FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1990152592> ?p ?o ?g. }

• W1990152592 endingPage "24468" @default.
• W1990152592 startingPage "24461" @default.
• W1990152592 abstract "Malonyl-CoA decarboxylase (MCD) catalyzes the proton-consuming conversion of malonyl-CoA to acetyl-CoA and CO2. Although defects in MCD activity are associated with malonyl-CoA decarboxylase deficiency, a lethal disorder characterized by cardiomyopathy and developmental delay, the metabolic role of this enzyme in mammals is unknown. A computer-based search for novel peroxisomal proteins led to the identification of a candidate gene for human MCD, which encodes a protein with a canonical type-1 peroxisomal targeting signal of serine-lysine-leucineCOOH. We observed that recombinant MCD protein has high intrinsic malonyl-CoA decarboxylase activity and that a malonyl-CoA decarboxylase-deficient patient has a severe mutation in the MCD gene (c.947–948delTT), confirming that this gene encodes human MCD. Subcellular fractionation experiments revealed that MCD resides in both the cytoplasm and peroxisomes. Cytoplasmic MCD is positioned to play a role in the regulation of cytoplasmic malonyl-CoA abundance and, thus, of mitochondrial fatty acid uptake and oxidation. This hypothesis is supported by the fact that malonyl-CoA decarboxylase-deficient patients display a number of phenotypes that are reminiscent of mitochondrial fatty acid oxidation disorders. Additional support for this hypothesis comes from our observation that MCD mRNA is most abundant in cardiac and skeletal muscles, tissues in which cytoplasmic malonyl-CoA is a potent inhibitor of mitochondrial fatty acid oxidation and which derive significant amounts of energy from fatty acid oxidation. As for the role of peroxisomal MCD, we propose that this enzyme may be involved in degrading intraperoxisomal malonyl-CoA, which is generated by the peroxisomal β-oxidation of odd chain-length dicarboxylic fatty acids. Malonyl-CoA decarboxylase (MCD) catalyzes the proton-consuming conversion of malonyl-CoA to acetyl-CoA and CO2. Although defects in MCD activity are associated with malonyl-CoA decarboxylase deficiency, a lethal disorder characterized by cardiomyopathy and developmental delay, the metabolic role of this enzyme in mammals is unknown. A computer-based search for novel peroxisomal proteins led to the identification of a candidate gene for human MCD, which encodes a protein with a canonical type-1 peroxisomal targeting signal of serine-lysine-leucineCOOH. We observed that recombinant MCD protein has high intrinsic malonyl-CoA decarboxylase activity and that a malonyl-CoA decarboxylase-deficient patient has a severe mutation in the MCD gene (c.947–948delTT), confirming that this gene encodes human MCD. Subcellular fractionation experiments revealed that MCD resides in both the cytoplasm and peroxisomes. Cytoplasmic MCD is positioned to play a role in the regulation of cytoplasmic malonyl-CoA abundance and, thus, of mitochondrial fatty acid uptake and oxidation. This hypothesis is supported by the fact that malonyl-CoA decarboxylase-deficient patients display a number of phenotypes that are reminiscent of mitochondrial fatty acid oxidation disorders. Additional support for this hypothesis comes from our observation that MCD mRNA is most abundant in cardiac and skeletal muscles, tissues in which cytoplasmic malonyl-CoA is a potent inhibitor of mitochondrial fatty acid oxidation and which derive significant amounts of energy from fatty acid oxidation. As for the role of peroxisomal MCD, we propose that this enzyme may be involved in degrading intraperoxisomal malonyl-CoA, which is generated by the peroxisomal β-oxidation of odd chain-length dicarboxylic fatty acids. malonyl-CoA decarboxylase carnitine palmitoyltransferase dicarboxylic fatty acid expressed sequence tag base pair(s) open reading frame polymerase chain reaction maltose-binding protein rapid amplification of cDNA ends type-1 peroxisomal targeting signal Malonyl-CoA decarboxylase activity (EC 4.1.1.9, SchemeFS1) has been described in a wide array of organisms, including prokaryotes, birds, and mammals (1Kim Y.S. Kolattukudy P.E. Arch. Biochem. Biophys. 1978; 190: 234-246Crossref PubMed Scopus (63) Google Scholar, 2Kim Y.S. Kolattukudy P.E. Arch. Biochem. Biophys. 1978; 190: 585-597Crossref PubMed Scopus (45) Google Scholar, 3An J.H. Kim Y.S. Eur. J. Biochem. 1998; 257: 395-402Crossref PubMed Scopus (108) Google Scholar). However, the physiological role of this enzyme is somewhat unclear. The only eukaryotic malonyl-CoA decarboxylase (MCD)1 gene that has been cloned was goose (Anser anser), which expresses two MCD transcripts from a single gene (4Jang S.H. Cheesbrough T.M. Kolattukudy P.E. J. Biol. Chem. 1989; 264: 3500-3505Abstract Full Text PDF PubMed Google Scholar). The longer of the two transcripts is ubiquitously expressed and encodes a mitochondrial enzyme, whereas the shorter form has a more restricted pattern of expression and encodes a cytosolic enzyme. The mitochondrial localization of goose MCD suggested that this enzyme may function in removing intramitochondrial malonyl-CoA that is produced by the adventitious activity of propionyl-CoA carboxylase on acetyl-CoA. As for the cytosolic form of the enzyme, it was hypothesized to participate in the synthesis of methyl branched-chain fatty acids (5Courchesne-Smith C. Jang S.-H. Shi Q. DeWille J. Sasaki G. Kolattukudy P.E. Arch. Biochem. Biophys. 1992; 298: 576-586Crossref PubMed Scopus (29) Google Scholar). Although these hypotheses may explain the localization and function of goose MCD, metabolic studies raise the possibility that there are additional physiologically relevant roles for this enzyme. In mammals, cytoplasmic malonyl-CoA is a potent inhibitor of carnitine palmitoyltransferase (CPT1) and, thus, of mitochondrial fatty acid oxidation (6McGarry J.D. Brown N.F. Eur. J. Biochem. 1997; 244: 1-14Crossref PubMed Scopus (1327) Google Scholar). In adipogenic tissues such as liver and white fat, acetyl-CoA carboxylase produces cytoplasmic malonyl-CoA under fed conditions as a precursor for fatty acid synthesis, and fatty acid synthase consumes this cytoplasmic pool of malonyl-CoA. However, nonadipogenic tissues such as cardiac and skeletal muscle also produce significant quantities of cytoplasmic malonyl-CoA from acetyl-CoA carboxylase (7Ha J. Lee J.-K. Kim K.-S. Witters L.A. Kim K.-H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11466-11470Crossref PubMed Scopus (132) Google Scholar), and the inhibitory effects of malonyl-CoA on CPT1 are roughly 100-fold greater in these tissues than in liver (6McGarry J.D. Brown N.F. Eur. J. Biochem. 1997; 244: 1-14Crossref PubMed Scopus (1327) Google Scholar). The synthesis of cytoplasmic malonyl-CoA by acetyl-CoA carboxylase in muscle is tightly regulated by the AMP-dependent protein kinase (8Vavvas D. Apazdis A. Saha A.K. Gamble J. Patel A. Kemp B.E. Witters L.A. Ruderman N.B. J. Biol. Chem. 1997; 272: 13255-13261Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar), which inactivates acetyl-CoA carboxylase and blocks malonyl-CoA synthesis under conditions which require that muscle use fatty acids as their primary energy source (i.e. fasted and/or exercised states). However, there is currently no model for how these tissues degrade the cytoplasmic malonyl-CoA that is produced by acetyl-CoA carboxylase, even though this process is critical for the activation of mitochondrial fatty acid oxidation. One possible model is that a cytoplasmic form of MCD could act to remove cytoplasmic malonyl-CoA under conditions that inhibit acetyl-CoA carboxylase, thereby allowing mitochondrial fatty acid oxidation to resume. An additional role for MCD could be in the oxidation of dicarboxylic fatty acids (DFAs). In contrast to long and medium chain fatty acids, which are oxidized primarily in the mitochondria, DFAs are oxidized only in peroxisomes (9Suzuki H. Yamada J. Watanabe T. Suga T. Biochim. Biophys. Acta. 1989; 990: 25-30Crossref PubMed Scopus (56) Google Scholar). DFA oxidation is poorly understood, and it is not clear whether peroxisomes degrade DFAs completely to malonyl-CoA (for odd chain-length DFAs) and oxalyl-CoA (for even chain-length DFAs). However, if the β-oxidation of DFAs in peroxisomes is complete, a peroxisomal form of MCD could function to eliminate this metabolic end-product of odd chain-length DFA oxidation. A key role for malonyl-CoA decarboxylase in mammalian metabolism is suggested by the severe phenotypes of patients who lack this enzyme activity. Malonyl-CoA decarboxylase deficiency, also known as malonic aciduria, is a genetic disorder characterized by developmental delay, cardiomyopathy, mental retardation, and in its more severe forms, neonatal death (10Brown G.K. Scholem R.D. Bankier A. Danks D.M. J. Inherit. Metab. Dis. 1984; 7: 21-26Crossref PubMed Scopus (59) Google Scholar, 11Haan E.A. Scholem R.D. Croll H.B. Brown G.K. Eur. J. Pediatr. 1986; 144: 567-570Crossref PubMed Scopus (41) Google Scholar, 12Yano S. Sweetman L. Thorburn D.R. Modifi S. Williams J.C. Eur. J. Pediatr. 1997; 156: 382-383Crossref PubMed Scopus (43) Google Scholar, 13MacPhee G. Logan R. Mitchell J. Howells D. Tsotsis E. Thorburn D. Arch. Dis. Child. 1993; 69: 433-436Crossref PubMed Scopus (33) Google Scholar, 14Matalon R. Michaels K. Kaul R. Whitman V. Rodriguez-Novo J. Goodman S. Thorburn D. J. Inherit. Metab. Dis. 1993; 16: 571-573Crossref PubMed Scopus (40) Google Scholar). These patients have several phenotypes that are reminiscent of mitochondrial fatty acid oxidation deficiencies, including diet-induced and infection-induced vomiting, seizures, hypoglycemia, and organic aciduria, as well as cardiomyopathy. Here we report the identification of a novel human gene encoding malonyl-CoA decarboxylase. Its role in malonic aciduria, its pattern of expression, the subcellular distribution of its product, and its possible roles in cellular metabolism are discussed. We searched the ExPASy TrEMBL/EMBL data bases for all proteins terminating in possible forms of the PTS1. This data base scan identified goose malonyl-CoA decarboxylase as a candidate peroxisomal protein because it ends in the canonical PTS1 of serine-lysine-leucineCOOH (15Gould S.J. Keller G.A. Hosken N. Wilkinson J. Subramani S. J. Cell Biol. 1989; 108: 1657-1664Crossref PubMed Scopus (880) Google Scholar). To determine whether the human gene also encoded a protein with a PTS1, and thus might encode a peroxisomal protein, we searched for human homologues of goose MCD. The goose MCD protein sequence was used as query in a BLAST search of the human data base of expressed sequence tags (ESTs) for any genes that had the potential to encode proteins with high sequence similarity to goose MCD. Multiple human ESTs were identified, all of which appeared to represent a single human gene. The longest of these at the time, GenBankTM accession number N52074, corresponded to the cDNA clone 282566 and was obtained from Genome Systems (St. Louis, MO). This clone was sequenced in its entirety. Based on its similarity to the goose isoforms of MCD, this human MCD clone lacked the first 170 codons relative to goose cytosolic MCD and the first 230 codons relative to goose mitochondrial MCD. To obtain additional sequence data for the 5′ end of the human MCD cDNA, we used an MCD-specific primer (5′-CTCCTTGACGACTCGCTTTATGAGG-3′) and a library vector-specific primer (5′-ATACCATTACAATGGATG-3′) to amplify 5′ fragments of the MCD gene from a human heart cDNA library (MCD expression is higher in muscle than in any other tissues). The largest band that was generated was approximately 900-bp long. Subcloning and sequencing of this clone revealed that it overlapped with our truncated MCD cDNA clone and added about 600 bp to the 5′ end of the MCD sequence. The assembled 2,121-bp MCD sequence contained a 95-bp 5′ untranslated region, a 1,362-bp open reading frame (ORF), and a 664-bp 3′ untranslated region. Its deduced protein product of 454 amino acids initiated at the same relative position as the goose cytosolic MCD (5Courchesne-Smith C. Jang S.-H. Shi Q. DeWille J. Sasaki G. Kolattukudy P.E. Arch. Biochem. Biophys. 1992; 298: 576-586Crossref PubMed Scopus (29) Google Scholar). Our most recent data base searches revealed the presence of 34 ESTs for human MCD. None of these are as long as our assembled “full-length”MCD cDNA. However, four of these (GenBankTMaccession numbers AI1284158, AI379572, AI123407, and AA015957) do appear to contain the entire MCD ORF and start at positions −50, −32, −31, and −1, respectively, relative to the A of the putative initiator ATG in the sequence we have assembled. We also identified a human genomic DNA clone for the MCD locus by screening a bacterial artificial chromosome library of human genomic DNAs with a 190-bp probe from the 5′ end of the MCD cDNA. An 8-kilobase Xba I fragment containing the 5′ end of the MCD gene was generated from the MCD bacterial artificial chromosome clone, and the structure of the MCD gene upstream of the 5′ end of the MCD cDNA was determined by sequence analysis of this DNA fragment. An oligonucleotide corresponding to the 22 bp immediately upstream of the 5′ end of the MCD cDNA (5′-ACCATGCGAGGCTTCGGGGCCAGGCTTG-3′) was used in PCR reactions to test for the expression of a longer cDNA product that might contain a second, upstream ATG. 5′-RACE experiments were also performed but failed to generate any MCD cDNA clones longer than the cDNA reported here. Additional BLAST sequence searches were used to identify a putative Caenorhabditis elegans form of MCD (GenBank accession number Z46242). A portion of the MCD ORF encoding amino acids 187 to 451 was amplified using the primers 5′-CCAGTCGACGGACATGAAGCGCCGCGTTGGGCC-3′ and 5′-CCAAGCGGCCGCTCAAAGCTTCCAGTTCTTTTGAAACTGGGCCACTAGGC-3′ and the cDNA clone 282566 as template. The resulting PCR product was cleaved with Sal I and Not I and cloned between the Sal I and Not I sites of pMBP, a variant of pMALc2 (New England Biolabs) that contains unique Sal I and Not I sites for insertion of fragments downstream of the maltose-binding protein (MBP) ORF (16Geisbrecht B.V. Zhu D. Schulz K. Nau K. Morrell J.C. Geraghty M. Schulz H. Erdmann R. Gould S.J. J. Biol. Chem. 1998; 273: 33184-33191Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). The resulting plasmid, pMBP-MCD/187–451, was the source for a recombinant MCD fragment that was used for immunization of rabbits and generation of affinity purified anti-MCD antibodies. Another set of primers (5′-CCAGTCGACGATGGACGAGCTGCTGCGCCG-3′ and 5′-CCAAGCGGCCGCTCAAAGCTTCGAGTTCTTTTGAAACTGGGCCACTAGGC-3′) was used to amplify the entire MCD ORF in the correct reading frame for fusion to MBP in pMBP. The template for this reaction was a human heart cDNA library. A product of the expected length was generated, cleaved with Sal I and Not I and cloned between the Sal I and Not I sites of pMBP. The insert in this plasmid (pMBP-MCD) was sequenced in its entirety, confirming the full-length sequence assembled from the overlapping MCD cDNAs described in the cDNA cloning section above. This plasmid was used for generating full-length recombinant MCD that was used for enzyme assays. To create pcDNA3-MCD, the Eco RI-Not I fragment containing the MCD cDNA was excised from pMBP-MCD and inserted between the Eco RI and Not I sites of pcDNA3 (Invitrogen, San Diego). Recombinant MCD lacking its first 186 amino acids and last 3 amino acids was expressed in fusion with MBP from an Escherichia coli strain DH10B (17Grant S.G. Jessee J. Bloom F.R. Hanahan D. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4645-4649Crossref PubMed Scopus (920) Google Scholar) carrying the plasmid pMBP-MCD/187–451. Full-length MCD was expressed in fusion with MBP from DH10B cells carrying the plasmid pMBP-MCD. MBP-LacZ was expressed from DH10B cells carrying pMALc2. All three proteins were expressed and purified as described by Geisbrecht et al. (16Geisbrecht B.V. Zhu D. Schulz K. Nau K. Morrell J.C. Geraghty M. Schulz H. Erdmann R. Gould S.J. J. Biol. Chem. 1998; 273: 33184-33191Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), with the exception that cells were induced overnight at room temperature. MBP-MCD/187–451 was used for the immunization of rabbits and affinity purification of anti-MCD antibodies, as described (18Crane D.I. Kalish J.E. Gould S.J. J. Biol. Chem. 1994; 269: 21835-21844Abstract Full Text PDF PubMed Google Scholar). Recombinant MBP-MCD and MBP-LacZ were used for the analysis of MCD enzyme activity. Malonyl-CoA decarboxylase activity was assayed by coupling the MCD reaction to malate dehydrogenase and citrate synthase and by measuring the production of NADH spectrophotometrically, as described by Kim and Kolattukudy (2Kim Y.S. Kolattukudy P.E. Arch. Biochem. Biophys. 1978; 190: 585-597Crossref PubMed Scopus (45) Google Scholar). Only initial rates were used in the calculation of Km and vmax. Assays for catalase (19Peters T.J. Muller M. de Duve C. J. Exp. Med. 1972; 136: 1117-1139Crossref PubMed Scopus (273) Google Scholar) and succinate dehydrogenase (20Pennington R.J. Biochem. J. 1961; 80: 649-654Crossref PubMed Scopus (836) Google Scholar) have been described. Whole cell protein extracts for use in immunoblotting were prepared from cultured human HepG2 cells, a hepatoblastoma cell line, and 5756T cells, a human skin fibroblast cell line. A nearly confluent flask (150 cm2) of cells was resuspended in a solution of 30 mm Tris, 1 mmEDTA, 1% SDS, pH 8.0. The suspension was incubated for 10 min on ice, then spun at 15,000 × g for 10 min. The supernatant containing soluble protein was removed and resuspended in SDS-polyacrylamide gel electrophoresis buffer. Coupled in vitro transcription and translation of human MCD was performed with TNT in vitro translation reagents according to the manufacturer's instructions (Promega, Madison, WI) and the pcDNA3-MCD plasmid. Subcellular fractionation of rat liver was performed as described (21Mihalik S.J. Prog. Clin. Biol. Res. 1992; 357: 239-244Google Scholar). Northern blot analysis was performed using standard protocols and human multitissue Northern blots from CLONTECH (Palo Alto, CA). Human fibroblast RNA was extracted from cultured fibroblast monolayers using PureScript reagents and protocols (Gentra Systems, Minneapolis, MN). Human genomic DNA was prepared from cultured human skin fibroblasts using PureGene reagents and protocols (Gentra). Synthesis of MCD first strand cDNA was performed as described (22Michaud J. Brody L. Steel G. Fontaine G. Martin L. Valle D. Mitchell G. Genomics. 1992; 13: 389-394Crossref PubMed Scopus (113) Google Scholar,23Warren D.S. Morrell J.C. Moser H.W. Valle D. Gould S.J. Am. J. Hum. Genet. 1998; 63: 347-359Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar) using the MCD-specific primer MCD-RT (5′-ACGGCTAAAGCAACCATCGC-3′). For analysis of the 5′ end of the MCD cDNA, the antisense oligonucleotides MCD-RACE.1 (5′-CCACCTGGCCGTGGTCCACGCCGAAGC-3′) and MCD-RACE.2 (5′-GCCCACCGTAGAAGCTCACG-3′) were used in combination with the MCDstart (5′-ATGGACGAGCTGCTGCGCCG-3′) and MCD-hypoATG (5′-ATGCGAGGCTTCGGGCCAGGC-3′) oligonucleotides. The human heart cDNA library was obtained from CLONTECH. For mutation detection studies, RNA was extracted from fibroblasts derived from a severely affected malonic aciduria patient (MA002), first strand cDNA was synthesized as noted above, and two overlapping fragments of the MCD cDNA were amplified from the MA002 first strand MCD cDNA by PCR using the following oligonucleotide pairs: MCDstart and MCD-N3 (5′-CCTTGACGACTCGCTTTATG-3′), and MCD-C5 (5′-GCAACATCCAGGCAATCGTG-3′) and MCD-C3 (5′-TGCGGGACAAGAACACAGTC-3′). The resulting PCR products were sequenced directly using the same oligonucleotides used in the PCR reactions. We previously described the development and application of context sensitive motif scanning for the in silico identification of novel peroxisomal proteins in the yeast Saccharomyces cerevisiae (24Geraghty M.T. Bassett D. Morrell J.C. Gatto G.J. Bai J. Geisbrecht B.V. Hieter P. Gould S.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2937-2942Crossref PubMed Scopus (49) Google Scholar). We applied a similar approach to identify candidate peroxisomal proteins in data bases from higher eukaryotes. One of the many candidates that were identified was the goose malonyl-CoA decarboxylase (A. anser MCD). This enzyme contains a perfect match to the consensus sequence for the type-1 peroxisomal targeting signal (PTS1), serine-lysine-leucineCOOH (15Gould S.J. Keller G.A. Hosken N. Wilkinson J. Subramani S. J. Cell Biol. 1989; 108: 1657-1664Crossref PubMed Scopus (880) Google Scholar) and suggested that MCD might contribute to peroxisomal metabolic processes. However, it seemed that an analysis of human MCD would have greater general relevance, because there is a larger body of work on the physiological effects of malonyl-CoA in mammalian tissues (6McGarry J.D. Brown N.F. Eur. J. Biochem. 1997; 244: 1-14Crossref PubMed Scopus (1327) Google Scholar) and because malonyl-CoA decarboxylase deficiency is associated with defects in this enzyme (10Brown G.K. Scholem R.D. Bankier A. Danks D.M. J. Inherit. Metab. Dis. 1984; 7: 21-26Crossref PubMed Scopus (59) Google Scholar, 11Haan E.A. Scholem R.D. Croll H.B. Brown G.K. Eur. J. Pediatr. 1986; 144: 567-570Crossref PubMed Scopus (41) Google Scholar, 12Yano S. Sweetman L. Thorburn D.R. Modifi S. Williams J.C. Eur. J. Pediatr. 1997; 156: 382-383Crossref PubMed Scopus (43) Google Scholar, 13MacPhee G. Logan R. Mitchell J. Howells D. Tsotsis E. Thorburn D. Arch. Dis. Child. 1993; 69: 433-436Crossref PubMed Scopus (33) Google Scholar, 14Matalon R. Michaels K. Kaul R. Whitman V. Rodriguez-Novo J. Goodman S. Thorburn D. J. Inherit. Metab. Dis. 1993; 16: 571-573Crossref PubMed Scopus (40) Google Scholar). We used a computer-based approach to identify the human malonyl-CoA decarboxylase gene. The BLAST algorithm was used to scan the data base of human expressed sequence tags for cDNAs capable of encoding proteins similar to goose malonyl-CoA decarboxylase. Multiple overlapping ESTs corresponding to a single gene were identified. The cDNA clone that appeared to have the longest 5′ end was obtained from a commercial vendor and sequenced in its entirety. This clone appeared to be missing several hundred base pairs from the 5′ end. Additional MCD cDNA clones containing another 600 bp at the 5′ end were obtained from a human heart cDNA library, allowing us to assemble an apparent full-length cDNA for human MCD. The compiled human MCD cDNA sequence (Fig.1) is 2,121-bp long and contains a 1,362-bp open reading frame. The presumptive initiator ATG has a good match to the consensus sequence for high efficiency translation initiation (25Kozak M. Mamm. Genome. 1996; 7: 563-574Crossref PubMed Scopus (755) Google Scholar, 26Kozak M. Annu. Rev. Cell Biol. 1992; 8: 197-225Crossref PubMed Scopus (415) Google Scholar), particularly because it has purines at both the −3 and +4 positions, relative to the A of the ATG. The deduced protein product is 454-amino acids long, has a predicted molecular mass of approximately 50 kDa, and starts at exactly the same relative position as the goose cytoplasmic MCD (Fig. 2). Furthermore, it also contains the canonical PTS1 of serine-lysine-leucineCOOH. We also identified partial cDNA clones for the mouse and rat forms of MCD, and they also encode proteins that contain a PTS1 (data not shown). Additional data base searches led to the identification of the putative C. elegans MCD, which does not contain a peroxisomal targeting signal-like sequence and is considerably shorter than the vertebrate proteins at its N terminus (Fig. 2).Figure 2Alignment of human, goose, and worm forms of MCD. The deduced human MCD protein sequence (Hs MCD) is presented on the top line, the goose MCD protein sequence (Aa MCD) is shown in the middle line, and the putative C. elegans MCD protein sequence (Ce MCD) is presented on the bottom line. Two forms of MCD are expressed in goose, a longer form that contains a mitochondrial leader sequence and a shorter cytoplasmic form that starts at position 51 of the mitochondrial precursor but is otherwise identical. The amino acids that are present only in the precursor of the goose mitochondrial MCD are italicized. Note that the human form of MCD corresponds precisely to that of the cytoplasmic form of goose MCD.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Additional searches of the human EST data base identified 34 MCD cDNA clones, 4 of which were deposited only recently and contained the entire MCD ORF. However, none were as long as the MCD cDNA described in this report. 5′-RACE also failed to provide evidence for a longer MCD transcript. Knowledge of gene structure can help in the analysis of gene transcripts, and we therefore identified an MCD genomic DNA bacterial artificial chromosome clone. A fragment of this clone that hybridized to the 5′ end of the MCD cDNA clone was obtained, and sequence analysis of this clone revealed the presence of an in-frame ATG just 22-bp upstream of the 5′ end of the MCD cDNA, as well as the absence of splice acceptor sites between this ATG and the ATG we designated as the beginning of the MCD open reading frame (Fig. 3). Any transcripts originating upstream of this ATG would have the potential to encode a longer form of human MCD, and we used PCR techniques to search for such transcripts. We prepared first strand MCD cDNA from human fibroblast mRNA and used this as template in PCR reactions containing either of two antisense MCD oligonucleotides (MCD-RACE.1 and MCD-RACE.2) and the MCD-hypoATG oligonucleotide, which spans this hypothetical upstream ATG. No detectable products were generated from these reactions even though (a) these same two combinations of primers amplified a fragment of the correct size from MCD genomic DNA and (b) the same cDNA sample and antisense oligonucleotides could be used to amplify a fragment of the correct size in PCR reactions using a 5′ primer (MCDstart), which spanned the ATG that we list at position +1 of the cDNA sequence. Similar results were obtained when these various primer pairs were used with human heart cDNA as the template. Given the sensitivity of PCR detection techniques, these data indicate that MCD transcripts containing the hypothetical upstream ATG are either of very low abundance or do not exist, at least in human fibroblasts and heart tissue. Although we find no evidence for MCD transcripts that contain the hypothetical upstream ATG, it is useful to consider whether an mRNA that contained this sequence would encode a protein analogous to goose mitochondrial MCD. We think that this is unlikely for two reasons. First, this putative upstream ATG is followed by a pyrimidine (Fig. 3), which lessens the probability that it would serve as an efficient initiator codon were it present in a mammalian mRNA (the +4 position is almost always a purine in highly expressed transcripts (25Kozak M. Mamm. Genome. 1996; 7: 563-574Crossref PubMed Scopus (755) Google Scholar, 26Kozak M. Annu. Rev. Cell Biol. 1992; 8: 197-225Crossref PubMed Scopus (415) Google Scholar)). Even more importantly, the region between the hypothetical upstream ATG and the ATG at position +1 of the cDNA clone encodes a peptide sequence that lacks features of a mitochondrial leader sequence and shares only slight similarity to the N-terminal mitochondrial targeting signal of goose mitochondrial MCD. As an independent test of whether the cDNA reported here is capable of encoding the full-length MCD protein we compared the mobility of endogenously synthesized MCD with that of MCD synthesized in vitro from the MCD cDNA clone. Affinity purified anti-MCD antibodies were generated and tested by immunoblot analysis of total cellular protein extracts from human fibroblasts and human hepatoblastoma cells. These antibodies detected a single polypeptide of approximately 50 kDa in both cell types (Fig.4 A), indicating that they are specific for MCD. We then synthesized MCD in vitro in a rabbit reticulocyte lysate and used immunoblot analysis to compare its mobility with that of endogenously synthesized human fibroblast MCD. A single protein was detected in each sample and their mobilities were indistinguishable from one another (Fig. 4 B). Control experiments confirmed that the level of rabbit MCD present in the in vitro translation lysates was below the limit of detection. Taken together, these various lines of evidence suggest that the MCD present in human fibroblasts corresponds to the product of the MCD cDNA clone. To test the hypothesis that the gene we had identified encoded human malonyl-CoA decarboxylase, we expressed the entire human MCD protein in bacteria as a fusion with MBP. The recombinant MBP-MCD fusion protein was purified by affinity chromatography on an amylose resin. Assessment of MBP-MCD purity by SDS-polyacrylamide gel electrophoresis showed a predominant band at 90 kDa (Fig. 5), the size predicted for a fusion containing MBP (42 kDa) and human MCD (46 kDa). Purified recombinant MBP-MCD and purified MBP-LacZ (expressed and purified by the identical protocol and from the same strain of E. coli) were assayed for their ability to convert malonyl-CoA to acetyl-CoA. MBP-MCD showed significant malonyl-CoA decarboxylase activity, with a specific activity of 3 units/mg and a Km of 220 μm for malonyl-CoA (Fig. 5). The MBP-LacZ protein lacked activity altogether, demonstrating that the activity of the MBP-MCD fusion protein was intrinsic to the portion derived from MCD and that E. coli MCD does not co-purify with MBP fusion proteins on amylose resin. Although human MCD displayed a high Km for malonyl-CoA, it is only slightly higher than the Km of the goose e" @default.
• W1990152592 created "2016-06-24" @default.
• W1990152592 creator A5010626005 @default.
• W1990152592 creator A5040743356 @default.
• W1990152592 creator A5042584066 @default.
• W1990152592 creator A5070776310 @default.
• W1990152592 creator A5079827681 @default.
• W1990152592 date "1999-08-01" @default.
• W1990152592 modified "2023-10-08" @default.
• W1990152592 title "MCD Encodes Peroxisomal and Cytoplasmic Forms of Malonyl-CoA Decarboxylase and Is Mutated in Malonyl-CoA Decarboxylase Deficiency" @default.
• W1990152592 cites W1542370090 @default.
• W1990152592 cites W1543589576 @default.
• W1990152592 cites W1570204140 @default.
• W1990152592 cites W1611736211 @default.
• W1990152592 cites W1630273828 @default.
• W1990152592 cites W168671060 @default.
• W1990152592 cites W1792892656 @default.
• W1990152592 cites W1964606633 @default.
• W1990152592 cites W1966348307 @default.
• W1990152592 cites W1971024458 @default.
• W1990152592 cites W1989988844 @default.
• W1990152592 cites W1992709343 @default.
• W1990152592 cites W1994238431 @default.
• W1990152592 cites W1996733925 @default.
• W1990152592 cites W2031846075 @default.
• W1990152592 cites W2038663142 @default.
• W1990152592 cites W2038881580 @default.
• W1990152592 cites W2042033533 @default.
• W1990152592 cites W2042342410 @default.
• W1990152592 cites W2056467041 @default.
• W1990152592 cites W2059496748 @default.
• W1990152592 cites W2071579812 @default.
• W1990152592 cites W2074952408 @default.
• W1990152592 cites W2079326336 @default.
• W1990152592 cites W2079653458 @default.
• W1990152592 cites W2101431784 @default.
• W1990152592 cites W2107163098 @default.
• W1990152592 cites W2170566884 @default.
• W1990152592 doi "https://doi.org/10.1074/jbc.274.35.24461" @default.
• W1990152592 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/10455107" @default.
• W1990152592 hasPublicationYear "1999" @default.
• W1990152592 type Work @default.
• W1990152592 sameAs 1990152592 @default.
• W1990152592 citedByCount "93" @default.
• W1990152592 countsByYear W19901525922012 @default.
• W1990152592 countsByYear W19901525922013 @default.
• W1990152592 countsByYear W19901525922014 @default.
• W1990152592 countsByYear W19901525922015 @default.
• W1990152592 countsByYear W19901525922016 @default.
• W1990152592 countsByYear W19901525922017 @default.
• W1990152592 countsByYear W19901525922018 @default.
• W1990152592 countsByYear W19901525922019 @default.
• W1990152592 countsByYear W19901525922020 @default.
• W1990152592 countsByYear W19901525922021 @default.
• W1990152592 countsByYear W19901525922022 @default.
• W1990152592 countsByYear W19901525922023 @default.
• W1990152592 crossrefType "journal-article" @default.
• W1990152592 hasAuthorship W1990152592A5010626005 @default.
• W1990152592 hasAuthorship W1990152592A5040743356 @default.
• W1990152592 hasAuthorship W1990152592A5042584066 @default.
• W1990152592 hasAuthorship W1990152592A5070776310 @default.
• W1990152592 hasAuthorship W1990152592A5079827681 @default.
• W1990152592 hasBestOaLocation W19901525921 @default.
• W1990152592 hasConcept C104317684 @default.
• W1990152592 hasConcept C127078168 @default.
• W1990152592 hasConcept C181199279 @default.
• W1990152592 hasConcept C185592680 @default.
• W1990152592 hasConcept C190062978 @default.
• W1990152592 hasConcept C2779364145 @default.
• W1990152592 hasConcept C2780627266 @default.
• W1990152592 hasConcept C55493867 @default.
• W1990152592 hasConcept C82714985 @default.
• W1990152592 hasConcept C86803240 @default.
• W1990152592 hasConceptScore W1990152592C104317684 @default.
• W1990152592 hasConceptScore W1990152592C127078168 @default.
• W1990152592 hasConceptScore W1990152592C181199279 @default.
• W1990152592 hasConceptScore W1990152592C185592680 @default.
• W1990152592 hasConceptScore W1990152592C190062978 @default.
• W1990152592 hasConceptScore W1990152592C2779364145 @default.
• W1990152592 hasConceptScore W1990152592C2780627266 @default.
• W1990152592 hasConceptScore W1990152592C55493867 @default.
• W1990152592 hasConceptScore W1990152592C82714985 @default.
• W1990152592 hasConceptScore W1990152592C86803240 @default.
• W1990152592 hasIssue "35" @default.
• W1990152592 hasLocation W19901525921 @default.
• W1990152592 hasOpenAccess W1990152592 @default.
• W1990152592 hasPrimaryLocation W19901525921 @default.
• W1990152592 hasRelatedWork W1990152592 @default.
• W1990152592 hasRelatedWork W2013944686 @default.
• W1990152592 hasRelatedWork W2020432769 @default.
• W1990152592 hasRelatedWork W2038156824 @default.
• W1990152592 hasRelatedWork W2101161344 @default.
• W1990152592 hasRelatedWork W2167914080 @default.
• W1990152592 hasRelatedWork W2992200842 @default.
• W1990152592 hasRelatedWork W4230463627 @default.
• W1990152592 hasRelatedWork W4242665269 @default.
• W1990152592 hasRelatedWork W836714657 @default.
• W1990152592 hasVolume "274" @default.

• next