Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues
Reexamination Certificate
1997-12-02
2001-07-10
Kunz, Gary L. (Department: 1646)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
C530S327000, C530S324000, C530S350000
Reexamination Certificate
active
06258929
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention is in the general field of proteins involved in Alzheimer's disease.
Various genes and gene products involved in the development of Alzheimer's disease have been identified. Neuritic plaques characteristic of the disease are composed of &bgr;-amyloid (A&bgr;), which are oligopeptides of about 40-43 amino acids in length derived from the &bgr;-amyloid precursor protein (&bgr;APP). Mutations in the gene encoding &bgr;APP are associated with some cases of familial Alzheimer's disease. Other cases of familial Alzheimer's disease have been associated with mutations in two other loci, presenilin-1 and presinilin-2.
SUMMARY OF THE INVENTION
The invention is based on the discovery of a heretofore undescribed protein, which has been named ALARM or &dgr;-catenin, on the basis of its interaction with presenilin 1. ALARM shows a striking sequence similarity to members of the armadillo (arm)-plakoglobin-&bgr; catenin protein family. In addition, ALARM transcripts are confined almost exclusively to brain tissue.
In addition to the specific human ALARM sequences provided (or cross-referenced) herein, molecules relevant to the invention include fragments of those sequences and related polypeptides, non-peptide mimetics, and nucleic acid sequences. The invention also includes antibodies to ALARM polypeptides. These polypeptides, as well as nucleic acid encoding them, can be used for a variety of diagnostic and therapeutic applications.
In one aspect the invention features a substantially pure vertebrate ALARM polypeptide, e.g, an ALARM polypeptide from a mammal such as the human ALARM polypeptide shown in
FIG. 1
(SEQ ID NO:2).
By “protein” and “polypeptide” is meant any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation).
Polypeptides include, but are not limited to: recombinant polypeptides, natural polypeptides, and synthetic polypeptides as well as polypeptides which are preproteins or proproteins.
One way to ascertain purity of a preparation is by per cent dry weight. Generally, useful preparations are at least 60% by weight (dry weight) the compound of interest, i.e., an ALARM polypeptide. Preferably the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight the compound of interest. Purity can be measured by any appropriate standard method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. A “mature human ALARM” is the amino acid sequence shown in
FIG. 1
(SEQ ID NO:2).
Polypeptides substantially identical to mature human ALARM may have an amino acid sequence which is at least 85%, preferably 90%, and most preferably 95% or even 99% identical to the amino acid sequence of the ALARM polypeptide of the
FIG. 1
(SEQ ID NO:2). When assessing sequence identity of polypeptides, the length of the reference polypeptide sequence will generally be at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably 35 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 110 nucleotides.
Sequence identity can be measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705).
In the case of polypeptide sequences which are less than 100% identical to a reference sequence, the non-identical positions are preferably, but not necessarily, conservative substitutions for the reference sequence. Conservative substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine and glutamine; serine and threonine; lysine and arginine; and phenylalanine and tyrosine.
Where a particular polypeptide is said to have a specific percent identity to a reference polypeptide of a defined length, the percent identity is relative to the reference peptide. Thus, a peptide that is 50% identical to a reference polypeptide that is 100 amino acids long can be a 50 amino acid polypeptide that is completely identical to a 50 amino acid long portion of the reference polypeptide. It might also be a 100 amino acid long polypeptide which is 50% identical to the reference polypeptide over its entire length. Of course, many other polypeptides will meet the same criteria.
Polypeptides corresponding to one or more domains of ALARM are also within the scope of the invention. Thus, also featured is a polypeptide including at least one antigenic determinant of ALARM, a polypeptide comprising at least one copy of the 42 amino acid arm repeat in the ALARM polypeptide, or a polypeptide comprising a &bgr;APP binding domain of ALARM. Preferred polypeptides are those which are soluble under normal physiological conditions.
The polypeptides of the invention can be expressed fused to another polypeptide, e.g., a marker polypeptide or fusion partner. For example, the polypeptide can be fused to a hexa-histidine tag to facilitate purification of bacterially expressed protein or a hemagglutinin tag to facilitate purification of protein expressed in eukaryotic cells.
In another aspect, the invention also features a substantially pure polypeptide which includes a first portion and a second portion; the first portion includes an ALARM polypeptide and the said second portion includes a detectable marker. The first portion can be either a full-length form of ALARM or one or more domains thereof. The first portion is fused to an unrelated protein or polypeptide (i.e., a fusion partner) to create a fusion protein.
The invention also includes a pharmaceutical composition which includes an ALARM polypeptide.
In still another aspect the invention features a recombinant nucleic acid encoding an ALARM polypeptide. In one preferred embodiments the nucleic acid encodes a soluble ALARM polypeptide.
The invention also features isolated nucleic acids encoding polypeptides corresponding to one or more domains of ALARM or ALARM-related polypeptides discussed above. ALARM-encoding nucleotides can include the nucleic acids shown in
FIG. 1
, (SEQ ID NO:1) e.g., nucleotides 366-2636 of FIG.
1
. Also encompassed within the invention are nucleic acid sequences that encode forms of ALARM in which sequences are altered or deleted.
By “isolated nucleic acid” is meant nucleic acid that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (one on the 5′ end and one on the 3′ end) in the naturally occurring genome of the organism from which it is derived. Thus, a recombinant nucleic acid could include some or all of the 5′ non-coding (e.,g., promoter) sequences which are immediately contiguous to the coding sequence. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus, such as a retrovirus; or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.
Nucleic acid sequences substantially identical to human ALARM sequences have a nucleotide sequence which is at least 85%, preferably 90%, and most preferably 95% or even 99% identical to the amino acid sequence of the ALARM polypeptide of
FIG. 1
(SEQ ID NO:2). For nucleic acids, the length of the reference nucleic acid sequence will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 110 nucleotides.
Also within the invention are nucleic acids encoding
Kosik Kenneth S.
Zhou Jianhua
Brigham & Women's Hospital
Fish & Richardson P.C.
Gucker Stephen
Kunz Gary L.
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