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WO1999047560A1 - Isoforme 2 de gene de type dimethylglycine dehydrogenase - Google Patents

Isoforme 2 de gene de type dimethylglycine dehydrogenase Download PDF

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Publication number
WO1999047560A1
WO1999047560A1 PCT/CN1998/000041 CN9800041W WO9947560A1 WO 1999047560 A1 WO1999047560 A1 WO 1999047560A1 CN 9800041 W CN9800041 W CN 9800041W WO 9947560 A1 WO9947560 A1 WO 9947560A1
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WIPO (PCT)
Prior art keywords
polypeptide
dehydrogenase
dimethylglycme
seq
polynucleotide
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PCT/CN1998/000041
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English (en)
Inventor
Jiahui Xia
Chunyu Liu
Kuanping Yu
Qian Pan
Duo Zheng
Heping Dai
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Hunan Medical University
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Application filed by Hunan Medical University filed Critical Hunan Medical University
Priority to PCT/CN1998/000041 priority Critical patent/WO1999047560A1/fr
Publication of WO1999047560A1 publication Critical patent/WO1999047560A1/fr

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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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0026—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on CH-NH groups of donors (1.5)

Definitions

  • This invention relates to newly identified polynucleotides, polypeptides encoded by them and to the use of such polynucleotides and polypeptides, and to their production More particularly, the polynucleotides and polypeptides of the present invention relate to the dimethylglycine dehydrogenase family, hereinafter referred to as dimethylglycme dehydrogenase-like 2 gene The invention also relates to inhibiting or activating the action of such polynucleotides and polypeptides
  • Sarcosine (N-methylglycine) is er__ymat ⁇ cally formed from dimethylglycine by dimethylglycine dehydrogenase (EC 1 5 99 2) and converted to glycine by sarcosine dehydrogenase (EC 1 5 99 1) Sarcosine dehydrogenase deficiency will cause sarcosinemia This indicates that the dimethylglycine dehydrogenase family has an established, proven history as therapeutic targets Clearly there is a need for identification and charactenzation of further members of the dimethylglycine dehydrogenase family which can play a role in preventing, ameliorating or correcting dysfunctions or diseases, including, but not limited to, sarcosinemia, cardiomyopathy, retinitis pigmentosa, deafness, neurological disease, cancer, metabolic defects and AIDS
  • the invention relates to dimethylglycine dehydrogenase-like 2 polypeptides and recombinant materials and methods for their production
  • Another aspect of the invention relates to methods for using such dimethylglycine dehydrogenase-like 2 polypeptides and polynucleotides
  • Such uses include the treatment of sarcosinemia, cardiomyopathy, retinitis pigmentosa, deafness, neurological disease, cancer, metabolic defects and AIDS, among others
  • the mvention relates to methods to identify agomsts and antagonists usmg the materials provided by the invention, and treatmg conditions associated with dimethylglycine dehydrogenase-like 2 gene imbalance with the identified compounds
  • Yet another aspect of the invention relates to diagnostic assays for detecting diseases associated with inappropriate dimethylglycine dehydrogenase-like 2 gene activity or levels DESCRIPTION OF THE INVENTION
  • Dimethylglycine dehydrogenase-like 2 gene refers, among others, generally to a polypeptide having the ammo acid sequence set forth in SEQ ID NO 2 or an allehc variant thereof "Dimethylglycine dehydrogenase-like 2 gene” activity or “dimethylglycine dehydrogenase-like 2 polypeptide activity” or "biological activity of the dimethylglycine dehydrogenase-like 2 gene” or “dimethylglycine dehydrogenase-like 2 polypeptide” refers to the metabolic or physiologic function of said dimethylglycine dehydrogenase-like 2 gene including similar activities or improved activities or these activities with decreased undesirable side-effects Also included are antigenic and lmmunogemc activities of said dimethylglycine dehydrogenase-like 2 gene
  • “Dimethylglycine dehydrogenase-like 2 gene” refers to a polynucleotide having the nucleotide sequence set forth in SEQ ID NO 1 or allehc variants thereof and/or their complements
  • Antibodies as used herein includes polyclonal and monoclonal antibodies, chime ⁇ c, single chain, and humanized antibodies, as well as Fab fragments, including the products of an Fab or other lmmunoglobulm expression library
  • Isolated means altered “by the hand of man” from the natural state If an "isolated” composition or substance occurs m nature, it has been changed or removed from its o ⁇ gmal environment, or both
  • a polynucleotide or a polypeptide naturally present in a living animal is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated”, as the term is employed herein
  • Polynucleotide generally refers to any poly ⁇ bonucleotide or polydeox ⁇ bonucleotide, which may be unmodified R A or DNA or modified RNA or DNA
  • Polynucleotides include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and double- stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comp ⁇ smg DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions
  • polynucleotide refers to triple-stranded regions comp ⁇ smg RNA or DNA or both RNA and DNA
  • polynucleotide also includes DNAs or RNAs contammg one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons "Modified" bases include, for example
  • Polypeptide refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, 1 e , peptide isosteres
  • Polypeptide refers to both short chains, commonly referred to as peptides, ohgopeptides or ohgomers, and to longer chains, generally referred to as proteins Polypeptides may contain amino acids other than the 20 gene-encoded amino acids
  • Polypeptides include amino acid sequences modified either by natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature Modifications can occur anywhere m a polypeptide, including the peptide backbone, the amino acid side-chains and the ammo or carboxyl termini It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide Also,
  • Identity is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences.
  • identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences.
  • Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al. , Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S.F. et al., J. Molec. Biol. 215: 403-410 (1990).
  • the BLAST X program is publicly available from NCBI and other sources (BLAST Manual , Altschul, S. , et al. , NCBI NLM NIH Bethesda, MD 20894; Altschul, S. , et ai , J. Mol. Biol. 215: 403-410 (1990).
  • the well known Smith Waterman algorithm may also be used to determine identity.
  • Preferred parameters for polypeptide sequence comparison include the following:
  • Preferred parameters for polynucleotide comparison include the following:
  • Gap Length Penalty 3 A program useful with these parameters is publicly available as the "gap" program from Genetics Computer Group, Madison WI. The aforementioned parameters are the default parameters for polynucleotide comparisons.
  • Preferred polynucleotide embodiments further include an isolated polynucleotide comprising a polynucleotide having at least a 50,60, 70, 80, 85, 90, 95, 97 or 100% identity to a polynucleotide reference sequence of SEQ ID NO: l, wherein said reference sequence may be identical to the sequence of SEQ ID NO: 1 or may include up to a certain integer number of nucleotide alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion, and wherein said alterations may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of nucleotide alterations is determined by multiplying the total number of nucleotides in SEQ ID NO:
  • n n is the number of nucleotide alterations
  • x n is the total number of nucleotides in SEQ ID NO: l
  • y is 0.50 for 50% , 0.60 for 60% , 0.70 for 70% , 0.80 for 80% , 0.85 for 85 % , 0.90 for 90%, 0.95 for 95%, 0.97 for 97% or 1.00 for 100%, and wherein any non- integer product of x n and y is rounded down to the nearest integer prior to subtracting it from x n .
  • Alterations of a polynucleotide sequence encoding the polypeptide of SEQ ID NO:2 may create nonsense, missense or frameshift mutations in this coding sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations.
  • Preferred polypeptide embodiments further include an isolated polypeptide comprising a polypeptide having at least a 50,60, 70, 80, 85, 90, 95, 97 or 100% identity to a polypeptide reference sequence of SEQ ID NO:2, wherein said reference sequence may be identical to the sequence of SEQ ID NO: 2 or may include up to a certain integer number of amino acid alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of amino acid alterations is determined by multiplying the total number of amino acids in SEQ ID NO: 2 by the numerical percent of the respective percent identity and subtracting that product from said total number of amino acids in SEQ ID NO:2, or
  • n a is the number of amino acid alterations
  • x a is the total number of amino acids in SEQ ID NO:2
  • y is 0.50 for 50% , 0.60 for 60% , 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%, 0.97 for 97% or 1.00 for 100%, and wherein any non- integer product of x a and y is rounded down to the nearest integer prior to subtracting it from
  • the present invention relates to dimethylglycine dehydrogenase-like 2 polypeptides (or dimethylglycine dehydrogenase-like 2 proteins)
  • the dimethylglycine dehydrogenase- like 2 polypeptides include the polypeptide of SEQ ID NO 2, as well as polypeptides comp ⁇ smg the amino acid sequence of SEQ ID NO 2, and polypeptides comprising the ammo acid sequence which have at least 80% identity to that of SEQ ID NO 2 over its entire length, and still more preferably at least 90% identity, and even still more preferably at least 95% identity to SEQ ID NO 2 Furthermore, those with at least 97-99% are highly preferred Also included within dimethylglycine dehydrogenase-like 2 polypeptides are polypeptides having the amino acid sequence which have at least 80% identity to the polypeptide having the ammo acid sequence of SEQ ID NO 2 over its entire length, and still more preferably at least 90% identity, and still more preferably
  • the dimethylglycine dehydrogenase-like 2 polypeptides may be in the form of the "mature" protein or may be a part of a larger protein such as a fusion protein It is often advantageous to include an additional amino acid sequence which contains secretory or leader se ⁇ uences, pro- sequences, sequences which aid in pu ⁇ fication such as multiple histidine residues, or an additional sequence for stability during recombinant production
  • fragments of the dimethylglycine dehydrogenase-like 2 polypeptides are also mcluded in the invention
  • a fragment is a polypeptide having an amino acid sequence that entirely is the same as part, but not all, of the amino acid sequence of the aforementioned dimethylglycine dehydrogenase-like 2 polypeptides
  • fragments may be "free- standing," or comp ⁇ sed within a larger polypeptide of which they form a part or region, most preferably as a single continuous region
  • Representative examples of polypeptide fragments of the invention include, for example, fragments from about amino acid number 1-20, 21-40, 41-60, 61-80, 81-100, and 101 to the end of dimethylglycine dehydrogenase-like 2 polypeptide
  • “about” includes the particularly recited ranges larger or smaller by several, 5, 4, 3, 2 or 1 amino acid at either extreme or at both extremes
  • Preferred fragments include, for example
  • Vanants of the defined sequence and fragments also form part of the present invention
  • Preferred vanants are those that vary from the referents by conservative amino acid substitutions - 1 e , those that substitute a residue with another of like charactenstics Typical such substitutions are among Ala, Val, Leu and lie, among Ser and Thr, among the acidic residues Asp and Glu, among Asn and Gin, and among the basic residues Lys and Arg, or aromatic residues
  • Particularly prefe ⁇ ed are vanants in which several, 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination
  • the dimethylglycine dehydrogenase-like 2 polypeptides of the invention can be prepared in any suitable manner
  • Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods Means for preparing such polypeptides are well understood in the art
  • Dimethylglycine dehydrogenase-hke 2 polynucleotides include isolated polynucleotides which encode the dimethylglycine dehydrogenase- ke 2 polypeptides and fragments, and polynucleotides closely related thereto More specifically, dimethylglycine dehydrogenase-hke 2 polynucleotides of the invention include a polynucleotide compnsing the nucleotide sequence contained m SEQ ID NO 1 encoding a dimethylglycine deh drogenase-l ⁇ ke 2 polypeptide of SEQ ID NO 2, and polynucleotide having the particular sequence of SEQ ID NO 1 Dimethylglycine dehydrogenase-like 2 polynucleotides further mclude a polynucleotide compnsing a nucleotide sequence that has at
  • the dimethylglycine dehydrogenase-like 2 gene of the invention is structurally related to other proteins of the dimethylglycine dehydrogenase family, as shown by the results of sequencing the cDNA of Table 1 (SEQ ID NO 1) encoding human dimethylglycme dehydrogenase-like 2 gene
  • the cDNA sequence of SEQ ID NO 1 contains an open reading frame (nucleotide number 133 to 1557) encoding a polypeptide of 475 amino acids of SEQ ID NO 2
  • the amino acid sequence of Table 2 (SEQ ID NO 2) has about 88 8% identity (usmg FASTA) in 278 ammo acid residues with partial rat dimethylglycine dehydrogenase-like protein (PJL Blache et al Swissprot Q64380 )
  • dimethyglycine dehydrogenase-like 2 protein is 35 2% identical to rat dimethylglycine dehydrogenase over 381 amino acid residues (H
  • dimethylglycine dehydrogenase-like 2 polypeptides and polynucleotides of the present invention are expected to have, inter aha, similar biological functions/properties to their homologous polypeptides and polynucleotides, and their utility is obvious to anyone skilled in the art
  • a nucleotide sequence of a human dimethylglycine dehydrogenase-like 2 gene (SEQ ID NO 1)
  • One polynucleotide of the present invention encoding dimethylglycine dehydrogenase-like 2 gene may be obtained usmg standard cloning and screening, from a cDNA library de ⁇ ved from mRNA in cells of human liver using the expressed sequence tag (EST) analysis (Adams, M D , et al Science (1991) 252 1651-1656, Adams, M D et al , Nature, (1992) 355 632-634.
  • EST expressed sequence tag
  • Polynucleotides of the invention can also be obtained from natural sources such as genomic DNA hbra ⁇ es or can be synthesized usmg well known and commercially available techniques The nucleotide sequence encoding dimethylglycine dehydrogenase-like 2 polypeptide of SEQ
  • ID NO 2 may be identical to the polypeptide encoding sequence contained in Table 1 (nucleotide number 133 to 1557 of SEQ ID NO 1), or it may be a sequence, which as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptide of SEQ ID NO 2
  • the polynucleotide may include the coding sequence for the mature polypeptide or a fragment thereof, by itself, the coding sequence for the mature
  • the marker sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc ) and descnbed m Gentz et al , Proc Natl Acad Set USA (1989) 86 821 -824, or is an HA tag
  • the polynucleotide may also contain non-coding 5 ' and 3 ' sequences, such as transcnbed, non-translated sequences, splicing and polyadenylation signals, nbosome binding sites and sequences that stabilize mRNA
  • polynucleotides encoding dimethylglycine dehydrogenase- like 2 gene vanants which compnse the amino acid sequence dimethylglycine dehydrogenase-like 2 polypeptide of Table 2 (SEQ ED NO 2) m which several, 5-10, 1-5, 1-3, 1-2 or 1 ammo acid residues are substituted, deleted or added, m any combination
  • the present invention further relates to polynucleotides that hybndize to the herein above- descnbed sequences
  • the present invention especially relates to polynucleotides which hyb ⁇ dize under stringent conditions to the herein above-descnbed polynucleotides
  • sh ⁇ ngent conditions means hybndization will occur only if there is at least 80%, and preferably at least 90%, and more preferably at least 95%, yet even more preferably 97-99% identity between the sequences
  • Polynucleotides of the invention which are identical or sufficiently identical to a nucleotide sequence contained in SEQ ID NO 1 or a fragment thereof, may be used as hybndization probes for cDNA and genomic DNA, to isolate full-length cDNAs and genomic clones encoding dimethylglycme dehydrogenase-like 2 polypeptide and to isolate cDNA and genomic clones of other genes (including genes encoding homologs and orthologs from species other than human) that have a high sequence similanty to the dimethylglycine dehydrogenase-like 2 gene
  • Such hybndization techniques are known to those of skill in the art Typically these nucleotide sequences are 80% identical, preferably 90% identical, more preferably 95% identical to that of the referent
  • the probes generally will compnse at least 15 nucleotides Preferably, such probes will have at least 30 nucleotides and may have at least 50 nucleotides Particularly
  • a method compnses the steps of screening an appropnate library under stingent hybndization conditions with a labeled probe having the SEQ ID NO 1 or a fragment thereof, and isolating full-length cDNA and genomic clones contammg said polynucleotide sequence
  • 11 polynucleotides of the present mvention further include a nucleotide sequence comp ⁇ smg a nucleotide sequence that hybndize under stringent condition to a nucleotide sequence having SEQ ID NO 1 or a fragment thereof Also included with dimethylglycme dehydrogenase-like 2 polypeptides are polypeptide compnsing amino acid sequence encoded by nucleotide sequence obtained by the above hybndization condition Such hybndization techniques are well known to those of skill m the art St ⁇ ngent hybndization conditions are as defined above or, alternatively, conditions under overnight mcubation at 42°C m a solution compnsing 50% formamide, 5xSSC (150mM NaCl, 15mM t ⁇ sodium citrate), 50 mM sodium phosphate (pH7 6), 5x Denhardt's solution, 10 % dextran sulfate, and 20 microgram/ml denatured,
  • the present invention also relates to vectors which compnse a polynucleotide or polynucleotides of the present mvention, and host cells which are genetically engmeered with vectors of the mvention and to the production of polypeptides of the mvention by recombinant techniques Cell-free translation systems can also be employed to produce such proteins usmg RNAs de ⁇ ved from the DNA constructs of the present mvention
  • host cells can be genetically engmeered to incorporate expression systems or portions thereof for polynucleotides of the present mvention
  • Introduction of polynucleotides into host cells can be effected by methods desc ⁇ bed m many standard laboratory manuals, such as Davis et al , BASIC METHODS IN MOLECULAR BIOLOGY (1986) and Sambrook et al , MOLECULAR CLONING A LABORATORY MANUAL, 2nd Ed , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N Y (1989) such as calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, catiomc lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection
  • appropnate hosts include bacte ⁇ al cells, such as streptococci, staphylococci, E cob, Streptomyces and Bacillus subtihs cells, fungal cells, such as yeast cells and Asperg ⁇ lus cells, insect cells such as Drosophila S2 and Spodoptera Sf9 cells, animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells, and plant cells
  • bacte ⁇ al cells such as streptococci, staphylococci, E cob, Streptomyces and Bacillus subtihs cells
  • fungal cells such as yeast cells and Asperg ⁇ lus cells
  • insect cells such as Drosophila S2 and Spodoptera Sf9 cells
  • animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells
  • Such systems include, among others, chromosomal, episomal and virus-de ⁇ ved systems, e g , vectors de ⁇ ved from bacte ⁇ al plasrmds, from bacte ⁇ ophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal
  • the expression systems may contain control regions that regulate as well as engender expression Generally, any system or vector suitable to maintain, propagate or express polynucleotides to produce a polypeptide m a host may be used.
  • the appropnate nucleotide sequence may be inserted mto an expression system by any of a va ⁇ ety of well-known and routine techniques, such as, for example, those set forth m Sambrook et al , MOLECULAR CLONING, A LABORATORY MANUAL (supra)
  • a va ⁇ ety of well-known and routine techniques, such as, for example, those set forth m Sambrook et al , MOLECULAR CLONING, A LABORATORY MANUAL (supra)
  • the dimethylglycme dehydrogenase-like 2 polypeptide is to be expressed for use m screening assays, generally, it is prefened that the polypeptide be produced at the surface of the cell In this event, the cells may be harvested prior to use m the screening assay If dimethylglycme dehydrogenase-like 2 polypeptide is secreted mto the medium, the medium can be recovered m order to recover and pu ⁇ fy the polypeptide, if produced intracellularly, the cells must first be lysed before the polypeptide is recovered Dimethylglycme dehydrogenase-hke 2 polypeptides can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography Most preferably, high performance liquid chromat
  • This mvention also relates to the use of dimethylglycme dehydrogenase-like 2 polynucleotides for use as diagnostic reagents Detection of a mutated form of the dimethylglycme dehydrogenase-hke 2 gene associated with a dysfunction will provide a diagnostic tool that can add to or define a diagnosis of a disease or susceptibility to a disease which results from under-expression, over-expression or altered
  • Nucleic acids for diagnosis may be obtained from a subject's cells, such as from blood, urine, saliva, tissue biopsy or autopsy matenal
  • the genomic DNA may be used directly for detection or may be amplified enzymatically by usmg PCR or other amplification techmques pnor to analysis RNA or cDNA may also be used in similar fashion
  • Deletions and insertions can be detected by a change m size of the amplified product m companson to the normal genotype
  • Pomt mutations can be identified by hybndizmg amplified DNA to labeled dimethylglycme dehydrogenase-like 2 gene nucleotide sequences Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences m meltmg temperatures
  • DNA sequence differences may also be detected by alterations m electrophoretic mobility of DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencmg See, e g
  • sarcosinemia, cardiomyopathy, retinitis pigmentosa, deafness, neurological disease, cancer, metabolic defects and AIDS can be diagnosed by methods comprising determining from a sample derived from a subject an abnormally decreased or mcreased level of dimethylglycme dehydrogenase-like 2 polypeptide or dimethylglycme dehydrogenase-like 2 gene mRNA Decreased or increased expression can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides, such as, for example, PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods Assay techmques that can be used to determine levels of a protein, such as a dimethylglycme dehydrogenase-like 2 polypeptide, m a sample de ⁇ ved from a host are well-known to those of skill m the art Such assay methods mclude radioimmunoassays,
  • the present invention relates to a diagonostic kit for a disease or suspectabihty to a disease, particularly sarcosinemia, cardiomyopathy, retinitis pigmentosa, deafness, neurological disease, cancer, metabolic defects and AIDS which comprises
  • a dimethylglycine dehydrogenase-like 2 polynucleotide preferably the nucleotide sequence of SEQ ID NO 1, or a fragment thereof ,
  • a dimethylglycme dehydrogenase-like 2 polypeptide preferably the polypeptide of SEQ ID NO 2, or a fragment thereof, or
  • the nucleotide sequences of the present mvention are also valuable for chromosome identification
  • the sequence is specifically targeted to and can hyb ⁇ dize with a particular location on an individual human chromosome
  • the mapping of relevant sequences to chromosomes according to the present mvention is an important first step m correlating those sequences with gene associated disease
  • Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be conelated with genetic map data Such data are found, for example, m V McKusick, Mende an Inhentance in Man (available on line through Johns Hopkins University Welch Medical Library)
  • the relationship between genes and ⁇ seases that have been mapped to the same chromosomal region are then identified through linkage analysis (comhentance of physically adjacent genes)
  • the differences in the cDNA or genomic sequence between affected and unaffected individuals can also be determined If a mutation is observed m some or all of the affected individuals but
  • the dimethylglycme dehydrogenase-like 2 gene is mapped to 9q34 where sarcosinemia was localized
  • polypeptides of the mvention or their fragments or analogs thereof, or cells expressmg them can also be used as immunogens to produce antibodies immunospecific for the dimethylglycme dehydrogenase-like 2 polypeptides
  • immunospecific means that the antibodies have substantiall greater affinity for the polypeptides of the mvention than their affinity for other related polypeptides m the pnor art
  • Antibodies generated against the dimethylglycme dehydrogenase-like 2 polypeptides can be obtained by administering the polypeptides or epitope-beanng fragments, analogs or cells to an animal, preferably a nonhuman, usmg routine protocols For preparation of monoclonal antibodies, any techmque which provides antibodies produced by continuous cell l e cultures can be used Examples mclude the hybndoma technique (Kohler, G and Milstein, C , Nature (1975) 256 495-497), the t ⁇ oma technique, the human B-cell hybndoma techmque (Kozbor et al , Immunology Today (1983) 4 72) and the EBV-hybndoma technique (Cole et al , MONOCLONAL ANTIBODIES AND CANCER THERAPY, pp 77-96, Alan R Liss, Inc , 1985) Techniques for the production of s gle cham antibodies (U S Patent No
  • the above-descnbed antibodies may be employed to isolate or to identify clones expressmg the polypeptide or to pu ⁇ fy the polypeptides by affinity chromatography
  • Antibodies against dimethylglycme dehydrogenase-like 2 polypeptides may also be employed to treat sarcosinemia, cardiomyopathy, retinitis pigmentosa, deafness, neurological disease, cancer, metabolic defects and AIDS, among others
  • Vaccines Another aspect of the invention relates to a method for mducing an immunological response m a mammal which comp ⁇ ses inoculating the mammal with a dimethylglycme dehydrogenase-like 2 polypeptide, or a fragment thereof, adequate to produce antibody and/or T cell immune response to protect said animal from sarcosinemia, cardiomyopathy, retinitis pigmentosa, deafness, neurological disease, cancer, metabolic defects and AIDS, among others
  • Yet another aspect of the invention relates to a method of mducing immunological response in a mammal which compnses delivering dimethylglycme dehydrogenase-like 2 polypeptide via a vector directing expression of dimethylglycme dehydrogenase- ke 2 polynucleotide in vivo m order to mduce such an immunological response to produce antibody to protect said animal from diseases
  • a further aspect of the invention relates to an immunological/vaccine formulation (composition) which, when introduced into a mammalian host, induces an immunological response in that mammal to a dimethylglycme dehydrogenase-like 2 polypeptide wherein the composition comprises a dimethylglycme dehydrogenase-like 2 polypeptide or dimethylglycme dehydrogenase-like 2 gene
  • the vaccine formulation may further comprise a suitable carrier Since dimethylglycme dehydrogenase-like 2 polypeptides may be broken down in the stomach, it is preferably administered parenterally (including subcutaneous, intramuscular, intravenous, intradermal etc injection)
  • Formulations suitable for parenteral administration mclude aqueous and non-aqueous ste ⁇ le injection solutions which may contain anti-oxidants, buffers, bacte ⁇ ostats and solutes which render the formulation lnstonic with the blood of the recipient, and aqueous and non-aqueous
  • the dimethylglycme dehydrogenase-like 2 polypeptides of the present mvention may be employed m a screening process for compounds which activate (agomsts) or inhibit activation of (antagonists, or otherwise called inhibitors) the dimethylglycme dehydrogenase-like 2 polypeptide of the present mvention
  • polypeptides of the mvention may also be used to assess identify agonist or antagonists from, for example, cells, cell-free preparations, chemical hbra ⁇ es, and natural product mixtures
  • agomsts or antagonists may be natural or modified substrates, gands, receptors, enzymes, etc , as the case may be, of the polypeptide of the present mvention, or may be structural or functional mimetics of the polypeptide of the present mvention See Cohgan et al , Current Protocols in Immunology 1(2) Chapter 5 (1991)
  • Dimethylglycme dehydrogenase-like 2 polypeptides are responsible for many biological functions, mcludmg many pathologies Accordingly, it is desirous to find compounds and drugs which stimulate dimethylglycme dehydrogenase-like 2 polypeptides on the one hand and which can inhibit the function of dimethylglycme dehydrogenase-like 2 polypeptides on the other hand
  • agomsts are employed for therapeutic and prophylactic purposes for such conditions as sarcosinemia
  • Such screening procedures may involve usmg appropnate cells which express the dimethylglycme dehydrogenase-like 2 polypeptide or respond to dimethylglycme dehydrogenase-like 2 polypeptides of the present mvention Such cells mclude cells from mammals, yeast, Drosophila or E coli Cells which express the dimethylglycme dehydrogenase-like 2 polypeptide (or cell membrane containing the expressed polypeptide) or respond to dimethylglycme dehydrogenase-like 2 polypeptides are then contacted with a test compound to observe bmdmg, or stimulation or inhibition of
  • the assays may simply test bmdmg of a candidate compound wherein adherence to the cells bearing the dimethylglycme dehydrogenase-like 2 polypeptide is detected by means of a label directly or indirectly associated with the candidate compound or in an assay mvolvmg competition with a labeled competitor Further, these assays may test whether the candidate compound results in a signal generated by activation of the dimethylglycme dehydrogenase-like 2 polypeptide, using detection systems appropriate to the cells bearing the dimethylglycme dehydrogenase- ke 2 polypeptide Inhibitors of activation are generally assayed m the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed
  • the assays may simply comprise the steps of mixing a candidate compound with a solution containing a dimethylglycme dehydrogenase-like 2 polypeptide to form a mixture, measuring dimethylglycme dehydrogenase-like 2 gene activity in the mixture, and comparing the dimethylglycme dehydrogenase-like 2 gene activity of the mixture to a standard
  • the dimethylglycme dehydrogenase-like 2 gene cDNA, protein and antibodies to the protein may also be used to configure assays for detecting the effect of added compounds on the production of dimethylglycme dehydrogenase-like 2 gene mRNA and protem in cells
  • an ELISA may be constructed for measuring secreted or cell associated levels of dimethylglycme dehydrogenase-like 2 protem usmg monoclonal and polyclonal antibodies by standard methods known m the art, and this can be used to discover agents which may inhibit or enhance the production of dimethylglycme dehydrogen
  • dimethylglycme dehydrogenase-hke 2 polypeptide antagonists examples include antibodies or, in some cases, oligonucleotides or proteins which are closely related to the gands, substrates, receptors, enzymes, etc , as the case may be, of the dimethylglycme dehydrogenase-like 2 polypeptide, e g , a fragment of the hgands, substrates, receptors, enzymes, etc , or small molecules which bind to the polypetide of the present mvention but do not elicit a response, so that the activity of the polypeptide is prevented
  • the present invention relates to a screenmg kit for identifying agonists, antagonists, hgands, receptors, substrates, enzymes, etc for dimethylglycme dehydrogenase-like 2 polypeptides, or compounds which decrease or enhance the production of dimethylglycme dehydrogenase-like 2 polypeptides, which comprises
  • This mvention provides methods of treatmg abnormal conditions such as sarcosinemia, cardiomyopathy, retinitis pigmentosa, deafness, neurological disease, cancer, metabolic defects and AIDS related to both an excess of and insufficient amounts of dimethylglycme dehydrogenase-like 2 polypeptide activity
  • dimethylglycme dehydrogenase-like 2 polypeptide is administered to a subject an inhibitor compound (antagonist) as hereinabove descnbed along with a pharmaceutically acceptable earner m an amount effective to inhibit the function of the dimethylglycme dehydrogenase-like 2 polypeptide, such as, for example, by blockmg the bmdmg of hgands, substrates, receptors, enzymes, etc , or by inhibiting a second signal, and thereby alleviating the abnormal condition
  • soluble forms of dimethylglycme dehydrogenase-like 2 polypeptides still capable of binding the hgand, substrate, enzymes, receptors, etc in competition with endogenous dimethylglycme dehydrogenase-like 2 polypeptides may be administered Typical embodiments of such competitors comprise fragments of the dimethylglycme dehydrogenase-like 2 polypeptide
  • expression of the gene encoding endogenous dimethylglycme dehydrogenase-like 2 polypeptide can be inhibited using expression blocking techniques Known such techniques involve the use of antisense sequences, either internally generated or separately administered See, for example, O'Connor, J Neurochem (1991) 56 560 m Ohgodeoxynucleotides as Antisense Inhibitors of Gene Expression.
  • oligonucleotides which form triple helices with the gene can be supplied See, for example, Lee et al , Nucleic Acids Res (1979) 6 3073, Cooney et al , Science (1988) 241 456, Dervan et al , Science (1991) 251 1360 These ohgomers can be administered per se or the relevant ohgomers can be expressed in vivo
  • gene therapy may be employed to effect the endogenous production of dimethylglycme
  • Peptides such as the soluble form of dimethylglycme dehydrogenase-hke 2 polypeptides, and agomsts and antagonist peptides or small molecules, may be formulated m combination with a suitable pharmaceutical earner
  • a suitable pharmaceutical earner Such formulations compnse a therapeutically effective amount of the polypeptide or compound, and a pharmaceutically acceptable earner or excipient
  • earners mclude but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof Formulation should suit the mode of administration, and is well within the skill of the art
  • the invention further relates to pharmaceutical packs and kits compnsmg one or more containers filled with one or more of the ingredients of the aforementioned compositions of the mvention
  • Polypeptides and other compounds of the present mvention may be employed alone or m conjunction with other compounds, such as therapeutic compounds
  • Prefened forms of systemic administration of the pharmaceutical compositions mclude injection, typically by mtravenous injection Other injection routes, such as subcutaneous, intramuscular, or intrapentoneal, can be used Alternative means for systemic administration mclude transmucosal and transdermal administration usmg penetrants such as bile salts or fusidic acids or other detergents.
  • oral administration may also be possible Administration of these compounds may also be topical and/or localized, m the form of salves, pastes, gels and the like
  • the dosage range required depends on the choice of peptide, the route of administration, the nature of the formulation, the nature of the subject's condition, and the judgment of the attending practitioner Suitable dosages, however, are m the range of 0 1-100 ⁇ g/kg of subject Wide vanations in the needed dosage, however, are to be expected m view of the vanety of compounds available and the differing efficiencies of vanous routes of administration For example, oral administration would be expected to require higher dosages than administration by mtravenous injection Vanations m these
  • 21 dosage levels can be adjusted usmg standard empincal routines for optimization, as is well understood m the art
  • Polypeptides used m treatment can also be generated endogenously m the subject, m treatment modalities often referred to as "gene therapy" as descnbed above
  • m treatment modalities often referred to as "gene therapy” as descnbed above
  • cells from a subject may be engmeered with a polynucleotide, such as a DNA or RNA, to encode a polypeptide ex vivo, and for example, by the use of a retroviral plasmid vector The cells are then introduced mto the subject
  • CAGAGCCCTA CCCGGGGCAT GGGGCCATGC AACCTGTCCA GCGCAGCTGG CCCCACAGCC 240
  • CTGCGTGTTC CAGGAGCGGC ATGGCTGGGA GCGACCGGGA TGGTTTCATC CCCGAGGNCC 1680
  • ACGCCTACCG CAGGCTGCTG GCAGACGAGT
  • ACACCTTCGC CTTCCCGCCC CACCACGACA 1800 CGATCAAGAA GGAGTGCCTG GCCTGCAGAG GGGCCGCCGC TGTGTTTGAC ATGTCCTACT 1860

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Abstract

L'invention concerne des polypeptides et des polynucléotides de type diméthylglycine déhdrogénase 2 et des procédés d'obtention de ces polypeptides par des techniques recombinantes. Font aussi l'objet de cette invention des procédés d'utilisation des polypeptides et des polynucléotides de type diméthylglycine déhydrogénase 2 dans la conception de protocoles pour le traitement de sarcosinémie, de myocardiopathies, de rétinite pigmentaire, de surdité, de cancers, d'affections neurologiques, de troubles métaboliques et du SIDA, entre autres, et des doses diagnostiques pour ces pathologies.
PCT/CN1998/000041 1998-03-18 1998-03-18 Isoforme 2 de gene de type dimethylglycine dehydrogenase WO1999047560A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN1998/000041 WO1999047560A1 (fr) 1998-03-18 1998-03-18 Isoforme 2 de gene de type dimethylglycine dehydrogenase

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN1998/000041 WO1999047560A1 (fr) 1998-03-18 1998-03-18 Isoforme 2 de gene de type dimethylglycine dehydrogenase

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Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
GENBANK, AC000388, 29 May 1997, HAWKINS T.L. et al., "Genomic Sequence from Human 9q34". *
GENBANK, U62481, 11 July 1996, BLACHE P. et al., "Rattus Norvegicus Dimethylglycine Dehydrogenase-Like Protein mRNA, Partial Cds". *
GENKBANK, AC002295, 10 June 1997, HAWKINS T.L. et al., "Genomic Sequence from Human 9q34". *

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