cDNA cloning of a novel autoantigen targeted by a minor subset of anti-centromere antibodies

Abstract

Using autoimmune serum from a patient with anti-centromere antibodies, we have identified and partially characterized a novel protein with a mol. wt of approximately 27 kD (hereafter referred to as p27). A cDNA expression library was screened with this serum, and two overlapping inserts were isolated among three positive clones other than clones corresponding to centromere protein (CENP)-B and CENP-C. Analysis of the sequence showed an open reading frame of approximately 0.6 kb encoding 199 amino acids with a predicted mol. wt of 21.5 kD. Immunoblotting analysis with bacterial recombinant p27 showed that approximately 2% of anti-centromere antibody-positive patients had autoantibodies to p27, whereas only one of 215 autoimmune patients without anti-centromere antibodies reacted with the recombinant. All five cases with anti-p27 antibodies, who were diagnosed as having scleroderma and/or Sjögren's syndrome, showed internal organ involvement. Although affinity-purified anti-p27 human or mouse polyclonal antibodies failed to stain any cellular structures in an immunofluorescence study, the potential association of anti-p27 with anti-centromere antibodies suggests that this novel autoantigen might play a role in mitosis.

Fig. 1

Nucleotide and deduced amino acid sequences of cDNA clone, λ12-1, encoding a novel autoantigen p27. The other clone, λ38-1, encompassed nucleotide 64 to the end of λ12-1. Regions of Pro-rich, Glu/Asp-rich, and Ser/Thr-rich regions are boxed. The combined cDNA nucleotide and amino acid sequences have been submitted to GenBank with accession number AB001740.

Fig. 2

Immunoblotting of HeLa cell extract reacting with sera from patient 1 and a mouse immunized with GST fusion protein. Patient 1 serum reacted with centromere protein (CENP)-A and -B polypeptides and 27-kD polypeptide (lane 1). Antibody affinity-purified from the membrane blotted with GST fusion recombinants reacted only with 27-kD polypeptide (lane 2), but that affinity-purified from unrelated portions of the membrane did not react with any peptides (lane 3). Mouse polyclonal antibodies against GST fusion recombinants also reacted with 27-kD polypeptide (lane 4). Lane 5, an anti-centromere antibody-positive serum containing anti-chromo specificity which reacted with p23 and p25.

Fig. 3

The size of recombinant protein and HeLa cellular protein (a), and immunoreactivity of recombinant protein against autoimmune sera (b). (a) Lane 1 shows Western blotting of a digested fusion protein with patient 1 serum and lane 2 shows the result of Western blotting of HeLa cellular protein with patient 1 sera. (b) Lanes 1–5 show positive reactivity for p27; lane 1, patient 1; lane 2, patient 2; lane 3, patient 3; lane 4, patient 4; lane 5, patient 5. Lanes 6–9 and N are negative; lanes 6–9, autoimmune sera; lane N, normal healthy subject.