BINDING OF ALZ-50 DEPENDS ON PHE(8) IN TAU SYNTHETIC PEPTIDES AND VARIES BETWEEN NATIVE AND DENATURED TAU PROTEINS

Alz 50 is a monoclonal antibody that in Western blotting analysis reco gnizes both normal tau as well as hyperphosphorylated tau proteins ass ociated with paired helical filaments (PHF-tau) in Alzheimer disease ( AD). Within tissue sections of AD brain, however, Alz 50 immunolabels only PHF, which suggests that the antibody recognizes a conformational epitope. Using competitive enzyme-linked immunosorbent assay, we demo nstrate that Alz 50 binds to tau synthetic peptides with low affinity( K-D between 0.27 to 2.7 x 10(-5) M) and that the binding is specific f or the RQEF sequence corresponding to N-terminal residues 5-8 of tau. The Alz 50 epitope appears to be largely dependent on Phe(8), a strong ly hydrophobic amino acid residue, since the substitution of phe(8) wi th Ala(8) in the synthetic peptide abolishes Alz 50 binding. The effec ts of tau conformation on Alz 50 binding were studied with various nor mal tau proteins with either low or high phosphate content (adult vs. fetal) and PHF-tau proteins. The normal tau fractions were isolated fr om both adult and fetal human brains using affinity chromatography (na tive form) and heat/perchloric acid treatments (denatured form). PHF-t au was isolated as Sarcosyl-insoluble fraction. With competitive ELISA , the denatured form of normal tau (fetal and adult) bound Alz 50 with the same high affinity as did PHF-tau (K-D between 1.3 to 1.8 X 10(-7 ) M). In contrast, the native form of tau from either brain was unable to fully compete for Alz 50 and at most only 50% of the Alz 50 bindin g sites in native tau were occupied. These results suggest that native tau may exist either in complexes with other proteins or in a form of dimers/oligomers, in which only some N-termini are available for bind ing (e.g. head-to-tail assembly). The results also suggest that denatu ration rather than phosphorylation of tau has more significant effect on interactions of tau with Alz 50.