AN ANTIBODY WHICH SPECIFICALLY RECOGNIZES PRELAMIN A BUT NOT MATURE LAMIN A - APPLICATION TO DETECTION OF BLOCKS - IN FARNESYLATION-DEPENDENT PROTEIN PROCESSING

A polyclonal antibody [anti-prelamin A antibody (alpha-PA)] has been o btained against the peptide LLGNSSPRTQSPQN which is proteolytically re moved during the farnesylation-dependent processing of prelamin A to m ature lamin A. We tested the ability of this antibody to detect inhibi tion of farnesylation-dependent protein processing of prelamin A. The alpha-PA antibody was shown to immunoprecipitate prelamin A from lovas tatin-treated HeLa cells but not mature Iamin A from untreated cells. Further studies were performed after antigen-affinity chromatographic purification of the antibody. Western blotting of lovastatin-treated H eLa cell extract demonstrated that the purified alpha-PA antibody reco gnizes prelamin A. Furthermore, this signal could be competed away by incubation with the peptide. Indirect immunofluorescence helped detect nuclear accumulation of the antigen in response to treatment of HeLa cells with lovastatin or in Chinese hamster ovary K1 cells transiently transfected with a prelamin A mutant blocked in farnesylation. This a ntibody should be useful for screening compounds that may block any of the three common steps in the farnesylation-dependent processing of p roteins (farnesylation, endoproteolysis, and carboxymethylation) since it appears that prelamin A undergoes all of these reactions prior to removal of the antigenic peptide. Inhibitors of these reactions have b een proposed as potential anticancer drugs, since they would be expect ed to block the biological activity of oncogenic p21(ras) proteins. Si nce such screening would be performed most efficiently by enzyme-linke d immunosorbent assays, we can detect the accumulation of prelamin A a fter treatment with lovastatin by performing this procedure as well. A pplication of alpha-PA in an enzyme-linked immunosorbent assay, which demonstrates the activity of a peptidomimetic farnesyltransferase inhi bitor, supports the use of this antibody in large scale screening for inhibitors of farnesylation-dependent protein processing.