Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving transferase
Reexamination Certificate
1994-11-28
2001-03-13
Duffy, Patricia A. (Department: 1645)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving transferase
C435S007100, C435S961000, C436S811000
Reexamination Certificate
active
06200768
ABSTRACT:
The invention relates to epitopes of the tau protein which are specifically occurring in a phosphorylated state in tau protein from Alzheimer paired helical filaments, to protein kinases which are responsible for the phosphorylation of the amino acids of the tau protein giving rise to said epitopes, and to antibodies specific for said epitopes. The invention further relates to pharmaceutical compositions for the treatment or prevention of Alzheimer's disease, to diagnostic compositions and methods for the detection of Alzheimer's disease and to the use of said epitopes for the generation of antibodies specifically detecting Alzheimer tau protein. Additionally, the invention relates to methods for testing drugs effective in dissolving Alzheimer paired helical filaments or preventing the formation thereof.
The brains of Alzheimer patients contain two characteristic types of protein deposits, the plaques and the tangles. These structures have been of peak importance in Alzheimer research during the last few years (for a recent review see Goedert et al., Current opinion in Neurobiology 1 (1991), 441 to 447). A prominent component of the tangles are the paired helical filaments (PHFs). It seems now clear that the PHFs are largely made up of the microtubule-associated protein tau which is normally attached to the neuronal microtubule network and, furthermore, particularly enriched in the axons.
There are six isoforms of tau in human brain that arise from alternative splicing of a single gene. All these isoforms also occur in PHFs (Goedert et al., Neuron 3 (1989), 519-526). The main biochemical differences between normal and Alzheimer PHF tau protein known so far may be summarized as follows:
(1) PHF tau protein is, in contrast to normal tau protein, highly insoluble which makes a biochemical analysis difficult;
(2) PHF tau protein reacts with certain antibodies in a phosphorylation dependent manner, suggesting a special phosphorylation status (Grundke-Iqbal et al., Proc. Natl. Acad. Sci. USA 83 (1986), 4913-4917, Nukina et al., Proc. Natl. Acad. Sci. USA 84 (1987), 3415-3419);
(3) PHF tau protein has a lower electrophoretic mobility in SDS gels, suggesting a higher M
r
value which may be related to its phosphorylation pattern (Steiner et al., EMBO J. 9 (1990), 3539-3544);
(4) PHF tau protein forms paired helical filaments with a characteristic 78 nm crossover repeat (Crowther and Wischik, EMBO J. 4 (1985), 3661-3665).
Tau protein purified from brain has very little secondary structure (as judged by CD spectroscopy), and a sedimentation constant of 2.6S, pointing to a highly asymmetric shape (Cleveland et al., J. Mol. Biol. 1161 (1977), 227-247, in agreement with electron microscopic data (Hirokawa et al., J. Cell. Biol. 107 (1988), 1449-1459. The C-terminal half contains 3 or 4 internal repeats which are involved in microtubule binding and promoting their assembly (hence “assembly domain”). This domain can be phosphorylated by several protein kinases (Steiner et al., EMBO J. 9 (1990), 3539-3544), a point that may be significant in view of the abnormal phosphorylation of Alzheimer tau (see, e.g. Grundke-Iqbal et al., ibid.). Moreover, the repeat region also lies in the core of Alzheimer paired helical filaments (see, e.g. Goedert et al., ibid.; Jakes et al. EMBO J. 10(1991), 2725-2729).
It has been hypothesized that PHF tau protein has a lower affinity for microtubules compared to normal tau proteins since a similar effect has been found when normal tau is phosphorylated in vitro by some kinases (Lindwall and Cole, J. Biol. Chem. 259 (1984), 5301-5305). Lack or reduced binding to microtubules might therefore be a result of abnormal phosphorylation of the tau protein. This abnormal state might lead to microtubule disassembly and interfere with vital neuronal processes, such as rapid axonal transport. The abnormally phosphorylated tau proteins might then aggregate into PHFs. As a consequence thereof the neurons would eventually die thus setting the stage for the generation of the Alzheimer's disease.
Up to now, it was not known which protein kinases are responsible for the abnormal phosphorylation. Ishiguro et al. (Neuroscience Letters 128, (1991), 195-198) have isolated a kinase fraction from bovine brain extracts which contain a protein kinase recognizing the serine/threonine proline motif. This kinase phosphorylated residues Ser 144, Thr 147, Ser 177 and Ser 315 of the tau protein. These residues differed from the ones reported by others (Lee et al., Science 251 (1991), 675-678). Therefore, it remains unclear which protein kinase and which target amino acid residue(s) are involved in the generation of Alzheimer's disease, if at all.
It is, moreover, of utmost importance for the diagnosis of Alzheimer's disease, in particular at an early stage of the disease process, to develop antibodies which are specifically directed to epitopes on the protein which are characteristic of the Alzheimer state. A monoclonal antibody, TAU1, has been isolated which is capable of distinguishing between phosphorylated and non-phosphorylated forms of the tau protein (see, e.g., Lee et al., ibid.). However, this antibody specifically recognizes dephosphorylated tau protein which is seemingly not associated with the Alzheimer state. Another antibody, Alz 50 (Ksiezak-Reding et al., J. Biol. Chem. 263 (1988), 7943-7947) reacts with PHFs as well as with tau protein. Sternberger et al., Proc. Natl. Acad. Sci. USA 82 (1985), 4774-4776, have isolated an antibody, SMI 34, which recognizes a phosphorylated epitope common to Alzheimer tau protein and neurofilament protein. Finally, Lee et al. (ibid.) made antibodies directed to a phosphorylated peptide comprising the KSPV motif in the C-terminal region of the tau protein. All these antibodies known in the art have the disadvantage that for none of them it is known whether they recognize an epitope which is uniquely characteristic for the Alzheimer's disease state.
Furthermore, no reliable data on the fine structure of Alzheimer paired helical filaments, nor on the the mode or regulation of their formation from tau proteins is available so far. For the prevention of the formation of PHFs it would be highly advantageous if the mode of assembly of PHFs from tau protein and the regulatory mechanisms underlying said assembly were known.
Thus, the technical problem underlying the present invention was to provide a phosporylated epitope characteristic for the Alzheimer tau protein, a kinase activity which specifically catalyzes this phosphorylation, pharmaceutical compositions comprising inhibitors to said kinases, antibodies for recognizing said epitopes, diagnostic compositions containing said epitopes, methods involving kinases and/or antibodies for the in vitro diagnosis of Alzheimer's disease, methods for the in vitro conversion of normal tau protein into Alzheimer tau protein and methods for testing drugs effective in dissolving Alzheimer PHFs or preventing the formation thereof.
The solution to the above technical problem is achieved by providing the embodiments characterized in the claims. Accordingly, the present invention relates to an epitope of the tau protein which is specifically occurring in a phosphorylated state in tau protein from Alzheimer paired helical filaments.
The term “phosphorylated state in tau proteins from Alzheimer paired helical filaments” refers to a state of the tau protein where tau shows an upward M
r
shift, has a reduced binding to microtubules and is phosphorylated at ser or thr followed by pro, or certain serines in the repeat region (see below).
Note: Amino acids are denoted by the one-letter or three-letter code; see e.g. Lehninger, Biochemistry, 2nd edition, Worth Publishers, New York, 1975, page 72.
There may be one or more epitopes of the tau protein which specifically occur in a phosphorylated state in Alzheimer paired helical filaments. These epitopes may, moreover, be phosphorylated by a single or different enzymes displaying phosphorylating activity.
In a preferred embodiment of the presen
Biernat Jacek
Drewes Gerard
Lichtenberg-Kraag Birgit
Mandelkow Eckhard
Mandelkow Eva-Maria
Duffy Patricia A.
Marshall O'Toole Gerstein Murray & Borun
Max-Planck-Gesellschaft zur Forderung der Wissenschaften
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