The Molecular Basis of Glycogen Storage Disease Type 1a
2002; Elsevier BV; Volume: 277; Issue: 7 Linguagem: Inglês
10.1074/jbc.m110486200
ISSN1083-351X
AutoresJeng‐Jer Shieh, Mügen Terzioglu, Hisayuki Hiraiwa, Julia Marsh, Chi‐Jiunn Pan, Liyuan Chen, Janice Y. Chou,
Tópico(s)Carbohydrate Chemistry and Synthesis
ResumoGlycogen storage disease type 1a is caused by a deficiency in glucose-6-phosphatase (G6Pase), a nine-helical endoplasmic reticulum transmembrane protein required for maintenance of glucose homeostasis. To date, 75 G6Pase mutations have been identified, including 48 mutations resulting in single-amino acid substitutions. However, only 19 missense mutations have been functionally characterized. Here, we report the results of structure and function studies of the 48 missense mutations and the ΔF327 codon deletion mutation, grouped as active site, helical, and nonhelical mutations. The 5 active site mutations and 22 of the 31 helical mutations completely abolished G6Pase activity, but only 5 of the 13 nonhelical mutants were devoid of activity. Whereas the active site and nonhelical mutants supported the synthesis of G6Pase protein in a manner similar to that of the wild-type enzyme, immunoblot analysis showed that the majority (64.5%) of helical mutations destabilized G6Pase. Furthermore, we show that degradation of both wild-type and mutant G6Pase is inhibited by lactacystin, a potent proteasome inhibitor. Taken together, we have generated a data base of residual G6Pase activity retained by G6Pase mutants, established the critical roles of transmembrane helices in the stability and activity of this phosphatase, and shown that G6Pase is a substrate for proteasome-mediated degradation. Glycogen storage disease type 1a is caused by a deficiency in glucose-6-phosphatase (G6Pase), a nine-helical endoplasmic reticulum transmembrane protein required for maintenance of glucose homeostasis. To date, 75 G6Pase mutations have been identified, including 48 mutations resulting in single-amino acid substitutions. However, only 19 missense mutations have been functionally characterized. Here, we report the results of structure and function studies of the 48 missense mutations and the ΔF327 codon deletion mutation, grouped as active site, helical, and nonhelical mutations. The 5 active site mutations and 22 of the 31 helical mutations completely abolished G6Pase activity, but only 5 of the 13 nonhelical mutants were devoid of activity. Whereas the active site and nonhelical mutants supported the synthesis of G6Pase protein in a manner similar to that of the wild-type enzyme, immunoblot analysis showed that the majority (64.5%) of helical mutations destabilized G6Pase. Furthermore, we show that degradation of both wild-type and mutant G6Pase is inhibited by lactacystin, a potent proteasome inhibitor. Taken together, we have generated a data base of residual G6Pase activity retained by G6Pase mutants, established the critical roles of transmembrane helices in the stability and activity of this phosphatase, and shown that G6Pase is a substrate for proteasome-mediated degradation. Glycogen storage disease type 1 (GSD-1), 1The abbreviations used are:GSD-1glycogen storage disease type 1G6Paseglucose-6-phosphataseWTwild-typeERendoplasmic reticulum also known as von Gierke disease, is a group of autosomal recessive metabolic disorders that occur approximately once in every 100,000 live births (reviewed in Refs. 1Chen Y.-T. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. Childs B. Kinzler K.W. Vogelstein B. The Metabolic and Molecular Bases of Inherited Disease. 8th Ed. McGraw-Hill Inc., New York2001: 1521-1551Google Scholar, 2Chou J.Y. Mansfield B.C. Trends Endocrinol. Metab. 1999; 10: 104-113Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 3Chou J.Y. Curr. Mol. Med. 2001; 1: 25-44Crossref PubMed Scopus (41) Google Scholar). GSD-1a (MIM 232 200), the major subtype representing over 80% of GSD-1 cases, is caused by a deficiency in glucose-6-phosphatase (G6Pase; EC 3.1.3.9), which catalyzes the hydrolysis of glucose-6-phosphate to glucose and phosphate, the terminal steps in gluconeogenesis and glycogenolysis. Patients afflicted with GSD-1a cannot maintain glucose homeostasis and manifest hypoglycemia, hepatomegaly, kidney enlargement, growth retardation, hyperlipidemia, hyperuricemia, and lactic acidemia. Long-term complications include gout, hepatic adenomas with risk for malignancy, osteoporosis, platelet dysfunction, pulmonary hypertension, and renal failure. glycogen storage disease type 1 glucose-6-phosphatase wild-type endoplasmic reticulum The cloning of the G6Pase gene has enabled researchers to show that GSD-1a individuals are homozygotes or compound heterozygotes for loss of function mutations in the gene (4Lei K.-J. Shelly L.L. Pan C.-J. Sidbury J.B. Chou J.Y. Science. 1993; 262: 580-583Crossref PubMed Scopus (320) Google Scholar, 5Lei K.-J. Shelly L.L. Lin B. Sidbury J.B. Chen Y.-T. Nordlie R.C. Chou J.Y. J. Clin. Invest. 1995; 95: 234-240Crossref PubMed Google Scholar, 6Lei K.-J. Chen Y.-T. Chen H. Wong L.-J.C. Liu J.-L. McConkie-Rosell A. Van Hove J.L.K., Ou, H.C.-Y. Yeh N.J. Pan L.Y. Chou J.Y. Am. J. Hum. Genet. 1995; 57: 766-771PubMed Google Scholar, 7Parvari R. Lei K.-J. Bashan N. Hershkovitz E. Korman S.H. Barash V. Lerman-Sagie T. Mandel H. Chou J.Y. Moses S.W. Am. J. Med. Genet. 1997; 72: 286-290Crossref PubMed Scopus (35) Google Scholar, 8Parvari R. Lei K.-J. Szonyi L. Narkis G. Moses S. Chou J.Y. Eur. J. Hum. Genet. 1997; 5: 191-195Crossref PubMed Scopus (18) Google Scholar, 9Bruni N. Rajas F. Montano S. Chevalier-Porst F. Maire I. Mithieux G. Ann. Hum. Genet. 1999; 63: 141-146Crossref PubMed Google Scholar, 10Weston B.W. Lin J.L. Muenzer J. Cameron H.S. Arnold R.R. Seydewitz H.H. Mayatepek E. Van Schaftingen E. Veiga-da-Cunha M. Matern D. Chen Y.T. Pediat. Res. 2000; 48: 329-334Crossref PubMed Scopus (25) Google Scholar, 11Akanuma J. Nishigaki T. Fujii K. Matsubara Y. Inui K. Takahashi K. Kure S. Suzuki Y. Ohura T. Miyabayashi S. Ogawa E. Iinuma K. Okada S. Narisawa K. Am. J. Med. Genet. 2000; 91: 107-112Crossref PubMed Scopus (36) Google Scholar). To date, 75G6Pase mutations (including 2 reported here) have been identified in GSD-1a patients on the basis of their absence from the normal population and/or their co-segregation with the disease phenotype (reviewed in Refs. 2Chou J.Y. Mansfield B.C. Trends Endocrinol. Metab. 1999; 10: 104-113Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar and 3Chou J.Y. Curr. Mol. Med. 2001; 1: 25-44Crossref PubMed Scopus (41) Google Scholar). Interestingly, 48 candidate mutations are missense mutations that result in single-amino acid substitutions. Characterization of these mutations will provide critical information on functionally important residues of the protein. In this study, we functionally characterize all 48 missense mutations by site-directed mutagenesis and transient expression assays. A data base of residual enzymatic activity retained by the G6Pase mutants will serve as a reference for evaluating genotype-phenotype relationships and the minimal G6Pase activity required to correct the GSD-1a phenotype. Sequence alignment suggests that mammalian G6Pases, lipid phosphatases, acid phosphatases, and vanadium haloperoxidases share a conserved phosphatase signature motif, and in G6Pase, this occurs between residues 76 and 180 (12Stukey J. Carman G.M. Protein Sci. 1997; 6: 469-472Crossref PubMed Scopus (222) Google Scholar, 13Hemrika W. Wever R. FEBS Lett. 1997; 409: 317-319Crossref PubMed Scopus (46) Google Scholar). The crystal structure of the vanadium-containing chloroperoxidase from the plant pathogenic fungusCurvularia inaequalis has been resolved (14Hemrika W. Renirie R. Dekker H.L. Barnett P. Wever R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2145-2149Crossref PubMed Scopus (172) Google Scholar). The results show the active site residues in vanadium-containing chloroperoxidase are contained within the phosphatase signature motif. Based on the crystal structure and mechanism of action of vanadium-containing chloroperoxidase (13Hemrika W. Wever R. FEBS Lett. 1997; 409: 317-319Crossref PubMed Scopus (46) Google Scholar, 14Hemrika W. Renirie R. Dekker H.L. Barnett P. Wever R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2145-2149Crossref PubMed Scopus (172) Google Scholar), the amino acids predicted to participate in G6Pase catalysis include Lys76, Arg83, His119, Arg170, and His176. Five mutations that alter active site residues in G6Pase, K76N, R83C, R83H, H119L, and R170Q, have been identified in GSD-1a patients (4Lei K.-J. Shelly L.L. Pan C.-J. Sidbury J.B. Chou J.Y. Science. 1993; 262: 580-583Crossref PubMed Scopus (320) Google Scholar, 6Lei K.-J. Chen Y.-T. Chen H. Wong L.-J.C. Liu J.-L. McConkie-Rosell A. Van Hove J.L.K., Ou, H.C.-Y. Yeh N.J. Pan L.Y. Chou J.Y. Am. J. Hum. Genet. 1995; 57: 766-771PubMed Google Scholar, 15Huner G. Podskarbi T. Schutz M. Baykal T. Sarbat G. Shin Y.S. Demirkol M. J. Inherit. Metab. Dis. 1998; 21: 445-446Crossref PubMed Scopus (15) Google Scholar, 16Kozak L. Francova H. Hirabincova E. Stastna S. Peskova K. Elleder M. Hum. Mutat. 2000; 16: 89Crossref PubMed Google Scholar, 17Wu M.-C. Tsai F.-J. Lee C.-C. Tsai C.-H. Wu J.-Y. Hum. Mutat. 2000; 16: 447Crossref PubMed Scopus (8) Google Scholar). R83C and R83H were shown to abolish phosphatase activity in transient expression assays (4Lei K.-J. Shelly L.L. Pan C.-J. Sidbury J.B. Chou J.Y. Science. 1993; 262: 580-583Crossref PubMed Scopus (320) Google Scholar, 6Lei K.-J. Chen Y.-T. Chen H. Wong L.-J.C. Liu J.-L. McConkie-Rosell A. Van Hove J.L.K., Ou, H.C.-Y. Yeh N.J. Pan L.Y. Chou J.Y. Am. J. Hum. Genet. 1995; 57: 766-771PubMed Google Scholar). In this study, we show that K76N, H119L, and R170Q also completely abolish G6Pase activity, demonstrating the importance of these residues in G6Pase catalysis. Very little is known about the structural requirements for the correct folding and catalytic activity of G6Pase. We have shown that human G6Pase is anchored to the endoplasmic reticulum (ER) by nine transmembrane helices with the amino terminus in the lumen and the carboxyl terminus in the cytoplasm (18Pan C.-J. Lei K.-J. Annabi B. Hemrika W. Chou J.Y. J. Biol. Chem. 1998; 273: 6144-6148Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 19Pan C.-J. Lei K.-J. Chou J.Y. J. Biol. Chem. 1998; 273: 21658-21662Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Therefore, the large collection of G6Pase mutations can now be studied in the context of their positions with respect to the ER and the cytoplasm. In this study, we undertake structure-function analysis of G6Pase. We show that amino acid residues that comprise the catalytic center and nonhelical regions in G6Pase play no essential role in the stability of the enzyme. On the other hand, the structural integrity of transmembrane helices is critical for the correct folding, stability, and enzymatic activity of G6Pase. Proteins with abnormal conformation are rapidly eliminated through intracellular protein degradation, which represents a quality control system in cells (20Wickner S. Maurizi M.R. Gottesman S. Science. 1999; 286: 1888-1893Crossref PubMed Scopus (918) Google Scholar). Cytosolic proteasomes are responsible for rapid degradation of many membrane proteins, including cystic fibrosis transmembrane conductance regulator (21Ward C.L. Omura S. Kopito R.R. Cell. 1995; 83: 121-127Abstract Full Text PDF PubMed Scopus (1133) Google Scholar, 22Jensen T.J. Loo M.A. Pind S. Williams D.B. Goldberg A.L. Riordan J.R. Cell. 1995; 83: 129-135Abstract Full Text PDF PubMed Scopus (775) Google Scholar), the major histocompatibility complex class I molecule, and the α-chains of T-cell antigen receptor (reviewed in Refs. 23Brodsky J.L. McCracken A.A. Trends Cell Biol. 1997; 7: 151-156Abstract Full Text PDF PubMed Scopus (185) Google Scholar, 24Lee D.H. Goldberg A.L. Trends Cell Biol. 1998; 8: 397-403Abstract Full Text Full Text PDF PubMed Scopus (1252) Google Scholar, 25Lecker S.H. Solomon V. Mitch W.E. Goldberg A.L. J. Nutr. 1999; 129: 227S-237SCrossref PubMed Google Scholar). The proteasome pathway can be inhibited by the Streptomyces metabolite, lactacystin (26Fenteany G. Standaert R.F. Lane W.S. Choi S. Corey E.J. Schreiber S.L. Science. 1995; 268: 726-731Crossref PubMed Scopus (1504) Google Scholar), which inhibits the proteasome specifically without inhibiting other proteases (reviewed in Ref. 27Fenteany G. Schriber S.L. J. Biol. Chem. 1998; 273: 8545-8548Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar). The availability of proteasome inhibitor allows a rapid analysis in intact cells of the possible contributions of protein breakdown by the proteasomes. In this study, we show that wild-type and mutant G6Pases are predominantly degraded in the ER through the proteasome pathway. The G6Pase gene in GSD-1a patients was characterized by single-strand conformational polymorphism analysis (28Orita M. Iwahana H. Kanazawa H. Hayashi K. Sekiya T. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2766-2770Crossref PubMed Scopus (3393) Google Scholar) on mutation detection enhancement gels (AT Biochem, Malvern, PA) containing 5% glycerol. Exon-containing fragments were amplified by PCR using primers containing intronic, 5′-, and 3′-untranslated sequences of the human G6Pase gene as described previously (6Lei K.-J. Chen Y.-T. Chen H. Wong L.-J.C. Liu J.-L. McConkie-Rosell A. Van Hove J.L.K., Ou, H.C.-Y. Yeh N.J. Pan L.Y. Chou J.Y. Am. J. Hum. Genet. 1995; 57: 766-771PubMed Google Scholar). The mutation-containing fragments identified by single-strand conformational polymorphism analysis were subcloned and characterized by DNA sequencing. Human G6Pase-DraIII (29Lei K.-J. Shelly L.L. Pan C.-J. Liu J.-L. Chou J.Y. J. Biol. Chem. 1995; 270: 11882-11886Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) cDNA was used as a template for mutant construction by PCR. The eight-amino acid FLAG marker peptide, DYKDDDDK (Scientific Imaging Systems, Eastman Kodak, CT) was used to tag the amino or carboxyl terminus of G6Pase as described previously (18Pan C.-J. Lei K.-J. Annabi B. Hemrika W. Chou J.Y. J. Biol. Chem. 1998; 273: 6144-6148Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). The two outside PCR primers for G6Pase mutants that contain mutations upstream of the DraIII site are nucleotides 77–96 (G1; sense) and nucleotides 625–602 of G6Pase-DraIII (I2; antisense) (29Lei K.-J. Shelly L.L. Pan C.-J. Liu J.-L. Chou J.Y. J. Biol. Chem. 1995; 270: 11882-11886Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), and the two outside PCR primers for mutants that contain mutations downstream of theDraIII site are nucleotides 611–634 (I1; sense) and nucleotides 1150–1133 of G6Pase-DraIII (G2; antisense). The two outside primers for G6Pase R170Q, G184E, G184V, G188D, and G188R mutants are G1 and G2. After PCR, the amplified fragment was ligated into either the pSVLhG6Pase-DraIII-3′ fragment, the pSVLhG6Pase-DraIII-5′ fragment, or the pSVL fragment. The mutant primers are: M5R (nucleotides 83–103), ATG→AGG at position 5; T16A (nucleotides 116–136), ACA→GCA at position 16; Q20R (nucleotides 128–148), CAG→CGG at position 20; Q54P (nucleotides 230–250), CAG→CCG at position 54; W63R (nucleotides 257–277), TGG→CGG at position 63; G68R (nucleotides 272–292), GGA→CGA at position 68; K76N (nucleotides 296–316), AAG→AAC at position 76; W77R (nucleotides 299–319), TGG→CGG at position 77; G81R (nucleotides 311–331), GGA→CGA at position 81; T108I (nucleotides 392–412), ACC→ATC at position 108; T111I (nucleotides 401–421), ACT→ATT at position 111; P113L (nucleotides 407–427), CCA→CTA at position 113; H119L (nucleotides 425–445), CAT→CTT at position 119; G122D (nucleotides 434–454), GGC→GAC at position 122; A124T (nucleotides 440–460), GCA→ACA at position 124; W156L (nucleotides 535–555), TGG→TTG at position 156; V166A (nucleotides 566–586), GTC→GCC at position 166; R170Q (nucleotides 578–598), CGA→CAA at position 170; H179P (nucleotides 605–625), CAT→CCT at position 179; G184E (nucleotides 620–640), GGA→GAA at position 184; G184V (nucleotides 620–640), GGA→GTA at position 184; G188D (nucleotides 632–652), GGC→GAC at position 188; G188R (nucleotides 632–652), GGC→CGC at position 188; L211P (nucleotides 701–721), CTC→CCC at position 211; A241T (nucleotides 791–811), GCC→ACC at position 241; T255I (nucleotides 833–853), ACC→ATC at position 255; P257L (nucleotides 839–859), CCC→CTC at position 257; N264K (nucleotides 860–880), AAC→AAA at position 264; L265P (nucleotides 863–883), CTG→CCG at position 265; G266V (nucleotides 866–886), GGC→GTC at position 266; G270R (nucleotides 878–898), GGC→CGC at position 270; S298P (nucleotides 962–982), TCT→CCT at position 298; F322L (nucleotides 1034–1054), TTC→CTC at position 322; V338F (nucleotides 1082–1102), GTC→TTC at position 338; and I341N (nucleotides 1091–1111), ATC→AAC at position 341. The antisense primer for each mutant has the corresponding complementary sequence. Bold letters indicate nucleotide changes. We have also constructed carboxyl-terminal FLAG-tagged human G6Pase mutants, R83C, R83H, E110K, E110Q, V166G, G188S, G222R, W236R, G270V, R295C, ΔF327, and L345R, as described previously (4Lei K.-J. Shelly L.L. Pan C.-J. Sidbury J.B. Chou J.Y. Science. 1993; 262: 580-583Crossref PubMed Scopus (320) Google Scholar, 5Lei K.-J. Shelly L.L. Lin B. Sidbury J.B. Chen Y.-T. Nordlie R.C. Chou J.Y. J. Clin. Invest. 1995; 95: 234-240Crossref PubMed Google Scholar, 6Lei K.-J. Chen Y.-T. Chen H. Wong L.-J.C. Liu J.-L. McConkie-Rosell A. Van Hove J.L.K., Ou, H.C.-Y. Yeh N.J. Pan L.Y. Chou J.Y. Am. J. Hum. Genet. 1995; 57: 766-771PubMed Google Scholar). The nucleotide sequence in all constructs was verified by DNA sequencing. D38V-3′-FLAG and P178S-3′-FLAG mutants have been described previously (19Pan C.-J. Lei K.-J. Chou J.Y. J. Biol. Chem. 1998; 273: 21658-21662Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). COS-1 cells were grown at 37 °C in HEPES-buffered Dulbecco's modified minimal essential medium supplemented with 4% fetal bovine serum. Cells in 25-cm2 flasks were transfected with 10 μg of wild-type (WT) or mutant construct in a pSVL vector by the DEAE-dextran/chloroquine method as described previously (30Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Greene Publishing and Wiley-Interscience, New York1992: 9.2.1-9.2.6Google Scholar). To correct for transfection efficiency, 2 μg of pCMVβ (BD Biosciences,CLONTECH) was cotransfected with WT or mutant G6Pase cDNA construct. After incubation at 37 °C for 2 days, the transfected cultures were harvested for phosphohydrolase and β-galactosidase assays, Western blot analysis, or RNA isolation. Phosphohydrolase activity was determined essentially as described previously (4Lei K.-J. Shelly L.L. Pan C.-J. Sidbury J.B. Chou J.Y. Science. 1993; 262: 580-583Crossref PubMed Scopus (320) Google Scholar). Reaction mixtures (100 μl) contained 50 mm cacodylate buffer, pH 6.5, 10 mmglucose-6-phosphate, 2 mm EDTA, and appropriate amounts of cell homogenates and were incubated at 30 °C for 10 min. Sample absorbance was determined at 820 nm and is related to the amount of phosphate released using a standard curve constructed by a stock of inorganic phosphate solution. Nonspecific phosphatase activity was estimated by preincubating cell homogenates at pH 5 for 10 min at 37 °C, a condition that inactivates the thermolabile G6Pase (31Hers H.G. Levine R. Luft L. Advances in Metabolic Disorders. 1. Academic Press, London1964: 1-44Google Scholar). β-Galactosidase activity was measured by the release ofO-nitrophenol fromO-nitrophenyl-β-galactopyranoside at 37 °C in a reaction mixture containing 100 mm sodium phosphate buffer, pH 7.3, 1 mm MgCl2, 50 mmβ-mercaptoethanol, and 0.665 mg/mlO-nitrophenyl-β-galactopyranoside. β-Galactosidase activity was estimated by absorbance at 420 nm using a β-galactosidase standard obtained from Promega Biotech. Total RNA was isolated using the RNeasy total RNA isolation kit (Qiagen), fractionated by electrophoresis through a 1.2% agarose gel containing 2.2m formaldehyde, and transferred to a Hybond-N+ membrane (Amersham Biosciences, Inc.) by electroblotting. The membranes were hybridized with either a G6Pase or β-actin probe labeled by random priming. For Western blot analysis of FLAG-tagged G6Pase, proteins in transfected COS-1 lysates were separated by electrophoresis through a 13% polyacrylamide-SDS gel and trans-blotted onto polyvinylidene fluoride membranes (Millipore). The membranes were first incubated with a monoclonal antibody against the FLAG epitope (Scientific Imaging Systems) and then incubated with goat anti-mouse IgG antibody (Kirkegarrd & Perry Laboratories, Gaithersburg, MD). The immunocomplex was detected with the horseradish peroxidase-linked chemiluminescence system containing the SuperSignal West Pico Chemiluminescent substrate obtained from Pierce. In vitro transcription-translation of G6Pase cDNA constructs in a pGEM-11Zf(+) vector was performed using the troponin T-coupled reticulocyte lysate system obtained from Promega Biotech (Madison, WI). l-[35S]methionine was used as the labeled precursor. The in vitro synthesized proteins were analyzed by 12% polyacrylamide-SDS gel electrophoresis and fluoro-autoradiography. Single-strand conformational polymorphism and DNA sequencing analyses were used to identify mutations in theG6Pase gene of four GSD-1a patients. Six different mutations were identified, including T255I, 158delC, 836delA, W70X, Q347X, and ΔF327 (Table I). Two novel mutations identified in this study are T255I and 836delA.Table IMutations identified in the G6Pase gene of GSD-1a patientsPatientExonMutationEffect on coding sequenceComment1V843C → T/T255IThr to Ile at 255Homozygote2I158delCFrameshift, stop at 35Compound heterozygoteV1118C → T/Q347XGln → Stop at 3473I288G → A/W70XTrp → Stop at 70Compound heterozygoteV836delAFrameshift, stop at 3004V1057delTTC/ΔF327Del Phe at 327Homozygote Open table in a new tab Characterization of missense mutations that result in single-amino acid substitutions will provide valuable information on functionally important residues in G6Pase. The 48 missense G6Pase mutations identified in GSD-1a patients are scattered throughout the primary sequence (Fig.1). Nineteen missense mutations, including D38V (8Parvari R. Lei K.-J. Szonyi L. Narkis G. Moses S. Chou J.Y. Eur. J. Hum. Genet. 1997; 5: 191-195Crossref PubMed Scopus (18) Google Scholar), W77R (9Bruni N. Rajas F. Montano S. Chevalier-Porst F. Maire I. Mithieux G. Ann. Hum. Genet. 1999; 63: 141-146Crossref PubMed Google Scholar), R83C (4Lei K.-J. Shelly L.L. Pan C.-J. Sidbury J.B. Chou J.Y. Science. 1993; 262: 580-583Crossref PubMed Scopus (320) Google Scholar), R83H (6Lei K.-J. Chen Y.-T. Chen H. Wong L.-J.C. Liu J.-L. McConkie-Rosell A. Van Hove J.L.K., Ou, H.C.-Y. Yeh N.J. Pan L.Y. Chou J.Y. Am. J. Hum. Genet. 1995; 57: 766-771PubMed Google Scholar), E110K (3Chou J.Y. Curr. Mol. Med. 2001; 1: 25-44Crossref PubMed Scopus (41) Google Scholar), E110Q (8Parvari R. Lei K.-J. Szonyi L. Narkis G. Moses S. Chou J.Y. Eur. J. Hum. Genet. 1997; 5: 191-195Crossref PubMed Scopus (18) Google Scholar), A124T (9Bruni N. Rajas F. Montano S. Chevalier-Porst F. Maire I. Mithieux G. Ann. Hum. Genet. 1999; 63: 141-146Crossref PubMed Google Scholar), V166G (7Parvari R. Lei K.-J. Bashan N. Hershkovitz E. Korman S.H. Barash V. Lerman-Sagie T. Mandel H. Chou J.Y. Moses S.W. Am. J. Med. Genet. 1997; 72: 286-290Crossref PubMed Scopus (35) Google Scholar), P178S (6Lei K.-J. Chen Y.-T. Chen H. Wong L.-J.C. Liu J.-L. McConkie-Rosell A. Van Hove J.L.K., Ou, H.C.-Y. Yeh N.J. Pan L.Y. Chou J.Y. Am. J. Hum. Genet. 1995; 57: 766-771PubMed Google Scholar), G184E (9Bruni N. Rajas F. Montano S. Chevalier-Porst F. Maire I. Mithieux G. Ann. Hum. Genet. 1999; 63: 141-146Crossref PubMed Google Scholar), G188S (6Lei K.-J. Chen Y.-T. Chen H. Wong L.-J.C. Liu J.-L. McConkie-Rosell A. Van Hove J.L.K., Ou, H.C.-Y. Yeh N.J. Pan L.Y. Chou J.Y. Am. J. Hum. Genet. 1995; 57: 766-771PubMed Google Scholar), G188R (10Weston B.W. Lin J.L. Muenzer J. Cameron H.S. Arnold R.R. Seydewitz H.H. Mayatepek E. Van Schaftingen E. Veiga-da-Cunha M. Matern D. Chen Y.T. Pediat. Res. 2000; 48: 329-334Crossref PubMed Scopus (25) Google Scholar), L211P (9Bruni N. Rajas F. Montano S. Chevalier-Porst F. Maire I. Mithieux G. Ann. Hum. Genet. 1999; 63: 141-146Crossref PubMed Google Scholar), G222R (5Lei K.-J. Shelly L.L. Lin B. Sidbury J.B. Chen Y.-T. Nordlie R.C. Chou J.Y. J. Clin. Invest. 1995; 95: 234-240Crossref PubMed Google Scholar), W236R (6Lei K.-J. Chen Y.-T. Chen H. Wong L.-J.C. Liu J.-L. McConkie-Rosell A. Van Hove J.L.K., Ou, H.C.-Y. Yeh N.J. Pan L.Y. Chou J.Y. Am. J. Hum. Genet. 1995; 57: 766-771PubMed Google Scholar), P257L (11Akanuma J. Nishigaki T. Fujii K. Matsubara Y. Inui K. Takahashi K. Kure S. Suzuki Y. Ohura T. Miyabayashi S. Ogawa E. Iinuma K. Okada S. Narisawa K. Am. J. Med. Genet. 2000; 91: 107-112Crossref PubMed Scopus (36) Google Scholar), G270V (6Lei K.-J. Chen Y.-T. Chen H. Wong L.-J.C. Liu J.-L. McConkie-Rosell A. Van Hove J.L.K., Ou, H.C.-Y. Yeh N.J. Pan L.Y. Chou J.Y. Am. J. Hum. Genet. 1995; 57: 766-771PubMed Google Scholar), R295C (4Lei K.-J. Shelly L.L. Pan C.-J. Sidbury J.B. Chou J.Y. Science. 1993; 262: 580-583Crossref PubMed Scopus (320) Google Scholar), and L345R (6Lei K.-J. Chen Y.-T. Chen H. Wong L.-J.C. Liu J.-L. McConkie-Rosell A. Van Hove J.L.K., Ou, H.C.-Y. Yeh N.J. Pan L.Y. Chou J.Y. Am. J. Hum. Genet. 1995; 57: 766-771PubMed Google Scholar), were shown to abolish or greatly reduced G6Pase activity by site-directed mutagenesis and transient expression assays. In this study, we constructed 35 mutants carrying G6Pase missense mutations, including 29 mutations that have not been characterized and 6 of the previously characterized mutations, W77R, A124T, G184E, L211P, G188R, and P257L. Glucose-6-phosphate hydrolytic activities of these mutants were examined after transient expression of WT or mutant G6Pase cDNA into COS-1 cells. We also included in this study the single codon deletion mutation, ΔF327, shown to be devoid of enzymatic activity (5Lei K.-J. Shelly L.L. Lin B. Sidbury J.B. Chen Y.-T. Nordlie R.C. Chou J.Y. J. Clin. Invest. 1995; 95: 234-240Crossref PubMed Google Scholar). To facilitate structure-function analysis, we have grouped these mutations into three categories (active site, helical, and nonhelical mutations) based on their predicted catalytic, transmembrane helical, luminal, and cytoplasmic locations in G6Pase (Fig. 1). The amino acids predicted to be critical to glucose-6-phosphate binding and hydrolysis include Lys76, Arg83, His119, Arg170, and His176(12Stukey J. Carman G.M. Protein Sci. 1997; 6: 469-472Crossref PubMed Scopus (222) Google Scholar, 13Hemrika W. Wever R. FEBS Lett. 1997; 409: 317-319Crossref PubMed Scopus (46) Google Scholar, 14Hemrika W. Renirie R. Dekker H.L. Barnett P. Wever R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2145-2149Crossref PubMed Scopus (172) Google Scholar). Five active site mutations, K76N (16Kozak L. Francova H. Hirabincova E. Stastna S. Peskova K. Elleder M. Hum. Mutat. 2000; 16: 89Crossref PubMed Google Scholar), R83C (4Lei K.-J. Shelly L.L. Pan C.-J. Sidbury J.B. Chou J.Y. Science. 1993; 262: 580-583Crossref PubMed Scopus (320) Google Scholar), R83H (6Lei K.-J. Chen Y.-T. Chen H. Wong L.-J.C. Liu J.-L. McConkie-Rosell A. Van Hove J.L.K., Ou, H.C.-Y. Yeh N.J. Pan L.Y. Chou J.Y. Am. J. Hum. Genet. 1995; 57: 766-771PubMed Google Scholar), H119L (17Wu M.-C. Tsai F.-J. Lee C.-C. Tsai C.-H. Wu J.-Y. Hum. Mutat. 2000; 16: 447Crossref PubMed Scopus (8) Google Scholar), and R170Q (15Huner G. Podskarbi T. Schutz M. Baykal T. Sarbat G. Shin Y.S. Demirkol M. J. Inherit. Metab. Dis. 1998; 21: 445-446Crossref PubMed Scopus (15) Google Scholar), have been identified in theG6Pase gene of GSD-1a patients. In earlier studies (4Lei K.-J. Shelly L.L. Pan C.-J. Sidbury J.B. Chou J.Y. Science. 1993; 262: 580-583Crossref PubMed Scopus (320) Google Scholar, 6Lei K.-J. Chen Y.-T. Chen H. Wong L.-J.C. Liu J.-L. McConkie-Rosell A. Van Hove J.L.K., Ou, H.C.-Y. Yeh N.J. Pan L.Y. Chou J.Y. Am. J. Hum. Genet. 1995; 57: 766-771PubMed Google Scholar), we have shown that R83C and R83H mutants were devoid of G6Pase activity (Table II). In this study, we demonstrate that K76N, H119L, and R170Q mutations also completely abolished phosphohydrolase activity (Table II), demonstrating the importance of these residues in G6Pase catalysis.Table IIPhosphohydrolase activity of G6Pase active site mutant constructsMutationNon-FLAG construct3′ FLAG construct(nmol/min/mg)(nmol/min/mg)Mock6.75 ± 0.2811.99 ± 0.15WT69.42 ± 5.7873.36 ± 1.82K76N5.44 ± 0.869.03 ± 0.68R83C6.32 ± 0.649.28 ± 0.24R83H5.18 ± 0.109.26 ± 0.90H119L6.32 ± 0.1610.86 ± 0.84R170Q4.94 ± 0.078.61 ± 0.62WT or mutant G6Pase construct was transfected into COS-1 cells, and phosphohydrolase activity was assayed as described under “Materials and Methods” using two independent isolates of each construct in three separate transfections. Data are presented as the mean ± S.E. Open table in a new tab WT or mutant G6Pase construct was transfected into COS-1 cells, and phosphohydrolase activity was assayed as described under “Materials and Methods” using two independent isolates of each construct in three separate transfections. Data are presented as the mean ± S.E. Twelve of the 13 nonhelical G6Pase mutations are situated inside the ER lumen, and 1 (Q54P) is located in cytoplasmic loop 1 (Fig. 1). Earlier studies have shown that the E110K (2Chou J.Y. Mansfield B.C. Trends Endocrinol. Metab. 1999; 10: 104-113Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) mutation totally inactivated G6Pase, but the E110Q (8Parvari R. Lei K.-J. Szonyi L. Narkis G. Moses S. Chou J.Y. Eur. J. Hum. Genet. 1997; 5: 191-19
Referência(s)