Analyses of exon 4a structure reveal unique properties of Big tau.

Tau is a microtubule-associated protein that modulates the dynamic properties of microtubules and is involved in neurodegenerative diseases known as tauopathies. Tau is expressed as multiple low molecular weight (LMW) isoforms in most neurons of the central nervous system but only as a high molecular weight isoform in neurons of the peripheral nervous system and in a few types of central neurons. Big tau is defined by the inclusion of the alternatively spliced exon 4a, which adds about 250 amino acids to the domain of tau that projects away from the microtubule. Despite low sequence conservation of exon 4a, its length remains remarkably consistent across vertebrates. Here, we analyzed the charge distribution, hydrophobicity, and aggregation propensity of the human sequences of LMW tau, Big tau and the stretch of amino acids encoded by exon 4a. The exon 4a amino acids display a pronounced net negative charge (acidic/basic ratio = 1.30), a consistently hydrophilic composition (average Kyte-Doolittle score = -0.9259) and low β-sheet content of 4.78%. This contrasts with LMW tau, which is more hydrophobic (-0.8930) and contains extended aggregation-prone motifs within the microtubule-binding domain including high β-sheet content of 17.33%. The inclusion of exon 4a in Big tau shifts the global hydrophobicity to intermediate values (-0.9036) and reduces predicted β-sheet content to 13.14%, suggesting decreased aggregation potential. Evolutionary analyses across mammals, birds, and amphibians (human, rat, zebra finch, frog) confirms the minimal sequence identity (16-24% identity in non-mammals) and conserved exon size but show preservation of net negative charge (acidic/basic ratio 1.3-2.3), indicating convergent retention of charge-based properties. Hydrophilicity was also broadly conserved, though less invariant across species. These results demonstrate that exon 4a introduces a highly acidic, hydrophilic module that counterbalances the aggregation-prone domains of LMW tau. The conservation of size and structural properties of the exon-4a-encoded stretch of amino acids, despite sequence divergence, implies strong evolutionary pressure to maintain biophysical properties that counteract pathogenic misfolding.