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ABSTRACT: Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethionine (AdoMet), the main alkylating agent in living cells. Additionally, in the liver, MAT is also responsible for up to 50% of methionine catabolism. Humans with mutations in the gene MAT1A, the gene that encodes the catalytic subunit of MAT I and III, have decreased MAT activity in liver, which results in a persistent hypermethioninemia without homocystinuria. The hypermethioninemic phenotype associated with these mutations is inherited as an autosomal recessive trait. The only exception is the dominant mild hypermethioninemia associated with a G-A transition at nucleotide 791 of exon VII. This change yields a MAT1A-encoded subunit in which arginine 264 is replaced by histidine. Our results indicate that in the homologous rat enzyme, replacement of the equivalent arginine 265 by histidine (R265H) results in a monomeric MAT with only 0.37% of the AdoMet synthetic activity. However the tripolyphosphatase activity is similar to that found in the wild type (WT) MAT and is inhibited by PP(i). Our in vivo studies demonstrate that the R265H MAT I/III mutant associates with the WT subunit resulting in a dimeric R265H-WT MAT unable to synthesize AdoMet. Tripolyphosphatase activity is maintained in the hybrid MAT, but is not stimulated by methionine and ATP, indicating a deficient binding of the substrates. Our data indicate that the active site for tripolyphosphatase activity is functionally active in the monomeric R265H MAT I/III mutant. Moreover, our results provide a molecular mechanism that might explain the dominant inheritance of the hypermethioninemia associated with the R264H mutation of human MAT I/III.
Journal of Biological Chemistry 05/2001; 276(17):13803-9. · 4.77 Impact Factor
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ABSTRACT: Methionine adenosyltransferase (MAT) I/III deficiency, caused by mutations in the MAT1A gene, is characterized by persistent hypermethioninemia without elevated homocysteine or tyrosine. Clinical manifestations are variable and poorly understood, although a number of individuals with homozygous null mutations in MAT1A have neurological problems, including brain demyelination. We analyzed MAT1A in seven hypermethioninemic individuals, to provide insight into the relationship between genotype and phenotype. We identified six novel mutations and demonstrated that mutations resulting in high plasma methionines may signal clinical difficulties. Two patients-a compound heterozygote for truncating and severely inactivating missense mutations and a homozygote for an aberrant splicing MAT1A mutation-have plasma methionine in the 1,226-1,870 microM range (normal 5-35 microM) and manifest abnormalities of the brain gray matter or signs of brain demyelination. Another compound heterozygote for truncating and inactivating missense mutations has 770-1,240 microM plasma methionine and mild cognitive impairment. Four individuals carrying either two inactivating missense mutations or the single-allelic R264H mutation have 105-467 microM plasma methionine and are clinically unaffected. Our data underscore the necessity of further studies to firmly establish the relationship between genotypes in MAT I/III deficiency and clinical phenotypes, to elucidate the molecular bases of variability in manifestations of MAT1A mutations.
The American Journal of Human Genetics 03/2000; 66(2):347-55. · 10.60 Impact Factor
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ABSTRACT: We have used site-directed mutagenesis to probe the structural requirements for catalysis and dimerization of human hepatic methionine adenosyltransferase (hMAT). We built a homology model of the dimeric hMAT III inferred by the crystal structure of the highly homologous Escherichia coli MAT dimer. The active sites of both enzymes comprise the same amino acids and are located in the inter-subunit interface. All of the amino acids predicted to be in the hMAT III active site were mutated, as well as residues in a conserved ATP binding region. All of the mutations except one severely affected catalytic activity. On the other hand, dimerization was affected only by single mutations of three different residues, all on one monomer. The homology model suggested that the side chains of these residues stabilized the monomer and participated in a bridge between subunits consisting of a network of metal and phosphate ions. In agreement with this observation, we demonstrated that dimerization cannot occur in the absence of phosphate.
Archives of Biochemistry and Biophysics 02/2000; 373(1):56-62. · 2.93 Impact Factor
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P K Chiang, M E Chamberlin,
D Nicholson,
S Soubes,
X Su,
G Subramanian,
D E Lanar,
S T Prigge,
J P Scovill,
L H Miller,
J Y Chou
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ABSTRACT: S-Adenosylmethionine (AdoMet) synthetase (SAMS: EC 2.5.1.6) catalyses the formation of AdoMet from methionine and ATP. We have cloned a gene for Plasmodium falciparum AdoMet synthetase (PfSAMS) (GenBank accession no. AF097923), consisting of 1209 base pairs with no introns. The gene encodes a polypeptide (PfSAMS) of 402 amino acids with a molecular mass of 44844 Da, and has an overall base composition of 67% A+T. PfSAMS is probably a single copy gene, and was mapped to chromosome 9. The PfSAMS protein is highly homologous to all other SAMS, including a conserved motif for the phosphate-binding P-loop, HGGGAFSGKD, and the signature hexapeptide, GAGDQG. All the active-site amino acids for the binding of ADP, P(i) and metal ions are similarly preserved, matching entirely those of human hepatic SAMS and Escherichia coli SAMS. Molecular modelling of PfSAMS guided by the X-ray crystal structure of E. coli SAMS indicates that PfSAMS binds ATP/Mg(2+) in a manner similar to that seen in the E. coli SAMS structure. However, the PfSAMS model shows that it can not form tetramers as does E. coli SAMS, and is probably a dimer instead. There was a differential sensitivity towards the inhibition by cycloleucine between the expressed PfSAMS and the human hepatic SAMS with K(i) values of 17 and 10 mM, respectively. Based on phylogenetic analysis using protein parsimony and neighbour-joining algorithms, the malarial PfSAMS is closely related to SAMS of other protozoans and plants.
Biochemical Journal 01/2000; 344 Pt 2:571-6. · 4.90 Impact Factor
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ABSTRACT: The Skn-1a POU transcription factor is primarily expressed in keratinocytes of murine embryonic and adult epidermis. Although some POU factors expressed in a tissue-specific manner are important for normal differentiation, the biological function of Skn-1a remains unknown. Previous in vitro studies indicate that Skn-1a has the ability to transactivate markers of keratinocyte differentiation. In this study, we have characterized Skn-1a's transactivation domain(s) and engineered a dominant negative protein that lacked this transactivation domain. Deletional analysis of the human homologue of Skn-1a with three target promoters revealed the presence of two functional domains: a primary C-terminal transactivation domain and a combined N-terminal inhibitory domain and transactivation domain. Skn-1a lacking the C-terminal region completely lost transactivation ability, irrespective of the promoter tested, and was able to block transactivation by normal Skn-1a in competition assays. Compared with full-length, Skn-1a lacking the N-terminal region demonstrated either increased transactivation (bovine cytokeratin 6 promoter), comparable transactivation (human papillomavirus type 1a long control region), or loss of transactivation (human papillomavirus type 18 long control region). The identification of a primary C-terminal transactivation domain enabled us to generate a dominant negative Skn-1a factor, which will be useful in the quest for a better understanding of this keratinocyte-specific gene regulator.
Journal of Biological Chemistry 10/1999; 274(37):26399-406. · 4.77 Impact Factor
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ABSTRACT: Methionine adenosyltransferase (MAT) I/III deficiency, characterized by isolated persistent hypermethioninemia, is caused by mutations in the MAT1A gene encoding MAT(alpha)1, the subunit of major hepatic enzymes MAT I ([alpha1]4) and III([alpha1]2). We have characterized 10 MAT1A mutations in MAT I/III-deficient individuals and shown that the associated hypermethioninemic phenotype was inherited as an autosomal recessive trait. However, dominant inheritance of hypermethioninemia, also hypothesized to be caused by MAT I/III deficiency, has been reported in two families. Here we show that the only mutation uncovered in one of these families, G, is a G-->A transition at nt 791 in exon VII of one MAT1A allele that converts an arginine at position 264 to a histidine (R264H). This single allelic R264H mutation was subsequently identified in two hypermethioninemic individuals in an additional family, C. Family C members were also found to inherit hypermethioninemia in a dominant fashion, and the available affected members analyzed carried the single allelic R264H mutation. Substitution of R-264 with histidine (R264H, the naturally occurring mutant), leucine (R264L), aspartic acid (R264D), or glutamic acid (R264E) greatly reduced MAT activity and severely impaired the ability of the MAT(alpha)1 subunits to form homodimers essential for optimal catalytic activity. On the other hand, when lysine was substituted for R-264 (R264K), the mutant alpha1 subunit was able to form dimers that retain significant MAT activity, suggesting that amino acid 264 is involved in intersubunit salt-bridge formation. Cotransfection studies show that R264/R264H MAT(alpha)1 heterodimers are enzymatically inactive, thus providing an explanation for the R264H-mediated dominant inheritance of hypermethioninemia.
The American Journal of Human Genetics 03/1997; 60(3):540-6. · 10.60 Impact Factor
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ABSTRACT: Individuals deficient in hepatic methionine adenosyltransferase (MAT) activity (MAT I/III deficiency) have been demonstrated to contain mutations in the gene (MATA1) that encodes the major hepatic forms, MAT I and III. MAT I/III deficiency is characterized by isolated persistent hypermethioninemia and, in some cases, unusual breath odor. Most individuals with isolated hypermethioninemia have been free of major clinical difficulties. Therefore a definitive diagnosis of MAT I/III deficiency, which requires hepatic biopsy, is not routinely made. However, two individuals with isolated hypermethioninemia have developed abnormal neurological problems, including brain demyelination, suggesting that MAT I/III deficiency can be deleterious. In the present study we have examined the MATA1 gene of eight hypermethioninemic individuals, including the two with demyelination of the brain. Mutations that abolish or reduce the MAT activity were detected in the MATA1 gene of all eight individuals. Both patients with demyelination are homozygous for mutations that alter the reading frame of the encoded protein such that the predicted MATalpha1 subunits are truncated and enzymatically inactive. The product of MAT, S-adenosylmethionine (AdoMet), is the major methyl donor for a large number of biologically important compounds including the two major myelin phospholipids, phosphatidylcholine and sphingomyelin. Both are synthesized primarily in the liver. Our findings demonstrate that isolated persistent hypermethioninemia is a marker of MAT I/III deficiency, and that complete lack of MAT I/III activity can lead to neurological abnormalities. Therefore, a DNA-based diagnosis should be performed for individuals with isolated hypermethioninemia to assess if therapy aimed at the prevention of neurological manifestations is warranted.
Journal of Clinical Investigation 09/1996; 98(4):1021-7. · 15.39 Impact Factor
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ABSTRACT: Human placentas express high levels of the placental alkaline phosphatase (PLAP) gene and low levels of a highly related gene, germ cell AP (GCAP). Malignant transformation of the placenta is accompanied by a reversal of this pattern of expression. Three Sp1-binding GC-rich DNA elements (sites I-III) located within the first 156 base pairs upstream of the GCAP gene have been shown to direct optimal GCAP gene expression in choriocarcinoma cells. Here we show that the first 100 base pairs upstream of the GCAP gene, which contains sites I and II, constitutes a minimal GCAP promoter. The simultaneous presence of both sites I and II is necessary for GCAP expression and its induction by sodium butyrate. The PLAP promoter directs only a very low level of gene expression in choriocarcinoma cells; the expression does not respond to butyrate. The -100/-1 DNA regions between the GCAP and PLAP promoters differ by only eight base pairs. However, the GC-rich stretches in sites I and II of the GCAP promoter are disrupted in the corresponding PLAP promoter. This disruption blocks or markedly reduces the binding of choriocarcinoma nuclear factors to the PLAP promoter, leading to a reduction in expression and a loss of butyrate response. We further demonstrate that nucleotides -75 to -58 in both AP promoters, which bind a human Y-box binding protein, appear to down-regulate GCAP expression.
Biochemistry 08/1996; 35(30):9807-14. · 3.42 Impact Factor
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ABSTRACT: Eleven pregnancy-specific glycoprotein (PSG) genes reside on human chromosome 19. The sequence of these genes is extremely similar and that similarity extends to their putative control regions. However, the expression pattern of each PSG gene differs in the placenta, the primary site of PSG synthesis. To understand the molecular mechanisms underlying differential PSG expression, we characterized promoter elements of six PSG genes. We have shown previously that nucleotides -172 to -34 with respect to the translation start site constitute a minimal promoter in the PSG12 gene (class 1). We now show that PSG1-I and PSG3 are also members of class 1 genes. In contrast, only nucleotides -172 to -80 are necessary for promoter activity in PSG5, PSG6, and PSG11 genes (class 2). Class 2 genes contain a perfect Sp1 recognition sequence (CCCCGCCC) at nucleotides -148 to -141 which is necessary for promoter activity. Placental cell extracts formed three protein-DNA complexes with nucleotides -172 to -80 of all six PSG genes. One of the components of these complexes is an Sp1-like molecule. We have previously reported activator sequences within nucleotides -83 to -34 in PSG12. We now show that a 50-kDa protein binds to this region of PSG12, and the resultant complex can be supershifted by a monoclonal antibody to PEA3.
Journal of Biological Chemistry 07/1994; 269(25):17152-9. · 4.77 Impact Factor
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ABSTRACT: The pregnancy-specific glycoproteins (PSGs) of the placenta, members of the immunoglobulin superfamily, are encoded by multiple linked genes located on chromosome 19. To study the control of PSG expression, we have immortalized differentiated human placental cells (HP-A1) temperature-sensitive for transformation by a recombinant adenovirus-(ori-)-SV40 tsA mutant virus. We now show that expression of the PSG gene in HP-A1 cells is temperature-sensitive. At the permissive temperature (33 degrees C), these cells expressed low levels of PSG mRNA and synthesized a 64-kDa PSG. Shifting HP-A1 cells to a nonpermissive temperature (39.5 degrees C) increased PSG mRNA expression and biosynthesis with preferential increase in the synthesis of a 54-kDa and a low level of a 72-kDa PSG. Moreover, PSG expression was greatly induced by 5-bromo-2'-deoxyuridine (BudR), which selectively increased synthesis of PSGs of 72 and 54 kDa. In the presence of BudR, HP-A1 synthesized PSGs of 72, 64, and 54 kDa, similar to the pattern seen with placental PSGs. Ribonuclease protection assays demonstrated that HP-A1 cells express the majority of PSG mRNAs and BudR stimulated expression of PSG1 and PSG1-like transcripts. Reverse transcription and polymerase chain reaction analysis using PSG gene-specific primers demonstrated that untreated HP-A1 cells expressed primarily PSG1, PSG2, PSG4, and PSG5 mRNAs. BudR stimulated the expression of all PSG transcripts except PSG4. Moreover, in transient expression assays, BudR increased chloramphenicol acetyltransferase (CAT) expression directed by PSG1-I, PSG4, PSG5, PSG6, and PSG11 promoter-CAT fusion genes.(ABSTRACT TRUNCATED AT 250 WORDS)
Biochemistry 07/1994; 33(23):7260-6. · 3.42 Impact Factor
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ABSTRACT: The pregnancy-specific glycoproteins (PSGs) of the human placenta are a group of proteins that together with the carcinoembryonic antigens comprise a subfamily within the immunoglobulin superfamily. To study the control of PSG expression, we isolated and characterized PSG genes and identified cis-acting DNA elements in the 5'-flanking gene regions essential for PSG expression. Two overlapping PSG cosmid clones, which contain two allelic variants of a PSG gene (PSG12 and PSG12 psi), were isolated from an unamplified library made from a single individual. Cosmid 1 contains exons 1 (5'/L) and 2 (L/N) of the PSG12 gene located downstream of a previously identified PSG1-I gene. Cosmid 6 contains a portion of the PSG1-I gene lacking exons 1 and 2 upstream of a complete PSG12 psi transcription unit. Sequence comparison indicates that exons 5'/L and L/N in PSG12 and PSG12 psi are 99% identical, except that the L/N exon in the PSG12 psi gene contains a stop codon. Both PSG12 and PSG12 psi transcripts were detected in the human placenta, indicating that both genes are actively transcribed. However, the PSG12 psi gene may represent an allelic pseudogene variant of the PSG12 gene, because all identified PSGs contain a functional N-domain. Primer extension analysis showed that the PSG12 gene starts at a cluster of sites located at -106 to -104 base pairs with respect to the translation start site. In transient transfection assays using a chloramphenicol acetyltransferase reporter gene, we demonstrated that the -835 to -34 DNA region upstream of the translation start site of PSG12 or PSG12 psi contained both positive and negative elements that control PSG expression. Deletion analysis showed that nucleotides -172 to -34 in the PSG12 gene could function as a promoter. Gel retardation analysis showed that protein factors in human placental cell extract formed four complexes (I, II, IIa, and III) with the PSG12(-172/-34) DNA. Site-directed mutagenesis that prevents protein factor binding to the PSG12 promoter resulted in a marked reduction in transcription activation, locating the core enhancers at nucleotides -148 to -141 and -60 to -55. Mutagenesis studies also showed that the ACAGC repeats at nucleotides -84 to -68 in the PSG12 5'-flanking are essential for expression of the PSG12 gene in human placental cells.
Journal of Biological Chemistry 09/1993; 268(23):17528-38. · 4.77 Impact Factor
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ABSTRACT: The human zona pellucida, composed of three glycoproteins (ZP1, ZP2, and ZP3), forms an extracellular matrix that surrounds ovulated eggs and mediates species-specific fertilization. The genes that code for at least two of the zona proteins (ZP2 and ZP3) cross-hybridize with other mammalian DNA. The recently characterized mouse sperm receptor gene (Zp-3) was used to isolate its human homolog. The human homolog spans approximately 18.3 kilobase pairs (kbp) (compared to 8.6 kbp for the mouse gene) and contains eight exons, the sizes of which are strictly conserved between the two species. Four short (8-15 bp) sequences within the first 250 bp of the 5' flanking region in the human Zp-3 homolog are also present upstream of mouse Zp-3. These elements may modulate oocyte-specific gene expression. By using the polymerase chain reaction, a full-length cDNA of human ZP3 was isolated from human ovarian poly(A)+ RNA and used to deduce the structure of human ZP3 mRNA. Certain features of the human and mouse ZP3 transcripts are conserved. Both have unusually short 5' and 3' untranslated regions, both contain a single open reading frame that is 74% identical, and both code for 424 amino acid polypeptides that are 67% the same. The similarity between the two proteins may define domains that are important in maintaining the structural integrity of the zona pellucida, while the differences may play a role in mediating the species-specific events of mammalian fertilization.
Proceedings of the National Academy of Sciences 09/1990; 87(16):6014-8. · 9.68 Impact Factor
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ABSTRACT: The zona pellucida is an extracellular matrix which surrounds mammalian oocytes and plays an important role in the species-specific fertilization of mammals. The mouse zona is composed of three sulfated glycoproteins one of which, ZP3, serves as the primary sperm receptor in the initial interaction between egg and sperm. We have previously cloned a ZP3 cDNA and shown that the gene for ZP3 is expressed in a precise temporal and spatial fashion only in growing oocytes. We now describe the genomic organization of this gene whose expression is germ-line and female sex-specific. The gene contains 8 exons spanning approximately 8.6 kilobases in the mouse genome where it appears to be present as a single copy gene. S1 analyses of the 5' and 3' ends of the gene define the transcription start and stop sites and show that ZP3 mRNA has very short untranslated regions. Sequence determination has revealed a novel tandem repeat which is reiterated six times in the 5' flanking region and five times in the seventh intron.
Developmental Biology 02/1989; 131(1):207-14. · 4.07 Impact Factor
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Progress in clinical and biological research 02/1989; 294:21-32.
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Annals of the New York Academy of Sciences 02/1989; 564:281-8. · 3.15 Impact Factor
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ABSTRACT: At fertilization, mammalian sperm bind is a species-specific manner to the extracellular zona pellucida that surrounds ovulated eggs. ZP3, an 83,000-85,000 Da glycoprotein of the murine zona pellucida, has been shown to inhibit sperm binding via its O-linked oligosaccharide side chains. We have recently isolated cDNA clones coding for ZP3 and have demonstrated that ZP3 transcripts are accumulated in oocytes where their expression is developmentally regulated during oogenesis. We now report that ZP3 mRNA is 1317 nt long with an estimated poly(A) tail of 200-300 nt. The short 29-nt 5' untranslated region is followed by a single open reading frame coding for a polypeptide chain of 46,307 Da which includes six possible sites for N-linked oligosaccharides. The N-terminus of ZP3 contains a potential 22-amino acid signal peptide which upon cleavage would result in a secreted core protein of 43,943 Da. The termination codon is a part of the AATAAA polyadenylation signal and is contained in an unusually short 16-nt 3' untranslated region. Sequences homologous to ZP3 are conserved among mammals and are expressed in ovarian tissue as mature transcripts with indistinguishable molecular weights.
Developmental Biology 07/1988; 127(2):287-95. · 4.07 Impact Factor
