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. 1999 Mar;58(3):169–174. doi: 10.1136/ard.58.3.169

Serum samples of patients with rheumatoid arthritis contain a specific autoantibody to "denatured" aldolase A in the osteoblast-like cell line, MG-63

F Ukaji 1, I Kitajima 1, T Kubo 1, C Shimizu 1, T Nakajima 1, I Maruyama 1
PMCID: PMC1752850  PMID: 10364915

Abstract

OBJECTIVE—To identify rheumatoid arthritis (RA) specific autoantibody and its antigen in the human osteoblast-like cell line, MG-63.
METHODS—MG-63 cell extract was subjected to western blotting by using RA and normal serum samples as probes. The autoantigen was purified and its N-terminal sequence was determined by automated Edman degradation. The reactivity of denatured aldolase A was evaluated by immunoblotting. Screening by enzyme linked immunosorbent assay (ELISA) using the autoantibody was performed.
RESULTS—40 kDa protein was found only in the RA serum samples and it was identified as aldolase A. A polyclonal antibody for rabbit muscle aldolase A bound to the 40 kDa protein and reacted in preference with the denatured enzyme. Using ELISA for denatured rabbit aldolase A, the autoantibody was found in approximately 10% of RA patients, whereas it was not found in the other arthropathy and healthy adults.
CONCLUSION—This 40 kDa anti-aldolase A autoantibody, which was identified only in serum samples of RA patients with severe bone erosion, could be related to a certain event that induces RA specific joint destructions.

 Keywords: autoantibody; aldolase A; denature; rheumatoid arthritis

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Figure 1  .

Figure 1  

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Detection of 40 kDa protein by using RA serum as a primary antibody. The MG-63 (20 µg/lane) was separated by SDS-PAGE on 10% polyacrylamide gel, and the obtained proteins were electroblotted. The membranes were incubated with serum of RA patients or healthy controls (1/100 dilution). The 40 kDa protein was chemiluminescently recognised only in the serum samples from six RA patients who were in a progressive bone destruction stage. RA: with serum of RA patients. Normal: with serum of healthy persons.

Figure 2  .

Figure 2  

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Purification of 40 kDa protein. The MG-63 cell extract (lane C) was applied to the TSKgel Super Q-5PW column and a flow through a fraction was collected (lane Q). After concentration, the sample was separated into three peaks by the TSKgel Phenyl-5PW RP column (lane 1-3). Aliquot of each fraction was resolved by electrophoresis on 10% SDS/polyacrylamide gels. Western blotting was performed by using RA serum (1/100 dilution) as the probe. Arrow: 40 kDa protein.

Figure 3  .

Figure 3  

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Identification of 40 kDa protein. (A) RA serum recognised rabbit muscle aldolase A. Purified rabbit muscle aldolase A (0.1 µg/lane) was applied to SDS-PAGE on 10% polyacrylamide gel and transferred to nitrocellulose membranes. The membranes were incubated with either RA or normal serum, and bound IgG were detected. Normal serum reacted weakly with rabbit aldolase A. (B) Immunoadsorption of RA serum with rabbit muscle aldolase A. RA serum samples were preincubated with BSA (30 mg/ml) or rabbit muscle aldolase A (5 or 30 mg/ml), and reactivity to the 40 kDa protein was examined by western blotting. Lane 1: no adsorption. Lane 2: adsorbed by BSA (30 mg/ml). Lane 3 (30 mg/ml) and Lane 4 (5 mg/ml): adsorbed by rabbit muscle aldolase A. 

Figure 4  .

Figure 4  

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Recognition of the RA serum reactive 40 kDa protein by antirabbit aldolase A antibody. The MG-63 cell extract (100 µl, 2 mg/ml) was immunoprecipitated with the antirabbit aldolase A antibody or with the control IgG. The immunoprecipitates were analysed by western blotting with RA or normal serum. Upper panel: immunoprecipitation (IP) with the anti-aldolase A antibody (Ab). Lower panel: IP with the control IgG. Lane 1: antirabbit muscle aldolase A antibody. Lane 2: control IgG. Lane 3-7: RA serum. Lane 8 and 9: normal serum. The arrow shows the position of 40 kDa protein.

Figure 5  .

Figure 5  

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Detection of anti-aldolase autoantibody in various serum samples by ELISA. Multititre wells were coated with 2 µg of denatured rabbit aldolase A. The diluted serum samples (1/11) were applied to the wells and the amounts of bound IgG were determined. The cut off value (A 405 nm = 0.3) was calculated from the data of seronegative normal serum samples (mean (2SD)). RA: rheumatoid arthritis. OA: osteoarthritis. SLE: systemic lupus erythematosus.

Figure 6  .

Figure 6  

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Reactivity of anti-aldolase autoantibody in RA serum samples with denatured and native aldolase A. The serum samples (1/11 dilution) were preincubated with the denatured or native aldolase A (10 µg/ml), and the reactivity were evaluated as the reactivity against coated aldolase A. The percentage of inhibition was calculated from absorbance value with and without each inhibitor. The autoantibody bound more tightly to the denatured aldolase A. RA1: patient 1 with RA. RA2: patient 2 with RA. RA3: patient 3 with RA. RA4: patient 4 with RA.

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