The Molecular Basis of Type 1 Glycogen Storage Diseases
Janice
Yang Chou*
Heritable
Disorders Branch, National Institute of Child Health and Human Development,
National Institutes of Health, Bethesda, MD 20892, USA
*Address
correspondence to this authors at the Building 10, Room 9S241, NIH, Bethesda,
MD 20892, USA; Tel: +1-301-496-1094; Fax: +1-301-402-6035; Email: chou@helix.nih.gov
Abstract: Glycogen
storage disease type 1 (GSD-1), also known as von Gierke disease, is a group
of autosomal recessive metabolic disorders caused by deficiencies in the
activity of the glucose-6-phosphatase (G6Pase) system that consists of at
least two membrane proteins, glucose-6-phosphate transporter (G6PT) and
G6Pase. G6PT translocates glucose-6-phosphate (G6P) from cytoplasm to the
lumen of the endoplasmic reticulum (ER) and G6Pase catalyzes the hydrolysis of
G6P to produce glucose and phosphate. Therefore, G6PT and G6Pase work in
concert to maintain glucose homeostasis. Deficiencies in G6Pase and
G6PT cause GSD-1a
and GSD-1b, respectively. Both
manifest functional G6Pase deficiency characterized by growth retardation,
hypoglycemia, hepatomegaly, kidney enlargement, hyperlipidemia, hyperuricemia,
and lactic acidemia. GSD-1b patients also suffer from chronic neutropenia and
functional deficiencies of neutrophils and monocytes, resulting in recurrent
bacterial infections as well as ulceration of the oral and intestinal mucosa.
The G6Pase gene maps to chromosome 17q21 and encodes a 36-kDa
glycoprotein that is anchored to the ER by 9 transmembrane helices with its
active site facing the lumen. Animal models of GSD-1a have been developed and
are being exploited to delineate the disease more precisely and to develop new
therapies. The G6PT gene maps to
chromosome 11q23 and encodes a 37-kDa protein that is anchored to the ER by 10
transmembrane helices. A functional assay for the recombinant G6PT protein has
been established, which showed that G6PT functions as a G6P transporter in the
absence of G6Pase. However, microsomal G6P uptake activity was markedly
enhanced in the simultaneous presence of G6PT and G6Pase. The cloning of the G6PT
gene now permits animal models of GSD-1b to be generated. These recent
developments are increasing our understanding of the GSD-1disorders and the
G6Pase system, knowledge that will facilitate the development of novel
therapeutic approaches for these disorders.
GSD-1 and the G6Pase System
Glycogen storage disease type 1 (GSD-1) is a group of autosomal recessive metabolic disorders that occur approximately once in every 100,000 live births [reviewed in 1, 2]. Patients afflicted with GSD-1 are unable to maintain glucose homeostasis and present with growth retardation, hypoglycemia, hepatomegaly, kidney enlargement, hyperlipidemia, hyperuricemia, and lactic acidemia. Long-term presentations include gout, hepatic adenomas with risk for malignancy, osteoporosis, platelet dysfunction, pulmonary hypertension, and renal failure. The GSD-1 abnormality is caused by a deficiency in the activity of microsomal glucose-6-phosphatase (G6Pase) [1, 2], a key enzyme in glucose homeostasis. G6Pase catalyzes the hydrolysis of glucose-6-phosphate (G6P) to glucose and phosphate [reviewed in 3], the terminal steps in gluconeogenesis and glycogenolysis (Fig. 1). Over the last 20 years, dietary therapies, which consist of continuous nasogastric infusion of glucose [4] or frequent oral administration of uncooked cornstarch [5], are directed toward controlling hypoglycemia.
While
these therapies have greatly improved the prognosis of GSD-1patients, the
underlying pathological process remains uncorrected and the efficacy of
dietary treatment is frequently limited by poor compliance. Therefore,
long-term complications still develop in adult patients. Currently,
alternative therapies that aim at a complete amelioration of the clinical
symptoms of GSD-1a are being developed.
The GSD-1 disorder was described first by von Gierke in 1929 [6]. Cori and Cori [7] in 1952 showed that GSD-1 is caused by the absence of G6Pase activity, establishing for the first time that metabolic disorders could arise from enzyme deficiencies. With the increasing number of patients identified, it became apparent that a number of patients presenting with the clinical symptoms of GSD-1 were not deficient in the G6Pase enzyme. Disrupted microsomal preparations from livers and kidneys of these patients had G6Pase activity even though they failed to metabolize G6P efficiently. To account for this biochemical heterogeneity, Senior and Loridan [8] classified GSD-1a as GSD-1 lacking G6Pase activity in disrupted microsomes from the liver and the kidneys and GSD-1b as GSD-1 with normal G6Pase activity. Narisawa et al. [9] and Lange et al. [10] proposed that GSD-1b is caused by a defect in the microsomal G6P transport system. This and kinetic analysis of G6Pase catalysis [11, 12] led to the concept of a G6Pase system [reviewed in 13] in which the G6Pase enzyme had to interact with other proteins to successfully hydrolyze G6P in vivo.
Figure
1. The primary anabolic and catabolic pathways of glycogen in the liver.
It has been known for several decades that G6Pase is tightly associated with the membrane of endoplasmic reticulum (ER) and nucleus and that enzymatic activity in intact microsomes is resistant to limited proteolysis [3, 14, 15]. This suggested that the active site of the enzyme is not exposed to the cytoplasm. Therefore, the G6P substrate must be translocated to the lumen of the ER before hydrolysis can occur. The products, glucose and phosphate, must be then transported back into the cytoplasm for release into the circulation. Based on kinetic studies of G6Pase catalysis, Arion and coworkers [11, 12] proposed that G6Pase is a multi-component enzyme complex and that hydrolysis of G6P in vivo involves several integral membrane proteins, including the G6Pase catalytic unit and associated substrate and product transporters (Fig. 2). The G6Pase system forms the basis of the translocase-catalytic unit model of G6Pase catalysis [13] and explains clinical heterogeneities seen in GSD-1 patients [1, 2]. Consistent with this model, the GSD-1 disorder was divided into four subtypes, corresponding to defects in: 1) the G6Pase catalytic unit (GSD-1a); 2) the G6P transporter, G6PT (GSD-1b); 3) a putative phosphate transporter, T2 (GSD-1c); and 4) a putative glucose transporter, T3 (GSD-1d). Molecular genetic studies have demonstrated that inactivating mutations in the G6Pase and G6PT genes cause GSD-1a [16] and GSD-1b [17], respectively. The GSD-1c locus, which has been characterized biochemically [18] and was shown to be distinct from that of GSD-1a [19] and GSD-1b [20], has not yet been characterized. To date, no GSD-1d patient has been identified.
Figure 2. The G6Pase system, a key enzyme complex in glucose homeostasis.
The G6Pase Gene and the Encoded Protein
GSD-1a, caused by a deficiency in the G6Pase catalytic unit, is the most prevalent form of GSD-1, representing over 80% of GSD-1 cases [1, 2]. The enzyme had eluded molecular characterization because of its hydrophobicity, tight association with the ER membranes, and difficulty of purification. In 1993, a murine G6Pase cDNA was isolated from a mouse liver library by differential screening [21] and a human G6Pase cDNA was subsequently isolated by its homology to the murine cDNA [16]. When expressed in COS-1 cells, the recombinant human and murine G6Pases had kinetic constants, inhibitor sensitivity, latency, and thermal lability identical to the respective liver microsomal enzymes, confirming the identity of the cloned genes. G6Pase cDNAs have since been characterized from the rat [22] and the dog [23]. All four mammalian G6Pases are polypeptides of 357 amino acid residues, sharing 87 to 91% amino acid sequence identity [2].
The structural organization of human (Fig. 3) and murine G6Pase genes have been determined [16, 21]. Both are single copy genes composed of 5 exons and spanning 10-12 kb of chromosomal DNA. In humans, the gene maps to chromosome 17q21 (Fig. 3).
Mammalian G6Pases are hydrophobic proteins containing an ER membrane protein retention motif, two adjacent lysine residues at the carboxyl (C)-terminus (Fig. 4) which is consistent with the known localization of the enzyme. The orientation of human G6Pase in the ER has been resolved by protease sensitivity assays using amino (N)- and C-terminal tagged proteins [24]. In intact microsomes of COS-1 cells transfected with the tagged-G6Pase constructs, the N-terminal tags are resistant to external protease digestion, while the C-terminal tags are cleaved. This implies that the N- and C-termini are on opposite sides of the membrane with the luminal N-terminus separated from the cytoplasmic C-terminus by an odd number of transmembrane helices. This favors the nine transmembrane helical model for G6Pase (Fig. 4) predicted by the algorithm of Hoffman and Stoffel [25] using the TMpred program.
The topology of human G6Pase was further explored by characterizing the Asn-linked glycosylation sites in G6Pase [26]. There are three conserved potential Asn-linked glycosylation sites at amino acid residues 96-98, 203-205, and 276-278 in human G6Pase (Fig. 4). A survey of mammalian multi-span membrane proteins suggests that Asn-linked oligosaccharides are preferentially localized to single extracytosolic segments that are at least 33 residues in length [27, 28]. According to the prediction of the nine-transmembrane model (Fig. 4), the glycosylation site at amino acid residues 96-98 situates in a 37-residue luminal loop, while the sites at amino acid residues 203-205 and 276-278 situate in a 12-residue cytoplasmic loop and transmembrane helix-7, respectively. If this were correct, only the site at amino acid residues 96-98 would be glycosylated. Using site-directed mutagenesis, in vitro translation, and immunodetection of G6Pase, only Asn-96 was found to be glycosylated [26], further supporting the nine-transmembrane topology model.
The Active Site of G6Pase
Sequence alignment suggests that mammalian G6Pases, lipid phosphatases, acid phosphatases, and vanadium haloperoxidases share a conserved phosphatase signature motif, K-X6-RP-(X12-54)-PSGH-(X31-54)-SR-X5-H-X3-D [29, 30]. In G6Pase, this occurs between residues 76 and 180 (Fig. 4). The crystal structure of the vanadium-containing chloroperoxidase (VCPO) from the plant pathogenic fungus Curvularia inaequalis has been resolved [31]. The results show that the enzyme molecule has an overall cylindrical shape with two four-helix bundles as main structural motifs of the secondary structure. Three domains that are far apart in the primary structure form the active site of VCPO and the active-site residues are contained within the phosphatase signature motif. In the VCPO active site, H496, which is predicted to bind covalently to vanadate or phosphate, acts as a nucleophile, K353, R360, as well as R490 stablize the penta-coordinated transition state, and H404 plays a role in leaving group protonation (Table 1). Site-directed mutagenesis and transient expression studies of phsophatase activity of active-site mutants of VCPO [32, 33] showed that the H496A mutant was inactive under all substrate concentrations examined. On the other hand, K353A, R360A, and R490A mutants exhibited decreased phsophatase activity. Kinetic analysis also shows that the phosphatase reaction of apoCPO is consistent with a two-step product release mechanism in which liberating the phosphate moiety from the phosphorylated enzyme is rate-limiting [33].
Based on the crystal structure of VCPO, the amino acids predicted to participate in G6Pase catalysis include K76, R83, H119, R170, and H176 (Table 1). The nine-transmembrane helical model places R83, H119, R170, and H176 on the luminal side of the ER (Fig. 4). Accordingly, in G6Pase, H176 acts as a nucleophile forming a phosphohistidine enzyme-intermediate, R83 and R170 could donate hydrogen bonds to phosphate and stablize the transition-state, and H119 could provide the proton needed to liberate the glucose moiety (Fig. 5). Unlike VCPO, which is a soluble protein, mammalian G6Pases are membrane-bound enzymes. While K353 in VCPO forms hydrogen bonds with phosphate and stablizes the trasnsition-state, the corresponding residue, K76, in G6Pase is located within transmembrane helix-2. Therefore, it is unclear whether K76 plays a similar role in G6Pase catalysis as that of R360 in VCPO. To date, 4 mutations, K76N, R83C, R83H, and R170Q, that alter active site residues in G6Pase have been identified in GSD-1a patients (see below). R83C and R83H were shown to abolish phosphatase activity in transient expression assays [16, 34]. Future studies should focus on the elucidation of the roles of K76 and R170 in G6Pase catalysis.
To determine whether R83, H119, and H176 play crucial roles in G6Pase catalysis, a large number of R83 (R83C, R83E, R83H, R83K, R83RL, R83M, R83N, R83Q, R83S, and R83T), H119 (H119A, H119I, H119K, H119M, H119N, H119R, and H119T) and H176 (H176A, H176M, H176N, H176I, H176S, H176K, and H176R) mutants were constructed. Transient expression studies showed that all mutations abolished G6Pase activity, demonstrating the importance of these residues in phsophatase reaction. The stringent requirements for R83 and H119 in G6Pase catalysis, however, differ from the results in VCPO where R360A and H404A mutants retained residual phosphatase activity [32, 33]. Therefore, it should be careful when comparisons are made between soluble and membrane-bound enzymes. The challenge ahead is to demonstrate that H176 in G6Pase is the amino acid residue that covalently binds the phosphate moiety to form the phosphorylated enzyme intermediate.
Tissue-Specific Expression of the G6Pase Gene
It has been well established that the G6Pase gene is expressed at high levels in the liver and in the kidneys [reviewed in 3]. However, reports of expression in other organs have been controversial [35, 36]. Immunological analyses can lead to erroneous conclusions because monoclonal or mono-specific antiserum to mammalian G6Pase is currently not available. Recently, in situ and Northern-blot hybridization analyses have demonstrated unequivocally that the G6Pase gene is expressed primarily in the liver, kidney, and intestine [37]. While the G6Pase mRNA is found in all hepatocytes, expression of the G6Pase transcript is restricted to the kidney cortex and to the intestinal mucosa (Fig. 6).
Figure
6. Localization of the G6Pase transcript in mouse tissues by in
situ hybridization analysis.
Using enzymatic assays, several studies reported the presence of G6Pase in mammalian pancreas [reviewed in 3]. However, the high phosphatase background activity in the tissue making the results inconclusive. Recently, it has been reported that the mouse pancreas expresses a G6Pase-related gene and a cDNA encoding this islet-specific G6Pase-related protein has been isolated and characterized [38, 39]. The islet-specific G6Pase-related protein is a hydrophobic polypeptide of 355 amino acid residues that shares 50% overall identity to murine G6Pase. While murine G6Pase is enzymatically active [21], the murine islet-specific G6Pase-related protein is devoid of phosphatase activity [38]. The role of this protein is unknown, and the human counterpart of islet-specific G6Pase-related protein has not been characterized.
The Molecular Basis of GSD-1a
Prior to the cloning of the G6Pase gene, GSD-1 was diagnosed primarily by clinical symptoms, supported by measurements of G6Pase activity in liver biopsy samples. Reliable carrier testing was not available. While prenatal diagnosis of a fetal liver biopsy is possible, it is rarely used because this invasive procedure presents a high risk to the fetus. With the cloning of the G6Pase gene, DNA-based diagnostic tests for this disorder have been developed from many laboratories and a data-base of G6Pase mutations have been established. The data-base provides the foundation for a gene-based diagnosis of carriers in at-risk families and a non-invasive prenatal screening test.
To date, 65 separate mutations (Fig. 7) have been identified in the G6Pase gene of approximately 346 GSD-1a patients. These include 42 missense, 12 insertion/deletion, 7 nonsense, 3 splicing, and 1 codon deletion mutations [16, 19, 34, 40-71]. Nineteen missense (D38V, W77R, R83C, R83H, E110K, E110Q, A124T, V166G, P178S, G184E, G188S, G188R, L211P, G222R, W236R, P257L, G270V, R295C, and L345R), 2 nonsense (R170X and Q347X), 1 codon deletion (DF327), and 1 insertion/deletion (813insG822delC) mutations (Fig. 7) have been shown to abolish or greatly reduce G6Pase activity when assayed by site-directed mutagenesis and transient expression analyses [16, 19, 34 40, 47, 48, 57, 65]. Moreover, a splicing G6Pase mutation (727GÆT) was shown to cause exon-skipping [42]. Taken together, these studies demonstrate that lesions in the G6Pase gene cause GSD-1a, firmly establishing the molecular basis of this disorder.
The mutations in the G6Pase gene appear to cluster according to the known ethnic background of patients (Table 2). Of the 65 mutations identified to date, 53 (81.5%) are found in Caucasian patients, presumably reflecting their greater ethnic diversity. Within this group, however, R83C (33.4%) and Q347X (20.1%) account for over 50% of all cases. Most Japanese patients carry the 727GÆT mutation, accounting for 88% of the 124 alleles analyzed and most patients of Jewish origin carry the R83C mutation, accounting for 94% of the 34 alleles analyzed (Table 2). In Chinese patients, two mutations, R83H and 727GÆT, each of which accounts for 36% of the 56 alleles analyzed. Although the number of American Hispanic (18 alleles) and Muslim Arab (8 alleles) patients examined is statistically too low to draw clear conclusions, it is interesting to note that there is an apparent bias in the prevalent mutations of these two groups. In American Hispanics, 78% of the 18 alleles analyzed are 459insTA and R83C and in Muslim Arabs, 50% of the 8 alleles analyzed are V166G (Table 2). So far, the Q347X mutation has been identified only in Caucasian/Jewish patients, the 459insTA mutation only in American Hispanic patients, and the V166G mutation only in Muslim Arab patients. The 727GÆT mutation was identified in patients of both Japanese and Chinese origins, which may reflect the genetic relatedness of the two populations.
Structure-Function Relationship of Human G6Pase
So far, 43 amino acid codon mutations have been uncovered in the G6Pase gene of GSD-1a patients (Fig. 7). The mutations cluster into 3 regions of the protein (Fig. 4): 1) at the active-site (K76N, R83C, R83H, and R170Q); 2) within transmembrane helices, 1 (D38V), 2 (W63R, G68R, W77R, G81R), 3 (G122D and A124T), 4 (W156L, V166A, and V166G), 5 (P178S, P179P, G184E, G184V, G188D, G188S, and G188R), 6 (L211P and G222R), 7 ( N264K, L265P, G266V, and G270V), 8 (R295C and S298P), and 9 (DF327, V338F, I341N, and L345R); and 3) in the two large luminal loops, 1 (T108I, E110K, E110Q, and P113L) and 3 (W236R, T255I, and P257L). As mentioned earlier, mutations in R83 abolish G6Pase activity [16, 34]. Mutational analysis of the transmembrane helical residues has shown that 13 of the 14 mutations (D38V, W77R, A124T, V166G, P178S, G184E, G188S, G188R, L211P, G270V, R295C, DF327, and L345R) totally abolish G6Pase activity, while one, G222R, retains 4% of WT activity [16, 19, 34, 40, 47, 48, 57]. Moreover, the D38V and P178S mutations, located in helix-1 and -5 (Fig. 4), respectively, were shown to destablize the enzyme [26]. Taken together, these data suggest that the structural integrity of transmembrane helices is critical for G6Pase activity.
While the E110K mutant is devoid of G6Pase activity, the other 3 luminal loop mutants, E110Q [48], W236R [34], and P257L [65], retained 18.2%, 4.2%, and 1.2% wild-type enzymatic activity, respectively. This suggests that structural requirements for luminal loops in G6Pase catalysis are not as stringent as those of transmembrane helices. To date, 2 mutations, T16A and Q20R, have been identified in the N-terminal domain of G6Pase and 1, Q54P, has been identified in cytoplasmic loop 1 (Fig. 4). It would be of interest to determine if these mutations abolish G6Pase activity. Future studies should focus on functional characterization of the additional mutations uncovered in the G6Pase gene of GSD-1a patients in order to increase our understanding of the structural requirements for the stability and G6P hydrolytic activity of G6Pase.
Genotype-Phenotype Relationship in GSD-1a
It is interesting to note that a Japanese patient homozygous for the P257L mutation, which retained 1.2% of wild-type G6Pase activity, had a very mild phenotype [65]. The patient experienced no hypoglycemic episodes and required no dietary therapy. The phenotypes of patients carrying E110Q, G222R, and W236R mutations, which also retain residual G6Pase enzymatic activities, have not been reported. One should bear in mind that it is important to document the results of phenotypic studies of additional GSD-1a patients carrying leaky G6Pase mutations. Knowledge of the minimal G6Pase activity needed to prevent hypoglycemic episodes in GSD-1a patients may facilitate the development of novel therapeutic approaches for this disorder.
A recent report described that patients homozygous for a G188R mutation located within helix-5 (Fig. 4) of G6Pase manifest an atypical GSD-1b phenotype, including mild chronic neutropenia, decreased oxidative metabolism, impaired chemotaxis, and defective killing of E. coli [70]. The G6PT gene, examined in one of the patients, is normal. It is unlikely that genetic heterogeneity could be the cause for this unusual phenotype because four GSD-1a patients carrying the homozygous G188R mutation and exhibiting this atypical phenotype are from three unrelated families. Since G6P uptake and hydrolysis are tightly coupled reactions [72], it is possible that G6Pase could regulate G6P import through functional interaction with G6PT or other membrane components as suggested by the authors [70]. However, human neutrophils and monocytes express low or undetectable level of the G6Pase gene [73]. The challenge ahead is to elucidate how a mutation in the G6Pase gene that is expressed primarily in the liver, kidney, and intestine could cause neutropenia and neutrophil dysfunctions, typical of G6PT deficiency. Characterization of animal models of GSD-1b generated by targeted disruption of the G6PT gene may yield clues to this unusual GSD-1b phenotype in GSD-1a patients.
Animal Models of GSD-1a and Gene Therapy
Two animal models of GSD-1a, a mouse and a dog, are currently available. The G6Pase-/- mice [72], generated by gene targeting, manifest a phenotype virtually identical to that of human GSD-1a patients, including hypoglycemia, growth retardation, hepatomegaly, kidney enlargement, hyperlipidemia, and hyperuricemia. Hematoxylin and eosin staining and electron microscopic analyses revealed that the G6Pase-/- mice have marked glycogen storage and lipid deposition in hepatocytes and glycogen storage in the tubular epithelial cells of the kidney [72], similar to that seen in human GSD-1a patients. The G6Pase-/- mouse is therefore an attractive model for the study of GSD-1a.
The G6Pase-/- mice have been used to evaluate the feasibility of gene replacement therapy for GSD-1a [74]. The results show that infusion of an adenoviral vector containing the murine G6Pase gene (Ad-mG6Pase) into G6Pase-/- mice restored 19% of normal (G6Pase+/+) hepatic G6Pase activity and markedly improved the survival rates of the G6Pase-/- mice. Ad-mG6Pase infusion also greatly improved the growth rate of G6Pase-/- mice and normalized plasma glucose, cholesterol, triglyceride, and uric acid profiles (Fig. 8). Further, liver and kidney enlargement were less pronounced with near normal levels of glycogen depositions in both organs (Fig. 9). Therefore, a single administration of a recombinant adenovirus vector can alleviate the clinical manifestations of GSD-1a in mice, suggesting that this disorder in humans can potentially be corrected by gene therapy.
A naturally occurring dog mutant carrying a 450GÆC mutation in the G6Pase gene, resulting in a Met to Ile substitution at amino acid 121 (M121I) has been reported [23]. The maltese puppies manifest the typical symptoms of human GSD-1a, including hypoglycemia, hepatomegaly, and failure to thrive. The two animal models may allow a clearer understanding of the biology and pathophysiology of GSD-1a and help to delineate the mechanisms of G6Pase catalysis. The knowledge obtained should facilitate the development of novel therapies for this disorder.
G6PT, the GSD-1b Gene
Two complementary approaches have been used to identify the GSD-1b locus. Linkage analysis has mapped the GSD-1b locus to chromosome 11q23 [75] and screening of an expressed sequence tag database using a sequence homologous to bacterial transporters of phosphate esters has identified a candidate human G6PT cDNA [76]. This candidate G6PT cDNA was subsequently mapped to chromosome 11q23 [17, 77, 78] where the GSD-1b locus is resided, confirming its identity. The G6PT cDNAs have since been characterized from the mouse and the rat [73]. All three mammalian G6PT proteins are polypeptides of 429 amino acid residues; each containing an ER transmembrane protein retention motif, two adjacent lysine residues at the C-terminus [73, 76], consistent with the localization of the transporter and its proposed relationship to the G6Pase enzyme. Mouse and rat G6PT proteins share 95% and 93% amino acid sequence identity, respectively, to the human protein [73].
The structural organization of human G6PT gene has been determined [17, 79, 80]. It is a single copy gene [17] composed of 9 exons, and spanning approximately 5.3 kb of chromosomal DNA (Fig. 10). Two alternately spliced transcripts, G6PT [76] and vG6PT [81] exist, differing in the absence or presence of exon-7 sequence [17, 78].
G6PT is a 37-kDa ER Transembrane Protein
Hydropathy profile analysis of the G6PT amino acid sequence predicts that this transporter is a hydrophobic protein anchored in the ER membrane by either ten (Fig. 11) or twelve [76] transmembrane helices. The orientation of human G6PT in the ER has been resolved recently by protease sensitivity and glycosylation scanning assays using N- and C-terminal tagged proteins [82]. Cleavage sites for factor Xa protease [83] were inserted between G6PT and its terminal tag, which allows the in situ removal of the tag without affecting the G6PT protein. In intact microsomes of COS-1 cells transfected with the tagged-G6PT constructs, both N- and C-terminal tags are sensitive to factor Xa cleavage [82]. This implies that both N- and C-termini face the cytoplasm, indicating that G6PT contains an even number of transmembrane helices.
The topology of human G6PT was further explored by glycosylation scanning analysis [82]. Sequence analysis has identified a potential Asn-linked glycosylation site at amino acid residues 354-356 that is conserved among human, mouse, and rat G6PT proteins [73, 76]. This glycosylation site is predicted to be located in a 17-amino acid loop in either the ten- (Fig. 11) or twelve-domain model, thus would not satisfy the criteria for an acceptor of oligosaccharides [27, 28]. In vitro transcription-translation assays demonstrated that mammalian G6PTs are nonglycoproteins of 37-kDa [82], further implying that G6PT contains an even number of transmembrane helices.
The major difference between the ten- and twelve-domain G6PT models is that amino acid residues 50 to 71, which constitute the transmembrane segment-2 in the twelve-domain model [76], are part of a 51-residue loop in the ten-domain model (Fig. 4). By generating a potential Asn-linked glycosylation site within the region spanning amino acid residues 50 to 71 in G6PT, it was shown that the newly introduced glycosylation sites, N53SS and N55QS, were utilized as acceptors for oligosaccharides [82]. This supports the ten-transmembrane helical model for the G6PT protein (Fig. 11).
The calculated molecular mass of mammalian G6PT proteins is 46-kDa [73, 76]. Since migration of hydrophobic proteins is known to be anomaly on polyacrylamide gels, an apparent molecular mass of 37-kDa was obtained when the in vitro synthesized human, mouse, and rat G6PT proteins were analyzed electrophoretically on 10% polyacrylamide-SDS gels [82]. Immonoblot analysis of N- or C-terminal tagged G6PT synthesized in a heterologous expression system using a monoclonal antibody against the tag further confirmed that human G6PT is a 37- kDa protein [82]. An apparent molecular mass of 46-kDa was reported for rat G6PT protein using a polyclonal antibody raised against a 20-amino acid peptide that is located at the N-terminus of G6PT [85]. The authors have observed discrepancies between changes in the expression of G6PT mRNA and the p46 protein. Since immunological analyses using a polyclonal antibody that is not mono-specific could generate confusing and misleading data, one should be extremely cautious in the interpretation of the results.]
Both G6PT and vG6PT are Microsomal G6P Transporters
Kinetic studies of G6P hydrolysis [10] and transport [86] have suggested that GSD-1b is caused by a deficiency in microsomal G6P transport. Using G6Pase-deficient mice, it has been shown that transport and hydrolysis of G6P are tightly coupled processes and that G6Pase activity is required for an efficient transport of G6P into the microsomes [72]. Based on this finding, a functional assay for the recombinant G6PT protein was established [17], which showed that G6PT can function as a G6P transporter in the absence of G6Pase (Fig. 12A). However, microsomal G6P uptake activity was markedly enhanced in the simultaneous presence of G6PT and G6Pase.
The vG6PT which contains an additional 22 amino acids encoded by exon-7 of the gene is also active in microsomal G6P transport [87] (Fig. 12A). The results indicate that the extra 22 amino acids in vG6PT, which constitute a part of a 30-amino acid luminal loop 4 [87], play no vital role in microsomal G6P transport. The G6PT transcript is expressed ubiquitously in all tissues and organs examined [73, 87], including the brain, heart, skeletal muscle, placenta, pancreas, liver, kidney, adrenal gland, lymph node, neutrophils/monocytes, intestine, and the lung (Fig. 12B). On the other hand, the vG6PT transcript [87] is expressed exclusively in the brain, heart and the skeletal muscle (Fig. 12B). These results raise the possibility that mutations in exon-7 of the G6PT gene, which would not perturb glucose homeostasis, might have other adverse effects on the tissues that express the vG6PT gene
The Molecular Basis of GSD-1b
GSD-1b is the second most prevalent form of GSD-1 [1, 2]. It presents with the same clinical symptoms of GSD-1a that reflect a defect in the metabolism of G6P. However, unlike other GSD-1 subgroups, GSD-1b patients suffer additional infectious complications due to chronic neutropenia and functional deficiencies of neutrophils and monocytes [88, 89] which add to the severity of the disease. Treatments of GSD-1b patients consist of a combination of a dietary therapy to correct the symptoms of G6Pase deficiency [1, 2] and a human granulocyte-macrophage colony stimulating factor therapy [90, 91], to restore neutrophil/monocyte functions and to reduce the frequency of infections.
With the cloning of the G6PT gene, DNA-based diagnostic tests for this disorder have been developed in many laboratories. To date, 66 separate mutations (Fig. 13) have been identified in the G6PT gene among approximately 126 GSD-1b patients [17, 76-78, 80, 84, 92-100]. These include 27 missense, 9 nonsense, 15 insertion/deletion, 13 splicing, and 2 codon-deletion (Fig. 13) mutations, which appear to scattered throughout the coding region (Figs. 11 and 13). Of the 66 mutations identified to date, 57 (86.4%) are found in Caucasian patients, presumably reflecting their greater ethnic diversity (Table 3). Within this group, 1211delCT (30.2%) and G339C (13.6%) are the prevalent mutations, accounting for over 40% of all cases (Table 3). The number of alleles in Japanese (26 alleles), Bedouin (18 alleles), and Pakistani (10 alleles) patients examined is statistically too low to draw clear conclusions (Table 3). However, it is interesting to note an apparent bias in the prevalent mutation of Japanese patients where 42.3% is W118R.
To demonstrate that G6PT mutations identified in GSD-1b patients cause the disease, a series of mutant G6PT constructs carrying missense or codon-deletion mutations were constructed and functionally characterized in transient expression assays [17, 84]. All mutations were shown to abolish microsomal G6P transport function of the encoded protein, firmly establishing the molecular basis of the GSD-1b disorder.
Structural-Function Relationship of Human G6PT
G6PT belongs to a family of transporters of phosphorylated metabolites that also includes the glycerol-3-phosphate transporter (101) and hexose-6-phosphate transporter (102, 103). A signature motif has been identified among these transporters. In human G6PT, this motif includes amino acids 133 to 149 (Fig. 11). Two mutations, Q133P [96] and G149E [17], have been identified in the G6PT gene of GSD-1b patients; the G149E mutation was shown to abolish microsomal G6P uptake activity [17]. Future studies will focus on the role(s) of this signature motif in substrate binding, recognition, or translocation.
To date, 16 missense (G20D, R28C, R28H, S55R, G68R, L85P, G88D, W118R, G149E, G150R, C183R, I278N, R300C, R300H, G339C, and A373D) and the DF93 mutations have been shown to abolish microsomal G6P uptake activity [17, 84]. Immunoblot analyses show that G20D, DF93, and I278N mutations, located in helix-1, -2, and -6, respectively, destablize the G6PT [84]. The results suggest that the structural integrity of transmembrane helices is critical for G6P transport activity.
The cytoplasmic tail at amino acid residues 415 to 429 in the C-domain of human G6PT is extended directly from helix-10 encompassing amino acid residues 396 to 414 (Fig. 11). Two nonsense mutations, W393X [84] and R415X [96], lacking helix-10 plus the cytoplasmic tail and the entire cytoplasmic tail, respectively, were identified in the G6PT gene of GSD-1b patients. To investigate the molecular basis of G6PT deficiency caused by these mutations, the W393X and R415X mutants were constructed and functionally characterized in transient expression assays [84]. Further, to examine the role of helix-10 and the cytoplasmic tail in the stability and microsomal transport activity of the G6PT, additional helix-10 (W393X, E401X and T408X) and cytoplasmic tail (N416X, I417X, R418X, T419X, K420X, K426X, and K427X) mutants were characterized [84]. The results show that helix-10 mutants (W393X, E401X and T408X) were devoid of microsomal G6P uptake activity [84] when assayed in a heterologous expression system, COS-1 cells (Fig. 14). Immunoblot analysis showed that mutants directed little or no protein synthesis in COS-1 cells (Fig. 14), indicating that the lack of transporter synthesis is the cause of the loss of G6P transport activity. However, in vitro transcription-translation assays showed that the three helix-10 mutants directed the synthesis of similar amounts of G6PT proteins as that of the wild-type transporter [84]. This indicates that W393X, E401X and T408X mutants are synthesized and degraded rapidly in cells, suggesting that helix-10 is important for the folding of the transporter into an active configuration.
Figure
13. Mutations identified in the G6PT
gene of GSD-1b patients. The G6PT protein encoded by the 8 exons of the gene
is shown. From left to right: insertion/deletion, splicing, missense, and
nonsense mutations. Mutations that have been functionally characterized are
shown in bold.
Unlike helix-10 mutants, R415X, N416X, I417X mutants, lacking 15 (the entire tail), 14, and 13 amino acids in the cytoplasmic tail of G6PT (Fig. 11) directed the synthesis of reduced levels of G6PT protein, leading to a corresponding decrease in microsomal G6P transport activity (Fig. 14). The results indicate that amino acids 415 to 417 in the cytoplasmic tail of G6PT also play a critical role in the correct folding of the transporter [84]. However, R418X, T419X, K420X, K426X, and K427X mutants behave like the wild-type trasnporter (Fig. 14), indicating that the last 12 amino acids of the cytoplasmic tail, including the ER membrane protein retention signal, play no essential role(s) in the functional integrity of G6PT [84].
G6PT Function and Neutropenia
In addition to functional G6Pase deficiency, GSD-1b patients also suffer from neutropenia and functional deficiencies of neutrophils and monocytes [88, 89]. Polymorphonuclear leukocytes from GSD-1b patients exhibit impaired mobility and chemotaxis as well as diminished respiratory burst, hexose monophosphate shunt, and phagocytotic activities [reviewed in 89]. Moreover, neutrophils and monocytes from GSD-1b patients are unable to sequester Ca2+ [104]. The results of a recent study suggest that GSD-1b patients carrying leaky mutations in the G6PT gene will manifest no neutropenia and suffer no recurrent bacterial infections [98]. Mutational analysis showed that GSD-1b patients carrying either a homozygous splicing (794GÆA) mutation or heterozygous G339D and R415X mutations suffer no impairment in their polymorphonuclear leukocyte functions. Reverse transcriptase-polymerase chain reaction analysis demonstrated that the 794GÆA mutation is leaky because the mutated G6PT gene of the patient directed the expression of both mature and exon-3 truncated G6PT transcripts [98]. Now, it is apparent that the patient carrying the G339D and R415X mutations also has residual G6P uptake activity because the R415X mutation only partially inactive the transporter [84]. These studies demonstrate a close relationship between the residual activity retained by the patient’s G6PT protein and the susceptibility of the GSD-1b patient to neutropenia as well as neutrophil and monocyte dysfunctions.
How a G6P transport defect impairs the functions of the patients' polymorphonuclear leukocytes is unclear. Human neutrophils and monocytes express the G6PT, but not the G6Pase gene [73], and the G6PT transcript expressed in polymorphonuclear leukocytes is identical to the liver transcript [87]. Moreover, liver allografts that correct the apparent G6P metabolic deficiencies, do not correct neutrophil dysfunctions in GSD-1b patients [105]. Therefore, the defect must be intrinsic to the cells of the myeloid lineage. It has been shown that G6P enhances ATP-dependent microsomal Ca2+ sequestration [106, 107] and the presence of ATP and Ca2+ also leads to a higher level of G6P accumulation in the ER lumen [107]. In neutrophils and monocytes, G6P stimulates glycolysis and hexose monophosphate shunt activity, which provide the major source of energy for chemotaxis and phagocytosis. It is interesting to note that the G6PT protein is structurally more similar to uhpC, a G6P-specific receptor, than it is to uhpT, a related phosphate ester transporter that transports a variety of phosphate esters [102, 103]. Thus, it is possible that the G6PT protein has dual roles, dependent upon the tissue or cells in which it is expressed. In gluconeogenic tissues that express high levels of the G6Pase gene, the primary function of the G6PT protein is to transport G6P to the ER to be hydrolyzed by G6Pase to produce glucose and phosphate. In other tissues or cells, including neutrophils and monocytes, the G6PT protein may function as a G6P receptor/sensor that regulates Ca2+ sequestration, glycolysis, and hexose monophosphate shunt activity. This could provide an explanation for the observations that despite possessing an intact G6Pase gene, GSD-1b patients manifest neutrophil and monocyte dysfunctions as well as a functional G6Pase deficiency.
Conclusion and Future Directions
Molecular and genetic evidence have unequivocally demonstrated that the two major GSD-1 subgroups, GSD-1a and GSD-1b, have different etiologies. Lesions in the G6Pase gene that abolish or greatly reduce enzymatic activity cause GSD-1a and lesions in the G6PT gene that abolish or greatly reduce microsomal G6P uptake activity cause GSD-1b. Animal model of GSD-1a are available and are being used to broaden our understanding of this disorder and to develop gene therapies. Animal models of GSD-1b are now being generated. Providing that they mimicking human GSD-1b patients, these animal models can increase our understanding of the biology and pathophysiology of GSD-1b and facilitate the development of novel therapeutic approaches.
Several areas of GSD-1 research need to be clarified: 1) whether the GSD-1d disorder exists. A cDNA encoding a putative glucose transporter, expressed primarily in the human liver and kidney, has recently been isolated (108). The encoded protein is predicted to have twelve membrane-spanning domains and contain a possible N-terminal ER targeting motif. It would be of interest to elucidate the function of the encoded protein and to determine if a deficiency of this putative glucose transporter elicits a GSD-1-like phenotype; 2) the molecular basis of the GSD-1c disorder. This can be accomplished providing that additional GSD-1c patients can be identified; 3) the structural requirements for folding and functional integrity of G6Pase and G6PT. This will be facilitated by functional characterization of all missense and codon-deletion mutations uncovered in the G6Pase gene of GSD-1a patients and in the G6PT gene of GSD-1b patients; 4) whether H176 in G6Pase is the acceptor for the phosphate moiety during G6Pase catalysis; 5) the role of the pancreatic islet-specific G6Pase-related protein in glucose homeostasis; 6) whether the G6PT protein is also a G6P receptor; and 7) whether there is a relationship between G6P metabolic defects and neutrophil and monocyte dysfunctions. The full understanding of type 1 GSDs will lead to the elucidation of the interrelationship between G6P metabolism and long-term complications of these disorders.
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![Figure 12. (A) Uptake of [U-14C]G6P into microsomes of G6PT (D), vG6PT (s), G6PT/G6Pase (O), vG6PT/G6Pase (l), and G6Pase («) transfected COS-1 cells. (B) Reverse transcriptase-polymerase chain reaction and Southern-blot analyses of G6PT and vG6PT mRNA expression in human tissues. (Modified from Ref. 87, with permission)](Chou-fig12-A-&-12-B-pg37.jpg){kind=link}
