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WO2005113761A2 - Enzymes alanine transaminase et procedes d'utilisation - Google Patents

Enzymes alanine transaminase et procedes d'utilisation Download PDF

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Publication number
WO2005113761A2
WO2005113761A2 PCT/US2005/013019 US2005013019W WO2005113761A2 WO 2005113761 A2 WO2005113761 A2 WO 2005113761A2 US 2005013019 W US2005013019 W US 2005013019W WO 2005113761 A2 WO2005113761 A2 WO 2005113761A2
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Prior art keywords
polypeptide
alt
seq
alt2
antibody
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PCT/US2005/013019
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WO2005113761A3 (fr
Inventor
Da-Wei Gong
Alan R. Shuldiner
Rong-Ze Yang
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University of Maryland Baltimore
University of Maryland College Park
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University of Maryland Baltimore
University of Maryland College Park
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Priority to US11/587,331 priority Critical patent/US7914985B2/en
Publication of WO2005113761A2 publication Critical patent/WO2005113761A2/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1096—Transferases (2.) transferring nitrogenous groups (2.6)

Definitions

  • ALT polypeptides alanine transaminase (ALT) polypeptides and the use thereof as a diagnostic marker to predict and monitor tissue damage and/or tissue malfunction. More specifically, the ALT polypeptides are murine and/or rattus ALT polypeptides and said ALT polypeptides are used to detect, predict and/or determine hepatic processes of an animal, particularly mice and/or rats.
  • the present invention additionally relates to assays for the ALT polypeptides to diagnose tissue damage and/or tissue malfunction having a range of etiologies that include but are not limited to hepatitis, nonalcoholic steatohepatitis (NASH), fatty liver, cirrhosis, and drug hepatotoxicity, and other disorders in muscle, brain, kidney, and adipose tissue, particularly in mice and/or rats.
  • tissue damage and/or tissue malfunction having a range of etiologies that include but are not limited to hepatitis, nonalcoholic steatohepatitis (NASH), fatty liver, cirrhosis, and drug hepatotoxicity, and other disorders in muscle, brain, kidney, and adipose tissue, particularly in mice and/or rats.
  • NASH nonalcoholic steatohepatitis
  • fatty liver fatty liver
  • cirrhosis cirrhosis
  • drug hepatotoxicity and other disorders in muscle, brain, kidney,
  • Alanine transaminase [EC 2.6.1.2., also known as glutamate pyruvate transaminase (GPT) and alanine aminotransferase] is a pyridoxal enzyme catalyzing reversible transamination between alanine and 2-oxoglutarate to form pyruvate and glutamate.
  • GPT glutamate pyruvate transaminase
  • alanine aminotransferase is a pyridoxal enzyme catalyzing reversible transamination between alanine and 2-oxoglutarate to form pyruvate and glutamate.
  • ALT transfers the ⁇ -amino group from glutamate to pyruvate to form alanine, which is a major amino acid in blood during fasting.
  • Alanine is taken up by the liver for generating glucose from pyruvate in a reverse ALT reaction, constituting the so-called alanine-glucose cycle. This cycle is also important during intensive exercise when skeletal muscles operate anaerobically, producing not only ammonia groups from protein breakdown but also large amounts of pyruvate from glycolysis.
  • ALT activities exist in many tissues, including liver, muscle, heart, kidney, and brain.
  • cDNAs complementary DNAs
  • hALTl and hALT2 Molecular cloning of the complementary DNAs (cDNAs) of two human ALT isoenzymes, hALTl and hALT2 have been disclosed in International Publication WO 02/092768, herein fully incorporated by reference in its entirety.
  • gptl and gpt2 tissue-dependent role for ALT isoenzymes.
  • the most well-known aspect of ALT is that it is used clinically as an index of liver integrity or hepatocellular damage.
  • Serum ALT activity is significantly elevated in a variety of liver damage conditions including viral infection, alcoholic steatosis, nonalcoholic steatohepatitis (NASH), and drug toxicity, although the underlying mechanism is generally not well understood. While low level of ALT is present in peripheral circulation because of normal cell turnover or release from nonvascular sources, the liver has been shown to contain the highest levels of ALT. The difference between ALT levels in liver and in blood has been shown to be about 2,000-3,000-fold. Hence, the increased ALT in serum, plasma, or blood is regarded as a marker of liver injury because of the "leakage" of hepatic ALT into the circulation. Usually, the nature of liver injury causes the blood ALT levels to vary greatly.
  • ALT alanine aminotransferase
  • mALTl and mALT2 Complementary DNAs of murine homologues of human alanine aminotransferase 1 and 2
  • rALTl and rALT2 rat homologues of human alanine aminotransferase 1 and 2
  • the polypeptides of murine ALT1 (mALTl) and ALT2 (mALT2) of this invention share 87% and 93% identity, respectively, with their human counterparts at the amino acid level.
  • the murine ALT genes of the two murine ALT isoenzymes localize to separate chromosomes, with the murine ALT1 gene (gptl) on chromosome 15 and the murine ALT2 gene (gpt2) on chromosome 8.
  • the murine gptl and gpt2 also differ in messenger RNA expression.
  • the murine gptl is mainly expressed in liver, bowel, and white adipose tissue (WAT) and the murine gpt2 is highly expressed in muscle, liver, and white adipose tissue.
  • WAT white adipose tissue
  • Expression of recombinant murine ALT1 and murine ALT2 proteins in Escherichia coli (E. coli) produced functional enzymes that catalyze alanine transamination.
  • Rat ALT1 polypeptide consists of 496 amino acids and shares 97% and 88% identity to murine and human ALTl, respectively, at the amino acid level.
  • Rat ALT2 polypeptide is composed of 522 amino acids and share 98% and 95% identity to murine and human ALT2, respectively, at the amino acid level.
  • Rat ALTl and rat ALT2 polypeptides have 68% sequence identity and 77% similarity.
  • the genes of rat ALTl and ALT2 reside on the chromosome 7 and 19, respectively.
  • a sequence alignment of murine ALTl and ALT2, human ALTl and ALT2 and rat ALTl and ALT2 is provided in FIG. 6.
  • the diagnostic value of murine ALT isoenzymes in liver disease was determined by an obese animal model. In fatty livers of obese mice, murine ALT2 gene expression is induced 2-fold, but murine ALTl remains the same.
  • murine ALT2 is responsible for the increased ALT activity in hepatic steatosis and allows for a murine ALT isoenzyme-specific assay having more diagnostic value than total ALT activity assays currently in clinical use.
  • the murine isoenzyme-specific assays of this invention provide improved assays for assessing preclinical toxicity of new medications.
  • Another embodiment of the present invention is directed to antibodies, particularly anti-ALTl antibodies and anti-ALT2 antibodies.
  • the antibody specifically binds to murine ALTl. In another specific embodiment, the antibody specifically binds to murine ALT2. In yet another specific embodiment, the antibody specifically binds to rat ALTl. In still yet another specific embodiment, the antibody specifically binds to rat ALT2. It is an object of this invention to have a murine ALT polypeptide which has the amino acid sequence of SEQ ID NO:l (murine ALTl) or an amino acid having about 95% homology thereto. In certain specific embodiments, the amino acid having about 95% homology to SEQ ID NO:l is SEQ ID NO:6.
  • the amino acid having about 95% homology to SEQ ID NO:2 is SEQ ID NO:5.
  • a polynucleotide which encodes for each of the murine ALT isoenzymes is SEQ ID NO:l and/or SEQ ID NO:2 or an amino acid sequence having about 95% homology to SEQ ID NO:l and or SEQ ID
  • polynucleotide sequence be the sequence of SEQ ID NO:3 or SEQ ID NO:4.
  • polynucleotide sequence is the polynucleotide sequence encoding for the homolog of SEQ ID NO:3.
  • polynucleotide sequences are SEQ ID NO:7 (rat ALTl) or
  • SEQ ID NO:8 (rat ALT2). It is another object of this invention to have a polynucleotide which encodes for each of the rat ALT isoenzymes. It is a further object of this invention that the polynucleotide encodes the amino acid sequence of SEQ ID NO:5 and/or SEQ ID NO:6 or an amino acid sequence having about 95% homology to SEQ ID NO: 5 and/or SEQ ID NO:6. It is a further object of this invention that the polynucleotide sequence be the sequence of SEQ ID NO:7 or SEQ ID NO:8. It is another object of this invention to have an antibody which binds specifically to one of the isoenzymes of ALT and not the other isoenzyme.
  • an antibody of one embodiment of this invention is specific for murine ALT2 and does not bind to murine ALTl.
  • an antibody of the present invention is specific for rat ALT2 polypeptide and not rat ALTl polypeptide.
  • the antibody of the present invention binds to the murine ALT2 sequence of SEQ ID NO:2 or an ALT2-specific fragment thereof or a homolog of SEQ ID NO:2, or, alternatively, to the protein encoded by the DNA sequence of SEQ ID NO:4 or a murine ALT2-specific fragment thereof.
  • an antibody of the present invention specifically binds a rat ALT2 amino acid sequence of SEQ ID NO: 5 or a fragment or homolog thereof.
  • the antibody of the present invention specifically binds a rat ALTl amino acid sequence of SEQ ID NO: 6 or a fragment or homolog thereof. It is an object of this invention to have an expression vector for each of the ALT isoenzymes.
  • the expression vector can be a plasmid, cosmid, or other type of vector where the DNA sequence encoding for the ALT is operatively linked to expression sequences, such as a promoter.
  • the DNA sequence for murine ALT can be the sequence of SEQ ID NO:3 and or SEQ ID NO:4, or can be a sequence which encodes for the amino acid sequence of SEQ ID NO:l and/or SEQ ID NO:2 or a homolog of SEQ ID NO:l and/or SEQ ID NO:2.
  • the DNA sequence for rat ALT can be the sequence of SEQ ID NO:7 (rALTl) and/or SEQ ID NO:8 (rALT2), or can be a sequence which encodes for the amino acid sequence of SEQ ID NO:5 and/or SEQ ID NO:6 or a homolog of SEQ ID NO:5 and/or SEQ ID NO:6.
  • the bodily fluids can be blood, serum, lymph, urine, sweat, mucus, sputum, saliva, semen, spinal fluid, interstitial fluid, synovial fluid, cerebrospinal fluid, gingival fluid, vaginal fluid, and pleural fluid.
  • the tissue can be liver, brain, muscle, adipose tissue, and kidney. It is another object of this invention to have a method for diagnosing or detecting injury or disease involving tissue which contains ALT2.
  • the method involves using antibodies (polyclonal or monoclonal) that specifically bind to a ALT2 polypeptide of the present invention to measure the level of ALT2 in bodily fluids from the animal. It is another object of this invention to use antibodies (polyclonal or monoclonal) that specifically bind to ALTl to measure the level of ALTl in bodily fluids from the animal and then to compare the level of ALT2 to ALTl. When the level of ALT2 is sufficiently higher than the level of ALTl or the level of ALT2 falls within a pre-determined range, then the animal is diagnosed with a specific disease or injury.
  • the bodily fluids can be blood, serum, lymph, urine, sweat, mucus, sputum, saliva, semen, spinal fluid, interstitial fluid, synovial fluid, cerebrospinal fluid, gingival fluid, vaginal fluid, and pleural fluid.
  • the tissue can be liver, brain, muscle, adipose tissue (white adipose tissue "WAT” or brown adipose tissue “BAT”), and kidney. It is an object of this invention to have a kit useful in diagnosing damage or disease in tissue containing ALT.
  • This kit has a measurer of ALT, either ALTl or ALT2, levels in a sample of bodily fluids and an indicator for determining if amount of ALT measured by the ALT measurer falls in a range associated with damage or a specific disease in the ALT containing tissue. It is further object of this invention that the kit may also contain a measurer for both ALTl and ALT2 levels in a sample of bodily fluids and an indicator for determining if amount of each of ALTl and ALT2 measured by the measurer(s) falls in a range associated with damage or a specific disease in the ALT containing tissue.
  • the ALTl measurer and the ALT2 measurer can be a biologic assay, an antibody-based assay, an enzyme linked immunosorbent assay, a Western blot, a rapid immunoassay, a radioimmunoassay, and combinations thereof. It is another object of this invention to have a diagnostic kit useful for diagnosing damage or disease to ALTl containing tissue and/or ALT2 containing tissue.
  • This diagnostic kit can contain ALTl specific antibodies (polyclonal or monoclonal), immunoassay reagents, and a positive and negative control. This kit can also have ALT2 specific antibodies (polyclonal or monoclonal).
  • This kit includes a means for determining if a measurement of ALTl and/or ALT2 indicates a diagnosis of damage or disease in ALTl containing tissue and/or ALT2 containing tissue.
  • the kit can also have instructions indicating when a level of ALTl and or ALT2 is indicative for diagnosis of damage or disease in tissue containing ALTl or ALT2.
  • This kit can have a measurer of ALT2 levels in a bodily fluids sample and an indicator for determining if the ALT2 level measured falls in a range associated with a specific condition.
  • the kit can determine when there are altered levels of ALTl in bodily fluids (altered can be higher than normal or lower than normal).
  • This kit can also have a measurer of ALTl levels in a bodily fluids sample and another indicator for determining if the ALTl level measured falls in a range associated with a specific condition.
  • this kit can have a third indicator for comparing the values of ALTl and ALT2 and determining if the levels of ALTl and ALT2 fall in a range associated with a specific condition.
  • the measurer of this kit can be selected from one or more of the following: a biologic assay, an antibody-based assay, an enzyme linked immunosorbent assay, a Western blot, a rapid immunoassay, a radioimmunoassay, and combinations thereof. It is an object of this invention to have a method for producing a ALT polypeptide of the present invention.
  • the ALT produced can be the same as the amino acid sequences of SEQ ID NO:l or SEQ ID NO:2 or SEQ ID NO:5 or SEQ ID NO:6, or a homolog, fragment, or variant thereof.
  • This method involves cloning the DNA encoding for ALT in an expression vector, introducing the expression vector into a host cell to produce a recombinant host cell, and subjecting to the recombinant host cell to conditions such that ALT is expressed. It is a further object of this invention that the ALT expressed can be isolated and purified.
  • the DNA sequence placed in the plasmid can be the nucleic acid sequence of SEQ ID NO:3 or SEQ ID NO:4 or SEQ ID NO:7 or SEQ ID NO:8, or a nucleic acid sequence which encodes for a variant, homolog, or fragment of murine ALTl or murine ALT2.
  • the DNA sequence inserted into the plasmid may be a nucleic acid sequence which hybridizes, such as, for example, under conditions of high stringency, to a nucleic acid encoding a polypeptide of SEQ ID NO:l or SEQ ID NO:2 or SEQ ID NO:5 or SEQ ID NO:6.
  • the high stringency conditions include 0.1 X SSC with 0.1% SDS wash buffer at hybridization temperature, such as, for example, at about 60, 61, 62, 63, 64, 65, 66, 67, or 68 degrees C.
  • This method involves contacting a sample of bodily fluids with at least one antibody which specifically binds to an ALT2 of the present invention, detecting the ALT2 antibody which is bound to ALT2, and comparing the amount of detected ALT2 antibody to a known quantity for an animal without the condition.
  • the quantity of detected ALT2 antibody differs sufficiently from the known quantity from an animal without the condition, then it indicates that the animal has the condition.
  • the method also can involve contacting the sample of bodily fluids with at least one antibody which specifically binds to ALTl, detecting the ALTl antibody which is bound to ALTl, and comparing said amount of detected ALT 1 antibody to a known quantity for an animal without the condition.
  • this method when the quantity of detected ALTl antibody differs sufficiently from the known quantity from an animal without the condition, then it indicates that the animal has the condition.
  • this method can also involve comparing the amount of ALT2 antibody detected to the total amount of antibody detected and/or to the amount of ALTl antibody detect; and/or the amount of ALTl antibody detected to the total amount of antibody detected and/or to the amount of ALT2 antibody detected.
  • the condition is indicated if the amount of ALT2 antibody detected when compared to the amount of ALT 1 antibody detected or the total amount of antibody detected falls within a certain range.
  • the condition is indicated if the amount of ALTl antibody detected when compared to the amount of ALT2 antibody detected or the total amount of antibody detected falls within a certain range.
  • the bodily fluids for this method can be selected from the following group: blood, serum, lymph, urine, sweat, mucus, sputum, saliva, semen, spinal fluid, interstitial fluid, synovial fluid, cerebrospinal fluid, gingival fluid, vaginal fluid, and pleural fluid.
  • One embodiment of the invention is an isolated and purified murine ALT polypeptide comprising the amino acid sequence selected from a group including SEQ ID NO:l and SEQ ID NO: 2.
  • Another embodiment of the invention is an isolated and purified polynucleotide encoding for the murine ALT polypeptide.
  • the isolated and purified polynucleotide of one embodiment comprises a polynucleotide sequence selected from a group including SEQ ED NO:3 and SEQ ID NO:4.
  • the invention also includes an isolated and purified antibody which binds specifically to the murine ALT polypeptides of the present invention.
  • the invention further includes an expression vector for murine ALT comprising the murine ALT polynucleotide sequence operatively linked to an expression sequence.
  • Another embodiment of the invention is an isolated and purified rat ALT polypeptide comprising the amino acid sequence selected from a group including SEQ ID NO:5 and SEQ ID NO: 6.
  • Another embodiment of the invention is an isolated and purified polynucleotide encoding for the rat ALT polypeptide.
  • the isolated and purified polynucleotide of one embodiment comprises a polynucleotide sequence of SEQ ID NO:7 or SEQ ID NO: 8.
  • the invention also includes an isolated and purified antibody which binds specifically to the rat ALT polypeptides of the present invention.
  • the invention further includes an expression vector for rat ALT comprising the rat ALT polynucleotide sequence operatively linked to an expression sequence.
  • Another embodiment of the invention is a method of detecting in a sample the presence of mRNA, wherein the mRNA encodes for an ALT polypeptide of the present invention.
  • the method comprises contacting the sample with a polynucleotide probe, wherein the polynucleotide probe is sufficient to specifically detect under stringent hybridization conditions the presence of the mRNA, and detecting the formation of a hybrid of the polynucleotide probe and the mRNA.
  • Another embodiment of the invention is a method of detecting an ALT polypeptide of the present invention in a sample, wherein the sample comprises a bodily fluid, the method comprising contacting a sample of the bodily fluids with at least one antibody that specifically binds to the ALT polypeptide and detecting the antibody which is bound to the ALT polypeptide in the sample.
  • Yet another embodiment of the invention is a method of diagnosing or detecting injury or disease involving tissue which contains an ALT polypeptide of the present invention in an animal suspected of having the injury or disease.
  • the method comprises: contacting a sample of bodily fluids from the animal with at least one first antibody, wherein the first antibody specifically binds to an ALTl polypeptide; detecting the first antibody which is bound to the ALTl polypeptide in the sample; contacting the sample of bodily fluids with at least one second antibody wherein the second antibody specifically binds to an ALT2 polypeptide; detecting the second antibody which is bound to the ALT2 polypeptide in the sample; and comparing the amount of the ALTl polypeptide bound to the first antibody and the amount of the ALT2 polypeptide bound to the second antibody; wherein when the amount of the bound ALT2 polypeptide is sufficiently higher than the amount of the bound ALTl polypeptide, it indicates that the animal has a disease or injury affecting tissue containing ALT2.
  • the sample of bodily fluids can comprise a fluid selected from a group comprising blood, serum, lymph, urine, sweat, mucus, sputum, saliva, semen, spinal fluid, interstitial fluid, synovial fluid, cerebrospinal fluid, gingival fluid, vaginal fluid, and pleural fluid.
  • the tissue can be selected from a group comprising liver, brain, muscle, adipose tissue, and kidney. Another embodiment of the invention is a method of diagnosing or detecting injury or disease involving tissue which contains an ALT polypeptide in an animal suspected of having the injury or disease.
  • the method comprises: contacting a sample of bodily fluids from the animal suspected of having the injury or disease with at least one first antibody wherein the first antibody specifically binds to an ALT polypeptide; detecting the first antibody which is bound to the ALT polypeptide in the sample; and comparing the amount of the ALT polypeptide in the sample of bodily fluids to an amount of ALT in the bodily fluids of an animal known not to have injury or disease involving tissue which contains ALT polypeptide; wherein when the amount of ALT in the bodily fluids of the sample is higher than the amount of ALT polypeptide in the bodily fluids of the animal known not to have injury or disease it indicates that the animal suspected has a disease or injury affecting tissue containing ALT.
  • the sample of bodily fluids can comprise a fluid selected from a group including blood, serum, lymph, urine, sweat, mucus, sputum, saliva, semen, spinal fluid, interstitial fluid, synovial fluid, cerebrospinal fluid, gingival fluid, vaginal fluid, and pleural fluid.
  • the tissue can be selected from a group comprising liver, brain, muscle, adipose tissue, and kidney.
  • a further embodiment of the invention is a diagnostic kit for use in diagnosing damage or disease in tissue containing an ALT polypeptide of the present invention.
  • the kit comprises a measurer for determining a measurement of an ALT polypeptide in a sample of bodily fluids and an indicator for determimng if the measurement falls in a range associated with damage or disease in the tissue containing the ALT of the present invention.
  • the ALT polypeptide is murine ALT2 of SEQ ID NO:2 or an amino acid sequence having about 95% homology thereto.
  • the homolog is an amino acid sequence of SEQ ID NO:5.
  • the ALT polypeptide is rat ALT2 of SEQ ID NO: 5 or an amino acid sequence having about 95% homology thereto.
  • the measurer can be selected from a group comprising a biologic assay, an antibody-based assay, an enzyme linked immunosorbent assay, a Western blot, a rapid immunoassay, a radioimmunoassay, and combinations thereof.
  • Another embodiment of the invention is a diagnostic kit for use in diagnosing damage or disease in tissue containing an ALT polypeptide of the present invention.
  • the kit comprises: an aliquot of antibodies that bind specifically to an ALT polypeptide of the present invention; immunoassay reagents; and a control for determining if a measurement of an ALT polypeptide of the present invention indicates a diagnosis of damage or disease in tissue containing an ALT polypeptide of the present invention.
  • the control comprises instructions indicating that an increase or decrease in the amount of the ALT polypeptide indicates a diagnosis for damage or disease in tissue containing the ALT polypeptide.
  • the kit further comprises an aliquot of antibodies that bind specifically to ALT2 polypeptides of the present invention and a control for determining if a measurement of said ALT2 polypeptide indicates a diagnosis of damage or disease in tissue containing said ALT2 polypeptide.
  • the control comprises instructions indicating that an increase or decrease in the amount of ALT2 indicates a diagnosis for damage or disease in tissue containing the ALT polypeptide.
  • Another embodiment of the invention is a diagnostic kit for use in a condition associated with altered levels of an ALT polypeptide 1 in bodily fluids.
  • the kit comprises a measurer for determining a measurement of an ALT 1 polypeptide in a sample of bodily fluids and an indicator for determining if the measurement falls in a range associated with the condition.
  • Another embodiment of the invention is a diagnostic kit for use in a condition associated with altered levels of an ALT 2 in bodily fluids.
  • the kit comprises a measurer for determining a measurement of an ALT 2 polypeptide in a sample of bodily fluids and an indicator for determining if the measurement falls in a range associated with the condition.
  • Another embodiment of the invention is a diagnostic kit for use in a condition associated with altered levels of at least one of an ALT 1 and an ALT2 in bodily fluids.
  • the kit comprises: a measurer for determining a first measurement of an ALT 1 polypeptide in a sample of bodily fluids; a measurer for determining a second measurement of ALT2 polypeptide in the sample of bodily fluids; and an indicator for determining if the first and second measurements fall in a range associated with the condition.
  • Still another embodiment of the invention is method of diagnosing a condition associated by altered levels of an ALT 1 in bodily fluids in an animal suspected of having the condition.
  • the method comprises: contacting a sample of bodily fluids from the animal with at least one antibody wherein the antibody specifically binds to an ALT 1 polypeptide; detecting the antibody which is bound to the ALT 1 polypeptide in the sample; and comparing the amount of the detected antibody to a known quantity for an animal without the condition; wherein if the quantity of the detected antibody differs sufficiently from the known quantity from an animal without the condition, indicates that the animal has the condition.
  • Another embodiment of the invention is a method of diagnosing a condition associated by altered levels of an ALT 2 in bodily fluids in an animal suspected of having the condition.
  • the method comprises: contacting a sample of bodily fluids from the animal with at least one antibody wherein the antibody specifically binds to an ALT 2 polypeptide; detecting the antibody which is bound to the ALT 2 polypeptide in the sample; and comparing the amount of the detected antibody to a known quantity for an animal without the condition; wherein if the quantity of the detected antibody differs sufficiently from the known quantity from an animal without the condition, indicates that the animal has the condition.
  • the sample of bodily fluids for the above methods and/or kits can comprise a fluid selected from a group comprising of blood, serum, lymph, urine, sweat, mucus, sputum, saliva, semen, spinal fluid, interstitial fluid, synovial fluid, cerebrospinal fluid, gingival fluid, vaginal fluid, and pleural fluid.
  • the measurer for above methods and/or kits can be selected from the group comprising a biologic assay, an antibody-based assay, an enzyme linked immunosorbent assay, a Western blot, a rapid immunoassay, a radioimmunoassay, and combinations thereof.
  • Another embodiment of the invention is a method of producing an ALT polypeptide.
  • the method comprises: providing an ALT polynucleotide sequence in an expression vector; introducing the expression vector into a host cell such that a recombinant host cell is produced; and subjecting to the recombinant host cell to conditions such that the ALT polypeptide is expressed.
  • the ALT polynucleotide sequence is selected from a group including SEQ ID NO:3 and SEQ ID NO:4.
  • the ALT polynucleotide sequence is selected from a group including SEQ ID NO:7 and SEQ ID NO:8.
  • FIG. 1A-B are a comparison of the sequence of murine ALT isoenzymes (mALTl and mALT2) of this invention and the polypeptide sequence of human ALT isoenzymes.
  • FIG. 1A is a comparison between murine ALTl and human ALTl.
  • FIG. IB is a comparison between murine ALT2 and human ALT2.
  • Peptide sequences are aligned using BESFIT from the GCG package and amino acids are numbered to the right of the sequence. Identical amino acids are denoted by a vertical bar, strongly similar amino acids by a colon (:), and weakly similar amino acids by a period (.).
  • FIG. 2 is Northern blots showing the expression of murine ALT mRNA.
  • FIG. 3A is an electrophoresis gel where thirty microliters of E. coli extracts containing 100 to 150 ⁇ g of protein were analyzed on 4%-20% SDS-PAGE and stained with Coomassie Blue. Arrows indicate IPTG induced protein bands corresponding to mALTl and mALT2.
  • FIG. 3A is an electrophoresis gel where thirty microliters of E. coli extracts containing 100 to 150 ⁇ g of protein were analyzed on 4%-20% SDS-PAGE and stained with Coomassie Blue. Arrows indicate IPTG induced protein bands corresponding to mALTl and mALT2.
  • FIG. 3B is a graph of ALT activity of soluble cell extracts of E. coli harboring plasmid pET28-mALTl (ALTl), ⁇ ET28-mALT2 (ALT2), or empty vector pET28 (control) after IPTG induction.
  • FIGS. 4A and 4B are Northern blots of total RNA extracted from fatty liver of obese (ob/ob) and lean (+/?) mice blotted with 32 P-labeled mALTl or mALT2 DNA probe.
  • FIG. 4C is a graph of murine ALT expression.
  • FIG. 5 is a graph of ALT and AST activities in obese (ob/ob) and lean (+/?) mice measured for their ALT and AST activities.
  • FIG. 6 is a sequence alignment of ALTl and ALT2 polypeptides from human, mouse and rat species. Highly conserved amino acids (> 90%) are in capital letters and less conserved ( ⁇ 90% and > 50%) are in small letters. Symbol "! is for any amino acids of I or V, "$" for L or M, "%” for F or Y, and "#” is for N, D, Q, E, B, or Z. DEFINITIONS Within the context of this specification, each term or phrase below will include the following meaning or meanings.
  • murine ALT polypeptide “murine ALT protein,” “murine ALT,” and “mALT” are interchangeable and generally refer to or include any and/or all murine ALT polypeptides or isoenzymes, including murine ALTl, murine ALT2, and any variant, homolog, or fragment of murine ALTl or murine ALT2.
  • rat ALT polypeptide “rat ALT protein,” “rat ALT,” and “rALT” are interchangeable and generally refer to or include any and/or all rat (rattus) ALT polypeptides or isoenzymes, including rat ALTl, rat ALT2, and any variant, homolog, or fragment of rat ALTl or rat ALT2.
  • protein and “polypeptide” are used interchangeably in both singular and plural forms, as are the terms “nucleic acid” and “polynucleotide.” These and additional terms may be defined with additional language in the remaining portions of the specification.
  • DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS The present invention covers the nucleotide and amino acid sequences of murine
  • ALT antibodies specific to murine ALT
  • antibodies specific to murine ALT and the use of these polypeptides, polynucleotides, and antibodies to diagnose various diseases and conditions in tissue that produce murine ALT, such as fatty liver, and to differentially diagnose liver injury caused by fatty liver (liver steatosis) and by alcohol, trauma, infection, toxicity, and other causes of liver damage.
  • This invention also includes homologs and functional fragments of murine ALT polypeptides as well as expression vectors containing murine ALT polynucleotide sequences and recombinant host cells which contain an expression vector containing murine ALT polynucleotide sequences.
  • This invention also includes homologs and functional fragments of rat ALT polypeptides as well as expression vectors containing rat ALT polynucleotide sequences and recombinant host cells which contain an expression vector containing rat ALT polynucleotide sequences.
  • homology is often measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705 or the NCBI BLAST program).
  • sequence analysis software e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705 or the NCBI BLAST program.
  • sequence analysis software e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705 or the NCBI BLAST program.
  • the term "functional fragments" include those fragments of SEQ ID NO:l and/or SEQ ID NO:2 and/or SEQ ID NO:5 and/or SEQ ID NO:6 and/or a polypeptide having about 95% sequence identity to that of the SEQ ID NO:l and/or SEQ ID NO:2 and/or SEQ ID NO: 5 and/or SEQ ID NO: 6 and that retains the function, activity, or immunobiological properties of said ALT polypeptide.
  • One of skill in the art can screen for the functionality of a fragment by using the examples provided herein, where full length ALTl and ALT2 are described.
  • substantially identical is also meant an amino acid sequence which differs only by conservative amino acid substitutions, for example, substitution of one amino acid for another of the same class (e.g., valine for glycine, arginine for lysine,etc.) or by one or more non-conservative substitutions, deletions, or insertions located at positions of the amino acid sequence which do not destroy the function of the protein assayed (e.g., as described herein).
  • such a sequence is at least 85%, and more preferably from 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, to 100% homologous at the amino acid level to SEQ ID NO: 1 or SEQ ID NO:2 or SEQ ID NO:5 or SEQ ID NO:6.
  • Functional Equivalence Modification and changes may be made in the structure of the peptides of the present invention and DNA segments which encode them and still obtain a functional molecule that encodes a protein or peptide with desirable characteristics. The following is a discussion based upon changing the amino acids of a protein to create an equivalent, or even an improved, second-generation molecule.
  • the amino acid changes may be achieved by changing the codons of the RNA sequence, according to the following codon table: TABLE 1 Amino Acids Codons AAllaanniinnee Ala A GCA GCC GCG GCU Cysteine Cys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAG Phenylalanine Phe F UUC UUU GGllvycciinnee Gly G GGA GGC GGG GGU Histidine His H CAC CAU Isoleucine He I AUA AUC AUU Lysine Lys K AAA AAG Leucine Leu L UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCU Glutamine Gin Q CAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGU SSeeririnnee Ser S AGC AGU UCA UCC UCG UCU Thre
  • hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.
  • Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics (Kyte and Doolittle, 1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (4.5).
  • hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0.+-.1); glutamate (+3.0.+-.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5.+-.1); alanine (-0.5); histidine (-0.5); cysteine (- 1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).
  • amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein.
  • substitution of amino acids whose hydrophilicity values are within .+-.2 is preferred, those which are within .+-.1 are particularly preferred, and those within .+-.0.5 are even more particularly preferred.
  • amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • amino acid sequences may include additional residues, such as additional N- or C-terminal amino acids and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned.
  • terminal sequences particularly applies to sequences, which may, for example, include various unnaturally occurring amino acid sequences flanking either of the N- or C- termini to allow for facile covalent linkage to another molecule, i.e., a reporter molecule.
  • the homolog may include a substitution in SEQ ID NO:l of C56R, F109L, D126N, R133K, I152V, P158L, Q165R, A200S, R204H, A205T, A222T, D227G, R249H, C253G, R258H, V263A, E291K, G293R, M297L, A328S(*), V339T, E352A, M387L, T393A, S395T, K400A, R406K, E408A, R421S, Q434R, L439P, K440R, Q443E, D447E(*), C459R, Q477R, M491L, V496L(*), R502S(*), H503R, H510L or a combination thereof.
  • the homolog may include a substitution in SEQ ID NO:6 of C56R, F109L, D126N, R133K, I152V, P158L, Q165R, S200A, R204H, A205T, A222T, D227G, R249H, C253G, R258H, V263A, K291E, G293R, M297L, S328A, V339T, E352A, M387L, T393A, S395T, K400A, R406K, E408A, R421S, Q434R, L439P, K440R, Q443E, E447D, C459R, Q477R, M491L, L496V, S502R, H503R, H510L or a combination thereof.
  • the homolog may include a substitution in SEQ ID NO:5 of H24Q, D45E, M77L, H99Q, N123D, L196I, I228V, D245N, L251V, R252Q, Q253E, Q321E, P326H, V392E, S407F, Q430H, L445F, S456A, K458Q, E501D, H508Q, L515I, K516N, K520Q, S522A or a combination thereof.
  • the homolog may include a substitution in SEQ ID NO:2 of H24Q, D45E, M77L, H99Q, N123D, L196L V228I, D245N, L251V, R252Q, Q253E, Q321E, H326P, E392V, F407S, Q430H, L445F, S456A, K458Q, D501E, H508Q, L515I, K516N, Q520K, S522A or a combination thereof.
  • a substantially pure polypeptide is meant a ALT polypeptide that has been separated from components that naturally accompany it.
  • the polypeptide is substantially pure when it is at least 60%, by weight, free from the proteins and other naturally occurring molecules with which it is typically associated.
  • the preparation is at least 75%, 80%, 90%, 95%, and most preferably at least 99%, by weight, an ALT polypeptide.
  • a substantially pure ALT polypeptide can be obtained, for example, by extraction from a natural source; by expression of a recombinant nucleic acid encoding the desired ALT polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.
  • a protein is substantially free of naturally associated components when it is separated from those contaminants that accompany it in its natural state.
  • a protein that is chemically synthesized or produced in a cellular system different from the cell from which it naturally originates is substantially free from its naturally associated components.
  • substantially pure polypeptides include those derived from eukaryotic organisms but synthesized in E. coli or other prokaryotes.
  • the polynucleotide molecules of the present disclosure can be expressed in a variety of prokaryotic and eukaryotic cells using regulatory sequences, vectors, and methods well established in the literature.
  • An ALT polypeptide produced according to the present description can be purified using a number of established methods such as affinity chromatography using anti-mALT antibodies coupled to a solid support. Fusion proteins of an antigenic tag and an ALT polypeptide can be purified using antibodies to the tag. Optionally, additional purification is achieved using conventional purification means such as liquid chromatography, gradient centrifugation, and gel electrophoresis, among others. Methods of protein purification are known in the art and can be applied to the purification of recombinant ALT polypeptide described herein. Construction of ALT encoded fusion proteins is also contemplated.
  • Fusion proteins typically contain additions, substitutions, or replacements of one or more contiguous amino acids of the native ALT polypeptide with amino acid(s) from a suitable fusion protein partner. Such fusion proteins are obtained using recombinant DNA techniques generally well known by one of skill in the art. Briefly, DNA molecules encoding the hybrid ALT protein of interest are prepared using generally available methods such as PCR mutagenesis, site directed mutagenesis, and/or restriction digestion and ligation. The hybrid DNA is then inserted into expression vectors and introduced into suitable host cells. Recombinant gene expression vectors comprising a nucleic acid encoding an ALT protein of interest, or portions thereof, can be constructed in a variety of forms well-known in the art.
  • Preferred expression vectors include plasmids and cosmids.
  • Expression vectors include one or more fragments of murine ALT.
  • an expression vector comprises a nucleic acid encoding an ALT polynucleotide sequence of SEQ ID NO:l or SEQ ID NO:2 or SEQ ID NO:5 or SEQ ID NO:6.
  • the phrase "operatively encode” refers to one or more protein coding regions associated with those regulatory sequences required for expression of the polypeptide encoded by the coding region. Examples of such regulatory regions including promoter binding sites, enhancer elements, ribosome binding sites, and the like.
  • regulatory sequences for use in various eukaryotic and prokaryotic systems are described in Ausubel, et al., Short Protocols in Molecular Biology, 3rd ed., John Wiley & Sons, fric, New York, 1997, which is hereby incorporated by reference in its entirety.
  • Expression vectors for use with ALT typically contain regulatory sequences derived from a compatible species for expression in the desired host cell. For example, when E.
  • coli is the host cell, the host cell population can be typically transformed using pBR322, a plasmid derived from an E. coli species. (Bolivar, et al., Gene, 2: 95,1977). pBR322 contains genes for ampicillin (AMPR) and tetracycline resistance and thus provides easy means for identifying transformed cells.
  • AMPR ampicillin
  • Promoters suitable for use with prokaryotic hosts illustratively include the betalactamase and lactose promoter systems (Chang, et al., Nature, 275: 615,1978; and Goeddel, et al., Nature, 281: 544,1979), alkaline phosphatase, the tryptophan (trp) promoter system (Goeddel, Nucleic Acids Res., 8: 4057,1980) and hybrid promoters such as the taq promoter (de Boer, et al., Proc. Natl. Acad. Sci. USA, 80: 21-25,1983). Other functional bacterial promoters are also suitable.
  • nucleotide sequences are generally known in the art, thereby enabling a skilled worker to ligate them to a polynucleotide which encodes the peptide of interest (Siebenlist, et al., Cell, 20: 269,1980) using linkers or adapters to supply any required restriction sites.
  • eukaryotic microbes such as yeast cultures can also be used as source for the regulatory sequences. Saccharomyces cerevisiae is a commonly used eukaryotic host microorganism. Suitable promoting sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase (Hitzeman, et al., J. Biol.
  • glycolytic enzymes Hess, et al. J. Adv. Enzyme Reg. 7: 149,1968; and Holland, Biochemistry, 17: 4900,1978, such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose- 6phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.
  • enolase glyceraldehyde-3-phosphate dehydrogenase
  • hexokinase hexokinase
  • pyruvate decarboxylase phosphofructokinase
  • glucose- 6phosphate isomerase 3-phosphoglycerate mutase
  • yeast promoters which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degraded enzymes associated with nitrogen metabolism, metallothionine, glyceraldehyde-3 -phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization.
  • Yeast enhancers also are advantageously used with yeast promoters.
  • a recombinant virus is used as the expression vector.
  • Exemplary viruses include the adeno viruses, adeno-associated viruses, herpes viruses, vaccinia, or an RNA virus such as a retrovirus or an alphavirus.
  • the retroviral vector is a derivative of a murine or avian retrovirus.
  • the alphavirus vector is derived from Sindbis or Semliki Forest Virus. All of these vectors can transfer or incorporate a gene for a selectable marker so that transduced cells can be identified and generated.
  • the vector is now target specific.
  • Retroviral vectors can be made target specific by inserting, for example, a polynucleotide encoding a sugar, a glycolipid, or a protein.
  • Preferred targeting is accomplished by using an antibody to target the retroviral vector, such as to the vicinity of a mucosal inductor site, using, for example, a mALT-specific antibody.
  • an antibody to target the retroviral vector such as to the vicinity of a mucosal inductor site
  • a mALT-specific antibody for example, a mALT-specific antibody.
  • Those of skill in the art know of, or can readily ascertain without undue experimentation, specific polynucleotide sequences which can be inserted into the retroviral genome to allow target specific delivery of the retroviral vector containing the polynucleotides of interest.
  • Construction of suitable vectors containing desired coding, non-coding and control sequences employ standard ligation techniques. Isolated plasmids or DNA fragments are cleaved, tailored, and re-ligated in the form desired to construct the plasmids required.
  • the ligation mixtures can be used to transform a host cell and successful transformants selected by antibiotic resistance where appropriate. Plasmids from the transformants are prepared, analyzed by restriction and/or sequenced by, for example, the method of Messing, et al., (Nucleic Acids Res., 9: 309,1981), the method of Maxam, et al., (Methods in Enzymology, 65: 499,1980), or other suitable methods which are known to those skilled in the art. Size separation of cleaved fragments is performed using conventional gel electrophoresis as described, for example, by Maniatis, et al., (Molecular Cloning, pp.
  • Host cells can be transformed with the expression vectors described herein and cultured in conventional nutrient media modified as is appropriate for inducing promoters, selecting transformants or amplifying genes.
  • the culture conditions such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan following the teachings herein provided.
  • peptide sequences of human ALTl and murine ALT2 are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan following the teachings herein provided.
  • ALT2 were used as probes to search the mouse murine expressed sequence tag (EST) database using tBLASTn.
  • EST mouse murine expressed sequence tag
  • IMAGE clones 4195300 and 5065322 were fully sequenced and revealed the highest homology to human ALTl and ALT2, respectively, in the entire protein-coding region.
  • the DNA nucleotide sequences of these two clones were confirmed by sequencing analysis and are predicted to encode proteins of 496 (clone 4195300) and 522 (clone 5065322) amino acids. As shown in FIG.
  • IMAGE clone 4195300 shares about 87% identity and about 89% similarity to human ALTl, but about 70% identity and about 72% similarity to human ALT2, whereas clone 5065322 shares about 93% identity and about 95% similarity with human ALT2, but about 69% identity to human ALTl .
  • the cDNA clone 4195300 and the clone 5065322 were thus determined to be murine ALTl and murine ALT2, respectively.
  • rat ALTl and ALT2 Sixty-seven percent of amino acids are identical in murine ALTl and murine ALT2; a similar degree of conservation, about 68%, is found between human ALTl and ALT2. Cloning of rat ALTl and ALT2 was achieved through bioinformatics by interrogating the closest homolog in GenBank with murine and human ALTl and ALT2 protein sequences.
  • the rat IMAGE clone 7113147 encodes a protein that shares 95% and 98% identity to human and murine ALT2, respectively, but 68% and 67% identity to human and murine ALTl, respectively. Thus, the cDNA clone 7113147 was determined as rat ALT2.
  • Rat ALTl cDNA was cloned from rat liver first-strand cDNA by PCR amplification with high-fidelity DNA polymerase using primers based rat expressed sequence tags (ESTs) which shares highest identity to murine and human ALTl sequences.
  • the resultant PCR fragment was cloned into TOPO cloning vector (frivitrogen) and sequenced in full.
  • the translated protein sequence shares high identities of 88% and 97% to human and murine ALTl, respectively, but low identities of 70% and 68% to human and murine ALT2, respectively. Thus, this clone was determined as rat ALTl .
  • the gene expression of murine ALTl and murine ALT2 was examined in mouse tissues by Northern analysis.
  • mice Male obese mice (ob/ob), littermate control (+/?), and C57BL/6J, 6 to 8 weeks old, were obtained from Jackson Laboratory and euthanized with CO 2 according to protocol approved by Institutional Animal Care and Use Committee. Tissues were immediately frozen in liquid nitrogen until use for RNA extraction or enzyme activity assay. Total RNA was prepared with Trizol, available from Life Technologies Inc., Gaithersburg, Maryland, from the snap-frozen tissues. For the tissue distribution study, pooled 15 ⁇ g of total RNA from 3 to 4 mice were electrophoresed on a 1.2% agarose gel and blotted to a Nitro-plus membrane, available from Schleicher & Schuell, Dassel, Germany.
  • the DNA probes of murine ALTl (1.4 kb) and murine ALT2 (2.4 kb) were derived from restriction enzyme digestion of IMAGE clone 4195300 (Sal I/Not I) and clone 5065322 (Sal ITNot I), respectively. Probes were random-labeled with 32 P-dCTP, hybridization was carried out at 65°C in Rapid-hyb buffer, available from Amersham Biosciences, Piscataway, New Jersey, and blots were washed twice with 0.5 x SSC/1% SDS at 65°C (stringent wash) and visualized by Phosphorhnager, available from Amersham Biosciences, or x-ray film. As shown in FIG.
  • the 3.3 kb murine ALT2 messenger RNA (mRNA) was expressed at high levels in muscle, liver, and white adipose tissue (WAT), at moderate levels in brain and kidney, and at a low level in heart.
  • WAT white adipose tissue
  • the 1.8 kb murine ALTl mRNA was highly expressed in liver and considerably in WAT, intestine, and colon tissue.
  • particular tissues selectively express one ALT isoenzyme over the other. For instance, murine ALT2 was significantly expressed, and murine ALTl barely expressed, in muscle and brain tissue. In contrast, bowel tissue generally expressed only murine ALTl, and not murine ALT2.
  • the coding region of mALTl cDNA was amplified by polymerase chain reaction (PCR) at 28 cycles at 94°C for 30 seconds, 56°C for 30 seconds, and 72°C for 1.5 minutes, with a final extension of 7 minutes at 72°C using the Turbo Pfu PCR system (Stratagene) with an Ndel-linked primer, pi 408 5'- GGAAGATCTCATATGGCCTCACAAAGGAATGAC-3' (nt ⁇ 106-126, BC022625; SEQ ID NO:9), and a NotI;-linked primer, pl409 5'-
  • AATGCGGCCGCTCAGGAGTACTCATGAGTGAA-3' (1596-1576, BC022625; SEQ ID NO: 10), using IMAGE clone 4195300 as a template.
  • the resulting PCR product was digested with Ndel/Notl and subcloned into pET28a, available from Novagen, Madison, Wisconsin, creating plasmid pET28-mALTl. The absence of mutations in the inserted murine ALTl cDNA was verified by DNA sequence analysis.
  • plasmid pET28-mALT2 AATGCGGCCGCTCATGAGTACTGCTCCAGGAA-3' (nt 1696-1676, BC034219; SEQ ID NO:12), creating plasmid pET28-mALT2.
  • plasmid ⁇ ET28-mALTl, ⁇ ET28-mALT2, or empty vector pET28 were used to transform competent E. coli. (Tuner DE3, available from Novagen).
  • a fresh colony of the transformants was grown in 50 ml LB media containing 30 ⁇ g/ml kanamycin to an OD 600 of 0.7, at which time isopropyl-beta-D- thiogalactopyranoside (IPTG) was added (1 mmol, final concentration) to induce expression of the recombinant proteins.
  • IPTG isopropyl-beta-D- thiogalactopyranoside
  • ALT activity of the bacterially expressed recombinant murine ALT proteins was confirmed using a GPT Optimized Alanine Aminotransferase kit, available from Sigma Diagnostics (catalog no. DG159-K), St. Louis, Missouri, according to manufacturer instructions. Briefly, 0.5 ml of cell lysate was incubated with a 2.5 ml mixture of reagent A and B containing L-alanine, nicotinamide adenine dinucleotide, Lactate Dehydrogenase, and 2-oxoglutarate at 25 °C. Absorbance at 340 mn was recorded at 1, 2, and 3 minutes after incubation. The slope of absorbance decrease is proportional to ALT activity.
  • Protein concentration of cell lysates were determined by Coomassie Brilliant Blue G250 (BioRad) using bovine serum albumin as a standard. Final ALT activities were corrected by protein concentration of cell lysates.
  • One unit of ALT activity was defined as the amount of enzyme that catalyzes the formation of 1 ⁇ mol/liter of nicotinamide adenine dinucleotide per minute at 25°C.
  • AST aspartate aminotransferase
  • the resulting homogenate was further sonicated (3 x 10 seconds, setting 4, using a Fisher 550 Sonic Dismembrator) followed by centrifugation at 10,000 rpm for 15 minutes at 4°C.
  • the supernatant was assayed for hepatic ALT using an L-type GPT J2 kit, available from Wako Chemicals, Osaka, Japan, according to the manufacturer's instructions.
  • AST activity was measured with an AST/GOT Liqui-UV kit, available from Stanbio Laboratories, Boerne, Texas, according to the manufacturer's instructions.
  • Murine ALT2 gene expression is specifically induced in fatty liver. The distinctive tissue distribution patterns of murine ALTl and ALT2 mRNAs are likely due to a difference in their gene regulation.
  • FIGS. 4A-C demonstrate the increased mALT2 gene expression in fatty liver.
  • FIGS. 4A and 4B are duplicate blots containing 15 ⁇ g of total RNA extracted from fatty liver of obese (ob/ob) and lean (+/?) mice blotted with 32 P- labeled mALTl or mALT2 DNA probe. Hybridization signals were visualized and quantitated by Phosphorlmager.
  • FIGS. 4A and 4B RNA loading from one of the duplicate gels is shown in lower blot.
  • ALT is an important intermediary enzyme involved in the metabolism of amino acids, glucose, and possibly fatty acids and is well known for its use as a surrogate marker for liver damage in clinical diagnostics.
  • the mouse genes reside on separate chromosomes and have distinct tissue distributions and possibly cellular localizations.
  • the murine ALT isoenzymes behave discordantly in various clinical conditions. In other words, under certain clinical conditions, one isoenzyme may be elevated but not the other, or vice versa. By virtue of this feature, individual murine ALT isoenzymes can be better diagnostic markers than a total murine ALT activity.
  • rat ALT isoenzymes can be better diagnostic markers than a total rat ALT activity.
  • ALT activities are present in many tissues, including liver, heart, kidney, muscle, brain, and adipose tissue in rodents.
  • Northern blot data indicate one or both of the murine ALT genes are expressed in the tissues where ALT activity has been observed.
  • Murine ALTl is mainly expressed in liver and bowels, whereas murine ALT2 is highly expressed in muscle, liver, fat, and kidney, a tissue pattern reminiscent of human ALTl and ALT2 tissue distribution.
  • ALT and AST activity levels have been used in clinic diagnostics for many years. Elevation of these two enzyme activities in serum are regarded as evidence of liver damage, as in viral hepatitis, NASH, or drug hepatotoxicity. However, the mechanism for the serum ALT increase has not been well understood and has been thought to be caused by "leakage" of the cellular enzyme into the systemic circulation.
  • ALT elevation in muscle disease may be due to a "leak" of ALT, presumably ALT2, from muscle, where ALT2, but not ALTl, is abundantly expressed.
  • a specific ALT isoenzyme may be induced in a given clinical condition.
  • hepatic murine ALTl and murine ALT2 gene expression were examined in obese mice because hepatic steatosis is associated with this genetically obese model. Indeed, murine ALT2, but not murine ALTl, gene expression is specifically induced. Furthermore, total murine ALT enzymatic activity is increased by 30% in fatty liver over nonfatty liver, suggesting that murine ALT2 may be primarily responsible for the increased serum ALT activity in liver steatosis. Interestingly, AST activity remains unchanged in the same condition.
  • the ALT isoenzymes may be present in different cellular compartments, which can also be utilized for diagnostic purposes: the release of a given ALT isoenzyme into circulation reflects the nature of the liver damage. It has been shown that serum mitochondrial AST content is specifically increased in patients treated with halothane, and suggested that this AST isoenzyme is a sensitive marker for halothane- induced hepatic injury. Both cytosolic and mitochondrial murine and rat ALT activities were found in liver, kidney, and skeletal and cardiac muscles. At present, which ALT isoenzyme is cytosolic or mitochondrial is not certainly clear.
  • ALT isoenzyme specific antibodies help to elucidate the cellular localization of ALT isoenzymes and their changes in disease states.
  • ALT isoform-specific antibody is required for establishment of isoform-specific detection, such as by enzyme-linked immunoabsorbant assay (ELISA).
  • ELISA enzyme-linked immunoabsorbant assay
  • a recombinant ALTl polypeptide and/or a recombinant ALT2 polypeptide is generated in bacteria and purified to homogeniety. About 2 mg of the purified protein is used to immunize mice for generating antibody specific for murine ALT or, duley, to immunize rats for generating antibody specific for rat ALT.
  • a monoclonal hybridoma against a isoform of an ALT polypeptide of the present invention is determined by determining binding specificity to said ALT polypeptide.
  • One such method is Western blot analysis in which about 20 ng of ALT2, of ALTl, and of bovine serum albumin are load onto an SDS-PAGE gel, electrophoresised, and blotted to PVDF membrane, which is incubated with cell culture media of the indicated hybridoma cells (1:100 dilution) and visualized by chemiluminescence. Cross-reaction of the antibody produced by the hyridoma cell identified above to the other isoform is also determined.
  • the identified isoform-specific antibody is employed in the methods of the present invention, such as an ALT isoform- specific ELISA.
  • Differences in ALT isoenzyme in tissue distribution, gene regulation, and possible cellular localization suggest that the ability to measure ALT isoenzyme-specific activity levels is a significant improvement over measurement of total ALT activity in clinical diagnostics.
  • the cloning of murine and rattus homologues of human ALT isoenzymes provides a novel tool for clinical applications of ALT isoenzymes as molecular markers for nonalcoholic fatty liver diseases as well as other clinical conditions. Data herein are generally presented as mean ⁇ standard deviation (SD). Statistical significance was determined by Student unpaired t test. P value less than .05 was considered significant.

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Abstract

L'invention concerne des polypeptides alanine transaminase (ALT) et leur utilisation comme marqueurs de diagnostic pour prévenir et contrôler les dégâts tissulaires et/ou les défaillances tissulaires. Ces polypeptides sont de type murin, souris et/ou rat, et ils permettent de déceler, prévoir et/ou déterminer les processus hépatiques d'un animal, en particulier la souris et/ou le rat.
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WO2007136822A3 (fr) * 2006-05-19 2008-02-28 George Mason Intellectual Prop Diagnostic de maladie du foie utilisant l'état d'activation de la voie akt/mtor/irs dans les adipocytes
US9757359B2 (en) 2008-01-11 2017-09-12 Reata Pharmaceuticals, Inc. Synthetic triterpenoids and methods of use in the treatment of disease
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