Cloning of novel soluble gp130 and detection of its neutralizing autoantibodies in rheumatoid arthritis
Masao Tanaka, … , Masao Murakami, Kazuwa Nakao
Masao Tanaka, … , Masao Murakami, Kazuwa Nakao
Published January 1, 2000
Citation Information: J Clin Invest. 2000;106(1):137-144. https://doi.org/10.1172/JCI7479.
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Article

Cloning of novel soluble gp130 and detection of its neutralizing autoantibodies in rheumatoid arthritis

Abstract

In an attempt to isolate disease-associated autoantigens in rheumatoid arthritis (RA), we cloned a new autoantigen named gp130-RAPS, which is a novel soluble form of the IL-6 signal–transducing molecule gp130. gp130-RAPS is a 50-kDa protein translated from alternatively spliced mRNA and has a truncated form of gp130 with a unique sequence, Asn-Ile-Ala-Ser-Phe (NIASF), in its COOH-terminus. We observed serum antibodies to this NIASF sequence frequently in patients with RA, but not in those with other systemic rheumatic diseases or in healthy subjects. In RA, detection of those antibodies was significantly associated with disease activity indices such as serum C–reactive protein (CRP) levels, erythrocyte sedimentation rate, blood platelet counts, and serum IL-6 concentration. In vitro experiments revealed that gp130-RAPS inhibited IL-6 activity, and this inhibition was neutralized by antibodies to the COOH-terminus of gp130-RAPS derived from patients with RA. Thus, autoantibody to gp130-RAPS may play an important role in the progression of RA by promoting IL-6 activity. Inspection of autoantibodies to gp130-RAPS may become a practical clinical test for RA. gp130-RAPS and its autoantibody provide a new clue to the complicated pathogenesis of RA.

Authors

Masao Tanaka, Masaaki Kishimura, Shoichi Ozaki, Fumio Osakada, Hidetaka Hashimoto, Mitsuo Okubo, Masao Murakami, Kazuwa Nakao

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Figure 1

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Schematic representations of gp130-RAPS cDNA and its partial genome stru...
Schematic representations of gp130-RAPS cDNA and its partial genome structure (a and b), as well as a demonstration of its mRNA expression (c) and translated products (d). (a) Top: An 83-bp fragment (hatched) was deleted in gp130-RAPS cDNA. Each gp130-RAPS molecule had three intact fibronectin type III modules (ovals) and lacked a cytoplasmic region (rounded rectangle). Bottom: The 83-bp deletion brought about a frame shift and addition of the unique amino acid sequence NIASF followed by a stop codon. Nucleotide numbers are the same in the membrane-bound gp130 (18). (b) The gp130 genomic sequence had at least two introns adjacent to the deleted 83-bp exon fragment (hatched) in gp130-RAPS. Each intron had a donor-acceptor structure compatible with the GT-AG rule (shown in the box, underlined nucleotides). It seemed that in mRNA splicing, loss of introns a and b, together with the 83-bp exon, produced gp130-RAPS mRNA, and that their loss kept that exon preserved and created gp130 mRNA. Arrows indicate primers for sequencing and RT-PCR. An Rsa I site was located in the 83-bp exon. (c) RT-PCR analysis demonstrated gp130-RAPS mRNA expression in IL-6–stimulated SF-1 synovial cells. The expression was upregulated after 3-hour stimulation by IL-6 (100 ng/mL) with sIL-6R (100 ng/mL). PCR was performed for up to 35 cycles. In 40-cycle PCR, the bands of gp130-RAPS differed little in density (data not shown). As shown in the control lanes, 115-bp and 198-bp bands represent the products of gp130-RAPS and gp130 mRNA, respectively. GAPDH gene expression, represented by 983-bp bands, was used as a control to ensure equivalent amounts of original templates. Rsa I digestion could not completely eliminate nonspliced gp130 mRNA derivatives. (d) Rabbit α-RAPC15 Ab (positive control, PC) and RA patient (RA1) α-RAPC15 Ab (RA) reacted to gp130-RAPS and immunoprecipitated it mainly from culture supernatant (S) of gp130-RAPS cDNA-transfected COS-7 cells (transfectant) (lanes 1 and 3), and a little from their lysate (L) (lanes 2 and 4). gp130-RAPS protein had an approximate molecular weight of 50 kDa under reducing conditions. An extra band with a lower molecular weight was probably formed by partially degraded molecules (lane 1). IgG from normal healthy subjects (negative control, NC) did not react to gp130-RAPS (lanes 5 and 6). Sample controls derived from nontransfected COS-7 cells were also examined (wild-type) (lanes 7–12). Immunoblotting was performed with rabbit α-RAPC15 Ab.