Glucose-6-phosphatase dependent substrate transport in the glycogen storage disease type-1a mouse.

Glycogen storage disease type 1a (GSD-1a) is caused by a deficiency in microsomal glucose-6-phosphatase (G6Pase), the key enzyme in glucose homeostasis. A G6Pase knockout mouse which mimics the pathophysiology of human GSD-1a patients was created to understand the pathogenesis of this disorder, to delineate the mechanisms of G6Pase catalysis, and to develop future therapeutic approaches. By examining G6Pase in the liver and kidney, the primary gluconeogenic tissues, we demonstrate that glucose-6-P transport and hydrolysis are performed by separate proteins which are tightly coupled. We propose a modified translocase catalytic unit model for G6Pase catalysis.

Inhibition of synthesis of alpha-fetoprotein by glucocorticoids in cultured hepatoma cells.

alpha-Fetoprotein (AFP) synthesis was studied in the presence and absence of glucocorticoids in rat hepatoma Mc-A-RH-7777 cells. Radioimmunoassay of media from cell cultures grown in the presence of glucocorticoid (dexamethasone or cortisol) showed a reduction in AFP, an increase in albumin, and no significant change in transferrin accumulation, as compared to controls. Labeling experiments with L-[35S]methionine indicated that in both cells and media of dexamethasone-treated cultures there was a 50--80% reduction in polypeptide precipitated by anti-AFP serum, as compared with controls; no change was seen in polypeptide precipitated by anti-transferrin serum. Pulse and pulse-chase experiments demonstrated that dexamethasone inhibited the synthesis of AFP but not its secretion. The half-time for secretion of AFP in the presence and absence of dexamethasone was 43 min.

Study of liver differentiation in vitro.

A clonal rat fetal liver cell line that expresses the functions of differentiated liver cells under controllable conditions has been established. Normal fetal liver cells were transformed by a temperature-sensitive A (tsA) mutant (tsA209) of simian virus 40. At the permissive temperature (33 degrees C), the tsA209-transformed liver cell line (RLA209-15) can be cultured indefinitely and cloned readily. The RLA209-15 cells were temperature sensitive for maintenance of the transformed phenotype. These transformed liver cells selectively lost four characteristics of the transformed phenotype at the restrictive temperature (40 degrees C): generation time of the cells increased, the saturation density decreased, the efficiency of growth on nontransformed cell layers decreased, and the ability to clone in soft agar was lost. The transformation can be reversed simply by a shift in temperature. RLA209-15 fetal liver cells synthesized alpha-fetoprotein albumin, and transferrin. At 33 degrees C, the levels of these liver proteins were relatively low. At 40 degrees C the transformed phenotype was lost and the levels of alpha-fetoprotein, albumin, and transferrin were greatly increased. At the restrictive temperature, maximal induction of the synthesis of alpha-fetoprotein, albumin, and transferrin was achieved 3-4 d after the upward shift in temperature. The synthesis of alpha-fetoprotein then decreased; the synthesis of albumin and transferrin, however, was maintained. A second phase of albumin and transferrin synthesis was observed in all cultures after 6 d or more at 40 degrees C. Alpha-Fetoprotein, albumin, and transferrin secreted by RLA209-15 cells were immunologically indistinguishable from authentic alpha-fetoprotein, albumin, and transferrin, respectively. RLA209-15 cells, like primary cultures of hepatocytes and a simian virus 40 tsA255-transformed fetal liver cell line (RLA255-4) reported earlier from this laboratory, responded to glucagon with markedly elevated levels of cyclic AMP. Thus, it appears that glucagon receptors characteristic of hepatocytes are retained in the simian virus 40 tsA-transformed fetal liver cells.

Mutations in the glucose-6-phosphatase gene that cause glycogen storage disease type 1a.

Glycogen storage disease (GSD) type 1a is caused by the deficiency of D-glucose-6-phosphatase (G6Pase), the key enzyme in glucose homeostasis. Despite both a high incidence and morbidity, the molecular mechanisms underlying this deficiency have eluded characterization. In the present study, the molecular and biochemical characterization of the human G6Pase complementary DNA, its gene, and the expressed protein, which is indistinguishable from human microsomal G6Pase, are reported. Several mutations in the G6Pase gene of affected individuals that completely inactivate the enzyme have been identified. These results establish the molecular basis of this disease and open the way for future gene therapy.

Angiotensin mediates renal fibrosis in the nephropathy of glycogen storage disease type Ia.

Patients with glycogen storage disease type Ia (GSD-Ia) develop renal disease of unknown etiology despite intensive dietary therapies. This renal disease shares many clinical and pathological similarities to diabetic nephropathy. We studied the expression of angiotensinogen, angiotensin type 1 receptor, transforming growth factor-beta1, and connective tissue growth factor in mice with GSD-Ia and found them to be elevated compared to controls. While increased renal expression of angiotensinogen was evident in 2-week-old mice with GSD-Ia, the renal expression of transforming growth factor-beta and connective tissue growth factor did not increase for another week; consistent with upregulation of these factors by angiotensin II. The expression of fibronectin and collagens I, III, and IV was also elevated in the kidneys of mice with GSD-Ia, compared to controls. Renal fibrosis was characterized by a marked increase in the synthesis and deposition of extracellular matrix proteins in the renal cortex and histological abnormalities including tubular basement membrane thickening, tubular atrophy, tubular dilation, and multifocal interstitial fibrosis. Our results suggest that activation of the angiotensin system has an important role in the pathophysiology of renal disease in patients with GSD-Ia.

Identification of mutations in the gene for glucose-6-phosphatase, the enzyme deficient in glycogen storage disease type 1a.

Glycogen storage disease (GSD) type 1a is an autosomal recessive inborn error of metabolism caused by a deficiency in microsomal glucose-6-phosphatase (G6Pase), the key enzyme in glucose homeostasis. Southern blot hybridization analysis using a panel of human-hamster hybrids showed that human G6Pase is a single-copy gene located on chromosome 17. To correlate specific defects with clinical manifestations of this disorder, we identified mutations in the G6Pase gene of GSD type 1a patients. In the G6Pase gene of a compound heterozygous patient (LLP), two mutations in exon 2 of one allele and exon 5 of the other allele were identified. The exon 2 mutation converts an arginine at codon 83 to a cysteine (R83C). This mutation, previously identified by us in another GSD type 1a patient, was shown to have no detectable phosphohydrolase activity. The exon 5 mutation in the G6Pase gene of LLP converts a glutamine codon at 347 to a stop (Q347SP). This Q347SP mutation was also detected in all exon 5 subclones (five for each patient) of two homozygous patients, KB and CB, siblings of the same parents. The predicted Q347SP mutant G6Pase is a truncated protein of 346 amino acids, 11 amino acids shorter than the wild type G6Pase of 357 residues. Site-directed mutagenesis and transient expression assays demonstrated that G6Pase-Q347SP was devoid of G6Pase activity. G6Pase is an endoplasmic reticulum (ER) membrane-associated protein containing an ER retention signal, two lysines (KK), located at residues 354 and 355. We showed that the G6Pase-K355SP mutant containing a lysine-355 to stop codon mutation is enzymatically active. Our data demonstrate that the ER protein retention signal in human G6Pase is not essential for activity. However, residues 347-354 may be required for optimal G6Pase catalysis.

Molecular mechanisms of an inborn error of methionine pathway. Methionine adenosyltransferase deficiency.

Methionine adenosyltransferase (MAT) is a key enzyme in transmethylation, transsulfuration, and the biosynthesis of polyamines. Genetic deficiency of alpha/beta-MAT causes isolated persistent hypermethioninemia and, in some cases, unusual breath odor or neural demyelination. However, the molecular mechanism(s) underlying this deficiency has not been clearly defined. In this study, we characterized the human alpha/beta-MAT transcription unit and identified several mutations in the gene of patients with enzymatically confirmed diagnosis of MAT deficiency. Site-directed mutagenesis and transient expression assays demonstrated that these mutations partially inactivate MAT activity. These results establish the molecular basis of this disorder and allow for the development of DNA-based methodologies to investigate and diagnose hypermethioninemic individuals suspected of having abnormalities at this locus.

Demyelination of the brain is associated with methionine adenosyltransferase I/III deficiency.

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.

Mutations in the glucose-6-phosphatase gene are associated with glycogen storage disease types 1a and 1aSP but not 1b and 1c.

Glycogen storage disease (GSD) type 1, which is caused by the deficiency of glucose-6-phosphatase (G6Pase), is an autosomal recessive disease with heterogenous symptoms. Two models of G6Pase catalysis have been proposed to explain the observed heterogeneities. The translocase-catalytic unit model proposes that five GSD type 1 subgroups exist which correspond to defects in the G6Pase catalytic unit (1a), a stabilizing protein (1aSP), the glucose-6-P (1b), phosphate/pyrophosphate (1c), and glucose (1d) translocases. Conversely, the conformation-substrate-transport model suggests that G6Pase is a single multifunctional membrane channel protein possessing both catalytic and substrate (or product) transport activities. We have recently demonstrated that mutations in the G6Pase catalytic unit cause GSD type 1a. To elucidate whether mutations in the G6Pase gene are responsible for other GSD type 1 subgroups, we characterized the G6Pase gene of GSD type 1b, 1c, and 1aSP patients. Our results show that the G6Pase gene of GSD type 1b and 1c patients is normal, consistent with the translocase-catalytic unit model of G6Pase catalysis. However, a mutation in exon 2 that converts an Arg at codon 83 to a Cys (R83C) was identified in both G6Pase alleles of the type 1aSP patient. The R83C mutation was also demonstrated in one homozygous and five heterogenous GSD type 1a patients, indicating that type 1aSP is a misclassification of GSD type 1a. We have also analyzed the G6Pase gene of seven additional type 1a patients and uncovered two new mutations that cause GSD type 1a.

Temperature-sensitive adult liver cell line dependent on glucocorticoid for differentiation.

A clonal rat adult hepatocyte cell line (RALA255-10G) was shown to be temperature sensitive (ts) for growth and differentiation. Glucocorticoid was necessary to maintain the maximal levels of differentiated functions in these cells. The RALA255-10G cell line was established by transforming primary adult hepatocytes with simian virus 40 tsA255 virus that is temperature sensitive for maintenance of transformation. At the permissive temperature (33 degrees C), RALA255-10G cells showed characteristics of malignant transformation, synthesized low levels of albumin and transferrin, and contained low levels of functional receptors for glucagon. At the nonpermissive temperature (40 degrees C), these cells regain the normal differentiated phenotype, and the levels of these three hepatic functions were increased. Induction of albumin and transferrin production by RALA255-10G cells at 40 degrees C was shown to be the result of the increase in the biosynthesis of these proteins. Furthermore, the albumin and transferrin produced by these cells were immunologically and electrophoretically indistinguishable from authentic rat albumin and transferrin. Glucocorticoid, which reduced the growth rate and saturation density of RALA255-10G cells at 33 degrees C, was absolutely required by these cells to synthesize albumin at both temperatures. This hormone also enhanced transferrin production and glucagon response. Our data indicate that glucocorticoid hormone is one of the factors that maintain adult hepatocytes in a differentiated state.

Regulation of rat liver maturation in vitro by glucocorticoids.

The biochemistry of liver maturation was studied by using the RLA209-15 fetal rat hepatocyte line that is temperature sensitive for maintenance of the differentiated fetal liver phenotype. At 33 degrees C these cells were dedifferentiated; but at 40 degrees C they were phenotypically differentiated and, like normal fetal hepatocytes, synthesized moderate levels of albumin and transferrin, high levels of authentic (69,000 and 73,000 molecular weight) rat fetal alpha-fetoprotein (AFP), and low levels of a 65,000-molecular-weight variant AFP. Our results indicated that administration of glucocorticoid hormones to RLA209-15 cells at 40 degrees C induced a series of events associated with normal hepatocyte maturation; synthesis of fetal AFP was inhibited, whereas the synthesis of variant AFP, albumin, transferrin, tyrosine aminotransferase, and alpha 1-acid glycoprotein was induced. The variant AFP was produced by RLA209-15 cells at both temperatures and was encoded by an mRNA of 1.7 kilobases (kb). The fetal AFP was encoded by an mRNA of 2.2 kb. Normal adult rat liver contained three AFP mRNAs of 2.2 (minor), 1.7, and 1.5 kb. The 1.7-kb adult liver AFP mRNA comigrated with the RNA found in RLA209-15 cells, and both directed the synthesis of a 50,000-molecular-weight precursor polypeptide of the variant AFP. Administration of glucocorticoids to RLA209-15 cells grown at 33 degrees C stimulated synthesis of both the fetal and variant AFPs, but the levels of the 2.2-kb AFP mRNA were preferentially increased. RLA209-15 cells contained two glucocorticoid receptor mRNAs of 6.8 and 4.5 kb. The glucocorticoid-mediated maturation described above was blocked by the antiglucocorticoid RU486.

Effects of adenosine cyclic nucleotides on the synthesis of human chorionic gonadotropin in transformed human placental cells.

The synthesis of human chorionic gonadotropin (HCG) and its subunits was studied in simian virus 40 (SV40) tsA mutant-transformed human first-trimester and term placental cells at 33 degrees (the temperature at which the cells have the transformed phenotype) and at 40 degrees (the temperature at which the tsA transformants regain their nontransformed phenotype). 8-Bromo cyclic adenosine 3':5'-monophosphate (8BrcAMP) and dibutyryl cyclic adenosine 3':5'-monophosphate (Bt2cAMP) greatly induced the synthesis of human chorionic gonadotropin alpha (HCG alpha) with little or no stimulation of the synthesis of HCG in transformed placental cells grown at 33 degrees or 40 degrees. The ratio of HCG alpha to HCG in these cells, therefore, increased in the presence of either nucleotide. 8BrcAMP and Bt2cAMP also greatly induced the synthesis of HCG alpha in nontransformed secondary placental cells (at 6th to 20th passage), although the synthesis of HCG was not detectable in these cells under the experimental conditions used. The synthesis of HCG as well as HCG alpha was stimulated in choriocarcinoma cells by 8BrcAMP and Bt2cAMP. The ratio of HCG alpha to HCG in uninduced choriocarcinoma cells increased during growth in culture. 8BrcAMP stimulated the synthesis of HCG preferentially in these cells, thus decreasing the ratio of HCG alpha to HCG. Our data indicate that adenosine cyclic nucleotides have different effects on the production of HCG but not on HCG alpha in SV40 tsA-transformed placental cells and choriocarcinoma cells.

Tyrosine aminotransferase gene expression in a temperature-sensitive adult rat liver cell line.

Regulation of tyrosine aminotransferase gene expression was studied in an adult rat hepatocyte line (RALA255-10G) which is temperature sensitive for the maintenance of the differentiated liver phenotype. Glucocorticoid hormones such as cortisol were necessary for expression of the aminotransferase gene. In the absence of these steroids, enzyme synthesis, activity, and mRNA accumulation were virtually abolished. In the presence of cortisol at 33 degrees C, RALA255-10G cells showed characteristics of malignant transformation and contained little tyrosine aminotransferase activity, synthesized low levels of this enzyme, and produced low levels of enzyme mRNA. At 40 degrees C, cells maintained in the presence of cortisol regained the normal, differentiated phenotype, and tyrosine aminotransferase synthesis and mRNA accumulation were greatly increased. This increase in aminotransferase synthesis paralleled the increase in the enzyme mRNA. However, after a temperature shift-up, tyrosine aminotransferase activity was increased only for the first 2 days, probably due to thermal inactivation of this enzyme at 40 degrees C. Dibutyryl cAMP alone was not sufficient to induce expression of the tyrosine aminotransferase gene, but it enhanced the induction caused by cortisol. Immunocytochemical studies revealed that the enhanced expression of the tyrosine aminotransferase gene at 40 degrees C and in the presence of cortisol or cortisol plus dibutyryl cAMP resulted from an increase in both the number of cells producing this enzyme and the quantity of tyrosine aminotransferase synthesized per cell.

Transcriptional regulation and the effects of sodium butyrate and glycosylation on catalytic activity of human germ cell alkaline phosphatase.

Human choriocarcinoma cells, the malignant trophoblasts, synthesize germ cell alkaline phosphatase (GCAP) which shares 98% sequence identity with the placental alkaline phosphatase (AP). The two isozymes are immunologically similar but react differentially toward inhibition by L-leucine or EDTA. Administration of sodium butyrate to choriocarcinoma cells greatly increased the transcription rate of the GCAP gene, resulting in an increase in mRNA expression and enzyme biosynthesis. The butyrate-modulated AP induction was blocked by cycloheximide, suggesting that a mediator protein may be involved. Protein sequence deduced from complementary DNA analysis suggests that GCAP contains two potential sites for asparagine (N)-linked glycosylation. The marked increase in GCAP expression by butyrate in choriocarcinoma cells allowed us to study the extent of N-linked glycosylation and its role on GCAP enzyme activity. After limited tunicamycin treatment, Mr 65,000 (fully processed), Mr 58,000 (nonglycosylated), and Mr 62,000 polypeptides were synthesized by these cells in the presence of butyrate. This suggests that the Mr 62,000 product may be the singly glycosylated GCAP monomer and that both sites are glycosylated in this phosphatase. The glycosylated and nonglycosylated GCAPs, synthesized by butyrate-treated choriocarcinoma cells in the absence or presence of tunicamycin, respectively, were similarly inhibited by L-leucine or EDTA. Moreover, the specific enzyme activity of glycosylated and nonglycosylated GCAP remained unchanged, indicating that AP lacking N-linked oligosaccharide side chains was catalytically active. This is supported by the finding that nonglycosylated GCAP incorporated inorganic phosphate which binds to the active site of AP. Since the active form of AP is a homodimer, our data indicate that the glycan moieties are not required for the dimerization and catalytic activity of GCAP.

Two alternative mRNAs coding for the angiogenic factor, placenta growth factor (PlGF), are transcribed from a single gene of chromosome 14.

We have previously reported on the identification of a cDNA (placenta growth factor, PlGF) coding for a novel angiogenic factor expressed in placental tissue that is similar to vascular permeability factor/vascular endothelial growth factor (VPF/VEGF). Biochemical and functional characterization of PlGF derived from transfected COS-1 cells revealed that it is a glycosylated dimeric secreted protein able to stimulate endothelial cell growth in vitro. Here, we report the isolation and characterization of the PlGF gene located on chromosome 14. At least two different mRNAs are produced from this single-copy gene in different cell lines and tissues. Sequence comparison of the polypeptides encoded by the two different isolated cDNAs indicates that they are identical except for the insertion of a highly basic 21 amino acid stretch at the carboxyl end of the protein. RNA expression analysis of several tissues, tumors and cell lines indicates differential distribution of the two PlGF mRNAs. Finally, preliminary results indicate that the PIGF gene has been conserved in evolution, since the human PlGF cDNA hybridizes to sequences present in the genomic DNA of Drosophila, Xenopus, chicken and mouse.

Modification of transmembrane electron transport activity in plasma membranes of simian virus 40 transformed pineal cells.

Changes have been found in the plasma membrane enzyme system which carries out transmembrane electron transport and associated proton transport in Simian virus 40 (SV40) temperature-sensitive A (tsA) mutant-transformed rat pineal cell line, RPN209-1. This cell line was temperature-sensitive for the maintenance of transformation. RPN209-1 cells expressed the transformed phenotype (rapid growth, high cell density, and cloning in soft agar) at the permissive temperature (33 degrees C) and the nontransformed phenotype (slower growth, lower saturation density, and lower cloning efficiency in soft agar) at the nonpermissive temperature (40 degrees C). The reduction of external ferricyanide, hexaammine ruthenium and diferric transferrin was used to measure the transmembrane redox activity. The transformed RPN209-1 cells expressed a lower transmembrane redox activity, which is more sensitive to the antitumor drug adriamycin, when compared to the cells with a nontransformed phenotype. The lower transmembrane redox activity is associated with a decrease in the affinity for ferricyanide and a change in Vmax of the enzyme. Since the transformed cells have 25% lower concentration of NADH, the decrease in Vmax may be partly based on substrate limitation. Ionic strength variation in the assay media shows that the change in activity with transformation is not based on change in cell-surface change. Treatment with neuraminidase, however, indicates that sialic acid is important for enzyme activity, consistent with previous proposals that the transmembrane enzyme is a glycoprotein. The proton extrusion associated with transplasma membrane electron transport is increased in transformed cells relative to the rate of ferricyanide reduction. A relation between proton pumping transplasma membrane electron transport and growth stimulation by external oxidants is discussed.

Reduction of diferric transferrin by SV40 transformed pineal cells stimulates Na+/H+ antiport activity.

Transplasmalemma electron transport by HeLa and pineal cells to reduce external ferricyanide is associated with proton release from the cells. Diferric transferrin also acts as an electron acceptor for the transmembrane oxidoreductase. We now show that reduction of external diferric transferrin by RPNA-209-1 SV40 transformed pineal cells is accompanied by proton release from the cells. The stoichiometry of proton release to electron transfer is much greater than would be expected from aniostropic electron flow across the membrane through protonated carriers. The proton release is not stimulated by apotransferrin and the diferric transferrin-stimulated activity is inhibited by apotransferrin. Apotransferrin also inhibits reduction of diferric transferrin by these cells. The proton release is dependent on external sodium ions and is inhibited by amiloride, which indicates that the proton release is mediated by the Na+/H+ antiport and that this antiport is activated by electron transport through the transmembrane dehydrogenase. Growth stimulation by diferric transferrin or other external oxidants can be based in part on activation of the Na+/H+ antiport.

Effect of glucocorticoids on the mouse methionine adenosyltransferase A1 gene expression, which is regulated by two promoters.

The methionine adenosyltransferase A1 (MATA1) gene encodes the hepatic forms of the enzyme MAT I and III. To determine the molecular mechanisms that regulate MATA1 gene expression, we characterized promoters and the 5'-flanking sequence of MATA1. Transient expression assays demonstrated the presence of two promoters for the MATA1 gene. The p1 promoter is contained in the -57 to -2 nucleotide region, and gives rise to the P1 transcript initiated at +1. The p2 promoter is contained in the -248 to -146 nucleotide region. The -229 to -213 nucleotide region of the MATA1 gene, which contains an Ets-binding-site sequence, was necessary for p2 promoter activity. Sequence analysis of 5'-RACE products indicated that there was a transcript (P2) initiated at -156. The -107 to +145 nucleotide region is missing from the mature P2 transcript, which suggests that the -107 to +145 nucleotide sequence is an intron of the P2 transcript. The p2 promoter may give rise to the P2 transcript. The p1 promoter activity was increased by glucocorticoids, but the p2 promoter activity was not affected by glucocorticoids.

Molecular genetics of hepatic methionine adenosyltransferase deficiency.

Hepatic methionine adenosyltransferase (MAT) deficiency is caused by mutations in the human MAT1A gene that abolish or reduce hepatic MAT activity that catalyzes the synthesis of S-adenosylmethionine from methionine and ATP. This genetic disorder is characterized by isolated persistent hypermethioninemia in the absence of cystathionine beta-synthase deficiency, tyrosinemia, or liver disease. Depending on the nature of the genetic defect, hepatic MAT deficiency can be transmitted either as an autosomal recessive or dominant trait. Genetic analyses have revealed that mutations identified in the MAT1A gene only partially inactivate enzymatic activity, which is consistent with the fact that most hepatic MAT-deficient individuals are clinically well. Two hypermethioninemic individuals with null MAT1A mutations have developed neurological problems, including brain demyelination, although this correlation is by no means absolute. Presently, it is recommended that a DNA-based diagnosis should be performed for isolated hypermethioninemic individuals with unusually high plasma methionine levels to assess if therapy aimed at the prevention of neurological manifestations is warranted.

Characterization of pregnancy-specific beta 1-glycoprotein synthesized by human placental fibroblasts.

We have previously demonstrated that human placental fibroblasts produce a pregnancy-specific beta 1-glycoprotein (PS beta G) immunologically indistinguishable from placental PS beta G. This was confirmed by the immunocytochemical localization of PS beta G in these fibroblasts. In addition, placental fibroblasts contain all three PS beta G mRNAs of 2.3, 2.2, and 1.7 kilobases which hybridize with the three PS beta G cDNAs (PSG16, PSG93, and PSG95) identified, although at 1.4-2.5% of the levels in human term placenta. The major PS beta G species synthesized by placental fibroblasts is a 62K glycopolypeptide formed from a 58K intracellular precursor polypeptide. However, the PS beta G species found in human placenta are one major glycoprotein of 72K and two minor ones of 64K and 54K. Poly(A)+ RNA from placental fibroblasts directed the synthesis of two polypeptides of 48K and 46K (major), whereas, poly(A)+ RNA from human placenta directed the synthesis of higher levels of four polypeptides of 50 K, 48 K (major), 46 K, and 36 K. Thus, the major PS beta G species found in fibroblasts and human placenta differ. The carbohydrate side-chains are essential for the stability of fibroblast PS beta G, because PS beta G synthesis in these fibroblasts could not be detected in the presence of tunicamycin, a protein glycosylation inhibitor which did not affect PS beta G mRNA expression. Our finding that a variant PS beta G species is produced in placental fibroblasts raises the possibility that the authentic placental PS beta G species may have different functions.