Autoantibodies from primary biliary cirrhosis patients with anti-p95c antibodies bind to recombinant p97/VCP and inhibit in vitro nuclear envelope assembly

Abstract

We have reported previously that p95c, a novel 95-kDa cytosolic protein, was the target of autoantibodies in sera of patients with autoimmune hepatic diseases. We studied 30 sera that were shown previously to immunoprecipitate a 95 kDa protein from [(35)S]-methionine-labelled HeLa lysates and had a specific precipitin band in immunodiffusion. Thirteen sera were available to test the ability of p95c antibodies to inhibit nuclear envelope assembly in an in vitro assay in which confocal fluorescence microscopy was also used to identify the stages at which nuclear assembly was inhibited. The percentage inhibition of nuclear envelope assembly of the 13 sera ranged from 7% to 99% and nuclear envelope assembly and the swelling of nucleus was inhibited at several stages. The percentage inhibition of nuclear assembly was correlated with the titre of anti-p95c as determined by immunodiffusion. To confirm the identity of this autoantigen, we used a full-length cDNA of the p97/valosin-containing protein (VCP) to produce a radiolabelled recombinant protein that was then used in an immunoprecipitation (IP) assay. Our study demonstrated that 12 of the 13 (93%) human sera with antibodies to p95c immunoprecipitated recombinant p97/VCP. Because p95c and p97 have similar molecular masses and cell localization, and because the majority of sera bind recombinant p97/VCP and anti-p95c antibodies inhibit nuclear assembly, this is compelling evidence that p95c and p97/VCP are identical.

Fig. 1

Relationship between percentage inhibition of nuclear assembly and titres of anti-p95c antibodies in the sera of PBC patients. Sera with low titre (<1 : 128) anti-p95c antibodies exhibit less inhibition of nuclear assembly whereas sera with high titres (>1/128) show the most marked inhibition of nuclear assembly.

Fig. 2

Confocal immunofluorescent microscopy of the nuclear reassembly assay. Nuclear envelope assembly was inhibited by the index serum (IK) but not by phosphate buffered saline or normal healthy serum. Nuclear assembly was judged to have occurred when the length of the long axis divided by the short axis was less than 2.

Fig. 3

Immunoprecipitation of p97/VCP recombinant protein with human anti-p95 sera. The p97/VCP protein was expressed as a [³⁵S]-labelled in vitro transcription and translation (TnT) product and then immunoprecipitated with the human sera. Thirteen sera with anti-p95c antibodies (lanes 1–13) and the index anti-p95c sera (I) immunoprecipitated the ∼97 kDa recombinant protein whereas normal human serum (N) and a control serum from a patient with antimitochondrial antibodies (C) did not. The reactivity of the serum in lane 10 is weak compared to other sera but was equivocally positive on the original imaging film. Molecular weight markers are indicated on the left.