SCD | SCTD | SDG | SGDDM | SHBG | UMD | Main Page
 
MOLECULAR GENETICS OF HERITABLE HUMAN DISORDERS
     
Janice Y. Chou, Ph.D., Head, Section on Cellular Differentiation
Li-Yuan Chen, Ph.D., Postdoctoral Fellow
Abhijit Ghosh, Ph.D., Postdoctoral Fellow
Jeng-Jer Shieh, Ph.D., Postdoctoral Fellow
Mao-Sen Sun, M.D., Ph.D., Postdoctoral Fellow
Brian C. Mansfield, Ph.D., Guest Researcher
Chi-Jiun Pan, Chemist
Shirley Fang, Technical Training Fellow
Janice Chou
 
Our goal is to understand the molecular genetics and pathogenesis of heritable metabolic human disorders with specific emphasis on type 1 glycogen storage diseases (GSD-I). Also known as von Gierke disease, GSD-I is a group of autosomal recessive disorders caused by deficiencies in the endoplasmic reticulum (ER)–bound glucose-6-phosphatase (G6Pase) system, a key enzyme complex in glucose homeostasis. The G6Pase system comprises at least two integral membrane proteins, G6Pase and the glucose-6-phosphate transporter (G6PT). G6PT translocates glucose-6-phosphate (G6P), the product of gluconeogenesis and glycogenolysis, from the cytoplasm to the lumen of the ER, and, inside the ER, G6Pase catalyzes the hydrolysis of G6P to produce glucose and phosphate. Over the last 20 years, dietary therapy has been used to alleviate some, but not all, metabolic abnormalities of GSD-1 patients and to slow disease progress. However, the underlying disease remains untreated, and poor compliance frequently limits the efficacy of dietary treatment. Therefore, long-term complications still develop in adult patients. An understanding of the molecular genetics and pathogenesis of GSD-1 is needed in order to develop therapies that can rectify the long-term complications of GSD-1.

Gene Therapy for Glycogen Storage Disease Type Ia
Sun, Pan, Shieh, Ghosh, Chen, Mansfield, Chou
Deficiencies in G6Pase and G6PT cause GSD1a and GSD-1b, respectively. Patients suffering from both disorders manifest the symptoms of G6Pase enzyme deficiency characterized by growth retardation, hypoglycemia, hepato-megaly, nephromegaly, hyperlipidemia, hyper-uricemia, and lactic acidemia. However, GSD-Ib patients also present with unique symptoms not so obviously associated with G6Pase activity, including chronic neutropenia and myeloid dysfunctions, which result in recurrent bacterial infections. The most prevalent from of GSD-Ia is caused by a deficiency in G6Pase, a highly hydrophobic protein anchored to the ER by 9transmembrane helices. The protein cannot be expressed in a soluble form and must embed correctly in the ER membrane and couple with other proteins to be functional. Therefore, enzyme replacement therapy is not an option, but somatic gene therapy, i.e., targeting G6Pase to the liver and the kidney, is an attractive possibility. To this end, we had previously generated G6Pase-deficient mice that manifest a metabolic profile and phenotype virtually identical to that of human GSD-Ia patients. Using neonatal GSD-Ia mice, we now demonstrate that gene transfer mediated by a combined adeno- and adeno-associated virus vector leads to sustained G6Pase expression in both the liver and kidney and corrects the murine GSD-Ia disease for at least 12 months. Our results suggest that human GSD-Ia would be treatable by gene therapy.

The Catalytic Center of Glucose-6-Phosphatase
Ghosh, Shieh, Pan, Sun, Chou
The amino acids constituting the catalytic center of G6Pase include Lys76, Arg83, His119, Arg170, and His176. During catalysis, a His residue in G6Pase becomes phosphorylated, generating an enzyme-phosphate intermediate. It was predicted that His176 would be the amino acid that acts as a nucleophile forming a phosphohistidine-enzyme intermediate and that His119 would be the amino acid that provides the proton needed to liberate the glucose moiety. However, the phosphate acceptor in G6Pase has eluded molecular characterization. To identify the His residue that covalently binds the phosphate moiety, we generated recombinant adenoviruses carrying wild-type G6Pase and active site mutants of this enzyme. A 40-kDa [32P]-phosphate-G6Pase intermediate was identified after incubating [32P]-glucose-6-phosphate with microsomes expressing wild-type enzyme but not with microsomes expressing either H119A or H176A mutant G6Pase. Human G6Pase contains five methionine residues at positions 1, 5, 121, 130, and 279. After cyanogen bromide cleavage, His119 is predicted to be within a 116 amino acid peptide of 13.5 kDa with an isoelectric point of 5.3 (residues 6–121) and His176 within a 149 amino acid peptide of 16.8 kDa with an isoelectric point of 9.3 (residues 131–279). We show that after digestion of a nonglycosylated [32P]-phosphate-G6Pase intermediate by cyanogen bromide, the [32P]-phosphate remains bound to a peptide of 17 kDa with an isoelectric point above 9, demonstrating that His176 is the phosphate acceptor in G6Pase.

Structure and Function Analysis of Mutations in Glucose-6-Phosphatase
Shieh, Pan, Chen, Chou
To date, 75 G6Pase mutations have been identified in GSD-Ia patients: 48 missense, nine nonsense, 15 insertion/deletion, and three splicing mutations. Interestingly, 64 percent of candidate mutations are missense mutations that result in single amino acid substitutions. Characterization of these mutations will provide valuable information on functionally important residues of the protein. Using site-directed mutagenesis and transient expression assays, we have characterized all 48 missense mutations. The database of residual activity retained by these mutants will serve as a reference in evaluating genotype-phenotype relationships and identifying the minimal G6Pase activity required to correct the GSD-Ia phenotype.

Structure-Function Analysis of the Glucose-6-Phosphate Transporter
Chen, Pan, Shieh, Chou
G6PT is anchored in the ER by 10transmem-brane helices. To date, 69 G6PT mutations, including 28 missenses and two codon-deletions, have been identified in GSDIb patients. We previously characterized one codon deletion and 15 missense mutations by using a pSVL-based expression assay. We now report the functional characterization of all 30 codon mutations by using an improved G6PT assay based on an adenoviral vector–mediated expression system. Twenty of the naturally occurring mutations completely abolish microsomal G6P uptake activity while the other 10 mutations partially inactivate the transporter. We also report a structure-function analysis of G6PT and show that wild-type and mutant G6PT are degraded in cells predominantly through the proteasome pathway.
.
 

PUBLICATIONS

  1. Chou JY, Matern D, Mansfield BC, Chen YT. Type I glycogen storage diseases: disorders of the glucose-6-phosphatase complex. Curr Mol Med. 2002;2:121-143.
  2. Ghosh A, Shieh J-J, Pan C-J, Sun M-S, Chou JY. The catalytic center of glucose-phosphatase: His176 is the nucleophile forming the phosphohistidine-enzyme intermediate during catalysis. J Biol Chem. 2002;277:32837-32842.
  3. Hiraiwa H, Pan C-J, Lin B, Akiyama TE, Gonzalez FJ, Chou JY. A molecular link between the common phenotypes of type 1 glycogen storage disease and HNF1a-null mice. J Biol Chem. 2001;276:7963-7967.
  4. Shieh J-J, Terzioglu M, Hiraiwa H, Marsh J, Pan CJ, Chen LY, Chou JY. The molecular basis of glycogen storage disease type 1a: structure and function analysis of mutations in glucose-6-phosphatase. J Biol Chem. 2001;277:5047-5053.
  5. Sun M-S, Pan C-J, Shieh JJ, Ghosh A, Chen LY, Mansfield BC, Ward JM, Byrne BJ, Chou JY. Sustained hepatic and renal glucose-6-phosphatase expression corrects glycogen storage disease type Ia in mice. Hum Mol Genet. 2002;11:2155-2164.

COLLABORATORS
Barry J. Byrne, M.D., Powell Gene Therapy Center and Department of Pediatrics and Molecular Genetics and Microbiology, University of Florida, Gainesville, FL
Jerrold M. Ward, Ph.D., Office of Laboratory Animal Science, NCI, Frederick, MD