Biochem. J. (2008) 415, 123–134 (Printed in Great Britain)
123
doi:10.1042/BJ20080722
Transaldolase deficiency influences the pentose phosphate pathway,
mitochondrial homoeostasis and apoptosis signal processing
Yueming QIAN*, Sanjay BANERJEE*, Craig E. GROSSMAN*†, Wendy AMIDON*, Gyorgy NAGY*, Maureen BARCZA‡,
Brian NILAND*†, David R. KARP§, Frank A. MIDDLETON‡, Katalin BANKI and Andras PERL*†1
*Department of Medicine, SUNY Upstate Medical University, Syracuse, NY 13210, U.S.A., †Department of Microbiology and Immunology, SUNY Upstate Medical University, Syracuse,
NY 13210, U.S.A., ‡Department of Physiology, SUNY Upstate Medical University, Syracuse, NY 13210, U.S.A., §University of Texas Southwestern Medical Center, Dallas, TX 75390,
U.S.A., and Department of Pathology, SUNY Upstate Medical University, Syracuse, NY 13210, U.S.A.
TAL (transaldolase) was originally described in the yeast as an
enzyme of the PPP (pentose phosphate pathway). However, certain organisms and mammalian tissues lack TAL, and the overall
reason for its existence is unclear. Recently, deletion of Ser171
(TALS171) was found in five patients causing inactivation,
proteasome-mediated degradation and complete deficiency of
TAL. In the present study, microarray and follow-up Westernblot, enzyme-activity and metabolic studies of TALS171 TD
(TAL-deficient) lymphoblasts revealed co-ordinated changes in
the expression of genes involved in the PPP, mitochondrial
biogenesis, oxidative stress, and Ca2+ fluxing. Sedoheptulose 7phosphate was accumulated, whereas G6P (glucose 6-phosphate)
was depleted, indicating a failure to recycle G6P for the oxidative
branch of the PPP. Nucleotide analysis showed depletion of
NADPH and NAD+ and accumulation of ADP-ribose. TD cells
have diminished ψ m (mitochondrial transmembrane potential)
and increased mitochondrial mass associated with increased pro-
duction of nitric oxide and ATP. TAL deficiency resulted in
enhanced spontaneous and H2 O2 -induced apoptosis. TD lymphoblasts showed increased expression of CD38, which hydrolyses
NAD+ into ADP-ribose, a trigger of Ca2+ release from the
endoplasmic reticulum that, in turn, facilitated CD20-induced
apoptosis. By contrast, TD cells were resistant to CD95/Fasinduced apoptosis, owing to a dependence of caspase activity on
redox-sensitive cysteine residues. Normalization of TAL activity
by adeno-associated-virus-mediated gene transfer reversed the elevated CD38 expression, ATP and Ca2+ levels, suppressed H2 O2 and CD20-induced apoptosis and enhanced Fas-induced cell
death. The present study identified the TAL deficiency as a modulator of mitochondrial homoeostasis, Ca2+ fluxing and apoptosis.
INTRODUCTION
Regeneration of GSH from its oxidized form, GSSG, is dependent
on NADPH produced by the PPP [1].
Understanding regulation of the PPP has been complicated
by the fact that the pathway is comprised of two separate,
oxidative and non-oxidative, branches [1]. Reactions in the oxidative branch are irreversible, whereas all reactions of the nonoxidative phase are fully reversible. TAL (transaldolase) was,
on the basis of metabolites and enzymatic activities detected in
yeast [3], originally described as an enzyme of the non-oxidative
branch of the PPP. However, certain organisms [4,5] and mammalian tissues do not express TAL [6] and the non-oxidative
branch can function without this enzyme [7,8]. Hence the overall
reason for the existence of TAL has not been clearly established.
Mutations in TAL have been associated with liver cirrhosis in
Metabolism of glucose through the PPP (pentose phosphate
pathway) fulfils two unique functions: formation of R5P (ribose
5-phosphate) for the synthesis of nucleotides, RNA and DNA, and
generation of NADPH as a reducing equivalent for biosynthetic
reactions and maintenance of a reducing environment [1]. ROI
(reactive oxygen intermediates) have long been considered as
toxic by-products of aerobic existence, but evidence is now
accumulating that controlled levels of ROI modulate cellular
function and are necessary for signal-transduction pathways,
including those mediating PCD (programmed cell death) [2].
A normal reducing atmosphere, required for cellular integrity
is provided by GSH, which protects cells from damage by ROI.
Key words: apoptosis, Ca2+ , mitochondrion, pentose phosphate
pathway, transaldolase.
Abbreviations used: AAV, adeno-associated virus; Ab, antibody; ALDC, aldolase C; annexin V-FITC, fluorescein-conjugated annexin V; annexin V-PE,
phycoerythrin-conjugated annexin V; 2-APB, 2-aminoethoxydiphenyl borane; BAPTA-AM, 1,2-bis-(o -aminophenoxy)ethane-N ,N ,N ,N -tetra-acetic acid
tetrakis(acetoxymethyl ester); BNIP3L, bcl-2-like interacting protein 3-like mitochondrial protein; [Ca2+ ]c , cytoplasmic Ca2+ concentration; [Ca2+ ]m , mitochondrial Ca2+ concentration; C-PTIO, carboxy-2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide; DAF-FM, 4-amino-5-methylamino-2 ,7 -difluorofluorescein diacetate; DCF, 5,6-carboxy-2 ,7 -dichlorofluorescein; DCFH-DA, 5,6-carboxy-2 ,7 -dichlorofluorescein diacetate; DHR, dihydrorhodamine 123;
DiOC6 , 3,3 -dihexyloxacarbocyanine iodide; EBV, Epstein–Barr virus; E4P, D-erythrose 4-phosphate; FBS, fetal bovine serum; F6P, fructose 6-phosphate; FB,
fibroblast; GGT, γ-glutamyltransferase; G6P, glucose 6-phosphate; G6PD, G6P dehydrogenase; GA3P, D-glyceraldehyde 3-phosphate; GLH, gluconolactone hydrolase; GPX, glutathione peroxidase; HE, hydroethidine; HMOX1, haem oxygenase; (hr)GFP, (humanized Renilla ) green fluorescent protein; IP3R,
Ins(1,4,5)P 3 receptor; IRES, internal ribosomal entry site; JC-1, 5,5 ,6,6 -tetrachloro-1,1 ,3,3 -tetraethylbenzimidazolocarbocyanine iodide; LB, lymphoblast;
MCB, monochlorobimane; mClCCP, carbonyl cyanide m-chlorophenylhydrazone; MTG, MitoTracker Green-FM; NAO, nonyl Acridine Orange; NPT,
neomycin phosphotransferase; NRF-1, nuclear respiratory factor 1; NUDT1, nucleotide diphosphatase type motif 1/8-oxo-7,8-dihydroguanosine triphosphatase; PCD, programmed cell death; PFKM, phosphofructokinase; 6PG, 6-phosphogluconate; PGC-1, peroxisome proliferator-activated receptor γ-coactivator 1.; 6PGD, 6-phosphogluconate dehydrogenase; PGK1, phosphoglycerate kinase 1; PGI, phosphoglucose isomerase; PPP, pentose phosphate
pathway; PTK2B, protein tyrosine kinase 2., Ca2+ -regulated; R5P, ribose 5-phosphate; ROI, reactive oxygen intermediates; SOD, superoxide dismutase;
S7P, sedoheptulose 7-phosphate; TAL, transaldolase; TD, TAL-deficient; TK, transketolase; TMRM, tetramethylrhodamine methyl ester; ψm , mitochondrial
transmembrane potential.
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2008 Biochemical Society
124
Y. Qian and others
children from three families [9–11]. Deletion of Ser171 was noted
in five out of six cases [9,11], and this was found to lead
to inactivation, proteasome-mediated degradation and complete
deficiency of TAL [12]. The pathogenesis of liver cirrhosis has
been associated with increased cell death of hepatocytes and
biliary epithelial cells [13]. To understand the consequences
of TAL deficiency on cell-death-signal processing, B-cell LBs
(lymphoblasts) available from the first TD (TAL-deficient) patient
were investigated. Relative to control LBs, TD B-cells exhibit
increased spontaneous, H2 O2 -, and CD20-induced apoptosis
and reduced CD95/Fas-dependent apoptosis. Altered cell-deathsignal processing is mediated by a unique pattern of metabolic
changes affecting (1) the PPP (characterized by elevated S7P
(sedoheptulose 7-phosphate) and diminished G6P (glucose 6phosphate) levels, which reflect blocked recycling of G6P
for the oxidative branch, (2) nucleotide metabolism (depletion
of NADPH and elevation of ADP-ribose), (3) Ca2+ fluxing
(increased cytoplasmic and mitochondrial Ca2+ levels and elevated
CD38 expresssion) and (4) decreased ψ m (mitochondrial
transmembrane potential) and increased NO (nitric oxide)
production, increased mitochondrial mass, increased ROI and
ATP production. Normalization of TAL activity reversed the
elevated CD38 expression and Ca2+ levels, the increased H2 O2 and CD20-induced apoptosis and the diminished Fas-induced
apoptosis. The results indicate that TAL regulates the PPP via
G6P recycling and thus controls NADPH production, nucleotide
metabolism, Ca2+ fluxing, the ψ m and pathway-specifically
influences apoptosis signal processing of human B-cells.
EXPERIMENTAL
Cell culture
Human EBV (Epstein–Barr virus)-transformed human B-cell LBs
were cultured in RPMI 1640 medium supplemented with 10 %
(v/v) FBS (fetal bovine serum), 2 mM L-glutamine, 100 units/ml
penicillin, 100 μg/ml streptomycin and 0.25 μg/ml amphotericin
B. EBV-transformed LBs from TD patients and human control
donors [GM13416 (Coriel Cell Repositories, Camden, NJ,
U.S.A.) and JAB (a B-cell line we established in our laboratory
from a human donor)] were established as described previously
[12]. Human FBs (fibroblasts) were maintained in Hams F10 medium supplemented with 20 % FBS, 2 mM L-glutamine,
100 units/ml penicillin, 100 μg/ml streptomycin and 0.25 μg/ml
amphotericin B. FBs were isolated from a skin biopsy obtained
from the lateral aspect of the left thigh from a TAL-deficient
patient [9] and age-matched female controls (#1 and #2). All
cell lines were maintained in a humidified atmosphere with 5 %
CO2 at 37 ◦C. Cell-culture products were purchased from Cellgro
(Mediatech, Herndon, VA, U.S.A.).
Induction of apoptosis and cell viability assays
Apoptosis was induced with H2 O2 (100 μM; Sigma, St Louis,
MO, U.S.A.) and Fas Ab (antibody) CH-11 (1 μg/ml; Upstate
Biotechnology, Saranac Lake, NY, U.S.A.) as previously
described [14,15]. CD20-mediated apoptosis was induced by
cross-linking with goat anti-human IgG. Tissue-culture wells
were pretreated with 100 μg/ml goat anti-human IgG (Jackson
ImmunoResearch Laboratories, West Grove, PA, U.S.A.) at 37 ◦C
for 2 h, washed twice with PBS, then treated with CD20 Ab (10,
100 or 1000 μg/ml rituximab; Genentech, South San Francisco,
CA, U.S.A.), and washed again twice with PBS before the addition
of cells at 106 /ml. Apoptosis was quantified by staining with
annexin V-Alexa 647 (Molecular Probes; excitation at 650 nm;
c The Authors Journal compilation c 2008 Biochemical Society
emission at 665 nm recorded in FL-5) and exclusion of propidium
iodide (excitation at 535 nm; emission at 617 nm recorded in
FL-2).
Flow-cytometric analysis of ψ m , mitochondrial mass, ROI and
NO production and cytoplasmic and mitochondrial Ca2+ level
ψ m was estimated by staining with 10 nM DiOC6 (3,3 -dihexyloxacarbocyanine iodide; Molecular Probes) [15] for 15 min at
37 ◦C in the dark before flow cytometry (excitation at 488 nm;
emission at 525 nm recorded in FL-1). ψ m was also quantified
using a potential-dependent J-aggregate-forming lipophilic
cation, JC-1 (5,5 ,6,6 -tetrachloro-1,1 ,3,3 -tetraethylbenzimidazolocarbocyanine iodide). JC-1 selectively incorporates into
mitochondria, where it forms monomers (fluoresces green
at 527 nm) or aggregates, at high transmembrane potentials
(fluoresces red at 590 nm). Co-treatment with a protonophore,
5 μM mClCCP (carbonyl cyanide m-chlorophenylhydrazone;
Sigma) for 15 min at 37 ◦C resulted in decreased DHR
(dihydrorhodamine 123), DiOC6 , and JC-1 fluorescence and
served as a positive control for disruption of ψ m [15]. ψ m was
also assessed by staining with 1 μM TMRM (tetramethylrhodamine methyl ester; excitation at 543 nm; emission at 567 nm
recorded in FL-2; all from Molecular Probes). Mitochondrial mass
was assessed by staining with potential-insensitive mitochondrial dyes 50 nM NAO (nonyl Acridine Orange; excitation at
490 nm; emission at 540 nm recorded in FL-1; Molecular Probes)
or with 100 nM MTG (MitoTracker Green-FM; excitation at
490 nm; emission at 516 nm recorded in FL-1; Molecular Probes).
Production of ROI was assessed using oxidation-sensitive fluorescent probes DCFH-DA (5,6-carboxy-2 ,7 -dichlorofluoresceindiacetate), DHR and HE (hydroethidine; Molecular Probes)
as previously described [14]. Although rhodamine 123, the
fluorescent product of DHR oxidation, binds selectively to the
mitochondrial inner membrane, ethidium and DCF (5,6-carboxy2 ,7 dichlorofluorescein) remain in the cytosol of living cells.
DCF and HE (hydroethidine) preferentially detect H2 O2 and
superoxide (O2 − ) respectively [16]. Intracellular glutathione
levels were assessed with 100 μM MCB (monochlorobimane;
excitation at 380 nm; emission at 461 nm; FL-UV). Production
of nitric oxide (NO) was assessed by using DAFFM (4-amino5-methylamino-2 ,7 -difluoroflourescein diacetate; Molecular
Probes). Measurement of NO was calibrated by incubating
cells with the NO donors NOC-18 {(Z)-1-[2-(2-aminoethyl)N-(2-ammonioethyl)amino]diazen-1-ium 1,2-diolate; 200 μM–
1.8 mM} and sodium nitroprusside (400 μM–10 mM). CPTIO
(carboxy-2-phenyl-4,4,5,5-tetramethyl-imidazoline-1oxyl-3-oxide; 500 μM, 24 h), a specific NO chelator [17], was
used to reduce NO levels and inhibit NO signalling. The [Ca2+ ]c
(cytoplasmic Ca2+ concentration) was measured by loading
the cells with 1 μM Fluo-3 acetoxymethyl ester (excitation
at 506 nm, emission at 526 nm recorded in FL-1; Molecular
Probes). The [Ca2+ ]m (mitochondrial Ca2+ concentration) was
estimated by loading the cells with 4 μM Rhod-2 acetoxymethyl
ester, which is compartmentalized into the mitochondria [18].
2-APB (2-aminoethoxydiphenyl borane), a membrane-permeant
antagonist of the IP3R [Ins(1,4,5)P3 receptor] and BAPTA-AM
acid
[1,2-bis-(o-aminophenoxy)ethane-N,N,N ,N -tetra-acetic
tetrakis(acetoxymethyl ester); 5 μM] were used to inhibit Ca2+
signalling [17].
Western-blot analysis
Cytosolic lysates of LBs and FBs were prepared by harvesting
cells in a mixture comprising 40 mM triethanolamine, pH 7.6,
10 mM EDTA, 1 mM sodium orthovanadate, 0.1 mM sodium
Influence of transaldolase deficiency
125
column packed with 3-μm-particle-size ODS-2 (Millipore Waters
Chromatography, Milford, MA, U.S.A.) attached to a
Waters Alliance HPLC chromatograph equipped with a Model
996 photodiode array detector set at 254 nm. The mobile
phase was made with buffers A and B. Buffer A consisted of
0.1 M KH2 PO4 , pH 5.5, and 8 mM tetrabutylammonium hydrogen
sulfate. Buffer B consisted of 70 % (v/v) buffer A and 30 % (v/v)
methanol. Gradient elution was performed as follows: from 10 %
B to 75 % B in 11 min; to 100 % B at 12 min; back to 10 % B at
21 min [25].
molybdate, 10 mM sodium pyrophosphate and 50 mM NaF. Protein concentrations were determined by the Bradford method using the Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules,
CA, U.S.A.). A 10 μg portion of protein lysates, unless otherwise
indicated, were separated on an SDS/12%-(w/v)-polyacrylamide
gel and electroblotted on to nitrocellulose. For whole-cell lysates,
2 × 105 cells were resuspended in 10 μl of sample buffer
and boiled at 95 ◦C for 5 min prior to loading. Nitrocellulose
strips were immunoblotted with anti-TAL Ab 170 and β. -actinspecific mouse Ab 1501R (Chemicon, Temecula, CA, U.S.A.)
that had been treated overnight with blocking reagents as
previously described [19]. G6PD (G6P dehydrogenase) was
detected with a rabbit Ab specific to G6PD (catalogue no.
A300-404A; Bethyl Laboratories, Montgomery, TX, U.S.A.).
Horseradish-peroxidase-conjugated goat anti-rabbit IgG (Jackson
ImmunoResearch Laboratories) was utilized as a secondary Ab
for TAL, whereas biotin-conjugated goat anti-mouse IgG and
streptavidin (Jackson ImmunoResearch Laboratories) were used
as secondary and tertiary reagents respectively for the detection
of β-actin. Protein bands were visualized by enhanced chemiluminescence using Western Lightning Chemiluminescence
Reagent Plus (PerkinElmer, Boston, MA, U.S.A.) using a Kodak
Image Station 440CF and quantified with Kodak 1D Image
Analysis Software (Eastman Kodak Company, Rochester, NY,
U.S.A.).
Cell pellets were fixed overnight in PBS with 2.5 % (w/v) glutaraldehyde, post-fixed in 1 % OsO4 , dehydrated in a graded series
of ethanols, infiltrated with propylene oxide and embedded in
Araldite 502 epoxy resin. Ultrathin sections were stained with
uranyl acetate and Reynold’s lead citrate prior to examination
using a Tecnai BioTWIN 12 transmission electron microscope
(FEI, Hillsboro, OR, U.S.A.).
Enzyme-activity assays
Microarray analysis of gene expression
TAL, TK (transketolase) and G6PD activities were measured
as described previously [14]. GPX (glutathione peroxidase)
activity was measured in the presence of a mixture containing
150 mM KH2 PO4 , pH 7.0, 1 mM GSH, 0.25 mM NADPH, 3 units
of glutathione reductase and 1 mM H2 O2 [20]. Catalase was
measured in the presence of 50 mM KH2 PO4 /Na2 HPO4 , pH 7.0,
and 10 mM H2 O2 at room temperature (20 ◦C) by continuous reading of A240 for 3 min [21]. SOD (superoxide dismutase) activity was measured in the presence of 0.05 M sodium pyrophosphate
buffer, pH 8.3, 186 μM phenazine methosulphate, 300 μM
Nitroblue Tetrazolium and 780 μM NADH [22].
For comparative gene-expression studies, RNA was extracted
from TD and control FBs and LBs using the RNeasy kit from
Qiagen. The mRNA fraction from approx. 5 μg of total RNA was
amplified and labelled using the MessageAmp kit (Ambion). A
20 μg portion of biotinylated cRNA was hydrolysed randomly
to 35–200 nucleotides in a fragmentation buffer solution (at
94 ◦C for 35 min). A 15 μg portion of the fragmented cRNA
was then added to a hybridization buffer (100 mM Mes, 1 M
Na+ (as NaCl), 20mM EDTA, 0.01 % Tween-20, 0.1 mg/ml
herring sperm DNA and 0.5 mg/ml acetylated BSA), containing
known concentrations of positive control genes (50 pM Oligo
B2, 1.5, 5, 25 and 100 pM Escherichia coli bioB, bioC, bioD
and cre provided by Affymetrix, Santa Clara, CA, U.S.A.).
The entire hybridization solution was heated at 99 ◦C for 5 min,
equilibrated at 45 ◦C for 5 min, then centrifuged at 15 000 g for
5 min before being injected into a U133A GeneChip (Affymetrix).
After washing and staining, fluorescent images were scanned at
2 μm resolution using the Agilent G2500A Gene Array Scanner.
The Microarray Analysis Suite, version 5.0 (Affymetrix), was
employed to generate the comparative analysis presented in this
study. Distinct algorithms of the software were used to determine
the absolute call that distinguishes the presence (P) or absence of a
transcript (A), the differential change in gene expression [increase
(I), decrease (D), marginal increase (MI), marginal decrease (MD)
and no change (NC)], and the magnitude (fold) change. Foldchange calculations were based on the average difference of each
probe set, since this output is directly related to its expression
level. After scanning the GeneChips, the Affymetrix software
(MicroArray Suite 5.0) was used to calculate the intensity of the
signal from each perfect match probe relative to the signal for
the mismatch probe and to determine whether or not the gene
was present in the sample [and a probability value (P < 0.05)
associated with this determination], as well as to measure the
expression level of the gene. The overall chip intensities for each
sample were scaled by linear adjustment to the same target value
(1000). Pivot tables containing the scaled data from each experiment were generated and imported into GeneSpring (Silicon
Measurement of phosphorylated sugars by HPLC
A total of 107 cells were resuspended in 1 ml of 10 % perchloric
acid, and after three freeze–thaw cycles the precipitate
was removed by centrifugation (15 000 g, 10 min, 4 ◦C). The
supernatant was neutralized with 120 μl of 10 M KOH. After
centrifugation, the supernatant was filtered through a 0.2-μmpore-size PVDF membrane, dried in a Speedvac apparatus and
dissolved in 100 μl of water. The total protein content of each
sample was determined using the Lowry assay [23]. A 25 μl
portion was injected into the HPLC chromatograph equipped with
a Dionex ED40 amperometric detector and the material analysed
on a Carbopack PAI column (Dionex, Sunnyvale, CA, U.S.A.)
using a gradient of 100 mM NaOH/1 M sodium acetate in 100 mM
NaOH [24].
Measurement of nucleotides by HPLC
Pellets from 107 cells washed in PBS were resuspended in 50 μl
of 0.16 M KCl/5 mM glucose at 4 ◦C. Then 100 μl of ice-cold
0.5 M KOH was added. The sample was mixed, diluted with
100 μl of ice-cold water and neutralized with 30 μl of cold 1 M
KH2 PO4. The extracts were centrifuged for 10 min at 15 000 g
at 4 ◦C, the supernatants were filtered through a 0.45-μmpore-size polypropylene membrane, and 10–20 μl aliquots were
injected into a 125 mm (length) × 4.6 mm (diameter) Hypersil
ATP measurement by chemiluminescence
Intracellular ATP levels were determined using the luciferin–
luciferase method [26].
Electron microscopy
c The Authors Journal compilation c 2008 Biochemical Society
126
Y. Qian and others
Figure 1 HPLC analysis of (A) G6P and S7P levels in FBs and LBs of a TD patient (TDP) and two healthy controls and (B) nucleotides in LBs of a TD patient
(TDP LB) and control LB JAB and GM13416
Results are means +
experiments. ∗ P < 0.05;
− S.E.M. for five or ∗more independent
∗∗
means +
− S.E.M. of four measurements. P < 0.05; P < 0.01.
∗∗
P < 0.01. (B) Bar charts show nucleotide levels in TDP and control LBs JAB and GM13416. Results are
Genetics, Redwood City, CA, U.S.A.). In GeneSpring, the individual gene chip data were normalized by adjusting the median
intensity of each array to a value of 1.0.
Flow cytometry of cell-surface antigen expression
TD and control FBs were freshly plated or 107 LBs/ml were
resuspended in fresh medium 24 h before analysis. A total of
c The Authors Journal compilation c 2008 Biochemical Society
107 cells were stained in 200 μl of PBS with 1 % (v/v) fetal-calf
serum with primary antibodies specific for CD95 (ZB4 and IgG1;
Upstate Biotechnology) or GGT (γ -glutamyl transferase; 3A8 and
IgG2a [27]) for 30 min at 4 ◦C. IgG1 CD38 Ab was obtained from
Caltag (Burlingame, CA, U.S.A.). ME0.5 IgG1 (Serotec, Oxford,
U.K.) and OKT3 IgG2a antibodies (A.T.C.C., Manassas, VA,
U.S.A.) were used as isotype controls. After washing them three
times, cells were stained with phycoerythrin-conjugated goat
Influence of transaldolase deficiency
Figure 2
127
Effect of TAL deficiency on the enzymatic activities of PPP and connected metabolic pathways
(A) Western-blot analysis of TAL, G6PD and β. -actin levels in cytosolic protein lysates (20 μg/lane) of FBs and LBs from TD patient and controls. G6PD/β. -actin levels relative to TD cells, normalized
at 1, were determined by densitometry as described in [12]. (B) Activities of PPP enzymes TAL, TK and G6PD and antioxidant enzymes GPX, CAT and SOD in TD and control LBs 1 (GM13416)
and 2 (JAB). (C) Activities of PPP enzymes TAL and G6PD and antioxidant enzymes CAT and SOD in TD FBs and control FBs 1 and 2. Results are means +
− S.E.M. for four or more independent
experiments.
anti-mouse or anti-human IgG for 30 min at 4 ◦C and analysed
by flow cytometry.
Transduction of wild-type TAL cDNA by AAV (adeno-associated
virus) vector (pAAV-IRES-hrGFP)
The full-length wild-type TAL cDNA [19] was inserted upstream
of the IRES (internal ribosomal entry site) of the pAAV-
IRES-hrGFP vector (Stratagene, La Jolla, CA, U.S.A.; where
hrGFP refers to humanized Renilla green fluorescent protein).
TAL-expressing AAV was produced by transfection of HEK293 (human embryonic kidney 293) cells with TAL-containing
pAAV-IRES-hrGFP, pAAV-RC (containing AAV replication and
capsid genes) and pHelper plasmids, which supply the necessary
exogenous gene products for virus production (Stratagene). At
a 1:1 multiplicity of infection, > 99 % of cells infected with
c The Authors Journal compilation c 2008 Biochemical Society
128
Y. Qian and others
pAAV-TAL-IRES-hrGFP or pAAV-IRES-hrGFP control virus
were GFP-positive. Maximal GFP expression was observed 24 h
after virus infection. GFP and TAL expression were diminished by
50 % after 48 h and became undetectable at 5 days post infection.
For flow-cytometric analysis with FL-1 fluorochrome excited at
488 nm, changes elicited by the wild-type TAL-producing AVV
were compared with those infected by AAV lacking TAL. In these
viruses, GFP was replaced with the 792-bp neomycin phosphotransferase gene transferred from pUHD172.1neo [28]. Using
Western-blot analysis, both TAL and NPT (neomycin phosphotransferase) were detected in pAAV-TAL-IRES-neo-transduced
cells using rabbit Ab to NPT II (United States Biological,
Swampscott, MA, U.S.A.; results not shown).
Table 1 Cumulative analysis of the ψ m by TMRM and JC-1 fluorescence,
mitochondrial mass by NAO and MTG fluorescence, cytoplasmic and mitochondrial ROI production by HE and DHR fluorescence, H2 O2 levels by DCF
fluorescence, cytoplasmic and mitochondrial Ca2+ levels by Fluo-3 and
Rhod-2 fluorescence, and NO production by DAF-FM fluorescence in annexin
V-negative cells using flow cytometry
A total of 5 × 106 LBs of a TD patient (TD LB) and normal donor cells (Normal LBs) were
cultured with or without 50 μM H2 O2 for 24 h. Results are expressed as relative fluorescence
values with respect to those of unstimulated normal cells adjusted to 1.0 for each experiment.
∗
Results are means +
− S.E.M. for eight independent experiments. P < 0.05 reflects comparison
of untreated TD LBs with untreated normal LBs, whereas #P < 0.05 reflects a comparison of
H2 O2 -treated TD LBs with H2 O2 -treated normal LBs for each parameter.
Relative fluorescence value
Normal LBs
TD LBs
Statistics
Fluorescing compound
Control
H2 O2
Control
H2 O 2
Results were analysed using the Student t test or Mann–Whitney
rank sum test for non-parametric data. ANOVA was assessed
with Graphpad (San Diego, CA, U.S.A.) software. Changes were
considered significant at P < 0.05.
TMRM
JC-1
NAO
MTG
HE
DHR
DCF
Fluo-3
Rhod-2
DAF-FM
MCB
1+
− 0.17
1+
− 0.13
1+
− 0.23
1+
− 0.12
+ 0.21
1−
1+
− 0.14
1+
− 0.07
1+
− 0.19
1+
− 0.14
1+
− 0.12
1+
− 0.08
1.67 +
− 0.14
1.44 +
− 0.09
1.76 +
− 0.37
0.86 +
− 0.08
+ 0.61
3.29 −
1.81 +
− 0.23
0.70 +
− 0.03
1.74 +
− 0.25
1.39 +
− 0.12
1.65 +
− 0.60
1.13 +
− 0.05
∗
0.77 +
− 0.04∗
0.79 +
0.03
−
∗
2.04 +
− 0.37∗
1.39 +
− 0.07∗
+ 0.08
0.85 −
∗
0.77 +
− 0.08∗
1.24 +
0.14
−
∗
1.53 +
− 0.11∗
1.29 +
− 0.08∗
1.61 +
− 0.14
0.98 +
− 0.03
0.84 +
− 0.16#
0.58 +
− 0.08#
1.09 +
− 0.01
1.34 +
− 0.03
+ 0.46
2.09 −
1.61 +
− 0.04
0.79 +
− 0.07
1.45 +
− 0.05
1.35 +
− 0.03
0.74 +
− 0.11#
0.94 +
− 0.04
Human experimentation
Our research was carried out in accordance with the Declaration
of Helsinki (2000) of the World Medical Association and had the
approval of the ethical committees of our institutions.
RESULTS
Effect of TAL deficiency on metabolites and enzyme activities of the
PPP and interconnected pathways
TAL catalyses the transfer of a three-carbon fragment, corresponding to dihydroxyacetone, to GA3P (D-glyceraldehyde 3phosphate) and E4P (D-erythrose 4-phosphate) [1]. In the forward
reaction of the PPP, TAL transfers dihydroxyacetone from S7P
to GA3P, thus producing E4P and F6P (fructose 6-phosphate).
The latter is converted by glucose-phosphate isomerase into G6P,
which, in turn, becomes a substrate of G6PD. In the reverse
reaction, E4P and G6P are converted into GA3P and S7P. To
assess the impact of TAL deficiency on the PPP, unique substrates
of the pathway were analysed by HPLC. As shown in Figure 1(A)
and Supplementary Figure S1 at http://www.BiochemJ.org/bj/
415/bj4150123add.htm, S7P was markedly accumulated both in
TD FBs (P = 0.012) and LBs (P = 0.0049). By contrast, G6P
levels were reduced in TD FBs (0.062 +
− 0.017 pmol/μg of protein) with respect to control FBs #1 (0.246 +
− 0.044 pmol/μg of
protein; P = 0.0132) or control FBs #2 (0.279 +
− 0.066 pmol/μg
of protein; P = 0.047). G6P levels were also diminished in TD
LBs relative to the control LBs #1 (P = 0.034) or control LBs #2
(P = 0.039) (Figure 1A).
Accumulation of S7P and depletion of G6P indicate that
TAL deficiency blocked the forward reaction, i.e. recycling of
G6P for the oxidative branch of the PPP. The oxidative PPP
generates NADPH [1]. NADPH, as well as NADP+ , AMP, cAMP
and NAD+ were diminished, whereas ADP-ribose levels were
elevated in TD cells (Figure 1B and Supplementary Figure S2 at
http://www.BiochemJ.org/bj/415/bj4150123add.htm). Following
the alkaline extraction required to preserve the integrity of
NADPH and NADH [14], ATP was degraded. Therefore, ATP was
measured by the luciferase assay [26]. The ATP content of TD LBs
was increased (2.8 +
− 0.3 pmol/μg of protein) in comparison with
JAB (0.9 +
− 0.2 pmol/μg of protein; P = 0.0002) and GM13416
control LBs (1.5 +
− 0.4 pmol/μg of protein; P = 0.004).
c The Authors Journal compilation c 2008 Biochemical Society
Profound depletion of G6P and NADPH suggested diminished
activity of the oxidative PPP. As previously reported [12], TAL
protein and enzyme activity were undetectable in LB and FB of
the TALS171 patient (Figure 2A). To assess the impact of TAL
deficiency on the oxidative PPP, expression and activity of G6PD,
the rate-limiting enzyme of NADPH production, was measured.
Both G6PD protein (Figure 2A) and enzyme activity levels were
markedly diminished in LBs and FBs of the TD patient relative
to control cells (Figures 2B and 2C). Although activities of TK
and GPX were not significantly affected, catalase and SOD were
reduced in TD LBs and FBs (Figures 2B and 2C).
Loss of ψ m and increased mitochondrial biogenesis characterize
mitochondrial dysfunction in TD cells
The oxidation–reduction equilibrium of pyridine nucleotides
(NADH/NAD+ +NADPH/NADP+ ) regulates ψ m [29]. Since
TAL deficiency may affect cell viability through controlling
ψ m [14,15,30], annexin V-negative live cells were assessed
for ψ m using potentiometric fluorescent dyes TMRM and JC1 (Supplementary Figure S3 at http://www.BiochemJ.org/bj/415/
bj4150123add.htm and Table 1). A significant decrease in ψ m
was detected by both TMRM and JC-1 fluorescence. As altered
incorporation of potentiometric dyes may represent changes
in mitochondrial mass, the latter was assessed by staining
with the potential-insensitive mitochondrial probes NAO and
MTG. Surprisingly, the mitochondrial mass in TD cells was
increased, as determined by both NAO (+ 104 %, P = 0.01)
and MTG (+ 39 %, P = 0.0003) fluorescence. Along the same
lines, electron microscopy showed increased numbers of mitochondria in TD LBs (29.2 +
− 5.2/cell) with respect to control
LBs (+ 10.2 +
2.2/cell,
P
=
0.0107).
Mitochondria also appeared
−
larger in TD cells (Figure 3), exhibiting features of megamitochondria [31]. Whereas intracytosolic and mitochondrial
ROI production, monitored by HE (− 15.0 %, P = 0.02) and
DHR (− 23.0 %, P < 0.01) fluorescence were reduced, DCF
Influence of transaldolase deficiency
Figure 3
129
Electron microscopy of control (A and B) and TD LBs (C and D)
The arrow indicates megamitochondrion in (D). Note: 1 micron=1 μm.
fluorescence, preferentially detecting H2 O2 [16], was enhanced
in TD LBs (+ 23.8 %, P = 0.0013). TD LBs also produced
increased amounts of NO, as monitored by DAF-FM fluorescence
(+ 61.3 %, P = 0.0081; Figure 3), which is a key trigger of
mitochondrial biogenesis [32]. Since mitochondria store Ca2+
[18], we investigated the [Ca2+ ]c and [Ca2+ ]m levels. [Ca2+ ]c
and [Ca2+ ]m of TD LBs were elevated with respect to control
LB, as determined by Fluo-3 (+ 53.0 %, P = 0.023) and Rhod2 fluorescence (+ 29.0 %, P = 0.001) respectively (Table 1 and
Supplementary Figure S3).
Mitochondrial function was further tested by exposure of TALdeficient and control LBs to a low dose (50 μM) of H2 O2 .
Although H2 O2 is freely diffusible, it is not, in itself, an
ROI. Induction of apoptosis by H2 O2 , requires mitochondrial
transformation into an ROI, e.g. OH− , through the Fenton
reaction [33]. After exposure to H2 O2 , the potentiometric
dyes showed mitochondrial hyperpolarization in accordance
with previous findings [26,34]. Mitochondrial hyperpolarization is dependent on NO production [17]. TD cells
exhibited a blunted response in H2 O2 -induced NO production
and mitochondrial hyperpolarization (Supplementary Figure S3 and Table 1). Maintenance of GSH in a reduced state
is dependent on the availability of NADPH [1]. Although
NADPH was profoundly depleted, GSH levels were normal
in TD cells, suggesting compensatory changes in GSH
metabolism.
c The Authors Journal compilation c 2008 Biochemical Society
130
Y. Qian and others
incubation with GGT Ab for 48 h reduced GSH content of
TAL-deficient LB measured by MCB fluorescence (results not
shown).
Expression of wild-type TAL reverses changes in ATP, Ca2+ fluxing
and apoptosis susceptibility of TD cells
Figure 4 Relative mRNA levels of genes significantly altered in TD LBs
(P < 0.05 using GeneSpring) and reversed by correction of TAL activity based
on microarray analysis
Gene expression was investigated in control and TD FBs and LBs as well as TD LBs infected with
control AAV (TD LB-pAAV) and TAL-producing AAV (TD-LB-pAAV/TAL). Abbreviation: CLCA2,
Ca2+ -regulated chloride channel 2.
TAL deficiency elicits co-ordinated changes in expression of genes
involved in the PPP, mitochondrial biogenesis, GSH metabolism
and Ca2+ fluxing
The impact of TAL deficiency on the PPP and connected metabolic pathways was investigated in the context of global gene
expression using the Human Genome U133A chip with probe
sets of 22 283 genes. The specificity of TAL-deficiency-induced
changes on gene expression was validated by analysis of TD
LBs transduced with an AAV vector expressing wild-type TAL.
Expression of 18 genes was significantly and specifically altered
by TAL deficiency, i.e., normalization of TAL expression by
AAV-mediated gene transfer reversed the changes in expression
of each of these genes (Figure 4). Reduced TAL transcription
was detected in both TD FBs and LBs in comparison with
control FBs and LBs, in agreement with earlier Northern-blot
studies [12]. G6PD mRNA levels were reduced in a manner that
correlated with diminished G6PD protein levels (Figure 2A).
G6PD, TK, 6PGD (6-phosphogluconate dehydrogenase), PGI
(phosphoglucose isomerase), PFKM (phosphofructokinase),
TPI1 (triosephosphate isomerase 1), ALDC (aldolase C) and
PGK1 (phosphoglycerate kinase 1) mRNA levels were all reduced
in TD LBs and FBs, following the pattern of TAL expression. On
the basis of ANOVA, expression of these enzymes was co-ordinately regulated in TD LBs and FBs (P = 0.005). Interestingly,
genes regulating mitochondrial biogenesis [PGC-1 (peroxisome
proliferator-activated receptor γ -co-activator 1)’, NRF-1 (nuclear
respiratory factor 1), HMOX1 (haem oxygenase), NUDT1 (nucleotide diphosphatase type motif 1/8-oxo-7,8-dihydroguanosine
triphosphatase), PTK2B (protein tyrosine kinase 2., Ca2+ regulated) and BNIP3L (bcl-2-like interacting protein 3-like
mitochondrial protein)] were also co-ordinately altered in TD FBs
and LBs. Expression of the Fas/Apo-1/CD95 cell death receptor
was only increased in TD FBs but not in LBs (results not shown).
Flow-cytometric staining with monoclonal Ab ZB4 confirmed
increased surface expression of the Fas/Apo-1/CD95 on FBs but
not on LBs of the TD patient (Figure 5A). Increased surface
expression levels of GGT (Figure 5B) and CD38/NAD+ hydrolase
were also confirmed on TD FBs and LBs by flow cytometry
(Figure 5C). Overexpression of GGT may help maintain GSH
levels in cells with diminished NADPH production. Indeed,
c The Authors Journal compilation c 2008 Biochemical Society
The phenotype of homozygous TALS171/TD patient’s cells
may be influenced by mutations in genes other than TAL. Therefore, we normalized TAL activity by transduction of wild-type
TAL cDNA using an AAV vector. At 1:1 multiplicity of infection,
> 99 % of AAV-infected cells were GFP-positive (Figure 6A)
and expressed functionally active TAL (Figures 6B and 6C).
After AAV infection, TAL expression (Figure 6B) and activity levels reached ∼ 100 % of those in control LB (Figure 6C).
Normalization of TAL protein expression and activity resulted in
reduced ATP (Figure 6D), increased GSH (Figure 6E) and reduced
[Ca2+ ]c in TD LBs (Figure 6F).
B-cell homoeostasis is maintained through H2 O2 - [35],
Fas/CD95- [36] or CD20-mediated apoptosis [37]. To investigate
the impact of TAL deficiency on the processing of these celldeath signals, cells were exposed to 100 μM H2 O2 or Ab to Fas/
CD95 or CD20 12 h after AAV-mediated transduction of wildtype TAL and survival was measured by annexin-V staining
24 h later. H2 O2 -induced apoptosis was enhanced in TD LBs
as compared with control LBs, a situation that was reversed
after infection with wild-type TAL-producing AAV (Figure 6G).
Although microarray analysis and cell-surface staining showed
similar CD20 expression levels (results not shown), cross-linking
of CD20 resulted in accelerated apoptosis of TD LBs (Figure 6H).
Enhanced CD20-induced cell death was reversed by expression
of wild-type TAL (Figure 6H). In accordance with its dependence
on Ca2+ fluxing [37], increased CD20-induced apoptosis of TD
cells was also reversed by pretreatment with 5 μM BAPTA-AM
for 30 min (results not shown). In contrast, Fas-induced apoptosis
was reduced in TD LB; which was also reversed by infection
with TAL-producing AAV (Figure 6I). These results reveal that
the apoptosis-susceptibility of B-cells is regulated by TAL in a
signal-dependent manner.
DISCUSSION
TAL is an enzyme of the non-oxidative branch of the PPP which
is the principal supplier (1) of NADPH for reductive biosynthesis
and regeneration of glutathione from its oxidized form, and (2)
of ribose 5-phosphate for nucleotide production. G6PD has been
uniformly regarded as the rate-controlling enzyme for both of
these functions of the PPP [1]. TAL deficiency has been found to
be associated with liver cirrhosis in children from three families
[9–11]. Utilizing LB and FB cells available from the first patient
[12], the present study provides evidence that deficiency of TAL
influences apoptosis signal processing in a death-pathway-specific
manner. TD LBs exhibit increased spontaneous and H2 O2 -induced
apoptosis. Fas-induced apoptosis was reduced in LBs of the TD
patient. Fas-induced caspase activity is dependent on maintaining
of active-site cysteine residues in a reduced state [38]. Coordinate up-regulation of GSH and Fas-dependent apoptosis by
normalization of TAL expression suggests that oxidative stress
limits activity of caspases in TD LBs. NADPH depletion may
underlie oxidative stress. Increased H2 O2 levels in TD cells is
also consistent with deficiency of NADPH, which is required for
conversion of H2 O2 into water by catalase [21].
The mechanism of oxidative stress in TD cells was investigated
at the level of PPP enzymes and metabolites. S7P, a substrate of
Influence of transaldolase deficiency
Figure 5
131
Flow-cytometric analyses
(A) Flow cytometry of cell-surface expression of the Fas/Apo-1/CD95 antigen on TD (grey areas) and control fibroblasts (control FBs 1 and 2; white areas) and LBs (control LBs 1 and 2; white areas).
(B) Flow cytometry of GGT expression on TD (grey areas) and control cells (control FBs 1 and 2 and control LBs 1 and 2; white areas). (C) Flow cytometry of CD38 expression on TD (grey areas)
and control cells (control FBs 1 and 2 and control LBs 1 and 2; white areas). The value mean channel fluorescence of TD cells is shown over each set of curves and the mean channel fluorescence of
control cells is shown in parenthesis (white area overlays).
the forward TAL reaction, was accumulated, whereas G6P was
depleted. Accumulation of S7P is compatible with a failure to
recycle R5P into G6P through the non-oxidative branch of the
PPP, thus reducing NADPH production by the oxidative branch.
ADP-ribose and ATP levels were clearly elevated, indicating that
nucleotide synthesis was not limited, but rather stimulated, by
the availability of R5P. The five-carbon sugars R5P, xylulose 5phosphate and ribulose-5-phosphate are likely to accumulate and
inhibit 6PGD [39]. Co-ordinate accumulation of ADP-ribose and
depletion of NAD+ may be attributed to overexpression of CD38
[40,41]. CD38 or NAD+ hydrolase metabolizes NAD+ into ADPribose and cADP-ribose, which, in turn, stimulate the release
of Ca2+ from the endoplasmic reticulum [40]. Normalization of
TAL activity reduced CD38 expression and [Ca2+ ]c , observations
that are consistent with the notion that oxidative stress is
responsible for overexpression of CD38 and the resultant NAD+
depletion, ADP-ribose accumulation and increased [Ca2+ ]c .
Moreover, mitochondria constitute major Ca2+ stores [42]; thus
enhanced mitochondrial mass also favours higher [Ca2+ ]m levels.
Increased mitochondrial mass allows for increased production of
ATP. In turn, excess ATP could also inhibit G6PD and 6PGD
[43].
Expression of genes associated with the PPP and mitochondrial
biogenesis were co-ordinately regulated with TAL deficiency
and normalized by AAV-mediated transduction of the wild-type
TAL gene. Overexpression of Fas/CD95, both at the mRNA
level and on the surface of TD FBs, has been associated
with increased Fas-dependent apoptosis of these cells (results not
shown). Fas expression was not enhanced, and Fas-dependent
apoptosis was diminished, in TD LBs. On the basis of microarray and confirmatory flow-cytometric studies, CD38 was
seen to be overexpressed on both TD LBs and FBs. The
promoter region of CD38 harbours several redox-sensitive
AP-1 (activator protein 1) motifs [44]. Thus oxidative stress
may account for increased expression of CD38 in TD B-cells.
Normalization of TAL activity reversed the elevated CD38
expression and Ca2+ levels, the increased H2 O2 - and CD20induced apoptosis and the diminished Fas-induced apoptosis of
c The Authors Journal compilation c 2008 Biochemical Society
132
Figure 6
Y. Qian and others
Effect of normalization of TAL protein and activity levels on ATP, Ca2+ , GSH and apoptosis-susceptibility of TD LBs
(A) Flow-cytometric analyses of uninfected control TD LBs (TDLB, black), TDLB infected with TAL-expressing AAV (TDLB-pAAV/TAL, first grey curve; produces TAL and GFP) and TD LBs infected
with GFP-expressing AAV (TD LB-pAAV, second grey curve; produces GFP only). (B) Western-blot analysis of TAL and β. -actin levels in TD LBs, TD LBs infected with control AAV (TDLB-pAAV),
and TD LBs infected with TAL-producing AAV (TDLB-pAAV/TAL) and control GM13416 and JAB LBs. (C) TAL enzyme activity levels in cells analysed in (B). (D) Measurement of ATP levels by the
luciferase assay. (E) Assessment of intracellular Ca2+ by Fluo-3 relative fluorescence (RF). (F) Assessment of intracellular GSH by MCB relative fluorescence. (G) Apoptosis induced by 100 μM
H2 O2 12 h after AAV infection and measured by annexin-V/propidium iodide staining 24 h later. (H) Apoptosis induced by CD20 Ab 6 h after AAV infection and measured by annexin-V/PI staining
48 h later. Results are means +
− S.E.M. for four experiments. (I) Apoptosis induced by CH11 antibody to CD95 12 h after AAV infection and measured by annexin-V/PI staining 24 h later.
B cells, clearly indicating that each of these changes resulted from
TAL deficiency. Increased CD20-induced apoptosis was also
reversed by the Ca2+ chelator BAPTA-AM, suggesting that the
c The Authors Journal compilation c 2008 Biochemical Society
enhancement of this cell-death pathway is mediated by
high Ca2+ levels. Although the role of B-cell apoptosis in human
TAL deficiency is presently unclear, enhanced spontaneous and
Influence of transaldolase deficiency
H2 O2 -induced apoptosis and inhibited CD95/Fas apoptosis could
contribute to the pathogenesis of liver disease. Clinical manifestations of TAL deficiency are dominated by the consequences of
liver cirrhosis [9–11], which results from increased cell death
of hepatocytes and biliary epithelial cells [8,13]. Increased
expression of CD38 [45], elevation of [Ca2+ ]c and [Ca2+ ]m , as
well as depletion of NAD+ and NADPH, have been implicated
in mitochondrial dysfunction, oxidative stress [46] and cirrhosis
of the liver [47]. There is growing evidence for the involvement
of the CD95/Fas cell-death-receptor-initiated apoptosis pathway
in physiological regulation of hepatocyte turnover [48], and
defective Fas signalling predisposes to hepatocarcinogenesis [49].
As oxidative stress leads to cirrhosis [47], whereas CD95/Fas
resistance facilitates hepatocarcinogenesis [49], the present study
identifying TAL as a signal-specific regulator of apoptosis is
likely to be relevant for the pathogenesis of TAL deficiency.
We thank Dr David Fernandez (Department of Medicine, SUNY Upstate Medical University,
Syracuse, NY 13210, U.S.A.) for helpful discussions and Dr Paul Phillips (Department of
Medicine, SUNY Upstate Medical University, Syracuse, NY 13210, U.S.A.) for continued
encouragement and support. This work was supported in part by grant DK 49221 from
the National Institutes of Health, the Central New York Community Foundation and the
Children’s Miracle Network.
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Received 7 April 2008/19 May 2008; accepted 22 May 2008
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Biochem. J. (2008) 415, 123–134 (Printed in Great Britain)
doi:10.1042/BJ20080722
SUPPLEMENTARY ONLINE DATA
Transaldolase deficiency influences the pentose phosphate pathway,
mitochondrial homoeostasis and apoptosis signal processing
Yueming QIAN*, Sanjay BANERJEE*, Craig E. GROSSMAN*†, Wendy AMIDON*, Gyorgy NAGY*, Maureen BARCZA‡,
Brian NILAND*†, David R. KARP§, Frank A. MIDDLETON‡, Katalin BANKI¶ and Andras PERL*†1
*Department of Medicine, SUNY Upstate Medical University, Syracuse, NY 13210, U.S.A., †Department of Microbiology and Immunology, SUNY Upstate Medical University, Syracuse,
NY 13210, U.S.A., ‡Department of Physiology, SUNY Upstate Medical University, Syracuse, NY 13210, U.S.A., §University of Texas Southwestern Medical Center, Dallas, TX 75390,
U.S.A., and ¶Department of Pathology, SUNY Upstate Medical University, Syracuse, NY 13210, U.S.A.
Figure S1 HPLC analysis of G6P and S7P levels in LBs of a TD patient
(TD LB) and in LBs of two healthy controls (LB 1 and LB 2)
Chromatograms indicate electric charge (nC) of sugar phosphates recorded on a
pulse-amperometric detector.
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2008 Biochemical Society
Y. Qian and others
Figure S2
HPLC analysis of nucleotides in LBs of a TD patient (TDP LB) and control LB JAB and GM13416
Chromatograms show the UV absorbance (AU) of nucleotide extracts recorded on a photodiode-array detector. Annotated peaks were positively identified by spiking TDP LB extracts separately with
each compound.
c The Authors Journal compilation c 2008 Biochemical Society
Influence of transaldolase deficiency
Figure S3
For legend see Figure S3(C)
c The Authors Journal compilation c 2008 Biochemical Society
Y. Qian and others
Figure S3
For legend see next page
c The Authors Journal compilation c 2008 Biochemical Society
Influence of transaldolase deficiency
Figure S3 Assessment of ψ m , mitochondrial mass, ROI levels, cytoplasmic and mitochondrial Ca2+ concentrations and NO production in control (GM13416)
and TD LBs by flow cytometry
A total of 5 × 106 cells were resuspended in fresh medium in the absence or presence of 100 μM H2 O2 and assayed after incubation at 37 ◦C for 24 h. For each dot-plot shown, the x-axis corresponds
to FL-1 (green: JC-1 monomers, NAO, MTG, DHR, DCF, Fluo-3, DAF-FM and annexin V-FITC), whereas the y -axis corresponds to FL-2 (red: JC-1 aggregates, HE, Rhod-2 and annexin V-PE)
fluorescence channel. (A) ψ m was measured by JC-1 (FL-1: lower right corner; FL-2: upper left corner) and DiOC6 (FL-1), mitochondrial mass was monitored by NAO (FL-2) and MTG fluorescence
(FL-1). (B) Cytoplasmic and mitochondrial ROI production was assessed by HE (FL-2) and DHR (FL-1) and H2 O2 levels were evaluated by DCF (FL-1). (C) Cytoplasmic and mitochondrial Ca2+
concentrations were assessed by Fluo-3 (FL-1) and Rhod-2 (FL-2), and NO production was monitored by DAF-FM fluorescence (FL-1). Cell viability was assessed by staining with annexin V-FITC,
annexin V-PE, or annexin V-Cy5 matched with emission spectra of potentiometric, oxidation-, NO- or Ca2+ -sensitive fluorescent probes. Values in dot-plots indicate percentage of annexin V-positive
cells and mean channel FL-1 and FL-2 fluorescence of annexin V-negative cells respectively. Results are representative of those obtained in eight independent experiments.
c The Authors Journal compilation c 2008 Biochemical Society
Y. Qian and others
Figure S4
Schematic diagram of the PPP in TAL deficiency
The oxidative phase produces two NADPH molecules (colored in green) per G6P molecule. TAL, an enzyme of the non-oxidative phase, catalyses the transfer of dihydroxyacetone from S7P and
F6P to GA3P and E4P respectively. The forward reaction of TAL favours generation of G6P (blue arrowhead); the reverse TAL reaction promotes metabolism of G6P into R5P (red arrowhead). TAL
deficiency leads to the accumulation of S7P and depletion of G6P (blue arrows). Thus TAL deficiency primarily blocks the forward reaction and inhibits recycling of G6P. The five-carbon sugars R5P,
xylulose 5-phosphate and RU5P are likely to accumulate (red arrows) and they inhibit 6PGD [1]. Accumulation of ADP-ribose enhances Ca2+ fluxing [2]. Excess ATP inhibits both G6PD and 6PGD
[3]. Inhibition of G6PD and 6PGD and the requirement of NADPH to metabolize GA3P by TPOR (triosephosphate oxidoreductase) deplete NADPH. Glutathione reductase uses NADPH to regenerate
GSH from GSSG. Abbreviation: 3K6PG, 3-oxo-6-phosphogluconate.
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monophosphate shunt in Rana ridibunda erythrocytes. Comp. Biochem. Physiol.
B Comp. Biochem. 95, 287–294
Received 7 April 2008/19 May 2008; accepted 22 May 2008
Published as BJ Immediate Publication 22 May 2008, doi:10.1042/BJ20080722
c The Authors Journal compilation c 2008 Biochemical Society