Binding of Alz 50 depends on Phe8 in tau synthetic peptides and varies between native and denatured tau proteins.
Alz 50 is a monoclonal antibody that in Western blotting analysis recognizes both normal tau as well as hyperphosphorylated tau proteins associated 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 demonstrate that Alz 50 binds to tau synthetic peptides with low affinity (KD between 0.27 to 2.7 x 10(-5) M) and that the binding is specific for the RQEF sequence corresponding to N-terminal residues 5-8 of tau. The Alz 50 epitope appears to be largely dependent on Phe8, a strongly hydrophobic amino acid residue, since the substitution of Phe8 with Ala8 in the synthetic peptide abolishes Alz 50 binding. The effects of tau conformation on Alz 50 binding were studied with various normal tau proteins with either low or high phosphate content (adult vs. fetal) and PHF-tau proteins. The normal tau fractions were isolated from both adult and fetal human brains using affinity chromatography (native form) and heat/perchloric acid treatments (denatured form). PHF-tau 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 (KD 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 binding 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 binding (e.g. head-to-tail assembly). The results also suggest that denaturation rather than phosphorylation of tau has more significant effect on interactions of tau with Alz 50.
Ksiezak-Reding
H
H
Department of Pathology, Albert Einstein College of Medicine, Bronx, NY, USA.
Leibowitz
R L
RL
Bowser
R
R
Davies
P
P
D000328:Adult; D000368:Aged; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000941:Antigens / Q000276:immunology*; D002417:Cattle; D004797:Enzyme-Linked Immunosorbent Assay; D005333:Fetus; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry / Q000276:immunology; D010649:Phenylalanine / Q000737:chemistry; D011489:Protein Denaturation; D016875:tau Proteins / Q000737:chemistry / Q000276:immunology*
C056167:Alzheimer's disease antigen; D000911:Antibodies, Monoclonal; D000941:Antigens; D010446:Peptide Fragments; D016875:tau Proteins; C085348:tau-1 monoclonal antibody; D010649:Phenylalanine