Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
1996-06-07
2001-04-24
Lankford, Jr., Leon B. (Department: 1651)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S212000, C435S219000, C530S387100, C530S388100, C530S388150, C530S388260
Reexamination Certificate
active
06221645
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the cleavage of &bgr;-amyloid precursor protein to produce &bgr;-amyloid peptide. More particularly, the present invention relates to isolated and purified compositions containing an enzyme responsible for such cleavage (&bgr;-secretase) and assays for identifying inhibitors of &bgr;-secretase.
Alzheimer's disease is characterized by the presence of numerous amyloid plaques and neurofibrillary tangles (highly insoluble protein aggregates) present in the brains of Alzheimer's disease patients, particularly in those regions involved with memory and cognition. &bgr;-amyloid peptide is a major constituent of amyloid plaque which is produced by cleavage of &bgr;-amyloid precursor protein. It is presently believed that a normal (non-pathogenic) processing of the &bgr;-amyloid precursor protein occurs via cleavage by a putative “&agr;-secretase” which cleaves between amino acids 16 and 17 of the &bgr;-amyloid peptide region within the protein. It is further believed that pathogenic processing occurs in part via a putative “&bgr;-secretase” which cleaves at the amino-terminus of the &bgr;-amyloid peptide region within the precursor protein. Heretofore, however, the existence of &bgr;-secretase has not been confirmed.
The identification, isolation, and characterization of novel biological molecules having unique activities is generally useful. For example, novel enzymes can be used to catalyze reactions of a type associated with their class. In particular, novel proteases can be used to cleave proteins for a variety of purposes, and the availability of new proteases provides unique capabilities. In addition to such uses associated with enzymes in general, the identification, isolation, and purification of the putative &bgr;-secretase enzyme would permit chemical modeling of a critical event in the pathology of Alzheimer's disease and would allow the screening of compounds to determine their ability to inhibit &bgr;-secretase activity.
For these reasons, it would be desirable to isolate, purify, and characterize the enzyme responsible for the pathogenic cleavage of &bgr;-amyloid precursor protein at the amino-terminus of the &bgr;-amyloid peptide region. In particular, it would be desirable to utilize such an enzyme (referred to hereinafter as &bgr;-secretase) in methods for screening candidate drugs for the ability to inhibit the activity of &bgr;-secretase in in vitro systems. It would be particularly desirable if such screening assays could be performed in a rapid format which would permit the screening of large numbers of test drugs in automated fashion.
2. Description of the Background Art
&bgr;-amyloid precursor protein (APP) is expressed in three differently-spliced forms of 695, 751, and 770 amino acids, and “normal” processing involves proteolytic cleavage at a site between residues Lys
16
and Leu
17
in the &bgr;-amyloid peptide. Kang et al. (1987)
Nature
325:773-776. Soluble &bgr;-amyloid peptide which has been cleaved at the putative &bgr;-secretase site has also been found in the culture medium of non-diseased cells (Haass et al. (1992)
Nature
359:322-325) and in CSF from healthy humans and animals (Seubert et al. (1992)
Nature
359:325-327). The possible existence of the putative &bgr;-secretase is discussed in, for example, Selkoe, “Cell Biology of the Amyloid &bgr;-Protein and the Mechanism of Alzheimer's Disease,” in
Annual Review of Cell Biology
, Spudich et al., eds., Annual Review, Inc., Palo Alto, Calif., vol. 10, 1994. The Swedish mutation of APP is also discussed in Selkoe, supra. See also, Esch et al. (1994) Science 248:1122.
SUMMARY OF THE INVENTION
The present invention provides novel &bgr;-secretase compositions comprising an isolated and purified enzyme which cleaves &bgr;-amyloid precursor protein (APP) at the amino-terminus of &bgr;-amyloid peptide (&bgr;AP) within APP, referred to hereinafter as “&bgr;-secretase activity.” The compositions of the present invention will generally have a &bgr;-secretase activity which is at least five-fold greater than that of a solubilized but unenriched membrane fraction from human 293 cells, preferably being at least ten-fold greater than that of the membrane fraction, and more preferably being at least 100-fold greater than that of the membrane fraction. The &bgr;-secretase enzyme is characterized by (1) an apparent molecular weight in the range from 260 kD to 300 kD as determined by gel exclusion chromatography, (2) a more accurate apparent molecular weight in the range from 60 kD to 148 kD determined by electrophoresis, (3) a net negative charge at pH 5 and a net negative charge at pH 7.5, and (4) binding to wheat germ agglutinin.
The compositions of the present invention are generally useful as proteolytic chemicals and specifically useful in assays for determining whether a test substance will inhibit proteolytic cleavage of APP resulting from the novel &bgr;-secretase. The method comprises exposing a polypeptide comprising the &bgr;-secretase site of APP (located at the amino-terminus of the &bgr;AP region within APP) to an at least partially purified &bgr;-secretase in the presence of the test substance under conditions such that the &bgr;-secretase would be expected to cleave the polypeptide into an amino-terminal fragment and a carboxy-terminal fragment in the absence of test substance which inhibits such cleavage. Test substances which inhibit such cleavage are thus identified as having &bgr;-secretase inhibition activity. Such test methods preferably employ the &bgr;-secretase compositions described above. Generation of fragments of APP-derived polypepetides is detected, e.g. by an antibody specific for the carboxy end of the amino terminal fragment or the amino end of the carboxy-terminal fragment. The polypeptide substrate for the &bgr;-secretase may comprise a fusion polypeptide including an amino-terminal portion having a binding epitope. Use of such a fusion polypeptide as the &bgr;-secretase substrate facilitates detection of cleavage by capture of the amino-terminal portion and labelling of the amino-terminal portion.
The compositions of the present invention further comprise purified &bgr;-secretase protein having an amino acid sequence substantially identical to at least a portion of the amino acid sequence of [SEQ ID No.:1]. Compositions still further comprise &bgr;-secretase protein analogs whose amino acid sequence is not naturally occurring or which is a fragment which comprise a contiguous sequence of at least ten amino acids from the amino acid sequence of native &bgr;-secretase [SEQ ID No.:1]. In one embodiment, the &bgr;-secretase analog or fragment, when presented as an immunogen, elicits the production of an antibody which specifically binds to natural &bgr;-secretase protein. In another embodiment, the &bgr;-secretase cleaves &bgr;-amyloid precursor protein at the &bgr;-amyloid peptide cleavage location.
The present invention still further provides recombinant nucleic acid molecules and compositions comprising a nucleotide sequence of at least 25 contiguous nucleotides from [SEQ ID No.:2]. In one embodiment, the recombinant nucleic acid molecule further comprises expression control sequences operatively linked to a nucleic acid sequence which encodes for the expression of a polypeptide whose amino acid sequence comprises a contiguous sequence of at least ten amino acids from [SEQ ID No.:1]. In another aspect, the recombinant nucleic acid molecules are transfected or otherwise introduced to recombinant host cells with an expression vector comprising the recombinant nucleic acid molecule having expression control sequences which provide for expression of &bgr;-secretase or a portion thereof having at least ten contiguous amino acids from [SEQ ID No.:1]. The recombinant nucleic acid molecules may further be provided as nucleic acid probes, typically including at least 15 contiguous nucleotides which specifically hybridize
Anderson John P.
Chrysler Susanna M. S.
Keim Pamela S.
McConlogue Lisa Clair
Sinha Sukanto
Elan Pharmaceuticals Inc.
Lankford , Jr. Leon B.
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