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 obtained against the peptide LLGNSSPRTQSPQN which is proteolytically removed during the farnesylation-dependent processing of prelamin A to mature lamin A. We tested the ability of this antibody to detect inhibition of farnesylation-dependent protein processing of prelamin A. The alpha-PA antibody was shown to immunoprecipitate prelamin A from lovastatin-treated HeLa cells but not mature lamin A from untreated cells. Further studies were performed after antigen-affinity chromatographic purification of the antibody. Western blotting of lovastatin-treated HeLa cell extract demonstrated that the purified alpha-PA antibody recognizes 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 antibody should be useful for screening compounds that may block any of the three common steps in the farnesylation-dependent processing of proteins (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 been proposed as potential anticancer drugs, since they would be expected to block the biological activity of oncogenic p21ras proteins. Since such screening would be performed most efficiently by enzyme-linked immunosorbent assays, we can detect the accumulation of prelamin A after treatment with lovastatin by performing this procedure as well. Application of alpha-PA in an enzyme-linked immunosorbent assay, which demonstrates the activity of a peptidomimetic farnesyltransferase inhibitor, supports the use of this antibody in large scale screening for inhibitors of farnesylation-dependent protein processing.
Sinensky
M
M
Eleanor Roosevelt Institute, Denver, Colorado 80206.
Fantle
K
K
Dalton
M
M
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D000918:Antibody Specificity; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D006367:HeLa Cells; D006801:Humans; D034904:Lamin Type A; D034882:Lamins; D008148:Lovastatin / Q000494:pharmacology; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000276:immunology* / Q000378:metabolism*; D011498:Protein Precursors / Q000276:immunology* / Q000378:metabolism*; D017368:Protein Prenylation
D000906:Antibodies; D034904:Lamin Type A; D034882:Lamins; D009687:Nuclear Proteins; D011498:Protein Precursors; D008148:Lovastatin