Mapping of the Alz 50 epitope in microtubule‐associated proteins tau

Alz 50 and seven other monoclonal antibodies have been shown to react with both tau and Alzheimer brain proteins of molecular mass 60–70 kDa. The location of some of the epitopes of these antibodies (Alz 50, Tau‐2, NP14, Ab 636.7) on the tau molecule is unknown, whereas those of others (Tau 60, Tau 14, Tau‐1, Tau 46) have recently been demonstrated in fetal human tau at amino acid residues 60–72, 83–120, 131–140, and 315–352. To determine the location of the unknown epitopes, human tau was digested with chymotrypsin and trypsin, and the bovine microtubule fraction was incubated with chymotrypsin. Comparison of the immunoblots of chymotryptic digested tau with those of untreated preparations showed that the Alz 50 epitope was more sensitive than other tau epitopes to proteolysis. Cleavage of a 3–4 kDa polypeptide from the periphery of tau was sufficient to remove the Alz 50 epitope, but not the epitopes of Tau 46 (C‐end) or Tau 60 (N‐end). The distribution of the Alz 50 epitope in endogenously degraded, chymotrypsin or trypsin digested tau fragments was different from that of the Tau 46 epitope known to be located within 38 residues from the C‐terminus of the tau molecule. Based on these observations Alz 50 epitope was considered to be located within 3–4 kDa of the N‐terminus of tau. A comparison of immunoblots of different tau‐reactive antibodies showed similarities between Tau 60 and Tau‐2, and between Tau 14, Tau‐1, NP14, and Ab 636.7. Microtubule fractions, after chymotryptic digestion, retained small fragments (<29 kDa) reactive with all tau antibodies except Alz 50 and Tau 60. The two smallest fragments (<20–22 kDa), which were bound to digested microtubules and presumably contained microtubule binding domains, were recognized by Ab 636.7 and Tau 46. Among the eight tau‐reactive antibodies, the epitopes of Tau‐1, Ab 636.7, NP14, and Tau 46 were conserved in tau from human, bovine, rat, and mouse. The epitopes of the other four antibodies were more species specific. These results support our conclusion that the Alz 50 epitope is located at the N‐terminus of tau, the Tau‐2 epitope is located between Tau 60 and Tau 14 epitopes, and the Ab 636.7 and NP14 epitopes are in the C‐half of tau molecule closer than Tau‐1 epitope to the C‐terminus.