Glycobiology Advance Access originally published online on June 29, 2005
Glycobiology 2005 15(11):1102-1110; doi:10.1093/glycob/cwi100
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© Published by Oxford University Press 2005.
Use of a cell-free system to determine UDP-N-acetylglucosamine 2-epimerase and N-acetylmannosamine kinase activities in human hereditary inclusion body myopathy
2 Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD; 3 Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC; 4 Neuromuscular Diseases Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD; and 5 Office of Rare Diseases, Intramural Program, Office of the Director, National Institutes of Health, Bethesda, MD
1 To whom correspondence should be addressed; e-mail: mhuizing{at}mail.nih.gov
Received on May 2, 2005; revised on June 17, 2005; accepted on June 20, 2005
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Abstract |
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Hereditary inclusion body myopathy (HIBM) is an autosomal recessive neuromuscular disorder associated with mutations in uridine diphosphate (UDP)-N-acetylglucosamine (GlcNAc) 2-epimerase (GNE)/N-acetylmannosamine (ManNAc) kinase (MNK), the bifunctional and rate-limiting enzyme of sialic acid biosynthesis. We developed individual GNE and MNK enzymatic assays and determined reduced activities in cultured fibroblasts of patients, with HIBM harboring missense mutations in either or both the GNE and MNK enzymatic domains. To assess the effects of individual mutations on enzyme activity, normal and mutated GNE/MNK enzymatic domains were synthesized in a cell-free in vitro transcription–translation system and subjected to the GNE and MNK enzymatic assays. This cell-free system was validated for both GNE and MNK activities, and it revealed that mutations in one enzymatic domain (in GNE, G135V, V216A, and R246W; in MNK, A631V, M712T) affected not only that domain’s enzyme activity, but also the activity of the other domain. Moreover, studies of the residual enzyme activity associated with specific mutations revealed a discrepancy between the fibroblasts and the cell-free systems. Fibroblasts exhibited higher residual activities of both GNE and MNK than the cell-free system. These findings add complexity to the tightly regulated system of sialic acid biosynthesis. This cell-free approach can be applied to other glycosylation pathway enzymes that are difficult to evaluate in whole cells because their substrate specificities overlap with those of ancillary enzymes.
Key words: cell-free transcription–translation / GNE / HIBM / MNK enzymes / sialic acid
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
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Hereditary inclusion body myopathy (HIBM; OMIM 600737 [OMIM] ) is an autosomal recessive neuromuscular disorder of adult onset, characterized by slowly progressive muscle weakness and atrophy that involves all limbs, but partially spares the quadriceps muscles (Sadeh et al., 1993
; Griggs et al., 1995; Sivakumar and Dalakas, 1996). Histologically, the muscle fibers degenerate and form cytoplasmic rimmed vacuoles and cytoplasmic or nuclear filamentous inclusions (Argov et al., 1998; Askanas and Engel, 1998).
Linkage analysis in several families of Persian–Jewish and Kurdish–Iranian–Jewish origin aided identification of uridine diphosphate N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE), located on 9p13.2, as the gene responsible for HIBM (Eisenberg et al., 2001). Many GNE mutations have since been determined in HIBM patients worldwide (Vasconcelos et al., 2002; Del Bo et al., 2003; Eisenberg et al., 2003; Broccolini et al., 2004). In addition, a Japanese disorder resembling HIBM, distal myopathy with rimmed vacuoles (DMRV) or Nonaka myopathy (MIM 605820
[OMIM]
), has been shown to also result from GNE mutations (Arai et al., 2002; Kayashima et al., 2002; Tomimitsu et al., 2002).
GNE encodes the bifunctional enzyme GNE (EC 5.1.3.14
[EC]
)/ N-acetylmannosamine (ManNAc) kinase (MNK, EC 2.7.1.60
[EC]
), which catalyzes the first two committed, rate-limiting steps in the biosynthesis of N-acetylneuraminic acid (NeuAc), the most abundant mammalian sialic acid (Hinderlich et al., 1997; Lucka et al., 1999). The N-terminal epimerase domain (GNE) converts uridine diphosphate (UDP)-N-acetylglucosamine (GlcNAc) to ManNAc, whereas the C-terminal kinase domain (MNK) converts ManNAc to ManNAc-6-phosphate (Figure 1A). A downstream product, cytidine 5'-monophosphate N-acetylneuraminic acid (CMP-NeuAc), feedback-inhibits GNE activity by binding to its allosteric site (codons 263–266), which is mutated in the dominant disorder sialuria (MIM 269921
[OMIM]
). Loss of feedback inhibition results in overproduction of cytoplasmic sialic acid (Weiss et al., 1989; Seppala et al., 1999).
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All reported GNE mutations in patients with HIBM are outside the allosteric site and within the GNE and/or MNK domains. HIBM patients have variably reduced GNE and MNK enzymatic activities, and the extent of the reduction is mutation dependent (Effertz et al., 1999; Hinderlich et al., 2004; Noguchi et al., 2004; Salama et al., 2005). Loss of GNE or MNK activity impairs sialic acid production and can interfere with proper sialylation of cell-surface glycoconjugates (Keppler et al., 1999). Some GNE mutations also cause hyposialylation of skeletal muscle proteins (Huizing et al., 2004; Noguchi et al., 2004; Saito et al., 2004; Salama et al., 2005). However, it has not been definitively determined that hyposialylation is directly responsible for the pathology of HIBM.
In this paper, the underlying biochemical abnormalities in HIBM are further characterized. Individual GNE and MNK enzyme activities were measured in cultured fibroblasts of controls and patients with HIBM and in normal and mutated GNE/MNK recombinant enzymes synthesized in a cell-free in vitro transcription–translation system. We demonstrate that such a cell-free system offers an attractive and convenient approach to study the direct effects of mutations on enzymatic activity, without the influence of other intracellular components.
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Results |
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HIBM patient mutation analyses
Mutation analyses were performed on genomic DNA of fibroblast cultures from three HIBM patients (Figure 1B). Patient 1 was compound heterozygous for two missense mutations in the GNE domain, G135V (exon 3, GGT>GTT) and R246W (exon 4, CGG>TGG). Patient 2 was compound heterozygous for one missense mutation in the GNE domain, V216A (exon 4, GTT>GCT), and one missense mutation in the MNK domain, A631V (exon 11, GCG>GTG). Patient 3 was homozygous for the Iranian–Jewish missense founder mutation in the MNK domain, M712T (exon 12, ATG>ACG). Real-time quantitative polymerase chain reaction (PCR) and northern blots using fibroblast RNA were performed to examine the relative expression levels of the GNE gene. In all the three HIBM patients, fibroblast GNE RNA expression was similar to that of controls (data not shown).
GNE/MNK fibroblast enzyme assays
The GNE assay employed in this study, previously described for assessing GNE activity in sialuria fibroblasts, is based on detection of radiolabeled substrate and product (UDP-[3H]GlcNAc and [3H]ManNAc, respectively) by high pH anion-exchange chromatography with pulsed amperometric detection (Weiss et al., 1989; Seppala et al., 1991, 1999). In addition, we developed a novel MNK (kinase) assay on the basis of similar detection methods for the enzymatic substrate ([3H]ManNAc) and product ([3H] N-acetylmannosamine-6-phosphate [ManNAc-6P]).
For each of the three patients with HIBM, GNE and MNK enzyme activities were determined in extracts of their cultured fibroblasts. Compared with controls, all three patients exhibited decreased, but not absent, GNE and MNK activities (Table I). Patient 1, with two GNE domain mutations, had the lowest residual GNE activity (38%). Patient 2, who harbors only one GNE domain mutation, had 48% residual activity, and Patient 3, with two MNK domain mutations, had 83% residual activity. Residual MNK enzyme activity was the highest for Patient 1 (72%), followed by that of Patient 2 (63%) and that of Patient 3 (55%).
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Cell-free expression of GNE/MNK
The residual GNE and MNK activities in patients’ fibroblasts could either be intrinsic to the mutant protein or related to some other protein present in the cells. To distinguish between these possibilities, we expressed recombinant GNE/MNK proteins carrying the specific mutations of our patients in a cell-free in vitro transcription–translation system to permit isolated measurements of GNE and MNK enzyme activities.
First, the translation efficiency of GNE/MNK in the cell-free system was determined by allowing [35S]-labeled methionine to incorporate into 1 µg of GNE-pET17b (normal and mutated) plasmid translated under standard conditions. Similar amounts of [35S] (normalized for the number of methionine residues in each translated product) were incorporated into each of the normal and mutated recombinant GNE/MNK proteins. On the basis of radiolabeled [35S]-methionine incorporation, a typical 50 µL translation mix yielded 55 ± 18 pmol GNE/MNK protein (N = 21). This converts to ~79 pg GNE/MNK protein/µL translation mix.
In addition, [35S]-labeled aliquots (3 µL) were electrophoresed on a polyacrylamide gel that was subsequently dried and exposed to X-ray film (Figure 2). Similar band intensities for each translated product again indicated equivalent translation efficiencies of normal and mutated GNE/MNK in the cell-free system.
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We further validated the cell-free system for the GNE and MNK enzyme assays by demonstrating a direct linear relationship between the amount of product formed and the amount of cell-free translated GNE/MNK enzyme supplied for both GNE activity (Figure 3A) and MNK activity (Figure 3B). This validates the use of 50 µL of translated product (~4 ng GNE/MNK protein) as standard for each enzyme assay.
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As the MNK assay was a novel assay, MNK enzyme kinetics were performed in the cell-free system. A decrease in radiolabeled substrate ([3H]ManNAc) and a corresponding increase in product ([3H]ManNAc-6P) with time of incubation was demonstrated for MNK activity, and 20 min appeared to be an optimum incubation time to employ for this assay (Figure 4A).
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Michaelis–Menten kinetics were applied to determine the Km and Vmax for MNK enzymatic activity in the cell-free system. The initial velocity, Vi (pmol of ManNAc-6P formed per minute), was determined at substrate (ManNAc) concentrations ranging from 0 to 200 µM (Figure 4B). A Lineweaver-Burk plot (Figure 4B, inset) yielded a Vmax of 48 pmol ManNAc-6P/(55 pmol of enzyme.min) and an apparent Km of 85 µM. This Km value is similar to that of the rat MNK enzyme, that is, ~93 µM (Hinderlich et al., 1997). These results indicated that the cell-free assay is valid for in vitro GNE/MNK enzyme measurements.
Using GNE/MNK recombinant proteins synthesized in the cell-free system, we determined the individual GNE and MNK activities associated with the mutations of Patients 1–3. As parallel experiments employing [35S]-methionine incorporation yielded equivalent translation efficiencies for normal and mutated GNE/MNK (Figure 2), results could be expressed as percentage of activity provided by the wild-type translated GNE/MNK protein (Table II). All the three proteins with mutations in the GNE domain (G135V, V216A, and R246W) exhibited markedly reduced (<2%) epimerase activities (Table II). In fact, the G135V and R246W mutations exhibited negligible GNE activities. Similarly, low amounts of residual epimerase activity (<5% of normal) were present in proteins bearing MNK domain mutations (A631V and M712T). Residual MNK activities were 8–20% for all GNE/MNK protein mutations.
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When the GNE domain alone (amino acids 1–303) was expressed, MNK activity was lost, as expected, but epimerase activity was also virtually eliminated. When the MNK domain alone (amino acids 409–722) was expressed, the epimerase activity was lost, as expected, but ~10% of the kinase activity remained.
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Discussion |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
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Aberrant sialic acid or sialic acid metabolism results in severe clinical conditions in humans (Gahl et al., 1996
; Sillanaukee et al., 1999; Strehle, 2003), including sialuria (Seppala et al., 1999), sialidosis (neuraminidase deficiency; Thomas, 2001), Salla disease and ISSD (lysosomal free sialic acid storage disorders because of transport defects; Schleutker et al., 1995; Aula and Gahl, 2001), and HIBM (Eisenberg et al., 2001; Huizing et al., 2004).
HIBM is associated with mutations in the key enzyme of sialic acid biosynthesis, GNE/MNK, which harbors two enzymatic functions (epimerase and kinase). This type of bifunctional enzyme is unusual in nature and commonly involves successive steps in a biochemical pathway (Wei et al., 1993; Suchi et al., 1997; Venkatachalam, 2003). The GNE/MNK enzyme is even more novel in that it contains an allosteric site within its epimerase domain.
The exact effects of GNE/MNK mutations on sialic acid metabolism and the pathophysiology of HIBM remain elusive (Huizing et al., 2004; Noguchi et al., 2004; Saito et al., 2004; Salama et al., 2004). Skin fibroblasts proved to be an excellent system to study the effects of HIBM-related GNE/MNK mutations; muscle cells are limited in availability, and leukocytes express insufficient levels of GNE/MNK enzyme for accurate activity measurements (Nishino et al., 2002; Hinderlich et al., 2004; Noguchi et al., 2004). Using fibroblasts cultured from three patients with HIBM, we found that GNE and MNK catalytic activities were both decreased and that the extent of the decrements correlated with the number of mutations in the respective enzymatic domain (Table I).
This finding could have several explanations. Our patients’ GNE missense mutations, all involving evolutionarily conserved amino acids, might influence GNE mRNA stability or epigenetics (Oetke et al., 2003; Giordanengo et al., 2004). However, this possibility was not supported by real-time quantitative PCR and northern blotting experiments demonstrating GNE expression levels in HIBM cells similar to those of control fibroblast (data not shown). Alternatively, our patients’ specific missense mutations could have caused aberrant post-translational modifications; although the GNE/MNK protein is not predicted to undergo glycosylation, it has several potential phosphorylation sites (Horstkorte et al., 2000). Against this explanation was the finding that our patients’ mutations were distant from the predicted phosphorylation consensus sites and did not change the predicted phosphorylation pattern (NetPhos 2.0 Server [http://www.cbs.dtu.dk/services/NetPhos/]). Finally, our patients’ missense mutations could involve the active sites of the GNE and/or MNK enzymes. This remains a possibility, because the exact locations of the active sites remain to be determined (Effertz et al., 1999; Blume et al., 2004).
Another important finding emanating from the fibroblast studies involves the large amount of GNE and MNK activity remaining in these cells, despite the presence of two GNE/MNK missense mutations. It may be that a complete absence of GNE and/or MNK activity is lethal. Null mutations on both GNE alleles have never been identified in patients, and a mouse knockout model lacking a functional GNE gene did not develop beyond the embryonic stage (Schwarzkopf et al., 2002). In contrast, missense GNE mutations may permit residual GNE/MNK enzymatic activities and residual production of sialic acid, leading to a progressive shortage of sialic acid manifesting later in life. This shortage could lead to the hyposialylation of glycoproteins that is detected in some patients (Huizing et al., 2004; Noguchi et al., 2004; Saito et al., 2004; Salama et al., 2004) and might explain the late onset of myopathy in HIBM.
There remained a question of the source of the residual GNE and MNK activity in patients’ fibroblasts. Did it derive from the mutant GNE/MNK protein itself, or were there contributions from other epimerases (e.g., GlcNAc 2-epimerase) or kinases (e.g., GlcNAc kinase) with overlapping substrate specificities, as previously proposed (Hinderlich et al., 1998; Takahashi et al., 1999; Samuel and Tanner, 2002; Luchansky et al., 2003)?
The answer could be determined by measuring the enzymatic activities of mutant GNE/MNK in a system outside of human cells. In previous studies, specific human GNE mutations were expressed in Sf6 insect cells using a baculovirus expression system (Effertz et al., 1999; Hinderlich et al., 2004) in COS-7 cells (Noguchi et al., 2004) and in bacterial or yeast strains (Blume et al., 2004). We chose a cell-free in vitro transcription–translation system, in part because it did not require the laborious steps of transfection, cell culture, and cell extraction. More importantly, a cell-free environment eliminates the effects of redundant cellular components.
The cell-free synthesized GNE/MNK recombinant proteins yielded GNE and MNK enzymatic activities that varied linearly with the amount of protein present (Figure 3) and, for MNK, displayed an apparent Km for ManNAc of 85 µM, similar to that reported for rat (93 µM) (Hinderlich et al., 1997). Using the cell-free system, each of the GNE domain mutations (G135V, V216A, and R246W) yielded strikingly low (<2% of normal) GNE activities (Table II), far less than the <38–48% of normal activity present in mutant fibroblasts (Table I). Similarly, in the cell-free translation system, the MNK mutant (M712T) exhibited significantly lower MNK activity (~8% of normal) than that present in fibroblasts homozygous for this mutation (55% of normal).
One possibility to explain the higher mutant GNE and MNK activities in fibroblasts compared with the cell-free system is that the mutant GNE/MNK proteins made in a living cell fold better or bind to a chaperone which allows them to have more activity. Another possibility is the presence of additional sugar epimerases and kinases in fibroblasts. For example, GlcNAc 2-epimerase (EC 5.1.3.8
[EC]
) catalyzes the reversible epimerization of GlcNAc and ManNAc (Maru et al., 1996; Takahashi et al., 1999; Samuel and Tanner, 2002; Luchansky et al., 2003), a reaction that thermodynamically favors the formation of GlcNAc with an equilibrium ratio of 3.9:1 (Samuel and Tanner, 2002). In our GNE (epimerase) assay system, the UDP-[3H]GlcNAc used as substrate partially decayed into [3H]GlcNAc (Seppala et al., 1991, 1999), which could produce [3H]ManNAc by virtue of the GlcNAc 2-epimerase present in fibroblasts. [3H]GlcNAc levels in our fibroblast assays were probably increased enough to push the GlcNAc 2-epimerase reaction toward ManNAc production.
Fibroblasts also contain kinases that may account for the excess ManNAc-6P production by cells compared with the cell-free translation system. For example, GlcNAc kinase (EC 2.7.1.59
[EC]
) has high intrinsic MNK activity (Hinderlich et al., 1998). We consider that GlcNAc kinase present in fibroblast extracts could convert [3H]ManNAc into [3H]ManNAc-6P in our MNK enzyme assays, increasing the apparent MNK enzyme activity. This was nicely illustrated by the M712T mutation, for which Patient 3 was homozygous (55% MNK activity in fibroblasts versus 8% activity in the cell-free system).
The two MNK domain mutants (A631V and M712T) had severely reduced (<5% of normal) GNE activities (Table II). Conversely, the GNE domain mutants (G135V, V216A, and R246W) showed substantially reduced MNK activity in the cell-free system (8–20% of normal). These findings were supported by the fibroblast studies, in which MNK domain mutations reduced GNE activity (as in Patient 3) and GNE domain mutations reduced MNK activity (as in Patient 1). This may have occurred because the missense mutations caused misfolding of the enzyme, changing its secondary structure.
It is known that the GNE/MNK protein forms a homohexamer by oligomerization and that GNE mutations can impair the oligomerization process and reduce both GNE and MNK enzyme activities (Hinderlich et al., 1997; Effertz et al., 1999). As a monomer, GNE/MNK has no enzymatic activity; its dimer exhibits only MNK activity, and the hexameric state displays both GNE and MNK activities (Hinderlich et al., 1997; Effertz et al., 1999). The hexamerization process in our cell-free system ought to have occurred in a fashion similar to that in fibroblast cells. The presence of GNE activity in our cell-free synthesized normal and mutated GNE/MNK requires that hexamers were formed. Moreover, GNE/MNK enzyme kinetics in the cell-free system (Figure 4) were similar to kinetics previously shown for cellular systems (Hinderlich et al., 1997). Finally, the secondary structure formation of GNE/MNK has been determined in several other systems such as human lymphoblast cell lines (Hinderlich et al., 2004) and COS-7 cells (Noguchi et al., 2004) or Sf9 cells (Effertz et al., 1999; Hinderlich et al., 2004) expressing recombinant GNE/MNK. These findings suggest that the oligomerization phenomenon cannot explain the lower percentages of residual enzyme activities in the cell-free system compared with the fibroblast system.
Aberrant oligomerization could explain why the expression of the GNE domain alone (amino acids 1–303) yielded neither MNK nor GNE enzymatic activity (Table II). Specifically, the presence of the MNK domain is necessary for GNE activity, because the MNK domain is required for formation of a functional hexamer (Hinderlich et al., 1997; Effertz et al., 1999). When the MNK domain alone (amino acids 409–722) was expressed, GNE activity was lost, but 
10% of MNK activity remained. This suggested that the MNK domains alone might form a functional unit and that MNK activity is not as sensitive as GNE activity to conformational changes. This is supported by the fact that the GNE/MNK dimer exhibits MNK activity, but lacks GNE activity (Hinderlich et al., 1997; Effertz et al., 1999).
Mutations in either the GNE or the MNK domain clearly affect GNE catalytic activity more than MNK activity. The fact that MNK domain mutations significantly reduce GNE catalytic activity can explain the clinical similarity between patients with GNE domain mutations and patients with MNK domain mutations.
Although results in fibroblasts and in a cell-free system may be recapitulated in muscles, the muscle cell’s contingent of ancillary epimerases and kinases may differ from that of the fibroblast. Such a scenario could explain the apparently contradictory findings regarding muscle hyposialylation in HIBM patients with different GNE mutations. Saito et al. (2004) and Salama et al. (2005) showed hyposialylation in muscle cells of patients homozygous for two GNE domain mutations, and Huizing et al. (2004) showed hyposialylation of 
-dystroglycan in muscles of patients with one GNE domain and one MNK domain missense mutation. However, Hinderlich et al. (2004) reported normal sialylation in muscle cells of patients homozygous for a mutation in the MNK domain (M712T, similar to our Patient 3), perhaps because these patients are able to produce sufficient sialic acid in muscles because of slightly higher residual GNE activities.
This study demonstrates the usefulness of a cell-free in vitro transcription–translation system to assess enzyme activities related to patients’ mutations. The system is powerful, convenient, and relatively simple to employ. It is particularly appropriate for enzymes that do not rely on post-translational modifications for their function. For example, enzymes involved in glycosylation pathways (sugar transferases, epimerases, and kinases) generally do not rely on post-translational modifications and contain, in many cases, overlapping in vivo substrate specificities with other ancillary enzymes. Therefore, this group of enzymes would be outstanding candidates for the study of individual activities and specificities in a cell-free system.
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Materials and methods |
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Patients and cells
All patients were enrolled in a clinical protocol approved by the institutional review board of either the National Human Genome Research Institute (NHGRI) or the National Institute of Neurological Disorders and Stroke (NINDS) to investigate inborn errors of metabolism such as HIBM. Written informed consent was obtained from each patient. The diagnosis of HIBM was based on clinical features, muscle pathology, and the presence of GNE gene mutations. Primary cultures of fibroblasts were obtained from a 4-mm skin biopsy and grown in Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum containing 100 U/mL penicillin and 0.1 mg/mL streptomycin.
Patient 1 was a 27-year-old American of English/Irish/Scottish descent who had progressive weakness beginning at 20 years of age, recently requiring crutches to ambulate. Patient 2 was a 37-year-old American patient of non-Jewish descent. She had onset of leg weakness in her early twenties and became wheelchair-bound in her mid thirties. She had an older brother affected with HIBM (Vasconcelos et al., 2002). Patient 3 was a 38-year-old male of Iranian–Jewish descent who first manifested upper extremity weakness at age 35 years. His younger sister developed weakness in her early twenties after the birth of her first child.
PCR and sequencing
For GNE mutation analyses, patients’ genomic DNA was PCR-amplified exon by exon (primer sequences available on request) and directly sequenced. Standard PCR amplification procedures were employed (Sambrook et al., 1989). Automated sequencing was performed on a Beckman CEQ 2000 using the CEQ Dye Terminator Cycle Sequencing kit according to the manufacturer’s protocols (Beckman Coulter, Fullerton, CA).
Cell-free in vitro transcription–translation
The human GNE coding sequence (GenBank accession number NM_005476
[GenBank]
) was amplified from cDNA of normal human fibroblasts and was transferred using EcoRI and XhoI restriction enzymes, into the pET17b vector, which contained a N-terminal T7-tag and a T7 promotor sequence (EMD Biosciences, San Diego, CA). This GNE-pET17b plasmid was used to create each of the patient-specific GNE mutations by site-directed mutagenesis using the QuickChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) according to the manufacturer’s instructions. pET17b containing only the GNE domain was created by introducing a termination at codon 303 (TGT>TGA) in the normal GNE-pET17b (Figure 1B). pET17b translating only the MNK domain was created by PCR amplification and subsequent subcloning (using EcoRI- and XhoI-restriction sites) of GNE codons 409 through the termination codon 722 (Figure 1B). All constructs were verified by sequencing before experimental use.
The GNE-pET17b constructs were translated into recombinant proteins using the TnT-T7 Quick Coupled Transcription–Translation System, essentially as described by the supplier (Promega, Madison, WI). Briefly, GNE-pET17b plasmid DNA (1 µg/50 µL TnT master-mix) and methionine (non-radiolabeled for GNE/MNK enzyme measurements, [35S]-labeled for parallel experiments to confirm translation efficiency) were added to manufacturer-provided TnT master-mix (containing RNA polymerase, nucleotides, amino acids, and reticulocyte lysate). Reactions were incubated at 30°C for 90 min, after which 50 µL aliquots were taken for direct GNE and MNK enzyme activity measurements.
Aliquots (2 µL) of each [35S]-methionine-labeled translation mix were subjected to trichloroacetic acid (TCA) precipitation. Two microliter translation mix was added to 98 µL of 1 M NaOH/2% H2O2 and incubated for 10 min at 37°C. Protein precipitation was performed by adding 900 µL of ice-cold 25% TCA/2% casamino acids (BD Biosciences, Palo Alto, CA) and incubating on ice for 30 min. Precipitated proteins were collected by vacuum filtering 250 µL of translation mix onto Whatman GF/A glass fiber filters (Whatman, Florham Park, NJ). Filters were washed and dried, and [35S] radioactivity was counted in a MicroBeta Scintillation Counter (Perkin Elmer, Boston, MA). Amounts of [35S] were converted into pmol of protein produced based on the specific [35S] radioactivity (1000 Ci/mmol) and the number of methionine residues present in each of the cell-free synthesized proteins.
Additional aliquots (3 µL) of each [35S]-methionine-labeled translation mix were electrophoresed on denaturing 4–12% Tris–Glycine polyacrylamide gels (Invitrogen, Carlsbad, CA). After electrophoresis, the gels were fixed for 30 min in solution A (50% methanol, 10% acetic acid, 40% water), soaked for 5 min in solution B (7% acetic acid, 7% methanol, 10% glycerol), and dried at 80°C for 30–90 min under vacuum in a geldryer (Labconco, Kansas City, MO). The dried gels were exposed 6–24 h to BIOMAX MR X-Ray film (Kodak Imaging Systems, Rochester, NY).
GNE enzyme assay
The conversion of UDP-[3H]GlcNAc to [3H]ManNAc was assayed as described (Weiss et al., 1989). Briefly, in a final reaction volume of 117.5 µL, 5 µL of UDP-[3H]GlcNAc (1 mCi/mL, 60 Ci/mmol) (American Radiolabeled Chemicals, Saint Louis, MO) was added to a mixture containing 64 µM UDP-GlcNAc, 10 mM MgCl2, and 75 µL of protein/enzyme mixture. The protein/enzyme mixture consisted of fibroblasts lysed in buffer A (pH 7.2, containing 0.2 M MES, 0.15 M NaCl, 10mM CaCl2, 1% Triton X, 4 µg/mL aprotinin, and 0.1 mM UDP). The total protein concentration in the fibroblast lysates was determined using the BCA Protein Assay Reagent Kit (Pierce, Rockford, IL). In the case of in vitro translated GNE/MNK proteins, 50 µL of the translated protein mix (see Cell-free in vitro transcription-translation) was added to 25 µL of buffer A. The GNE (epimerase) reactions were performed by incubation for 6 min at 37°C and stopped by boiling the sample for 1 min. After incubation on ice, the samples were centrifuged at 13,000 x g for 15 min at 4°C. The supernatants were removed and frozen at –20°C until analyzed by high pH anion exchange chromatography on a BioLC carbohydrate analyzer as described below.
MNK enzyme assay
To analyze the conversion of [3H]ManNAc to [3H]ManNAc-6P by MNK, 5 µL of [3H]ManNAc (1 mCi/mL, 15–30Ci/mmol in 90% ethanol) (American Radiolabeled Chemicals) was pipetted to the very bottom of a test tube and allowed to evaporate. In a total reaction volume of 117.5 µL, 75 µL of the protein/enzyme mixture (see GNE enzyme assay) was added to 64 µM ManNAc, 10 mM MgCl2, and 1.1 mM Mg-ATP (Boston Biochem, Cambridge, MA). The samples were briefly mixed by swirling and incubated at 37°C for 20 min. Samples were then boiled for 1 min to stop the reaction, placed on ice, and centrifuged for 15 min at 13,000 x g at 4°C. The supernatant was removed and frozen at –20°C until analyzed by high pH anion-exchange chromatography on a BioLC carbohydrate analyzer as described below.
Chromatography separation and radiolabel analysis
The BioLC carbohydrate analyzer (Dionex, Sunnyvale, CA) employs high pH anion-exchange chromatography with pulsed amperometric detection to separate oligosaccharides based on charge as well as size and composition. Thirty microlitrer of each supernatant was applied to a CarboPac PA-100 column, after which UDP-GlcNAc, GlcNAc, ManNAc, and ManNAc-6 phosphate were separated by eluting an isocratic solution of 10 mM NaOH, followed by an increasing (0–900 mM) NaOAc gradient. Twenty microliter of either GlcNAc/ManNAc (for the GNE assay) or ManNAc/ManNAc-6P standards (for the MNK assay) was co-injected to identify migration times of the monosaccharides. ManNAc-6P standard was kindly provided by Dr S. Hinderlich (Berlin, Germany). Fractions were collected at 30 s (for GNE assay) or 1 min (for MNK assay) intervals. Eluted fractions were measured for [3H] radioactivity using a MicroBeta Scintillation Counter (Perkin Elmer) and were converted into pmol based on the specific radioactivity of the substrate.
Enzyme kinetics
All kinetic experiments were performed using GNE-pET17b in the cell-free system under the standard conditions described above. To correct for ancillary enzymatic activity within the rabbit reticulocyte lysate present in the transcription/translation mix, GNE and MNK enzyme assays were performed on translated "empty" pET17b plasmids (without GNE inserted). Negligible GNE and MNK activities were detected in this lysate (data not shown). To validate that the amounts of radiolabeled product formed ([3H]ManNAc for GNE activity and [3H]ManNAc-6P for MNK activity) were linear with the amounts of translated GNE/MNK enzyme added, 0–50 µL of translated enzyme was employed (Figure 3A and B). For MNK activity, the decrease in substrate ([3H]ManNAc) and the increase in product ([3H]ManNAc-6P) were recorded after 0–30 min of incubation (Figure 4A).
For Michaelis–Menten analysis of MNK activity, non-radioactive ManNAc substrate was added to [3H]ManNAc to a specific concentration of 78 nCi/µmol ManNAc. Final concentrations of 10–500 µM were mixed with the translated protein for the MNK enzyme assay. The reactions were carried out for 10 min to obtain initial velocities. Radioactivity (desintegrations per minute [dpm]) was converted to pmol of ManNAc-6P product. The Km and Vmax were determined using a Lineweaver–Burk plot.
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Acknowledgments |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
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We thank Carlos Sanchez and Emily Gottlieb for excellent technical assistance.
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Abbreviations |
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CMP-NeuAc, cytidine 5'-monophosphate N-acetylneuraminic acid; DMRV, distal myopathy with rimmed vacuoles; dpm, desintegrations per minute; GlcNAc, N-acetylglucosamine; GNE, uridine diphosphate N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase; HIBM, hereditary inclusion body myopathy; ManNAc, N-acetylmannosamine; ManNAc-6P, N-acetylmannosamine-6-phosphate; MNK, N-acetylmannosamine kinase; TCA, trichloroacetic acid; UDP, uridine diphosphate
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