Intracellular vitamin D binding protein

Details Intracellular vitamin D binding protein Intracellular vitamin D binding protein

1997-02-11

2001-07-31

Kunz, Gary L. (Department: 1646)

Chemistry: natural resins or derivatives; peptides or proteins;

Proteins, i.e., more than 100 amino acid residues

C536S023500, C530S358000

Reexamination Certificate

active

06268478

ABSTRACT:

FIELD OF THE INVENTION
The invention is in the field of vitamin D and steroid hormone signaling.
BACKGROUND
Most genera of new world primates exhibit vitamin D resistance. This resistance is biochemically characterized by the maintenance of a high circulating concentration of the active vitamin D hormone 1,25 dihydroxyvitamin D(1,25-(OH)2D) and the prohormone 25-hydroxyvitamin D(25-OHD). Clinical manifestations of vitamin D resistance include rickets in rapidly growing adolescent animals deprived of adequate sunlight exposure. Old world primates, including humans, do not exhibit vitamin D resistance. Levels of 25-OHD may be as much as ten fold lower, and levels of 1,25-(OH)
2
D as much as 100 fold lower, in old world primates than those observed in most new world primates. Vitamin D resistance phenomenon in new world primates also correlates with high circulating levels of other steroid hormones including glucocorticoid (Chrousos et al.
Endocrinology
115:25-32 (1984), Lipsett et al.
Recent Prog. Hormone Res.
42:199-246 (1985), Brandon et al.
Cancer Res.
49:2203-2213 (1989)), mineral corticoid, progesterone, testosterone, 17&bgr;-estradiol (Chrousos et al.
J. Clin. Endocrinol. Metab.
58:516-920 (1984)), 1,25-(OH)
2
D (Takahashi et al.
Biochem S.
227:555-563 (1985)).
Unlike the majority of resistant states described for other steroid hormones and vitamin D in humans, resistance to vitamin D in new world primates does not appear to be related to a mutation in the vitamin D receptor (VDR) protein. Rather, the vitamin D resistant state in new world primates is associated with the apparent high expression of an intracellular vitamin D binding protein (IDBP). IDBP is distinct from members of the serum vitamin D binding protein/albumin families of proteins in that IDBP is cysteine poor. Additionally, while serum vitamin D binding protein/albumin and vitamin D/steroid receptor protein families are principally confined to the extracellular domain and nucleus of the cell, respectively, IDBP predominantly localizes in the cell cytoplasm. However, like these other sterol/steroid binding proteins, IDBP preferentially binds 25-hydroxylated vitamin D metabolites.
IDBP activity has been enriched from extracts of new world primate cells. Nevertheless, numerous attempts to purify IDBP to homogeneity were unsuccessful. Therefore, the precise biochemical characteristics and primary molecular structure of IDBP remained elusive.
By gaining an understanding of the biochemical mechanisms behind vitamin D resistance and the high levels of circulating steroid hormones in new world primates, new opportunities for treating and diagnosing diseases related to either over-production or under-production of vitamin D and other steroidal hormones may be achieved. Such diseases include osteoporosis, hypercalcemia, and vitamin D intoxications.
SUMMARY OF THE INVENTION
The invention relates to the discovery and purification of novel intracellular vitamin D binding proteins (hereinafter “IDBP”) and the isolation of polynucleotide sequences encoding the proteins. Surprising, these IDBPs are heat shock proteins. The human homolog of IDBP is more commonly known as the heat shock protein hsp70. However, the property of steroid hormone binding has not been previously recognized in heat shock proteins. IDBPs are of interest because they mediate the vitamin D resistance, i.e., insensitivity, observed in new world primates. IDBPs are distinct from the vitamin D receptor and other intracellular receptors such as the estrogen receptor. IDBPs can interfere with the biological activity of the vitamin D receptor and other related intracellular receptor proteins. IDBPs of the invention can competitively bind to a variety of steroid compounds that normally bind to intracellular receptor proteins. These steroids include vitamin D, 17&bgr;-estradiol, testosterone, and progesterone. By binding to such steroid compounds, IDBPs may both prevent the ligands from interacting with their cognate intracellular receptor and serve to concentrate the intracellular receptors. Thus by regulating the intracellular levels of the subject IDBPs, desirable physiological effects may be obtained. Such effects may be used to treat a variety of diseases involving signaling at intracellular receptors including osteoporosis, glucocorticoid mediated disorders, and hypercalcemia associated with vitamin D overproduction, granuloma forming diseases.
One aspect of the invention is to provide compositions of purified IDBPs for use as mediators of steroid compound activity. The purified proteins may be obtained from either recombinant cells or naturally occurring cells. The purified intracellular vitamin D binding proteins of the invention may be mammalian in origin. Primate, including human and
Callithrix jacchus
(common marmoset), derived IDBPs are examples of the various IDBPs specifically provided for. The invention also provides allelic variants and biologically active derivatives of naturally occurring IDBPs.
Another aspect of the invention is to provide polynucleotides encoding the IDBPs of the invention and to provide polynucleotides complementary to the polynucleotide coding strand. The polynucleotides of the invention may be used to provide for the recombinant expression of IDBPs. The polynucleotides of the invention may also be used for genetic therapy purposes so as to treat diseases related to intracellular receptors that bind ligands that bind to IDBPs. The invention also provides polynucleotides for use as hybridization probes and amplification primers for the detection of naturally occurring polynucleotides encoding IDBPS. Preferred polynucleotides are those which encode the
Callithrix jacchus
-derived IDBP.
Another aspect of the invention is to provide antibodies capable of binding to the IDBPs of the invention for use in altering steroid compound activity. The antibodies may be polyclonal or monoclonal. The invention also provides methods of using the subject antibodies to detect and measure expression of vitamin D binding protein either in vitro or in vivo.
Another aspect of the invention is to provide assays for the detection or screening of therapeutic compounds that interfere with the interaction between IDBP and vitamin D (or other ligands that bind to IDBP). The assays of the invention comprise the step of measuring the effect of a compound of interest on binding between IDBP and vitamin (or other ligands that bind to IDBP). Binding may be measured in a variety of ways, including the use of labeled IDBP or labeled ligands.


REFERENCES:
Gacad et al. Identification and Partial characterization of a Non-Receptor Competitive Binding Protein for 1,25 Dihydroxy-Vitamin D3in Vitamin D-Resistant New World Primate Cells. Clinical Research 40(2): 207A, 1st col. (Abstract) (1992).
Gacad et al. Specificity of Steroid Binding in New World Primate B95-8 Cells with a Vitamin D-Resistant Phenotype. Endocrinology 131(6): 2581-2587 (1992).
Arbelle et al. Specificity of Vitamin D Sterol Binding to the Vitamin D Resistance-Causing Intracellular Binding Protein in New World Primates. Clinical Research 41(2): 238A. 1st col. (Abstract) (1993).
Gacad et al. Purification of a Novel Vitamin D Binding Protein From Vitamin D-Resistant New World Primate Cells. Clinical Research 41(2): 153A, 2nd col. (Abstract) (1993).
Arbelle et al., “Structural determinants of ligand binding to the vitamin D resistance-causing intracellular vitamin D binding protein in new world primates”,J. Bone Min. Res., 8:S226 (1993).
Gacad and Adams, “Partial characterization of an intracellular non-receptor vitamin D binding protein in vitamin D-resistant new world primate cells”,J. Bone Min. Res., 7:151S (1992).
Gacad and Adams, “Identification of competitive binding component in vitamin D-resistant new world primate cells with a low affinity but high capacity for 1,25-dihydroxyvitamin D3”,J. Bone Min. Res., 8:27-35 (1993).
Gacad and Adams, “The intracellular vitamin D binding protein (IDBP) in vitamin D-resistant new world cells in a member of the heat shock protein-70 (hsp-70

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