Human Methionine Synthase
1997; Elsevier BV; Volume: 272; Issue: 6 Linguagem: Inglês
10.1074/jbc.272.6.3628
ISSN1083-351X
AutoresLinda H. Chen, Mei‐Lan Liu, Hye‐Yeon Hwang, Sam Li‐Sheng Chen, Julie R. Korenberg, Barry Shane,
Tópico(s)Metabolism and Genetic Disorders
ResumoHuman cDNAs for methionine synthase (5-methyltetrahydrofolate:L-homocysteine S-transmethylase; EC 2.1.1.13) have been isolated from fetal and adult liver and HepG2 libraries. The cDNAs span 7.2 kilobases (kb) and consist of a 394-base pair upstream untranslated region, a 3795-base pair open reading frame encoding a 1265-residue 140.3-kDa protein, and about 3 kb of 3′ region. The deduced protein sequence shares 53 and 63% identity with the Escherichia coli and the presumptive Caenorhabditis elegans proteins, respectively, and contains all residues implicated in B12 binding to the E. coli protein. Several potential polymorphisms and a cryptic splice deletion were detected in the coding region of the cDNAs. A polymorphism that results in a D919G modification in the protein is fairly common in human DNA samples. Northern analyses of poly(A) mRNA indicated two major species of about 8 and 10 kb in human tissues and some minor, partially spliced species. mRNA levels were highest in the pancreas, skeletal muscle, and heart of the adult and in the kidney in the fetus and were low in adult liver. Genomic clones were isolated and the 5′ region was analyzed. Exon 1 is preceded by a number of potential promoter sites, including an E box, CAAT boxes, and a GC box, but this region lacks a TATA element. The human methionine synthase gene was localized to chromosome region 1q42.3-43 by in situ hybridization. Human cDNAs for methionine synthase (5-methyltetrahydrofolate:L-homocysteine S-transmethylase; EC 2.1.1.13) have been isolated from fetal and adult liver and HepG2 libraries. The cDNAs span 7.2 kilobases (kb) and consist of a 394-base pair upstream untranslated region, a 3795-base pair open reading frame encoding a 1265-residue 140.3-kDa protein, and about 3 kb of 3′ region. The deduced protein sequence shares 53 and 63% identity with the Escherichia coli and the presumptive Caenorhabditis elegans proteins, respectively, and contains all residues implicated in B12 binding to the E. coli protein. Several potential polymorphisms and a cryptic splice deletion were detected in the coding region of the cDNAs. A polymorphism that results in a D919G modification in the protein is fairly common in human DNA samples. Northern analyses of poly(A) mRNA indicated two major species of about 8 and 10 kb in human tissues and some minor, partially spliced species. mRNA levels were highest in the pancreas, skeletal muscle, and heart of the adult and in the kidney in the fetus and were low in adult liver. Genomic clones were isolated and the 5′ region was analyzed. Exon 1 is preceded by a number of potential promoter sites, including an E box, CAAT boxes, and a GC box, but this region lacks a TATA element. The human methionine synthase gene was localized to chromosome region 1q42.3-43 by in situ hybridization. INTRODUCTIONMethionine synthase, one of two B12-dependent mammalian enzymes, catalyzes the remethylation of homocysteine to methionine and the concurrent demethylation of 5-methyltetrahydrofolate to tetrahydrofolate (1Matthews R.G. Blakley R.L. Benkovik S.J. Folates and Pterins.Wiley Interscience. 1984; 1: 497-553Google Scholar). Under conditions of B12-depletion, such as pernicious anemia, loss of methionine synthase activity leads to a "methyl folate trap." The depletion of other folate coenzymes results in defective DNA synthesis and the development of megaloblastic anemia (1Matthews R.G. Blakley R.L. Benkovik S.J. Folates and Pterins.Wiley Interscience. 1984; 1: 497-553Google Scholar, 2Shane B. Stokstad E.L.R. Annu. Rev. Nutr. 1985; 5: 115-141Crossref PubMed Scopus (266) Google Scholar, 3Shane B. Vitam. Horm. 1989; 45: 263-335Crossref PubMed Scopus (289) Google Scholar). Recently, homocysteine has received considerable attention as elevations in plasma homocysteine have been implicated as a risk factor for vascular disease (4Brattstrom L. Lindgren A. Israelsson B. Malinow M.R. Norrving B. Upson B. Hamfelt A. Eur. J. Clin. Invest. 1992; 22: 214-221Crossref PubMed Scopus (259) Google Scholar, 5Selhub J. Jacques P.F. Bostom A.G. D'Agostino R.B. Wilson P.W. Belanger A.J. O'Leary D.H. Wolf P.A. Schaefer E.J. Rosenberg I.H. New Engl. J. Med. 1995; 332: 286-291Crossref PubMed Scopus (1141) Google Scholar). Polymorphisms in methylenetetrahydrofolate reductase, the enzyme that catalyzes the synthesis of 5-methyltetrahydrofolate, and in cystathionine β-synthase, which catalyzes the removal of homocysteine via the transsulfuration pathway, have been implicated in elevated homocysteine levels and in vascular disease risk (6Frost P. Blom H.J. Milos R. Goyette P. Sheppard C.A. Matthews R.G. Boers G.J.H. den Heijer M. Kluijtmans L.A.J. van den Heuval L.P. Rozen R. Nat. Genet. 1995; 10: 111-113Crossref PubMed Scopus (5085) Google Scholar, 7Jacques P.F. Bostom A.G. Williams R.R. Ellison R.C. Eckfeldt J.H. Rosenberg I.H. Selhub J. Rozen R. Circulation. 1996; 93: 7-9Crossref PubMed Scopus (1269) Google Scholar).Little is known about the regulation or properties of eukaryotic methionine synthases, partly because of the very limited distribution of B12-dependent enzymes in eukaryotes. The Escherichia coli methionine synthase gene has been cloned and the protein purified to homogeneity, and the structure of its B12-binding domain has been elucidated (8Banerjee R.V. Johnston N.L. Sobeski J.K. Datta P. Matthews R.G. J. Biol. Chem. 1989; 264: 13888-13895Abstract Full Text PDF PubMed Google Scholar, 9Drennan C.L. Huang S. Drummond J.T. Matthews R.G. Ludwig M.L. Science. 1994; 266: 1669-1674Crossref PubMed Scopus (555) Google Scholar, 10Jarrett J.T. Amaratunga M. Drennan C.L. Scholten J.D. Sands R.H. Ludwig M.L. Matthews R.G. Biochemistry. 1996; 35: 2464-2475Crossref PubMed Scopus (86) Google Scholar). Other bacterial genes and the Caenorhabditis elegans methionine synthase gene have been tentatively identified by homology to the E. coli gene (Ref. 11Wilson R. Ainscough R. Anderson K. Baynes C. Berks M. Bonfield J. Burton J. Connell M. Copsey T. Cooper J. Coulson A. Craxton M. Dear S. Du Z. Durbin R. Favello A. Fulton L. Gardner A. Green P. Hawkins T. Hillier L. Jier M. Johnston L. Jones M. Kershaw J. Kirsten J. Laister N. Latreille P. Lightning J. Lloyd C. McMurray A. Mortimore B. O'Callaghan M. Parsons J. Percy C. Rifken L. Roopra A. Saunders D. Shownkeen R. Smaldon N. Smith A. Sonnhammer E. Staden R. Sulston J. Thierry-Mieg J. Thomas K. Vaudin M. Vaughan K. Waterston R. Watson A. Weinstock L. Wilkinson-Sproat J. Wohldman P. Nature. 1994; 368: 32-38Crossref PubMed Scopus (1439) Google Scholar; accession number Z46828). The pig liver enzyme has recently been purified to near homogeneity and some of its kinetic properties have been characterized (12Chen Z. Crippen K. Gulati S. Banerjee R. J. Biol. Chem. 1994; 269: 27193-27197Abstract Full Text PDF PubMed Google Scholar). We are interested in the metabolic control of the folate-dependent methionine resynthesis pathway in mammalian tissues and the potential role of polymorphisms in the enzymes involved in this cycle in disturbances of one-carbon metabolism. As a prelude we have isolated and characterized various human methionine synthase cDNAs. In this report, we describe the molecular cloning of human methionine synthase cDNAs and the localization, expression, and partial characterization of its gene. INTRODUCTIONMethionine synthase, one of two B12-dependent mammalian enzymes, catalyzes the remethylation of homocysteine to methionine and the concurrent demethylation of 5-methyltetrahydrofolate to tetrahydrofolate (1Matthews R.G. Blakley R.L. Benkovik S.J. Folates and Pterins.Wiley Interscience. 1984; 1: 497-553Google Scholar). Under conditions of B12-depletion, such as pernicious anemia, loss of methionine synthase activity leads to a "methyl folate trap." The depletion of other folate coenzymes results in defective DNA synthesis and the development of megaloblastic anemia (1Matthews R.G. Blakley R.L. Benkovik S.J. Folates and Pterins.Wiley Interscience. 1984; 1: 497-553Google Scholar, 2Shane B. Stokstad E.L.R. Annu. Rev. Nutr. 1985; 5: 115-141Crossref PubMed Scopus (266) Google Scholar, 3Shane B. Vitam. Horm. 1989; 45: 263-335Crossref PubMed Scopus (289) Google Scholar). Recently, homocysteine has received considerable attention as elevations in plasma homocysteine have been implicated as a risk factor for vascular disease (4Brattstrom L. Lindgren A. Israelsson B. Malinow M.R. Norrving B. Upson B. Hamfelt A. Eur. J. Clin. Invest. 1992; 22: 214-221Crossref PubMed Scopus (259) Google Scholar, 5Selhub J. Jacques P.F. Bostom A.G. D'Agostino R.B. Wilson P.W. Belanger A.J. O'Leary D.H. Wolf P.A. Schaefer E.J. Rosenberg I.H. New Engl. J. Med. 1995; 332: 286-291Crossref PubMed Scopus (1141) Google Scholar). Polymorphisms in methylenetetrahydrofolate reductase, the enzyme that catalyzes the synthesis of 5-methyltetrahydrofolate, and in cystathionine β-synthase, which catalyzes the removal of homocysteine via the transsulfuration pathway, have been implicated in elevated homocysteine levels and in vascular disease risk (6Frost P. Blom H.J. Milos R. Goyette P. Sheppard C.A. Matthews R.G. Boers G.J.H. den Heijer M. Kluijtmans L.A.J. van den Heuval L.P. Rozen R. Nat. Genet. 1995; 10: 111-113Crossref PubMed Scopus (5085) Google Scholar, 7Jacques P.F. Bostom A.G. Williams R.R. Ellison R.C. Eckfeldt J.H. Rosenberg I.H. Selhub J. Rozen R. Circulation. 1996; 93: 7-9Crossref PubMed Scopus (1269) Google Scholar).Little is known about the regulation or properties of eukaryotic methionine synthases, partly because of the very limited distribution of B12-dependent enzymes in eukaryotes. The Escherichia coli methionine synthase gene has been cloned and the protein purified to homogeneity, and the structure of its B12-binding domain has been elucidated (8Banerjee R.V. Johnston N.L. Sobeski J.K. Datta P. Matthews R.G. J. Biol. Chem. 1989; 264: 13888-13895Abstract Full Text PDF PubMed Google Scholar, 9Drennan C.L. Huang S. Drummond J.T. Matthews R.G. Ludwig M.L. Science. 1994; 266: 1669-1674Crossref PubMed Scopus (555) Google Scholar, 10Jarrett J.T. Amaratunga M. Drennan C.L. Scholten J.D. Sands R.H. Ludwig M.L. Matthews R.G. Biochemistry. 1996; 35: 2464-2475Crossref PubMed Scopus (86) Google Scholar). Other bacterial genes and the Caenorhabditis elegans methionine synthase gene have been tentatively identified by homology to the E. coli gene (Ref. 11Wilson R. Ainscough R. Anderson K. Baynes C. Berks M. Bonfield J. Burton J. Connell M. Copsey T. Cooper J. Coulson A. Craxton M. Dear S. Du Z. Durbin R. Favello A. Fulton L. Gardner A. Green P. Hawkins T. Hillier L. Jier M. Johnston L. Jones M. Kershaw J. Kirsten J. Laister N. Latreille P. Lightning J. Lloyd C. McMurray A. Mortimore B. O'Callaghan M. Parsons J. Percy C. Rifken L. Roopra A. Saunders D. Shownkeen R. Smaldon N. Smith A. Sonnhammer E. Staden R. Sulston J. Thierry-Mieg J. Thomas K. Vaudin M. Vaughan K. Waterston R. Watson A. Weinstock L. Wilkinson-Sproat J. Wohldman P. Nature. 1994; 368: 32-38Crossref PubMed Scopus (1439) Google Scholar; accession number Z46828). The pig liver enzyme has recently been purified to near homogeneity and some of its kinetic properties have been characterized (12Chen Z. Crippen K. Gulati S. Banerjee R. J. Biol. Chem. 1994; 269: 27193-27197Abstract Full Text PDF PubMed Google Scholar). We are interested in the metabolic control of the folate-dependent methionine resynthesis pathway in mammalian tissues and the potential role of polymorphisms in the enzymes involved in this cycle in disturbances of one-carbon metabolism. As a prelude we have isolated and characterized various human methionine synthase cDNAs. In this report, we describe the molecular cloning of human methionine synthase cDNAs and the localization, expression, and partial characterization of its gene.
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