RESUMEN
AIMS: To conduct a direct comparison regarding the non-coplanar positioning accuracy between the optical surface imaging system Catalyst HDTM and non-coplanar cone-beam computed tomography (NC-CBCT) in intracranial single-isocentre non-coplanar stereotactic radiosurgery (SRS) and hypofractionated stereotactic radiotherapy (HSRT). MATERIALS AND METHODS: Twenty patients with between one and five brain metastases who underwent single-isocentre non-coplanar volumetric modulated arc therapy (NC-VMAT) SRS or HSRT were enrolled in this study. For each non-zero couch angle, both Catalyst HDTM and NC-CBCT were used for set-up verification prior to beam delivery. The set-up error reported by Catalyst HDTM was compared with the set-up error derived from NC-CBCT, which was defined as the gold standard. Additionally, the dose delivery accuracy of each non-coplanar field after using Catalyst HDTM and NC-CBCT for set-up correction was measured with SRS MapCHECKTM. RESULTS: The median set-up error differences (absolute values) between the two positioning methods were 0.30 mm, 0.40 mm, 0.50 mm, 0.15°, 0.10° and 0.10° in the vertical, longitudinal, lateral, yaw, pitch and roll directions, respectively. The largest absolute set-up error differences regarding translation and rotation were 1.5 mm and 1.1°, which occurred in the longitudinal and yaw directions, respectively. Only 35.71% of the pairs of measurements were within the tolerance of 0.5 mm and 0.5° simultaneously. In addition, the non-coplanar field with NC-CBCT correction yielded a higher gamma passing rate than that with Catalyst HDTM correction (P < 0.05), especially for evaluation criteria of 1%/1 mm with a median increase of 12.8%. CONCLUSIONS: Catalyst HDTM may not replace NC-CBCT for non-coplanar set-up corrections in single-isocentre NC-VMAT SRS and HSRT for single and multiple brain metastases. The potential role of Catalyst HDTM in intracranial SRS/HSRT needs to be further studied in the future.
Asunto(s)
Neoplasias Encefálicas , Radiocirugia , Humanos , Radiocirugia/métodos , Neoplasias Encefálicas/diagnóstico por imagen , Neoplasias Encefálicas/radioterapia , Neoplasias Encefálicas/cirugía , Tomografía Computarizada de Haz Cónico , Carmustina , Etopósido , Planificación de la Radioterapia Asistida por Computador/métodosRESUMEN
AIMS: To evaluate the clinical feasibility of single-isocentre non-coplanar volumetric modulated arc therapy (NC-VMAT) with non-coplanar cone beam computed tomography (NC-CBCT) in hypofractionated stereotactic radiotherapy (HSRT) for five or fewer multiple brain metastases. MATERIALS AND METHODS: Ten patients with multiple brain metastases who underwent single-isocentre NC-VMAT HSRT with limited couch rotations (within ±45°) and NC-CBCT with a limited scanning range (150-200°) were included in the current analysis. Conventional single-isocentre coplanar VMAT (C-VMAT) plans were generated and compared with NC-VMAT plans. The intracranial response and toxicities of single-isocentre NC-VMAT HSRT were also evaluated. RESULTS: Compared with C-VMAT, NC-VMAT generated better target conformity (P < 0.05), a lower gradient index (P < 0.05) and better normal brain tissue sparing, especially for volume ≥12 Gy, with a median reduction of 12.65 cm3. For 45° couch rotation, NC-CBCT produced sufficient image quality to differentiate bony anatomy, even with a 150° scanning range, which could be successfully used for patient set-up correction. After NC-CBCT, 57.1% of the measured non-coplanar set-up errors exceeded the threshold value. The median gamma passing rate of NC-VMAT was higher than that of C-VMAT plans (P < 0.05). The non-coplanar beam of NC-VMAT with NC-CBCT corrections exhibited superior gamma passing rate to that without NC-CBCT corrections. The intracranial objective response rate and disease control rate for all patients were 80% (8/10) and 100% (10/10), respectively, and the most common toxicities were headache (20%) and dizziness (20%). CONCLUSION: NC-VMAT with limited couch rotation (within ±45°) combined with NC-CBCT with a limited scanning range (150-200°) markedly improves the plan quality and set-up accuracy in single-isocentre multiple-target HSRT.
Asunto(s)
Neoplasias Encefálicas , Radiocirugia , Radioterapia de Intensidad Modulada , Humanos , Radioterapia de Intensidad Modulada/métodos , Dosificación Radioterapéutica , Estudios de Factibilidad , Planificación de la Radioterapia Asistida por Computador/métodos , Radiocirugia/métodos , Neoplasias Encefálicas/diagnóstico por imagen , Neoplasias Encefálicas/radioterapia , Neoplasias Encefálicas/secundario , Tomografía Computarizada de Haz CónicoRESUMEN
A deficiency in microsomal glucose-6-phosphatase (G6Pase) activity causes glycogen storage disease type 1 (GSD-1), a clinically and biochemically heterogeneous group of diseases. It has been suggested that catalysis by G6Pase involves multiple components, with defects in the G6Pase catalytic unit causing GSD-1a and defects in the putative substrate and product translocases causing GSD-1b, 1c, and 1d. However, this model is open to debate. To elucidate the G6Pase system, we have examined G6Pase mRNA expression, G6Pase activity, and glucose 6-phosphate (G6P) transport activity in the murine liver and kidney during normal development. In the liver, G6Pase mRNA and enzymatic activity were detected at 18 days gestation and increased markedly at parturition, before leveling off to adult levels. In the kidney, G6Pase mRNA and enzymatic activity appeared at 19 days gestation and peaked at weaning, suggesting that kidney G6Pase may have a different metabolic role. In situ hybridization analysis demonstrated that, in addition to the liver and kidney, the intestine expressed G6Pase. Despite the expression of G6Pase in the embryonic liver, microsomal G6P transport activity was not detectable until birth, peaking at about age 4 weeks. Our study strongly supports the multicomponent model for the G6Pase system.
Asunto(s)
Desarrollo Embrionario y Fetal , Glucosa-6-Fosfatasa/metabolismo , Glucosa-6-Fosfatasa/fisiología , Factores de Edad , Animales , Animales Recién Nacidos , Transporte Biológico , Catálisis , Desarrollo Embrionario y Fetal/genética , Regulación del Desarrollo de la Expresión Génica , Glucosa-6-Fosfatasa/genética , Intestinos/enzimología , Intestinos/crecimiento & desarrollo , Riñón/enzimología , Riñón/crecimiento & desarrollo , Ratones , Microsomas Hepáticos/enzimología , Especificidad de Órganos/genéticaRESUMEN
Deficiency of glucose-6-phosphatase (G6Pase), an endoplasmic reticulum transmembrane glycoprotein, causes glycogen storage disease type 1a. We have recently shown that human G6Pase contains an odd number of transmembrane segments, supporting a nine-transmembrane helical model for this enzyme. Sequence analysis predicts the presence of three potential asparagine (N)-linked glycosylation sites, N96TS, N203AS, and N276SS, conserved among mammalian G6Pases. According to this model, Asn96, located in a 37-residue luminal loop, is a potential acceptor for oligosaccharides, whereas Asn203 and Asn276, located in a 12-residue cytoplasmic loop and helix 7, respectively, would not be utilized for this purpose. We therefore characterized mutant G6Pases lacking one, two, or all three potential N-linked glycosylation sites. Western blot and in vitro translation studies showed that G6Pase is glycosylated only at Asn96, further validating the nine-transmembrane topology model. Substituting Asn96 with an Ala (N96A) moderately reduced enzymatic activity and had no effect on G6Pase synthesis or degradation, suggesting that oligosaccharide chains do not play a major role in protecting the enzyme from proteolytic degradation. In contrast, mutation of Asn276 to an Ala (N276A) destabilized the enzyme and markedly reduced enzymatic activity. We present additional evidence suggesting that the integrity of transmembrane helices is essential for G6Pase stability and catalytic activity.
Asunto(s)
Asparagina/química , Glucosa-6-Fosfatasa/química , Oligosacáridos/química , Secuencia de Aminoácidos , Western Blotting , Línea Celular , Glucosa-6-Fosfatasa/genética , Glicosilación , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Estructura Secundaria de Proteína , Relación Estructura-ActividadRESUMEN
Glycogen-storage disease type 1 (GSD-1), also known as "von Gierke disease," is caused by a deficiency in microsomal glucose-6-phosphatase (G6Pase) activity. There are four distinct subgroups of this autosomal recessive disorder: 1a, 1b, 1c, and 1d. All share the same clinical manifestations, which are caused by abnormalities in the metabolism of glucose-6-phosphate (G6P). However, only GSD-1b patients suffer infectious complications, which are due to both the heritable neutropenia and the functional deficiencies of neutrophils and monocytes. Whereas G6Pase deficiency in GSD-1a patients arises from mutations in the G6Pase gene, this gene is normal in GSD-1b patients, indicating a separate locus for the disorder in the 1b subgroup. We now report the linkage of the GSD-1b locus to genetic markers spanning a 3-cM region on chromosome 11q23. Eventual molecular characterization of this disease will provide new insights into the genetic bases of G6P metabolism and neutrophil-monocyte dysfunction.
Asunto(s)
Cromosomas Humanos Par 11 , Glucosa-6-Fosfatasa/genética , Enfermedad del Almacenamiento de Glucógeno Tipo I/genética , Deleción Cromosómica , Mapeo Cromosómico , Consanguinidad , Etnicidad , Familia , Femenino , Genes Recesivos , Marcadores Genéticos , Enfermedad del Almacenamiento de Glucógeno Tipo I/enzimología , Humanos , Escala de Lod , Masculino , Repeticiones de Microsatélite , Microsomas/enzimología , Linaje , Polimorfismo GenéticoRESUMEN
Deficiency of microsomal glucose-6-phosphatase (G6Pase), the key enzyme in glucose homeostasis, causes glycogen storage disease type 1a, an autosomal recessive disorder. Characterization of the transmembrane topology of G6Pase should facilitate the identification of amino acid residues contributing to the active site and broaden our understanding of the effects of mutations that cause glycogen storage disease type 1a. Using N- and C-terminal tagged G6Pase, we show that in intact microsomes, the N terminus is resistant to protease digestion, whereas the C terminus is sensitive to such treatment. Our results demonstrate that G6Pase possesses an odd number of transmembrane helices, with its N and C termini facing the endoplasmic reticulum lumen and the cytoplasm, respectively. During catalysis, a phosphoryl-enzyme intermediate is formed, and the phosphoryl acceptor in G6Pase is a His residue. Sequence alignment suggests that mammalian G6Pases, lipid phosphatases, acid phosphatases, and a vanadium-containing chloroperoxidase (whose tertiary structure is known) share a conserved phosphatase motif. Active-site alignment of the vanadium-containing chloroperoxidase and G6Pases predicts that Arg-83, His-119, and His-176 in G6Pase contribute to the active site and that His-176 is the residue that covalently binds the phosphoryl moiety during catalysis. This alignment also predicts that Arg-83, His-119, and His-176 reside on the same side of the endoplasmic reticulum membrane, which is supported by the recently predicted nine-transmembrane helical model for G6Pase. We have previously shown that Arg-83 is involved in positioning the phosphate during catalysis and that His-119 is essential for G6Pase activity. Here we demonstrate that substitution of His-176 with structurally similar or dissimilar amino acids inactivates the enzyme, suggesting that His-176 could be the phosphoryl acceptor in G6Pase during catalysis.
Asunto(s)
Glucosa-6-Fosfatasa/química , Proteínas de la Membrana/química , Microsomas/enzimología , Secuencia de Aminoácidos , Sitios de Unión , Endopeptidasa K/farmacología , Glucosa-6-Fosfatasa/efectos de los fármacos , Glucosa-6-Fosfatasa/metabolismo , Enfermedad del Almacenamiento de Glucógeno Tipo I/enzimología , Histidina , Humanos , Proteínas de la Membrana/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Mutagénesis , Conformación Proteica , Eliminación de Secuencia , Tripsina/farmacologíaRESUMEN
Glycogen storage disease type 1a (von Gierke disease, GSD 1a) is caused by the deficiency of microsomal glucose-6-phosphatase (G6Pase) activity which catalyzes the final common step of glycogenolysis and gluconeogenesis. The recent cloning of the G6Pase cDNA and characterization of the human G6Pase gene enabled the characterization of the mutations causing GSD 1a. This, in turn, allows the introduction of a noninvasive DNA-based diagnosis that provides reliable carrier testing and prenatal diagnosis. In this study, we report the biochemical and clinical characteristics as well as mutational analyses of 12 Israeli GSD 1a patients of different families, who represent most GSD 1a patients in Israel. The mutations, G6Pase activity, and glycogen content of 7 of these patients were reported previously. The biochemical data and clinical findings of all patients were similar and compatible with those described in other reports. All 9 Jewish patients, as well as one Muslim Arab patient, presented the R83C mutation. Two Muslim Arab patients had the V166G mutation which was not found in other patients' populations. The V166G mutation, which was introduced into the G6Pase cDNA by site-directed mutagenesis following transient expression in COS-1 cells, was shown to cause complete inactivation of the G6Pase. The characterization of all GSD 1a mutations in the Israeli population lends itself to carrier testing in these families as well as to prenatal diagnosis, which was carried out in 2 families. Since all Ashkenzai Jewish patients harbor the same mutation, our study suggests that DNA-based diagnosis may be used as an initial diagnostic step in Ashkenazi Jews suspected of having GSD 1a, thereby avoiding liver biopsy.
Asunto(s)
Enfermedad del Almacenamiento de Glucógeno Tipo I/genética , Árabes/genética , Análisis Mutacional de ADN , Femenino , Glucosa-6-Fosfatasa/análisis , Glucosa-6-Fosfatasa/genética , Enfermedad del Almacenamiento de Glucógeno Tipo I/etnología , Humanos , Islamismo , Israel , Judíos/genética , Hígado/enzimología , Glucógeno Hepático/análisis , Masculino , Polimorfismo Conformacional Retorcido-Simple , Diagnóstico PrenatalRESUMEN
Glycogen storage disease type 1a (von Gierke disease, GSD-1A) is caused by the deficiency of microsomal glucose-6-phosphatase (G6Pase) activity which catalyzes the final common step of glycogenolysis and gluconeogenesis. The cloning of the G6Pase cDNA and characterization of the human G6Pase gene enabled the identification of the mutations causing GSD-1a. This, in turn, allows the development of non-invasive DNA-based diagnosis that provides reliable carrier testing and prenatal diagnosis. Here we report on two new mutations E110Q and D38V causing GSD-1a in two Hungarian patients. The analyses of these mutations by site-directed mutagenesis followed by transient expression assays demonstrated that E110Q retains 17% of G6Pase enzymatic activity while the D38V abolishes the enzymatic activity. The patient with the E110Q has G222R as his other mutation. G222R was also shown to preserve about 4% of the G6Pase enzymatic activity. Nevertheless, the patient presented with the classical severe symptomatology of the GSD-1a.
Asunto(s)
Glucosa-6-Fosfatasa/genética , Enfermedad del Almacenamiento de Glucógeno Tipo I/enzimología , Mutación , Niño , Glucosa-6-Fosfatasa/metabolismo , Enfermedad del Almacenamiento de Glucógeno Tipo I/genética , Humanos , Hungría , Masculino , Monoéster Fosfórico Hidrolasas/metabolismoRESUMEN
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.
Asunto(s)
Glucosa-6-Fosfatasa/genética , Glucosa-6-Fosfatasa/metabolismo , Enfermedad del Almacenamiento de Glucógeno Tipo I/enzimología , Enfermedad del Almacenamiento de Glucógeno Tipo I/etiología , Animales , Animales Recién Nacidos , Secuencia de Bases , Transporte Biológico , Glucemia/análisis , Glucosa-6-Fosfato , Glucofosfatos/genética , Glucofosfatos/metabolismo , Enfermedad del Almacenamiento de Glucógeno Tipo I/genética , Riñón/metabolismo , Riñón/patología , Hígado/metabolismo , Hígado/patología , Ratones , Ratones Noqueados , Modelos Biológicos , Datos de Secuencia Molecular , FenotipoRESUMEN
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.
Asunto(s)
Errores Innatos del Metabolismo de los Aminoácidos/genética , Metionina Adenosiltransferasa/deficiencia , Metionina/metabolismo , Secuencia de Bases , Femenino , Humanos , Metionina Adenosiltransferasa/genética , Datos de Secuencia Molecular , Mutación , Polimorfismo Conformacional Retorcido-Simple , Transcripción GenéticaRESUMEN
Diagnosis of glycogen storage disease (GSD) type 1a currently is established by demonstrating the lack of glucose-6-phosphatase (G6Pase) activity in the patient's biopsied liver specimen. Recent cloning of the G6Pase gene and identification of mutations within the gene that causes GSD type 1a allow for the development of a DNA-based diagnostic method. Using SSCP analysis and DNA sequencing, we characterized the G6Pase gene of 70 unrelated patients with enzymatically confirmed diagnosis of GSD type 1a and detected mutations in all except 17 alleles (88%). Sixteen mutations were uncovered that were shown by expression to abolish or greatly reduce G6Pase activity and that therefore are responsible for the GSD type 1a disorder. R83C and Q347X are the most prevalent mutations found in Caucasians, 130X and R83C are most prevalent in Hispanics, and R83H is most prevalent in Chinese. The Q347X mutation has thus far been identified only in Caucasian patients, and the 130X mutation has been identified only in Hispanic patients. Our results demonstrate that the DNA-based analysis can accurately, rapidly, and noninvasively detect the majority of mutations in GSD type 1a. This DNA-based diagnosis now permits prenatal diagnosis among at-risk patients and serves as a database in screening and counseling patients clinically suspected of having this disease.
Asunto(s)
Glucosa-6-Fosfatasa/genética , Enfermedad del Almacenamiento de Glucógeno Tipo I/genética , Mutación , Alelos , Secuencia de Bases , Etnicidad/genética , Humanos , Datos de Secuencia Molecular , Polimorfismo Conformacional Retorcido-Simple , PrevalenciaRESUMEN
Glucose-6-phosphatase (G6Pase) is the enzyme deficient in glycogen storage disease type 1a, an autosomal recessive disorder. We have previously identified six mutations in the G6Pase gene of glycogen storage disease type 1a patients and demonstrated that these mutations abolished or greatly reduced enzymatic activity of G6Pase, a hydrophobic protein of 357 amino acids. Of these, four mutations (R83C, R295C, G222R, and Q347X) are missense and one (Q347X) generates a truncated G6Pase of 346 residues. To further understand the roles and structural requirements of amino acids 83, 222, 295, and those at the carboxyl terminus in G6Pase catalysis, we characterized mutant G6Pases generated by near-saturation mutagenesis of the aforementioned amino acids. Substitution of Arg-83 with amino acids of diverse structures including Lys, a conservative change, yielded mutant G6Pase with no enzymatic activity. On the other hand, substitution of Arg-295 with Lys (R295K) retained high activity, and R295N, R295S, and R295Q exhibited moderate activity. All other substitutions of Arg-295 had no G6Pase activity, suggesting that the role of Arg-295 is to stabilize the protein either by salt bridge or hydrogen-bond formation. Substitution of Gly-222, however, remained functional unless a basic (Arg or Lys), acidic (Asp), or large polar (Gln) residue was introduced, consistent with the hydrophobic requirement of codon 222, which is predicted to be in the fourth membrane-spanning domain. It is possible that Arg-83 is involved in stabilizing the enzyme (His)-phosphate intermediate formed during G6Pase catalysis. There exist 9 conserved His residues in human G6Pase. His-9, His-119, His-252, and His-353 reside on the same side of the endoplasmic reticulum membrane as Arg-83. Whereas H119A mutant G6Pase had no enzymatic activity, H9A, H252A, and H353A mutant G6Pases retained significant activity. Substitution of His-119 with amino acids of diverse structures also yielded mutant G6Pase with no activity, suggesting that His-119 is the phosphate acceptor in G6Pase catalysis. We also present data demonstrating that the carboxyl-terminal 8 residues in human G6Pase are not essential for G6Pase catalysis.
Asunto(s)
Glucosa-6-Fosfatasa/química , Enfermedad del Almacenamiento de Glucógeno Tipo I/enzimología , Secuencia de Aminoácidos , Aminoácidos/química , Animales , Secuencia de Bases , Catálisis , Línea Celular Transformada , Chlorocebus aethiops , Codón/genética , Retículo Endoplásmico/enzimología , Glucosa-6-Fosfatasa/genética , Glucosa-6-Fosfatasa/metabolismo , Enfermedad del Almacenamiento de Glucógeno Tipo I/genética , Calor , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes de Fusión/metabolismo , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Glucosa-6-Fosfatasa/genética , Enfermedad del Almacenamiento de Glucógeno Tipo I/clasificación , Enfermedad del Almacenamiento de Glucógeno Tipo I/genética , Mutación/genética , Alelos , Secuencia de Aminoácidos , Secuencia de Bases , Células Cultivadas , Clonación Molecular , Genoma Humano , Enfermedad del Almacenamiento de Glucógeno Tipo I/enzimología , Heterocigoto , Homocigoto , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN , TransfecciónRESUMEN
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.
Asunto(s)
Regulación de la Expresión Génica , Glicoproteínas/genética , Proteínas Gestacionales/genética , Glicoproteínas beta 1 Específicas del Embarazo , Regiones Promotoras Genéticas , Secuencia de Bases , Cromosomas Humanos Par 19 , Reactivos de Enlaces Cruzados , Proteínas de Unión al ADN/metabolismo , Humanos , Sustancias Macromoleculares , Datos de Secuencia Molecular , Sondas de Oligonucleótidos/química , ARN Mensajero/genética , Alineación de Secuencia , Homología de Secuencia de Ácido Nucleico , Factor de Transcripción Sp1/metabolismo , Factores de Transcripción/metabolismoRESUMEN
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.
Asunto(s)
Glucosa-6-Fosfatasa/genética , Enfermedad del Almacenamiento de Glucógeno Tipo I/genética , Mutación , Secuencia de Bases , Cromosomas Humanos Par 17 , Retículo Endoplásmico/metabolismo , Exones/genética , Femenino , Enfermedad del Almacenamiento de Glucógeno Tipo I/enzimología , Heterocigoto , Humanos , Células Híbridas , Masculino , Datos de Secuencia Molecular , Linaje , Eliminación de SecuenciaRESUMEN
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.
Asunto(s)
Glucosa-6-Fosfatasa/genética , Enfermedad del Almacenamiento de Glucógeno Tipo I/genética , Mutación , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , ADN Complementario/genética , Exones , Glucosa-6-Fosfatasa/metabolismo , Enfermedad del Almacenamiento de Glucógeno Tipo I/enzimología , Glicosilación , Humanos , Hígado/enzimología , Ratones , Datos de Secuencia Molecular , Conformación Proteica , TransfecciónRESUMEN
Glycogen storage disease (GSD) type 1a (von Gierke disease) is caused by a deficiency in glucose-6-phosphatase, the key enzyme in glucose homeostasis catalyzing the terminal step in gluconeogenesis and glycogenolysis. Despite its clinical importance, this membrane-bound enzyme has eluded molecular characterization. Here we report the cloning and characterization of a murine glucose-6-phosphatase cDNA by screening a mouse liver cDNA library differentially with mRNA populations representing the normal and the albino deletion mouse known to express markedly reduced glucose-6-phosphatase activity. Additionally, we identified the gene that consists of 5 exons. Biochemical analyses indicate that the in vitro expressed enzyme is indistinguishable from mouse liver microsomal glucose-6-phosphatase exhibiting essentially identical kinetic constants, latency, thermal lability, and vanadate sensitivity. The characterization of the murine glucose-6-phosphatase gene opens the way for studying the molecular basis of GSD type 1a in humans and its etiology in an animal model.
Asunto(s)
Glucosa-6-Fosfatasa/genética , Enfermedad del Almacenamiento de Glucógeno Tipo I/enzimología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células Cultivadas , ADN Complementario , Glucosa-6-Fosfatasa/metabolismo , Humanos , Hidrólisis , Cinética , Ratones , Ratones Mutantes , Microsomas Hepáticos/enzimología , Datos de Secuencia Molecular , ARN Mensajero/metabolismoRESUMEN
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.
Asunto(s)
Variación Genética , Glicoproteínas/genética , Proteínas Gestacionales/genética , Glicoproteínas beta 1 Específicas del Embarazo , Alelos , Secuencia de Aminoácidos , Secuencia de Bases , ADN , Glicoproteínas/biosíntesis , Humanos , Datos de Secuencia Molecular , Placenta/metabolismo , Proteínas Gestacionales/biosíntesis , Regiones Promotoras Genéticas , Seudogenes , Caperuzas de ARN , Mapeo Restrictivo , Homología de Secuencia de Ácido Nucleico , Transcripción GenéticaRESUMEN
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.
Asunto(s)
Cromosomas Humanos Par 14 , Sustancias de Crecimiento/genética , Neovascularización Patológica , Placenta/fisiología , Proteínas Gestacionales/genética , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Secuencia Conservada , Expresión Génica , Genes , Humanos , Intrones , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/química , Factor de Crecimiento Placentario , ARN Mensajero/genética , Especificidad de la EspecieRESUMEN
The pregnancy-specific glycoproteins (PSGs) of the human placenta and the carcinoembryonic antigens comprise a subfamily within the immunoglobulin superfamily. There may be as many as 20 different PSG genes which are predominantly expressed in the placenta. As an initial step toward understanding the control of PSG expression, we isolated and characterized two nearly identical PSG genes, PSG1 and PSG1-I. PSG1, which lacks exon 1 (5'/L), but contains exons 2 (L/N), 3 (A1), 4 (A2), and 5 (B2-C), encodes five previously identified type I transcripts, PSG1a, 1b, 1c, 1d, and 1e in a L/N-A1-A2-B2-C domain arrangement. PSG1-I, which contains a complete transcriptional unit consisting of exons 5'/L, L/N, A1, and B2-C, encodes type II PSG transcripts in a L/N-A1-B2-C domain arrangement. The predicted PSG1-I-encoded proteins share nearly complete sequence identity with the PSG1-encoded members, except the latter contain extra A domains. Amplification by polymerase chain reaction of placental or hydatidiform mole cDNA demonstrates that PSG1-I is a functional type II PSG gene. Using transient expression assays, we demonstrated that the -834/-34 region upstream of the translational start site of the PSG1-I gene contained the PSG promoter elements and the -834 to -456 region contained negative control elements. Sodium butyrate, an inducer of PSG synthesis, greatly stimulated expression of all PSG1-I-chloramphenicol acetyltransferase (CAT) fusion gene constructs. However, butyrate was at least 2-fold more effective in stimulating CAT activity of fusion genes containing upstream sequences (-834 to -576) than those containing proximal sequences (-456 to -172), suggesting two regions in the PSG1-I gene that mediate the butyrate response.