was performed according to a method described previously [6] before immunoblotting with the 9D7 mAb. The Immobiline DryStrip gel (pH 310 L, 11 cm; Amersham Pharmacia Biotech, Uppsala, Sweden) was used for isoelectric focusing in horizontal dimensional electrophoresis and 13% SDS-PAGE was used for vertical electrophoresis. Carbamylated creatine kinase (BDH, Poole, UK) was employed as the isoelectric point calibration marker. After electrotransferring the proteins in the 2D gel to a PVDF membrane, the proteins were stained with 0.1% fast green FCF (Sigma) and immunoblotted with mAb 9D7. The cognate proteins of anti-DNA mAb were then isolated from the 2D gel, dried, and sent to the Beckman Center (Stanford University Medical Center, San Francisco, CA, USA) for matrix assisted laser desorption ionization-mass spectrometry (MALDI-MS) analysis, peptide mapping and microsequencing.
Cloning and mutation of hnRNP A2
The full-length cDNA of human hnRNP A2 was cloned from Jurkat T cells by reverse transcriptionpolymerase chain reaction (RT-PCR) and inserted into the expression vector pET-23a (Novagen, Madison, WI, USA) [20]. The forward primer sequence for the hnRNP A2 gene was 5'-ATCGGTACCAATGGAGAGAGAAAAGGAACAGTTCCG-3' and the reverse primer sequence was 5'-GATAGCTGTGCTCGAGGTATCGGCTCCTCCCACCATAACC-3'. The reverse primer sequences for the mutant hnRNP A2
768 and hnRNP A2
531 were 5'-GATAGCTGTGCTCGAGTCCTCCTCTTCCTCCTCCATAACC-3' and 5'-GATAGCTGTGCTCGAGAGACAAAGCCTTTCTTACTTCTGC-3' respectively. The forward primer sequence for the hnRNP A2
RBD1 was 5'-ATCGGTACCAAAACCAGGGGCTCATGTAACTGTG-3'. The nucleotide sequence of this gene was sequenced and confirmed (Mission Biotech, Taipei, Taiwan). To increase the production of recombinant proteins in bacteria, hnRNP A2 was expressed as a thioredoxin (Trx) fusion protein. Trx was fused to the N-terminus of hnRNP A2. After transforming and expressing the hnRNP A2 gene in BL21(DE3)pLysS cells (Novagen), the protein was purified with a histidine-binding metal chelation resin chromatography kit (Novagen).
HnRNP A2 binding assay
Polystyrene microtitre plates (Corning) were prepared by coating them with 100 µl of 2 µg/ml hnRNP A2 or Trx in 0.05 M carbonate buffer (pH 9.6) per well before incubating them at 4°C overnight. After washing with PBS (pH 7.2), 2% BSA in PBS (200 µl) was added to the wells and incubation was continued for another 2 h. Subsequent procedures of the assay were same as those of the DNA binding assay.
Determination of binding and internalization capacity of anti-DNA mAb
The binding and internalization capacities of the anti-DNA mAb were studied by flow cytometry and fluorescence microscopy [18]. Cells (3x106) were incubated with the culture supernatant of anti-DNA hybridoma (10 µg/ml) or mouse IgG2b (10 µg/ml) in an ice bath for 1 h. After washing with ice-cooled PBS, the cells were incubated with 200x diluted fluorescein isothiocyanate (FITC)-labelled goat anti-mouse IgG (Jackson Immunoresearch) in an ice bath for 40 min. The percentage and mean fluorescence intensity of positive cells were determined using a flow cytometer (Becton Dickinson, San Jose, CA, USA), after washing with ice-cooled PBS.
Internalization of anti-P mAb into the cells was determined according to Avrameas et al. [8]. Cells (3x105/ml) were incubated with the culture supernatants of anti-DNA hybridomas (10 µg/ml) or mouse IgG2b (10 µg/ml) in a 5% CO295% air incubator at 37°C for 18 h. After washing with PBS, the cells were fixed with 2% paraformaldehyde in PBS (pH 7.4) for 25 min, washed with PBS, and permeabilized with 0.1% Triton X-100 in PBS for 5 min. The fixed cells were then incubated with 200x diluted FITC-labelled goat anti-mouse IgG antibodies at room temperature for 40 min. After washing with PBS, intracellular mAbs were observed under a fluorescence microscope (Leica, Heidelberg, Germany). The percentage and mean fluorescence intensity of the positive cells were determined by flow cytometry.
Methylation of hnRNP A2 by PRMT1 in the presence of anti-DNA mAb
The methylation status of hnRNP A2 proteins was determined according to Lin et al. [21]. GST-PRMT1 (glutathione S-transferaseprotein arginine methyltransferase 1) was expressed in Escherichia coli and purified on glutathioneSepharose (Amersham Pharmacia Biotech). Methylation substrate (hnRNP A2, 2 µg) was preincubated with anti-DNA mAb or mouse IgG2b for 15 min and then incubated at 30°C for 30 min after adding recombinant GST-PRMT1 (1 µg) in the presence of S-(5'-adenosyl)-L-methionine-(methyl-3H) (Sigma) (2.8 µCi). Reactions were terminated by adding an equal volume of sample buffer and heating to 100°C for 5 min. Radiolabelled proteins were resolved by SDS-PAGE, stained with Coomassie blue and fluorographed. Trx-hnRNP A2 was excised from the gels and 3H incorporation was measured by liquid scintillation counting.
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Results
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Proteins in cells and tissues reactive with anti-DNA mAbs
Proteins with molecular masses of 35 and 50 kDa in colon cancer cells (Fig. 1A
, lane 1), normal brain astrocytes (lane 2), Jurkat T cells (lane 3), J774 monocytic cells (lane 4) and lung cancer cells (lane 5) were found to be reactive with 9D7 anti-DNA mAb. Reactions between these proteins and 9D7 anti-DNA mAb were also detected in preparations of normal tissues from the liver, kidney, brain, lung, heart and spleen of BALB/c mice by Western blotting (data not shown). Although acidic ribosomal phosphoproteins (P0, P1 and P2) [5] and DNase I [22] may react with anti-DNA and the molecular masses of P0 and DNase I are similar to that of the 35-kDa protein, 9D7 anti-DNA mAbs did not react with these proteins (Fig. 1B
).

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FIG. 1. Western blotting of cell lysates and proteins with 9D7 anti-DNA mAb. (A) Cells (5x105) were blotted with 9D7 anti-DNA mAb (10 µg/ml). Lane 1, RKO; lane 2, RBA-1; lane 3, Jurkat; lane 4, J774A.1; lane 5, H1299. (B) Proteins (2 µg) were blotted with 9D7 anti-DNA mAb (10 µg/ml). Lane 1, DNase I; lane 2, recombinant human P0 protein; lane 3, recombinant human P1 protein; lane 4, recombinant human P2 protein; lane 5, Jurkat T-cell lysate (1x105).
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Identity of the 35-kDa cognate antigen of anti-DNA
To identify the cognate proteins of anti-DNA mAbs, 2D gel electrophoresis of Jurkat cell lysate was performed (Fig. 2C
) before immunoblotting with 9D7 mAb (Fig. 2B
). The 50-kDa protein was not detectable using Coomassie brilliant blue staining. The 35-kDa cognate protein was isolated from the 2D gel, dried, digested with trypsin and identified by peptide mapping and sequencing. MALDI-MS analysis of the peptides map and mass indicated the presence of hnRNP A2 (data not shown). However, it was also possible that glyceraldehyde 3-phosphate dehydrogenase was present. To verify the results, peptide separation and sequencing were performed (Fig. 3
). The protein was found to have 100% homology with hnRNP A2, but not with glyceraldehyde 3-phosphate dehydrogenase. To confirm the reactivity of anti-DNA mAb with hnRNP A2, the cDNA of human hnRNP A2 was cloned and expressed in E. coli. The recombinant hnRNP A2 fusion protein was demonstrated to be reactive with anti-DNA (Fig. 4B
, lane 1).

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FIG. 2. Isolation of cognate proteins of anti-DNA mAb from Jurkat cell lysate by 2D gel electrophoresis. (A) The pI calibration marker (creatine kinase, CPK) was stained with silver nitrate. (B) 2D electrophoresis of the cell lysate was blotted with anti-DNA mAb by isoelectric focusing in the horizontal dimension followed by 13% SDS-PAGE in the vertical dimension. The separated proteins were probed with 9D7 anti-DNA mAb (10 µg/ml) after being electrotransferred to PVDF membrane. (C) 2D electrophoresis of the cell lysate was stained with Coomassie brilliant blue. The arrow shows the 35-kDa cognate protein of anti-DNA. This cognate protein was isolated and sequenced.
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FIG. 3. Amino acid sequence analysis of the 35-kDa protein. Sequences of peptides derived from the digested 35-kDa protein were isolated by 2D gel electrophoresis. The two peptides derived from the 35-kDa protein are completely homologous with heterogeneous nuclear ribonucleoprotein A2.
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Reactivity of affinity-purified 9D7 anti-DNA mAb with calf thymus DNA and hnRNP A2 fusion protein
The reactivity of affinity-purified 9D7 mAb with calf thymus DNA and hnRNP A2 fusion protein was assessed by ELISA (Fig. 5
). Although 10 µg/ml of 9D7 mAb showed an optical density at 410 nm (OD410) of 1 in the hnRNP A2 binding assay (Fig. 5B
), only 1 µg/ml of 9D7 mAb was required to give an OD410 of 1 in the DNA binding assay (Fig. 5A
). Similar findings were obtained for the cultured supernatants of the anti-DNA mAbs 9A4, 9D7D2, 5E3F5 and 6E11E3 (Table 1
). However, 12B3H2 was only reactive with DNA, whereas the control antibodies 9B6-4 and WH27 did not react with DNA or hnRNP A2 fusion protein. Moreover, there was no binding between monoclonal antibodies and Trx (Fig. 5B
).

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FIG. 5. The reactivity of affinity-purified monoclonal antibodies for calf thymus DNA and hnRNP A2 fusion protein was assessed by ELISA. Polystyrene microtitre plates were precoated with (A) calf thymus DNA or (B) hnRNP A2 fusion protein or Trx (0.2 µg/100 µl/well), and 100 µl of various concentrations of purified antibodies was added. The immune complexes were detected by adding horseradish peroxidase-conjugated goat anti-mouse IgG and freshly prepared substrate solution. Optical density was measured at 410 nm after incubation for 30 min. 9D7, anti-DNA mAb (IgG2b); 9B6-4, anti-ribosomal P protein mAb (IgG1); WH27, anti-Der p7 mAb (IgG2b).
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Epitope of hnRNP A2 reacted with anti-DNA mAb was mapped to RGG
To investigate the reaction between the epitope of hnRNP A2 and 9D7 anti-DNA, different deletion mutants of hnRNP A2 (Fig. 4A
) were cloned, expressed and subjected to Western blotting with anti-DNA (Fig. 4B
). The mutants hnRNP A2
768 (lane 2) and hnRNP A2
RBD1 (lane 4), which contain the RGG domain, were demonstrated to be reactive with anti-DNA. However, anti-DNA did not react with the mutants hnRNP A2
531 (lane 3) and hnRNP A2-RBD2 (lane 5), which contain only the RNA-binding domain (RBD1 and/or RBD2).
Cellular binding and internalization capabilities of anti-DNA mAb
The cellular binding and internalization capabilities of anti-DNA mAb were determined by incubating various types of cells with anti-DNA or mouse IgG2b at 4°C for 1 h (Fig. 6A
, left panel) and 37°C for 18 h (Fig. 6A
, right panel). Anti-DNA mAb (Fig. 6A
, filled area) was found to bind significantly with RBA-1 cells and internalize into RBA-1 and Jurkat cells. After entering the cells, anti-DNA mAb was observed to interact with proteins in the cytoplasm or inside the nucleus (Fig. 6B
).

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FIG. 6. Binding and internalization of anti-DNA to the cells was analysed by flow cytometry and fluorescence microscopy. (A) Cells (3x105/ml) were incubated with the cultured supernatant of 9D7 anti-DNA hybridoma (10 µg/ml, filled area) or mouse IgG2b (10 µg/ml, open area) in an ice bath for 1 h for the binding assay (left panels) and in a 5% CO295% air incubator at 37°C for 18 h for the internalization assay (right panels). In the binding assay, the cells were then incubated with FITC-labelled goat anti-mouse IgG in an ice bath for 40 min. In the internalization assay, the cells were fixed and permeabilized with 0.1% Triton X-100. The fixed cells were then incubated with FITC-labelled goat anti-mouse IgG antibodies at room temperature for 40 min. The percentage and mean fluorescence intensity of the positive cells were determined using a flow cytometer. (B) Internalization of monoclonal anti-DNA antibody (9D7) into RBA-1 cells was analysed by fluorescence microscopy.
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Increased methylation of hnRNP A2 by PRMT1 in the presence of 9D7 anti-DNA mAb
Anti-DNA may internalize into cells and react with the RGG domain of hnRNP A2. To investigate the effect of anti-DNA on methylation of hnRNP A2, recombinant PRMT1 was used (Fig. 7
). In the presence of anti-DNA (lane 3), methylation of hnRNP A2 by PRMT1 increased to about 3.5 times the control level. The potentiation effect of anti-DNA was dose-dependent (lane 2 and 3). However, mouse IgG2b only slightly elevated the methylation of hnRNP A2 by PRMT1 (lanes 4 and 5).

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FIG. 7. Methylation of hnRNP A2 by PRMT1 in the presence of 9D7 anti-DNA mAb. (A) Methylation substrate (Trx-hnRNP A2, 2 µg) was incubated with recombinant GST-PRMT1 (1 µg) and anti-DNA mAb or mouse IgG2b in the presence of [3H]AdoMet (2.8 µCi) at 30°C for 30 min. After termination of the reactions, the radiolabelled proteins were resolved by SDS-PAGE and fluorographed. Lane 1, control (no antibody added); lane 2, 0.04 µg anti-DNA mAb; lane 3, 0.16 µg anti-DNA mAb; lane 4, 0.04 µg mouse IgG2b; lane 5, 0.16 µg mouse IgG2b. (B) TrxhnRNPA2 was excised from the gels and 3H incorporation was measured by liquid scintillation counting. Column 1, control (no antibody added); column 2, 0.04 µg anti-DNA mAb; column 3, 0.16 µg anti-DNA mAb; column 4, 0.04 µg mouse IgG2b; column 5, 0.16 µg mouse IgG2b. cpm, counts per minute.
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Discussion
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Polyreactivity is a common property of natural and disease-associated human autoantibodies [23]. Anti-DNA may cross-react with myosin 1 [4], DNase I [22], ribosomal P protein [5], ribosomal protein S1 [24], HP8/HEVIN [14], nucleosome [16] and SnRNP proteins A and D [25]. In this study, we have demonstrated the cross-reaction between anti-DNA and hnRNP A2 by several approaches. These findings confirm the hypothesis of the heterogeneity of SLE by showing that the cross-reactive antigens for anti-DNA are heterogeneous and that different antigens may mediate different cellular fates for anti-DNA [17].
The hnRNPs are nuclear RNA-binding proteins and participate in a wide range of cellular activities from transcription and pre-mRNA processing in the nucleus to mRNA translation and trafficking in the cytoplasm [26]. hnRNP A is one of 20 major hnRNPs, and it may shuttle between the nucleus and cytoplasm and take part in the export of mRNA to the cytoplasm [27]. These proteins consist of two highly conserved RBD domains in the N-terminus and an RGG domain in the C-terminus. The RBD domain may interact with specific RNA proteins and are essential for the modulation of alternative splicing. The RGG domain may mediate proteinprotein interaction and is necessary for alternative splicing activity and the stabilization of RNA binding [28]. Because hnRNP A2 is a substrate for the arginine methyltransferase PRMT1, methylation of the RGG domain has been considered to be critical for nuclear localization of the proteins [29].
In addition to acting as a pre-mRNA binding protein in nuclear mRNA processing, hnRNPs A may also participate in the regulation of gene expression [3033]. This protein may interact specifically with the reiterated AUUUA sequences in the 3'-UTR (untranslated region) of labile mRNAs [30] and enhance the stability of IL-2 [33] and GM-CSF (granulocyte/macrophage colony-stimulating factor) [31] mRNA in T cells. In this study, the epitope of hnRNP A2 reacted with anti-DNA have been mapped in the RGG domain. Moreover, a significant increase in the methylation of hnRNP A2 by PRMT1 has also been observed in the presence of anti-DNA. After internalization, anti-DNA may interact with the RGG domain of hnRNP A2 and promote methylation. We have demonstrated that anti-DNA has a dual effect on normal human mononuclear cells (MNC). This effect not only enhances the release of proinflammatory cytokines IL-1ß, IL-8 and TNF-
from MNC to augment inflammatory reaction but also polarizes the immune reaction towards the T helper 2 (Th2) (increased IL-10 production) pathway [18]. The methylation of hnRNP A2 may in turn modulate the expression of proinflammatory cytokines and play a role in the pathogenesis of lupus by augmenting inflammatory reactions.
It has been demonstrated that the antigen-binding regions of anti-DNA resemble the nuclear localization sequences in several cell lines [8]. However, we determined the interaction between anti-DNA and hnRNP A2. Moreover, hnRNP A2 may shuttle between the cytoplasm and nucleus [27]. Because internalization of anti-DNA into the cytoplasm and nucleus has been demonstrated by fluorescence microscopy (Fig. 6B
), it is possible that hnRNP A2 may take part in the internalization process. However, anti-DNA mAb may only enter into a fraction of the cells (Fig. 6
). This may be because of the recycling of intracellular immunoglobulins back to the cell surface and their dissociation from the cell membrane into the medium [4]. In our preliminary study, we have observed the interaction of anti-DNA and hnRNP A2 expressed on the cell surface by the biotinylation of membrane proteins (data not shown). This possible cognate antigen on the cell surface may also vary at different stages of the cell cycle.
In addition to the 35-kDa hnRNP A2, a 50-kDa protein was also found to be reactive with anti-DNA (Fig. 1
). Although this protein was not detectable by Coomassie brilliant blue staining (Fig. 2C
), we demonstrated its reaction with anti-DNA by Western blotting (Fig. 2B
). These findings indicate that the 50-kDa protein may have potent reactivity with anti-DNA. Further studies are needed to determine its identity and its role in the pathogenesis of SLE.
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Acknowledgments
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This work was supported by grants from the National Science Council (90-2320-B-010-103), the VTY Joint Research Program, Tsou's Foundation (VTY90-G5-05, VTY91-P5-41) and the Yen Tjing Ling Medical Research Foundation (CI-88-3-2, CI-90-3-2), ROC. We thank Dr Horng-Der Shen for the kind donation of the WH27 monoclonal antibody.
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Notes
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Correspondence to: K.-H. Sun, Faculty of Medical Technology and Institute of Biotechnology in Medicine, National Yang-Ming University, 155 Section 2, Lie-Nong Street, Shih-Pai, Taipei, Taiwan 112, ROC. E-mail: ksun{at}ym.edu.tw 
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Submitted 1 May 2002;
Accepted 28 June 2002