ABSTRACT
In previous studies it has been shown that neural cells undergoing programmed cell death display strongly positive cytoplasmic immunoreactivity to polyclonal antibodies directed against a c-Jun N-terminal peptide. It was later found that c-Jun-like immunoreactivity in apoptosis was due to cross-reactivity with proteins other than c-JUN: We have analysed the biochemical counterpart of this property in neuroblastoma cell lines treated to induce apoptosis. Using the c-Jun/sc-45 antibody, several bands with apparent molecular masses distinct from c-Jun were detected in extracts in parallel with both the degree of apoptosis and the appearance of the cytoplasmic signal after immunostaining. c-Jun/sc-45 immunostaining was prevented by caspase inhibitors and did not require de novo protein synthesis. One of the antigens recognized by the c-Jun/sc-45 antibody was identified as seryl-tRNA synthetase. We provide evidence that seryl-tRNA synthetase is a substrate of caspase-3 in vitro and that the digested form turns highly immunoreactive towards the antibody. A carboxy-terminus epitope of the protein that constitutes a consensus site for caspase-3 is involved in c-Jun/sc-45 recognition. This epitope shares some amino acids with the peptide used as the immunogen and this could explain the cross-reactivity observed. In conclusion, we demonstrate here that cytoplasmic c-Jun/sc-45-like immunoreactivity specific to apoptosis is due to post-translational changes which occur in seryl-tRNA synthetase and probably also in other proteins as a consequence of caspase mediated proteolysis.
Subject(s)
Antibodies/immunology , Apoptosis , Caspases/metabolism , Epitopes/immunology , Peptide Fragments/immunology , Proto-Oncogene Proteins c-jun/immunology , Amino Acid Chloromethyl Ketones/pharmacology , Antibody Specificity , Caspase Inhibitors , Cycloheximide/pharmacology , Enzyme Inhibitors/pharmacology , Humans , Immunohistochemistry , Immunosorbent Techniques , Microscopy, Fluorescence , Neuroblastoma/chemistry , Neuroblastoma/pathology , Proto-Oncogene Proteins c-jun/analysis , Proto-Oncogene Proteins c-jun/antagonists & inhibitors , Serine-tRNA Ligase/immunology , Serine-tRNA Ligase/metabolism , Tumor Cells, CulturedABSTRACT
We have sequenced and expressed in Escherichia coli the gene encoding the seryl-tRNA synthetase from the pathogenic bacterium Staphylococcus aureus. The overexpressed and purified recombinant enzyme was able to aminoacylate unfractionated tRNA from E. coli. Its activity was not affected by antibodies raised against and inhibiting the E. coli seryl-tRNA synthetase.
Subject(s)
Genes, Bacterial , Serine-tRNA Ligase/genetics , Staphylococcus aureus/genetics , Amino Acid Sequence , Base Sequence , Escherichia coli/genetics , Immunoglobulin G/pharmacology , Molecular Sequence Data , Recombinant Proteins , Sequence Alignment , Sequence Analysis, DNA , Serine-tRNA Ligase/immunology , Staphylococcus aureus/enzymologyABSTRACT
Monospecific polyclonal antibodies (pAbs) against highly purified bovine seryl-tRNA synthetase (SerRS, EC 6.1.1.1) were prepared and their specificity tested. The interactions of pAbs with SerRS from different organisms were investigated by protein immunoblotting and ELISA methods. pAbs inhibit eukaryotic SerRS aminoacylating activity and exert no effect on SerRS activity from prokaryotes. It is proposed that prokaryotic and eukaryotic SerRS evolve from different ancestor genes.
