WO2000078788A1 - Nouvel adn complementaire associe a une pathologie renale - Google Patents

Nouvel adn complementaire associe a une pathologie renale Download PDF

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WO2000078788A1
WO2000078788A1 PCT/US2000/017434 US0017434W WO0078788A1 WO 2000078788 A1 WO2000078788 A1 WO 2000078788A1 US 0017434 W US0017434 W US 0017434W WO 0078788 A1 WO0078788 A1 WO 0078788A1
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seq
nucleic acid
protein
acid molecule
sequence
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WO2000078788B1 (fr
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Hong-Wei Sun
William Munger
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Gene Logic, Inc.
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Publication of WO2000078788B1 publication Critical patent/WO2000078788B1/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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the invention relates generally to differences in gene expression in renal tissue from humans with diseased and normal kidneys.
  • the invention relates specifically to isoforms of a novel human gene product which have modified expression levels in renal biopsy samples from patients with kidney diseases such as necrotizing crescentic glomerulonephritis (NCGN) and IgA nephropathy (IgAN).
  • NCGN necrotizing crescentic glomerulonephritis
  • IgAN IgA nephropathy
  • IgA nephropathy is the most common type of immunologically mediated glomerulonephritis (GN) and is characterized by deposition in the glomerular mesangium of IgA together with C3, C5b-9, and properdin.
  • GN immunologically mediated glomerulonephritis
  • IgA together with IgG and/or IgM can lead to a more progressive course of disease.
  • Fifteen to forty percent of primary glomerulonephritis in parts of Europe, Asia and Japan has been linked to IgAN and it is well accepted that IgAN can lead to ESRD.
  • IgAN often presents either as asymptomatic microscopic hematuria and/or proteinuria (most common in adults), or episodic gross hematuria following upper respiratory and other infections or exercise.
  • the course of IgAN is variable, with some patients showing no decline in glomerular filtration rate (GFR) over decades and other developing the nephrotic syndrome, hypertension and renal failure.
  • GFR glomerular filtration rate
  • the glomeruli are the portions of the internal kidney structures where the blood flows through very small capillaries and is filtered through membranes to form urine. Rapidly progressive glomerulonephritis (inflammation of the glomerulus) includes any type of
  • the disorder occurs in about 1 out of 10,000 people. While it is most common in people
  • vascular diseases such as vasculitis or polyarteritis, abscess of any
  • collagen vascular disease such as lupus nephritis and Henoch-Schonlein purpura, Goodpasture's syndrome, IgA nephropathy, membranoproliferative GN, anti-glomerular basement membrane antibody disease, a history of malignant tumors or blood or lymphatic system disorders, and exposure to hydrocarbon solvents.
  • collagen vascular disease such as lupus nephritis and Henoch-Schonlein purpura
  • Goodpasture's syndrome IgA nephropathy
  • membranoproliferative GN membranoproliferative GN
  • anti-glomerular basement membrane antibody disease a history of malignant tumors or blood or lymphatic system disorders
  • hydrocarbon solvents a history of malignant tumors or blood or lymphatic system disorders
  • edema swelling of the face, eyes, ankles, feet, extremities, abdomen, or generalized swelling
  • urine can be dark or smoke colored.
  • Symptoms that may also appear include: fever, myalgia (muscle aches), arthralgia (joint aches), shortness of breath, cough, malaise (general ill feeling), abdominal pain, loss of appetite, and diarrhea.
  • Signs and tests include an examination that reveals edema. Also, circulatory overload, with associated abnormal heart and lung sounds, may be present and the blood pressure may be elevated. Rapid, progressive loss of kidney function may be present. Urinalysis may be abnormal, showing blood in the urine, urine protein, white blood cells, casts, or other abnormalities. The BUN and creatinine may rise rapidly and the creatinine clearance decreases. Anti-glomerular basement membrane antibody tests may be positive in some cases. Complement levels may be decreased in some cases. Other tests for suspected causes may be performed, however a kidney biopsy confirms crescentic glomerulonephritis.
  • the treatment varies with the suspected cause.
  • the treatment goals may be a cure of the causative disorder, the control of symptoms, or the treatment of renal failure.
  • the causative disorders should be treated as is appropriate based on the cause.
  • Corticosteroids may relieve symptoms in some cases.
  • Other medications may include immunosuppressive agents including cyclophosphamide and azathioprine, anticoagulant (prevent the blood from clotting) or thrombolytic (clot-dissolving) medications, and others depending on the cause of the disorder.
  • Plasmapheresis may relieve the symptoms in some cases.
  • the blood plasma (the fluid portion of blood) containing antibodies is removed and replaced with intravenous fluids or donated plasma (without antibodies). The removal of antibodies may reduce inflammation in the kidney tissues. Dialysis or a kidney transplant may ultimately be necessary.
  • crescentic glomerulonephritis may progress to renal failure and end- stage renal disease in 6 months or less, although a few cases may resolve spontaneously.
  • the probable outcome improves with treatment, with as many as 75% of the cases showing attenuation of the symptoms.
  • the disorder may recur. If the disease occurs in childhood, it is likely that renal failure will eventually develop.
  • Complications of NCGN include congestive heart failure, pulmonary edema, hyperkalemia, acute renal failure, chronic renal failure, and end- stage renal disease.
  • Diabetic nephropathy is also a common cause of end-stage renal disease and accounts for 35% of the ESRD population in the United States. It results in considerable morbidity, mortality, and expense.
  • the average cost of managing one diabetic patient with ESRD is approximately $50,000 a year (Kobrin SM, Kidney Int Suppl 1997 Dec;63:S144-150).
  • Approximately 5.8 million people in the United States have been diagnosed by a physician as being diabetic, and an additional 4 to 5 million people have undiagnosed diabetes.
  • the incidence of diabetes appears to be declining from a peak of 300 per 100,000 population in 1973, to 230 per 100,000 in 1981, its prevalence continues to rise, due to a 19 percent decline since 1970 in deaths caused by diabetes.
  • diabetes In 1982, 34,583 deaths were attributed to diabetes, resulting in diabetes being ranked as the seventh leading underlying cause of death. Medical and surgical complications of diabetes due to macro- and microvascular disease result in 5,800 new cases of blindness, 4,500 perinatal deaths, 40,000 lower extremity amputations and 3,000 deaths due to diabetic coma (ketotic and hyperosmolar) and at least 4,000 new cases of end-stage renal disease (Verh K, Acad Geneeskd Belg 1989;51(2):81-149; discussion 149-151). A characteristic and early manifestation of diabetes in humans is renal hypertrophy (Bakris GL et al, Dis Mon (1993) 39(8):573-611).
  • This compensation mechanism is a physiological response in which the cells of the kidney increase in size and protein content without synthesizing DNA or dividing (Kujubu et al, Am J Physiol (1991) 260(6 Pt 2): F823- 5 827).
  • the compensatory growth of the kidney is a highly regulated process (Wolf et al, Kidney Int (1991) 39(3): 401-420).
  • Early studies demonstrated significant increases in ribosome synthesis, including increased rDNA transcription rates (Ouellette et al, Am J Physiol (1987) 253(4 Pt 1): C506-513).
  • Renal hypertrophy also involves a gradual and progressive increase in mRNA levels resulting in the coordinate expression of positive and negative growth control elements.
  • sustained message amplification observed during renal compensation represents a distinct cellular response which is distinguishable from the responses that characterize several pathways of cell differentiation (e.g., proto-oncogene expression patterns in hyperplasia in liver).
  • intervention is expected to be successful in halting or slowing down renal disease progression, means of accurately assessing the early manifestations of renal disease need to be established.
  • One way to accurately assess the early manifestations of renal disease is to identify markers which are uniquely associated with disease progression.
  • the development of therapeutics to prevent or repair kidney damage relies on the identification of kidney genes 5 responsible for kidney cell growth.
  • the present invention is based on our discovery of isoforms of a gene product which are present at different levels in healthy and diseased human kidneys.
  • one of the 0 isoforms of the invention is down-regulated in renal biopsy samples from patients with NCGN and IgAN.
  • the invention includes isolated nucleic acid molecules selected from the group consisting of an isolated nucleic acid molecule that encodes the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, an isolated nucleic acid molecule that encodes a fragment of at least 6 amino acids of SEQ ID NO: 2 or SEQ ID NO: 4, an isolated nucleic acid molecule which hybridizes to a nucleic acid molecule comprising SEQ ID NO: 1 or SEQ ID NO: 3 under conditions of sufficient stringency to produce a clear signal, an isolated nucleic acid molecule which hybridizes to a nucleic acid molecule that encodes the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 under conditions of sufficient stringency to produce a clear signal and an isolated nucleic acid molecule that exhibits at least about 60% nucleotide sequence identity over the open reading frames of SEQ ID NO: 1 or SEQ ID NO: 3.
  • the present invention further includes the nucleic acid molecules operably linked to one or more expression control elements, including vectors comprising the isolated nucleic acid molecules.
  • the invention further includes host cells transformed to contain the nucleic acid molecules of the invention and methods for producing a protein comprising the step of culturing a host cell transformed with a nucleic acid molecule of the invention under conditions in which the protein is expressed.
  • the invention further provides an isolated polypeptide selected from the group consisting of an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, an isolated polypeptide comprising a fragment of at least 10 amino acids of SEQ ED NO: 2 or SEQ ID NO: 4, an isolated polypeptide comprising conservative amino acid substitutions of SEQ ID NO: 2 or SEQ ID NO: 4, naturally occurring amino acid sequence variants of SEQ ID NO: 2 or SEQ ID NO: 4 and an isolated polypeptide that exhibits at least about 50% amino acid sequence identity with SEQ ID NO: 2 or SEQ ID NO: 4.
  • the invention further provides an isolated antibody that binds to a polypeptide of the invention, including monoclonal and polyclonal antibodies.
  • the invention further provides methods of identifying an agent which modulates the expression of a nucleic acid encoding the protein having the sequence of SEQ ID NO: 2 or SEQ ID NO: 4, comprising the steps of: exposing cells which express the nucleic acid to the agent; and determining whether the agent modulates expression of said nucleic acid, thereby identifying an agent which modulates the expression of a nucleic acid encoding the protein having the sequence of SEQ ID NO: 2 or SEQ ID NO: 4.
  • the invention further provides methods of identifying an agent which modulates at least one activity of a protein comprising the sequence of SEQ ID NO: 2 or SEQ ID NO: 4, comprising the steps of: exposing cells which express the protein to the agent; and determining whether the agent modulates at least one activity of said protein, thereby identifying an agent which modulates at least one activity of a protein comprising the sequence of SEQ ID NO: 2 or SEQ ID NO: 4.
  • the invention further provides methods of identifying binding partners for a protein comprising the sequence of SEQ ID NO: 2 or SEQ ID NO: 4, comprising the steps of: exposing said protein to a potential binding partner; and determining if the potential binding partner binds to said protein, thereby identifying binding partners for a protein comprising the sequence of SEQ ID NO: 2 or SEQ ID NO: 4.
  • the present invention further provides methods of modulating the expression of a nucleic acid encoding the protein having the sequence of SEQ ID NO: 2 or SEQ ID NO: 4, comprising the step of: administering an effective amount of an agent which modulates the expression of a nucleic acid encoding the protein having the sequence of SEQ ED NO: 2 or SEQ ID NO: 4.
  • the invention also provides methods of modulating at least one activity of a protein comprising the sequence of SEQ ED NO: 2 or SEQ ED NO: 4, comprising the step of: administering an effective amount of an agent which modulates at least one activity of a protein comprising the sequence of SEQ ED NO: 2 or SEQ ED NO: 4.
  • Figure 1 shows READS-based differential display used to evaluate differential gene expression in various human renal biopsy samples. These data show that the mRNA species encoding the nucleic acid of SEQ ID NO: 3 (detected as a band on the READS gel) was down-regulated in all the tested disease samples relative to normal samples. Tissue samples were obtained from patients with IgA nephropathy (IgAN), focal segmental glomerulosclerosis (FSGS), diabetic nephropathy, necrotizing crescentic glomerulonephritis
  • IgA nephropathy IgAN
  • FSGS focal segmental glomerulosclerosis
  • diabetic nephropathy necrotizing crescentic glomerulonephritis
  • Figure 2 shows the expression levels of mRNA encoding the protein of SEQ ED NO: 4 (short form) as determined by quantitative PCR analysis in various tissues relative to the expression of GAPDH.
  • Figure 3 shows the expression levels of mRNA encoding the protein of
  • SEQ ED NO: 2 (long form) as determined by quantitative PCR analysis in various tissues relative to the expression of GAPDH.
  • Figure 4 shows the total expression levels of mRNA encoding the proteins of SEQ ED NO: 2 and SEQ ID NO: 4 as determined by quantitative PCR analysis in various tissues relative to the expression of GAPDH.
  • Figure 5 shows the expression levels of mRNA encoding the protein of
  • SEQ ID NO: 4 (short form), relative to the expression of GAPDH, as determined by quantitative PCR analysis of renal biopsy samples from patients with IgAN (IgA nephropathy), necrotizing crescentic glomerulonephritis (NCGN) or minimal change disease.
  • Figure 6 shows the expression levels of mRNA encoding the protein of
  • SEQ ID NO: 2 (long form), relative to the expression of GAPDH, as determined by quantitative 5 PCR analysis of renal biopsy samples from patients with various kidney diseases (abbreviations are defined in the description of Figure 5).
  • Figure 7 shows the total expression levels of mRNA encoding the proteins of SEQ ID NO: 2 and SEQ ID NO: 4, relative to the expression of GAPDH, as determined by quantitative PCR analysis of renal biopsy samples from patients with various kidney diseases 10 (abbreviations are defined in the description of Figure 5).
  • Figure 8 shows the GAPDH-normalized expression levels of mRNA encoding the protein of SEQ ED NO: 4 (short form) as a percent of total levels (both short and long forms) as determined by quantitative PCR analysis of renal biopsy samples from patients with various kidney diseases (abbreviations are defined in the description of Figure 5).
  • Figure 9 shows the GAPDH-normalized expression levels of mRNA encoding the protein of SEQ ED NO: 2 (long form) as a percent of total levels (both short and long forms) as determined by quantitative PCR analysis of renal biopsy samples from patients with various kidney diseases (abbreviations are defined in the description of Figure 5).
  • Figure 10 shows the total GAPDH-normalized expression levels of 0 mRNA encoding the proteins of SEQ ID NO: 2 and SEQ ED NO: 4 as determined by quantitative PCR analysis of renal biopsy samples from patients with various kidney diseases (abbreviations are defined in the description of Figure 5).
  • the present invention is based in part on identifying two new human gene products that have altered expression levels in diseased renal tissue. Based on the number of nucleotides in the coding and non-coding regions, the two cDNAs are referred to as the "long" form (2505 bp) and the “short” form (1820 bp). These cDNAs are predicted to encode proteins consisting of 30 359 amino acids (from the longer cDNA) and 502 amino acids (from the shorter cDNA) and have a homologous region within the coding sequence.
  • the proteins of the present invention can serve as targets for agents that can be used to modulate the expression or activity of the proteins.
  • agents may be identified that modulate biological processes associated with renal disease, kidney transplantation and kidney regeneration.
  • the present invention is further based on the development of methods for isolating binding partners that bind to the protein.
  • Probes based on the protein are used as capture probes to isolate potential binding partners, such as other proteins.
  • Dominant negative proteins, DNAs encoding these proteins, antibodies to these proteins, peptide fragments of these proteins or mimics of these proteins may be introduced into cells to affect function. Additionally, these proteins provide a novel target for screening of synthetic small molecules and combinatorial or naturally occurring compound libraries to discover novel therapeutics to regulate kidney function.
  • the present invention provides isolated protein, allelic variants of the protein, and conservative amino acid substitutions of the protein.
  • the protein or polypeptide refers to a protein that has the human amino acid sequence depicted in SEQ ED NO: 2 or SEQ ED NO: 4.
  • the invention includes naturally occurring allelic variants and proteins that have a slightly different amino acid sequence than that specifically recited above.
  • amino acid number 176 of SEQ ID NO: 2 may be alanine instead of glycine
  • amino acid number 240 of SEQ D NO: 4 may be glycine instead of alanine.
  • Allelic variants, though possessing a slightly different amino acid sequence than those recited above, will still have the same or similar biological functions associated with the 359 or 502 amino acid protein.
  • the family of proteins related to the human amino acid sequence of SEQ ED NO: 2 or SEQ ED NO: 4 refers to proteins that have been isolated from organisms in addition to humans. The methods used to identify and isolate other members of the family of proteins related to the 359 and 502 amino acid proteins are described below.
  • the proteins of the present invention are preferably in isolated form.
  • a protein is said to be isolated when physical, mechanical or chemical methods are employed to remove the protein from cellular constituents that are normally associated with the protein. A skilled artisan can readily employ standard purification methods to obtain an isolated protein.
  • the proteins of the present invention further include conservative amino acid substitution variants (i.e., conservative) of the proteins herein described.
  • conservative variant refers to alterations in the amino acid sequence that do not adversely affect the biological functions of the protein.
  • a substitution, insertion or deletion is said to adversely affect the protein when the altered sequence prevents or disrupts a biological function associated with the protein.
  • the overall charge, structure or hydrophobic/hydrophilic properties of the protein can be altered without adversely affecting a biological activity.
  • the amino acid sequence can be altered, for example to render the peptide more hydrophobic or hydrophilic, without adversely affecting the biological activities of the protein.
  • the allelic variants, the conservative substitution variants, and the members of the protein family will have an amino acid sequence having at least about 50%, 60% or 75% amino acid sequence identity with the sequences set forth in SEQ ED NO: 2 or SEQ ED NO: 4, more preferably at least about 80%, even more preferably at least about 90%, and most preferably at least about 95% sequence identity.
  • Identity or homology with respect to such sequences is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the known peptides, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology, and not considering any conservative substitutions as part of the sequence identity. N-terminal, C-terminal or internal extensions, deletions, or insertions into the peptide sequence shall not be construed as affecting homology (see section B).
  • the proteins of the present invention include molecules having the amino acid sequence disclosed in SEQ ED NO: 2 or SEQ ED NO: 4; fragments thereof having a consecutive sequence of at least about 3, 4, 5, 6, 10, 15, 20, 25, 30, 35 or more amino acid residues of the human proteins; amino acid sequence variants wherein an amino acid residue has been inserted N- or C-terminal to, or within, the disclosed sequence; and amino acid sequence variants of the disclosed sequence, or their fragments as defined above, that have been substituted by another residue.
  • Contemplated variants further include those containing predetermined mutations by, e.g., homologous recombination, site-directed or PCR mutagenesis, and the corresponding proteins of other animal species, including but not limited to rabbit, rat, porcine, bovine, ovine, equine and non-human primate species, and the alleles or other naturally occurring variants of the family of proteins; and derivatives wherein the protein has been covalently modified by substitution, chemical, enzymatic, or other appropriate means with a moiety other than a naturally occurring amino acid (for example a detectable moiety such as an enzyme or radioisotope).
  • a detectable moiety such as an enzyme or radioisotope
  • members of the family of proteins can be used: 1) to identify agents which modulate at least one activity of the protein; 2) to identify binding partners for the protein, 3) as an antigen to raise polyclonal or monoclonal antibodies, and 4) as a therapeutic agent.
  • nucleic acid molecules that encode the protein having SEQ ID NO: 2 or SEQ ID NO: 4 and the related proteins herein described, preferably in isolated form.
  • nucleic acid is defined as RNA or DNA that encodes a protein or peptide as defined above, is complementary to a nucleic acid sequence encoding such peptides, hybridizes to such a nucleic acid and remains stably bound to it under appropriate stringency conditions, or encodes a polypeptide sharing at least about 50%, 60% or 75% sequence identity, preferably at least about 80%, and more preferably at least about 85%, with the peptide sequences.
  • genomic DNA e.g., genomic DNA, cDNA, mRNA and antisense molecules, as well as nucleic acids based on alternative backbones or including alternative bases whether derived from natural sources or synthesized.
  • hybridizing or complementary nucleic acids are defined further as being novel and unobvious over any prior art nucleic acid including that which encodes, hybridizes under appropriate stringency conditions, or is complementary to nucleic acid encoding a protein according to the present invention.
  • Homology or identity is determined by BLAST (Basic Local Alignment Search Tool) analysis using the algorithm employed by the programs blastp, blastn, blasts, tblastn and tblastx (Karlin et al. Proc. Natl. Acad. Sci.
  • the search parameters for histogram, descriptions, alignments, expect i.e., the statistical significance threshold for reporting matches against database sequences
  • cutoff, matrix and filter are at the default settings.
  • the default scoring matrix used by blastp, blastx, tblastn, and tblastx is the BLOSUM62 matrix (Henikoff et al. Proc. Natl. Acad. Sci. USA 89: 10915-10919 (1992), fully incorporated by reference).
  • the scoring matrix is set by the ratios of M (i.e., the reward score for a pair of matching residues) to N (i.e., the penalty score for mismatching residues), wherein the default values for M and N are 5 and -4, respectively.
  • “Stringent conditions” are those that (1) employ low ionic strength and high temperature for washing, for example, 0.015M NaCl/0.0015M sodium titrate/0.1% SDS at 50°C, or (2) employ during hybridization a denaturing agent such as formamide, for example, 50% (vol/vol) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42°C.
  • a denaturing agent such as formamide, for example, 50% (vol/vol) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42°C.
  • Another example is use of 50% formamide, 5 x SSC (0.75M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 ⁇ g/ml), 0.1% SDS, and 10% dextran sulfate at 42°C, with washes at 42°C. in 0.2 x SSC and 0.1% SDS.
  • a skilled artisan can readily determine and vary the stringency conditions appropriately to obtain a clear and detectable hybridization signal.
  • nucleic acid molecule is said to be "isolated” when the nucleic acid molecule is substantially separated from contaminant nucleic acid encoding other polypeptides from the source of nucleic acid.
  • the present invention further provides fragments of the encoding nucleic acid molecule.
  • a fragment of an encoding nucleic acid molecule refers to a small portion of the entire protein coding sequence. The size of the fragment will be determined by the intended use. For example, if the fragment is chosen so as to encode an active portion of the protein, the fragment will need to be large enough to encode the functional region(s) of the protein. If the fragment is to be used as a nucleic acid probe or PCR primer, then the fragment length is chosen so as to obtain a relatively small number of false positives during probing/priming.
  • Fragments of the encoding nucleic acid molecules of the present invention i.e., synthetic oligonucleotides
  • PCR polymerase chain reaction
  • Fragments of the encoding nucleic acid molecules of the present invention can easily be synthesized by chemical techniques, for example, the phosphotri ester method of Matteucci, et al, (J. Am. Chem. Soc. 103:3185-3191, 1981) or using automated synthesis methods.
  • larger DNA segments can readily be prepared by well known methods, such as synthesis of a group of oligonucleotides that define various modular segments of the gene, followed by ligation of oligonucleotides to build the complete modified gene.
  • the encoding nucleic acid molecules of the present invention may further be modified so as to contain a detectable label for diagnostic and probe purposes.
  • a detectable label for diagnostic and probe purposes.
  • a variety of such labels are known in the art and can readily be employed with the encoding molecules herein described. Suitable labels include, but are not limited to, biotin, radiolabeled nucleotides and the like. A skilled artisan can employ any of the art known labels to obtain a labeled encoding nucleic acid molecule.
  • the identification and characterization of the human nucleic acid molecule having SEQ ED NO: 1 or SEQ ED NO: 3 allows a skilled artisan to isolate nucleic acid molecules that encode other members of the protein family in addition to the sequences herein described. Further, the presently disclosed nucleic acid molecules allow a skilled artisan to isolate nucleic acid molecules that encode other members of the family of proteins in addition to the proteins having SEQ ED NO: 2 or SEQ ID NO: 4.
  • SEQ ED NO: 2 or SEQ ED NO: 4 can readily use the amino acid sequence of SEQ ED NO: 2 or SEQ ED NO: 4 to generate antibody probes to screen expression libraries prepared from appropriate cells.
  • polyclonal antiserum from mammals such as rabbits immunized with the purified protein (as described below) or monoclonal antibodies can be used to probe a mammalian cDNA or genomic expression library, such as lambda gtll library, to obtain the appropriate coding sequence for other members of the protein family.
  • the cloned cDNA sequence can be expressed as a fusion protein, expressed directly using its own control sequences, or expressed by constructions using control sequences appropriate to the particular host used for expression of the enzyme.
  • coding sequence herein described can be synthesized and used as a probe to retrieve DNA encoding a member of the protein family from any mammalian organism. Oligomers containing approximately 18-20 nucleotides (encoding about a 6-7 amino acid stretch) are prepared and used to screen genomic DNA or cDNA libraries to obtain hybridization under stringent conditions or conditions of sufficient stringency to eliminate an undue level of false positives.
  • pairs of oligonucleotide primers can be prepared for use in a polymerase chain reaction (PCR) to selectively clone an encoding nucleic acid molecule.
  • PCR polymerase chain reaction
  • a PCR denature/anneal/extend cycle for using such PCR primers is well known in the art and can readily be adapted for use in isolating other encoding nucleic acid molecules.
  • the present invention further provides recombinant DNA molecules (rDNAs) that contain a coding sequence.
  • a rDNA molecule is a DNA molecule that has been subjected to molecular manipulation in situ. Methods for generating rDNA molecules are well known in the art, for example, see Sambrook et al, Molecular Cloning (1989).
  • a coding DNA sequence is operably linked to expression control sequences and/or vector sequences.
  • the choice of vector and/or expression control sequences to which one of the protein family encoding sequences of the present invention is operably linked depends directly, as is well known in the art, on the functional properties desired, e.g., protein expression, and the host cell to be transformed.
  • a vector contemplated by the present invention is at least capable of directing the replication or insertion into the host chromosome, and preferably also expression, of the structural gene included in the rDNA molecule.
  • Expression control elements that are used for regulating the expression of an operably linked protein encoding sequence are known in the art and include, but are not limited to, inducible promoters, constitutive promoters, secretion signals, and other regulatory elements.
  • the inducible promoter is readily controlled, such as being responsive to a nutrient in the host cell's medium.
  • the vector containing a coding nucleic acid molecule will include a prokaryotic replicon, i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extrachromosomally in a prokaryotic host cell, such as a bacterial host cell, transformed therewith.
  • a prokaryotic replicon i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extrachromosomally in a prokaryotic host cell, such as a bacterial host cell, transformed therewith.
  • a prokaryotic host cell such as a bacterial host cell, transformed therewith.
  • vectors that include a prokaryotic replicon may also include a gene whose expression confers a detectable marker such as a drug resistance.
  • Typical bacterial drug resistance genes are those that confer resistance to ampicillin or tetracycline.
  • Vectors that include a prokaryotic replicon can further include a prokaryotic or bacteriophage promoter capable of directing the expression (transcription and translation) of the coding gene sequences in a bacterial host cell, such as E. coli.
  • a promoter is an expression control element formed by a DNA sequence that permits binding of RNA polymerase and transcription to occur. Promoter sequences compatible with bacterial hosts are typically provided in plasmid vectors containing convenient restriction sites for insertion of a DNA segment of the present invention.
  • Typical of such vector plasmids are pUC8, pUC9, pBR322 and pBR329 available from Biorad Laboratories, (Richmond, CA), pPL and pKK223 available from Pharmacia, Piscataway, N.J.
  • Expression vectors compatible with eukaryotic cells can also be used to form a rDNA molecules that contains a coding sequence.
  • Eukaryotic cell expression vectors are well known in the art and are available from several commercial sources. Typically, such vectors are provided containing convenient restriction sites for insertion of the desired DNA segment. Typical of such vectors are pSVL and pKSV-10 (Pharmacia), pBPV-l/pML2d (International Biotechnologies, Inc.), pTDTl (ATCC, #31255), the vector pCDM8 described herein, and the like eukaryotic expression vectors.
  • Eukaryotic cell expression vectors used to construct the rDNA molecules of the present invention may further include a selectable marker that is effective in an eukaryotic cell, preferably a drug resistance selection marker.
  • a preferred drug resistance marker is the gene whose expression results in neomycin resistance, i.e., the neomycin phosphotransferase (neo) gene. (Southern et al, J. Mol. Anal. Genet. 1:327-341, 1982.)
  • the selectable marker can be present on a separate plasmid, and the two vectors are introduced by co- transfection of the host cell, and selected by culturing in the appropriate drug for the selectable marker.
  • the present invention further provides host cells transformed with a nucleic acid molecule that encodes a protein of the present invention.
  • the host cell can be either prokaryotic or eukaryotic.
  • Eukaryotic cells useful for expression of a protein of the invention are not limited, so long as the cell line is compatible with cell culture methods and compatible with the propagation of the expression vector and expression of the gene product.
  • Preferred eukaryotic host cells include, but are not limited to, yeast, insect and mammalian cells, preferably vertebrate cells such as those from a mouse, rat, monkey or human cell line.
  • Preferred eukaryotic host cells include Chinese hamster ovary (CHO) cells available from the ATCC as CCL61, NEH Swiss mouse embryo cells NEH/3T3 available from the ATCC as CRL 1658, baby hamster kidney cells (BHK), and the like eukaryotic tissue culture cell lines. Any prokaryotic host can be used to express a rDNA molecule encoding a protein of the invention.
  • the preferred prokaryotic host is E. coli.
  • Transformation of appropriate cell hosts with a rDNA molecule of the present invention is accomplished by well known methods that typically depend on the type of vector used and host system employed.
  • electroporation and salt treatment methods are typically employed, see, for example, Cohen et al, Proc. Natl. Acad. Sci. USA 69:2110, 1972; and Maniatis et al, Molecular Cloning. A Laboratory Mammal. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982).
  • electroporation, cationic lipid or salt treatment methods are typically employed, see, for example, Graham et al, Virol. 52:456, 1973; Wigler et al, Proc. Natl. Acad. Sci. USA 76:1373-76, 1979.
  • Successfully transformed cells i.e., cells that contain a rDNA molecule of the present invention
  • cells resulting from the introduction of an rDNA of the present invention can be cloned to produce single colonies. Cells from those colonies can be harvested, lysed and their DNA content examined for the presence of the rDNA using a method such as that described by Southern, J. Mol Biol. 98:503, 1975, or Berent et al, Biotech. 3:208, 1985 or the proteins produced from the cell assayed via an immunological method.
  • the present invention further provides methods for producing a protein of the invention using nucleic acid molecules herein described.
  • the production of a recombinant form of a protein typically involves the following steps:
  • nucleic acid molecule that encodes a protein of the invention, such as the nucleic acid molecule depicted in SEQ ED NO: 1 or SEQ ED NO: 3, nucleotides 198-1274 of SEQ ED NO: 1 or nucleotides 5-1510 of SEQ ID NO: 3. If the encoding sequence is uninterrupted by introns, it is directly suitable for expression in any host.
  • the nucleic acid molecule is then preferably placed in operable linkage with suitable control sequences, as described above, to form an expression unit containing the protein open reading frame.
  • the expression unit is used to transform a suitable host and the transformed host is cultured under conditions that allow the production of the recombinant protein.
  • the recombinant protein is isolated from the medium or from the cells; recovery and purification of the protein may not be necessary in some instances where some impurities may be tolerated.
  • the desired coding sequences may be obtained from genomic fragments and used directly in appropriate hosts.
  • the construction of expression vectors that are operable in a variety of hosts is accomplished using appropriate replicons and control sequences, as set forth above.
  • the control sequences, expression vectors, and transformation methods are dependent on the type of host cell used to express the gene and were discussed in detail earlier.
  • Suitable restriction sites can, if not normally available, be added to the ends of the coding sequence so as to provide an excisable gene to insert into these vectors.
  • a skilled artisan can readily adapt any host/expression system known in the art for use with the nucleic acid molecules of the invention to produce recombinant protein.
  • Another embodiment of the present invention provides methods for use in isolating and identifying binding partners of proteins of the invention
  • a protein of the invention is mixed with a potential binding partner or an extract or fraction of a cell under conditions that allow the association of potential binding partners with the protein of the invention.
  • peptides, polypeptides, proteins or other molecules that have become associated with a protein of the invention are separated from the mixture.
  • the binding partner that bound to the protein of the invention can then be removed and further analyzed.
  • the entire protein for instance a protein comprising the entire amino acid sequence of SEQ ED NO: 2 or SEQ ED NO: 4 can be used.
  • a fragment of the protein can be used.
  • a cellular extract refers to a preparation or fraction which is made from a lysed or disrupted cell.
  • the preferred source of cellular extracts will be cells derived from human kidney tissue, for instance, renal biopsy tissue or tissue culture cells.
  • cellular extracts may be prepared from normal human kidney tissue or available cell lines, particularly kidney derived cell lines.
  • a variety of methods can be used to obtain an extract of a cell.
  • Cells can be disrupted using either physical or chemical disruption methods.
  • physical disruption methods include, but are not limited to, sonication and mechanical shearing.
  • chemical lysis methods include, but are not limited to, detergent lysis and enzyme lysis.
  • a skilled artisan can readily adapt methods for preparing cellular extracts in order to obtain extracts for use in the present methods.
  • the extract is mixed with the protein of the invention under conditions in which association of the protein with the binding partner can occur.
  • conditions can be used, the most preferred being conditions that closely resemble conditions found in the cytoplasm of a human cell.
  • Features such as osmolarity, pH, temperature, and the concentration of cellular extract used, can be varied to optimize the association of the protein with the binding partner.
  • the bound complex is separated from the mixture.
  • a variety of techniques can be utilized to separate the mixture. For example, antibodies specific to a protein of the invention can be used to immunoprecipitate the binding partner complex. Alternatively, standard chemical separation techniques such as chromatography and density/sediment centrifugation can be used. After removal of non-associated cellular constituents found in the extract, the binding partner can be dissociated from the complex using conventional methods. For example, dissociation can be accomplished by altering the salt concentration or pH of the mixture.
  • the protein of the invention can be immobilized on a solid support.
  • the protein can be attached to a nitrocellulose matrix or acrylic beads. Attachment of the protein to a solid support aids in separating peptide/binding partner pairs from other constituents found in the extract.
  • the identified binding partners can be either a single protein or a complex made up of two or more proteins. Alternatively, binding partners may be identified using a Far- Western assay according to the procedures of Takayama et al. (1997) Methods Mol. Biol. 69:171-84 or Sauder et al. J Gen. Virol. 77(5):991 -6 or identified through the use of epitope tagged proteins or GST fusion proteins.
  • the nucleic acid molecules of the invention can be used in a yeast two- hybrid system.
  • the yeast two-hybrid system has been used to identify other protein partner pairs and can readily be adapted to employ the nucleic acid molecules herein described.
  • Another embodiment of the present invention provides methods for identifying agents that modulate the expression of a nucleic acid encoding a protein of the invention such as a protein having the amino acid sequence of SEQ ID NO: 2 or SEQ ED NO: 4. Such assays may utilize any available means of monitoring for changes in the expression level of the nucleic acids of the invention.
  • an agent is said to modulate the expression of a nucleic acid of the invention, for instance a nucleic acid encoding the protein having the sequence of SEQ ED NO: 2 or SEQ ED NO: 4, if it is capable of up- or down-regulating expression of the nucleic acid in a cell.
  • cell lines that contain reporter gene fusions between the open reading frame defined by nucleotides 198-1274 of SEQ ID NO: 1 or nucleotides 5-1510 of SEQ ED NO: 3 and any assayable fusion partner may be prepared.
  • Numerous assayable fusion partners are known and readily available including the firefly luciferase gene and the gene encoding chloramphenicol acetyltransferase (Alam et al. (1990) Anal. Biochem. 188:245-254).
  • Cell lines containing the reporter gene fusions are then exposed to the agent to be tested under appropriate conditions and time. Differential expression of the reporter gene between samples exposed to the agent and control samples identifies agents which modulate the expression of a 5 nucleic acid encoding a protein having the sequence of SEQ ED NO: 2 or SEQ ED NO: 4.
  • Additional assay formats may be used to monitor the ability of the agent to modulate the expression of a nucleic acid encoding a protein of the invention such as the protein having SEQ ED NO: 2 or SEQ ID NO: 4.
  • mRNA expression may be monitored directly by hybridization to the nucleic acids of the invention. Cell lines are exposed to the agent to be
  • RNA or mRNA is isolated by standard procedures such those disclosed in Sambrook et al. (Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, 1989).
  • Probes to detect differences in RNA expression levels between cells exposed to the agent and control cells may be prepared from the nucleic acids of the invention. It is preferable, but
  • Probes may be designed from the nucleic acids of the invention through methods known in the art. For instance, the G+C content of the probe and the probe length can affect probe binding to its target sequence. Methods to optimize probe specificity are commonly available in Sambrook et al. (Molecular Cloning: A Laboratory Approach. Cold Spring Harbor Press, NY,
  • Hybridization conditions are modified using known methods, such as those described by Sambrook et al. and Ausubel et al. as required for each probe.
  • Hybridization of total cellular RNA or RNA enriched for polyA RNA can be accomplished in any available format.
  • total cellular RNA or RNA enriched for polyA RNA can be affixed to a solid support and the solid support exposed to at least one probe comprising at least one, or part of one of the sequences of the invention under conditions in which the probe will specifically hybridize.
  • nucleic acid fragments comprising at least one, or part of one of the sequences of the invention can be affixed to a solid support, such as a porous glass wafer. The glass wafer can then be exposed to total cellular RNA or polyA RNA from a sample under conditions in which the affixed sequences will specifically hybridize.
  • Such glass wafers and hybridization methods are widely available, for example, those disclosed by Beattie (WO 95/11755).
  • agents which up or down regulate the expression of a nucleic acid encoding the protein having the sequence of SEQ ED NO: 2 or SEQ ED NO: 4 are identified.
  • Hybridization for qualitative and quantitative analysis of mRNAs may also be carried out by using a RNase Protection Assay (i.e., RPA, see Ma et al. (1996) Methods 10: 273-238).
  • RPA RNase Protection Assay
  • an expression vehicle comprising cDNA encoding the gene product and a phage specific DNA dependent RNA polymerase promoter (e.g., T7, T3 or SP6 RNA polymerase) is linearized at the 3' end of the cDNA molecule, downstream from the phage promoter, wherein such a linearized molecule is subsequently used as a template for synthesis of a labeled antisense transcript of the cDNA by in vitro transcription.
  • a phage specific DNA dependent RNA polymerase promoter e.g., T7, T3 or SP6 RNA polymerase
  • the labeled transcript is then hybridized to a mixture of isolated RNA (i.e., total or fractionated mRNA) by incubation at 45 °C overnight in a buffer comprising 80% formamide, 40 mM Pipes, pH 6.4, 0.4 M NaCl and 1 mM EDTA.
  • the resulting hybrids are then digested in a buffer comprising 40 ⁇ g/ml ribonuclease A and 2 ⁇ g/ml ribonuclease. After deactivation and extraction of extraneous proteins, the samples are loaded onto urea/polyacrylamide gels for analysis.
  • cells or cell lines are first identified which express said gene products physiologically (e.g., using assays of tissue distribution via Northern blot, although RPAs may serve the identical purpose of expression selection).
  • Cell and/or cell lines so identified would be expected to comprise the necessary cellular machinery such that the fidelity of modulation of the transcriptional apparatus is maintained with regard to exogenous contact of agent with appropriate surface transduction mechanisms and/or the cytosolic cascades.
  • an expression vehicle e.g., a plasmid or viral vector
  • an expression vehicle e.g., a plasmid or viral vector
  • an operable non-translated 5'-promoter containing end of the structural gene encoding the instant gene products fused to one or more antigenic fragments, which are peculiar to the instant gene products, wherein said fragments are under the transcriptional control of said promoter and are expressed as polypeptides whose molecular weight can be distinguished from the naturally occurring polypeptides or may further comprise an immunologically distinct tag.
  • the agent comprises a pharmaceutically acceptable excipient and is contacted with cells comprised in an aqueous physiological buffer such as phosphate buffered saline (PBS) at physiological pH, Eagles balanced salt solution (BSS) at physiological pH, PBS or BSS comprising serum or conditioned media comprising PBS or BSS and/or serum incubated at 37°C .
  • PBS phosphate buffered saline
  • BSS Eagles balanced salt solution
  • Said conditions may be modulated as deemed necessary by one of skill in the art.
  • said cells are disrupted and the polypeptides of the disruptate are fractionated such that a polypeptide fraction is pooled and contacted with an antibody to be further processed by immunological assay (e.g., ELISA, immunoprecipitation or Western blot).
  • immunological assay e.g., ELISA, immunoprecipitation or Western blot.
  • the pool of proteins isolated from the "agent contacted” sample is then compared with a control sample where only the excipient is contacted with the cells and an increase or decrease in the immunologically generated signal from the "agent contacted" sample compared to the control is used to distinguish the effectiveness of the agent.
  • Another embodiment of the present invention provides methods for identifying agents that modulate at least one activity of a protein of the invention such as the protein having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.
  • Such methods or assays may utilize any means of monitoring or detecting the desired activity.
  • the relative amounts of a protein of the invention between a cell population that has been exposed to the agent to be tested compared to an un-exposed control cell population may be assayed.
  • probes such as specific antibodies are used to monitor the differential expression of the protein in the different cell populations.
  • Cell lines or populations are exposed to the agent to be tested under appropriate conditions and time.
  • Cellular lysates may be prepared from the exposed cell line or population and a control, unexposed cell line or population. The cellular lysates are then analyzed with the probe.
  • Antibody probes are prepared by immunizing suitable mammalian hosts in appropriate immunization protocols using the peptides, polypeptides or proteins of the invention if they are of sufficient length, or, if desired, or if required to enhance immunogenicity, conjugated to suitable carriers. Methods for preparing immunogenic conjugates with carriers such as BSA, KLH, or other carrier proteins are well known in the art. In some circumstances, direct conjugation using, for example, carbodiimide reagents may be effective; in other instances linking reagents such as those supplied by Pierce Chemical Co., Rockford, IL, may be desirable to provide accessibility to the hapten.
  • the hapten peptides can be extended at either the amino or carboxy terminus with a Cys residue or interspersed with cysteine residues, for example, to facilitate linking to a carrier.
  • Administration of the immunogens is conducted generally by injection over a suitable time period and with use of suitable adjuvants, as is generally understood in the art.
  • titers of antibodies are taken to determine adequacy of antibody formation.
  • Immortalized cell lines which secrete the desired monoclonal antibodies may be prepared using the standard method of Kohler and Milstein or modifications which effect immortalization of lymphocytes or spleen cells, as is generally known.
  • the immortalized cell lines secreting the desired antibodies are screened by immunoassay in which the antigen is the peptide hapten. polypeptide or protein.
  • the cells can be cultured either in vitro or by production in ascites fluid.
  • the desired monoclonal antibodies are then recovered from the culture supernatant or from the ascites supernatant.
  • Fragments of the monoclonals or the polyclonal antisera which contain the immunologically significant portion can be used as antagonists, as well as the intact antibodies.
  • Use of immunologically reactive fragments, such as the Fab, Fab', of F(ab') 2 fragments is often preferable, especially in a therapeutic context, as these fragments are generally less immunogenic than the whole immunoglobulin.
  • the antibodies or fragments may also be produced, using current technology, by recombinant means.
  • Antibody regions that bind specifically to the desired regions of the protein can also be produced in the context of chimeras with multiple species origin.
  • Agents that are assayed in the above method can be randomly selected or rationally selected or designed.
  • an agent is said to be randomly selected when the agent is chosen randomly without considering the specific sequences involved in the association of the a protein of the invention alone or with its associated substrates, binding partners, etc.
  • An example of randomly selected agents is the use a chemical library or a peptide combinatorial library, or a growth broth of an organism.
  • "Mimic” used herein refers to the modification of a region or several regions of a peptide molecule to provide a structure chemically different from the parent peptide but topographically and functionally similar to the parent peptide. (See Grant, GA., in Molecular Biology and Biotechnology. 1995, Meyers (ed.) pp 659-664, VCH Publishers, Inc., New York, NY).
  • an agent is said to be rationally selected or designed when the agent is chosen on a nonrandom basis which takes into account the sequence of the target site and/or its conformation in connection with the agent's action.
  • Agents can be rationally selected or rationally designed by utilizing the peptide sequences that make up these sites.
  • a rationally selected peptide agent can be a peptide whose amino acid sequence is identical to the EF-Hand motifs at amino acids 129-151 and 168-190 of SEQ ID NO: 2 or at amino acids 193-215 and 232-254 of SEQ ID NO: 4.
  • the agents of the present invention can be, as examples, peptides, small molecules, vitamin derivatives, as well as carbohydrates.
  • the peptide agents of the invention can be prepared using standard solid phase (or solution phase) peptide synthesis methods, as is known in the art.
  • the DNA encoding these peptides may be synthesized using commercially available oligonucleotide synthesis instrumentation and produced recombinantly using standard recombinant production systems. The production using solid phase peptide synthesis is necessitated if non-gene-encoded amino acids are to be included.
  • Another class of agents of the present invention are antibodies immunoreactive with critical positions of proteins of the invention.
  • Antibody agents are obtained by immunization of suitable mammalian subjects with peptides, containing as antigenic regions, those portions of the protein intended to be targeted by the antibodies.
  • the proteins and nucleic acids of the invention are expressed in renal tissue in NCGN, IgAN, FSGS and minimal change disease.
  • Agents that modulate, up-or- down-regulate the expression of the protein or agents such as agonists or antagonists of at least one activity of the protein may be used to modulate biological and pathologic processes associated with the protein's function and activity.
  • a subject can be any mammal, so long as the mammal is in need of modulation of a pathological or biological process mediated by a protein of the invention.
  • mammal is meant an individual belonging to the class Mammalia.
  • the invention is particularly useful in the treatment of human subjects.
  • Pathological processes refer to a category of biological processes which produce a deleterious effect.
  • expression of a protein of the invention may be associated with kidney cell growth regeneration and/or recovery from kidney disease.
  • an agent is said to modulate a pathological process when the agent reduces the degree or severity of the process.
  • kidney damage or ESRD may be prevented or disease progression modulated by the administration of agents which up-regulate or modulate in some way the expression or at least one activity of a protein of the invention.
  • the agents of the present invention can be provided alone, or in combination with other agents that modulate a particular pathological process.
  • an agent of the present invention can be administered in combination with other known drugs or may be combined with dialysis or anti -rejection drugs used during transplantation.
  • two agents are said to be administered in combination when the two agents are administered simultaneously or are administered independently in a fashion such that the agents will act at the same time.
  • the agents of the present invention can be administered via parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, or buccal routes. Alternatively, or concurrently, administration may be by the oral route.
  • the dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.
  • the present invention further provides compositions containing one or more agents which modulate expression or at least one activity of a protein of the invention. While individual needs vary, determination of optimal ranges of effective amounts of each component is within the skill of the art. Typical dosages comprise 0.1 to 100 ⁇ g/kg body wt. The preferred dosages comprise 0.1 to 10 ⁇ g/kg body wt. The most preferred dosages comprise 0.1 to 1 ⁇ g/kg body wt.
  • compositions of the present invention may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically for delivery to the site of action.
  • suitable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form, for example, water-soluble salts.
  • suspensions of the active compounds as appropriate oily injection suspensions may be administered.
  • Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension include, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran.
  • the suspension may also contain stabilizers. Liposomes can also be used to encapsulate the agent for delivery into the cell.
  • the pharmaceutical formulation for systemic administration according to the invention may be formulated for enteral, parenteral or topical administration. Indeed, all three types of formulations may be used simultaneously to achieve systemic administration of the active ingredient.
  • Suitable formulations for oral administration include hard or soft gelatin capsules, pills, tablets, including coated tablets, elixirs, suspensions, syrups or inhalations and controlled release forms thereof.
  • the compounds of this invention may be used alone or in combination, or in combination with other therapeutic or diagnostic agents.
  • the compounds of this invention may be coadministered along with other compounds typically prescribed for these conditions according to generally accepted medical practice.
  • the compounds of this invention can be utilized in vivo, ordinarily in mammals, such as humans, sheep, horses, cattle, pigs, dogs, cats, rats and mice, or in vitro.
  • Transgenic animals containing and mutant, knock-out or modified genes corresponding to the cDNA sequences of SEQ ID NO: 1 or SEQ ED NO: 3 are also included in the invention.
  • Transgenic animals are genetically modified animals into which recombinant, exogenous or cloned genetic material has been experimentally transferred. Such genetic material is often referred to as a "transgene".
  • the nucleic acid sequence of the transgene in this case a form of SEQ ID NO: 1 or SEQ ID NO: 3, may be integrated either at a locus of a genome where that particular nucleic acid sequence is not otherwise normally found or at the normal locus for the transgene.
  • the transgene may consist of nucleic acid sequences derived from the genome of the same species or of a different species than the species of the target animal.
  • germ cell line transgenic animal refers to a transgenic animal in which the genetic alteration or genetic information was introduced into a germ line cell, thereby conferring the ability of the transgenic animal to transfer the genetic information to offspring. If such offspring in fact possess some or all of that alteration or genetic information, then they too are transgenic animals.
  • the alteration or genetic information may be foreign to the species of animal to which the recipient belongs, foreign only to the particular individual recipient, or may be genetic information already possessed by the recipient. In the last case, the altered or introduced gene may be expressed differently than the native gene.
  • Transgenic animals can be produced by a variety of different methods including transfection, electroporation, microinjection, gene targeting in embryonic stem cells and recombinant viral and retroviral infection (see, e.g., U.S. Patent No. 4,736,866; U.S. Patent No. 5,602,307; Mullins et al, Hypertension (1993) 22(4):630-633; Bremn et al, Surg. Oncol (1997) 6(2)99-110; Tuan (ed.), Recombinant Gene Expression Protocols, Methods in Molecular Biology No. 62, Humana Press (1997)).
  • a number of recombinant or transgenic mice have been produced, including those which express an activated oncogene sequence (U.S. Patent No.
  • Alzheimer's disease U.S. Patent No. 5,720,936
  • have a reduced capacity to mediate cellular adhesion U.S. Patent No. 5,602,307
  • possess a bovine growth hormone gene Clutter et al, Genetics (1996) 143(4): 1753- 1760
  • are capable of generating a fully human antibody response McCarthy The Lancet (1997) 349(9049):405). While mice and rats remain the animals of choice for most transgenic experimentation, in some instances it is preferable or even necessary to use alternative animal species.
  • the method of introduction of nucleic acid fragments into recombination competent mammalian cells can be by any method which favors co-transformation of multiple nucleic acid molecules.
  • Detailed procedures for producing transgenic animals are readily available to one skilled in the art, including the disclosures in U.S. Patent No. 5,489,743 and U.S. Patent No. 5,602,307.
  • Kidney tissue was obtained from mice and from patients suffering from a number of kidney diseases, such as IgAN and NCGN.
  • Total cellular RNA was prepared from the kidney tissue described above as well as from control kidney tissue using the procedure of Newburger et al, J. Biol. Chem. (1981) 10 266(24): 16171-7 and Newburger et al, Proc. Natl. Acad. Sci. USA (1988) 85:5215-5219.
  • cDNA was synthesized according to the protocol described in the GEBCO/BRL kit for cDNA synthesis.
  • the reaction mixture for first-strand synthesis included 6 ⁇ g of total RNA, and 200 ng of a 15 mixture of 1 -base anchored oligo(dT) primers with all three possible anchored bases
  • reaction mixture may include lO ⁇ g of total 20 RNA, and 2 pmol of 1 of the 2-base anchored oligo(dT) primers a heel such as RP5.0 (CTCTCAAGGATCTTACCGCTT lg AT (SEQ ID NO: 7)), or
  • RP6.0 TAATACCGCGCCACATAGCAT ⁇ 8 CG (SEQ ID NO: 8)
  • RP9.2 CAGGGTAGACGACGCTACGCTigGA (SEQ ID NO: 9)
  • This mixture was then layered with 25 mineral oil and incubated at 65 C for 7 min followed by 50 C for another 7 min.
  • 2 1 of Superscript® reverse transcriptase 200 units/ 1; GIBCO/BRL
  • Second-strand synthesis was performed at 16 C for 2 hr.
  • the cDNAs were precipitated with ethanol and the yield of cDNA was calculated.
  • 200 ng of cDNA was obtained from 10 g of 30 total RNA.
  • the adapter oligonucleotide sequences were Al (TAGCGTCCGGCGCAGCGACGGCCAG (SEQ ID NO: 10)) and A2 (GATCCTGGCCGTCGGCTGTCTGTCGGCGC (SEQ ID NO: 11)).
  • One microgram of oligonucleotide A2 was first phosphorylated at the 5 end using T4 polynucleotide kinase (PNK). After phosphorylation, PNK was heated denatured, and 1 g of the oligonucleotide Al was added along with 10x annealing buffer (1 M NaCl/100 mM Tris-HCl, pH8.0/10 mM EDTA, pH8.0) in a final vol of 20 1.
  • This mixture was then heated at 65 C for 10 min followed by slow cooling to room temperature for 30 min, resulting in formation of the Y adapter at a final concentration of 100 ng/ 1.
  • About 20 ng of the cDNA was digested with 4 units oiBgl II in a final vol of 10 1 for 30 min at 37 C.
  • Two microliters ( 4 ng of digested cDNA) of this reaction mixture was then used for ligation to 100 ng ( 50-fold) of the Y-shaped adapter in a final vol of 5 1 for 16 hr at 15 C.
  • reaction mixture was diluted with water to a final vol of 80 1 (adapter ligated cDNA concentration, 50 pg/ 1) and heated at 65 C for 10 min to denature T4 DNA ligase, and 2- 1 aliquots (with 100 pg of cDNA) were used for PCR.
  • oligonucleotide Al or Al.l was 5 - end-labeled using 15 1 of [ - 32 P]ATP (Amersham; 3000 Ci/mmol) and PNK in a final volume of 20 1 for 30 min at 37 C. After heat denaturing PNK at 65 C for 20 min, the labeled oligonucleotide was diluted to a final concentration of 2 M in 80 1 with unlabeled oligonucleotide Al.l.
  • the PCR mixture (20 1) consisted of 2 1 ( 100 pg) of the template, 2 1 of 10 ⁇ PCR buffer (100 mM Tris-HCl, pH 8.3/500 mM KC1), 2 1 of 15 mM MgCl 2 to yield 1.5 mM final Mg 2+ concentration optimum in the reaction mixture, 200 M dNTPs, 200 nM each 5 and 3 PCR primers, and 1 unit of Amplitaq Gold®. Primers and dNTPs were added after preheating the reaction mixture containing the rest of the components at 85 C.
  • PCR This "hot start" PCR was done to avoid artefactual amplification arising out of arbitrary annealing of PCR primers at lower temperature during transition from room temperature to 94 C in the first PCR cycle.
  • PCR consisted of 5 cycles of 94 C for 30 sec, 55 C for 2 min, and 72 C for 60 sec followed by 25 cycles of 94 C for 30 sec, 60 C for 2 min, and 72 C for 60 sec. A higher number of cycles resulted in smeary gel patterns.
  • PCR products (2.5 1) were analyzed on 6% polyacrylamide sequencing gel. For double or multiple digestion following adapter ligation, 13.2 1 of the ligated cDNA sample was digested with a secondary restriction enzyme(s) in a final vol of 20 1.
  • 3 1 was used as template for PCR.
  • This template vol of 3 1 carried 100 pg of the cDNA and 10 mM MgCl 2 (from the 10* enzyme buffer), which diluted to the optimum of 1.5 mM in the final PCR vol of 20 1. Since Mg" + comes from the restriction enzyme buffer, it was not included in the reaction mixture when amplifying secondarily cut cDNA. Individual cDNA fragments corresponding to mRNA species were separated by denaturing polyacrylamide gel electrophoresis and visualized by autoradiography. Bands were extracted from the display gels as described by Liang et al. (1995 Curr. Opin. Immunol.
  • the sequence of the band is: gtcttctggt acaactacct gcctgatggg caaggttggg tgggtgacgt agacgactac 60 tcgctgcacg ggggctgcct ggtcacgcgc ggcaccaagt ggattgccaa caactggatt 120 aatgtggacc ccagccgagc gcggcaagcg ctgttccaac aggagatggc ccgccttgcc 180 cgagaagggg gcaccgactc acagcccgag tgggctctgg accgggccta ccgcgatgcg 240 cgcgtggaac tctgagggaa gagttagcccc cggttcccag ccgcgggtcg
  • the full length cDNA isoforms corresponding to SEQ ID NO: 5 were obtained by the oligo-pulling method. Briefly, a gene-specific oligo was designed based on the sequence of SEQ ID NO: 5. The oligo was labeled with biotin and used to hybridize with 2 ⁇ g of single strand plasmid DNA (cDNA recombinants) from a human kidney cDNA library following the procedures of Sambrook et al. The hybridized cDNAs were separated by streptavidin-co ⁇ jugated beads and eluted by heating. The eluted cDNA was converted to double strand plasmid DNA and used to transform E. coli cells (DH10B) and the longest cDNA was screened. After positive selection was confirmed by PCR using gene-specific primers, the cDNA clone was subjected to DNA sequencing.
  • cDNA recombinants single strand plasmid DNA
  • DH10B E. coli cells
  • the nucleotide sequence of the full-length cDNA isoforms corresponding to the differentially regulated band having the sequence of SEQ ID NO: 5 are set forth in SEQ ID NOs: 1 and 3.
  • the cDNA comprises 2505 base pairs with an open reading frame encoding a protein predicted to contain 359 amino acids.
  • the predicted amino acid sequence for the long form is presented in SEQ ID NOs: 1 and 2.
  • the cDNA comprises 1820 base pairs with an open reading frame encoding a protein predicted to contain 502 amino acids.
  • the predicted amino acid sequence for the short form is presented in SEQ ID NOs: 3 and 4.
  • Analysis of the amino acid sequence of SEQ D NO: 2 predicts regions resembling EF
  • RNA was isolated from human kidney, adrenal, pancreas, salivary gland, liver, prostate, thyroid, cerebellum, fetal brain, fetal liver, placenta, spinal tissue, colon, small intestine, stomach, bone marrow, thymus, spleen, heart, lung, testis, uterus, trachea, mammary gland, glomeruli, medulla and cortex using standard procedures.
  • Northern blots were prepared using a probe derived from SEQ ID NOS: 1 or 3 with hybridization conditions as described by
  • FIGS. 3-10 show the results of the quantitative PCR analysis of expression levels of mRNA corresponding to SEQ ED NOS: 1 and 3 in various human tissue samples.
  • Real time PCR detection was accomplished by the use of the ABI PRISM 7700 Sequence Detection System. The 7700 measures the fluorescence intensity of the sample each cycle and is able to detect the presence of specific amplicons within the PCR reaction.
  • Each sample was assayed for the level of GAPDH and mRNA corresponding to SEQ ID NO: 1, or SEQ ID NO: 3 , or to both SEQ ID NO: 1 and SEQ ID NO: 3 (using a primer against a sequence found in both the long and short forms).
  • GAPDH detection was performed using Perkin Elmer part#402869 according to the manufacturer's directions. Primers were designed from SEQ ID NOS: 1 and 3 using Primer Express, a program developed by PE to efficiently find primers and probes for specific sequences. These primers were used in conjunction with SYBR green (Molecular Probes), a nonspecific double stranded DNA dye, to measure the expression level mRNA corresponding to SEQ ID NOS: 1 and 3, which was normalized to the GAPDH level in each sample.
  • SYBR green Molecular Probes
  • Tissue distribution analyses indicate that the sequences of SEQ ED NO: 1 and SEQ ID NO: 3 are expressed throughout the human body. For example (see Figures 2-4), high levels of mRNA are observed in the pancreas, adrenal glands, prostate, cerebellum and other tissues, including kidney.
  • Figures 7 and 10 show that the quantity of total message (encompassing both forms) does not appear to significantly increase or decrease as IgAN progresses except in certain patients who may actually represent distinct sub-categories. Similarly, compared to samples of normal tissue, there was little change in the level of the total message in the biopsy samples from patients with NCGN, though these patients have the most acute pathology.

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Abstract

La présente invention concerne de façon générale des différences d'expression génique au niveau du tissu rénal humain entre reins atteints et reins normaux. L'invention concerne plus particulièrement des isoformes d'un nouveau produit génique humain dont les niveaux d'expression sont altérés dans les prélèvements de biopsies rénales faits sur des patients souffrant de pathologies rénales telles que la néphrite glomérulaire falciforme nécrosante ou NCGN (necrotizing crescentic glomerulonephritis) et la néphropathie de Berger (IgAN).
PCT/US2000/017434 1999-06-24 2000-06-26 Nouvel adn complementaire associe a une pathologie renale WO2000078788A1 (fr)

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AU57651/00A AU5765100A (en) 1999-06-24 2000-06-26 Novel cdna associated with renal disease

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010038974A2 (fr) * 2008-10-01 2010-04-08 경북대학교 산학협력단 Composition et trousse de diagnostic pour néphropathie à dépôts d’immunoglobulines a et néphropathie tgbm

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ADAMS M.D. ET AL.: "Initial assessment of human gene diversity and expression paterns based upon 83 million nucleotides of cDNA sequences", NATURE, vol. 377, September 1995 (1995-09-01), pages 3 - 17, SUPP. 28, XP002922157 *
HELAAKOSKI T. ET AL.: "Cloning, baculovirus expression and characterization of a second mouse prolyl 4-hydroxylase alpha-subunit isoform: Formation of an alpha2beta2 tetramer with the protein disulfide-isomerase/beta subunit", PROC. NATL. ACAD. SCI. USA, vol. 92, May 1995 (1995-05-01), pages 4427 - 4431, XP002931918 *
LEFKOWITH J.B. ET AL.: "Polyunsaturated fatty acids and renal disease", PROCEEDINGS OF THE SOCIETY FOR EXPERIMENTAL BIOLOGY AND MEDICINE, vol. 213, no. 1, 1996, pages 13 - 23, XP002931920 *
SAKAI H. ET AL.: "Molecular biology and the kidney molecular approaches to the study of renal disease", NEPHROLOGY, vol. 2, 1996, pages S107 - S110, SUP. 1, XP002931919 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010038974A2 (fr) * 2008-10-01 2010-04-08 경북대학교 산학협력단 Composition et trousse de diagnostic pour néphropathie à dépôts d’immunoglobulines a et néphropathie tgbm
WO2010038974A3 (fr) * 2008-10-01 2010-08-26 경북대학교 산학협력단 Composition et trousse de diagnostic pour néphropathie à dépôts d'immunoglobulines a et néphropathie tgbm

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