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molecular genetics of heritable human disorders

Janice Y. Chou, PhD, Head, Section on Cellular Differentiation Abhijit Ghosh, PhD, Postdoctoral Fellow So Youn Kim, PhD, Postdoctoral Fellow Jeng-Jer Shieh, PhD, Postdoctoral Fellow Wai Han Yiu, PhD, Postdoctoral Fellow Brian C. Mansfield, PhD, Guest Researcher Chi-Jiun Pan, BS, Senior Research Assistant Yuk Yin Cheung, MS, Graduate Student Mohammad Allamarvdasht, BS, Technical Training Fellow Andres Nguyen, MS, Technical Training Fellow

Glycogen storage disease type I (GSD-I) is caused by deficiencies in the glucose-6-phosphatase-alpha (G6Pase-alpha) complex, which is expressed primarily in the liver, kidney, and intestine. The complex consists of a glucose-6-phosphate transporter (G6PT), which translocates G6P from cytoplasm to the lumen of the endoplasmic reticulum (ER), and G6Pase-alpha, which hydrolyzes G6P to glucose. Together, they are responsible for interprandial glucose homeostasis. Deficiencies in G6Pase-alpha and G6PT cause GSD-Ia and GSD-Ib, respectively. Both manifest the symptoms of G6Pase-alpha deficiency, which is characterized by growth retardation, hypoglycemia, hepatomegaly, nephromegaly, hyperlipidemia, hyperuricemia, and lactic acidemia. GSD-Ib patients also present with neutropenia and myeloid dysfunctions. The currently available dietary therapy cannot treat the underlying disease, resulting in long-term complications in adult patients. An understanding of the molecular genetics and pathogenesis of GSD-I is needed in order to develop therapies that can rectify such long-term complications. Currently, only liver, kidney, and intestine are thought to be involved in interprandial glucose homeostasis, given the absence of G6Pase-alpha outside these organs. We identified a ubiquitously expressed G6P hydrolase, G6Pase-beta, that couples functionally with G6PT to form an active G6Pase complex. Our findings challenge the current dogma that only liver, kidney, and intestine can contribute to blood glucose homeostasis. Murine model of GSG-Ib Chen, Shieh, Lin,^a Pan, Mansfield, Chou; in collaboration with Gao, Lee, Moses, Murphy, Roe, Ward, Westphal In addition to disrupted glucose homeostasis, GSD-Ib patients have unexplained and unexpected defects in neutrophil respiratory burst, chemotaxis, and calcium flux in response to the bacterial peptide f-Met-Leu-Phe, along with intermittent neutropenia. We generated a G6PT knockout (G6PT^–/–) mouse that mimics all known defects of the human disorder and used the model to further our understanding of the pathogenesis of GSD-Ib. We showed that the neutropenia is caused directly by the loss of G6PT activity; that chemotaxis and calcium flux, induced by the chemokines KC and macrophage inflammatory protein-2, are defective in G6PT^–/–neutrophils; and that local production of these chemokines and the resultant neutrophil trafficking in vivo are depressed in G6PT^–/– ascites during an inflammatory response. The bone and spleen of G6PT^–/–mice are developmentally delayed and accompanied by marked hypocellularity of the bone marrow, elevation of myeloid progenitor cell frequencies in both organs, and a corresponding dramatic increase in granulocyte colony stimulating factor levels in both GSD-Ib mice and humans. Thus, in addition to transient neutropenia, a sustained defect in neutrophil trafficking, attributable to both the resistance of neutrophils to chemotactic factors and reduced local production of neutrophil-specific chemokines at sites of inflammation, may underlie the myeloid deficiency in GSD-Ib. These findings demonstrate that G6PT is not solely a G6P transport protein but also an important immunomodulatory protein, whose activities need to be addressed in treating the myeloid complications in GSD-Ib patients. Chen L-Y, Shieh J-J, Lin B, Pan C-J, Gao J-L, Murphy PM, Roe TF, Moses S, Ward JM, Westphal H, Lee EJ, Mansfield BC, Chou JY. Impaired glucose homeostasis, neutrophil trafficking and function in mice lacking the glucose-6-phosphate transporter. Hum Mol Genet 2003;12:2547-2558. Chou JY, Mansfield BC. Glucose-6-phosphate transporter: the key to glycogen storage disease type Ib. In: Broer S, Wagner CA, eds. Membrane Transporter Diseases. New York: Kluwer Academic Plenum Publishers, 2003; 191-205. Gene therapy for GSD-Ia Ghosh, Chou; in collaboration with Byrne G6Pase-alpha is a highly hydrophobic, multiple domain transmembrane ER protein, which cannot be expressed in soluble forms. To be functional, G6Pase-alpha and G6PT must be co-expressed and embedded correctly in the ER membrane. Therefore, protein replacement therapy for GSD-Ia is not an option, although somatic gene therapy, targeting G6Pase-alpha to the liver and kidney, is an attractive possibility that has recently received interest. Using the G6Pase-alpha knockout (G6Pase-alpha^–/–) mouse model generated in our laboratory, we have shown that gene transfer mediated by a combined adeno (Ad-mG6Pase-alpha) and adeno-associated virus (AAV) serotype 2 (AAV2-mG6Pase-alpha) leads to sustained G6Pase-alpha expression in both the liver and kidney and corrects the murine GSD-Ia disease for at least 12 months. The critical clinical presentation in GSD-Ia is life-threatening hypoglycemia. Because AAV2-mG6Pase-alpha–mediated transgene expression increases slowly, typically peaking five to seven weeks post-infusion, we co-infused AAV2-mG6Pase-alpha with Ad-mG6Pase-alpha, which allows the infused animals to survive weaning. We have now demonstrated that neonatal G6Pase-alpha^–/–mice infused only with AAV1-mG6Pase-alpha, a recombinant AAV of serotype 1, not only survived weaning but also lived to 12 months of age. The AAV1-mG6Pase-alpha–mediated gene transfer led to sustained G6Pase-alpha expression in both the liver and kidney and corrected the murine GSD-Ia disorder for the full 12 months of the study. Our results suggest that human GSD-Ia would be treatable by gene therapy. G6Pase-beta, a ubiquitously expressed G6P hydrolase Shieh, Pan, Mansfield, Chou Fine control of the blood glucose level is essential to avoid the hyper- or hypoglycemic shocks that are associated with many metabolic disorders, including diabetes mellitus and GSD-I. Between meals, the primary source of blood glucose is gluconeogenesis and glycogenolysis. In the final step of both pathways, G6P is hydrolyzed to glucose by the G6Pase-alpha complex. Given that G6Pase-alpha is expressed only in the liver, kidney, and intestine, it has been thought that most other tissues cannot contribute to interprandial blood glucose homeostasis. We demonstrate that, like G6Pase-alpha, PAP2.8/UGRP (renamed G6Pase-beta), a novel, widely expressed G6Pase-related protein, is an acid-labile, vanadate-sensitive, ER-associated phosphohydrolase. Both enzymes have the same active site structure and exhibit a similar Km toward G6P, but the Vmax of G6Pase-alpha is about six times higher than that of G6Pase-beta. Most important, G6Pase-beta couples with the G6P transporter to form an active G6Pase complex that can hydrolyze G6P to glucose. Our findings challenge the current dogma that only liver, kidney, and intestine can contribute to blood glucose homeostasis and explain why GSD-Ia patients, who lack a functional liver/kidney/intestine G6Pase-alpha complex, are still capable of endogenous glucose production. Shieh J-J, Pan C-J, Mansfield BC, Chou JY. Glucose-6-phosphate hydrolase, widely expressed outside the liver, can explain age-dependent resolution of hypoglycemia in glycogen storage disease type Ia. J Biol Chem 2003;278:47098-47103. Active site of G6Pase-beta Ghosh, Shieh, Pan, Chou It has been shown that, during G6P hydrolysis, G6Pase-alpha, a nine-transmembrane domain protein, forms a covalently bound phosphoryl enzyme intermediate through His^176, which lies on the lumenal side of the ER membrane. We have now shown that G6Pase-beta is also a nine-transmembrane domain protein that forms a covalently bound phosphoryl enzyme intermediate during G6P hydrolysis. However, the intermediate is not detectable in the G6Pase-beta active site mutants R79A, H114A, and H176A. Using [^32P]-G6P labeling coupled with cyanogen bromide mapping, we demonstrated that the phosphate acceptor in G6Pase-beta is His^167 and that it lies inside the ER lumen with the active site residues Arg^79 and His^114. Therefore, G6Pase-alpha and G6Pase-beta share a similar active site structure, topology, and mechanism of action. Ghosh A, Shieh J-J, Pan C-J, Chou JY. Histidine-167 is the phosphate acceptor in glucose-6-phosphatase-beta forming a phosphohistidine-enzyme intermediate during catalysis. J Biol Chem 2004;279:12479-12483. Potential new role for muscle in blood glucose homeostasis Shieh, Pan, Mansfield, Chou The demonstration of a significant, specific G6P hydrolase that can couple to G6PT outside the liver raises interesting questions about the ability of other tissues to cycle glucose and contribute to blood glucose homeostasis. Of particular interest is muscle, which expresses an elevated level of G6Pase-beta and stores the large share of body glycogen. We have now demonstrated that muscle expresses both G6Pase-beta and G6PT and that they can couple to form an active G6Pase complex. Our data suggest that muscle may have a previously unrecognized role in interprandial glucose homeostasis. Shieh J-J, Pan C-J, Mansfield BC, Chou JY. A potential new role for muscle in blood glucose homeostasis. J Biol Chem 2004;279:26215-26219. Topology of the islet-specific G6Pase-related protein Shieh, Pan, Mansfield, Chou The islet-specific G6Pase-related protein (IGRP) is a member of the G6Pase family that is expressed primarily in the pancreatic islets and has low or nondetectable hydrolase activity. Recently, the fragment of IGRP consisting of amino acids 206 to 214 was identified as the nona-peptide recognized by a prevalent population of pathogenic CD8^+ T cells in nonobese diabetic mice, a modelof type 1 diabetes. To understand more fully the potential roles of this protein in diabetes mellitus, we examined the subcellular localization and membrane topography of human IGRP. We showed that IGRP is a glycoprotein held in the endoplasmic reticulum by a nine-transmembrane domain and that the glycoprotein is degraded in cells predominantly through the proteasome pathway that generates the major histocompatibility complex class I–presented peptides. The divergence between amino acids 206 to 214 in IGRP and the corresponding sequence in mammalian G6Pase-alpha may explain why G6Pase-alpha, processed by the same pathway, is not presented to the immune system in an antigenic manner. Shieh J-J, Pan C-J, Mansfield BC, Chou JY. The islet-specific glucose-6-phosphatase-related protein, implicated in diabetes, is a glycoprotein embedded in the endoplasmic reticulum membrane. FEBS Lett 2004;562:160-164. ^aBaochuan Lin, PhD, former Postdoctoral Fellow COLLABORATORS Barry J. Byrne, MD, Powell Gene Therapy Center and Department of Pediatrics and Molecular Genetics & Microbiology, University of Florida, Gainesville, FL Ji-Liang Gao, PhD, Molecular Signaling Section, Laboratory of Host Defenses, NIAID, Bethesda, MD Eric J. Lee, DVM, Laboratory of Mammalian Genes and Development, NICHD, Bethesda, MD Shimon Moses, MD, Ben-Gurion University of the Negev, Beer-Sheva, Israel Philip M. Murphy, MD, Laboratory of Host Defenses, NIAID, Bethesda, MD Thomas F. Roe, MD, University of Southern California School of Medicine, Los Angeles, CA Jerrold M. Ward, PhD, Office of Laboratory Animal Science, NCI, Frederick, MD Heiner Westphal, MD, Laboratory of Mammalian Genes and Development, NICHD, Bethesda, MD For further information, contact chouja@mail.nih.gov