Purification, characterization and molecular cloning of muscle paranemin

Abstract

<p>Paranemin is an incompletely characterized ~280 kilodalton protein previously identified and immunolocalized in embryonic chick skeletal muscle. Paranemin has been purified from the same tissue source, has the same molecular weight by SDS-PAGE, and has the same antibody localization at the Z-lines of adult avian cardiac muscle. The method developed for preparation of purified paranemin from embryonic (chick) skeletal muscle includes homogenization, centrifugation and gel filtration, hydroxyapatite, and DEAE-cellulose chromatography. By using this method, ~2 mg of purified paranemin was routinely obtained. Amino acid analysis revealed that paranemin has a high acidic to basic amino acid ratio, which agrees with the measured pI range of 4.1-4.5. When the purified protein was stained with a cationic carbocyanine dye, Stains-all, paranemin stained an intense blue, indicating it is a phosphoprotein and/or a glycoprotein. Further testing determined that paranemin is a glycoprotein. A monoclonal antibody (4D3) was made to use in one-and two-dimensional Western blots, which were used to identify paranemin throughout the purification procedure, and for immunofluorescence studies. Double-label confocal immunofluorescence showed colocalization of paranemin with desmin at the Z-lines of adult cardiac and skeletal muscle cells and at cardiac muscle intercalated disks;I determined the full-length cDNA sequence of paranemin by immunoscreening a [lambda]gt22 cDNA library from embryonic chick skeletal muscle with a monoclonal antibody specific for paranemin (4D3) and by hybridization screening. Northern blot analysis reveals a single transcript of 5.3 kb, which is much smaller than predicted from the size of paranemin (~280 kDa) by SDS-PAGE. The pI and molecular weight, predicted from the deduced amino acid sequence of paranemin, are 4.17 and 178,161 Daltons, respectively. I found that paranemin is a novel intermediate filament (IF) protein, which may be classified as a type VI IF protein. Paranemin contains the conserved IF rod domain (308 amino acids), which is 63.3% identical in amino acid sequence to the rod domain of tanabin and 45.5% identical to the rod domain of nestin. The partial cDNA sequences of two proteins, namely EAP-300 and IFAPa-400, which overlap each other by 402 nucleotides, are almost identical to parts of the cDNA sequence of paranemin.</p>