WO2002064626A2 - Proteines humaines secretees isolees, molecules d'acides nucleiques codant pour des proteines humaines secretees et utilisations associees - Google Patents

Proteines humaines secretees isolees, molecules d'acides nucleiques codant pour des proteines humaines secretees et utilisations associees Download PDF

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WO2002064626A2
WO2002064626A2 PCT/US2001/042802 US0142802W WO02064626A2 WO 2002064626 A2 WO2002064626 A2 WO 2002064626A2 US 0142802 W US0142802 W US 0142802W WO 02064626 A2 WO02064626 A2 WO 02064626A2
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nucleic acid
amino acid
nos
seq
peptide
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PCT/US2001/042802
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WO2002064626A3 (fr
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Steven I. Ladunga
Maureen E. Higgins
Eugene Spier
Simon Greenberg
Yu Wang
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Pe Corporation (Ny)
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Priority claimed from US09/711,681 external-priority patent/US6503743B1/en
Priority claimed from US09/708,725 external-priority patent/US6489456B1/en
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Priority to AU2002258355A priority Critical patent/AU2002258355A1/en
Priority to JP2002564955A priority patent/JP2005503117A/ja
Priority to CA002427112A priority patent/CA2427112A1/fr
Priority to EP01273044A priority patent/EP1530586A2/fr
Publication of WO2002064626A2 publication Critical patent/WO2002064626A2/fr
Publication of WO2002064626A3 publication Critical patent/WO2002064626A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/18—Drugs for disorders of the alimentary tract or the digestive system for pancreatic disorders, e.g. pancreatic enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the present invention is in the field of secreted proteins that are related to the Wnt, lactate dehydrogenase, calgizzarin, retinoic acid receptor responder, MAP, and ferritin secreted protein subfamilies, recombinant DNA molecules, and protein production.
  • the present invention specifically provides novel secreted peptides and proteins and nucleic acid molecules encoding such secreted peptide and protein molecules, all of which are useful in the development of human therapeutics and diagnostic compositions and methods.
  • human proteins serve as pharmaceutically active compounds.
  • Several classes of human proteins that serve as such active compounds include hormones, cytokines, cell growth factors, and cell differentiation factors.
  • Most proteins that can be used as a pharmaceutically active compound fall within the family of secreted proteins. It is, therefore, important in developing new pharmaceutical compounds to identify secreted proteins that can be tested for activity in a variety of animal models.
  • the present invention advances the state of the art by providing many novel human secreted proteins.
  • Secreted proteins are generally produced within cells at rough endoplasmic reticulum, are then exported to the golgi complex, and then move to secretory vesicles or granules, where they are secreted to the exterior of the cell via exocytosis.
  • Secreted proteins are particularly useful as diagnostic markers. Many secreted proteins are found, and can easily be measured, in serum. For example, a 'signal sequence trap' technique can often be utilized because many secreted proteins, such as certain secretory breast cancer proteins, contain a molecular signal sequence for cellular export. Additionally, antibodies against particular secreted serum proteins can serve as potential diagnostic agents, such as for diagnosing cancer. Secreted proteins play a critical role in a wide array of important biological processes in humans and have numerous utilities; several illustrative examples are discussed herein. For example, fibroblast secreted proteins participate in extracellular matrix formation. Extracellular matrix affects growth factor action, cell adhesion, and cell growth.
  • Structural and quantitative characteristics of fibroblast secreted proteins are modified during the course of cellular aging and such aging related modifications may lead to increased inhibition of cell adhesion, inhibited cell stimulation by growth factors, and inhibited cell proliferative ability (Eleftheriou et al, Mutat Res 1991 Mar-Nov;256(2-6): 127-38).
  • amyloid beta/A4 protein precursor functions as a growth and/or differentiation factor.
  • the secreted form of APP can stimulate neurite extension of cultured neuroblastoma cells, presumably through binding to a cell surface receptor and thereby triggering intracellular transduction mechanisms.
  • Secreted APPs modulate neuronal excitability, counteract effects of glutamate on growth cone behaviors, and increase synaptic complexity.
  • secreted APPs play a major role in the process of natural cell death and, furthermore, may play a role in the development of a wide variety of neurological disorders, such as stroke, epilepsy, and Alzheimer's disease (Mattson et al, Perspect Dev Neurobiol 1998; 5(4):337-52).
  • PF4 platelet factor 4
  • beta- thromboglobulin beta- thromboglobulin
  • VEGF Vascular endothelial growth factor
  • the Wnt family of secreted, cysteine-rich glycoproteins is a family of highly conserved intercellular signaling proteins/Iigands that control a variety of developmental processes including cell fate, cell proliferation, cell polarity, cell migration, and epithelial-mesenchymal interactions. Through control of these cellular processes, Wnts contribute to the development of tissues and organs such as the limbs, the brain, the reproductive tract, and the kidney.
  • the Wnt signaling pathway regulates cell proliferation and differentiation in species as divergent as nematodes, flies, frogs, and humans.
  • Wnt signal transduction represents a fundamental mechanism for the generation of diverse cell fates during animal embryogenesis, and plays important roles in development, cellular proliferation, and differentiation. Mis- regulation of Wnt signaling can cause developmental defects and is implicated in the genesis of several human cancers. Many Wnt genes in the mouse have been mutated, leading to very specific developmental defects. Furthermore, Wnts are involved in processes as diverse as segmentation, CNS patterning, and control of asymmetric cell divisions.
  • the Wnt ligand binds to Frizzled family receptors on the cell surface to initiate a signal transduction cascade. Binding of Wnt to Frizzled receptors sends a signal that stabilizes cytoplasmic beta-catenin by do wnregulating the activity of a beta-catenin turnover complex. Beta-catenin can then translocate to the nucleus where it functions as a transcriptional activator and stimulates the expression of Wnt target genes including c-myc, c-jun, fra-1, and cyc ⁇ n DI. Some of the Wnt target genes, such as c-myc and cyclin DI, are oncogenes. Axin and its homolog Axil, which have recently been identified as components of the Wnt signaling pathway, negatively regulate this pathway.
  • Inappropriate activation of the Wnt signaling pathway has been implicated as a major factor in the development of human neoplasia.
  • Such oncogenic activation of the Wnt signaling pathway can occur at many levels.
  • inappropriate expression of the Wnt ligand and Wnt binding proteins have been observed in numerous human tumors.
  • Lack of regulation of the beta-catenin turnover complex, such as through activating mutations of beta-catenin has been observed in several tumors, and is likely to be a key contributing factor in neoplastic progression.
  • Activation of the Wnt signalling pathway by various means can therefore be a primary cause of oncogenesis, such as by affecting cell proliferation, morphology, and contact inhibition.
  • the Wnt signalling pathway may cooperate with other oncogenes in multistep tumour progression.
  • the Wnt signaling pathway is likely to play an important role in brain development and has been implicated in Alzheimer's Disease. Sustained loss of function of Wnt signaling components may trigger a series of misrecognition events, thereby participating in the onset and development of Alzheimer's Disease.
  • the Wnt gene family may also play an important role in various reproductive tract pathologies including cancer.
  • Wnt-7a one member of the Wnt gene family, Wnt-7a, plays a definitive role in uterine development and adult uterine function.
  • Wnt-7a is deregulated in response to pre-natal exposure to the synthetic estrogenic compound, DES.
  • DES synthetic estrogenic compound
  • Lactate dehydrogenase (LDH), sometimes referred to as lactic dehydrogenase, is the most clinically important dehydrogenase occurring in human serum. LDH is clinically important because serum level of certain isozymes reflect pathological conditions in particular tissues. Consequently, LDH is most often measured to evaluate the presence of tissue damage. LDH serves as an indicator suggestive of disturbances of the cellular integrity induced by pathological conditions. Since LDH is an enzyme present in essentially all major organ systems, serum LDH activity is abnormal in a large number of disorders. LDH is found in the cytoplasm of cells and catalyzes the following reversible reaction for the interconversion of pyruvate and lactate: Pyruvate + NADH ⁇ -» Lactate + NAD
  • This bidirectional reaction can be monitored spectrophotometrically by measuring either the increase in NADH at 340 nm produced in the lactate-to-pyruvate reaction, or by measuring the decrease in NADH at 340 nm produced in the pyruvate-to-lactate reaction.
  • Mammalian LDH exists as five tetrameric isozymes composed of combinations of two different polypeptide subunits with a molecular weight of 136,700 ⁇ 2,100 daltons per tetramer. These tetrameric isozymes each differ in catalytic, physical, and immunological properties.
  • H heart muscle
  • M muscle
  • H4 and M4 three hybrids
  • H3M H2M2 and HM3
  • H4 is the most negatively charged at pH 7 and appears nearest the anode upon zone electrophoresis.
  • Subunit H predominates in heart muscle and facilitates the aerobic oxidation of pyruvate.
  • the M subunit predominates in skeletal muscle and liver and is predominantly involved with anaerobic metabolism and pyruvate reduction.
  • LDH inhibits LDH; p-mercuribenzoate also inhibits LDH, but at a slower rate.
  • LDH is activated by a number of organic compounds that stabilize the enzyme, such as dimethyl sulfoxide, ethanol, and methanol. Diethystilbestrol and several of its derivatives also stabilize the enzyme. Normal total LDH levels are approximately 105 to 333 IU/L (international units per liter).
  • LDH levels may be indicative of such conditions as cerebrovascular accident (e.g., CVA, stroke), myocardial infarction, hemolytic anemia (as well as other forms of anemia), hypotension, infectious mononucleosis, intestinal ischemia and infarction, liver disease (e.g., hepatitis), muscle injury, muscular dystrophy, neoplastic conditions, pancreatitis, and pulmonary infarction.
  • CVA cerebrovascular accident
  • myocardial infarction e.g., myocardial infarction
  • hemolytic anemia as well as other forms of anemia
  • hypotension infectious mononucleosis
  • intestinal ischemia and infarction e.g., hepatitis
  • muscle injury e.g., muscular dystrophy
  • neoplastic conditions e.g., pancreatitis, and pulmonary infarction.
  • LDH isoenzyme analysis is for the diagnosis of myocardial infarction and other myocardial diseases. Serum LDH levels are elevated in various myocardial diseases and a certain pattern of LDH isoenzyme elevation occurs in myocardial disease. In the absence of hemolysis, an LDH-l/LDH-2 ratio greater than 1 (referred to as a "flipped" ratio) is usually indicative of acute myocardial infarction (AMI). This flipped ratio is observed in about 80% of AMI patients. The normal ratio rarely exceeds 0.80 in the absence of AMI. LDH activity begins to rise 8-12 hrs after the onset of chest pain, peaking at 24-48 hrs, and elevated levels may persist for 1 week or more.
  • LDH activity and its isoenzyme pattern are also useful indicators of pathological conditions in the lungs, such as cell damage or inflammation. Measurement of LDH activity levels and its isoenzyme pattern in pleural effusion and in bronchoalveolar lavage fluid may provide substantial information about lung and pulmonary endothelial cell injury.
  • LDH isoenzymes are also useful for evaluating other physiological conditions and disorders such as muscle trauma, liver damage, and a variety of malignancies. In these cases, " variations of isoenzyme distribution are useful diagnostic indicators. Serum LDH levels are also useful for diagnosing ovarian dysgerminoma and testicular germ cell tumors. Furthermore, LDH is also useful for establishing the survival duration and rate in Hodgkin's disease and non-Hodgkin's lymphoma, and in the follow-up of ovarian dysgerminoma. Additionally, LDH is also useful for a variety of NAD, NADH, NADP and NADPH related studies.
  • Calgizzarin also referred to as S100C and S100A11 protein, is a member of the SI 00 calcium-binding protein group.
  • the SI 00 proteins comprising at least 19 members, malce up a large subclass of the EF-hand family of calcium-binding proteins and exhibit diverse functions.
  • SI 00 proteins consist of two EF-hand calcium-binding motifs, connected by a flexible loop.
  • a number of SI 00 proteins form complexes with annexins, another family of calcium-binding proteins that also bind to phospholipids.
  • SI 00 proteins are important in intracellular calcium metabolism, and altered expression of SI 00 proteins is associated with several human disorders such as cancer, neurodegenerative diseases, cardiomyopathies, inflammatary disorders, diabetes, and allergies.
  • Calgizzarin in particular, may also function in cytoskeleton assembly and dynamics.
  • SI 00 protein family are involved in the calcium-dependent (and, in some instances, Zn2+- or Cu2+-dependent) regulation of a variety of intracellular activities including, but not limited to, protein phosphorylation, various enzyme activities, cell proliferation and differentiation (including tumorigenesis), cytoskeleton assembly and dynamics, the structural organization of membranes, intracellular calcium homeostasis, inflammation, and in protection from oxidative cell damage.
  • Some SI 00 members are released or secreted into the extracellular space where they may exert trophic or toxic effects depending on their concentration, act as chemoattractants for leukocytes, modulate cell proliferation, or regulate macrophage activation (Donato, Biochim Biophys Acta 1999 Jul 8;1450(3):191-231).
  • SI 00 dimers may expose two binding surfaces on opposite sides. Consequently, homodimeric SI 00 proteins are ideal for crossbridging two homologous or heterologous target proteins (Donato, Biochim Biophys Acta 1999 Jul 8;1450(3):191-231).
  • S100C forms a homodimer.
  • a stoichiometry of one peptide per S100C monomer has been observed, with the entire complex structure consisting of two peptides per S100C dimer. Each peptide interacts with both monomers of the S100C dimer.
  • the two S100C molecules of the dimer are linked by a disulphide bridge (Rety et al, Structure Fold Des 2000 Feb 15;8(2): 175-84).
  • S 100 proteins are differentially expressed in a large number of cell types and each member of the S 100 family exhibits a unique pattern of tissue/cell type specific expression. For example, several members of this family are widely distributed in the central nervous system of vertebrates and have been implicated in nervous system development, function, and disease.
  • SI 00 proteins may play a role in the regulation of glial proliferation and neuronal differentiation (Marks et al, Bioessays 1990 Aug;12(8):381-3), and may also be involved in the development of Down Syndrome. It has been suggested that abnormalities in SI 00 protein gene dosage at a critical period during development may be responsible for some of the neurologic abnormalities associated with Down Syndrome (Marks et al, Bioessays 1990 Aug;12(8):381-3).
  • S100C RNA levels varied from high in the placenta, through intermediate in heart, lung, kidney, and most muscle samples, to barely detectable in brain tissue. S100C was found to be predominantly localized in the nucleus, which is distinct from other SI 00 proteins, with slight variations among different glioblastoma cell types (Inada et al, Biochem Biophys Res Commun 1999 Sep 16;263(1):135- 8). Calcium-binding proteins may endow tumor cells with properties related to their malignancy and metastatic phenotype.
  • S100C has been found to be associated with uveal melanoma, the primary ocular tumor of adults (Van Ginkel et al, Biochim Biophys Acta 1998 Dec 10;1448(2):290-7).
  • Antibodies reactive with S100 protein are useful as diagnostic markers for cutaneous tumors.
  • S100C is dramatically down-regulated in immortalized human fibroblasts compared with their normal counterparts (Sakaguchi et al, J Cell Biol 2000 Jun 12; 149(6): 1193-206), indicating a possible involvement of nuclear S100C in the contact inhibition of cell growth.
  • S100C as well two isoforms of S100A9, S100A8, and S100A6, are significantly up-regulated in transformed colon mucosa (Stulik et al, Electrophoresis 1999 Dec;20(18):3638-46), suggesting a role of S100C in colorectal tumorigenesis. S100C expression is also remarkably elevated in colorectal cancers compared with normal colorectal mucosa (Tanaka et al, Cancer Lett 1995 Mar 2;89(2): 195-200).
  • S 100 A7 (also referred to as psoriasin) has been found to be expressed in breast cancer cell lines and in cancer cells of some breast carcinomas but not expressed in non-cancerous tissues, with the exception of skin, further suggesting that SI 00 genes may be involved in the regulation of cell transformation and/or differentiation (Moog-Lutz et al, Int J Cancer 1995 Oct 9;63(2):297-303).
  • S 100 protein is an early marker of cerebral damage and is useful as a marker for diagnosing cerebral injury following cardiac surgery.
  • SI 00 is released after cardiac surgery performed under cardiopulmonary bypass (CPB) and is an indicator of the degree of brain injury.
  • the level of SI 00 is correlated with the duration of CPB, deep circulatory arrest, and aortic cross-clamping.
  • Increased levels of SI 00 protein are correlated with the age of the patient and the number of microemboli, especially during aortic cannulation.
  • Perioperative cerebral complications such as stroke, delayed awakening, and confusion are associated with increased levels of SI 00 protein directly after bypass and from 15 to 48 hours afterwards.
  • increased levels of SI 00 protein are related to neuropsychological dysfunction following cardiac surgery.
  • Retinoids or vitamin A metabolites/derivatives
  • RARs retinoic acid receptors
  • 9cRA 9-cis retinoic acid
  • RXRs retinoid X receptors
  • RXR/RAR heterodimers modulate ligand-dependent gene expression by interacting as RXR/RAR heterodimers or RXR homodimers on specific target gene DNA sequences known as hormone response elements.
  • RXRs also serve as heterodimeric partners of nuclear receptors for vitamin D, thyroid hormone, and peroxisome proliferators (reviewed by Mangelsdorf et al., at pages 319-349 of The Retinoids, Second Edition, Sporn et al. (Raven Press, New York, 1994)).
  • retinoids are essential for normal growth, vision, tissue homeostasis, reproduction and overall survival (for reviews and references, See Sporn et al., The Retinoids, Vols. 1 and 2, Sporn et al., eds., Academic Press, Orlando, Fla. (1984)).
  • retinoids have been shown to be vital to the maintenance of skin homeostasis and barrier function in mammals (Fisher, G. J., and Voorhees, J. J., FASEB J. 10:1002-1013 (1996)).
  • Retinoids are also apparently crucial during embryogenesis, since offspring of dams with vitamin A deficiency (VAD) exhibit a number of developmental defects (Wilson, J.
  • retinoids synthetic structural analogues of all-trans retinoic acid or 9-cis-retinoic acid, commonly termed "retinoids", have been described in the literature to date. Some of these molecules are able to bind to, and specifically activate, the RARs or, on the other hand, the RXRs. Furthermore, some analogues are able to bind to, and activate a particular RAR receptor subtype (.alpha., .beta, or .gamma.). Finally, other analogues do not exhibit any particular selective activity with regard to these different receptors.
  • 9-cis-retinoic acid activates the RARs and the RXRs at one and the same time without any noteworthy selectivity for either of these receptors (nonspecific agonist ' ligand), whereas all-trans retinoic acid selectively activates the RARs (RAR-specific agonist ligand), with all subtypes being included.
  • a given substance or ligand is said to be specific for a given family of receptors (or, respectively, for a particular receptor of this family) when the said substance exhibits an affinity for all the receptors of this family (or, respectively, for the particular receptor of this family) which is stronger than that which it otherwise exhibits for all the receptors of any other family (or, respectively, for all the other receptors, of this same family or not).
  • the genetic activities of the RA signal are mediated through the two families of receptors—the RAR family and the RXR family— which belong to the superfamily of ligand- inducible transcriptional regulatory factors that include steroid/thyroid hormone and vitamin D3 receptors (for reviews see Leid et al., TIBS 17:427-433 (1992); Chambon, P., Semin. Cell Biol. 5:115-125 (1994); Chambon, P., FASEB J. 10:940-954 (1996); Giguere, V., Endocrinol. Rev. 15:61-79 (1994); Mangelsdorf, D. J., and Evans, R. M., Cell 83:841-850 (1995); Gronemeyer, H., and Laudet, V., Protein Profile 2:1 173-1236 (1995)).
  • RARs are the critical factors in tissue differentiation and development. They are up- regulated in rapidly dividing cells and tumors. RARs play an important role in lymphocyte activation. Synthetic antagonists of retinoic acid receptors can inhibit delayed type hypersensitivity (DTH). Growth factors and carotene regulate RXR expression levels. For example, granulocyte macrophage colony-stimulating factor induces retinoic acid receptors in myeloid leukemia cells.
  • Retinoic acid receptors can form heterodimers with other nuclear receptors.
  • the protein provided by the present invention can be used as a probe to detect possible interactions in the two-hybrid assay.
  • Synthetic peptides that mimic dimerization surface can disrupt intermolecular interactions between these receptors.
  • RAR gene rearrangements are the primary causes of some types of leukemia and provide a convenient genetic marker for malignant cell lines.
  • a number of retinoic acid derivatives are used in treatment of myelodysplastic disorders. They are designed to bind and activate RXRs.
  • Beta-carotene can prevent skin tumor formation in mouse models. N-(4-hydroxyphenyl)retinamide can delay onset of dysplasia in bronchi.
  • Different chemopreventive drugs can be designed to target individual retinoic receptors.
  • the sequences provided by the present invention may be used to design high affinity chemopreventive compounds.
  • the receptors differ in several important aspects.
  • the RARs and RXRs are significantly divergent in primary structure (e.g., the ligand binding domains of RAR.alpha. and RXR.alpha. have only 27% amino acid identity). These structural differences are reflected in the different relative degrees of responsiveness of RARs and RXRs to various vitamin A metabolites and synthetic retinoids.
  • distinctly different patterns of tissue distribution are seen for RARs and RXRs.
  • RXR.alpha. mRNA has been shown to be most abundant in the liver, kidney, lung, muscle and intestine.
  • RARs and RXRs have different target gene specificity.
  • response elements have recently been identified in the cellular retinal binding protein type II (CRBPII) and apolipoprotein Al genes which confer responsiveness to RXR, but not RAR.
  • CBPII retinal binding protein type II
  • apolipoprotein Al genes which confer responsiveness to RXR, but not RAR.
  • RAR has also been recently shown to repress RXR-mediated activation through the CRBPII
  • RXR response element (Manglesdorf et al, Cell, 66:555-61 (1991)). These data indicate that two retinoic acid responsive pathways are not simply redundant, but instead manifest a complex interplay. Recently, Heyman et al. (Cell, 68:397-406 (1992)) and Levin et al. (Nature, 355:359-61 (1992)) independently demonstrated that 9-cis-retinoic acid is a natural endogenous ligand for the RXRs. 9-cis-retinoic acid was shown to bind and transactivate the RXRs, as well as the RARs, and therefore appears to act as a "bifunctional" ligand. RAR Receptors
  • Receptors belonging to the RAR family are activated by both all-trans- and 9-cis-RA (Leid et al., TIBS 17:427-433 (1992); Chambon, P., Semin. Cell Biol. 5:115-125 (1994); Dolle, P., et al., Mech. Dev. 45:91-104 (1994); Chambon, P., FASEB J. 10:940-954 (1996)).
  • the DNA binding (C) and the ligand binding (E) domains of the three RAR types are highly similar, whereas the C-terminal domain F and the middle domain D exhibit no or little similarity.
  • the amino acid sequences of the three RAR types are also notably different in their B regions, and their main isoforms (.alpha.1 and .alpha.2, .beta.l to .beta.4, and .gamma.1 and .gamma.2) further differ in their N-terminal A regions (Leid et al., TIBS 17:427-433 (1992)).
  • RAR isoforms contain two transcriptional activation functions (AFs) located in the N-terminal A/B region (AF-1) and in the C-terminal E region (AF-2), which can synergistically, and to some extent differentially, activate various RA-responsive promoters (Leid et al, TIBS 17:427-433 (1992); Nagpal, S., et al., Cell 70:1007-1019 (1992); Nagpal, S., et al., EMBO J. 12:2349-2360 (1993)).
  • AFs transcriptional activation functions located in the N-terminal A/B region
  • AF-2 C-terminal E region
  • RXR.alpha., .beta members of the retinoid X receptor family (RXR.alpha., .beta, and
  • RXRs characterized to date are similar to the RARs in that the different RXR types also differ markedly in their N-terminal A/B regions (Leid et al, TIBS 17:427-433 (1992); Leid et al., Cell 68:377-395 (1992); Mangelsdorf et al., Genes and Dev.
  • RXR.alpha. and RXR.beta. have a widespread (possibly ubiquitous) expression pattern during mouse development and in the adult animal, being found in all fetal and adult tissues thus far examined (Mangelsdorf, D. J., et al., Genes & Devel.
  • RXR.gamma. transcripts appear to have a more restricted distribution, being expressed in developing skeletal muscle in the embryo (where their expression persists throughout life), in the heart (after birth), in sensory epithelia of the visual and auditory systems, in specific structures of the central nervous system, and in tissues involved in thyroid hormone homeostasis, e.g., the thyroid gland and thyrotrope cells in the pituitary (Mangelsdorf, D. J., et al., Genes & Devel.
  • RXR.beta. in the mouse have provided some insight into the physiological functions of these receptors.
  • the ocular and cardiac malformations observed in RXR.alpha.. sup. -/- fetuses are similar to those found in the fetal VAD syndrome, thus suggesting an important function of RXR.alpha. in the transduction of a retinoid signal during development.
  • RXRs in retinoid signaling are further supported by studies of compound RXR.alpha./RAR mutants, which reveal defects that are either absent or less severe in the single mutants (Kastner, P., et al., Cell 78:987-1003 (1994); Kastner, P., et al.,
  • RXR.gamma. sup.-/- homozygotes which are also RXR. alpha.. sup.-/- or RXR.beta.. sup.-/- exhibit no additional abnormalities beyond those seen in RXR.alpha.. sup.-/-, RXR.beta.. sup.-/- and fetal VAD syndrome fetuses (Krezel, W., et al., Proc. Natl. Acad. Sci.
  • RXR allele is sufficient to carry out all of the vital developmental and postnatal functions of the RXR family of receptors, particularly all of the developmental functions which depend on RARs and may require RXR partnership (Dolle, P., et al., Mech. Dev. 45:91-104 (1994); Kastner, P., et al., Cell 83:859-869 (1995)). Furthermore, the finding that RXR.alpha.. sup.-/- /RXR. gamma.. sup.-/- double mutant embryos are not more affected than are single RXR. alpha.. sup.-/- mutants (Krezel et al., Proc. Natl. Acad. Sci.
  • Nuclear receptors are members of a superfamily of ligand-inducible transcriptional regulatory factors that include receptors for steroid hormones, thyroid hormones, vitamin D3 and retinoids (Leid, M., et al., Trends Biochem. Sci. 17:427-433 (1992); Leid, M., et al., Cell 68:377-395 (1992); and Linney, E. Curr. Top. Dev. Biol.,
  • NRs exhibit a modular structure which reflects the existence of several autonomous functional domains.
  • the highly conserved region C contains two zinc fingers and corresponds to the core of the DNA-binding domain (DBD), which is responsible for specific recognition of the cognate response elements.
  • DBD DNA-binding domain
  • Region E is functionally complex, since in addition to the ligand-binding domain (LBD), it contains a ligand-dependent activation function (AF-2) and a dimerization interface.
  • An autonomous transcriptional activation function (AF-1) is present in the non-conserved N-terminal A/B regions of the steroid receptors.
  • AF-1 and AF-2 of steroid receptors exhibit differential transcriptional activation properties which appear to be both cell type and promoter context specific (Gronemeyer, H. Annu. Rev. Genet. 25:89-123 (1991)).
  • T-RA and 9-cis (9C-RA) retinoic acid signals are transduced by two families of nuclear receptors, RAR .alpha., .beta, and .gamma, (and their isoforms) are activated by both T-RA and 9C-RA, whereas RXR .alpha., .beta, and .gamma, are exclusively activated by 9C-RA (Allenby, G. et al., Proc. Natl. Acad. Sci. USA 90:30-34 (1993)).
  • the three RAR types differ in their B regions, and their main isoforms (.alpha.1 and .alpha.2, .beta.1-4, and .gamma.1 and .gamma.2) have different N-terminal A regions (Leid, M. et al., Trends Biochem. Sci. 17:427-433 (1992)).
  • the RXR types differ in their A/B regions (Mangelsdorf, D. J. et al., Genes Dev. 6:329-344 (1992)).
  • the E-region of RARs and RXRs has also been shown to contain a dimerization interface (Yu, V. C. et al., Curr. Opin. Biotechnol.
  • RAR/RXR heterodimers bind much more efficiently in vitro than homodimers of either receptor to a number of RA response elements (RAREs, also known as retinoic acid receptor responders) (Yu, V. C. et al., Cell 67:1251-1266 (1991); Berrodin, T. J. et al., Mol. Endocrinol 6:1468-1478 (1992); Bugge, T. H. et al., EMBO J. 1 1 :1409-1418
  • RAR and RXR heterodimers are also preferentially formed in solution in vitro (Yu, V. C. et al., Cell 67:1251-1266 (1991); Leid, M. et al, Cell 68:377-395 (1992); Marks, M. S. et al., EMBO J. 11 :1419-1435 (1992)), although the addition of 9C-RA appears to enhance the formation of RXR homodimers in vitro (Lehman, J. M. et al., Science 258:1944-1946 (1992); Zhang, X. K. et al., Nature 358:587-591 (1992b)). It has been shown that activation of RA-responsive promoters likely occurs through
  • the basis for the highly pleiotropic effect of retinoids may reside, at least in part, in the control of different subsets of retinoid-responsive promoters by cell-specifically expressed heterodimeric combinations of RAR:RXR types (and isoforms), whose activity may be in turn regulated by cell-specific levels of all-trans- and 9-cis-RA (Leid et al., TIBS 17:427-433 (1992)).
  • the RXR receptors may also be involved in RA-independent signaling.
  • the observation of aberrant lipid metabolism in the Sertoli cells of RXR.beta..sup.-/- mutant animals suggests that functional interactions may also occur between RXR.beta. and the peroxisomal proliferator-activated receptor signaling pathway (WO 94/26100; Kastner, P., et al., Genes & Devel. 10:80-92 (1996)).
  • Microtubule-associated proteins regulate microtubule stability and play critical roles in neuronal development and the balance between neuronal plasticity and rigidity.
  • Microtubule-associated proteins 1 A MAPI A
  • MAPI IB are abundant neuronal MAPs thought to be involved in neurite formation and stabilization.
  • MAPI A and MAP IB are two microtubule-associated proteins expressed at high levels in developing and mature neurons. These MAPs are synthesized as polyproteins that are proteolytically processed to form a heavy chain (-250 kDa) and a light chain (-30 kDa).
  • Light chain 3 (“LC3") is a subunit of both MAP 1 A and MAP 1 B .
  • MAPI A stabilizes microtubules in postnatal axons.
  • MAP IB is expressed in neurons and glial cells predominantly during a period of extension of neurites in the developing brain, suggesting an important role for MAP IB during neurite formation and axon guidance.
  • the relative levels of MAPI A and MAP IB change dramatically during development, with MAP IB expression highest in forming neurons, and MAPI A expression highest in mature neurons.
  • Light chain 3 (LC3) is a subunit of the neuronal microtubule-associated proteins
  • MAPs MAPI A
  • MAP1B MAPI A
  • cDNAs for MAPI A and MAP IB encode polyproteins that contain the MAPI A or MAP IB heavy chain and an LC2 or LCI subunit, respectively.
  • the cDNA encoding rat LC3 has been sequenced, and its sequence is not found in the MAPI A/LC2 or MAPlB/LClpolyprotein cDNAs.
  • the deduced amino acid sequence of LC3 is highly conserved between rat and mouse.
  • Rat LC3 is a 16.4- kDa protein with a predicted pi of 9.2. It is encoded on a 1.7-kilobase mRNA. Purified recombinant rat LC3 retains the ability to associate with microtubules assembled in the presence of brain MAPs and with microtubules assembled from purified tubulin.
  • Anti-LC3 immunohistochemistry reveals that LC3 in rat brain is restricted to neurons that are expressing either the MAPI A or MAP IB heavy chain. Although LC3 is expressed exclusively in cells expressing heavy chains, developmental changes in the total amount of LC3 protein are not proportional to changes in the amount of either the MAPI A or MAP IB heavy chain. LC3 protein expression measured by quantitiative immunoblotting is twice as high in postnatal brain as in embryonic and adult brain. The localization of the LC3 gene to human chromosome 20cen-ql3 demonstrates that LC3 is the only MAPI subunit that is not linked to the heavy chain genes. Because LC3 is a component of both the MAPI A and MAP IB microtubule-binding domains, the heavy-chain independent regulation of LC3 expression might modify MAPI microtubule-binding activity during development.
  • Iron is an essential element to metabolism, yet can be toxic in excessive amounts. Therefore, iron homeostasis —the ability to store and release iron in a controlled manner — is essential. Excess iron is stored as ferritin, primarily in the liver and spleen. Ferritins are a family of highly conserved iron-storage proteins that sequester iron inside a protein coat as a hydrous ferric oxide-phosphate mineral similar in structure to the mineral ferrihydrite.
  • a typical ferritin is composed of 24 subunits which interact noncovalently, forming a spherical shell.
  • the 24 subunits are either heavy (“H") or light (“L") chain peptides.
  • the H subunit contains ferroxidase activity and is responsible for converting soluble ferrous (Fe+2) iron to the storable (Fe+3) form. It has been proposed that the L subunit facilitates mineralization of iron at the ferritin core.
  • the ratio of H/L subunits in a ferritin is organ- specific. For example, the liver contains ferritin predominantly made of L subunits, whereas the heart contains predominantly H-ferritin.
  • H-ferritin subunits In the adult human brain, H-ferritin subunits have been observed to be roughly twice as abundant as L-subunits. The ratio of H/L ferritin in the brain also differs with cell type. Neurons contain predominantly H-ferritin, microglia contain predominantly L-ferritin, and oligodendrocytes contain a mixture of both ferritin subunits. Astrocytes in general do not contain ferritin except in mice and in the human striatum (an iron-rich area), where the astrocytes immunostain intensely for L-ferritin subunits. The differential cellular distribution of H- and L-ferritin indicates that neurons have a high iron requirement with little capacity to store iron, whereas the predominance of L-ferritin in microglia is consistent with their role as scavenger cells.
  • iron homeostasis disorders include hemachromatosis and thalassemia.
  • Deregulation of iron homeostasis has also been implicated in Alzheimer's disease.
  • Secreted proteins particularly members of the Wnt, lactate dehydrogenase, calgizzarin, retinoic acid receptor responder, MAP, and ferritin secreted protein subfamilies, are a major target for drug action and development. Accordingly, it is valuable to the field of pharmaceutical development to identify and characterize previously unknown members of this subfamily of secreted proteins.
  • the present invention advances the state of the art by providing previously unidentified human secreted proteins that have homology to members of the Wnt, lactate dehydrogenase, calgizzarin, retinoic acid receptor responder, MAP, and ferritin secreted protein subfamilies.
  • the present invention is based in part on the identification of amino acid sequences of human secreted peptides and proteins that are related to the Wnt, lactate dehydrogenase, calgizzarin, retinoic acid receptor responder, MAP, and ferritin secreted protein subfamilies, as well as allelic variants and other mammalian orthologs thereof.
  • These unique peptide sequences, and nucleic acid sequences that encode these peptides can be used as models for the development of human therapeutic targets, aid in the identification of therapeutic proteins, and serve as targets for the development of human therapeutic agents that modulate secreted protein activity in cells and tissues that express the secreted protein.
  • FIGURE 1 provides the nucleotide sequence of a cDNA molecule or transcript sequence that encodes the secreted protein of the present invention. (SEQ ID NOS: 1-6)
  • structure and functional information is provided, such as ATG start, stop and tissue distribution, where available, that allows one to readily determine specific uses of inventions based on this molecular sequence.
  • FIGURE 2 provides the predicted amino acid sequence of the secreted protein of the present invention. (SEQ ID NOS:7-12) In addition structure and functional information such as protein family, function, and modification sites is provided where available, allowing one to readily determine specific uses of inventions based on this molecular sequence.
  • FIGURE 3 provides genomic sequences that span the gene encoding the secreted protein of the present invention.
  • SEQ ID NOS:13-18 structure and functional information, such as intron/exon structure, promoter location, etc., is provided where available, allowing one to readily determine specific uses of inventions based on this molecular sequence.
  • 9 SNPs, including 1 indel have been identified in the gene encoding the Wnt protein provided by the present invention.
  • 4 SNPs have been identified in the gene encoding the calgizzarin protein provided by the present invention.
  • SNPs As indicated in Figure 3D, 30 SNPs, including 4 indel s, have been identified in the gene encoding the retinoic acid receptor responder protein provided by the present invention. As indicated in Figure 3E, 8 SNPs have been identified in the gene encoding the MAP protein provided by the present invention. As indicated in Figure 3F, 1 SNP has been identified in the gene encoding the ferritin protein provided by the present invention.
  • the present invention is based on the sequencing of the human genome.
  • sequencing and assembly of the human genome analysis of the sequence information revealed previously unidentified fragments of the human genome that encode peptides that share structural and/or sequence homology to protein/peptide/domains identified and characterized within the art as being a secreted protein or part of a secreted protein and are related to the Wnt, lactate dehydrogenase, calgizzarin, retinoic acid receptor responder, MAP, and ferritin secreted protein subfamilies. Utilizing these sequences, additional genomic sequences were assembled and transcript and/or cDNA sequences were isolated and characterized.
  • the present invention provides amino acid sequences of human secreted peptides and proteins that are related to the Wnt, lactate dehydrogenase, calgizzarin, retinoic acid receptor responder, MAP, and ferritin secreted protein subfamilies, nucleic acid sequences in the form of transcript sequences, cDNA sequences and/or genomic sequences that encode these secreted peptides and proteins, nucleic acid variation (allelic information), tissue distribution of expression, and information about the closest art known protein/peptide/domain that has structural or sequence homology to the secreted protein of the present invention.
  • the peptides that are provided in the present invention are selected based on their ability to be used for the development of commercially important products and services. Specifically, the present peptides are selected based on homology and/or structural relatedness to known secreted proteins of the Wnt, lactate dehydrogenase, calgizzarin, retinoic acid receptor responder, MAP, and ferritin secreted protein subfamilies and the expression pattern observed. The art has clearly established the commercial importance of members of this family of proteins and proteins that have expression patterns similar to that of the present gene.
  • the present invention provides nucleic acid sequences that encode protein molecules that have been identified as being members of the secreted protein family of proteins and are related to the Wnt, lactate dehydrogenase, calgizzarin, retinoic acid receptor responder, MAP, and ferritin secreted protein subfamilies (protein sequences are provided in Figure 2, transcript/cDNA sequences are provided in Figure 1 and genomic sequences are provided in Figure 3).
  • the peptide sequences provided in Figure 2 as well as the obvious variants described herein, particularly allelic variants as identified herein and using the information in Figure 3, will be referred herein as the secreted peptides of the present invention, secreted peptides, or peptides/proteins of the present invention.
  • the present invention provides isolated peptide and protein molecules that consist of, consist essentially of, or comprise the amino acid sequences of the secreted peptides disclosed in the Figure 2, (encoded by the nucleic acid molecule shown in Figure 1 , transcript/cDN A or Figure 3, genomic sequence), as well as all obvious valiants of these peptides that are within the art to make and use. Some of these variants are described in detail below.
  • a peptide is said to be "isolated” or “purified” when it is substantially free of cellular material or free of chemical precursors or other chemicals.
  • the peptides of the present invention can be purified to homogeneity or other degrees of purity. The level of purification will be based on the intended use. The critical feature is that the preparation allows for the desired function of the peptide, even if in the presence of considerable amounts of other components (the features of an isolated nucleic acid molecule is discussed below).
  • substantially free of cellular material includes preparations of the peptide having less than about 30% (by dry weight) other proteins (i.e., contaminating protein), less than about 20% other proteins, less than about 10% other proteins, or less than about 5% other proteins.
  • the peptide when it is recombinantly produced, it can also be substantially free of culture medium, i.e., culture medium represents less than about 20% of the volume of the protein preparation.
  • the language "substantially free of chemical precursors or other chemicals” includes preparations of the peptide in which it is separated from chemical precursors or other chemicals that are involved in its synthesis.
  • the language "substantially free of chemical precursors or other chemicals” includes preparations of the secreted peptide having less than about 30% (by dry weight) chemical precursors or other chemicals, less than about 20% chemical precursors or other chemicals, less than about 10% chemical precursors or other chemicals, or less than about 5% chemical precursors or other chemicals.
  • the isolated secreted peptide can be purified from cells that naturally express it, purified from cells that have been altered to express it (recombinant), or synthesized using known protein synthesis methods.
  • a nucleic acid molecule encoding the secreted peptide is cloned into an expression vector, the expression vector introduced into a host cell and the protein expressed in the host cell.
  • the protein can then be isolated from the cells by an appropriate purification scheme using standard protein purification techniques. Many of these techniques are described in detail below.
  • the present invention provides proteins that consist of the amino acid sequences provided in Figure 2 (SEQ ID NOS:7-12), for example, proteins encoded by the transcript/cDNA nucleic acid sequences shown in Figure 1 (SEQ ID NOS: 1-6) and the genomic sequences provided in Figure 3 (SEQ ID NOS: 13- 18).
  • the amino acid sequence of such a protein is provided in Figure 2.
  • a protein consists of an amino acid sequence when the amino acid sequence is the final amino acid sequence of the protein.
  • the present invention further provides proteins that consist essentially of the amino acid sequences provided in Figure 2 (SEQ ID NOS:7-12), for example, proteins encoded by the transcript/cDNA nucleic acid sequences shown in Figure 1 (SEQ ID NOS: 1-6) and the genomic sequences provided in Figure 3 (SEQ ID NOS:13-18).
  • a protein consists essentially of an amino acid sequence when such an amino acid sequence is present with only a few additional amino acid residues, for example from about 1 to about 100 or so additional residues, typically from 1 to about 20 additional residues in the final protein.
  • the present invention further provides proteins that comprise the amino acid sequences provided in Figure 2 (SEQ ID NOS: 7- 12), for example, proteins encoded by the transcript/cDNA nucleic acid sequences shown in Figure 1 (SEQ ID NOS: 1-6) and the genomic sequences provided in Figure 3 (SEQ ID NOS:13-18).
  • a protein comprises an amino acid sequence when the amino acid sequence is at least part of the final amino acid sequence of the protein. In such a fashion, the protein can be only the peptide or have additional amino acid molecules, such as amino acid residues (contiguous encoded sequence) that are naturally associated with it or heterologous amino acid residues/peptide sequences. Such a protein can have a few additional amino acid residues or can comprise several hundred or more additional amino acids.
  • the preferred classes of proteins that are comprised of the secreted peptides of the present invention are the naturally occurring mature proteins. A brief description of how various types of these proteins can be made/isolated is provided below.
  • the secreted peptides of the present invention can be attached to heterologous sequences to form chimeric or fusion proteins.
  • Such chimeric and fusion proteins comprise a secreted peptide operatively linked to a heterologous protein having an amino acid sequence not substantially homologous to the secreted peptide. "Operatively linked" indicates that the secreted peptide and the heterologous protein are fused in-frame.
  • the heterologous protein can be fused to the N-terminus or C-terminus of the secreted peptide.
  • the fusion protein does not affect the activity of the secreted peptide per se.
  • the fusion protein can include, but is not limited to, enzymatic fusion proteins, for example beta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-His fusions, MYC- tagged, Hl-tagged and Ig fusions.
  • Such fusion proteins, particularly poly-His fusions can facilitate the purification of recombinant secreted peptide.
  • expression and/or secretion of a protein can be increased by using a heterologous signal sequence.
  • a chimeric or fusion protein can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different protein sequences are ligated together in- frame in accordance with conventional techniques.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and re-amplified to generate a chimeric gene sequence (see Ausubel et al, Current Protocols in Molecular Biology, 1992).
  • many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST protein).
  • a secreted peptide-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the secreted peptide.
  • the present invention also provides and enables obvious variants of the amino acid sequence of the proteins of the present invention, such as naturally occurring mature forms of the peptide, allelic/sequence variants of the peptides, non-naturally occurring recombinantly derived variants of the peptides, and orthologs and paralogs of the peptides. Such variants can readily be generated using art-known techniques in the fields of recombinant nucleic acid technology and protein biochemistry. It is understood, however, that variants exclude any amino acid sequences disclosed prior to the invention.
  • variants can readily be identified/made using molecular techniques and the sequence information disclosed herein. Further, such variants can readily be distinguished from other peptides based on sequence and/or structural homology to the secreted peptides of the present invention. The degree of homology/identity present will be based primarily on whether the peptide is a functional variant or non-functional variant, the amount of divergence present in the paralog family and the evolutionary distance between the orthologs.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
  • at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more of the length of a reference sequence is aligned for comparison purposes.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid "homology”).
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
  • the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (Devereux, J., et al, Nucleic Acids Res.
  • the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of E. Myers and W. Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • nucleic acid and protein sequences of the present invention can further be used as a "query sequence" to perform a search against sequence databases to, for example, identify other family members or related sequences. Such searches can be performed using the
  • NBLAST and XBLAST programs (version 2.0) of Altschul, et al (J. Mol. Biol. 215:403-10 (1990)).
  • Gapped BLAST can be utilized as described in Altschul et al (Nucleic Acids Res. 25(17):3389-3402 (1997)).
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • Full-length pre-processed forms, as well as mature processed forms, of proteins that comprise one of the peptides of the present invention can readily be identified as having complete sequence identity to one of the secreted peptides of the present invention as well as being encoded by the same genetic locus as the secreted peptide provided herein.
  • the map position of the Wnt gene provided by the present invention was determined to be at chromosomal location 17q21 by ePCR.
  • the map position of the lactate dehydrogenase gene provided by the present invention was determined to be on chromosome 15 by ePCR.
  • the map position of the calgizzarin gene provided by the present invention was determined to be on chromosome 7 by ePCR.
  • the map position of the retinoic acid receptor responder gene provided by the present invention was determined to be on chromosome 16 by ePCR.
  • the map position of the MAP gene provided by the present invention was determined to be on chromosome 12 by ePCR.
  • the ferritin gene provided by the present invention maps to public BAC AC011181.
  • Allelic variants of a secreted peptide can readily be identified as being a human protein having a high degree (significant) of sequence homology/identity to at least a portion of the secreted peptide as well as being encoded by the same genetic locus as the secreted peptide provided herein. Genetic locus can readily be determined based on the genomic information provided in Figure 3, such as the genomic sequence mapped to the reference human. As indicated by the data presented in Figure 3 A, the map position of the Wnt gene provided by the present invention was determined to be at chromosomal location 17q21 by ePCR.
  • the map position of the lactate dehydrogenase gene provided by the present invention was determined to be on chromosome 15 by ePCR.
  • the map position of the calgizzarin gene > provided by the present invention was determined to be on chromosome 7 by ePCR.
  • the map position of the retinoic acid receptor responder gene provided by the present invention was determined to be on chromosome 16 by ePCR.
  • the map position of the MAP gene provided by the present invention was determined to be on chromosome 12 by ePCR.
  • the ferritin gene provided by the present invention maps to public BAC AC011181.
  • two proteins or a region of the proteins
  • a significantly homologous amino acid sequence, according to the present invention will be encoded by a nucleic acid sequence that will hybridize to a secreted peptide encoding nucleic acid molecule under stringent conditions as more fully described below.
  • Figure 3A provides information on SNPs that have been identified in a gene encoding the Wnt proteins of the present invention.
  • SNP variants were found, including 1 indel (indicated by a "-") and 2 SNPs in exons, of which 1 of these cause changes in the amino acid sequence (i.e., nonsynonymous SNPs).
  • Figure 3C provides information on SNPs that have been identified in a gene encoding the calgizzarin proteins of the present invention. 4 SNP variants were found.
  • Figure 3D provides information on SNPs that have been identified in a gene encoding the retinoic acid receptor responder proteins of the present invention.
  • 30 SNP variants were found, including 4 indels (indicated by a "-").
  • Figure 3E provides information on SNPs that have been identified in a gene encoding the MAP proteins of the present invention.
  • Figure 3F provides information on SNPs that have been identified in a gene encoding the ferritin proteins of the present invention. One SNP variant was found. The changes in the amino acid sequence that these SNPs cause is indicated in Figures 3 A-3F and can readily be determined using the universal genetic code and the protein sequence provided in Figures 2A-2F as a reference.
  • Paralogs of a secreted peptide can readily be identified as having some degree of significant sequence homology/identity to at least a portion of the secreted peptide, as being encoded by a gene from humans, and as having similar activity or function.
  • Two proteins will typically be considered paralogs when the amino acid sequences are typically at least about 60% or greater, and more typically at least about 70% or greater homology through a given region or domain.
  • Such paralogs will be encoded by a nucleic acid sequence that will hybridize to a secreted peptide encoding nucleic acid molecule under moderate to stringent conditions as more fully described below.
  • Orthologs of a secreted peptide can readily be identified as having some degree of significant sequence homology/identity to at least a portion of the secreted peptide as well as being encoded by a gene from another organism.
  • Preferred orthologs will be isolated from mammals, preferably primates, for the development of human therapeutic targets and agents. Such orthologs will be encoded by a nucleic acid sequence that will hybridize to a secreted peptide encoding nucleic acid molecule under moderate to stringent conditions, as more fully described below, depending on the degree of relatedness of the two organisms yielding the proteins.
  • Non-naturally occurring variants of the secreted peptides of the present invention can readily be generated using recombinant techniques.
  • Such variants include, but are not limited to deletions, additions and substitutions in the amino acid sequence of the secreted peptide.
  • one class of substitutions are conserved amino acid substitution.
  • Such substitutions are those that substitute a given amino acid in a secreted peptide by another amino acid of like characteristics.
  • conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu, and He; interchange of the hydroxyl residues Ser and Thr; exchange of the acidic residues Asp and Glu; substitution between the amide residues Asn and Gin; exchange of the basic residues Lys and Arg; and replacements among the aromatic residues Phe and Tyr.
  • Variant secreted peptides can be fully functional or can lack function in one or more activities, e.g. ability to bind substrate, ability to phosphorylate substrate, ability to mediate signaling, etc.
  • Fully functional variants typically contain only conservative variation or variation in non-critical residues or in non-critical regions.
  • Figure 2 provides the result of protein analysis and can be used to identify critical domains/regions.
  • Functional variants can also contain substitution of similar amino acids that result in no change or an insignificant change in function. Alternatively, such substitutions may positively or negatively affect function to some degree.
  • Non-functional variants typically contain one or more non-conservative amino acid substitutions, deletions, insertions, inversions, or truncation or a substitution, insertion, inversion, or deletion in a critical residue or critical region.
  • Amino acids that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham et al. , Science 2 4:1081-1085 (1989)), particularly using the results provided in Figure 2.
  • the latter procedure introduces single alanine mutations at every residue in the molecule.
  • the resulting mutant molecules are then tested for biological activity such as secreted protein activity or in assays such as an in vitro proliferative activity.
  • Sites that are critical for binding partner/substrate binding can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al, J. Mol Biol 224:899-904 (1992); de Vos et al Science 255:306-312 (1992)).
  • the present invention further provides fragments of the secreted peptides, in addition to proteins and peptides that comprise and consist of such fragments, particularly those comprising the residues identified in Figure 2.
  • the fragments to which the invention pertains are not to be construed as encompassing fragments that may be disclosed publicly prior to the present invention.
  • a fragment comprises at least 8, 10, 12, 14, 16, or more contiguous amino acid residues from a secreted peptide.
  • Such fragments can be chosen based on the ability to retain one or more of the biological activities of the secreted peptide or could be chosen for the ability to perform a function, e.g. bind a substrate or act as an immunogen.
  • Particularly important fragments are biologically active fragments, peptides that are, for example, about 8 or more amino acids in length.
  • Such fragments will typically comprise a domain or motif of the secreted peptide, e.g., active site or a substrate-binding domain.
  • fragments include, but are not limited to, domain or motif containing fragments, soluble peptide fragments, and fragments containing immunogenic structures.
  • Predicted domains and functional sites are readily identifiable by computer programs well known and readily available to those of skill in the art (e.g., PROSITE analysis). The results of one such analysis are provided in Figure 2.
  • Polypeptides often contain amino acids other than the 20 amino acids commonly referred to as the 20 naturally occurring amino acids. Further, many amino acids, including the terminal amino acids, may be modified by natural processes, such as processing and other post- translational modifications, or by chemical modification techniques well known in the art. Common modifications that occur naturally in secreted peptides are described in basic texts, detailed monographs, and the research literature, and they are well known to those of skill in the art (some of these features are identified in Figure 2).
  • Known modifications include, but are not limited to, acetylation, acylation, ADP- ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.
  • the secreted peptides of the present invention also encompass derivatives or analogs in which a substituted amino acid residue is not one encoded by the genetic code, in which a substituent group is included, in which the mature secreted peptide is fused with another compound, such as a compound to increase the half-life of the secreted peptide (for example, polyethylene glycol), or in which the additional amino acids are fused to the mature secreted peptide, such as a leader or secretory sequence or a sequence for purification of the mature secreted peptide or a pro-protein sequence.
  • a substituted amino acid residue is not one encoded by the genetic code, in which a substituent group is included, in which the mature secreted peptide is fused with another compound, such as a compound to increase the half-life of the secreted peptide (for example, polyethylene glycol), or in which the additional amino acids are fused to the mature secreted peptide, such as a leader or secretory sequence or a
  • the proteins of the present invention can be used in substantial and specific assays related to the functional information provided in the Figures; to raise antibodies or to elicit another immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its binding partner or ligand) in biological fluids; and as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state).
  • the protein binds or potentially binds to another protein or ligand (such as, for example, in a secreted protein-effector protein interaction or secreted protein- ligand interaction)
  • the protein can be used to identify the binding partner/ligand so as to develop a system to identify inhibitors of the binding interaction. Any or all of these uses are capable of being developed into reagent grade or kit format for commercialization as commercial products.
  • secreted proteins isolated from humans and their human/mammalian orthologs serve as targets for identifying agents for use in mammalian therapeutic applications, e.g. a human drug, particularly in modulating a biological or pathological response in a cell or tissue that expresses the secreted protein.
  • a large percentage of pharmaceutical agents are being developed that modulate the activity of secreted proteins, particularly members of the Wnt, lactate dehydrogenase, calgizzarin, retinoic acid receptor responder, MAP, and ferritin secreted protein subfamilies (see Background of the Invention).
  • the proteins of the present invention are useful for biological assays related to secreted proteins that are related to members of the Wnt, lactate dehydrogenase, calgizzarin, retinoic acid receptor responder, MAP, and ferritin secreted protein subfamilies.
  • Such assays involve any of the known secreted protein functions or activities or properties useful for diagnosis and treatment of secreted protein-related conditions that are specific for the subfamily of secreted proteins that the one of the present invention belongs to, particularly in cells and tissues that express the secreted protein.
  • the proteins of the present invention are also useful in drug screening assays, in cell- based or cell-free systems.
  • Cell-based systems can be native, i.e., cells that normally express the secreted protein, as a biopsy or expanded in cell culture.
  • cell-based assays involve recombinant host cells expressing the secreted protein.
  • the polypeptides can be used to identify compounds that modulate secreted protein activity of the protein in its natural state or an altered form that causes a specific disease or pathology associated with the secreted protein.
  • Both the secreted proteins of the present invention and appropriate variants and fragments can be used in high-throughput screens to assay candidate compounds for the ability to bind to the secreted protein. These compounds can be further screened against a functional secreted protein to determine the effect of the compound on the secreted protein activity. Further, these compounds can be tested in animal or invertebrate systems to determine activity/effectiveness. Compounds can be identified that activate (agonist) or inactivate (antagonist) the secreted protein to a desired degree.
  • the proteins of the present invention can be used to screen a compound for the ability to stimulate or inhibit interaction between the secreted protein and a molecule that normally interacts with the secreted protein, e.g. a substrate or a component of the signal pathway that the secreted protein normally interacts (for example, another secreted protein).
  • a molecule that normally interacts with the secreted protein e.g. a substrate or a component of the signal pathway that the secreted protein normally interacts (for example, another secreted protein).
  • Such assays typically include the steps of combining the secreted protein with a candidate compound under conditions that allow the secreted protein, or fragment, to interact with the target molecule, and to detect the formation of a complex between the protein and the target or to detect the biochemical consequence of the interaction with the secreted protein and the target.
  • Candidate compounds include, for example, 1) peptides such as soluble peptides, including Ig-tailed fusion peptides and members of random peptide libraries (see, e.g., Lam et al, Nature 354:82-84 (1991); Houghten et al, Nature 554:84-86 (1991)) and combinatorial chemistry-derived molecular libraries made of D- and/or L- configuration amino acids; 2) phosphopeptides (e.g., members of random and partially degenerate, directed phosphopeptide libraries, see, e.g., Songyang et al, Cell 72:161-118 (1993)); 3) antibodies (e.g., polyclonal, monoclonal, humanized, anti-idiotypic, chimeric, and single chain antibodies as well as Fab, F(ab ' )2, Fab expression library fragments, and epitope-binding fragments of antibodies); and 4) small organic and inorganic molecules (e.g.,
  • One candidate compound is a soluble fragment of the receptor that competes for substrate binding.
  • Other candidate compounds include mutant secreted proteins or appropriate fragments containing mutations that affect secreted protein function and thus compete for substrate. Accordingly, a fragment that competes for substrate, for example with a higher affinity, or a fragment that binds substrate but does not allow release, is encompassed by the invention.
  • any of the biological or biochemical functions mediated by the secreted protein can be used as an endpoint assay. These include all of the biochemical or biochemical/biological events described herein, in the references cited herein, incorporated by reference for these endpoint assay targets, and other functions lcnown to those of ordinary skill in the art or that can be readily identified using the information provided in the Figures, particularly Figure 2. Specifically, a biological function of a cell or tissues that expresses the secreted protein can be assayed.
  • Binding and/or activating compounds can also be screened by using chimeric secreted proteins in which the amino terminal extracellular domain, or parts thereof, the entire transmembrane domain or subregions, such as any of the seven transmembrane segments or any of the intracellular or extracellular loops and the carboxy terminal intracellular domain, or parts thereof, can be replaced by heterologous domains or subregions.
  • a substrate- binding region can be used that interacts with a different substrate then that which is recognized by the native secreted protein. Accordingly, a different set of signal transduction components is available as an end-point assay for activation. This allows for assays to be performed in other than the specific host cell from which the secreted protein is derived.
  • the proteins of the present invention are also useful in competition binding assays in methods designed to discover compounds that interact with the secreted protein (e.g. binding partners and/or ligands).
  • a compound is exposed to a secreted protein polypeptide under conditions that allow the compound to bind or to otherwise interact with the polypeptide.
  • Soluble secreted protein polypeptide is also added to the mixture. Ifthe test compound interacts with the soluble secreted protein polypeptide, it decreases the amount of complex formed or activity from the secreted protein target.
  • This type of assay is particularly useful in cases in which compounds are sought that interact with specific regions of the secreted protein.
  • the soluble polypeptide that competes with the target secreted protein region is designed to contain peptide sequences corresponding to the region of interest.
  • a fusion protein can be provided which adds a domain that allows the protein to be bound to a matrix.
  • glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtitre plates, which are then combined with the cell lysates (e.g., 3D S-labeled) and the candidate compound, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH).
  • the beads are washed to remove any unbound label, and the matrix immobilized and radiolabel determined directly, or in the supernatant after the complexes are dissociated.
  • the complexes can be dissociated from the matrix, separated by SDS-PAGE, and the level of secreted protein- binding protein found in the bead fraction quantitated from the gel using standard electrophoretic techniques.
  • the polypeptide or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin using techniques well known in the art.
  • antibodies reactive with the protein but which do not interfere with binding of the protein to its target molecule can be derivatized to the wells of the plate, and the protein trapped in the wells by antibody conjugation.
  • Preparations of a secreted protein-binding protein and a candidate compound are incubated in the secreted protein-presenting wells and the amount of complex trapped in the well can be quantitated.
  • Methods for detecting such complexes include immunodetection of complexes using antibodies reactive with the secreted protein target molecule, or which are reactive with secreted protein and compete with the target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the target molecule.
  • Agents that modulate one of the secreted proteins of the present invention can be identified using one or more of the above assays, alone or in combination. It is generally preferable to use a cell-based or cell free system first and then confirm activity in an animal or other model system. Such model systems are well known in the art and can readily be employed in this context.
  • Modulators of secreted protein activity identified according to these drug screening assays can be used to treat a subject with a disorder mediated by the secreted protein pathway, by treating cells or tissues that express the secreted protein. These methods of treatment include the steps of administering a modulator of secreted protein activity in a pharmaceutical composition to a subject in need of such treatment, the modulator being identified as described herein.
  • the secreted proteins can be used as "bait proteins" in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Patent No. 5,283,317; Zervos et al (1993) Cell 72:223-232; Madura et al.
  • the two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains.
  • the assay utilizes two different DNA constructs.
  • the gene that codes for a secreted protein is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4).
  • a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein (“prey" or "sample”) is fused to a gene that codes for the activation domain of the known transcription factor.
  • the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with the secreted protein.
  • a reporter gene e.g., LacZ
  • This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein in an appropriate animal model.
  • an agent identified as described herein e.g., a secreted protein-modulating agent, an antisense secreted protein nucleic acid molecule, a secreted protein-specific antibody, or a secreted protein- binding partner
  • an agent identified as described herein can be used in an animal or other model to determine the efficacy, toxicity, or side effects of treatment with such an agent.
  • an agent identified as described herein can be used in an animal or other model to determine the mechanism of action of such an agent.
  • this invention pertains to uses of novel agents identified by the above- described screening assays for treatments as described herein.
  • the secreted proteins of the present invention are also useful to provide a target for diagnosing a disease or predisposition to disease mediated by the peptide. Accordingly, the invention provides methods for detecting the presence, or levels of, the protein (or encoding mRNA) in a cell, tissue, or organism. The method involves contacting a biological sample with a compound capable of interacting with the secreted protein such that the interaction can be detected. Such an assay can be provided in a single detection format or a multi-detection format such as an antibody chip array.
  • One agent for detecting a protein in a sample is an antibody capable of selectively binding to protein.
  • a biological sample includes tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject.
  • the peptides of the present invention also provide targets for diagnosing active protein activity, disease, or predisposition to disease, in a patient having a variant peptide, particularly activities and conditions that are l ⁇ iown for other members of the family of proteins to which the present one belongs.
  • the peptide can be isolated from a biological sample and assayed for the presence of a genetic mutation that results in aberrant peptide. This includes amino acid substitution, deletion, insertion, rearrangement, (as the result of aberrant splicing events), and inappropriate post-translational modification.
  • Analytic methods include altered electrophoretic mobility, altered tryptic peptide digest, altered secreted protein activity in cell-based or cell-free assay, alteration in substrate or antibody-binding pattern, altered isoelectric point, direct amino acid sequencing, and any other of the known assay techniques useful for detecting mutations in a protein.
  • Such an assay can be provided in a single detection format or a multi-detection fomiat such as an antibody chip array.
  • In vitro techniques for detection of peptide include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence using a detection reagent, such as an antibody or protein binding agent.
  • the peptide can be detected in vivo in a subject by introducing into the subject a labeled anti -peptide antibody or other types of detection agent.
  • the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. Particularly useful are methods that detect the allelic variant of a peptide expressed in a subject and methods which detect fragments of a peptide in a sample.
  • the peptides are also useful in pharmacogenomic analysis.
  • Pharmacogenomics deal with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See, e.g., Eichelbaum, M. (Clin. Exp. Pharmacol Physiol 23(10-11):983-985 (1996)), and Linder, M.W. (Clin. Chem. 43(2):254-266 (1997)).
  • the clinical outcomes of these variations result in severe toxicity of therapeutic drugs in certain individuals or therapeutic failure of drugs in certain individuals as a result of individual variation in metabolism.
  • the genotype of the individual can determine the way a therapeutic compound acts on the body or the way the body metabolizes the compound.
  • the activity of drug metabolizing enzymes effects both the intensity and duration of drug action.
  • the pharmacogenomics of the individual permit the selection of effective compounds and effective dosages of such compounds for prophylactic or therapeutic treatment based on the individual's genotype.
  • the discovery of genetic polymorphisms in some drug metabolizing enzymes has explained why some patients do not obtain the expected drug effects, show an exaggerated drag effect, or experience serious toxicity from standard drug dosages. Polymorphisms can be expressed in the phenotype of the extensive metabolizer and the phenotype of the poor metabolizer. Accordingly, genetic polymorphism may lead to allelic protein variants of the secreted protein in which one or more of the secreted protein functions in one population is different from those in another population.
  • polymorphism may give rise to amino terminal extracellular domains and/or other substrate- binding regions that are more or less active in substrate binding, and secreted protein activation. Accordingly, subsfrate dosage would necessarily be modified to maximize the therapeutic effect within a given population containing a polymorphism.
  • specific polymorphic peptides could be identified.
  • the peptides are also useful for treating a disorder characterized by an absence of, inappropriate, or unwanted expression of the protein. Accordingly, methods for treatment include the use of the secreted protein or fragments.
  • the invention also provides antibodies that selectively bind to one of the peptides of the present invention, a protein comprising such a peptide, as well as variants and fragments thereof
  • an antibody selectively binds a target peptide when it binds the target peptide and does not significantly bind to unrelated proteins.
  • An antibody is still considered to selectively bind a peptide even if it also binds to other proteins that are not substantially homologous with the target peptide so long as such proteins share homology with a fragment or domain of the peptide target of the antibody. In this case, it would be understood that antibody binding to the peptide is still selective despite some degree of cross-reactivity.
  • an antibody is defined in terms consistent with that recognized within the art: they are multi-subunit proteins produced by a mammalian organism in response to an antigen challenge.
  • the antibodies of the present invention include polyclonal antibodies and monoclonal antibodies, as well as fragments of such antibodies, including, but not limited to, Fab or F(ab') 2 , and Fv fragments.
  • an isolated peptide is used as an immunogen and is administered to a mammalian organism, such as a rat, rabbit or mouse.
  • a mammalian organism such as a rat, rabbit or mouse.
  • the full-length protein, an antigenic peptide fragment or a fusion protein can be used.
  • Particularly important fragments are those covering functional domains, such as the domains identified in Figure 2, and domain of sequence homology or divergence amongst the family, such as those that can readily be identified using protein alignment methods and as presented in the Figures.
  • Antibodies are preferably prepared from regions or discrete fragments of the secreted proteins. Antibodies can be prepared from any region of the peptide as described herein. However, preferred regions will include those involved in function/activity and/or secreted protein/binding partner interaction. Figure 2 can be used to identify particularly important regions while sequence alignment can be used to identify conserved and unique sequence fragments.
  • An antigenic fragment will typically comprise at least 8 contiguous amino acid residues. The antigenic peptide can comprise, however, at least 10, 12, 14, 16 or more amino acid residues. Such fragments can be selected on a physical property, such as fragments correspond to regions that are located on the surface of the protein, e.g., hydrophilic regions or can be selected based on sequence uniqueness (see Figure 2).
  • Detection on an antibody of the present invention can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance.
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
  • an example of a luminescent material includes luminol;
  • examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125 I, 13I I, 35 S or 3 H.
  • the antibodies can be used to isolate one of the proteins of the present invention by standard techniques, such as affinity chromatography or immunoprecipitation.
  • the antibodies can facilitate the purification of the natural protein from cells and recombinantly produced protein expressed in host cells.
  • such antibodies are useful to detect the presence of one of the proteins of the present invention in cells or tissues to determine the pattern of expression of the protein among various tissues in an organism and over the course of normal development.
  • such antibodies can be used to detect protein in situ, in vitro, or in a cell lysate or supernatant in order to evaluate the abundance and pattern of expression.
  • such antibodies can be used to assess abnormal tissue distribution or abnormal expression during development or progression of a biological condition.
  • Antibody detection of circulating fragments of the full length protein can be used to identify turnover. Further, the antibodies can be used to assess expression in disease states such as in active stages of the disease or in an individual with a predisposition toward disease related to the protein's function. When a disorder is caused by an inappropriate tissue distribution, developmental expression, level of expression of the protein, or expressed/processed fonn, the antibody can be prepared against the normal protein. If a disorder is characterized by a specific mutation in the protein, antibodies specific for this mutant protein can be used to assay for the presence of the specific mutant protein.
  • the antibodies can also be used to assess normal and aberrant subcellular localization of cells in the various tissues in an organism.
  • the diagnostic uses can be applied, not only in genetic testing, but also in monitoring a treatment modality. Accordingly, where treatment is ultimately aimed at correcting expression level or the presence of aberrant sequence and aberrant tissue distribution or developmental expression, antibodies directed against the protein or relevant fragments can be used to monitor therapeutic efficacy. Additionally, antibodies are useful in pharmacogenomic analysis. Thus, antibodies prepared against polymo ⁇ hic proteins can be used to identify individuals that require modified treatment modalities.
  • the antibodies are also useful as diagnostic tools as an immunological marker for aberrant protein analyzed by electrophoretic mobility, isoelectric point, tryptic peptide digest, and other physical assays l ⁇ iown to those in the art.
  • the antibodies are also useful for tissue typing. Thus, where a specific protein has been correlated with expression in a specific tissue, antibodies that are specific for this protein can be used to identify a tissue type.
  • the antibodies are also useful for inhibiting protein function, for example, blocking the binding of the secreted peptide to a binding partner such as a substrate. These uses can also be applied in a therapeutic context in which treatment involves inhibiting the protein's function.
  • An antibody can be used, for example, to block binding, thus modulating (agonizing or antagonizing) the peptides activity.
  • Antibodies can be prepared against specific fragments containing sites required for function or against intact protein that is associated with a cell or cell membrane. See Figure 2 for structural information relating to the proteins of the present invention.
  • kits for using antibodies to detect the presence of a protein in a biological sample can comprise antibodies such as a labeled or labelable antibody and a compound or agent for detecting protein in a biological sample; means for determining the amount of protein in the sample; means for comparing the amount of protein in the sample with a standard; and instructions for use.
  • Such a kit can be supplied to detect a single protein or epitope or can be configured to detect one of a multitude of epitopes, such as in an antibody detection array. Arrays are described in detail below for nuleic acid arrays and similar methods have been developed for antibody arrays. Nucleic Acid Molecules
  • the present invention further provides isolated nucleic acid molecules that encode a secreted peptide or protein of the present invention (cDNA, transcript and genomic sequence).
  • Such nucleic acid molecules will consist of, consist essentially of, or comprise a nucleotide sequence that encodes one of the secreted peptides of the present invention, an allelic variant thereof, or an ortholog or paralog thereof.
  • an "isolated" nucleic acid molecule is one that is separated from other nucleic acid present in the natural source of the nucleic acid.
  • an “isolated” nucleic acid is free of sequences which naturally flanlc the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
  • flanking nucleotide sequences for example up to about 5KB, 4KB, 3KB, 2KB, or 1KB or less, particularly contiguous peptide encoding sequences and peptide encoding sequences within the same gene but separated by introns in the genomic sequence.
  • nucleic acid is isolated from remote and unimportant flanking sequences such that it can be subjected to the specific manipulations described herein such as recombinant expression, preparation of probes and primers, and other uses specific to the nucleic acid sequences.
  • an "isolated" nucleic acid molecule such as a transcript/cDNA molecule
  • a transcript/cDNA molecule can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized.
  • the nucleic acid molecule can be fused to other coding or regulatory sequences and still be considered isolated.
  • recombinant DNA molecules contained in a vector are considered isolated.
  • isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution.
  • isolated RNA molecules include in vivo or in vitro RNA transcripts of the isolated DNA molecules of the present invention.
  • Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically. Accordingly, the present invention provides nucleic acid molecules that consist of the nucleotide sequence selected from the sequences shown in Figure 1 or 3 (SEQ ID NOS: 1-6, transcript sequence and SEQ ID NOS: 13-18, genomic sequence), or any nucleic acid molecule that encodes the protein provided in Figure 2, SEQ ID NOS:7-12.
  • a nucleic acid molecule consists of a nucleotide sequence when the nucleotide sequence is the complete nucleotide sequence of the nucleic acid molecule.
  • the present invention further provides nucleic acid molecules that consist essentially of the nucleotide sequence selected from the sequences shown in Figure 1 or 3 (SEQ ID NOS: 1-6, transcript sequence and SEQ ID NOS:13-18, genomic sequence), or any nucleic acid molecule that encodes the protein provided in Figure 2, SEQ ID NOS:7-12.
  • a nucleic acid molecule consists essentially of a nucleotide sequence when such a nucleotide sequence is present with only a few additional nucleic acid residues in the final nucleic acid molecule.
  • the present invention further provides nucleic acid molecules that comprise the nucleotide sequences shown in Figure 1 or 3 (SEQ ID NOS: 1 -6, transcript sequence and SEQ ID NOS: 13-18, genomic sequence), or any nucleic acid molecule that encodes the protein provided in Figure 2, SEQ ID NOS:7-12.
  • a nucleic acid molecule comprises a nucleotide sequence when the nucleotide sequence is at least part of the final nucleotide sequence of the nucleic acid molecule. In such a fashion, the nucleic acid molecule can be only the nucleotide sequence or have additional nucleic acid residues, such as nucleic acid residues that are naturally associated with it or heterologous nucleotide sequences.
  • nucleic acid molecule can have a few additional nucleotides or can comprises several hundred or more additional nucleotides.
  • Figures 1 and 3 both coding and non-coding sequences are provided. Because of the source of the present invention, humans genomic sequence ( Figure 3) and cDNA/transcript sequences ( Figure 1), the nucleic acid molecules in the Figures will contain genomic intronic sequences, 5' and 3' non-coding sequences, gene regulatory regions and non-coding intergenic sequences. In general such sequence features are either noted in Figures 1 and 3 or can readily be identified using computational tools l ⁇ iown in the art.
  • the isolated nucleic acid molecules can encode the mature protein plus additional amino or carboxyl-terminal amino acids, or amino acids interior to the mature peptide (when the mature form has more than one peptide chain, for instance).
  • Such sequences may play a role in processing of a protein from precursor to a mature form, facilitate protein trafficking, prolong or shorten protein half- life or facilitate manipulation of a protein for assay or production, among other things.
  • the additional amino acids may be . processed away from the mature protein by cellular enzymes.
  • the isolated nucleic acid molecules include, but are not limited to, the sequence encoding the secreted peptide alone, the sequence encoding the mature peptide and additional coding sequences, such as a leader or secretory sequence (e.g., a pre-pro or pro-protein sequence), the sequence encoding the mature peptide, with or without the additional coding sequences, plus additional non-coding sequences, for example introns and non-coding 5' and 3' sequences such as transcribed but non-translated sequences that play a role in transcription, mRNA processing (including splicing and polyadenylation signals), ribosome binding and stability of mRNA.
  • the nucleic acid molecule may be fused to a marker sequence encoding, for example, a peptide that facilitates purification.
  • Isolated nucleic acid molecules can be in the form of RNA, such as mRNA, or in the form DNA, including cDNA and genomic DNA obtained by cloning or produced by chemical synthetic techniques or by a combination thereof.
  • the nucleic acid, especially DNA can be double-stranded or single-stranded.
  • Single-stranded nucleic acid can be the coding strand (sense strand) or the non-coding strand (anti-sense strand).
  • the invention further provides nucleic acid molecules that encode fragments of the peptides of the present invention as well as nucleic acid molecules that encode obvious variants of the secreted proteins of the present invention that are described above.
  • nucleic acid molecules may be naturally occurring, such as allelic variants (same locus), paralogs (different locus), and orthologs (different organism), or may be constructed by recombinant DNA methods or by chemical synthesis.
  • non-naturally occurring variants may be made by mutagenesis techniques, including those applied to nucleic acid molecules, cells, or organisms. Accordingly, as discussed above, the variants can contain nucleotide substitutions, deletions, inversions and insertions. Variation can occur in either or both the coding and non-coding regions.
  • the variations can produce both conservative and non-conservative amino acid substitutions.
  • the present invention further provides non-coding fragments of the nucleic acid molecules provided in Figures 1 and 3.
  • Preferred non-coding fragments include, but are not limited to, promoter sequences, enhancer sequences, gene modulating sequences and gene termination sequences. Such fragments are useful in controlling heterologous gene expression and in developing screens to identify gene-modulating agents.
  • a promoter can readily be identified as being 5' to the ATG start site in the genomic sequence provided in Figure 3.
  • a fragment comprises a contiguous nucleotide sequence greater than 12 or more nucleotides. Further, a fragment could at least 30, 40, 50, 100, 250 or 500 nucleotides in length. The length of the fragment will be based on its intended use.
  • the fragment can encode epitope bearing regions of the peptide, or can be useful as DNA probes and primers.
  • Such fragments can be isolated using the l ⁇ iown nucleotide sequence to synthesize an oligonucleotide probe.
  • a labeled probe can then be used to screen a cDNA library, genomic DNA library, or mRNA to isolate nucleic acid corresponding to the coding region.
  • primers can be used in PCR reactions to clone specific regions of gene.
  • a probe/primer typically comprises substantially a purified oligonucleotide or oligonucleotide pair.
  • the oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, 20, 25, 40, 50 or more consecutive nucleotides.
  • Orthologs, homologs, and allelic variants can be identified using methods well known in the art. As described in the Peptide Section, these variants comprise a nucleotide sequence encoding a peptide that is typically 60-70%, 70-80%, 80-90%, and more typically at least about 90-95% or more homologous to the nucleotide sequence selected from the sequences shown in the Figure sheets or a fragment of this sequence. Such nucleic acid molecules can readily be identified as being able to hybridize under moderate to stringent conditions, to the nucleotide sequence selected from the sequences shown in the Figure sheets or a fragment of the sequence. Allelic variants can readily be determined by genetic locus of the encoding gene.
  • the map position of the Wnt gene provided by the present invention was determined to be at chromosomal location 17q21 by ePCR.
  • the map position of the lactate dehydrogenase gene provided by the present invention was determined to be on chromosome 15 by ePCR.
  • the map position of the calgizzarin gene provided by the present invention was determined to be on chromosome 7 by ePCR.
  • the map position of the retinoic acid receptor responder gene provided by the present invention was determined to be on chromosome 16 by ePCR.
  • the map position of the MAP gene provided by the present invention was determined to be on chromosome 12 by ePCR.
  • the ferritin gene provided by the present invention maps to public BAC AC01 1 181.
  • Figure 3 A provides information on SNPs that have been identified in a gene encoding the Wnt proteins of the present invention. 9 SNP variants were found, including 1 indel (indicated by a "-") and 2 SNPs in exons, of which 1 of these cause changes in the amino acid sequence (i.e., nonsynonymous SNPs).
  • Figure 3C provides information on SNPs that have been identified in a gene encoding the calgizzarin proteins of the present invention. 4 SNP variants were found.
  • Figure 3D provides information on SNPs that have been identified in a gene encoding the retinoic acid receptor responder proteins of the present invention. 30 SNP variants were found, including 4 indels (indicated by a "-").
  • Figure 3E provides information on SNPs that have been identified in a gene encoding the MAP proteins of the present invention. 8 SNP variants were found.
  • Figure 3F provides information on SNPs that have been identified in a gene encoding the ferritin proteins of the present invention. One SNP variant was found. The changes in the amino acid sequence that these SNPs cause is indicated in Figures 3A-3F and can readily be determined using the universal genetic code and the protein sequence provided in Figures 2A-2F as a reference.
  • hybridizes under stringent conditions is intended to describe conditions for hybridization and washing under which nucleotide sequences encoding a peptide at least 60-70% homologous to each other typically remain hybridized to each other.
  • the conditions can be such that sequences at least about 60%, at least about 70%, or at least about 80% or more homologous to each other typically remain hybridized to each other.
  • stringent conditions are l ⁇ iown to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
  • stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45C, followed by one or more washes in 0.2 X SSC, 0.1% SDS at 50-65C. Examples of moderate to low stringency hybridization conditions are well known in the art.
  • the nucleic acid molecules of the present invention are useful for probes, primers, chemical intermediates, and in biological assays.
  • the nucleic acid molecules are useful as a hybridization probe for messenger RNA, transcript/cDNA and genomic DNA to isolate full- length cDNA and genomic clones encoding the peptide described in Figure 2 and to isolate cDNA and genomic clones that correspond to variants (alleles, orthologs, etc.) producing the same or related peptides shown in Figure 2.
  • 9 SNPs, including 1 indel have been identified in the gene encoding the Wnt protein provided by the present invention.
  • SNPs have been identified in the gene encoding the calgizzarin protein provided by the present invention.
  • 30 SNPs, including 4 indels, have been identified in the gene encoding the retinoic acid receptor responder protein provided by the present invention.
  • 8 SNPs have been identified in the gene encoding the MAP protein provided by the present invention.
  • 1 SNP has been identified in the gene encoding the ferritin protein provided by the present invention.
  • the probe can correspond to any sequence along the entire length of the nucleic acid molecules provided in the Figures. Accordingly, it could be derived from 5' noncoding regions, the coding region, and 3' noncoding regions. However, as discussed, fragments are not to be construed as encompassing fragments disclosed prior to the present invention.
  • the nucleic acid molecules are also useful as primers for PCR to amplify any given region of a nucleic acid molecule and are useful to synthesize antisense molecules of desired length and sequence.
  • the nucleic acid molecules are also useful for constructing recombinant vectors.
  • Such vectors include expression vectors that express a portion of, or all of, the peptide sequences.
  • Vectors also include insertion vectors, used to integrate into another nucleic acid molecule sequence, such as into the cellular genome, to alter in situ expression of a gene and/or gene product.
  • an endogenous coding sequence can be replaced via homologous recombination with all or part of the coding region containing one or more specifically introduced mutations.
  • the nucleic acid molecules are also useful for expressing antigenic portions of the proteins.
  • the nucleic acid molecules are also useful as probes for determining the chromosomal positions of the nucleic acid molecules by means of in situ hybridization methods.
  • the map position of the Wnt gene provided by the present invention was determined to be at chromosomal location 17q21 by ePCR.
  • the map position of the lactate dehydrogenase gene provided by the present invention was determined to be on chromosome 15 by ePCR.
  • the map position of the calgizzarin gene provided by the present invention was determined to be on chromosome 7 by ePCR.
  • the map position of the retinoic acid receptor responder gene provided by the present invention was determined to be on chromosome 16 by ePCR.
  • the map position of the MAP gene provided by the present invention was determined to be on chromosome 12 by ePCR.
  • the ferritin gene provided by the present invention maps to public BAC AC011181.
  • the nucleic acid molecules are also useful in making vectors containing the gene regulatory regions of the nucleic acid molecules of the present invention.
  • the nucleic acid molecules are also useful for designing ribozymes corresponding to all, or a part, of the mRNA produced from the nucleic acid molecules described herein.
  • the nucleic acid molecules are also useful for making vectors that express part, or all, of the peptides.
  • the nucleic acid molecules are also useful for constructing host cells expressing a part, or all, of the nucleic acid molecules and peptides.
  • the nucleic acid molecules are also useful for constructing transgenic animals expressing all, or a part, of the nucleic acid molecules and peptides.
  • the nucleic acid molecules are also useful as hybridization probes for determining the, presence, level, form and distribution of nucleic acid expression. Accordingly, the probes can be used to detect the presence of, or to determine levels of, a specific nucleic acid molecule in cells, tissues, and in organisms.
  • the nucleic acid whose level is determined can be DNA or RNA. Accordingly, probes corresponding to the peptides described herein can be used to assess expression and/or gene copy number in a given cell, tissue, or organism. These uses are relevant for diagnosis of disorders involving an increase or decrease in secreted protein expression relative to normal results.
  • In vitro techniques for detection of mRNA include Northern hybridizations and in situ hybridizations.
  • In vitro techniques for detecting DNA include Southern hybridizations and in situ hybridization.
  • Probes can be used as a part of a diagnostic test kit for identifying cells or tissues that express a secreted protein, such as by measuring a level of a secreted protein-encoding nucleic acid in a sample of cells from a subject e.g., mRNA or genomic DNA, or determining if a secreted protein gene has been mutated.
  • Nucleic acid expression assays are useful for drug screening to identify compounds that modulate secreted protein nucleic acid expression.
  • the invention thus provides a method for identifying a compound that can be used to treat a disorder associated with nucleic acid expression of the secreted protein gene, particularly biological and pathological processes that are mediated by the secreted protein in cells and tissues that express it.
  • the method typically includes assaying the ability of the compound to modulate the expression of the secreted protein nucleic acid and thus identifying a compound that can be used to treat a disorder characterized by undesired secreted protein nucleic acid expression.
  • the assays can be performed in cell-based and cell-free systems.
  • Cell-based assays include cells naturally expressing the secreted protein nucleic acid or recombinant cells genetically engineered to express specific nucleic acid sequences.
  • modulators of secreted protein gene expression can be identified in a method wherein a cell is contacted with a candidate compound and the expression of mRNA determined.
  • the level of expression of secreted protein mRNA in the presence of the candidate compound is compared to the level of expression of secreted protein mRNA in the absence of the candidate compound.
  • the candidate compound can then be identified as a modulator of nucleic acid expression based on this comparison and be used, for example to treat a disorder characterized by aberrant nucleic acid expression.
  • expression of mRNA is statistically significantly greater in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of nucleic acid expression.
  • nucleic acid expression is statistically significantly less in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of nucleic acid expression.
  • the invention further provides methods of treatment, with the nucleic acid as a target, using a compound identified through drug screening as a gene modulator to modulate secreted protein nucleic acid expression in cells and tissues that express the secreted protein.
  • Modulation includes both up-regulation (i.e. activation or agonization) or down-regulation (suppression or antagonization) or nucleic acid expression.
  • a modulator for secreted protein nucleic acid expression can be a small molecule or drug identified using the screening assays described herein as long as the drug or small molecule inhibits the secreted protein nucleic acid expression in the cells and tissues that express the protein.
  • the nucleic acid molecules are also useful for monitoring the effectiveness of modulating compounds on the expression or activity of the secreted protein gene in clinical trials or in a treatment regimen.
  • the gene expression pattern can serve as a barometer for the continuing effectiveness of treatment with the compound, particularly with compounds to which a patient can develop resistance.
  • the gene expression pattern can also serve as a marker indicative of a physiological response of the affected cells to the compound. Accordingly, such monitoring would allow either increased administration of the compound or the administration of alternative compounds to which the patient has not become resistant. Similarly, ifthe level of nucleic acid expression falls below a desirable level, administration of the compound could be commensurately decreased.
  • the nucleic acid molecules are also useful in diagnostic assays for qualitative changes in secreted protein nucleic acid expression, and particularly in qualitative changes that lead to pathology.
  • the nucleic acid molecules can be used to detect mutations in secreted protein genes and gene expression products such as mRNA.
  • the nucleic acid molecules can be used as hybridization probes to detect naturally occurring genetic mutations in the secreted protein gene and thereby to determine whether a subject with the mutation is at risk for a disorder caused by the mutation. Mutations include deletion, addition, or substitution of one or more nucleotides in the gene, chromosomal rearrangement, such as inversion or transposition, modification of genomic DNA, such as aberrant methylation patterns or changes in gene copy number, such as amplification.
  • Detection of a mutated form of the secreted protein gene associated with a dysfunction provides a diagnostic tool for an active disease or susceptibility to disease when the disease results from overexpression, underexpression, or altered expression of a secreted protein.
  • Individuals carrying mutations in the secreted protein gene can be detected at the nucleic acid level by a variety of techniques.
  • Figure 3A provides information on SNPs that have been identified in a gene encoding the Wnt proteins of the present invention. 9 SNP variants were found, including 1 indel (indicated by a "-") and 2 SNPs in exons, of which 1 of these cause changes in the amino acid sequence (i.e., nonsynonymous SNPs).
  • Figure 3C provides information on SNPs that have been identified in a gene encoding the calgizzarin proteins of the present invention. 4 SNP variants were found.
  • Figure 3D provides information on SNPs that have been identified in a gene encoding the retinoic acid receptor responder proteins of the present invention. 30 SNP variants were found, including 4 indels (indicated by a "-").
  • Figure 3E provides information on SNPs that have been identified in a gene encoding the MAP proteins of the present invention. 8 SNP variants were found.
  • Figure 3F provides information on SNPs that have been identified in a gene encoding the ferritin proteins of the present invention. One SNP variant was found.
  • the map position of the retinoic acid receptor responder gene provided by the present invention was determined to be on chromosome 1 by ePCR.
  • the map position of the MAP gene provided by the present invention was determined to be on chromosome 12 by ePCR.
  • the ferritin gene provided by the present invention maps to public B AC ACO 1 1181.
  • Genomic DNA can be analyzed directly or can be amplified by using PCR prior to analysis.
  • RNA or cDNA can be used in the same way.
  • detection of the mutation involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g.
  • This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to a gene under conditions such that hybridization and amplification of the gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. Deletions and insertions can be detected by a change in size of the amplified product compared to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to normal RNA or antisense DNA sequences.
  • nucleic acid e.g., genomic, mRNA or both
  • mutations in a secreted protein gene can be directly identified, for example, by alterations in restriction enzyme digestion patterns determined by gel electrophoresis.
  • sequence-specific ribozymes can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site. Perfectly matched sequences can be distinguished from mismatched sequences by nuclease cleavage digestion assays or by differences in melting temperature.
  • Sequence changes at specific locations can also be assessed by nuclease protection assays such as RNase and SI protection or the chemical cleavage method.
  • sequence differences between a mutant secreted protein gene and a wild-type gene can be determined by direct DNA sequencing.
  • a variety of automated sequencing procedures can be utilized when performing the diagnostic assays (Naeve, C.W., (1995) Biotechniques 79:448), including sequencing by mass spectrometry (see, e.g., PCT International Publication No. WO 94/16101; Cohen et al., Adv. Chromatogr. 36:121-162 (1996); and Griffin et al., Appl. Biochem. Biotechnol. 55:147-159 (1993)).
  • RNA/RNA or RNA/DNA duplexes Other methods for detecting mutations in the gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA duplexes (Myers et al, Science 250:1242 (1985)); Cotton et al, PNAS 55:4397 (1988); Saleeba et al, Meth. Enzymol. 277:286-295 (1992)), electrophoretic mobility of mutant and wild type nucleic acid is compared (Orita et al, PNAS 86:2766 (1989); Cotton et al, Mutat. Res. 255:125-144 (1993); and Hayashi et al, Genet. Anal. Tech. Appl.
  • the nucleic acid molecules are also useful for testing an individual for a genotype that while not necessarily causing the disease, nevertheless affects the treatment modality.
  • the nucleic acid molecules can be used to study the relationship between an individual's genotype and the individual's response to a compound used for treatment (pharmacogenomic relationship).
  • the nucleic acid molecules described herein can be used to assess the mutation content of the secreted protein gene in an individual in order to select an appropriate compound or dosage regimen for treatment.
  • Figure 3 A provides information on SNPs that have been identified in a gene encoding the Wnt proteins of the present invention.
  • SNP variants were found, including 1 indel (indicated by a "-") and 2 SNPs in exons, of which 1 of these cause changes in the amino acid sequence (i.e., nonsynonymous SNPs).
  • Figure 3C provides information on SNPs that have been identified in a gene encoding the calgizzarin proteins of the present invention. 4 SNP variants were found.
  • Figure 3D provides information on SNPs that have been identified in a gene encoding the retinoic acid receptor responder proteins of the present invention.
  • 30 SNP variants were found, including 4 indels (indicated by a "-").
  • Figure 3E provides information on SNPs that have been identified in a gene encoding the MAP proteins of the present invention.
  • Figure 3F provides information on SNPs that have been identified in a gene encoding the ferritin proteins of the present invention. One SNP variant was found. The changes in the amino acid sequence that these SNPs cause is indicated in Figures 3 A-3F and can readily be determined using the universal genetic code and the protein sequence provided in Figures 2A-2F as a reference.
  • nucleic acid molecules displaying genetic variations that affect treatment provide a diagnostic target that can be used to tailor treatment in an individual. Accordingly, the production of recombinant cells and animals containing these polymorphisms allow effective clinical design of treatment compounds and dosage regimens.
  • the nucleic acid molecules are thus useful as antisense constructs to control secreted protein gene expression in cells, tissues, and organisms.
  • a DNA antisense nucleic acid molecule is designed to be complementary to a region of the gene involved in transcription, preventing transcription and hence production of secreted protein.
  • An antisense RNA or DNA nucleic acid molecule would hybridize to the mRNA and thus block translation of mRNA into secreted protein.
  • a class of antisense molecules can be used to inactivate mRNA in order to decrease expression of secreted protein nucleic acid. Accordingly, these molecules can treat a disorder characterized by abnormal or undesired secreted protein nucleic acid expression.
  • This technique involves cleavage by means of ribozymes containing nucleotide sequences complementary to one or more regions in the mRNA that attenuate the ability of the mRNA to be translated. Possible regions include coding regions and particularly coding regions corresponding to the catalytic and other functional activities of the secreted protein, such as substrate binding.
  • the nucleic acid molecules also provide vectors for gene therapy in patients containing cells that are aberrant in secreted protein gene expression.
  • kits for detecting the presence of a secreted protein nucleic acid in a biological sample can comprise reagents such as a labeled or labelable nucleic acid or agent capable of detecting secreted protein nucleic acid in a biological sample; means for determining the amount of secreted protein nucleic acid in the sample; and means for comparing the amount of secreted protein nucleic acid in the sample with a standard.
  • the compound or agent can be packaged in a suitable container.
  • the kit can further comprise instructions for using the kit to detect secreted protein mRNA or DNA.
  • the present invention further provides nucleic acid detection kits, such as arrays or microarrays of nucleic acid molecules that are based on the sequence information provided in Figures 1 and 3 (SEQ ID NOS:l and 3).
  • Arrays or “Microarrays” refers to an array of distinct polynucleotides or oligonucleotides synthesized on a substrate, such as paper, nylon or other type of membrane, filter, chip, glass slide, or any other suitable solid support.
  • the microarray is prepared and used according to the methods described in US Patent 5,837,832, Chee et al, PCT application W095/11995 (Chee et al), Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) and Schena, M. et al (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all of which are incorporated herein in their entirety by reference.
  • such arrays are produced by the methods described by Brown et al. , US Patent No. 5,807,522.
  • the microarray or detection kit is preferably composed of a large number of unique, single-stranded nucleic acid sequences, usually either synthetic antisense oligonucleotides or fragments of cDNAs, fixed to a solid support.
  • the oligonucleotides are preferably about 6-60 nucleotides in length, more preferably 15-30 nucleotides in length, and most preferably about 20-25 nucleotides in length. For a certain type of microarray or detection kit, it may be preferable to use oligonucleotides that are only 7-20 nucleotides in length.
  • the microarray or detection kit may contain oligonucleotides that cover the known 5', or 3', sequence, sequential oligonucleotides which cover the full length sequence; or unique oligonucleotides selected from particular areas along the length of the sequence.
  • Polynucleotides used in the microarray or detection kit may be oligonucleotides that are specific to a gene or genes of interest.
  • the gene(s) of interest (or an ORF identified from the contigs of the present invention) is typically examined using a computer algorithm which starts at the 5' or at the 3' end of the nucleotide sequence.
  • Typical algorithms will then identify oligomers of defined length that are unique to the gene, have a GC content within a range suitable for hybridization, and lack predicted secondary structure that may interfere with hybridization. In certain situations it may be appropriate to use pairs of oligonucleotides on a microarray or detection kit.
  • the "pairs" will be identical, except for one nucleotide that preferably is located in the center of the sequence.
  • the second oligonucleotide in the pair serves as a control.
  • the number of oligonucleotide pairs may range from two to one million.
  • the oligomers are synthesized at designated areas on a substrate using a light-directed chemical process.
  • the substrate may be paper, nylon or other type of membrane, filter, chip, glass slide or any other suitable solid support.
  • an oligonucleotide may be synthesized on the surface of the substrate by using a chemical coupling procedure and an ink jet application apparatus, as described in PCT application W095/251 1 16 (Baldeschweiler et al.) which is incorporated herein in its entirety by reference.
  • a "gridded" array analogous to a dot (or slot) blot may be used to arrange and link cDNA fragments or oligonucleotides to the surface of a substrate using a vacuum system, thermal, UV, mechanical or chemical bonding procedures.
  • An array such as those described above, may be produced by hand or by using available devices (slot blot or dot blot apparatus), materials (any suitable solid support), and machines (including robotic instruments), and may contain 8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or any other number between two and one million which lends itself to the efficient use of commercially available instrumentation.
  • RNA or DNA from a biological sample is made into hybridization probes.
  • the mRNA is isolated, and cDNA is produced and used as a template to make antisense RNA (aRNA).
  • aRNA is amplified in the presence of fluorescent nucleotides, and labeled probes are incubated with the microarray or detection kit so that the probe sequences hybridize to complementary oligonucleotides of the microarray or detection kit. Incubation conditions are adjusted so that hybridization occurs with precise complementary matches or with various degrees of less complementarity. After removal of nonhybridized probes, a scanner is used to determine the levels and patterns of fluorescence.
  • the scanned images are examined to determine degree of complementarity and the relative abundance of each oligonucleotide sequence on the microarray or detection kit.
  • the biological samples may be obtained from any bodily fluids (such as blood, urine, saliva, phlegm, gastric juices, etc.), cultured cells, biopsies, or other tissue preparations.
  • a detection system may be used to measure the absence, presence, and amount of hybridization for all of the distinct sequences simultaneously. This data may be used for large-scale correlation studies on the sequences, expression patterns, mutations, variants, or polymorphisms among samples.
  • the present invention provides methods to identify the expression of the secreted proteins/peptides of the present invention.
  • such methods comprise incubating a test sample with one or more nucleic acid molecules and assaying for binding of the nucleic acid molecule with components within the test sample.
  • Such assays will typically involve arrays comprising many genes, at least one of which is a gene of the present invention and or alleles of the secreted protein gene of the present invention.
  • Figure 3A provides information on SNPs that have been identified in a gene encoding the Wnt proteins of the present invention.
  • SNP variants were found, including 1 indel (indicated by a "-") and 2 SNPs in exons, of which 1 of these cause changes in the amino acid sequence (i.e., nonsynonymous SNPs).
  • Figure 3C provides information on SNPs that have been identified in a gene encoding the calgizzarin proteins of the present invention. 4 SNP variants were found.
  • Figure 3D provides information on SNPs that have been identified in a gene encoding the retinoic acid receptor responder proteins of the present invention.
  • 30 SNP variants were found, including 4 indels (indicated by a "-").
  • Figure 3E provides information on SNPs that have been identified in a gene encoding the MAP proteins of the present invention.
  • Figure 3F provides information on SNPs that have been identified in a gene encoding the ferritin proteins of the present invention. One SNP variant was found. The changes in the amino acid sequence that these SNPs cause is indicated in Figures 3A-3F and can readily be determined using the universal genetic code and the protein sequence provided in Figures 2A-2F as a reference.
  • Conditions for incubating a nucleic acid molecule with a test sample vary. Incubation conditions depend on the format employed in the assay, the detection methods employed, and the type and nature of the nucleic acid molecule used in the assay.
  • any one of the commonly available hybridization, amplification or array assay formats can readily be adapted to employ the novel fragments of the Human genome disclosed herein. Examples of such assays can be found in Chard, T, An Introduction to Radioimmunoassay and Related Techniques, Elsevier Science Publishers, Amsterdam, The Netherlands (1986); Bullock, G. R. et al, Techniques in Immunocytochemistry, Academic Press, Orlando, FL Vol. 1 (1 982), Vol. 2 (1983), Vol. 3 (1985); Tijssen, P., Practice and Theory of Enzyme Immunoassays: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985).
  • test samples of the present invention include cells, protein or membrane extracts of cells.
  • the test sample used in the above-described method will vary based on the assay format, nature of the detection method and the tissues, cells or extracts used as the sample to be assayed. Methods for preparing nucleic acid extracts or of cells are well lcnown in the art and can be readily be adapted in order to obtain a sample that is compatible with the system utilized.
  • kits which contain the necessary reagents to carry out the assays of the present invention.
  • the invention provides a compartmentalized kit to receive, in close confinement, one or more containers which comprises: (a) a first container comprising one of the nucleic acid molecules that can bind to a fragment of the Human genome disclosed herein; and (b) one or more other containers comprising one or more of the following: wash reagents, reagents capable of detecting presence of a bound nucleic acid.
  • a compartmentalized kit includes any kit in which reagents are contained in separate containers. Such containers include small glass containers, plastic containers, strips of plastic, glass or paper, or arraying material such as silica.
  • Such containers allows one to efficiently transfer reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated, and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another.
  • Such containers will include a container which will accept the test sample, a container which contains the nucleic acid probe, containers which contain wash reagents (such as phosphate buffered saline, Tris-buffers, etc.), and containers which contain the reagents used to detect the bound probe.
  • wash reagents such as phosphate buffered saline, Tris-buffers, etc.
  • the invention also provides vectors containing- the nucleic acid molecules described herein.
  • the term "vector” refers to a vehicle, preferably a nucleic acid molecule, which can transport the nucleic acid molecules.
  • the vector is a nucleic acid molecule, the nucleic acid molecules are covalently linked to the vector nucleic acid.
  • the vector includes a plasmid, single or double stranded phage, a single or double stranded RNA or DNA viral vector, or artificial chromosome, such as a BAC, PAC, YAC, OR MAC.
  • a vector can be maintained in the host cell as an extrachromosomal element where it replicates and produces additional copies of the nucleic acid molecules.
  • the vector may integrate into the host cell genome and produce additional copies of the nucleic acid molecules when the host cell replicates.
  • the invention provides vectors for the maintenance (cloning vectors) or vectors for expression (expression vectors) of the nucleic acid molecules.
  • the vectors can function in prokaryotic or eukaryotic cells or in both (shuttle vectors).
  • Expression vectors contain cis-acting regulatory regions that are operably linked in the vector to the nucleic acid molecules such that franscription of the nucleic acid molecules is allowed in a host cell.
  • the nucleic acid molecules can be introduced into the host cell with a separate nucleic acid molecule capable of affecting transcription.
  • the second nucleic acid molecule may provide a trans-acting factor interacting with the cis-regulatory control region to allow transcription of the nucleic acid molecules from the vector.
  • a trans-acting factor may be supplied by the host cell.
  • a trans-acting factor can be produced from the vector itself. It is understood, however, that in some embodiments, transcription and/or translation of the nucleic acid molecules can occur in a cell-free system.
  • the regulatory sequence to which the nucleic acid molecules described herein can be operably linked include promoters for directing mRNA transcription. These include, but are not limited to, the left promoter from bacteriophage ⁇ , the lac, TRP, and TAC promoters from E. coli, the early and late promoters from SV40, the CMV immediate early promoter, the adenovirus early and late promoters, and retrovirus long-terminal repeats. In addition to control regions that promote transcription, expression vectors may also include regions that modulate transcription, such as repressor binding sites and enhancers. Examples include the SV40 enhancer, the cytomegalovirus immediate early enhancer, polyoma enhancer, adenovirus enhancers, and retrovirus LTR enhancers.
  • expression vectors can also contain sequences necessary for transcription tennination and, in the transcribed region a ribosome binding site for translation.
  • Other regulatory control elements for expression include initiation and termination codons as well as polyadenylation signals.
  • the person of ordinary skill in the art would be aware of the numerous regulatory sequences that are useful in expression vectors. Such regulatory sequences are described, for example, in Sambrook et al, Molecular Cloning: A Laboratory Manual. 2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1989).
  • a variety of expression vectors can be used to express a nucleic acid molecule.
  • Such vectors include chromosomal, episomal, and virus-derived vectors, for example vectors derived from bacterial plasmids, from bacteriophage, from yeast episomes, from yeast chromosomal elements, including yeast artificial chromosomes, from viruses such as baculoviruses, papovaviruses such as SV40, Vaccinia viruses, adenovirases, poxviruses, pseudorabies viruses, and retro viruses.
  • Vectors may also be derived from combinations of these sources such as those derived from plasmid and bacteriophage genetic elements, e.g. cosmids and phagemids.
  • Appropriate cloning and expression vectors for prokaryotic and eukaryotic hosts are described in Sambrook et al, Molecular Cloning: A Laboratory Manual. 2nd. ed., Cold Spring Harbor
  • the regulatory sequence may provide constitutive expression in one or more host cells
  • tissue specific i.e. tissue specific
  • inducible expression in one or more cell types such as by temperature, nutrient additive, or exogenous factor such as a hormone or other ligand.
  • a variety of vectors providing for constitutive and inducible expression in prokaryotic and eukaryotic hosts are well l ⁇ iown to those of ordinary skill in the ait.
  • the nucleic acid molecules can be inserted into the vector nucleic acid by well-known methodology.
  • the DNA sequence that will ultimately be expressed is joined to an expression vector by cleaving the DNA sequence and the expression vector with one or more restriction enzymes and then ligating the fragments together. Procedures for restriction enzyme digestion and ligation are well l ⁇ iown to those of ordinary skill in the art.
  • Bacterial cells include, but are not limited to, E. coli, Streptomyces, and Salmonella typhimurium.
  • Eukaiyotic cells include, but are not limited to, yeast, insect cells such as Drosophila, animal cells such as COS and CHO cells, and plant cells.
  • the invention provides fusion vectors that allow for the production of the peptides.
  • Fusion vectors can increase the expression of a recombinant protein, increase the solubility of the recombinant protein, and aid in the purification of the protein by acting for example as a ligand for affinity purification.
  • a proteolytic cleavage site may be introduced at the junction of the fusion moiety so that the desired peptide can ultimately be separated from the fusion moiety.
  • Proteolytic enzymes include, but are not limited to, factor Xa, thrombin, and enterokinase.
  • Typical fusion expression vectors include pGEX (Smith et al.
  • GST glutathione S-transferase
  • suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al, Gene 59:301-315 (1988)) and pET 1 Id (Studier et al, Gene Expression Technology: Methods in Enzymology 755:60-89 (1990)).
  • Recombinant protein expression can be maximized in host bacteria by providing a genetic background wherein the host cell has an impaired capacity to proteolytically cleave the recombinant protein.
  • the sequence of the nucleic acid molecule of interest can be altered to provide preferential codon usage for a specific host cell, for example E. coli. (Wada et al, Nucleic Acids Res. 20:2111-2118 (1992)).
  • the nucleic acid molecules can also be expressed by expression vectors that are operative in yeast.
  • yeast e.g., S. cerevisiae
  • vectors for expression in yeast include pYepSecl (Baldari, et al, EMBOJ. 5:229-234 (1987)), pMFa (Kurjan et al, Cell 50:933-943(1982)), pJRY88 (Schultz el al, Gene 54:113-123 (1987)), and pYES2 (Invitrogen Corporation, San Diego, CA).
  • the nucleic acid molecules can also be expressed in insect cells using, for example, baculovirus expression vectors.
  • Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series (Smith et al, Mol Cell Biol 5:2156- 2165 (1983)) and the pVL series (Lucklow et al, Virology 170:31-39 (1989)).
  • the nucleic acid molecules described herein are expressed in mammalian cells using mammalian expression vectors.
  • mammalian expression vectors include pCDM8 (Seed, B. Nature 529:840(1987)) and pMT2PC (Kaufman et al, EMBOJ. 5:187-195 (1987)).
  • the expression vectors listed herein are provided by way of example only of the well-known vectors available to those of ordinary skill in the art that would be useful to express the nucleic acid molecules.
  • the person of ordinary skill in the art would be aware of other vectors suitable for maintenance propagation or expression of the nucleic acid molecules described herein. These are found for example in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.
  • the invention also encompasses vectors in which the nucleic acid sequences described herein are cloned into the vector in reverse orientation, but operably linked to a regulatory sequence that permits transcription of antisense RNA.
  • an antisense transcript can be produced to all, or to a portion, of the nucleic acid molecule sequences described herein, including both coding and non-coding regions. Expression of this antisense RNA is subject to each of the parameters described above in relation to expression of the sense RNA (regulatory sequences, constitutive or inducible expression, tissue-specific expression).
  • the invention also relates to recombinant host cells containing the vectors described herein. Host cells therefore include prokaryotic cells, lower eukaryotic cells such as yeast, other eukaryotic cells such as insect cells, and higher eukaryotic cells such as mammalian cells.
  • the recombinant host cells are prepared by introducing the vector constructs described herein into the cells by techniques readily available to the person of ordinary skill in the art. These include, but are not limited to, calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, lipofection, and other techniques such as those found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). Host cells can contain more than one vector. Thus, different nucleotide sequences can be introduced on different vectors of the same cell.
  • nucleic acid molecules can be introduced either alone or with other nucleic acid molecules that are not related to the nucleic acid molecules such as those providing trans-acting factors for expression vectors.
  • the vectors can be introduced independently, co- introduced or joined to the nucleic acid molecule vector.
  • bacteriophage and viral vectors these can be introduced into cells as packaged or encapsulated virus by standard procedures for infection and transduction.
  • Viral vectors can be replication-competent or replication-defective. In the case in which viral replication is defective, replication will occur in host cells providing functions that complement the defects.
  • Vectors generally include selectable markers that enable the selection of the subpopulation of cells that contain the recombinant vector constructs.
  • the marker can be contained in the same vector that contains the nucleic acid molecules described herein or may be on a separate vector. Markers include tetracycline or ampicillin-resistance genes for prokaryotic host cells and dihydrofolate reductase or neomycin resistance for eukaryotic host cells. However, any marker that provides selection for a phenotypic trait will be effective.
  • RNA derived from the DNA constructs described herein can also be used to produce these proteins using RNA derived from the DNA constructs described herein.
  • secretion of the peptide is desired, which is difficult to achieve with ulti- transmembrane domain containing proteins such as kinases, appropriate secretion signals are incorporated into the vector.
  • the signal sequence can be endogenous to the peptides or heterologous to these peptides.
  • the protein can be isolated from the host cell by standard disruption procedures, including freeze thaw, sonication, mechanical disruption, use of lysing agents and the like.
  • the peptide can then be recovered and purified by well-known purification methods including ammonium sulfate precipitation, acid extraction, anion or cationic exchange chromatography, phosphocellulose chromatography, hydrophobic-interaction chromatography, affinity chromatography, hydroxylapatite chromatography, lectin chromatography, or high performance liquid chromatography.
  • the peptides can have various glycosylation patterns, depending upon the cell, or maybe non-glycosylated as when produced in bacteria.
  • the peptides may include an initial modified methionine in some cases as a result of a host-mediated process.
  • the recombinant host cells expressing the peptides described herein have a variety of uses. First, the cells are useful for producing a secreted protein or peptide that can be further purified to produce desired amounts of secreted protein or fragments. Thus, host cells containing expression vectors are useful for peptide production.
  • Host cells are also useful for conducting cell-based assays involving the secreted protein or secreted protein fragments, such as those described above as well as other formats l ⁇ iown in the art.
  • a recombinant host cell expressing a native secreted protein is useful for assaying compounds that stimulate or inhibit secreted protein function.
  • Host cells are also useful for identifying secreted protein mutants in which these functions are affected. Ifthe mutants naturally occur and give rise to a pathology, host cells containing the mutations are useful to assay compounds that have a desired effect on the mutant secreted protein (for example, stimulating or inhibiting function) which may not be indicated by their effect on the native secreted protein.
  • a transgenic animal is preferably a mammal, for example a rodent, such as a rat or mouse, in which one or more of the cells of the animal include a transgene.
  • a transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal in one or more cell types or tissues of the transgenic animal. These animals are useful for studying the function of a secreted protein and identifying and evaluating modulators of secreted protein activity.
  • Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, and amphibians.
  • a transgenic animal can be produced by introducing nucleic acid into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal.
  • Any of the secreted protein nucleotide sequences can be introduced as a transgene into the genome of a non-human animal, such as a mouse.
  • Any of the regulatory or other sequences useful in expression vectors can fonn part of the transgenic sequence. This includes intronic sequences and polyadenylation signals, if not already included.
  • a tissue-specific regulatory sequence(s) can be operably linked to the transgene to direct expression of the secreted protein to particular cells.
  • transgenic founder animal can be identified based upon the presence of the transgene in its genome and/or expression of transgenic mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene.
  • transgenic animals carrying a transgene can frirther be bred to other transgenic animals carrying other transgenes.
  • a transgenic animal also includes animals in which the entire animal or tissues in the animal have been produced using the homologously recombinant host cells described herein.
  • transgenic non-human animals can be produced which contain selected systems that allow for regulated expression of the transgene.
  • a system is the cre/loxP recombinase system of bacteriophage PI .
  • cre/loxP recombinase system of bacteriophage PI .
  • FLP recombinase system of S. cerevisiae (O'Gorman et al. Science 251 :1351-1355 (1991).
  • mice containing transgenes encoding both the Cre recombinase and a selected protein is required.
  • Such animals can be provided through the construction of "double" transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.
  • Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut, I. et al. Nature 555:810-813 (1997) and PCT International Publication Nos. WO 97/07668 and WO 97/07669.
  • a cell e.g., a somatic cell
  • the quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated.
  • the reconstructed oocyte is then cultured such that it develops to morala or blastocyst and then transfeired to pseudopregnant female foster animal.
  • the offspring bom of this female foster animal will be a clone of the animal from which the cell, e.g., the somatic cell, is isolated.
  • Transgenic animals containing recombinant cells that express the peptides described herein are useful to conduct the assays described herein in an in vivo context. Accordingly, the various physiological factors that are present in vivo and that could effect substrate binding, secreted protein activation, and signal transduction, may not be evident from in vitro cell-free or cell-based assays. Accordingly, it is useful to provide non-human transgenic animals to assay in vivo secreted protein function, including substrate interaction, the effect of specific mutant secreted proteins on secreted protein function and substrate interaction, and the effect of chimeric secreted proteins. It is also possible to assess the effect of null mutations, that is, mutations that substantially or completely eliminate one or more secreted protein functions.

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Abstract

L'invention concerne des séquences d'acides aminés de peptides codés par des gènes du génome humain, les peptides sécrétés de l'invention. Elle concerne spécifiquement un peptide isolé, des molécules d'acides nucléiques, des procédés d'identification d'orthologues et de paralogues des peptides sécrétés, ainsi que des procédés d'identification de modulateurs des peptides sécrétés.
PCT/US2001/042802 2000-10-27 2001-10-26 Proteines humaines secretees isolees, molecules d'acides nucleiques codant pour des proteines humaines secretees et utilisations associees WO2002064626A2 (fr)

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AU2002258355A AU2002258355A1 (en) 2000-10-27 2001-10-26 Isolated human secreted proteins, nucleic acid molecules encoding human secreted proteins, and uses thereof
JP2002564955A JP2005503117A (ja) 2000-10-27 2001-10-26 単離ヒト分泌タンパク質、ヒト分泌タンパク質をコード化する核酸分子、及びそれらの使用
CA002427112A CA2427112A1 (fr) 2000-10-27 2001-10-26 Proteines humaines secretees isolees, molecules d'acides nucleiques codant pour des proteines humaines secretees et utilisations associees
EP01273044A EP1530586A2 (fr) 2000-10-27 2001-10-26 Proteines humaines secretees isolees, molecules d'acides nucleiques codant pour des proteines humaines secretees et utilisations associees

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US24347600P 2000-10-27 2000-10-27
US24342800P 2000-10-27 2000-10-27
US24347700P 2000-10-27 2000-10-27
US24343600P 2000-10-27 2000-10-27
US24346700P 2000-10-27 2000-10-27
US24342900P 2000-10-27 2000-10-27
US60/243,477 2000-10-27
US60/243,428 2000-10-27
US60/243,476 2000-10-27
US60/243,429 2000-10-27
US60/243,436 2000-10-27
US60/243,467 2000-10-27
US70872400A 2000-11-09 2000-11-09
US70870000A 2000-11-09 2000-11-09
US70872300A 2000-11-09 2000-11-09
US70870100A 2000-11-09 2000-11-09
US09/708,725 2000-11-09
US09/708,724 2000-11-09
US09/711,681 2000-11-09
US09/708,701 2000-11-09
US09/708,700 2000-11-09
US09/711,681 US6503743B1 (en) 2000-10-27 2000-11-09 Isolated nucleic acid encoding a human lactate dehydrogenase and uses thereof
US09/708,723 2000-11-09
US09/708,725 US6489456B1 (en) 2000-10-27 2000-11-09 Isolated human secreted proteins, nucleic acid molecules encoding human secreted proteins, and uses thereof

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2190870A1 (fr) * 2007-08-28 2010-06-02 Auckland Uniservices Limited Marqueur cellulaire d'une lignée de mélanocytes et ses utilisations

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BARTON G J: "PROTEIN SEQUENCE ALIGMENT AND DATABASE SCANNING" PROTEIN STRUCTURE PREDICTION. A PRACTICAL APPROACH, XX, XX, 1996, pages 31-63, XP000829540 *
BERGSTEIN I ET AL: "Isolation of two novel WNT genes, WNT14 and WNT15, one of which (WNT15) is closely linked to WNT3 on human chromosome 17q21" GENOMICS, ACADEMIC PRESS, SAN DIEGO, US, vol. 46, no. 3, 15 December 1997 (1997-12-15), pages 450-458, XP002110186 ISSN: 0888-7543 *
GEORGE D G ET AL: "CURRENT METHODS IN SEQUENCE COMPARISON AND ANALYSIS" MACROMOLECULAR SEQUENCING AND SYNTHESIS SELECTED METHODS AND APPLICATIONS, XX, XX, 1988, pages 127-149, XP000829541 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2190870A1 (fr) * 2007-08-28 2010-06-02 Auckland Uniservices Limited Marqueur cellulaire d'une lignée de mélanocytes et ses utilisations
EP2190870A4 (fr) * 2007-08-28 2010-12-01 Auckland Uniservices Ltd Marqueur cellulaire d'une lignée de mélanocytes et ses utilisations

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CA2427112A1 (fr) 2002-08-22
AU2002258355A1 (en) 2002-08-28
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WO2002064626A3 (fr) 2005-02-10
EP1530586A2 (fr) 2005-05-18

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