Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – 15 to 23 amino acid residues in defined sequence
Reexamination Certificate
2000-03-17
2002-02-26
Park, Hankyel T. (Department: 1648)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
15 to 23 amino acid residues in defined sequence
C424S185100, C424S186100, C424S193100, C424S204100, C424S139100, C435S004000, C435S007100, C530S387100, C530S350000, C530S388200, C530S388300, C530S826000
Reexamination Certificate
active
06350854
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the identification and isolation of proteins which bind with prion protein, referred to hereafter as PrP. More particularly, the isolated proteins of the invention has a molecular weight of from about 55 kD to about 72 kD as determined by SDS-PAGE, and are referred to hereafter as anti-PrP proteins or PrP binding protein. Also described is an isolated peptide consisting of an amino acid sequence from said binding protein/antiPrP protein. Both the peptide and the protein have various diagnostic efficacies. In the case of the peptide, it can be used, e.g., to produce antibodies which are in turn used to identify the anti-PrP protein. Also, the peptide can bind, itself, to PrP. Similarly, the full protein may be used in the same way. Various diseases associated with prions can thus be diagnosed or screened using these materials. Further, one can screen for the presence of PrP in a sample using the protein of the invention.
BACKGROUND AND PRIOR ART
“Prions” or “protein infectious particles”, have been implicated in a number of pathological conditions. Known prion associated diseases are referred to generally as spongiform encephalopathies, due to a common feature of the diseases, i.e., the formation of “holes” in cranial tissue.
By far the most commonly recognized disease associated with prions is “scrapie”, found in sheeps and goats. Afflicted animals lose coordination, and eventually become unable to stand. Additional animal disorders associated with prions include transmissible mink encephalopathy; chronic wasting disease of mule, deer and elk; feline spongiform encephalopathy; and bovine spongiform encephalopathy (“mad cow disease”). Among humans, Kuru, or “laughing death” has been associated with cannibalism. By far the most serious human disorder associated with prions, however, is Creutzfeldt-Jakob disease. This condition generally becomes evident via the development of dementia in the subject. It is a cause of great concern because it can be transmitted iatrogenically, such as by corneal transplantation, use of contaminated surgical instruments, injection of purified growth hormones or other pituitary based materials, as well as via implantation of dura mater or electrodes in the brain. Additional pathological conditions associated with prions include Gerstmann-Sträussler-Scheinker disease (lataxia and cerebellum damage), and fatal familial insomnia. Both of these conditions are inheritable, and typically appear in midlife.
At first, the aforementioned conditions were believed to be caused by a slow acting virus found in cerebral tissue. This hypothesis was based upon the observation that the diseases could be transmitted by injection of brain extracts of afflicted animals into healthy animals. This hypothesis, however, is generally no longer accepted, because a virus has not been isolated in spite of concerted efforts to do so.
What has been found about these conditions is that, although inheritable, they are caused by proteinaceous material, rather than by nucleic acids. The proteinaceous material is referred to as the prion. Among the experiments which led to the hypothesis that protein material was implicated was the treatment of materials from infected animals to inactivate proteins but not nucleic acids. Under these conditions, the disease was not transmitted.
Elaborations on this hypothesis have identified a single protein in scrapie prions. This protein, “prion protein”, will be referred to as—PrP—hereafter. It is used, generically, to refer to the protein which forms the prion. See, e.g., Prusener, Science 252: 1512-1522 (Jun. 14, 1991) (“Molecular Biology of Prion Diseases”); Prusiner, et al, ed.,
Prion Diseases of Humans And Animals
(Ellis Horwood, 1992).
As with all proteins, PrP is encoded by a gene; however, expression of PrP is not sufficient to cause a prion associated condition. It has been determined that PrP may undergo changes in its three dimensional structure, leading to its prion form. To elaborate, the benign form of PrP shows a multiple alpha helix geometry. In the form of infective prions, however, the three dimensional structure “elongates”, forming beta sheets. In summary, the difference between the normal, harmless form of PrP and the form associated with diseases, e.g., appears to be completely conformational.
“Complementary hydropathy”, a concept critical to understanding the invention described herein, was first suggested by Biro, Medical Hypothesis 7: 981 (1981). The concept Biro set forth was based upon an observation that protein/protein interactions were observed to be specific. He argued that complementary coding, i.e., coding by both sense and “anti-sense” strands of nucleic acid molecules could lead to the required specificity. Work on the interaction between ACTH, &ggr;-endorphin, angiotensin II, luteinizing hormone release hormone, and fibronectin, and their receptors, supports this hypothesis. See Bost, et al, Mol. Cell Endocrin 44:1 (1986) (ACTH); Carr, et al, J. Neuroimmunol 12: 329 (1986) (&ggr;-endorphin), Elton, et al, Proc. Natl. Acad. Sci. USA 85: 2518 (1988); (angiotensin II); Mulchahey, et al, Proc. Natl. Acad. Sci. USA 83: 9714 (1986) (luteinizing hormone-releasing hormone); and Brentani, et al, Proc. Natl. Acad. Sci. USA 85: 364 (1988) (fibronectin).
All of this work supported a concept hypothesized by Blalock, et al, Biochem. Biophys. Res. Commun. 121: 203 (1984). Their observation was that when the codons for hydropathic amino acids were compared to their complementary codons, these complementary codons were generally codons which code hydrophilic amino acids. Blalock, et al observed a significant correlation (r=0.74) between hydropathic coefficients of amino acids encoded for by opposing DNA strands, and thus postulated that peptides encoded by complementary DNA strands would bind one another. As indicated, supra, this hypothesis is supported for a number of peptides.
In 1991, Goldgaber, Nature 351: 106 (May 9, 1991), reported on the possible application of complementarily to PrP. Goldgaber reported analyzing PrP complementary DNA sequences, and the identification of a large, overlapping open reading frame in the DNA “antisense” strand for the PrP gene. When Goldgaber analyzed the deduced amino acid sequence for this complementary coding region, he found it to be nearly a mirror image of PrP. Goldgaber is incorporated by reference in its entirety. While Manson, et al, Nature 352: 291 (Jul. 25, 1991), questioned this work, Hewinson, et al, Nature 352: 291 (Jul. 25, 1991) noted that it confirmed their own work. Prusiner, et al, Nature 362: 213 (Mar. 3, 1993), provided an interesting “wrinkle” on this research, when they reported that they did find an RNA unit of the proper size (4.5 kb) for hybridizing to PrP sense strands, but it was not derived from the antisense PrP strand.
The reports discussed supra, as well as a report by Moser, et al, Nature 362: 213 (Mar. 18, 1993), discuss the possibility of the anti-PrP protein, as it will be referred to hereafter, in prion associated diseases. Hewinson, et al suggested that the complementary protein might be a PrP receptor.
The work which follows presents, for the first time, the identification and characterization of an anti-PrP binding protein. This material may be used to identify the presence of PrP in samples, thus providing a method of screening and/or diagnosis, especially when other symptoms characteristic of prion associated disorder are observed. In view of the prevalence of prion associated disorders in livestock, e.g., there are both human and veterinary uses for the invention.
REFERENCES:
patent: 5679530 (1997-10-01), Brentani et al.
Brentani, “Biological Implications of Complementary Hydropathy of Amino Acids,”J. Theor. Biol., 133:495-499 (1988).
Brentani, “Complementary Hydropathy and the Evolution of Interactive Polypeptides,”J. Mol. Evol., 31:239-243 (1990).
Goldgaber, “Anticipating the Anti-Prion Protein?”,Nature, 351:106 (1991).
Manson et al., “Anti-Prions and Other Agents,”Nature, 352:291 (
Brentani Ricardo R.
de Souza Sandro J.
Martins Vilma J.
Brown Stacy S.
Fulbright & Jaworski
Ludwig Institute for Cancer Research
Park Hankyel T.
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