Differences in specificities of anti-centromere sera for the monomeric and dimeric C-terminal peptides of human centoromere protein C

Yoshinori Hayashi, Yoshinao Muro, Kenji Kuriyama1, Yasushi Tomita and Kenji Sugimoto1

Division of Connective Tissue Disease & Autoimmunity, Department of Dermatology, Nagoya University School of Medicine, Nagoya, Japan
1 Laboratory of Applied Molecular Biology, Department of Applied Biochemistry, University of Osaka Prefecture, Osaka, Japan

Correspondence to: Y. Muro


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Centromere protein-C (CENP-C), one of the centromere autoantigens and components of the inner plate of the kinetochore, is suggested to make a dimer at the C-terminus. In order to investigate the presence of conformation-specific anti-centromere antibodies (ACA) to the dimer form, the C-terminal 124 amino acids (CF-124) were expressed in Escherichia coli, affinity purified and chemically cross-linked. Immunoblotting was utilized to compare the reactivities between the dimers and the monomers against 58 ACA+ sera. The reactivities of the dimers were obviously higher in both IgG and IgM responses. The dimer was still more reactive than the glutathione S-transferase-fused monomer in some sera. Two kinds of CF-124 mutant (each contained one amino acid change at the N-terminal region of CF-124) and two cut segments of CF-124 (67 N-terminal amino acids and 58 C-terminal amino acids) were also examined. The former two mutants decreased the dimerization activity. The latter two mutants lost both activities except for the faint dimerization activity of the N-terminal half. Affinity-purified antibodies with CF-124 in a liquid phase containing the co-purified GroE protein of E. coli, GroEL, reacted to the centromere in culture cells. In conclusion, there are heterogeneous autoepitopes including some conformational epitopes at the C-terminal CENP-C.

Keywords: autoantibody, conformation, epitope, GroEL


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Three centromere proteins of 17, 80 and 140 kDa generally recognized by autoimmune sera from patients with a limited type of systemic scleroderma are designated centromere protein (CENP)-A, CENP-B and CENP-C respectively (13). CENP-C consists of 943 amino acids and is a presumptive component of the inner plate of the kinetochore (4). Recent reports have shown that CENP-C has three functional units, an N-terminal oligomerization domain, an internal DNA-binding domain and a C-terminal dimerization domain (57). In addition, the three major autoepitopes are almost overlapped with the three functional domains (6,8). In a previous report, we showed that the C-terminal region produced a dimer with a chemical cross-linker and also dimer formation in the native state by gel filtration (5). In the purification of the C-terminal region, a GroE protein of Escherichia coli, GroEL, was found to be co-purified (5). In this study, we examined the reactivity of several dozens of anti-centoromere antibodies (ACA) to the dimer form, comparing them with the monomer in immunoblotting analysis. With several mutated constructs derived from the C-terminal region, we determined that dimerization activity and antigenicity required quite close portions. We report here the probable existence of neoepitopes formed by protein dimerization.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Patient sera
Sera from 58 ACA+ patients were used. ACA was identified by indirect immunofluorescence (IIF) and immunoblotting as reported previously (3). Sera from 20 healthy individuals were used as negative controls.

Human CENP-C cDNA clones and their derivatives
The plasmid pCENP-CF-124 has been described elsewhere (5). The plasmid pCENP-CC6G was constructed by inserting a NotI stop codon linker (Nippon Gene, Tokyo, Japan) into the BalI site (at the position of amino acid 886) of the parental plasmid pCENP-CF-124. The plasmid pCENP-CC7G was constructed by inserting a SalI linker (pGGTCGACC) at the BalI site and recloning the 0.4 kb SalI–NotI fragment into the same sites of pGEX-4T-1 (Fig. 1AGo). PCR products of the CF-124 coding sequence using a 5' primer, 5'-TTCCTCGAGATCCTTTGCAGCCAAC-3', and a 3' primer, 5'-TTGACACCAGACCAACTGGTAATGG-3', were inserted into a T-vector (Promega, Madison, WI) (5). After sequencing of several clones, two kinds of 1-amino-acid-changed mutants (D839G or E831G) were selected, and pCENP-CF-124m1 and pCENP-CF-124m4 were constructed by inserting the 0.6 kb XhoI–NotI fragment of the clones into the SalI–NotI site of pGEX4T-1 (Fig. 1AGo). The mutant CF-124 cDNAs cloned into the pGEX vector were transformed into E. coli JM109 cells. In CF-124m1, the 20th amino acid from the N-terminus was changed from aspartic acid to glycine and the 12th one in CF-124m4 was changed from glutamic acid to glycine.



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Fig. 1. Structures of CF-124 and its derivatives. (A) CF-124 is the C-terminal polypeptide (amino acids 820–943) of CENP-C. In CF124-m1, the 20th amino acid is changed from aspartic acid to glycine and the 12th one in CF124-m4 is changed from glutamic acid to glycine. CC6G and CC7G are the cut segments of CF-124. CC6G is the 67 amino acid N-terminal (amino acids 820–886). CC7G is the 58 amino acid C-terminal (amino acids 886û943). (B) The coding sequences of CF-124, and its corresponding region in human, mouse (19) and chicken (GenBank accession no. AB004649).

 
The 67 N-terminal amino acids (amino acids 820–886) were expressed as recombinant protein in E. coli and named CC6G, and the recombinant protein of the 58 C-terminal amino acids (amino acids 886–943) was named CC7G (Fig. 1AGo).

Purification of recombinant proteins
The recombinants for each construct were expressed as glutathione S-transferase (GST)-fused protein in E. coli JM109 cells harboring pGEX-derived plasmid, crudely extracted, mixed with glutathione–Sepharose 4B (Pharmacia, Uppsala, Sweden) and washed with the washing buffer as described previously (5). To obtain the purified non-fused recombinants, the collected resin was incubated with the buffer containing thrombin as described previously (5).

Chemical cross-linking
CF-124 has been shown to make a dimer in the presence of a cross-linker. In this study also, the dimerization of recombinants was performed with a chemical cross-linker, disuccinimidyl suberate (DSS) (Pierce, Rockford, IL), as described (5). In some experiments, cross-linking with 1,4-di-[3'-(2'-pyridyldithio)proinamido]butane (DPDPB) (Pierce) was also performed according to the manufacturer's protocol.

Immunoblotting and protein staining
To compare the reactivities of ACA+ sera against the monomer and the dimer, immunoblotting was repeatedly performed more than twice. SDS–PAGE and the transfer of the recombinant proteins from a 17.5 or 20.0% polyacrylamide gel onto a PVDF membrane (Millipore, Bedford, MA) were done as described previously (3,9). Strips of membrane were incubated with human sera at a 1:50 dilution. The antibody–antigen complexes were detected with horseradish peroxidase-conjugated rabbit anti-human IgG or IgM (Dako, Glostrup, Denmark) at a 1:1000 dilution. Color development was carried out with Konica Immunostain (Konica, Tokyo, Japan). The quantities of the monomer and the dimer loaded on the gel were adjusted to be roughly equal, using a silver staining kit (Wako, Tokyo, Japan). That is, the monomer on the gel was about twice the amount of the dimer in terms of the molar ratio. Colloidal gold blotting kit (BioRad, Richmond, CA) was also used in order to compare the amounts of the polypeptides on the membrane.

Purification of the antibodies in a liquid phase
CF-124 was expressed as GST-fused protein in 50 ml culture of E. coli and purified by using 400 µl of glutathione–Sepharose without thrombin treatment. To purify the antibodies against the dimer CF-124 on the resin, an ACA+ serum (300 µl) was mixed with the resin (400 µl) in 600 µl of PBS for 1 h at 4°C, the resin was washed with cold PBS 6 times, and the antibodies were separated by glycine–HCl buffer, neutralized with 1 M Tris and concentrated (10). The purified antibodies were subjected to immunoblotting analysis in order to examine their reactivity against the CF-124 monomer, dimer and the protein extract from HeLa cells (3,9). In addition, IIF studies with HEp-2 cells were performed as described previously (3,10) to determine whether the purified antibodies reacted to centromere of fixed culture cells.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Immunoblotting analysis for comparison of the reactivities between monomer and dimer forms of CF-124
The recombinant protein was separated in 17.5% SDS–PAGE after cross-linking of CF-124 and then the gel was stained with silver (Fig. 2aGo). The 60 kDa polypeptide was the GroEL protein of E. coli, which was reported to be co-purified with CF-124 (5). The 28 kDa protein was the dimer and the 14 kDa was the monomer. Since, in our previous study (8), reactivities against the C-terminus of CENP-C were different among patients between IgG and IgM classes, we also tested the reactivities of the two classes in this study. Figure 2Go(b and c) shows the representative results of immunoblotting analyses with anti-IgG or -IgM secondary antibodies respectively. Out of 58 ACA+ sera, 27 sera contained anti-monomer IgG (47%), 42 sera contained anti-dimer IgG (72%), 17 sera contained anti-monomer IgM (29%) and 29 sera contained anti-dimer IgM (50%). In the same condition, no sera from 20 healthy controls reacted to the dimer or the monomer. The results show that both positive rates and reactivities were obviously higher in the dimer than in the monomer.



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Fig. 2. Protein staining and immunoblotting of monomer and dimer of CF-124 in SDS–PAGE. (a) Silver staining of the recombinant proteins separated in SDS–PAGE after dimerization. The 60 kDa polypeptide is E. coli protein GroEL, which is known to be co-purified with the C-terminus of CENP-C. The 28 kDa protein is the dimer of CF-124 and the 14 kDa protein, the monomer. (b and c) Representative immunoblotting data with IgG (b) or IgM (c) class antibodies. Lane 1, normal human serum. Lanes 2–9, ACA+ sera. (d) Lane 1, colloidal gold staining of the protein blotted on a membrane. Lanes 2 and 3, immunoblotting analysis with two ACA+ sera.

 
Figure 3Go summarizes the reactivities of the 58 sera against the monomer, the dimer and the GST-fused monomer (GST fusion) both in IgG and IgM. Among sera which reacted to the dimer but not to the monomer, many sera did not react to the GST-fused monomer. Also, in most of the sera which reacted to both the monomer and the dimer, the dimer was more reactive than the GST-fused monomer. These results showed that dimer-dominant reactivities were not merely induced by the stabilization of the structure on the antigenic epitope formed by cross-linking. Moreover, some sera had much higher reactivity for the CF-124 dimer even though both the molar concentration and protein density of the dimer were definitely lower than those of the monomer by protein staining on the membrane (Fig. 2dGo).



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Fig. 3. Reactivities of ACA against CF-124. The 58 ACA+ sera are categorized by the reactivities against the monomer (m), the dimer (d) and the GST-fused monomer (f): ±, presence or absence of immunogenicity; <, comparison of immunoreactivity, i.e. `m– d+' means that sera did not react to the monomer but reacted to the dimer and `f < d' means that sera reacted to the dimer more strongly than to the GST-fused monomer. Numbers in parenthesis indicate the numbers of patients. Bold numbers suggest the dimer-specific antibody.

 
Comparison of wild-type and mutants of CF-124 in antigenicity and dimerization efficiency
To map the linear autoepitope(s) of CF-124 finely, recombinant CF-124m1, CF-124m4 and wild CF-124 monomers were first subjected to immunoblotting analysis. Data are summarized in Table 1Go. From 27 anti-CF124 monomer-positive sera, the randomly selected 11 sera were tested against the three constructs—CF124, CF124m1 and CF124m4. Although the amount of proteins on the membrane was confirmed to be almost the same by the gold colloid method, nine out of 11 sera obviously reacted to CF124 more strongly than to CF124m1 and CF124m4. Notable differences in reactivities between the two mutants were not detected (data not shown).


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Table 1. Summary of activities in immunoblotting, dimerization and co-purification with GroEL of various constructs
 
Similar amounts of the three kinds of CF-124 monomer were cross-linked with various concentrations of DSS and subjected to silver staining and immunoblotting analysis (Fig. 4Go). Dimerization efficiencies were obviously decreased in the two mutants, suggesting a strong relationship between the reactivity of the C-terminal epitope and the dimerization efficiency. CF-124m1 had less dimerization activity than CF-124m4. Judging from the intensities of the corresponding dimer bands in silver staining and immunoblotting, the antigenicity of the dimers seemed to be similar among the three constructs.



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Fig. 4. Chemical cross-linking of CF-124, CF-124m1 and CF-124m4. (a) Similar amounts of the three kinds of recombinants were cross-linked with various concentrations of DSS and detected by silver-staining after separation in 17.5% SDS–PAGE. The concentrations of DSS were 0 (lanes 1), 3 (lanes 2), 12 (lanes 3) and 48 (lanes 4) µM. Arrowheads show the position of dimer bands. (b) Representative data of immunoblotting analysis using the same protein sets in (a).

 
Dimerization of two deletion mutants and their antigenicity
Chemical cross-linking of CC6G, CC7G and CF-124 was carried out as shown in Fig. 5Go. The 14 kDa monomer of CF-124 made a 28 kDa dimer with the cross-linker and the 7.5 kDa monomer of CC6G also made a 15 kDa dimer. However, as for CC7G, we could not determine whether the 6 kDa monomer of CC7G made a 12 kDa dimer because a 12 kDa band was already there without cross-linking by DSS. The intensities of the 6 and 12 kDa bands in the four lanes seemed almost the same, suggesting that no dimer form of CC7G was made. These data indicate that the 67 N-terminal amino acid stretch between positions 820 and 886 is necessary for the dimerization activity of CF-124, but the efficiency of the limited stretch alone is much lower than that of the whole CF-124.



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Fig. 5. Chemical cross-linking of CC6G, CC7G and CF-124. CC6G, CC7G and CF-124 were cross-linked with various concentrations of DSS and detected by silver staining after separation in 20.0% SDS–PAGE. The concentration of DSS was 0 (lanes 1), 3 (lanes 2), 12 (lanes 3) and 48 (lanes 4) µM.

 
The immunogenicity of CC6G and CC7G was examined in immunoblotting analysis. Purified recombinant CC6G and CC7G, after treatment with or without DSS, were transferred to the membrane and probed with 26 and 58 ACA+ sera respectively. However, all the sera were negative for the recombinant proteins and the presumptive dimers. Interestingly, GroEL was also co-purified with the recombinants in both cases, but the size of the GroEL co-purified with CC6G was a little smaller, probably due to proteolysis with thrombin, as confirmed by the reactivities against mAb for GroEL (StressGen, Victoria, Canada) (data not shown). We could not find the bands corresponding to GroES in these purification procedures. These results are summarized in Table 1Go.

DPDPB, which is a unique cross-linker with a disulfide bond, was also used in order to see if it is possible to make dimers for CF-124, CC6G and CC7G. Although CC6G has no cysteine residue, CF-124 has one cysteine residue in the region of CC7G. At concentrations of up to 2.88 mM, there was no dimer detected for all three constructs by SDS–PAGE even in a non-reduced condition (data not shown).

Purified antibodies against CF-124 in the presence of GroEL
We tried a purification of the antibody specific for the dimer structure of the cross-linked CF-124 on the membrane. Since the purified antibodies did not stain the centromere structure in IIF (data not shown), we next tried an affinity purification using the recombinant protein in a liquid phase. ACA#66, which reacted to the CF-124 monomer, dimer and GroEL in immunoblotting, was used for the affinity purification. When the purified antibodies against the dimer in a liquid phase containing GroEL were applied to immunoblotting with HeLa cell extract, the antibodies against CENP-A or CENP-B were negative, but those against CENP-C were slightly positive (data not shown). When the antibodies were applied to immunoblotting with the recombinants, the monomer or the dimer was hardly stained and GroEL was stained only a little (data not shown). By IIF staining analysis (Fig. 6Go), the antibodies showed a discrete speckled pattern corresponding to centromere staining. As a control, anti-U1-RNP antibodies from a lupus patient were also used for affinity purification and applied to IIF studies, but no staining was observed. Another control, the GST recombinant (not CF-124) was also used for the affinity purification of ACA#66 in a liquid phase. These purified antibodies produced no staining at all.



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Fig. 6. Centromere staining with purified autoimmune sera using undenatured CF-124 in a liquid phase containing GroEL. Staining of HEp-2 cells with ACA whole serum (ACA#66) (A), affinity-purified serum from #66 (B), anti-RNP whole serum (patient KN) (C) and affinity-purified serum from KN (D).

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
As proteins, which work as antigens in situ, are three-dimensional, it is reasonable that some autoantibodies can recognize specific three-dimensional structures. Although the existence of such autoantibodies has not yet been proven directly, some suggestive data have been reported. For example, human thyroid peroxidase, which is one of the major autoantigens in autoimmune thyroid disease, has a conformational B cell epitope on the C-terminal end, as demonstrated by enzymatic reduction and alkylation (11). Some antibodies from lupus patients are reported to recognize the proliferating cell nuclear antigen. These antibodies do not detect the monomer on SDS–PAGE but react to the native three-dimensional form as a trimer in immunoprecipitation (12).

The C-terminal end of CENP-C was reported to make a dimer easily in vitro (5), although the dimer form of CENP-C has not been demonstrated in vivo. We assumed the existence of antibodies that recognize the three-dimensional structures of a CENP-C dimer generated by a chemical cross-linker (13,14). In the responses of both IgG and IgM, the dimer was much more highly reactive than the monomer. Although the differences in binding in this study may simply reflect differences in valence induced by cross-linking, this would not be the explanation for what we observed for several reasons. (i) Although the monomer transferred to the membrane was about twice the amount of the dimer according to the protein silver staining, the positive rate and the reactivity of the monomer were lower than those of the dimer. (ii) Judging from the intensity of the protein bands on the membranes, since the antigen density of the dimer was not as high as that of the monomer, our results are different from the phenomenon derived from a dependence on the antigen-density suggested by the anti-ß2-glycoprotein I antibody (15). (iii) It is already known that recombinant CENP-B increases its reactivity in immunoblotting when it is fused with GST (16). Also in this study, the positive rate of the GST-fused monomer against ACA in immunoblotting was higher than that of the monomer. However, the reactivities of the dimer were significantly higher than those of the GST fusion. (iv) There were various differences in increasing ratios of reactivities of the dimer between IgG and IgM classes among patients. (v) Although the C-terminus of CENP-B also formed dimer, there was no increase of reactivities of ACA against the dimer (our unpublished results). Therefore, we assumed the neoepitope(s) derived from the dimers of CF-124.

Using several kinds of derivatives of CF-124, one linear autoepitope is considered to include the mutation site (amino acids 831 and 839), and extend into both the CC6G and CC7G regions. This epitope seems to be quite close to the dimerization domain. With DPDPB, which is a cross-linker making cysteine bonds, neither CF-124 nor CC7G made a dimer, thereby excluding the possibility of a relationship between the dimerization of CF-124 and a disulfide bond, even though Cys901 is conserved between human and mouse but not chicken (Fig. 1BGo). Because we failed to find characteristic amino acid motifs at this region, we now believe ionic interaction is the cause of dimerization.

We tried a purification of the antibody specific for the dimer structure of the cross-linked CF-124 on the membrane as well as that for the monomer. Purified antibodies both from the dimer and the monomer weakly reacted to each other in immunoblotting (data not shown), indicating the existence of cross-reactive antibodies. Neither of the purified antibodies stained the centromere structure in IIF. The condition for affinity purification might not have been proper for the antibodies because of weak affinity and the small amount of the antibodies in the whole serum. Otherwise, some epitopes formed by the C-terminus of CENP-C might be cryptic. On the other hand, the antibody against glutathione-beads-bonded GST fusion of CF-124 was purified in a liquid phase. In IIF analysis, the purified antibody reacted to the centromeric region. However, the antibody hardly reacted to the monomer or the dimer on the membrane and it reacted only slightly to GroEL in immunoblotting. Therefore, this antibody that recognized the newly made three-dimensional structure in the presence of GroEL is considered to be different from the one gained from the membrane. This antibody may reflect the more native form of CF-124. GroEL protein of E. coli, the bacterial chaperonine which is necessary for protein folding in vivo and is the homologue of mammalian heat shock protein 60 (17,18), might contribute to CENP-C folding. Efficient purification of the antibodies in the absence of ATP and the result that ATP hardly influenced the co-purification of GroEL with CF-124 (K. Kuriyama, unpublished observation) suggest that the interaction between CF-124 and GroEL might be ATP independent.

In conclusion, there are several autoimmune epitopes on CF-124; one of them is the linear epitope which includes the two mutation sites and extends into the CC6G and CC7G regions, another one is the three-dimensional structure recognized as a dimer and another still is the epitope which is in the more native macromolecular complex in the presence of GroEL. It has not been proven directly that CENP-C makes a dimer in vivo. However, the facts that there were some autoantibodies against the dimer of CENP-C in vitro and that the purification efficiency of antibodies to the C-terminus in a liquid phase was quite high suggest the important role of the C-terminus in the three-dimensional structure of CENP-C.


    Acknowledgments
 
This work was supported in part by grants 09770628 and 11670823 from the Ministry of Education, Science, Sports and Culture of Japan (Y. M.).


    Abbreviations
 
ACA anti-centromere antibody
CENP centromere protein
DPDPB 1,4-di-[3'-(2'-pyridyldithio)proinamido]butane
DSS disuccinimidyl suberate
GST glutathione S-transferase
IIF indirect immunofluorescence

    Notes
 
Transmitting editor: K. Sugamura

Received 10 March 2000, accepted 26 June 2000.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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