Human Molecular Genetics, 2002, Vol. 11, No. 25 3199-3207
© 2002 Oxford University Press
Structure–function analysis of the glucose-6-phosphate transporter deficient in glycogen storage disease type Ib
Section on Cellular Differentiation, Heritable Disorders Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, 20892
Received August 10, 2002; Revised September 25, 2002; Accepted September 30, 2002
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Glycogen storage disease type Ib (GSD-Ib) is caused by a deficiency in the glucose-6-phosphate transporter (G6PT), a 10 transmembrane domain endoplasmic reticulum protein. To date, 69 G6PT mutations, including 28 missenses and 2 codon deletions, have been identified in GSD-Ib patients. We previously characterized 15 of the missense and one codon deletion mutations using a pSVL-based expression assay. A lack of sensitivity in this assay limited the discrimination between mutations that lead to loss of function and mutations that leave a low residual activity. We now report an improved G6PT assay, based on an adenoviral vector-mediated expression system and its use in the functional characterization of all 30 codon mutations found in GSD-Ib patients. Twenty of the naturally occurring mutations completely abolish microsomal G6P uptake activity while the other 10 mutations, including 5 previously characterized ones, partially inactivate the transporter. This information should greatly facilitate genotype–phenotype correlation. We also report a structure–function analysis of G6PT. In addition to the 3 destabilizing mutations reported previously, we now show that the G50R, C176R, V235del, G339C and G339D mutations also compromise the G6PT stability. Mutation analysis of the amino-terminal domain of G6PT shows that it is required for optimal G6P uptake activity. Finally, we show that degradation of both wild-type and mutant G6PT is inhibited by a potent proteasome inhibitor, lactacystin, demonstrating that G6PT is a substrate for proteasome-mediated degradation.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Glycogen storage disease type I (GSD-I) is a group of autosomal recessive disorders caused by a failure to generate glucose and phosphate from glucose-6-phosphate (G6P). This metabolism is performed by a group of membrane-associated activities in the endoplasmic reticulum (ER) of the liver and kidney, known collectively as the glucose-6-phosphatase (G6Pase) system (reviewed in 1,2). The G6Pase system consists of 4 activities: G6P translocation, G6P hydrolysis, glucose transport and phosphate transport. A failure in any one of these activities results in one of the four subtypes of GSD-I: GSD-Ia; GSD-Ib; GSD-Ic; or GSD-Id. Two of the proteins in the G6Pase system, G6Pase and G6P transporter (G6PT), have been characterized at the molecular genetic level. A deficiency in G6PT, which translocates G6P from the cytoplasm into the lumen of the ER, results in GSD-Ib. A deficiency of G6Pase, which hydrolyzes G6P in the ER lumen into glucose and phosphate, results in GSD-Ia. Together, G6Pase and G6PT maintain glucose homeostasis. Both GSD-Ia and GSD-Ib share the phenotype characteristic of G6Pase deficiency, namely growth retardation, hypoglycemia, hepatomegaly, kidney enlargement, hyperlipidemia, hyperuricemia and lactic academia (1,2). In addition, GSD-Ib patients 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 (3,4). An understanding of the structure and function of G6PT will provide additional insight into the G6Pase system and help elucidate the relationship between G6PT deficiency and myeloid dysfunctions.
Human G6PT is a single copy gene consisting of 9 exons (5), spanning approximately 5.3 kb of DNA at chromosome 11q23 (6). To date, 69 separate G6PT mutations have been identified in GSD-Ib patients including 28 missense, 2 codon deletion, 15 insertion/deletion, 10 nonsense and 14 splicing mutations (reviewed in 1). Using a functional assay for G6P transport, developed previously, based on a pSVL-expression system, we showed that the F93del and 15 missense mutations abolish microsomal G6P transport activity (5,7), while the G20D, F93del, and I278N mutations destabilize the G6PT (7). This established that deficiencies in G6P transport cause GSD-Ib.
Human (8), mouse (9) and rat (9) G6PT proteins are polypeptides of 429 amino acids sharing 93–95% sequence identity. Protease sensitivity and glycosylation scanning analyses (10) have shown that human G6PT is anchored in the ER by 10 transmembrane helices with both the amino (N)- and carboxyl (C)-termini facing outwards into the cytoplasm. Using G6Pase-deficient mice, we have shown that transport and hydrolysis of G6P appear to be tightly coupled processes in which G6Pase activity is required for an efficient transport of G6P into the microsomes (11). Based on this finding, we have developed a functional assay for the recombinant G6PT using a pSVL-based expression system and shown that G6PT's function as a G6P transporter is enhanced markedly in the presence of G6Pase (5).
The pSVL-based assay lacks sufficient sensitivity to identify G6PT mutants retaining low G6P uptake activities because it can not express sufficiently high levels of G6PT (12) to measure low, residual activities. The finding that GSD-Ib patients carrying leaky G6PT mutations manifest no myeloid dysfunction (7,13) suggests that there is a close relationship between residual G6PT activity and the susceptibility of a GSD-Ib patient to neutropenia and neutrophil/monocyte dysfunctions. Therefore, an improved G6PT assay is required to examine this hypothesis. In this study, we adapt a recombinant adenoviral vector-mediated expression system to increase the level of expression of G6PT mutants and enhance the sensitivity of the G6P transport assay. Using this assay, we analyse functionally all 30 naturally occurring G6PT codon mutations and the structure–function relationship for V235del and the 13 previously uncharacterized missense G6PT mutations.
Proteins with abnormal conformations that arise by mutations or intracellular denaturation are rapidly eliminated by intracellular protein degradation (14,15). Cytosolic proteasomes are responsible for rapid degradation of many membrane proteins (reviewed in 16–18), including the cystic fibrosis transmembrane conductance regulator (19,20) and G6Pase (21). The contribution of the proteasome pathway to protein turnover can be examined using the Streptomyces-derived inhibitor, lactacystin (22,23). In this study, we show that degradation of wild-type (WT) and mutant G6PT is predominantly mediated by the proteasome pathway.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Microsomal G6P uptake activity of Ad-G6PT and Ad-G6Pase co-infected COS-1 cells
We have previously developed a functional assay for recombinant G6PT by transient co-transfection of COS-1 cells with G6PT and G6Pase in the pSVL vector (5). However, this assay does not have sufficient sensitivity to identify partial loss of activity mutants because the pSVL system is inadequate to direct very high levels of G6PT expression (12). To improve the sensitivity of the assay, we have adapted a recombinant adenoviral vector-mediated expression system, which has been widely used for high-level protein expression in mammalian cells (reviewed in 24). Results in Figure 1 show that microsomal G6P uptake activity in Ad-G6PT and Ad-G6Pase co-infected COS-1 cells was at least 3-fold higher than the activity in pSVLG6PT and pSVLG6Pase co-transfected cells.
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) or pSVLG6PT-WT and pSVLG6Pase co-transfected (•) COS-1 cells.G6PT mutations that cause GSD-Ib
Characterization of missense and codon deletion mutations, that result in single amino acid alterations, provides valuable information on functionally important residues in G6PT. To date, 28 missense and 2 codon deletion G6PT mutations scattered throughout the primary amino acid sequence (Fig. 2) have been identified in GSD-Ib patients (reviewed in 1). Previously, we have shown that F93del and 15 missense mutations, G20D, R28C, R28H, S55R, G68R, L85P, G88D, W118R, G149E, G150R, C183R, I278N, R300H, G339C and A373D (Fig. 2 shown in italic) abolished microsomal G6P uptake activity (5,7). In this study, we constructed Ad-G6PT mutants carrying the above mentioned mutations as well as V235del and the other 13 missense mutations, including MIV, N27K, G50R, S54R, Q133P, P153L, C176R, P191L, R300C, H301P, G339D, A367T and G376S (Fig. 2). To facilitate structure–function analysis, we grouped these mutations into helical (16 mutations), non-helical (13 mutations) and N-terminal (1 mutation) based on their predicted locations in human G6PT (Fig. 2).
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Microsomal G6P uptake activities were examined after co-infection of COS-1 cells with WT or mutant Ad-G6PT and WT Ad-G6Pase. Ten of the 16 helical G6PT mutants, G20D, L85P, F93del, G149E, G150R, C176R, C183R, V235del, G339D and A373D had no detectable microsomal G6P uptake activity and G88D, P153L, I278N, G339C, A367T and G376S retained 2.2, 8.6, 10.4, 4.9, 23.1 and 5.6% of WT activity, respectively (Table 1). The residual activities retained by G88D, I278N and G339C mutations were missed by the pSVL-based expression assays (7).
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Nine of the 13 non-helical G6PT mutants, N27K, R28C, R28H, G50R, S54R, S55R, W118R, Q133P and P191L, lacked detectable microsomal G6P uptake activity and G68R, R300C, R300H, H301P retained 8.1, 5.2, 7.1, and 24.2% of WT activity, respectively (Table 2). Six of the 7 luminal loop 1 mutations, N27K, R28C, R28H, G50R, S54R and S55R, were nonfunctional, suggesting that this loop plays a vital role in microsomal G6P uptake. Again, the residual activities retained by G68R and R300H mutations were identified only by using the adenoviral vector-mediated expression system.
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Using N-terminal FLAG-tagged G6PT constructs, we have shown that helical mutants, L85P, G88D, G149E, G150R, C183R, G339C and A373D and non-helical mutants, R28C, R28H, S55R, G68R, W118R and R300H accumulated proteins to a similar level as the WT construct in a heterologous expression system (7). On the other hand, G20D, F93del and I278N constructs located within helix-1, -2, and -6, respectively, accumulated lower amounts of proteins than the WT G6PT (7). This was largely confirmed using the adenoviral vector-mediated expression system (Fig. 3A and data not shown) with the exception of the G339C construct. Our earlier study, using a pSVL-based assay, had shown that both the G339C and WT constructs directed the accumulation of similar amounts of G6PT proteins in COS-1 cells (7). Using the adenoviral vector-mediated expression assay, we now show that the G339C mutant accumulated less G6PT protein than the WT construct (Fig. 3A). Therefore, we re-examined G339C synthesis in COS-1 cells co-transfected with pSVLG6PT-WT, pSVLG339C, pSVLG6PT-WT-5FLAG, or pSVLG339C-5FLAG, and pSVLG6Pase. Again, the results in Fig. 3B confirm that the G339C construct directed the accumulation of a lower level of protein than the WT construct.
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Among the 6 new helical mutations the P153L, A367T and G376S mutants accumulated proteins to similar levels as the WT G6PT (Fig. 3A). In contrast, the C176R, V235del and G339D mutants accumulated lower levels of G6PT (Fig. 3A). In summary, 7 of the 16 helical mutations, G20D, F93del, C176R, V235del, I278N, G339C and G339D, directed the accumulation of reduced amounts of G6PT compared to the WT construct.
G50R was the only non-helical G6PT mutant identified that accumulated a lower level of G6PT protein compared to the WT construct (Fig. 3C). The additional 6 new non-helical constructs, N27K, S54R, Q133P, P191L, R300C and H301P (Fig. 3C), like the 6 previously characterized non-helical mutants (5,7), directed the accumulation of similar amounts of G6PT proteins as the WT construct.
Northern blot analysis confirmed that similar levels of G6Pase transcripts were expressed in WT, G20D, G50R, F93del, C176R, I278N, V235del, G339C, or G339D G6PT-infected COS-1 cells (Fig. 4A), demonstrating that the decrease in G6PT biosynthesis is not due to a decrease in efficiency of expression of the mutant construct. Moreover, G20D, WT, G50R, F93del, C176R, V235del, I278N, G339C, or G339D constructs directed the synthesis of similar amounts of G6PT proteins in a cell-free transcription–translation system (Fig. 4B). The difference in expression between the in vitro expression system and the cell-based expression system suggests that the lower accumulation levels of the mutant proteins in COS-1 cells may result from misfolding and their rapid degradation.
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The role of the N-terminal domain in microsomal G6P uptake
The N-terminal domain of human G6PT consists of 7 amino acids (Fig. 2). The naturally occurring MIV mutation, predicted to encode a G6PT lacking amino acids 1–16, was devoid of microsomal G6P transport activity (Table 3). To examine the role of the N-domain in the transport function of G6PT, we constructed Ad-G6PT mutants by sequential deletions of the N-terminal residues in human G6PT. The A3M, Q4M, G5M, Y6M, G7M and Y8M mutants retained 44.5%, 20.2%, 15.1%, 17.4%, 16.5% and 8.7% of WT activity, respectively (Table 3), demonstrating that the N-domain is essential for optimal microsomal G6P uptake by the G6PT protein.
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The G6PT mutation MIV, which lacks the N-domain and part of helix 1, accumulated less protein in the heterologous COS-1 assay than the WT construct (Fig. 5) even though similar levels of G6PT transcripts were expressed (Fig. 4A). The MIV mutant also directed the accumulation of less G6PT protein than the WT construct in a cell-free transcription–translation system (Fig. 4B). This suggests that the observed decrease in MIV synthesis may result, in part, from a decreased translational efficiency of the mutant. On the other hand, A3M, Q4M, G5M, Y6M, G7M and Y8M directed the accumulation of similar amounts of G6PT proteins as WT G6PT in COS-1 cells (Fig. 5), suggesting that the N-terminal domain is not essential for the stability of G6PT.
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The proteasome inhibitor lactacystin induces the accumulation of WT and mutant G6PT
The effect of a proteasome inhibitor, lactacystin (22,23) on steady-state levels of G6PT was assessed by western blot analysis. In WT Ad-G6PT-infected cells, the steady-state level of the 37 kDa protein was increased in the presence of lactacystin (Fig. 6), indicating that G6PT is degraded, at least in part, through the proteasome pathway in cells.
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In the absence of lactacystin, the steady-state levels of mutant proteins in C176R, V235del, I278N, G339C and G339D-infected cells were lower than that in WT G6PT-infected cells (Fig. 6). In the presence of lactacystin, a marked increase in G6PT accumulation was observed, indicating that proteasomes are a major pathway for the turnover and degradation of G6PT mutants. We also investigated the effects of lactacystin on steady-state levels of W393X (7), a nonsense mutant that supported little or no G6PT synthesis in COS-1 cells in the absence of lactacystin (Fig. 6). In the presence of lactacystin, the steady-state level of the W393X mutant protein was comparable to that in WT G6PT infected cells, suggesting that the proteasome pathway is a principal regulatory pathway for G6PT proteins.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Glycogen storage disease type-Ib, which is characterized by phenotypic G6Pase deficiency and myeloid dysfunctions, is caused by a deficiency in the activity of the hydrophobic G6PT protein anchored in the ER (1,2). Kinetic studies of G6P hydrolysis (25,26) and transport (27) have suggested that G6PT mediates microsomal G6P uptake. Using a pSVL-based expression system, we had previously developed a functional assay for the recombinant G6PT and shown that G6PT functions as a G6P transporter (5). In this study, we adapted an adenoviral vector-mediated expression system to develop a more sensitive transport assay for G6P. Using the improved assay, we examined microsomal G6P uptake activities of G6PT mutants carrying 28 missense and 2 codon deletion mutations. We show that 20 of the 30 codon mutations completely abolished microsomal G6P uptake activity and 10 retained residual activity. The leaky mutations, G68R, G88D, I278N, G339C and R300H which retained 8.1, 2.2, 10.4, 4.9 and 7.1% of WT activity, respectively, were identified only by the improved assays. Seven of the 16 helical mutations and 1 of the 13 non-helical G6PT mutations destabilized the G6P transporter. Furthermore, 7 mutations were identified within the 51 residue luminal loop 1 in G6PT and 6 completely inactivated this transporter, indicating that this loop plays a vital role in microsomal G6P uptake. We also show that the cytoplasmic N-terminal domain played an essential role in optimal microsomal G6P uptake function of G6PT. Finally, we have provided evidence indicating that G6PT is degraded in cells predominantly through the proteasome pathway.
GSD-Ib is an autosomal recessive disorder and GSD-Ib carriers having 50% of normal G6PT uptake activity manifest no symptoms associated with G6PT deficiency. A recent study showed that two GSD-Ib patients carrying either a homozygous splicing (625G>A) or heterozygous G339D and R415X mutations manifest no neutropenia and suffer no recurrent bacterial infections (13). The 625G>A mutation is leaky because the mutated G6PT gene of the patient directed the expression of both mature and exon 3 truncated G6PT transcripts (13). The G415X mutations retained 47% of WT activity (7), suggesting that if the activities are additive, the 23.5% of normal G6PT activity retained in the compound heterozygote is sufficient to maintain the patient's myeloid functions. This suggests that there is a close relationship between the residual activity retained by the patient's G6PT protein and the susceptibility of the GSD-Ib patient to neutropenia as well as myeloid dysfunctions. The database of residual G6P uptake activity retained by the 30 G6PT codon mutants generated in this study should facilitate future genotype–phenotype delineations and the threshold of G6PT activity required to prevent myeloid dysfunctions.
The G20D, F93del, C176R, V235del, I278N, G339C and G339D mutations located in helix 1, 2, 4, 5, 6, 8 and 8, respectively and G50R within luminal loop 1 directed the accumulation of reduced amounts of G6PT proteins compared to the WT construct in a heterologous expression system. However, WT as well as mutant G6PT transcripts direct the synthesis of similar amounts of G6PT proteins in a cell-free translation system. Since folding of nascent proteins occurs during their in vitro synthesis by rabbit reticulocyte ribosomes, our data strongly suggest that these mutations cause misfolding and degradation of mutant proteins. It is interesting to note that substitution of Gly339 in helix 8 with either a Cys (G339C) or an Asp (G339D) greatly decreases G6P uptake activity and destabilizes the G6PT protein. G339C is the prevalent mutation in Caucasian GSD-Ib patients (reviewed in 1) and G339D was identified in a Japanese patient (13). The stringent structural requirement of Gly339 suggests that amino acid 339 in helix 8 plays an essential role in the transport function and stability of the G6PT protein.
Proteins with abnormal conformations that arise by mutations or intracellular denaturation are rapidly degraded, which represents a quality control system in the cell (14,15). Many membrane proteins, including cystic fibrosis transmembrane conductance regulator (19,20) and G6Pase (21) are degraded in cells by the proteasome system. The proteasome-mediated protein degradation can be blocked by lactacystin, a specific proteasome inhibitor that blocks proteasome action by becoming covalently linked to the active site nucleophil of the ß subunit of proteasome (22,23). In this study, we show that the steady-state levels of WT and mutant G6PT were markedly increased by lactacystin, indicating that degradation of G6PT, like G6Pase (21), is predominantly mediated by the proteasome pathway.
In summary, using a newly developed sensitive assay for G6PT, we have generated a database of residual microsomal G6P uptake activity retained by 30 codon mutations to facilitate genotype–phenotype delineation. Furthermore, we have elucidated a number of structural requirements for the stability and G6P transport activity of G6PT and demonstrated that proteasomes mediate degradation of this transporter.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Construction of G6PT mutants
The PCR template for the construction of G6PT mutants was nucleotides 166–1496 of the human G6PT cDNA (8), in a pSVL vector (Pharmacia, Piscataway, NJ). This contains the entire coding region, nucleotides 170–1459 and includes a BstEII site at nucleotides 863–869. The PCR primers for mutants that contained mutations located upstream of the BstEII site are nucleotides 166–186 (sense) and 851–878 (antisense) in the G6PT cDNA. The primers for mutants that contained mutations located downstream of the BstEII site are nucleotides 851–878 (sense) and 1476–1496 (antisense). PCR was carried out using Pwo polymerase for a total of 30 cycles in a GeneAmp PCR System 9700 (PE Applied Biosystems, Foster, CA) thermo-cycler. The cycles are: cycle 1: 94°C for 30 sec, cycles 2–29: 94°C for 30 sec, 55°C for 30 sec and 72°C for 45 sec, and the last cycle: 72°C for 7 min. The amplified fragments were ligated into either the pSVLhG6PT-BstEII-3' or the pSVLhG6PT-BstEII-5' fragment as previously described (5).
The sense and antisense mutant primers were 21 nucleotides in length with the codon to be mutated in the middle. The nucleotide changes in the mutant constructs include: MIV (nucleotides 170–172, ATG to GTG); N27K (nucleotides 248–250, AAT to AAA); G50R (nucleotides 317–319, GGG to CGG); S54R (nucleotides 329–331, AGC to AGA); Q133P (nucleotides 566–568, CAG to CCG); P153L (nucleotides 626–628, CCT to CTT); C176R (nucleotides 695–697, TGT to CGT); P191L (nucleotides 740–742, CCT to CTT); V235del (nucleotides 872–874, deleting GTG); R300C (nucleotides 1067–1069, CGC to TGC); H301P (nucleotides 1070–1072, CAT to CCT); G339D (nucleotides 1184–1186, GGT to GAT); A367T (nucleotides 1268–1270, GCC to ACC); and G376S (nucleotides 1295–1297, GGC to AGC).
The sense primers for constructing N-terminal deletion mutants were 20 nucleotides in length. The deletions are: A3M (deletion of amino acids 1–2, Met replaces Ala3), Q4M (deletion of amino acids 1–3, Met replaces Gln4), G5M (deletion of amino acids 1–4, Met replaces Gly5), Y6M (deletion of amino acids 1–5, Met replaces Tyr6), G7M (deletion of amino acids 1–6. Met replaces Gly7), and Y8M (deletion of amino acids 1–7, Met replaces Tyr8). The antisense primer contained nucleotides 851–878 of the G6PT cDNA. The amplified fragments were ligated into the pSVLhG6PT-BstEII-5' fragment. The nucleotide sequences of all constructs in their entirety were verified by DNA sequencing.
Construction of recombinant adenoviral G6PT mutants
Recombinant Adenovirus containing WT or mutant human G6PT (Ad-G6PT) or human G6Pase (Ad-G6Pase) was generated by the Cre-lox recombination system described by Hardy et al. (28). The cDNA construct was sublconed into in the pAdlox shuttle vector (28) and the resultant pAdlox construct was digested with SfiI. Two µg of SfiI-digested pAdlox construct and 6 µg of 
5 viral DNA, an E1- and E3-deleted Ad5 containing loxP sites flanking the packaging site 
, were co-transfected into CRE8 cells which contains the Cre recombinase gene (28) by the calcium phosphate-DNA coprecipitation method (29). The resultant recombinant virus was plaque purified and amplified to produce viral stocks with titers of 
5 to 10x109 plaque forming unit (PFU) per ml.
Expression in COS-1 cells and G6P uptake assays
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 150 cm2 flasks were co-infected with Ad-G6PT and Ad-G6Pase at multiplicity of infection of 50 and 25 PFU/cell, respectively. After incubation at 37°C for 24 h, the cell lysates were used for western blot analysis of G6PT. In addition, the infected cultures were used to isolate microsomes for G6P uptake and western blot analysis or for RNA isolation.
G6P uptake measurements were performed essentially as described before (5). Briefly, microsomes (40 µg) were incubated in a reaction mixture (100 µl) containing 50 mM sodium cacodylate buffer, pH 6.5, 250 mM sucrose and 0.2 mM [U-14C]G6P (50 µCi/µmol). The reaction was stopped at the appropriate time by filtering immediately through a nitrocellulose membrane (BA85, Schleicher & Schuell, Keene, NH) and washed with an ice-cold solution containing 50 mM Tris–HCl, pH 7.4 and 250 mM sucrose. Microsomes permeabilized with 0.2% deoxycholate, which abolished G6P uptake, were used as negative controls. Four independent experiments were conducted, and at least four G6P uptake studies were performed for each microsomal preparation. Statistical analysis using the unpaired t test was performed with The GraphPad Prism Program (GraphPad Software, San Diego, CA). Data are presented as the mean±SEM.
Generation of polyclonal anti-G6PT antisera and western blot analysis
To generate antibodies against human G6PT, nucleotides 241–405, containing amino acids 25–79 of human G6PT, encompassing the entire luminal loop 1 (amino acids 27–77) (10), were cloned into the pET-41a (+) vector (Novagen, Madison, WI). The G6PT peptide was produced as a glutathione S-transferase fusion protein containing a 6xHis tag at the C-terminus. Recombinant G6PT peptide was affinity purified on a nickel chelate column, and used to raise antibodies in NZ white rabbits.
For western blot analysis, microsomal proteins or total cellular proteins from infected COS-1 cells were resolved by electrophoresis through a 12% polyacrylamide–SDS gel and trans-blotted onto polyvinylidene fluoride membranes (Millipore Co., Bedford, MA). The membranes were incubated overnight with the rabbit anti-G6PT antibody, and then with horseradish peroxidase-conjugated goat anti-rabbit IgG (Kirkegarrd & Perry Laboratories, Gaithersburg, MD). The immunocomplex was visualized using the SuperSignal West Pico Chemiluminescent substrate from Pierce (Rockford, IL). Similar results were obtained in western blot analysis using either microsomal or total cellular proteins.
Northern blot and in vitro transcription–translation analyses
Total RNA was isolated by the guanidinium thiocyanate/CsCl method (30), fractionated by electrophoresis through a 1.2% agarose gel containing 2.2 M formaldehyde and transferred to a Nytran membrane by electrobloting. The membranes were hybridized with either a G6PT or a ß-actin probe labeled by random priming.
In vitro transcription–translation was performed using the TnT coupled reticulocyte lysate system obtained from Promega Biotech (Madison, WI) using G6PT constructs in a pGEM-11Zf(+) vector. L-[35S] methionine was used as the labeled precursor. The in vitro synthesized proteins were analysed by 12% polyacrylamide–SDS gel electrophoresis and fluorography.
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ACKNOWLEDGEMENT |
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We thank Dr Brian C. Mansfield for critical reading of the manuscript.
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FOOTNOTES |
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* To whom correspondence should be addressed at: Building 10, Room 9S241, NIH, Bethesda, MD 20892-1830, USA. Tel: +3014961094; Fax: +3014026035; Email: chou{at}helix.nih.gov
The authors wish it to be known that, in their opinion, these authors should be considered as joint First Authors.
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REFERENCES |
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