Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1999-04-20
2001-05-15
Huff, Sheela (Department: 1642)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C435S004000, C435S007100
Reexamination Certificate
active
06232440
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to nucleic acid and amino acid sequences of an annexin binding protein and to the use of these sequences in the diagnosis, prevention, and treatment of cancer, immune disorders, and neurological disorders.
BACKGROUND OF THE INVENTION
Annexins are a family of some 10 structurally related proteins found in diverse eukaroytic organisms such as fruit fly, sponges, slime molds, higher plants, and mammals (Towle, C. A. and Treadwell, B. V. (1992) J. Biol. Chem. 267:5416-23). Proteins in this family reversibly bind to negatively charged phospholipids (phosphatidylcholine and phosphatidylserine) in a calcium dependent manner. Many of the functions attributed to annexins are believed to be the result of this binding property. These functions include; (1) regulation of phospholipase A2 activity, (2) anticoagulant activity, (3) roles in cellular exocytosis, (4) membrane trafficking, (5) cytoskeletal organization, (6) phosphohydrolase activity, (7) various aspects of cell proliferation, and (8) calcium channel activity (Towle et al. supra).
Annexin V is a specific family member found in a variety of species including human. It is widely distributed in various cells and tissues and is particularly abundant in brain, where it is believed to act as a paracrine-type neurotrophic factor (Ohsawa, K. et al. (1996) J. Neurochem. 67:89-97). It is also known to possess anticoagulant activity, transport Ca
2+
ions across phospholipid membranes, and inhibit phospholipase A2 and protein kinase C.
Various proteins bind members of the annexin family and are implicated in mediating their activity. These include several cytoskeletal proteins such as actin, calspectin, synapsin I, and glial fibrillary protein. In an attempt to elucidate the mechanism underlying neurotrophic activity in rat brain, Oshawa et al. (supra) sought and found four specific annexin binding proteins, ABP-2, ABP-4, ABP-7, and ABP-10. They also demonstrated that Ca
2+
and phospholipid were necessary for maximum binding to occur.
No common binding motifs were found among the four proteins; however, all four proteins were highly hydrophilic, showed no hydrophobic regions, and contained high proportions (10-20%) of lysine, glutamate, and serine. Arginine or aspartate was also prevalent in three of the four proteins. Some or all of these common properties may be asocciated with annexin binding.
In addition, two of the proteins, ABP-2 and ABP-4, each demonstrated high homlogy with the known human proteins, X-linked helicase (XH2) and DNA methyltransferase (DMTase), respectively. XH2 is a transcriptional regulator the mutations of which affects the expression of several genes and results in a form of X-chromosome linked mental retardation (Gibbons, R. J. et al. (1995) Cell 80:837-845). Helicases such as XH2 are involved in a variety of cellular functions including mitotic chromosome segregation, DNA recombination and repair, and regulation of transcription. DMTase is also a key enzyme in controlling gene expression and regulating cell differentiation and maturation (Ohsawa et al. supra).
The discovery of a new annexin binding protein and the polynucleotides encoding it satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention and treatment of cancer, immune disorders, and neurological disorders.
SUMMARY OF THE INVENTION
The invention features a substantially purified polypeptide, annexin binding protein (NABP-1), having the amino acid sequence shown in SEQ ID NO:1, or fragments thereof
The invention further provides an isolated and substantially purified polynucleotide sequence encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or fragments thereof and a composition comprising said polynucleotide sequence. The invention also provides a polynucleotide sequence which hybridizes under stringent conditions to the polynucleotide sequence encoding the amino acid sequence SEQ ID NO:1, or fragments of said polynucleotide sequence. The invention further provides a polynucleotide sequence comprising the complement of the polynucleotide sequence encoding the amino acid sequence of SEQ ID NO:1, or fragments or variants of said polynucleotide sequence.
The invention also provides an isolated and purified sequence comprising SEQ ID NO:2 or variants thereof. In addition, the invention provides a polynucleotide sequence which hybridizes under stringent conditions to the polynucleotide sequence of SEQ ID NO:2. The invention also provides a polynucleotide sequence comprising the complement of SEQ ID NO:2, or fragments or variants thereof.
The present invention further provides an expression vector containing at least a fragment of any of the claimed polynucleotide sequences. In yet another aspect, the expression vector containing the polynucleotide sequence is contained within a host cell.
The invention also provides a method for producing a polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment thereof, the method comprising the steps of: a) culturing the host cell containing an expression vector containing at least a fragment of the polynucleotide sequence encoding NABP-1 under conditions suitable for the expression of the polypeptide; and b) recovering the polypeptide from the host cell culture.
The invention also provides a pharmaceutical composition comprising a substantially purified NABP-1 having the amino acid sequence of SEQ ID NO:1 in conjunction with a suitable pharmaceutical carrier.
The invention also provides a purified antagonist of the polypeptide of SEQ ID NO:1. In one aspect the invention provides a purified antibody which binds to a polypeptide comprising the amino acid sequence of SEQ ID NO:1.
Still further, the invention provides a purified agonist of the polypeptide of SEQ ID NO:1.
The invention also provides a method for treating or preventing a neurological disorder comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising a substantially purified NABP-1.
The invention also provides a method for treating or preventing cancer comprising administering to a subject in need of such treatment an effective amount of a substantially purified antagonist of NABP-1.
The invention also provides a method for treating or preventing an immune disorder comprising administering to a subject in need of such treatment an effective amount of a substantially purified anatagonist of NABP-1.
The invention also provides a method for detecting a polynucleotide which encodes NABP-1 in a biological sample comprising the steps of: a) hybridizing the complement of the polynucleotide sequence which encodes SEQ ID NO:1 to nucleic acid material of a biological sample, thereby forming a hybridization complex; and b) detecting the hybridization complex, wherein the presence of the complex correlates with the presence of a polynucleotide encoding NABP-1 in the biological sample. In one aspect the nucleic acid material of the biological sample is amplified by the polymerase chain reaction prior to hybridization.
REFERENCES:
Towle, C.A., et al., “Identification of a Novel Mammalian Annexin”,The Journal of Biological Chemistry, 267(8): 5416-5423 (1992).
Ohsawa,K., et al., “Molecular cloning and Characterization of Annexin V-Binding Proteins with Highly Hydrophilic Peptide Structure”,The Journal of Neurochemistry, 67(1): 89-97 (1996). (GI 1514949) (GI 1514948).
Gibbons, R.J., et al., “Mutations in a Putative Global Transcriptional Regulator Cause X-Linked Mental Retardation with a -thalassemia (ATR-X Syndrome)”,Cell, 80: 837-845 (1995).
Ohsawa, K.,(GI 1514949) GenBank Sequence Database (Accession D64061), National Center for Biotechnology Information: National Library of Medicine, Bethesda, Maryland 20849. (GI 1514948) Aug. 29, 1996.
Ohsawa, K., (GI 1514948) GenBank Sequence Database (Accession D64061), National Center for Biotechnology Information: National Library of Medicine, Bethesda, Maryland 20849. (GI 1514949) Aug. 29, 1996.
Keiko, O. et al., “Mole
Corley Neil C.
Hillman Jennifer L.
Shah Purvi
Harris Alana M.
Huff Sheela
Incyte Genomics Inc.
Incyte Genomics, Inc.
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