Phosphorylation-dependent epitopes of neurofilament antibodies on tau protein and relationship with Alzheimer tau.
We have studied the phosphorylation of tau protein from Alzheimer paired helical filaments, of tau from normal human brain, and of recombinant tau isoforms. As a tool we used monoclonal antibodies against neurofilament protein [Sternberger, N., Sternberger, L. & Ulrich, J. (1985) Proc. Natl. Acad. Sci. USA 82, 4274-4276] that crossreact with tau in a phosphorylation-dependent manner. This allowed us to deduce the state of phosphorylation in normal and pathological tau, as well as antibody epitopes. The epitope of antibody SMI33 is at the first Lys-Ser-Pro sequence motif (residues 234-236) and requires an unphosphorylated Ser-235. Antibody SMI31 binds between Ser-396 (in the second Lys-Ser-Pro motif) and Ser-404, both of which must be phosphorylated. SMI34 has a conformational epitope that depends on the interaction between regions on either side of the microtubule-binding region; it also requires phosphorylation. The phosphorylatable serines detected by the SMI antibodies are part of Ser-Pro motifs and can be phosphorylated by a protein kinase activity that can be used to induce a paired helical filament-like state in human brain tau in vitro. The phosphates are incorporated in several stages that can be identified by antibody reactivity and gel shift. This suggests a role for the phosphorylation sites in Alzheimer disease, as well as the involvement of a Ser-Pro-directed protein kinase.
Lichtenberg-Kraag
B
B
Max-Planck-Unit for Structural Molecular Biology, DESY, Hamburg, Federal Republic of Germany.
Mandelkow
E M
EM
Biernat
J
J
Steiner
B
B
Schröter
C
C
Gustke
N
N
Meyer
H E
HE
Mandelkow
E
E
D000544:Alzheimer Disease / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001921:Brain / Q000378:metabolism*; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D012016:Reference Values; D013997:Time Factors; D016875:tau Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*
D000906:Antibodies; D000939:Epitopes; D011994:Recombinant Proteins; D016875:tau Proteins; D011494:Protein Kinases