Alzheimer disease proteins (A68) share epitopes with tau but show distinct biochemical properties

Alz 50, a monoclonal antibody raised against Alzheimer brain homogenate, reacts with neurofibrillary tangles, microtubule-associated proteins tau, and Alzheimer brain proteins of molecular weight 70-60 kDa (A68). To study the relationship between A68 and normal human tau we compared the biochemical properties of these proteins and tested the reactivity of A68 with eight antibodies (Alz 50, Tau 60, Tau-2, Tau 14, Tau-1, Ab 636.7, NP14, Tau 46) that bind to various regions of tau molecule. On Western blots, all tau-reactive antibodies, except Tau-1, recognized A68. Pretreatment with alkaline phosphatase was required for the Tau-1 binding to A68. A68 consisted of three polypeptides of 68, 64, and 60 kDa, while tau contained 4-6 polypeptides of 50-65 kDa. A68 was less heterogenous than tau in the number of pI variants on two-dimensional gels. All A68 variants were more acidic (pI 5.5-6.5) than human tau (pI 6.5-8.5). Phosphatase treatment had only a minor effect on the pI and mobility of A68. Limited proteolysis of A68 with trypsin or chymotrypsin generated large fragments of 56-66 kDa (chymotrypsin) and 40-45 kDa (trypsin). While none of the fragments was recognized by Alz 50, the chymotryptic fragments were reactive with all the other tau antibodies, and the tryptic fragments were positive with five of the antibodies (Tau 14, Tau-1, Ab 636.7, NP14, and Tau 46). The peptide maps of A68 differed from that of tau in the number and the size of the peptide fragments. The differences in biochemical properties of these proteins and the sharing multiple epitopes suggest that A68 is a modified form of tau. The modification in part may be due to phosphorylation, although other changes rendering different isoelectrical properties and susceptibility to proteases need to be considered. The removal of the Alz 50 epitope by a cleavage of a 2-3 kDa fragment which does not contain the most C-terminal epitope (Tau 46) indicates that the Alz 50 epitope is located at the N-terminal periphery of the A68 molecule.