Identification of citrullinated alpha-enolase as a candidate autoantigen in rheumatoid arthritis

Abstract

Antibodies against citrullinated proteins are highly specific for rheumatoid arthritis (RA), but little is understood about their citrullinated target antigens. We have detected a candidate citrullinated protein by immunoblotting lysates of monocytic and granulocytic HL-60 cells treated with peptidylarginine deiminase. In an initial screen of serum samples from four patients with RA and one control, a protein of molecular mass 47 kDa from monocytic HL-60s reacted with sera from the patients, but not with the serum from the control. Only the citrullinated form of the protein was recognised. The antigen was identified by tandem mass spectrometry as alpha-enolase, and the positions of nine citrulline residues in the sequence were determined. Serum samples from 52 patients with RA and 40 healthy controls were tested for presence of antibodies against citrullinated and non-citrullinated alpha-enolase by immunoblotting of the purified antigens. Twenty-four sera from patients with RA (46%) reacted with citrullinated alpha-enolase, of which seven (13%) also recognised the non-citrullinated protein. Six samples from the controls (15%) reacted with both forms. Alpha-enolase was detected in the RA joint, where it co-localised with citrullinated proteins. The presence of antibody together with expression of antigen within the joint implicates citrullinated alpha-enolase as a candidate autoantigen that could drive the chronic inflammatory response in RA.

Figure 1

Screen to identify undeiminated and deiminated proteins reacting with RA and non-RA serum samples. Proteins from HL60 lysates incubated (+) or without (-) peptidylarginine deiminase (PAD) blotted with an antibody specific for modified citrulline residues (anti-citrulline) and a screening panel of rheumatoid arthritis (RA1 to RA4) and non-RA (control) serum. The rectangular box indicates a citrullinated protein reacting strongly with each of the RA serum samples but not the control.

Figure 2

Intracellular expression of immunogenic citrullinated 47 kDa protein. Presence of citrullinated 47 kDa protein reactive with rheumatoid arthritis serum 1 in different subcellular fractions of undifferentiated HL60s (U) and HL60 monocytes (M) (S100, cytosolic; P100, membrane; Nuc, nuclear) showing enrichment in the S100 (cytosolic) fraction.

Figure 3

Characterisation of the 47 kDa protein by two-dimensional electrophoresis. Proteins in the 47 kDa rich monocytic S100 fraction were separated by two-dimensional electrophoresis according to charge (x-axis) and molecular mass (y-axis). (a) The full complement of proteins was observed by silver staining. (c,d) Proteins reacting with rheumatoid arthritis serum samples 1 (c) and 4 (d) were highlighted by immunoblotting. (b) The highly reactive 47 kDa protein was confirmed as α-enolase by immunoblotting with the goat anti-α-enolase antibody. CAP1, adenyl cyclase-associated protein 1; EF1α, elongation factor 1α.

Figure 4

Prevalence of antibodies against deiminated and undeiminated α-enolase in RA patients and healthy controls. Immunoblotting of in vitro citrullinated and untreated α-enolase with serum samples from patients with rheumatoid arthritis (RA) and normal controls, showing that about half of the serum samples from the RA group contain antibodies with selectivity for citrullinated α-enolase.

Figure 5

Localisation of α-enolase in synovial membranes. Immunohistochemistry of biopsy sections from patients with osteoarthritis (a) and rheumatoid arthritis (RA) (b) with the goat anti-α-enolase antibody showing expression of α-enolase (stained brown) in cells in the subsynovium of the patient with RA and in endothelial cells in the patient with osteoarthritis. Cell nuclei are counterstained blue.

Figure 6

Localisation of citrullinated proteins in synovial membranes. (a) Immunostaining of citrullinated proteins by the anti-modified citrulline kit was mainly confined to the subsynovium. (b) No staining was visible on the control. (c) Staining produced by the anti-α-enolase antibody on an adjacent section was much stronger, and included the subsynovial cells which also stained for citrullinated antigens. Original magnification × 20 in all cases.

Figure 7

Presence of α-enolase in synovial cells from patients with rheumatoid arthritis (RA). Immunoblotting of lysates from RA synovial cells with anti-α-enolase (+PAD, deiminated α-enolase; – PAD, undeiminated α-enolase) showing a 47 kDa protein in synoviocytes, from three patients with RA, reacting with the goat anti-α-enolase antibody, which co-migrates with purified α-enolase.