C1D is a major autoantibody target in patients with the polymyositis–scleroderma overlap syndrome

Abstract

Objective

To assess whether the recently discovered exosome-associated proteins MPP6, C1D, KIAA0052/hMtr4, hSki2, and hSki8 are targeted by autoantibodies, and to determine whether these autoantibodies are accompanied by antibodies directed to the established exosome-associated autoantigens PM–Scl-75 and PM–Scl-100.

Methods

Complementary DNAs encoding the recently identified human exosome–associated proteins were expressed as His-tagged fusion proteins in Escherichia coli cells. Sera obtained from patients with several different autoimmune diseases were analyzed for the presence of autoantibodies directed to these proteins, in an enzyme-linked immunosorbent assay (ELISA). The ELISA data obtained for C1D were confirmed by Western blot analysis, using recombinant C1D.

Results

All exosome-associated proteins were found to be targeted by autoantibodies, although the frequency with which such antibodies occurred in patient sera was relatively low, with the exception of anti-C1D antibodies. Autoantibodies recognizing C1D were detected in 7 of 30 patients (23%) with the polymyositis (PM)–scleroderma overlap syndrome; this frequency was similar to the frequencies for the established autoantigens PM–Scl-75c (27%) and PM–Scl-100 (23%). Importantly, several patients with the PM–scleroderma overlap syndrome had anti-C1D antibodies but no anti–PM-Scl antibodies. Anti-C1D autoantibodies were observed in only 2 of 204 patients with other diseases, including PM, dermatomyositis, and scleroderma.

Conclusion

Our results demonstrate that the recently identified exosome-associated protein C1D is a major autoantigen in patients with the PM–scleroderma overlap syndrome and suggest that the use of recombinant C1D as an autoantibody target may aid in diagnosis of the PM–scleroderma overlap syndrome.

Idiopathic inflammatory myopathies (IIMs) represent a heterogeneous group of autoimmune diseases characterized by skeletal muscle weakness, elevated serum levels of muscle enzymes, and chronic inflammatory infiltrates. IIMs include 3 major diseases: polymyositis (PM), dermatomyositis (DM), and inclusion body myositis (1). In patients with clinical symptoms of PM or DM and scleroderma (the so-called PM–scleroderma overlap syndrome), disease-specific autoantibodies have been shown to recognize a nucleolar complex consisting of 11–16 proteins ranging from 18 kd to 110 kd. These autoantibodies serve as important markers for the PM–scleroderma overlap syndrome and are present in approximately one-fourth of patient sera. Anti–PM-Scl autoantibodies are also present in 6% of patients with PM or DM and in 2% of patients with scleroderma (2).

Further characterization of anti–PM-Scl–positive sera led to identification of the main autoantigenic proteins, PM–Scl-100 (a 100–110-kd protein) and PM–Scl-75 (a 50-kd protein), which migrate aberrantly in sodium dodecyl–polyacrylamide gel electrophoresis (SDS-PAGE) due to a highly charged C-terminus (3, 4). The major reactivity of anti–PM–Scl-100 antibodies appeared to be directed to an α-helical element of this protein (5). Indeed, an enzyme-linked immunosorbent assay (ELISA) based on this autoantigenic epitope proved to be a sensitive method to facilitate the diagnosis of the PM–scleroderma overlap syndrome (6). The autoantigenic epitope(s) of the PM–Scl-75 protein are less well defined, although a recent study provided evidence that a newly identified isoform of PM–Scl-75, termed PM–Scl-75c, contains a previously unidentified N-terminal region that is important for the autoantigenicity of the protein (7).

In 1999, the PM-Scl complex was shown to be the human counterpart of the yeast exosome, a complex consisting of RNA-binding proteins and 3′–5′ exoribonucleases, including Rrp45 (the yeast homolog of PM–Scl-75) and Rrp6p (the yeast homolog of PM–Scl-100), which is required for the processing and/or degradation of different types of RNAs (2). Besides PM–Scl-75 and PM–Scl-100, human homologs of all other yeast exosome components have been identified (8), and several of these components are also recognized by anti–PM-Scl antibodies, although to a lesser extent compared with PM–Scl-75 and PM–Scl-100 (9).

In recent years, several exosome-associated proteins have been identified that might be involved in processes such as the recruitment of the exosome to substrate RNAs. These proteins include MPP6, C1D, and KIAA0052 and were found to be required for efficient ribosomal RNA processing by the exosome. Because KIAA0052 was reported to be a human homolog of yeast Mtr4p, it will hereafter be referred to as hMtr4 (10–12). The recently described hSki2 and hSki8 proteins are part of the hSki complex, which was proposed to regulate cotranscriptional events such as messenger RNA (mRNA) quality control together with the exosome and human PAF complex (13). The hSki complex is related to the yeast Ski complex, which, together with the exosome, mediates 3′–5′ mRNA degradation in cytoplasm (2).

The aim of this study was to assess whether the human exosome–associated proteins MPP6, C1D, hMtr4, hSki2, and hSki8 are targeted by autoantibodies. The presence and prevalence of such autoantibodies were investigated using sera from patients with the PM–scleroderma overlap syndrome and those with various other diseases, in a recombinant protein ELISA.

PATIENTS AND METHODS

Patients.

Most patient sera were obtained from the departments of rheumatology and neurology of the University Medical Center St. Radboud, Nijmegen. Patients with PM, DM, scleroderma, or overlap syndromes of scleroderma with PM or DM (both of which are referred to as PM–scleroderma in this study) were classified according to the classification scheme described by Bohan and Peter, as modified by Cronin et al (14). Diagnoses were made according to established criteria, and the same sera were also included in a previous study that assessed the prevalence of anti-exosome antibodies in these patients (7). A separate group of sera from patients with IIM was selected, based on the presence of anti–PM–Scl-100 and/or anti–PM–Scl-75c antibodies. To assess the disease specificity of the antibodies detected, sera from patients with systemic lupus erythematosus (SLE), patients with multiple sclerosis (MS), and patients with rheumatoid arthritis (RA) were analyzed.

Expression and purification of recombinant proteins.

The complementary DNA (cDNA) encoding MPP6 (GenBank accession no. NM_005792) was cloned into the pQE32 vector (Qiagen, Hildon, Germany); the cDNAs encoding C1D (GenBank accession no. NM_006333) and hSki8 (GenBank accession no. CR457333) were cloned into suitable pET30 vectors (Novagen, Madison, WI). Due to low expression levels of full-length recombinant hMtr4 and hSki2 proteins, deletion mutant constructs were generated using appropriate internal restriction sites. The cDNA fragment of hMtr4 (clone IRATp970F0129D6, provided by the I.M.A.G.E. consortium [online at http://image.llnl.gov/image/html/idistributors.shtml]) was cloned into the pET30b vector using the internal restriction site Hind III (this construct encodes the N-terminal 800 amino acids of hMtr4); the cDNA fragment of hSki2 (GenBank accession no. Z48796) was cloned into the pET30b vector using the internal restriction site Sal I (this construct encodes the N-terminal 808 amino acids of hSki2).

The histidine-tagged recombinant proteins were expressed in Escherichia coli BL21/pLysS cells and purified by Ni²⁺ affinity-column chromatography using nickel–nitriloacetic acid Sepharose (Qiagen). The purity of the recombinant proteins was determined by SDS-PAGE and quantified by a BCA Protein Assay kit (Pierce, Rockford, IL). The proteins His-hSki8, His-hSki2, and His-hMtr4 appeared to accumulate in the insoluble fraction of the corresponding bacterial lysate and were purified in the presence of 7M urea.

ELISAs.

ELISAs were performed as described previously (9), with the exception that the microtiter plates were blocked with 400 μl of ELISA blocking buffer (5% skimmed milk, phosphate buffered saline [PBS], 0.2% Tween 20). Serum samples were diluted 100-fold in ELISA blocking buffer. Test results were considered to be positive if the optical density values exceeded the calculated cutoff value, which was determined by analyzing at least 3 negative control sera per plate with 99.9% confidence (15). All patient sera were tested in duplicate.

Western blot analysis.

For Western blot analysis, His-tagged recombinant C1D was separated by SDS-PAGE and transferred to nitrocellulose membranes. The blots were blocked with Western buffer (5% skimmed milk, PBS, 0.05% NP40) and incubated with 1,000-fold–diluted autoimmune patient sera in the same buffer. Subsequently, blots were incubated with a secondary antibody, rabbit anti-human IgG (Dako, Copenhagen, Denmark) diluted 2,500-fold in Western buffer, and a tertiary antibody, goat anti-rabbit IRDye 800CW (Li-Cor, Lincoln, NE) diluted 10,000-fold in Western buffer. Bound antibodies were visualized with the Odyssey Infrared Imaging System (Li-Cor).

RESULTS

Role of C1D in patients with the PM–scleroderma overlap syndrome.

The autoantibody response to the recently identified exosome-associated proteins MPP6, C1D, hMtr4, hSki2, and hSki8 was studied by ELISAs, using sera from 44 patients with PM, 43 patients with DM, 51 patients with scleroderma, 30 patients with the PM–scleroderma overlap syndrome, 22 patients with MS, 22 patients with RA, and 22 patients with SLE. Additionally, sera from 87 patients with IIMs were analyzed; these sera were selected based on their reactivity with the established PM-Scl autoantigens, PM–Scl-75c (n = 37), PM–Scl-100 (n = 81), or both (n = 31). Table 1 summarizes the reactivity of these patient sera with each of the exosome-associated proteins analyzed. Autoantibodies to these proteins appeared to be most prevalent in the IIM and the PM–scleroderma overlap groups. In total, 1.2% (4 of 321) of the sera were reactive with MPP6, 4.4% (14 of 321) were reactive with C1D, 2.5% (8 of 321) were reactive with hMtr4, 2.2% (7 of 321) were reactive with hSki2, and 0.9% (3 of 321) were reactive with hSki8.

Table 1. Reactivity of patient sera with exosome-associated proteins

Disease	No. of samples tested	Protein, no. (%) positive
		MPP6	C1D	hMtr4	hSki2	hSki8	PM–Scl-75c	PM–Scl-100
* PM = polymyositis; IIM = idiopathic inflammatory myopathy.
Polymyositis	44	0 (0)	2 (4.5)	1 (2.3)	2 (4.5)	0 (0)	0 (0)	1 (2.3)
Dermatomyositis	43	1 (2.3)	0 (0)	0 (0)	1 (2.3)	1 (2.3)	1 (2.3)	1 (2.3)
Scleroderma	51	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	5 (10)	1 (2)
Polymyositis–scleroderma overlap	30	0 (0)	7 (23)	2 (6.7)	0 (0)	1 (3.3)	8 (27)	7 (23)
Multiple sclerosis	22	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	1 (4.5)
Rheumatoid arthritis	22	0 (0)	0 (0)	0 (0)	1 (4.5)	0 (0)	1 (4.5)	2 (9.1)
Systemic lupus erythematosus	22	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	1 (4.5)	0 (0)
IIM (PM-Scl–positive)*	87	3 (3.4)	5 (5.7)	5 (5.7)	3 (3.4)	1 (1.1)	37 (43)	81 (93)
Total	321	4 (1.2)	14 (4.4)	8 (2.5)	7 (2.2)	3 (0.9)	53 (17)	94 (29)

It is noteworthy that the recombinant hSki2, hSki8, and hMtr4 proteins were isolated in the presence of urea, which may lead to a loss of autoantigenic epitopes due to (partial) denaturation of the protein. As a result, we cannot exclude the possibility that our data underestimate the autoantigenicity of these proteins. Interestingly, 7 of the sera that were reactive with C1D were from patients with the PM–scleroderma overlap syndrome (23%), which is comparable with the frequency by which PM–Scl-75c (27%) and PM–Scl-100 (23%) are targeted in this patient group. Anti-C1D autoantibodies were found in only 2 of 204 patients with other diseases, including PM, DM, and scleroderma. Figure 1A illustrates the autoantibody levels to C1D in PM–scleroderma sera compared with control sera (the antibody ratio was calculated by dividing the observed reactivity by the cutoff value).

Figure 1. Reactivity of patient sera with recombinant C1D. A, Results of enzyme-linked immunosorbent assay (ELISA) showing reactivity of CD1 with sera obtained from individual patients with different autoimmune diseases. The cutoff used for positivity (broken line) was determined based on the signals obtained with normal human sera. The antibody ratio was calculated using the following equation: (observed reactivity/cutoff value) × 10. B, Western blot analysis of patient sera that recognized C1D in ELISA and some sera that were not reactive with C1D in ELISA. The blots contain His-tagged recombinant C1D protein. Lanes 1–9, Sera from patients with the polymyositis–scleroderma (PM/Scl) overlap syndrome. Lanes 10–15, Sera from patients with idiopathic inflammatory myopathy (IIM). Lanes 16–18, Sera from patients with PM. The reactivity of these sera with C1D in an ELISA is indicated above the lanes. Arrow indicates the position of full-length His-tagged recombinant C1D. Values on the left indicate the positions of molecular mass markers (in kilodaltons). DM = dermatomyositis; MS = multiple sclerosis; RA = rheumatoid arthritis; SLE = systemic lupus erythematosus.

To confirm the presence of anti-C1D autoantibodies in these sera, the sera were also analyzed by Western blotting using His-tagged recombinant C1D. Figure 1B shows that the ELISA data obtained using sera from patients with PM–scleroderma overlap syndrome (lanes 1–7) and sera from patients with PM (lanes 16 and 17) were confirmed by Western blotting. In contrast, only 4 of the 5 anti-C1D–positive IIM sera (lanes 10–14) were reactive by Western blotting. As a control, several anti-C1D–negative sera were also analyzed by Western blotting but did not react with C1D (lanes 8, 9, 15, and 18). In general, sera displaying a high antibody ratio in ELISA also strongly reacted with C1D on Western blotting.

Co-occurrence of autoantibodies directed to different exosome-associated proteins.

Because anti–PM–Scl-75c autoantibodies are often observed in combination with anti–PM–Scl-100 autoantibodies, we investigated the co-occurrence of autoantibodies directed to the exosome-associated proteins. However, most patient sera recognized only 1 of the exosome-associated proteins, suggesting that there is no relationship between the generation of antibodies to the proteins that interact in vivo and their targeting by autoantibodies. Six patient sera recognized ≥2 exosome-associated proteins. Interestingly, the most frequently observed combination was the recognition of hMtr4 and hSki2 (4 of 6 sera), both of which are members of a subfamily of putative RNA helicases and are 37% identical at the amino acid sequence level (data not shown).

The co-occurrence of anti–PM-Scl and anti-C1D antibodies in sera from patients with anti– PM-Scl–positive IIM and patients with the PM–scleroderma overlap syndrome is illustrated in Figure 2. The majority of PM-Scl–positive sera from patients with IIM were selected based on their reactivity with PM–Scl-100, whereas some of these sera were selected because they contained autoantibodies to PM–Scl-75c. Two of 5 anti-C1D–positive sera from patients in the anti–PM-Scl–positive IIM cohort were also reactive with PM–Scl-100, whereas the other 3 sera were reactive with both PM–Scl-100 and PM–Scl-75c. As expected, no sera were reactive with C1D alone, because the sera from patients with IIMs were selected for their reactivity with either PM–Scl-100 and/or PM–Scl-75c (Figure 2A).

Figure 2. Reactivity of patient sera with PM–Scl-100, PM–Scl-75c, and C1D and co-occurrence of autoantibodies that recognize the recombinant proteins, in sera obtained from patients with anti–PM-Scl–positive IIM (A) and patients with the PM–scleroderma overlap syndrome (B). The asterisk indicates that no sera from patients with anti–PM-Scl–positive IIM were reactive with C1D alone, because these sera were selected for their reactivity with either PM–Scl-100 and/or PM–Scl-75c. Values are the number of positive sera. See Figure 1 for definitions.

Among the 30 sera from patients with the PM–scleroderma overlap syndrome, 11 were reactive with at least 1 of the antigens. Five of these sera appeared to contain autoantibodies to PM–Scl-75c and PM–Scl-100, as well as C1D. One serum sample was reactive with both PM–Scl-75c and PM–Scl-100, whereas no sera were reactive with a combination of C1D and PM–Scl-75c or PM–Scl-100. More importantly, the other 5 PM–scleroderma sera were reactive with only 1 of the 3 antigens, PM–Scl-100 (1 serum), PM–Scl-75c (2 sera), or C1D (2 sera). When the total group of patients with the PM–scleroderma overlap syndrome analyzed in this study was taken into account, 9 of 30 sera (30%) were reactive with PM–Scl-75c and/or PM–Scl-100. The inclusion of anti-C1D antibodies increased the frequency of PM–scleroderma-specific autoantibodies to 37%.

DISCUSSION

In this study, we showed that the exosome-associated proteins MPP6, C1D, hMtr4, hSki2, and hSki8 are targeted by autoantibodies, although patient sera containing such autoantibodies are rare. C1D was targeted relatively frequently by autoantibodies present in sera from patients with the PM–scleroderma overlap syndrome. For these patients, the PM–Scl-100 and PM–Scl-75c proteins are thought to carry the main autoantigenic epitopes, making anti–PM-Scl antibodies an important serologic marker. On Western blot, however, many PM–scleroderma sera stained several other proteins ranging from ∼18 kd to ∼110 kd. Interestingly, C1D is a nucleolar protein (16) of ∼18 kd, and our data showed that in some patients with the PM–scleroderma overlap syndrome, anti-C1D antibodies were present in the absence of anti–PM–Scl-75c and anti–PM–Scl-100 antibodies.

The presence of anti-PM/Scl antibodies in patient sera is often monitored by ELISA using a recombinant PM–Scl-100 protein. The results of recent studies, however, suggested that the inclusion of full-length recombinant PM–Scl-75c or a peptide based on an autoantigenic epitope of PM–Scl-100 can yield a more sensitive ELISA result in comparison with use of recombinant PM–Scl-100 alone (6, 7). Although more PM–scleroderma sera have to be tested for reactivity with the C1D antigen, our data also indicate that the use of C1D for autoantibody detection in sera from patients with the PM–scleroderma overlap syndrome may improve the sensitivity. Besides, anti-C1D reactivity was detected in only 4.5% of 44 PM sera and 5.7% of anti–PM-Scl–positive IIM sera (n = 87). None of the 160 DM, scleroderma, MS, RA, and SLE sera contained detectable amounts of anti-C1D autoantibodies. These data strongly suggest that production of these antibodies is specifically associated with the PM–scleroderma overlap syndrome.

The initial events that trigger an autoantibody response are still largely unknown. However, modifications occurring during cell death may generate neoepitopes that elicit a primary autoimmune response. Indeed, PM–Scl-75c has been shown to be cleaved by caspases during apoptosis (16), whereas PM–Scl-100 was found to be cleaved by purified granzyme B in vitro (17). It would be interesting to investigate whether or not the C1D protein is also modified during cell death, which might explain why autoantibodies to this protein are generated. Alternatively, the initial immune response might be directed to PM–Scl-100, which directly interacts with both the exosome core and C1D in vivo (16). In that scenario, intermolecular epitope spreading may lead to the generation of autoantibodies directed to C1D. In patients with anti-C1D antibodies but no apparent PM–Scl-100 autoantibodies, the latter may remain undetected because the relevant epitope(s) are absent or masked in the recombinant PM–Scl-100 protein.

Taken together, the results suggest that C1D protein is a major autoantigen in patients with the PM–scleroderma overlap syndrome, and that the use of recombinant C1D may increase the number of patients with PM–scleroderma whose sera are reactive in disease-specific autoantibody tests. Additional research is required to clarify the precise role of the C1D protein in the generation of the autoimmune response in patients with the PM–scleroderma overlap syndrome.

AUTHOR CONTRIBUTIONS

Dr. Pruijn had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study design. Schilders, Raijmakers, Pruijn.

Acquisition of data. Schilders, Vree Egberts.

Analysis and interpretation of data. Schilders, Raijmakers, Pruijn.

Manuscript preparation. Schilders, Raijmakers, Pruijn.

Statistical analysis. Schilders.

Acknowledgements

We thank Drs. G. Hengstman and F. van den Hoogen (University Medical Center St. Radboud, Nijmegen, The Netherlands) for collecting most of the patient sera, Dr. H. P. Seelig for the recombinant PM–Scl-75c protein, and Dr. B. Liedvogel for the recombinant PM–Scl-100 protein.