HYPERPURIFICATION OF PAIRED HELICAL FILAMENTS REVEALS ELEVATIONS IN HYDROXYPROLINE CONTENT AND A CORE STRUCTURE RELATED PEPTIDE FRAGMENT

Abstract

We have subjected conventionally-purified Alzheimer's paired helical filaments (PHF) to electrophoresis in a Tris/borate/SDS buffer and obtained the separation of PHF core protein(s) (PHFi) from solubilized PHF-associated proteins (PHFs). Electron microscopy revealed intact PHF structures before and after this separation, but no evidence of any other structures in the PHFi fraction. The percent mass of hydroxyproline and glycine increased in the PHFi fraction after 4.5 hr of electrophoresis to account for 5.8% and 13.6% of the total mass, respectively. ELISA data confirmed that our PHFi and PHFs fractions were reactive with several putative PHF-specific antibodies. These data suggest that inappropriate hydroxylation of proline residues occurs in precursor PHF protein(s), resulting in the polymerization and subsequent insolubility of PHF in brain regions affected with Alzheimer's disease. Neurofibrillary tangles (NFT), one of the primary neuropathological features of Alzheimer's disease, are comprised of cytosolic bundles of uniform proteins which microscopically appear to be paired helical filaments (PHF). PHF are thought to be responsible for the cellular necrosis associated with the clinical symptoms of Alzheimer's disease (Dayan, 1970; Hirano and Zimmerman, 1962). Optical reconstruction of PHF has recently indicated that the true structure is more accurately described as a twisted ribbon of 30 A in the axial direction (Wischik et al., 1988). Immunological studies have suggested that tubulin (Grundke-Iqbal et al., 1979), microtubule associated proteins (Grundke-Iqbal et al., 1986; Kosik et al., 1986; Wood et al., 1986; Ksiezak-Reding et al., 1987), intermediate filaments (Yen et al., 1983), neurofilaments (Anderton et al., 1982), and ubiquitin (Mori et al., 1987; Perry et al., 1987), form part of the PHF core protein. To date, however, no study has been able to definitively show that any one of these purported PHF components is contained in the PHF core structure, rather than being non-covalently bound proteins or co-purifying contaminants. The present study extends previously published purification procedures, resulting in an isolated stable PHF core structure which is indistinguishable from previously published descriptions of PHF. The amino acid composition (Bidlingmeyer et al., 1984) of the PHF core structure appears to include hydroxyproline, an amino acid not commonly found in cytosolic proteins. We also demonstrate that some monoclonal antibodies previously raised against semi-purified PHF recognize determinants which are not related to the PHF core structure but rather recognize PHF-associated proteins or contaminants of the purification process.