The tau proteins are a family of brain microtubule binding proteins that are required during axonal outgrowth and are found in neurofibrillary tangles in AMzeimer disease. A protein of higher molecular weight, immunologically related to tan, is expressed in the adult peripheral system and in cultured neuronal cell lines of neural crest origin. The predicted amino acid sequence ofthe high molecular weight tau from N115 cells has been determined from the sequence of its 2340-base-pair cDNA. High molecular weight tau contains an open reading frame encoding 733 amino acid residues. It contains sequences homologous to those present in the N-, middle, and C-terminal domains of adult brain tau proteins, incinding four homologous repeats, which are the tubulin binding sites, and an amino acid stretch, which is present only in the N-terminal domain of the mature brain variants. The middle region contains a previously unidentified nonhomol gous stretch of 237 amino acid residues as well as a domain of 66 residues homologous to exon 6 of the bovine gene that is absent in all bovine, rat, and mouse tau cDNAs sequenced so far. A cDNA probe specific to the nonhomologous tau insert hybridizes to the 8-to 9-kilobase tau mRNA in N115 cells but not to the 6-kilobase tan mRNA in brain. Probes for the domains common to brain tau isoforms hybridize to both messages. The sequence of hig molecular weight tan protein also suggests that it, like low molecular weight tan, is an elongated hydrophilic molecule. This cDNA should allow us to study the role of the do specific to these tau forms in the specialization of the peripheral nervous system and for study of their expression in normal and pathological states.Tau and MAP2 proteins are microtubule-associated proteins (MAPs) that are able to promote polymerization of tubulin into microtubules in vitro (1-3). Although the microtubules formed in the presence of these two MAPs are similar (4), several lines of evidence suggest that MAP2 and tau proteins play an essential role in the establishment of neuronal polarity. MAP2 is preferentially localized in dendrites, whereas tau is almost exclusively targeted to the axons and its expression is required during axonal outgrowth (5)(6)(7)(8). Additional interest in tau protein has stemmed from its identification as a major component of the paired helical filaments, the abnormal structures characteristic of the Alzheimer disease (9-12).
T proteins are microtubule-associated proteins that promote microtubule polymerization in vitro and in vivo . They are a family of neuronal proteins with apparent molecular weights in the range 50,000-68,000 determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Recently, a new member of this family has been described and its cDNA has been cloned . It has an apparent molecular weight of 116,000 and has been called high-molecular-weight 4 (HMW T) . All the T proteins are encoded by a single gene, which undergoes complex alternative splicing . In the present study, we have cloned into the baculovirus a cDNA fully encoding HMW T as well as a truncated cDNA encoding a protein beginning 13 amino acids in front of the T microtubulebinding domain . HMW T-recombinant-virus-infected Sf9 cells overexpressed HMW T, which induced the polymerization of microtubules and the formation of long cellular processes similar to those induced by low-molecularweight T (LMW T) overexpression . Process cross sections revealed a larger spacing (~35 nm) between microtubules when induced by HMW T than when induced by LMW T (~20 nm) . The truncated construct also induces processes, where microtubules were packed far more closely together (~10 nm) . Although branching did not occur in processes induced by intact Ts, 10% of the processes induced by the truncated T protein branched .
Using epitope mapping we have demonstrated that a high molecular weight protein (Mr approximately 115 × 10(3)) present in brain and spinal cord is a member of the tau family of microtubule-associated proteins. Antibodies directed against the amino-terminal, middle and carboxyl-terminal portions of tau recognize this protein. A limited survey of neuronal tissues has shown that this high molecular weight tau protein is present in brain, spinal cord, dorsal root ganglia, dorsal and ventral roots and peripheral nerves. High molecular weight tau protein is expressed at higher levels in spinal cord than in brain and is the only form of tau detected in the adult peripheral nervous system.
Using a novel PCR approach, we have cloned a cDNA encoding the entire high molecular weight tau molecule from rat dorsal root ganglia. The resulting 2080 bp cDNA differs from low molecular weight rat brain tau by the insertion of a novel 762 bp region (exon 4a) between exons 4 and 5. This cDNA clone is identical in sequence with a high molecular weight tau (HMW) cDNA from rat PC12 tumor cells and is closely related to a HMW tau cDNA from mouse N115 tumor cells. In vitro transcription/translation produces a protein that migrates on SDS-PAGE with the same apparent molecular weight as HMW tau purified from rat sciatic nerve. The HMW tau protein is generated from an 8 kb mRNA, which can be detected by northern blots in peripheral ganglia, but not in brain. A more sensitive assay using PCR and Southern blot analysis demonstrates the presence of exon 4a in spinal cord and in retina. In combination with immunohistochemical studies of spinal cord, these data suggest that HMW tau, though primarily in the peripheral nervous system, is also expressed in limited areas of the central nervous system, although its presence cannot be detected in the cerebral cortices.
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