Autoantibodies from primary biliary cirrhosis patients with anti-p95c antibodies bind to recombinant p97/VCP and inhibit in vitro nuclear envelope assembly.
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.
Miyachi
K
K
Health Sciences Research Institute, Yokohama, Kanagawa, Japan. mkiyomitsumd_8@hotmail.com
Hirano
Y
Y
Horigome
T
T
Mimori
T
T
Miyakawa
H
H
Onozuka
Y
Y
Shibata
M
M
Hirakata
M
M
Suwa
A
A
Hosaka
H
H
Matsushima
S
S
Komatsu
T
T
Matsushima
H
H
Hankins
R W
RW
Fritzler
M J
MJ
D000251:Adenosine Triphosphatases; D000937:Antigen-Antibody Reactions; D001323:Autoantibodies / Q000276:immunology*; D018797:Cell Cycle Proteins / Q000276:immunology*; D002467:Cell Nucleus / Q000276:immunology; D005260:Female; D006801:Humans; D008099:Liver / Q000473:pathology; D008105:Liver Cirrhosis, Biliary / Q000276:immunology* / Q000473:pathology; D008297:Male; D018613:Microscopy, Confocal; D011233:Precipitin Tests; D000074405:Valosin Containing Protein
D001323:Autoantibodies; D018797:Cell Cycle Proteins; D000251:Adenosine Triphosphatases; C000614178:VCP protein, human; D000074405:Valosin Containing Protein