WO1999047559A1 - Isoform 1 of dimethylglycine dehydrogenase-like gene - Google Patents

Abstract

Dimethylglycine dehydrogenase-like polypeptides and polynucleotides and methods for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods for utilizing dimethylglycine dehydrogenase-like polypeptides and polynucleotides in the design of protocols for the treatment of sarcosinemia, cardiomyopathy, retinitis pigmentosa, deafness, neurological disease, cancer, metabolic defects and AIDS, among others, and diagnostic assays for such conditions.

Description

ISOFORM 1 OF DIMETHYLGLYCINE DEHYDROGENASE-LIKE GENE

FIELD OF INVENTION 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 dimethylglycine dehydrogenase-like gene. The invention also relates to inhibiting or activating the action of such polynucleotides and polypeptides.

BACKGROUND OF THE INVENTION

Sarcosine (N-methylglycine) is enzymatically 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 characterization 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.

SUMMARY OF THE INVENTION

In one aspect, the invention relates to dimethylglycine dehydrogenase-like polypeptides and recombinant materials and methods for their production. Another aspect of the invention relates to methods for using such dimethylglycine dehydrogenase-like polypeptides and polynucleotides. Such uses include the treatment of sarcosinemia, cardiomyopathy, retinitis pigmentosa, deafness, neurological disease, cancer, metabolic defects and AIDS, among others. In still another aspect, the invention relates to methods to identify agonists and antagonists using the materials provided by the invention, and treating conditions associated with dimethylglycine dehydrogenase-like 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 gene activity or levels. DESCRIPTION OF THE INVENTION Definitions

The following definitions are provided to facilitate understanding of certain terms used frequently herein "Dimethylglycine dehydrogenase-like 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 gene activity" or "dimethylglycine dehydrogenase-like polypeptide activity" or "biological activity of the dimethylglycine dehydrogenase-like gene" or "dimethylglycine dehydrogenase-like polypeptide" refers to the metabolic or physiologic function of said dimethylglycine dehydrogenase-like gene including similar activities or improved activities or these activities with decreased undesirable side-effects Also mcluded are antigemc and lmmunogemc activities of said dimethylglycine dehydrogenase-hke gene

"Dimethylglycine dehydrogenase-hke 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 herem mcludes polyclonal and monoclonal antibodies, chimeπc, single cham, 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 oπgmal environment, or both For example, 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 polynbonucleotide or polydeoxπbonucleotide, which may be unmodified RNA 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 comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions In addition, "polynucleotide" refers to tπple-stranded regions comprising RNA or DNA or both RNA and DNA The term polynucleotide also mcludes 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, tπtylated bases and unusual bases such as mosme A variety of modifications has been made to DNA and RNA, thus, "polynucleotide" embraces chemically, enzymatically or metabohcally modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. "Polynucleotide" also embraces relatively short polynucleotides, often referred to as oligonucleotides.

"Polypeptide" refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres. "Polypeptide" refers to both short chains, commonly referred to as peptides, ohgopeptides or oligomers, 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 in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino 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, a given polypeptide may contain many types of modifications. Polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma- carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. See, for instance, PROTEINS - STRUCTURE AND

MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York, 1993 and Wold, F., Posttranslational Protein Modifications: Perspectives and Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York, 1983; Seifter et al, "Analysis for protein modifications and nonprotein cofactors", Meth Enzymol ( 1990) 182:626-646 and Rattan et al. , "Protein Synthesis: Posttranslational Modifications and Aging", Ann NY AcadSci (1992) 663:48-62.

"Variant" as the term is used herein, is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties. A typical variant of a.polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the ammo acid sequence of a polypeptide encoded by the reference polynucleotide Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below A typical variant of a polypeptide differs in ammo acid sequence from another, reference polypeptide Generally, differences are limited so that the sequences of the reference polypeptide and the vanant are closely similar overall and, in many regions, identical A vanant and reference polypeptide may differ in ammo acid sequence by one or more substitutions, additions, deletions in any combmation. A substituted or inserted ammo acid residue may or may not be one encoded by the genetic code A vanant of a polynucleotide or polypeptide may be a naturally occurring such as an allehc vanant, or it may be a vanant that is not known to occur naturally Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis

"Identity," as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, "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. "Identity" and "similarity" can be readily calculated by known methods, including but not limited to those described in (Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). 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 al. , 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: 1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970) Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl. Acad. Sci. USA. 89: 10915-10919 (1992) Gap Penalty: 12 Gap Length Penalty: 4

A program useful with these parameters is publicly available as the "gap" program from Genetics Computer Group, Madison Wl. The aforementioned parameters are the default parameters for polypeptide comparisons (along with no penalty for end gaps).

Preferred parameters for polynucleotide comparison include the following: 1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970) Comparison matrix: matches = + 10, mismatch = 0 Gap Penalty: 50 Gap Length Penalty: 3

A program useful with these parameters is publicly available as the "gap" program from Genetics Computer Group, Madison W 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:l by the numerical percent of the respective percent identity and subtracting that product from said total number of nucleotides in SEQ ID NO:l, or:

n_n < x_n - (x_n • y), wherein n_n is the number of nucleotide alterations, x_n is the total number of nucleotides in SEQ ID NO: l , and 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:

ⁿa ^{≤ x}a ^" ( a * ^v)>

wherein n_a is the number of amino acid alterations, x_a is the total number of amino acids in SEQ ID NO:2, and 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

Polypeptides of the Invention

In one aspect, the present invention relates to dimethylglycine dehydrogenase-like polypeptides (or dimethylglycine dehydrogenase-like proteins). The dimethylglycine dehydrogenase- like polypeptides include the polypeptide of SEQ ID NO 2, as well as polypeptides compnsmg the amino acid sequence of SEQ ID NO 2, and polypeptides compnsmg 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 prefened Also mcluded within dimethylglycine dehydrogenase-like polypeptides are polypeptides having the ammo 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 at least 95% identity to SEQ ID NO 2 Furthermore, those with at least 97-99% are highly preferred Preferably dimethylglycine dehydrogenase-like polypeptide exhibit at least one biological activity of dimethylglycine dehydrogenase-hke gene

The dimethylglycine dehydrogenase-hke polypeptides may be m the form of the "mature" protein or may be a part of a larger protem such as a fusion protein It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro- sequences, sequences which aid m punfication such as multiple histidine residues, or an additional sequence for stability dunng recombinant production

Fragments of the dimethylglycine dehydrogenase-like polypeptides are also included in the invention A fragment is a polypeptide having an ammo acid sequence that entirely is the same as part, but not all, of the amino acid sequence of the aforementioned dimethylglycine dehydrogenase-hke polypeptides As with dimethylglycine dehydrogenase-hke polypeptides, fragments may be "freestanding," or compnsed 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 dimethylglycme dehydrogenase- ke polypeptide In this context "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, truncation polypeptides having the amino acid sequence of dimethylglycine dehydrogenase-like polypeptides, except for deletion of a continuous seπes of residues that includes the ammo terminus, or a continuous senes of residues that mcludes the carboxyl terminus or deletion of two continuous senes of residues, one including the ammo terminus and one including the carboxyl terminus Also preferred are fragments characterized by structural or functional attnbutes such as fragments that compnse alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and tum-forming regions, coil and coil-forming regions, hydropfulic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-formmg regions, substrate binding region, and high anϋgenic mdex regions Other preferred fragments are biologically active fragments Biologically active fragments are those that mediate dimethylglycme dehydrogenase-like gene activity, including those with a similar activity or an improved activity, or with a decreased undesirable activity Also mcluded are those that are antigenic or lmmunogenic m an animal, especially m a human

Preferably, all of these polypeptide fragments retain the biological activity of the dimethylglycme dehydrogenase-like gene, including antigenic activity 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 characteπstics Typical such substitutions are among Ala, Val, Leu and He, 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 Phe and Tyr Particularly preferred are vanants in which several, 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination

The dimethylglycme dehydrogenase-like polypeptides of the invention can be prepared m any suitable manner Such polypeptides mclude 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

Polynucleotides of the Invention Another aspect of the invention relates to dimethylglycme dehydrogenase-like polynucleotides

Dimethylglycme dehydrogenase-like polynucleotides mclude isolated polynucleotides which encode the dimethylglycme dehydrogenase-like polypeptides and fragments, and polynucleotides closely related thereto More specifically, the dimethylglycme dehydrogenase-like polynucleotides of the invention include a polynucleotide compnsmg the nucleotide sequence contained in SEQ ID NO 1 encoding a dimethylglycme dehydrogenase-like polypeptide of SEQ ID NO 2, and polynucleotides havmg the particular sequence of SEQ ID NO 1 Dimethylglycme dehydrogenase-like polynucleotides further include a polynucleotide compnsmg a nucleotide sequence that has at least 80% identity over its entire length to a nucleotide sequence encoding the dimethylglycme dehydrogenase-like polypeptide of SEQ ID NO 2, and a polynucleotide compnsmg a nucleotide sequence that is at least 80% identical to of SEQ ID NO 1 over its entire length In this regard, polynucleotides at least 90% identical are particularly preferred, and those with at least 95% are especially preferred Furthermore, those with at least 97% are highly preferred and those with at least 98-99% are most highly preferred, with at least 99% being the most preferred Also mcluded under dimethylglycme dehydrogenase-like polynucleotides are a nucleotide sequence which has sufficient identity to a nucleotide sequence contained in SEQ ID NO:l to hybridize under conditions useable for amplification or for use as a probe or marker. The invention also provides polynucleotides which are complementary to such dimethylglycine dehydrogenase-like polynucleotides.

Dimethylglycine dehydrogenase-like 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 dimethylglycine dehydrogenase-like gene. The cDNA sequence of SEQ ID NO: 1 contains an open reading frame (nucleotide number 133 to 1425) encoding a polypeptide of 431 amino acids of SEQ ID NO:2. The amino acid sequence of Table 2 (SEQ ID NO:2) has about 88.8% identity (using FASTA) in 278 amino acid residues with partial rat dimethylglycine dehydrogenase-like protein (PJL Blache et al. Swissprot:Q64380 ). Furthermore, dimethyglycine dehydrogenase-like protein (SEQ ID NO:2) is 35.2% identical to rat dimethylglycine dehydrogenase over 381 amino acid residues (H Lang et al. Eur J. Biochem. 198:793-799, 1991). The nucleotide sequence of Table 1 (SEQ ID NO: 1) has about 86.2%identity (using FASTA) in 836 nucleotide residues with rat dimethylglycine dehydrogenase-like gene (PJL Blache et al. Genbank: L79910 ). Furthermore, dimethylglycine dehydrogenase-like gene (SEQ ID NO : 1 ) is 54.1 % identical to rat dimethylglycine dehydrogenase over 440 nucleotide base residues (H Lang et al. Eur J. Biochem. 198:793-799, 1991). Thus, dimethylglycine dehydrogenase-like 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. Table 1'

CCTGGAGTTCCGGCCAGGCCACTGCTTGGGAAGCAAGAAGGTGAAGGCACCTCTGCTGGGCCAAGCACTCTTAGGGCCGA GGGGCACTGCAGCTGACAAGAGCTCCCTGTTTTGCTGAGGCCTGGAGCCCCCATGGCCTCACTGAGCCGAGCCCTACGTG TGGCTGCTGCCCACCCTCGCCAGAGCCCTACCCGGGGCATGGGGCCATGCAACCTGTCCAGCGCAGCTGGCCCCACAGCC GAGAAGAGTGTGCCATATCAGCGGACCCTGAAGGAGGGACAGGGCACCTCGGTGGTGGCCCAAGGCCCAAGCCGGCCCCT GCCCAGCACGGCCAACGTGGTGGTCATTGGTGGAGGCAGCTTGGGCTGCCAGACCCTGTACCACCTGGCCAAGCTGGGCA TGAGTGGGGCGGTGCTGCTGGAGCGGGAGCGGCTGACCTCCGGGACCACCTGGCACACGGCAGGCCTGCTGTGGCAGCTG CGGCCCAGTGACGTGGAGGTGGAGCTTCTGGCCCACACTCGGCGGGTGGTGAGCCGGGAGCTGGAGGAGGAGACGGGACT ACACACGGGCTGGATCCAGAATGGGGGCCTCTTCATCGCGTCCAACCGGCAGCGCCTGGACGAGTACAAGAGGCTCATGT CGCTGGGCAAGGCGTATGGTGTGGAATCCCATGTGCTGAGCCCGGCAGAGACCAAGACTCTGTACCCGCTGATGAATGTG GACGACCTCTACGGGACCCTGTATGTGCCGCACGACGGTACCATGGACCCCGCTGGCACCTGTACCACCCTCGCCAGGGC AGCTTCTGCCCGAGGAGCACAGGTCATTGAGAACTGCCCAGTGACCGGCATTCGTGTGTGGACGGATGATTTTGGGGTGC GGCGGGTCGCGGGTGTGGAGACTCAGCATGGTTCCATCCAGACACCCTGCGTGGTCAATTGTGCAGGAGTGTGGGCAAGT GCTGTGGGCCGGATGGCTGGAGTCAAGGTCCCGCTGGTGGCCATGCACCATGCCTATGTCGTCACCGAGCGCATCGAGGG GATTCAGAACATGCCCAATGTCCGTGATCATGATGCCTCTGTCTACCTCCGCCTCCAAGGGGATGCCTTGTCTGTGGGTG GCTATGAGGCCAACCCCATCTTTTGGGAGGAGGTGTCAGACAAGTTTGCCTTCGGCCTCTTTGACCTGGACTGGGAGGTG- TTCACCCAGCACATTGAAGGCGCCATCAACAGGGTCCCCGTGCTGGAGAAGACAGGAATCAAGTCCACGGTCTGCGGCCC TGAATCCTTCACGCCCGACCACAAGCCCCTGATGGGGGAGGCACCTGAGCTCCGAGGGTTCTTCCTGGGCTGTGGCTTCA ACAGCGCAGGGAAGGTCCAGACAGTCCTGCCACTCCTGTTTACCGTCAACGTCTATCTGTATCTGTAGGTCAGGAGGACA AACATAGGTCAATAAATATGTAATGTTAGTGAACG

A nucleotide sequence of a human dimethylglycine dehydrogenase-like gene (SEQ ID NO: 1).

Table 2^b MASLΞRALRVAAAH PRQS PTRGMGPCNLSSAAGPTAEKSVPYQRTLKEGQGTSWAQGPSRPLPSTAN WIGGGSLGCQ TLYHLAKLGMSGAVLLERERLTSGTTWHTAGLLWQLRPSDVEVELLAHTRRWSRELEEETGLHTGWIQNGGLFIASNRQ RLDEYKRLMSLGKAYGVEΞHVLS PAET TLY PLMNVDDLYGTLYVPHDGTMDPAGTCTTLARAASARGAQVIENCPVTGI RVWTDDFGVRRVAGVETQHGSIQTPCWNCAGVWASAVGRMAGVKVPLVAMHHAYWTERIEGIQNMPNVRDHDASVYLR LQGDALSVGGYEANPI FWEEVSDKFAFGLFDLDWEVFTQHIEGAINRVPVLEKTGI ΞTVCGPESFTPDHKPLMGEAPEL RGFFLGCGFNSAGKVQTVLPLLFTVNVYLYL

An ammo acid sequence of a human dimethylglycme dehydrogenase-like gene (SEQ ID NO. 2)

One polynucleotide of the present mvention encoding dimethylglycme dehydrogenase-like gene may be obtained using standard cloning and screening, from a cDNA library denved from rnRNA in cells of human fetal liver usmg the expressed sequence tag (EST) analysis (Adams, M.D , et al

Science (1991) 252.1651-1656, Adams, M.D. et al , Nature, (1992) 555.632-634, Adams, M.D., et al , Nature (1995) 377 Supp.3-174) Polynucleotides of the mvention can also be obtained from natural sources such as genomic DNA libranes or can be synthesized usmg well known and commercially available techniques The nucleotide sequence encoding dimethylglycme dehydrogenase-like polypeptide of SEQ

ID NO 2 may be identical to the polypeptide encoding sequence contained in Table 1 (nucleotide number 133 to 1425 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

When the polynucleotides of the invention are used for the recombinant production of dimethylglycme dehydrogenase-hke polypeptide, the polynucleotide may include the codmg sequence for the mature polypeptide or a fragment thereof, by itself; the codmg sequence for the mature polypeptide or fragment m reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protein sequence, or other fusion peptide portions For example, a marker sequence which facilitates purification of the fused polypeptide can be encoded In certam preferred embodiments of this aspect of the mvention, the marker sequence is a hexa-histidme peptide, as provided m the pQE vector (Qiagen, Inc.) and descnbed in Gentz et al. , Proc Nad Acad Set USA (1989) 86:821-824, or is an HA tag. The polynucleotide may also contain non-codmg 5' and 3' sequences, such as transcnbed, non-translated sequences, splicing and polyadenylation signals, πbosome binding sites and sequences that stabilize mRNA. Further preferred embodiments are polynucleotides encoding dimethylglycme dehydrogenase- like gene variants comprise the ammo acid sequence dimethylglycine dehydrogenase-like polypeptide of Table 2 (SEQ ID NO:2) in which several, 5-10, 1-5, 1-3, 1-2 or 1 ammo acid residues are substituted, deleted or added, in any combination

The present invention further relates to polynucleotides that hybridize to the herein above- described sequences. In this regard, the present mvention especially relates to polynucleotides which hybridize under stringent conditions to the herein above-described polynucleotides. As herem used, the term "stnngent 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 mvention, which are identical or sufficiently identical to a nucleotide sequence contained m 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 polypeptides and to isolate cDNA and genomic clones of other genes (mcludmg genes encoding homologs and orthologs from species other than human) that have a high sequence similanty to the dimethylglycme dehydrogenase-like 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 comprise at least 15 nucleotides Preferably, such probes will have at least 30 nucleotides and may have at least 50 nucleotides Particularly prefened probes will range between 30 and 50 nucleotides

In one embodiment, to obtain a polynucleotide encoding dimethylglycme dehydrogenase-like polypeptides, mcludmg homologs and orthologs from species other than human, a method compnses the steps of screening an appropnate library under stingent hybndization conditions with a labeled probe havmg the SEQ ID NO 1 or a fragment thereof, and isolating full-length cDNA and genomic clones contammg said polynucleotide sequence Thus m another aspect, dimethylglycme dehydrogenase-like polynucleotides of the present mvention further mclude a nucleotide sequence compnsmg a nucleotide sequence that hybndize under stnngent condition to a nucleotide sequence havmg SEQ ID NO 1 or a fragment thereof Also mcluded with dimethylglycme dehydrogenase- ke polypeptides are polypeptides compnsmg ammo 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 Stnngent hybndization conditions are as defined above or, alternatively, conditions under overnight incubation at 42°C in a solution compnsmg 50% formamide, 5xSSC (150mM NaCl, 15mM tπsodium citrate), 50 mM sodium phosphate (pH7 6), 5x Denhardt's solution, 10 % dextran sulfate, and 20 microgram/ml denatured, sheared salmon sperm DNA, followed by washing the filters m 0 lx SSC at about 65°C The polynucleotides and polypeptides of the present mvention may be employed as research reagents and mateπals for discovery of treatments and diagnostics to animal and human disease

Vectors, Host Cells, Expression

The present mvention also relates to vectors which compnse a polynucleotide or polynucleotides of the present mvention, and host cells which are genetically engineered 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 denved from the DNA constructs of the present mvention

For recombinant production, host cells can be genetically engineered to incorporate expression systems or portions thereof for polynucleotides of the present mvention Introduction of polynucleotides mto host cells can be effected by methods descπbed 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

Representative examples of appropnate hosts mclude bactenal cells, such as streptococci, staphylococci, E cob, Streptomyces and Bacillus subtibs cells, fungal cells, such as yeast cells and Aspergdlus cells, insect cells such as Drosophύa S2 and Spodoptera Sf9 cells, animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells, and plant cells A great vanety of expression systems can be used Such systems mclude, among others, chromosomal, episomal and virus-denved systems, e g , vectors denved from bactenal plasmids, from bactenophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculovmises, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retrovrruses, and vectors denved from combinations thereof, such as those denved from plasmid and bactenophage genetic elements, such as cosmids and phagemids 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 appropπate nucleotide sequence may be inserted mto an expression system by any of a vanety of well-known and routine techniques, such as, for example, those set forth m Sambrook et al , MOLECULAR CLONING, A LABORATORY MANUAL (supra)

For secretion of the translated protem mto the lumen of the endoplasπuc reticulum, mto the peπplasmic space or mto the extracellular environment, appropnate secretion signals may be incorporated mto the desired polypeptide These signals may be endogenous to the polypeptide or they may be heterologous signals

If the dimethylglycme dehydrogenase-like polypeptide is to be expressed for use m screening assays, generally, it is preferred that the polypeptide be produced at the surface of the cell In this event, the cells may be harvested pπor to use in the screening assay If a dimethylglycme dehydrogenase-like polypeptide is secreted mto the medium, the medium can be recovered m order to recover and purify the polypeptide, if produced mtracellularly, the cells must first be lysed before the polypeptide is recovered

Dimethylglycme dehydrogenase-like polypeptides can be recovered and purified from recombinant cell cultures by well-known methods mcludmg 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 chromatography is employed for punfication Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured duπng isolation and or punfication

Diagnostic Assays

This mvention also relates to the use of dimethylglycme dehydrogenase-like polynucleotides for use as diagnostic reagents Detection of a mutated form of a dimethylglycme dehydrogenase-like 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 expression of dimethylglycme dehydrogenase-hke gene Individuals carrying mutations m the dimethylglycme dehydrogenase-like gene may be detected at the DNA level by a vanety of techmques 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 techniques pnor to analysis RNA or cDNA may also be used m similar fashion Deletions and insertions can be detected by a change m size of the amplified product m compaπson to the normal genotype Point mutations can be identified by hybndizing amplified DNA to labeled dimethylglycme dehydrogenase- ke 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 m gels, with or without denaturing agents, or by direct DNA sequencing See, e g , Myers et al , Science (1985) 230 1242 Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and S 1 protection or the chemical cleavage method See Cotton et al , Proc Natl Acad Sci USA ( 1985) 85 4397-4401 In another embodiment, an array of ohgonucleotide probes compnsmg the dimethylglycme dehydrogenase-hke gene nucleotide sequence or fragments thereof can be constructed to conduct efficient screening of e g , genetic mutations Array technology methods are well known and have general applicability and can be used to address a vanety of questions m molecular genetics mcludmg gene expression, genetic linkage, and genetic vanability (See for example M Chee et al , Science, Vol 274, pp 610-613 (1996)) The diagnostic assays offer a process for diagnosing or determinmg a susceptibility to sarcosinemia, cardiomyopathy, retinitis pigmentosa, deafness, neurological disease, cancer, metabolic defects and AIDS through detection of mutation m the dimethylglycme dehydrogenase-like gene by the methods descπbed In addition, sarcosinemia, cardiomyopathy, retinitis pigmentosa, deafness, neurological disease, cancer, metabolic defects and ALDS can be diagnosed by methods compnsmg determining from a sample derived from a subject an abnormally decreased or increased level of dimethylglycme dehydrogenase-like polypeptide or dimethylglycme dehydrogenase-like mRNA Decreased or increased expression can be measured at the RNA level usmg 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 hybndization methods Assay techniques that can be used to determine levels of a protein, such as a dimethylglycme dehydrogenase-hke polypeptide, m a sample denved from a host are well-known to those of skill m the art Such assay methods mclude radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays Thus in another aspect, the present mvention 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 compnses

(a) a dimethylglycme dehydrogenase-like polynucleotide, preferably the nucleotide sequence of SEQ ID NO 1, or a fragment thereof , (b) a nucleotide sequence complementary to that of (a),

(c) a dimethylglycme dehydrogenase-like polypeptide, preferably the polypeptide of SEQ ID NO 2, or a fragment thereof, or

(d) an antibody to a dimethylglycme dehydrogenase-like polypeptide, preferably to the polypeptide of SEQ ID NO. 2 It will be appreciated that m any such kit, (a), (b), (c) or (d) may compnse a substantial component

Chromosome Assays

The nucleotide sequences of the present mvention are also valuable for chromosome identification The sequence is specifically targeted to and can hybndize 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 Dosition of the sequence on the chromosome can be correlated with genetic map data Such data are found, for example, V McKusick, Mende an Inhentance m Man (available on line through Johns Hopkins Umversity Welch Medical Library) The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinhentance 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 not in any normal individuals, then the mutation is likely to be the causative agent of the disease

The dimethylglycme dehydrogenase-like gene is mapped to 9q34 where sarcosinemia was localized

Antibodies

The polypeptides of the mvention or their fragments or analogs thereof, or cells expressmg them can also be used as lmmunogens to produce antibodies immunospecific for the dimethylglycme dehydrogenase-like polypeptides The term "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-hke 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 technique which provides antibodies produced by continuous cell line cultures can be used Examples mclude the hybndoma technique (Kohler, G and Milstein, C , Nature ( 1975) 256495-497), the tπoma technique, the human B-cell hybndoma technique (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)

Techmques for the production of single cham antibodies (U S Patent No 4,946,778) can also be adapted to produce single cham antibodies to polypeptides of this mvention Also, transgemc mice, or other organisms mcludmg other mammals, may be used to express humanized antibodies

The above-descπbed antibodies may be employed to isolate or to identify clones expressing the polypeptide or to purify the polypeptides by affinity chromatography

Antibodies against dimethylglycme dehydrogenase-hke 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 mvention relates to a method for inducing an lmmunological response in a mammal which compnses inoculating the mammal with dimethylglycme dehydrogenase-like polypeptide, or a fragment thereof, adequate to produce antibody and/or T cell immune response to protect said animal from sarcosmemia, cardiomyopathy, retinitis pigmentosa, deafness, neurological disease, cancer, metabolic defects and AIDS, among others Yet another aspect of the invention relates to a method of inducing lmmunological response m a mammal which comprises, dehvenng dimethylglycme dehydrogenase-hke polypeptide via a vector directing expression of dimethylglycme dehydrogenase-like polynucleotide in vivo m order to induce such an lmmunological response to produce antibody to protect said animal from diseases

Further aspect of the invention relates to an lmmunological/vaccine formulation (composition) which, when introduced into a mammalian host, induces an lmmunological response in that mammal to a dimethylglycme dehydrogenase-like polypeptide wherem the composition comprises a dimethylglycme dehydrogenase-hke polypeptide or dimethylglycme dehydrogenase-hke gene The vaccine formulation may further comprise a suitable earner Smce dimethylglycme dehydrogenase-hke polypeptides may be broken down m the stomach, it is preferably administered parenterally (mcludmg subcutaneous, intramuscular, mtravenous, intradermal etc injection) Formulations suitable for parenteral administration mclude aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bactenostats and solutes which render the formulation instonic with the blood of the recipient, and aqueous and non-aqueous stenle suspensions which may include suspending agents or thickening agents The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dπed condition requiring only the addition of the stenle liquid earner immediately prior to use The vaccine formulation may also include adjuvant systems for enhancing the immunogemcity of the formulation, such as oil-in water systems and other systems known in the art The dosage will depend on the specific activity of the vaccine and can be readily determined by routine expenmentation

Screening Assays The dimethylglycme dehydrogenase-hke polypeptide of the present mvention may be employed in a screening process for compounds which activate (agonists) or inhibit activation of (antagonists, or otherwise called inhibitors) the dimethylglycme dehydrogenase-hke polypeptide of the present mvention Thus, polypeptides of the mvention may also be used to assess identify agonist or antagonists from, for example, cells, cell-free preparations, chemical hbranes, and natural product mixtures These agonists or antagonists may be natural or modified substrates, ligands, receptors, enzymes, etc., as the case may be, of the polypeptide of the present invention; or may be structural or functional mimetics of the polypeptide of the present invention. See Coligan et al. , Current Protocols in Immunology l(2):Chapter 5 (1991). Dimethylglycine dehydrogenase-like polypeptides are responsible for many biological functions, including many pathologies. Accordingly, it is desirous to find compounds and drugs which stimulate dimethylglycine dehydrogenase-like polypeptides on the one hand and which can inhibit the function of dimethylglycine dehydrogenase-like polypeptides on the other hand.. In general, agonists are employed for therapeutic and prophylactic purposes for such conditions as sarcosinemia, cardiomyopathy, retinitis pigmentosa, deafness, neurological disease, cancer, metabohc defects and AIDS. Antagonists may be employed for a variety of therapeutic and prophylactic purposes for such conditions as sarcosinemia, cardiomyopathy, retinitis pigmentosa, deafness, neurological disease, cancer, metabolic defects and AIDS.

In general, such screening procedures may involve using appropriate cells which express the dimethylglycine dehydrogenase-like polypeptide or respond to dimethylglycine dehydrogenase-like polypeptides of the present invention. Such cells mclude cells from mammals, yeast, Drosophila or E. coli. Cells which express the dimethylglycine dehydrogenase-like polypeptide (or cell membrane containing the expressed polypeptide) or respond to dimethylglycine dehydrogenase-like polypeptide are then contacted with a test compound to observe binding, or stimulation or inhibition of a functional response. The abihty of the cells which were contacted with the candidate compounds is compared with the same cells which were not contacted for dimethylglycine dehydrogenase-like activity.

The assays may simply test binding of a candidate compound wherein adherence to the cells bearing the dimethylglycine dehydrogenase-like polypeptide is detected by means of a label directly or indirectly associated with the candidate compound or in an assay involving competition with a labeled competitor. Further, these assays may test whether the candidate compound results in a signal generated by activation of the dimethylglycine dehydrogenase-like polypeptide, using detection systems appropriate to the cells bearing the dimethylglycine dehydrogenase-like polypeptide. Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed. Further, the assays may simply comprise the steps of mixing a candidate compound with a solution containing a dimethylglycine dehydrogenase-like polypeptide to form a mixture, measuring dimethylglycine dehydrogenase-like gene activity in the mixture, and comparing the dimethylglycine dehydrogenase-like gene activity of the mixture to a standard. The dimethylglycme dehydrogenase-like gene cDNA, protem and antibodies to the protem may also be used to configure assays for detectmg the effect of added compounds on the production of dimethylglycme dehydrogenase-like gene mRNA and protein in cells For example, an ELISA may be constructed for measuring secreted or cell associated levels of dimethylglycme dehydrogenase-like protein using monoclonal and polyclonal antibodies by standard methods known in the art, and this can be used to discover agents which may inhibit or enhance the production of dimethylglycme dehydrogenase-like gene (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues

The dimethylglycme dehydrogenase-like protein may be used to identify membrane bound or soluble receptors, if any, through standard receptor bmdmg techniques known m the art These include, but are not limited to, hgand bmdmg and crosslinking assays m which the dimethylglycme dehydrogenase-like gene is labeled with a radioactive isotope (eg 1251), chemically modified (eg biotinylated), or fused to a peptide sequence suitable for detection or punfication, and mcubated with a source of the putative receptor (cells, cell membranes, cell supernatants, tissue extracts, bodily fluids) Other methods mclude biophysical techmques such as surface plasmon resonance and spectroscopy In addition to bemg used for punfication and clonmg of the receptor, these binding assays can be used to identify agonists and antagonists of dimethylglycme dehydrogenase- like genes which compete with the binding of the dimethylglycme dehydrogenase-like gene to its receptors, if any Standard methods for conducting screening assays are well understood in the art Examples of potential dimethylglycme dehydrogenase-hke polypeptide antagonists mclude antibodies or, m some cases, o gonucleotides or proteins which are closely related to the hgands, substrates, receptors, enzymes, etc , as the case may be, of the dimethylglycme dehydrogenase-hke polypeptide, e g , a fragment of the hgands, substrates, receptors, enzymes, etc , or small molecules which bmd to the polypetide of the present mvention but do not elicit a response, so that the activity of the polypeptide is prevented

Thus in another aspect, the present invention relates to a screening kit for identifying agomsts, antagonists, hgands, receptors, substrates, enzymes, etc for dimethylglycme dehydrogenase-like polypeptides, or compounds which decrease or enhance the production of dimethylglycme dehydrogenase-like polypeptides, which compnses (a) a dimethylglycme dehydrogenase-hke polypeptide, preferably that of SEQ ID NO 2,

(b) a recombinant cell expressing a dimethylglycme dehydrogenase-like polypeptide, preferably that of SEQ ID N0 2,

(c) a cell membrane expressing a dimethylglycme dehydrogenase-like polypeptide, preferably that of SEQ ID NO 2, or (d) antibody to a dimethylglycme dehydrogenase-hke polypeptide. preferably that of SEQ ID NO 2 It will be appreciated that in any such kit, (a), (b), (c) or (d) may comprise a substantial component

Prophylactic and Therapeutic Methods

This mvention provides methods of treating abnormal conditions such as, sarcosmemia, cardiomyopathy, retinitis pigmentosa, deafness, neurological disease, cancer, metabohc defects and AIDS, related to both an excess of and insufficient amounts of dimethylglycme dehydrogenase-like polypeptide activity If the activity of dimethylglycme dehydrogenase-like polypeptide is m excess, several approaches are available One approach compnses administering to a subject an inhibitor compound (antagonist) as hereinabove descπbed along with a pharmaceutically acceptable earner m an amount effective to inhibit the function of the dimethylglycme dehydrogenase-hke polypeptide, such as, for example, by blocking the bmdmg of hgands, substrates, receptors, enzymes, etc , or by inhibiting a second signal, and thereby alleviating the abnormal condition In another approach, soluble forms of dimethylglycme dehydrogenase-hke polypeptides still capable of bmdmg the hgand, substrate, enzymes, receptors, etc m competition with endogenous dimethylglycme dehydrogenase-hke polypeptides may be administered Typical embodiments of such competitors compnse fragments of the dimethylglycme dehydrogenase-hke polypeptide In still another approach, expression of the gene encodmg endogenous dimethylglycme dehydrogenase-like polypeptides can be inhibited usmg expression blocking techmques 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 in Ohgodeoxynucleotides as Antisense Inhibitors of Gene Expression. CRC Press, Boca Raton, FL (1988) Alternatively, ohgonucleotides which form tnple 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 oligomers can be administered per se or the relevant oligomers can be expressed in vivo

For treating abnormal conditions related to an under-expression of dimethylglycme dehydrogenase-hke gene and its activity, several approaches are also available One approach compnses administering to a subject a therapeutically effective amount of a compound which activates the dimethylglycme dehydrogenase-like polypeptide, 1 e , an agonist as descπbed above, m combination with a pharmaceutically acceptable earner, to thereby alleviate the abnormal condition Alternatively, gene therapy may be employed to effect the endogenous production of dimethylglycme dehydrogenase- ke gene by the relevant cells in the subject For example, a polynucleotide of the mvention may be engmeered for expression a replication defective retroviral vector, as discussed above The retroviral expression construct may then be isolated and introduced mto a packagmg cell transduced with a retroviral plasmid vector contammg RNA encoding a polypeptide of the present mvention such that the packagmg cell now produces infectious viral particles contammg the gene of mterest These producer cells may be administered to a subject for engineering cells in vivo and expression of the polypeptide in vivo For overview of gene therapy, see Chapter 20, Gene Therapy and other Molecular Genetic-based Therapeutic Approaches, (and references cited therein) m Human Molecular Genetics, T Strachan and A P Read, BIOS Scientific Publishers Ltd (1996) Another approach is to administer a therapeutic amount of dimethylglycme dehydrogenase-like polypeptides m combination with a suitable pharmaceutical earner

Formulation and Administration

Peptides, such as the soluble form of dimethylglycme dehydrogenase-like polypeptides, and agomsts and antagomst peptides or small molecules, may be formulated in combmation with a suitable pharmaceutical earner Such formulations compnse a therapeutically effective amount of the polypeptide or compound, and a pharmaceutically acceptable earner or excipient Such 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 mvention 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 intravenous injection Other injection routes, such as subcutaneous, intramuscular, or lntrapeπtoneal, 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 In addition, if properly formulated in enteπc or encapsulated formulations, 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 in the range of 0 1 - 100 μg/kg of subject Wide vanations m the needed dosage, however, are to be expected in view of the vanety of compounds available and the differing efficiencies of vanous routes of administration For example, oral admmistration would be expected to require higher dosages than admmistration by intravenous injection Variations m these 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 refened to as "gene therapy" as descπbed above Thus, for example, 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 mtroduced mto the subject

All publications, mcludmg but not limited to patents and patent applications, cited m this specification are herem mcorporated by reference as if each mdividual publication were specifically and individually indicated to be incorporated by reference herem as though fully set forth

Claims

What is claimed is:

1 An isolated polynucleotide compnsmg a nucleotide sequence that has at least 80% identity over its entire length to a nucleotide sequence encoding the dimethylglycme dehydrogenase-like polypeptide of SEQ ID NO 2, or a nucleotide sequence complementary to said isolated polynucleotide

2 The polynucleotide of claim 1 wherein said polynucleotide compnses the nucleotide sequence contained m SEQ ID NO 1 encoding the dimethylglycme dehydrogenase-like polypeptide of SEQ ID N02

3 The polynucleotide of claim 1 wherem said polynucleotide compnses a nucleotide sequence that is at least 80% identical to that of SEQ ID NO 1 over its entire length

4 The polynucleotide of claim 3 which is polynucleotide of SEQ ID NO 1

5 The polynucleotide of claim 1 which is DNA or RNA

6 A DNA or RNA molecule compnsmg an expression system, wherem said expression system is capable of producmg a dimethylglycme dehydrogenase-hke polypeptide compnsmg an ammo acid sequence, which has at least 80% identity with the polypeptide of SEQ ID NO 2 when said expression system is present m a compatible host cell

7 A host cell comprising the expression system of claim 6

8 A process for producing a dimethylglycme dehydrogenase-like polypeptide comprising cultuπng a host of claim 7 under conditions sufficient for the production of said polypeptide and recovering the polypeptide from the culture

9 A process for producmg a cell which produces a dimethylglycme dehydrogenase-hke polypeptide thereof compnsmg transformmg or transfectmg a host cell with the expression system of claim 6 such that the host cell, under appropnate culture conditions, produces a dimethylglycme dehydrogenase-hke polypeptide

10 A dimethylglycme dehydrogenase-like polypeptide compnsmg an ammo acid sequence which is at least 80% identical to the ammo acid sequence of SEQ ID NO 2 over its entire length

11 The polypeptide of claim 10 which compnses the ammo acid sequence of SEQ ID

NO 2

12 An antibody immunospecific for the dimethylglycme dehydrogenase-like polypeptide of claim 10

13 A method for the treatment of a subject in need of enhanced activity or expression of the dimethylglycme dehydrogenase-like polypeptide of claim 10 compnsmg

(a) admimstenng to the subject a therapeutically effective amount of an agomst to said polypeptide, and or (b) providing to the subject an isolated polynucleotide compnsmg a nucleotide sequence that has at least 80% identity to a nucleotide sequence encodmg the dimethylglycme dehydrogenase-like polypeptide of SEQ ID NO 2 over its entire length, or a nucleotide sequence complementary to said nucleotide sequence m a form so as to effect production of said polypeptide activity in vivo

14 A method for the treatment of a subject havmg need to inhibit activity or expression of the dimethylglycme dehydrogenase-hke polypeptide of claim 10 compnsmg

(a) admimstenng to the subject a therapeutically effective amount of an antagomst to said polypeptide, and/or

(b) administering to the subject a nucleic acid molecule that inhibits the expression of the nucleotide sequence encoding said polypeptide, and/or

(c) admimstenng to the subject a therapeutically effective amount of a polypeptide that competes with said polypeptide for its hgand, substrate , or receptor

15 A process for diagnosing a disease or a susceptibility to a disease m a subject related to expression or activity of the dimethylglycme dehydrogenase-like polypeptide of claim 10 in a subject compnsmg

(a) determining the presence or absence of a mutation m the nucleotide sequence encoding said dimethylglycme dehydrogenase-hke polypeptide m the genome of said subject, and/or (b) analyzing for the presence or amount of the dimethylglycine dehydrogenase-like polypeptide expression in a sample derived from said subject.

16. A method for identifying compounds which inhibit (antagonize) or agonize the dimethylglycine dehydrogenase-like polypeptide of claim 10 which comprises :

(a) contacting a candidate compound with cells which express the dimethylglycine dehydrogenase-like polypeptide (or cell membrane expressing dimethylglycine dehydrogenase-like polypeptide) or respond to the dimethylglycine dehydrogenase-like polypeptide; and

(b) observing the binding, or stimulation or inhibition of a functional response; or comparing the abihty of the cells (or cell membrane) which were contacted with the candidate compounds with the same cells which were not contacted for dimethylglycine dehydrogenase-like polypeptide activity.

17. An agonist identified by the method of claim 16.

18. An antagonist identified by the method of claim 16.

19. A recombinant host cell produced by a method of Claim 9 or a membrane thereof expressing a dimethylglycine dehydrogenase-like polypeptide.