Biomedical subjects

C J Pan

Publications and source records attributed to C J Pan.

At least 19 recordsLinked to original sources

PubMed2000

A molecular link between the common phenotypes of type 1 glycogen storage disease and HNF1alpha-null mice.

The clinical manifestations of type 1 glycogen storage disease (GSD-1) in patients deficient in the glucose-6-phosphatase (G6Pase) system (e.g. growth retardation, hepatomegaly, hyperlipidemia, and renal dysfunction) are shared by Hnf1alpha(-/-) mice deficient of a transcriptional activator, hepatocyte nuclear factor 1alpha (HNF1alpha). However, the molecular mechanism is unknown. The G6Pase system, essential for the maintenance of glucose homeostasis, is comprised of glucose 6-phosphate transporter (G6PT) and G6Pase. G6PT translocates G6P from the cytoplasm to the lumen of the endoplasmic reticulum where it is metabolized by G6Pase to glucose and phosphate. Deficiencies in G6Pase and G6PT cause GSD-1a and GSD-1b, respectively. Hnf1alpha(-/-) mice also develop noninsulin-dependent diabetes mellitus caused by defective insulin secretion. In this study, we sought to determine whether there is a molecular link between HNF1alpha deficiency and function of the G6Pase system. Transactivation studies revealed that HNF1alpha is required for transcription of the G6PT gene. Hepatic G6PT mRNA levels and microsomal G6P transport activity are also markedly reduced in Hnf1alpha(-/-) mice as compared with Hnf1alpha(+/+) and Hnf1alpha(+/-) littermates. On the other hand, hepatic G6Pase mRNA expression and activity are up-regulated in Hnf1alpha(-/-) mice, consistent with observations that G6Pase expression is increased in diabetic animals. Taken together, the results strongly suggest that metabolic abnormalities in HNF1alpha-null mice are caused in part by G6PT deficiency and by perturbations of the G6Pase system.

Animals↗

PubMed2000

Structural requirements for the stability and microsomal transport activity of the human glucose 6-phosphate transporter.

Deficiencies in glucose 6-phosphate (G6P) transporter (G6PT), a 10-helical endoplasmic reticulum transmembrane protein of 429 amino acids, cause glycogen storage disease type 1b. To date, only three missense mutations in G6PT have been shown to abolish microsomal G6P transport activity. Here, we report the results of structure-function studies on human G6PT and demonstrate that 15 missense mutations and a codon deletion (delta F93) mutation abolish microsomal G6P uptake activity and that two splicing mutations cause exon skipping. While most missense mutants support the synthesis of G6PT protein similar to that of the wild-type transporter, immunoblot analysis shows that G20D, delta F93, and I278N mutations, located in helix 1, 2, and 6, respectively, destabilize the G6PT. Further, we demonstrate that G6PT mutants lacking an intact helix 10 are misfolded and undergo degradation within cells. Moreover, amino acids 415-417 in the cytoplasmic tail of the carboxyl-domain, extending from helix 10, also play a critical role in the correct folding of the transporter. However, the last 12 amino acids of the cytoplasmic tail play no essential role(s) in functional integrity of the G6PT. Our results, for the first time, elucidate the structural requirements for the stability and transport activity of the G6PT protein.

Amino Acid Sequence↗

PubMed2000

Correction of glycogen storage disease type 1a in a mouse model by gene therapy.

Glycogen storage disease type 1a (GSD-1a), characterized by hypoglycemia, liver and kidney enlargement, growth retardation, hyperlipidemia, and hyperuricemia, is caused by a deficiency in glucose-6-phosphatase (G6Pase), a key enzyme in glucose homeostasis. To evaluate the feasibility of gene replacement therapy for GSD-1a, we have infused adenoviral vector containing the murine G6Pase gene (Ad-mG6Pase) into G6Pase-deficient (G6Pase(-/-)) mice that manifest symptoms characteristic of human GSD-1a. Whereas <15% of G6Pase(-/-) mice under glucose therapy survived weaning, a 100% survival rate was achieved when G6Pase(-/-) mice were infused with Ad-mG6Pase, 90% of which lived to 3 months of age. Hepatic G6Pase activity in Ad-mG6Pase-infused mice was restored to 19% of that in G6Pase(+/+) mice at 7-14 days post-infusion; the activity persisted for at least 70 days. Ad-mG6Pase infusion also greatly improved growth of G6Pase(-/-) mice and normalized plasma glucose, cholesterol, triglyceride, and uric acid profiles. Furthermore, liver and kidney enlargement was less pronounced with near-normal levels of glycogen depositions in both organs. Our data demonstrate that a single administration of a recombinant adenoviral vector can alleviate the pathological manifestations of GSD-1a in mice, suggesting that this disorder in humans can potentially be corrected by gene therapy.

Adenoviridae↗

PubMed2000

Human variant glucose-6-phosphate transporter is active in microsomal transport.

Glycogen storage disease type lb (GSD-lb) is caused by deficiencies in the glucose-6-phosphate transporter (G6PT), which works together with glucose-6-phosphatase to maintain glucose homeostasis. In humans, there are two alternatively spliced transcripts, G6PT and variant G6PT (vG6PT), differing by the inclusion of a 66-bp exon-7 sequence in vG6PT. We have previously shown that the G6PT protein functions as a microsomal glucose-6-phosphate (G6P) transporter, which is anchored to the endoplasmic reticulum by ten transmembrane helices. Here, we demonstrate that vG6PT is also active in microsomal G6P transport. The additional 22 amino acids in vG6PT is predicted to constitute a part of the luminal loop 4. Our data indicate that this loop plays no vital role in microsomal G6P transport. Further, we show that G6PT mRNA is expressed in all organs and tissues examined, but that the vG6PT transcript is expressed exclusively in the brain, heart, and skeletal muscle. These results raise the possibility that mutations in exon-7 of the G6PT gene, which would not perturb glucose homeostasis, might have other deleterious effects.

Alternative Splicing↗

PubMed1999

Transmembrane topology of human glucose 6-phosphate transporter.

Glycogen storage disease type 1b is caused by a deficiency in a glucose 6-phosphate transporter (G6PT) that translocates glucose 6-phosphate from the cytoplasm to the endoplasmic reticulum lumen where the active site of glucose 6-phosphatase is situated. Using amino- and carboxyl-terminal tagged G6PT, we demonstrate that proteolytic digestion of intact microsomes resulted in the cleavage of both tags, indicating that both termini of G6PT face the cytoplasm. This is consistent with ten and twelve transmembrane domain models for G6PT predicted by hydropathy analyses. A region of G6PT corresponding to amino acid residues 50-71, which constitute a transmembrane segment in the twelve-domain model, are situated in a 51-residue luminal loop in the ten-domain model. To determine which of these two models is correct, we generated two G6PT mutants, T53N and S55N, that created a potential Asn-linked glycosylation site at residues 53-55 (N53SS) or 55-57 (N55QS), respectively. N53SS or N55QS would be glycosylated only if it is situated in a luminal loop larger than 33 residues as predicted by the ten-domain model. Whereas wild-type G6PT is not a glycoprotein, both T53N and S55N mutants are glycosylated, strongly supporting the ten-helical model for G6PT.

Amino Acid Sequence↗

PubMed1999

Inactivation of the glucose 6-phosphate transporter causes glycogen storage disease type 1b.

Glycogen storage disease type 1b (GSD-1b) is proposed to be caused by a deficiency in microsomal glucose 6-phosphate (G6P) transport, causing a loss of glucose-6-phosphatase activity and glucose homeostasis. However, for decades, this disorder has defied molecular characterization. In this study, we characterize the structural organization of the G6P transporter gene and identify mutations in the gene that segregate with the GSD-1b disorder. We report the functional characterization of the recombinant G6P transporter and demonstrate that mutations uncovered in GSD-1b patients disrupt G6P transport. Our results, for the first time, define a molecular basis for functional deficiency in GSD-1b and raise the possibility that the defective G6P transporter contributes to neutropenia and neutrophil/monocyte dysfunctions characteristic of GSD-1b patients.

Animals↗

PubMed1999

Type-1c glycogen storage disease is not caused by mutations in the glucose-6-phosphate transporter gene.

Glycogen storage disease type 1 (GSD-1) is a group of autosomal recessive disorders caused by deficiencies in glucose-6-phosphatase (G6Pase) and the associated substrate/product transporters. Molecular genetic studies have demonstrated that GSD-1a and GSD-1b are caused by mutations in the G6Pase enzyme and a glucose-6-phosphate transporter (G6PT), respectively. While kinetic studies of G6Pase catalysis predict that the index GSD-1c patient is deficient in a pyrophosphate/phosphate transporter, the existence of a separate locus for GSD-1c remains unclear. We have previously shown that the G6Pase gene of the index GSD-1c patient is intact; we now show that the G6PT gene of this patient is normal, strongly suggesting the existence of a distinct GSD-1c locus.

Antiporters↗

PubMed1998

Cloning and characterization of cDNAs encoding a candidate glycogen storage disease type 1b protein in rodents.

Glycogen storage disease type 1 (GSD-1) is a group of genetic disorders caused by a deficiency in the activity of the enzyme glucose-6-phosphatase. (G6Pase). GSD-1a and GSD-1b, the two major subgroups, have been confirmed at the molecular genetic level. The gene responsible for GSD-1b maps to human chromosome 11q23 and a candidate human GSD-1b cDNA that encodes a microsomal transmembrane protein has been identified. In this study, we show that this cDNA maps to chromosome 11q23; thus it is a strong candidate for GSD-1b. Furthermore, we isolated and characterized candidate murine and rat GSD-1b cDNAs. Both encode transmembrane proteins sharing 93-95% sequence homology to the human GSD-1b protein. The expression profiles of murine GSD-1b and G6Pase differ both in the liver and in the kidney; the GSD-1b transcript appears before the G6Pase mRNA during development. In addition to G6Pase deficiency, GSD-1b patients suffer neutropenia, neutrophil dysfunction, and recurrent bacterial infections. Interestingly, although the G6Pase mRNA is expressed primarily in the liver, kidney, and intestine, the GSD-1b mRNA is expressed in numerous tissues, including human neutrophils/monocytes.

Adult↗

PubMed1998

Ontogeny of the murine glucose-6-phosphatase system.

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.

Age Factors↗

PubMed1998

Asparagine-linked oligosaccharides are localized to a luminal hydrophilic loop in human glucose-6-phosphatase.

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.

Amino Acid Sequence↗

PubMed1998

Transmembrane topology of glucose-6-phosphatase.

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.

Amino Acid Sequence↗

PubMed1996

Differential expression and butyrate response of human alkaline phosphatase genes are mediated by upstream DNA elements.

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.

Alkaline Phosphatase↗

PubMed1996

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.

Animals↗

PubMed1995

Structure-function analysis of human glucose-6-phosphatase, the enzyme deficient in glycogen storage disease type 1a.

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.

Amino Acid Sequence↗

PubMed1994

Pregnancy-specific glycoprotein gene expression and the induction by 5-bromo-2'-deoxyuridine.

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)

Base Sequence↗

PubMed1994

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.

Base Sequence↗

PubMed1993

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.

Amino Acid Sequence↗

PubMed1993

Isolation of the gene for murine glucose-6-phosphatase, the enzyme deficient in glycogen storage disease type 1A.

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.

Amino Acid Sequence↗