MOLECULAR GENETICS OF HERITABLE HUMAN DISORDERS
, Head, Section on Cellular Differentiation
Shih-Yin Chen, PhD, Visiting Fellow
Hyun-Sik Jun, PhD, Visiting Fellow
So Youn Kim, PhD, Visiting Fellow
Wai Han Yiu, PhD, Visiting Fellow
Wentao Peng, PhD, Staff Scientist
Brian C. Mansfield, PhD, Guest Researcher
Chi-Jiunn Pan, BS, Senior Research Assistant
Yuk Yin Cheung, MS, Graduate Student
Robert A. Ruef, BS, Postbaccalaureate Fellow
Glycogen storage disease type I (GSD-I) is caused by deficiencies in the glucose-6-phosphatase-alpha (G6Pase-alpha) complex, which consists of a glucose-6-phosphate transporter (G6PT) and a G6Pase-alpha catalytic unit. G6PT translocates G6P from cytoplasm to the lumen of the endoplasmic reticulum (ER), and G6Pase-alpha hydrolyzes G6P to glucose. Together, the complex ensures interprandial glucose homeostasis. Deficiencies in G6Pase-alpha cause GSD-Ia, and deficiencies in G6PT cause GSD-Ib; both manifest the symptoms of disturbed glucose homeostasis. There is no known cure for GSD-I, and the current dietary therapy cannot prevent long-term complications in adult patients. Recently developed animal disease models now provide an opportunity for more precisely delineating the disease and developing therapies that target the underlying disease process. GSD-Ib patients exhibit neutropenia and neutrophil dysfunctions. The most noticeable difference between GSD-Ia and GSD-Ib, which might explain the dysfunction, lies in their expression patterns. G6Pase-alpha expression is restricted to the liver, kidney, and intestine, whereas G6PT is expressed ubiquitously. Recently a second ubiquitously expressed G6P hydrolase, G6Pase-beta, was reported, suggesting that the G6Pase-beta-G6PT complex might be functional in neutrophils and that the myeloid defects in GSD-Ib are attributable to the loss of activity of that complex.
Increased cellular cholesterol efflux in GSD-Ia
GSD-Ia patients manifest a pro-atherogenic lipid profile characterized by hypercholesterolemia, hypertriglyceridemia, reduced cholesterol in HDL, and increased cholesterol in LDL and VLDL fractions, yet they are not at elevated risk for developing atherosclerosis. We investigate cellular cholesterol efflux, the first step in reverse cholesterol transport, and antioxidant capacity—both are protective against atherosclerosis—in the sera of GSD-Ia patients. We show that sera from GSD-Ia patients are more efficient than sera from control subjects in promoting the scavenger receptor class B type I (SR-BI)–mediated cellular cholesterol efflux, which correlates with an increase in phospholipid and the ratio of HDL-phospholipid to HDL, the major determinants of the SR-BI–mediated cholesterol efflux, in the sera of GSD-Ia patients. Moreover, sera from GSD-Ia patients have higher total antioxidant capacity than do the sera of controls; the increase correlates with elevated levels of uric acid, a powerful plasma antioxidant. Taken together, the results suggest that the increase in SR-BI–mediated cellular cholesterol efflux and antioxidant capacity in the sera of GSD-Ia patients may contribute to protection against premature atherosclerosis.
Nguyen AD, Pan C-J, Shieh J-J, Chou JY. Increased cellular cholesterol efflux in glycogen storage disease type Ia mice: a potential mechanism that protects against premature atherosclerosis. FEBS Lett 2005;579:4713-8.
Nguyen AD, Pan C-J, Weinstein WA, Chou JY. Increased scavenger receptor class B type I-mediated cellular cholesterol efflux and antioxidant capacity in the sera of glycogen storage disease type Ia patients. Mol Genet Metab 2006;89:233-8.
Neutrophilia and elevated serum cytokines in GSD-Ia
GSD-Ia patients deficient in G6Pase-alpha manifest a disturbed glucose homeostasis, which, we hypothesized, might affect myeloid functions. Using a mouse model of GSD-Ia that mimicked the human disorder, we showed that GSD-Ia mice exhibit normal neutrophil activities but have elevated myeloid progenitor cells in the bone marrow and spleen. Interestingly, GSD-Ia mice exhibit a persistent increase in peripheral blood neutrophil counts along with elevated serum levels of granulocyte colony stimulating factor (G-CSF) and keratinocyte-derived chemokine (KC). Taken together, our results suggest that a loss of glucose homeostasis can compromise the immune system, resulting in neutrophilia and possibly explaining some of the unexpected clinical manifestations seen in GSD-Ia.
Kim SY, Chen L-Y, Weinstein DA, Chou JY. Neutrophilia and elevated serum cytokines are implicated in glycogen storage disease type Ia. FEBS Lett 2007;581:3833-8.
G6PT required for normal myeloid functions
In addition to disturbed glucose homeostasis, GSD-Ib patients manifest neutropenia and impaired neutrophil respiratory burst, chemotaxis, and calcium flux activities. In view of the ongoing controversy as to whether G6PT deficiency in the bone marrow underlies myeloid dysfunctions in GSD-Ib, we generated chimeric BM-G6PT−/− mice by transferring bone marrow from G6PT-deficient (G6PT−/−) mice to wild-type mice. While the wild-type mice have normal myeloid functions, the BM-G6PT−/− mice manifest myeloid abnormalities characteristic of G6PT−/− mice. Both types of mice have impaired neutrophil respiratory burst, chemotaxis response, and calcium flux activities and exhibit neutropenia. In the bone marrow of the G6PT-BM−/− and G6PT−/− mice, the number of myeloid progenitor cells and serum levels of G-CSF and KC are elevated. Moreover, in an experimental model of peritoneal inflammation, local production of KC and the related chemokine macrophage inflammatory protein-2 is depressed in both the BM-G6PT−/− and G6PT−/− mice, as is peritoneal neutrophil accumulation. Our findings demonstrate that myeloid dysfunctions in GSD-Ib are intrinsically linked to G6PT deficiency in the bone marrow and neutrophils.
Kim SY, Nguyen AD, Gao J-L, Murphy PM, Mansfield BC, Chou JY. Bone-marrow derived cells require a functional glucose-6-phosphate transporter for normal myeloid functions. J Biol Chem 2006;281:28794-801.
Gene therapy for murine GSD-Ib
G6PT is a hydrophobic protein anchored to the ER by 10 transmembrane helices. The protein cannot be expressed in a soluble form, and, to be functional, G6PT must embed correctly in the ER membrane. Therefore, protein replacement therapy is not an option for the treatment of GSD-Ib, but somatic gene therapy—targeting the G6PT gene to the gluconeogenic and myeloid tissues—is an attractive possibility. To evaluate the feasibility of gene replacement therapy for GSD-Ib, we infused adenoviral (Ad) vector containing human G6PT (Ad-hG6PT) into G6PT−/− mice. Ad-hG6PT infusion restored significant levels of G6PT mRNA expression in the liver, bone marrow, and spleen and corrected metabolic as well as myeloid abnormalities in G6PT−/− mice. The G6PT−/− mice receiving gene therapy exhibited improved growth; normalized serum profiles for glucose, cholesterol, triglyceride, uric acid, and lactic acid; and reduced hepatic glycogen deposition. The therapy also corrected neutropenia and lowered the elevated serum levels of G-CSF. The development of bone and spleen in the infused G6PT−/− mice improved and was accompanied by increased cellularity and normalized myeloid progenitor cell frequencies in both tissues. This effective use of gene therapy to correct metabolic imbalances and myeloid dysfunctions in GSD-Ib mice holds promise for the future of gene therapy in humans.
Chou JY, Mansfield BC. Gene therapy for type I glycogen storage diseases. Curr Gene Ther 2007;7:79-88.
Yiu WH, Pan C-J, Allamarvdasht A, Kim SY, Chou JY. Glucose-6-phosphate transporter gene therapy corrects metabolic and myeloid abnormalities in glycogen storage disease type Ib mice. Gene Ther 2006;14:219-26.
Mice lacking G6Pase-beta manifest neutropenia and neutrophil dysfunctions
Neutropenia and neutrophil dysfunction are common in many diseases, although their etiology is often unclear. Previous views held that a single ER enzyme, G6Pase-alpha, whose activity is limited to the liver, kidney, and intestine, was solely responsible for the final stages of gluconeogenesis and glycogenolysis in which G6P is hydrolyzed to glucose for release to the blood. Recently, we characterized a second G6Pase activity, G6Pase-beta. The kinetic properties of G6Pase-beta and G6Pase-alpha are similar, and the ubiquitous expression pattern of G6Pase-beta is similar to that of G6PT. In addition, G6Pase-beta is an integral ER membrane protein, containing nine transmembrane domains as does G6Pase-alpha, and it couples functionally with G6PT, in a similar manner to G6Pase-alpha, to form an active G6Pase complex that hydrolyzes G6P to glucose. These findings suggest that the G6Pase-beta-G6PT complex might be functional in neutrophils and that the myeloid defects in GSD-Ib result from the loss of activity of that complex. We hypothesized that a knockout mutation of G6Pase-beta should exhibit the neutrophil dysfunctions of GSD-Ib and lack the metabolic abnormalities of GSD-Ia and GSD-Ib. To test our hypothesis, we generated G6Pase-beta–deficient mouse strains by gene targeting. We showed that G6Pase-beta null mice manifest myeloid dysfunctions mimicking those of GSD-Ib patients. The absence of G6Pase-beta leads to neutropenia; defects in neutrophil respiratory burst, chemotaxis, and calcium flux; and increased susceptibility to bacterial infection. Consistent with these defects, neutrophils from G6Pase-beta–deficient mice undergo ER stress and an enhanced rate of apoptosis. The results demonstrate that G6P translocation and metabolism in the ER are critical for normal neutrophil functions and define a molecular pathway to neutropenia and neutrophil dysfunction of previously unknown etiology, providing a potential model for the treatment of these dysfunctions.
Cheung YY, Kim SY, Yiu WH, Pan CJ, Jun HS, Ruef RA, Lee EJ, Westphal H, Mansfield BC, Chou JY. Impaired neutrophil activity and increased susceptibility to bacterial infection in mice lacking glucose-6-phosphatase-beta. J Clin Invest 2007;117:784-93.
1 Andrew D. Nguyen, MS, former Postbaccalaureate Fellow
2 Jeng-Jer Shieh, PhD, former Postdoctoral Fellow
3 Li-Yuan Chen, PhD, former Postdoctoral Fellow
4 Mohammad Allamarvdasht, BS, former Postbaccalaureate Fellow
COLLABORATOR
Ji-Liang Gao, PhD, Laboratory of Molecular Immunology, NIAID, Bethesda, MD
Eric J. Lee, DVM, Program in Genomics of Differentiation, NICHD, Bethesda, MD
Philip M. Murphy, MD, Laboratory of Molecular Immunology, NIAID, Bethesda, MD
David A. Weinstein, MD, University of Florida College of Medicine, Gainesville, FL
Heiner Westphal, MD, Program in Genomics of Differentiation, NICHD, Bethesda, MD
For further information, contact chouja@mail.nih.gov.
