Non-oxidative pentose phosphate pathway controls regulatory T cell function by integrating metabolism and epigenetics.

Regulatory T (Treg) cells are critical for maintaining immune homeostasis and preventing autoimmunity. Here, we show that the non-oxidative pentose phosphate pathway (PPP) regulates Treg function to prevent autoimmunity. Deletion of transketolase (TKT), an indispensable enzyme of non-oxidative PPP, in Treg cells causes a fatal autoimmune disease in mice, with impaired Treg suppressive capability despite regular Treg numbers and normal Foxp3 expression levels. Mechanistically, reduced glycolysis and enhanced oxidative stress induced by TKT deficiency triggers excessive fatty acid and amino acid catabolism, resulting in uncontrolled oxidative phosphorylation and impaired mitochondrial fitness. Reduced α-KG levels as a result of reductive TCA cycle activity leads to DNA hypermethylation, thereby limiting functional gene expression and suppressive activity of TKT-deficient Treg cells. We also find that TKT levels are frequently downregulated in Treg cells of people with autoimmune disorders. Our study identifies the non-oxidative PPP as an integrator of metabolic and epigenetic processes that control Treg function.

Biomarkers Apo10 and TKTL1: Epitope-detection in monocytes (EDIM) as a new diagnostic approach for cholangiocellular, pancreatic and colorectal carcinoma.

OBJECTIVE: The EDIM (Epitope detection in monocytes) blood test is based on two biomarkers Apo10 and TKTL1. Apo10 is responsible for cell proliferation and resistance to apoptosis. TKTL1 plays a major role in anaerobic glycolysis of tumor cells, leading to destruction of the basal membrane and metastasis as well as in controlling cell cycle. For the first time we analyzed Apo10 and TKLT1 in patients with cholangiocellular (CCC), pancreatic (PC), and colorectal carcinoma (CRC). METHODS: Blood samples of 62 patients with CCC, PC, and CRC were measured and compared to 29 control patients. We also investigated 13 patients with inflammatory conditions, because elevated TKTL1 and Apo10 have been previously described in affected individuals. Flow cytometry was used to detect surface antigens CD14+/CD16+ (activated monocytes/macrophages). Percentages of macrophages harboring TKTL1 and Apo10 were determined. A combined EDIM score (EDIM-CS: TKTL1 plus Apo10) was calculated. Results were correlated with serum tumor markers CEA and CA19-9. RESULTS: Patients with CCC had 100% positive EDIM-CS but CEA and CA19-9 were positive in only 22.2% and 70%, respectively. Patients with PC had 100% positive EDIM-CS but positive tumor markers in only 37.5% (CEA) and 72.7% (CA19-9). Patients with CRC had 100% positive EDIM-CS but only 50% positive CEA. EDIM-CS was positive in 100% (62/62) of all cancer patients and in 0% of healthy individuals. Of the individuals with inflammation, 7.7% had a positive EDIM-CS. CONCLUSION: The sensitivity of the EDIM blood test and the comparison with traditional tumor markers indicate that this new test might improve the detection of carcinomas (CCC, PC and, CRC) and might be relevant for the diagnosis of all tumor entities.

[Cloning, expression and immunity analysis of transketolase of Echinococcus granulosus].

OBJECTIVE: To obtain the prokaryotic expression of transketolase genes and analyze its value as a diagnostic antigen for echinococcosis. METHODS: TK gene was amplified by PCR and cloned into prokaryotic vector pMD19-EgTK, and then subcloned into the expression vector pET-28a. The target gene TK prokaryotic expression plasmid pET-28a was constructed and transferred into BL21. The purified protein was identified by SDS-PAGE and Western blotting. The blood samples of patients with cystic echinococcosis (CE group), alveolar echinococcosis (AE group) and healthy people (healthy group) were collected and detected by ELISA with the recombinant EgTK protein as a diagnostic antigen. RESULTS: The recombinant plasmid pET-28a (+)-EgTK was constructed successfully, and there was a band around 70 kDa by using Western blotting. ELISA showed that the difference among the 3 groups of sera reaction A450 was significantly different (F = 44.47, P < 0. 01), and the A450 values ofthe CE group (1.46±0.41) and AE group (1.28±0.29) were higher than that of the healthy group (0.66±0.23), but there was no significant difference between the former two. CONCLUSIONS: The recombinant EgTK protein is better to distinguish the echinococcosis group and healthy group, but it can't do a differential diagnosis between CE and AE cases.

A biomarker based detection and characterization of carcinomas exploiting two fundamental biophysical mechanisms in mammalian cells.

BACKGROUND: Biomarkers allowing the characterization of malignancy and therapy response of oral squamous cell carcinomas (OSCC) or other types of carcinomas are still outstanding. The biochemical suicide molecule endonuclease DNaseX (DNaseI-like 1) has been used to identify the Apo10 protein epitope that marks tumor cells with abnormal apoptosis and proliferation. The transketolase-like protein 1 (TKTL1) represents the enzymatic basis for an anaerobic glucose metabolism even in the presence of oxygen (aerobic glycolysis/Warburg effect), which is concomitant with a more malignant phenotype due to invasive growth/metastasis and resistance to radical and apoptosis inducing therapies. METHODS: Expression of Apo10 and TKTL1 was analysed retrospectively in OSCC specimen (n = 161) by immunohistochemistry. Both markers represent independent markers for poor survival. Furthermore Apo10 and TKTL1 have been used prospectively for epitope detection in monocytes (EDIM)-blood test in patients with OSCC (n = 50), breast cancer (n = 48), prostate cancer (n = 115), and blood donors/controls (n = 74). RESULTS: Positive Apo10 and TKTL1 expression were associated with recurrence of the tumor. Multivariate analysis demonstrated Apo10 and TKTL1 expression as an independent prognostic factor for reduced tumor-specific survival. Apo10+/TKTL1+ subgroup showed the worst disease-free survival rate in OSCC.EDIM-Apo10 and EDIM-TKTL1 blood tests allowed a sensitive and specific detection of patients with OSCC, breast cancer and prostate cancer before surgery and in after care. A combined score of Apo10+/TKTL1+ led to a sensitivity of 95.8% and a specificity of 97.3% for the detection of carcinomas independent of the tumor entity. CONCLUSIONS: The combined detection of two independent fundamental biophysical processes by the two biomarkers Apo10 and TKTL1 allows a sensitive and specific detection of neoplasia in a noninvasive and cost-effective way. Further prospective trials are warranted to validate this new concept for the diagnosis of neoplasia and tumor recurrence.

Diagnostic use of epitope detection in monocytes blood test for early detection of colon cancer metastasis.

A follow-up strategy in cancer aftercare can result in early detection of metastasis and/or recurrence. Therefore, sensitive and reliable diagnostic tests that are easy to perform are needed. Here, the authors present the combined use of the epitope detection in monocytes (EDIM)-TKTL1 and EDIM-Apo10 blood test in aftercare monitoring of a patient with colon carcinoma. Whereas the established tumor markers CEA and CA19-9 did not indicate metastasis even at a timepoint where clinical signs and imaging techniques already demonstrated metastasis, the combined application of the EDIM-TKTL1 and the EDIM-Apo10 blood tests was positive 9 months before detection of metastasis. These findings - taken together with recently published evaluation data of the EDIM-TKTL1 blood test - suggest that the combined application of the EDIM-TKTL1 and the EDIM-Apo10 blood tests might indicate metastasis earlier than established tumor markers and could serve as sensitive and noninvasive methods that might be used for early detection of colon cancer metastasis.

Evidence against a major role for TKTL-1 in hypoxic and normoxic cancer cells.

Based on studies performed with a selected mouse monoclonal anti-transketolase- like (TKTL)-1 antibody (clone JFC12T10), overexpression of TKTL-1 has been shown to be correlated with poor survival and increased metastatic spread in several human tumor entities. Since the clinical aggressiveness mediated by TKTL-1 has been partially related to resistance to hypoxia,we originally aimed to explore the influence of hypoxia on the expression of TKTL-1. Unexpectedly, results of our experiments indicated that the antibody clone JFC12T10 lacks target specificity. Since the majority of data on the role of TKTL-1 in human cancer is based upon studies performed with this antibody clone, we subsequently re-evaluated the expression of TKTL-1 in six different cancer cell lines (HeLa, MCF-7, A549, HT-1080, M21 and TF-1). Using RT-PCR and consecutive sequence analysis, we show that transketolase (TKT), not TKTL-1, is the dominant isoform of transketolases in the cell lines analyzed. Our data argue against a major role of TKTL-1 for the metabolism of cancer cells.

Transketolase and 2',3'-cyclic-nucleotide 3'-phosphodiesterase type I isoforms are specifically recognized by IgG autoantibodies in multiple sclerosis patients.

The presence of autoantibodies in multiple sclerosis (MuS) is well known, but their target antigens have not been clearly identified. In the present study, IgG autoreactivity to neural antigens of normal human white matter separated by bidimensional electrophoresis was assessed in serum and cerebrospinal fluid of 18 MuS and 20 control patients. Broad IgG autoreactivity was detected by two-dimensional immunoblotting in all cases to neural antigens, most of which were identified by mass spectrometry. The comparative analysis of MuS and non-MuS reactive spots showed that a restricted number of neural protein isoforms were specifically recognized by MuS IgG. Almost all MuS patients had cerebrospinal fluid IgG directed to isoforms of one of the oligodendroglial molecules, transketolase, 2',3'-cyclic-nucleotide 3'-phosphodiesterase type I, collapsin response mediator protein 2, and tubulin beta 4. Interestingly 50% of MuS IgG recognized transketolase, which was mostly localized on oligodendrocytes in human white matter from normal and MuS samples. IgG autoreactivity to cytoskeletal proteins (radixin, sirtuin 2, and actin-interacting protein 1) was prevalent in secondary progressive MuS patients. Among the proteins recognized by serum IgG, almost all MuS patients specifically recognized a restricted number of neuronal/cytoskeletal proteins, whereas 2',3'-cyclic-nucleotide 3'-phosphodiesterase type I was the oligodendroglial antigen most frequently recognized (44%) by MuS seric IgG. Our immunomics approach shed new light on the autoimmune repertoire present in MuS patients revealing novel oligodendroglial and/or neuronal putative autoantigens with potential important pathogenic and diagnostic implications.

Contribution of the antibodies response induced by a low virulent Candida albicans strain in protection against systemic candidiasis.

A low virulent Candida albicans mutant, CNC13, deleted in the Mitogen Activated Protein (MAP) kynase HOG1 was used to immunize BALB/c mice. Hog1p is essential for the oxidative stress and hyperosmolarity responses. Several doses and immunization procedures were employed. The protection capacity of the different sera generated was analyzed in a murine model of systemic candidiasis. Using a proteomic approach (two-dimensional gel electrophoresis followed by Western blotting), we were able to distinguish two categories of serum: protective and nonprotective, which showed different titres of total Immunoglobulins (Igs) and IgG2a (analyzed by enzyme-linked immunosorbent assay). The levels of Igs and IgG2a in protective sera were significantly higher compared to nonprotective sera. The pattern of a "nonprotective" profile was composed of enolase (Eno1p), transketolase, heat shock protein and methionine synthase. Only antibodies against enolase are the IgG2a isotype. The pattern of a "protective" sera, on the other hand, was composed of antibodies against the following antigens: several isoforms of Eno1p, pyruvate decarboxylase, pyruvate kynase, a protein of the 40S ribosomal subunit, triosephosphate isomerase, DL-glycerol phosphatase and fructose-bisphosphate aldolase. All these antibodies are the IgG2a isotype. The proteins described in the protective sera might be useful for future vaccine development.

Exclusive expression of transketolase in the vanadocytes of the vanadium-rich ascidian, Ascidia sydneiensis samea.

Ascidians, especially those belonging to the Ascidiidae, are known to accumulate extremely high levels of vanadium in vanadocytes, one type of blood (coelomic) cell. Vanadium, which exists in the +5 oxidation state in seawater, is accumulated in the vanadocytes and reduced to the +3 oxidation state. We have been trying to characterize all of the polypeptides specific to vanadocytes and to specify the proteins that participate in the accumulation and reduction of vanadium. To date, we have localized three enzymes in vanadocytes: 6-phosphogluconate dehydrogenase (6-PGDH: EC 1.1.1.44), glucose-6-phosphate dehydrogenase (G6PDH: EC 1.1.1.49), and glycogen phosphorylase (GP: EC 2.4.1.1), all of which are involved in the pentose phosphate pathway. In the current study, we cloned a cDNA for transketolase, an essential and rate-limiting enzyme in the non-oxidative part of the pentose phosphate pathway, from vanadocytes. The cDNA encoded a protein of 624 amino acids, which showed 61.8% identity to the human adult-type transketolase gene product. By immunocytochemistry and immunoblot analyses, the transketolase was revealed to be a protein that was expressed only in vanadocytes and not in any of the more than ten other types of blood cell. This finding, taken together with the localized expression of the other three enzymes, strongly supports the hypothesis that the pentose phosphate pathway functions exclusively in vanadocytes.

High control coefficient of transketolase in the nonoxidative pentose phosphate pathway of human erythrocytes: NMR, antibody, and computer simulation studies.

The degree of control exerted by transketolase over metabolite flux in the nonoxidative pentose phosphate pathway in human erythrocytes was investigated using transketolase antiserum to modulate the activity of that enzyme. 31P NMR enabled the simultaneous measurement of the levels of pentose phosphate pathway metabolites following incubation of hemolysates with ribose 5-phosphate. The variations in metabolic flux which occurred as the transketolase activity of hemolysate samples was altered indicated that a high degree of control was exerted by transketolase. Investigations using transaldolase-depleted hemolysates showed that transaldolase exhibits a lesser degree of control over pathway flux. Experimental data were compared with simulations generated by a computer model encompassing the reactions of the classical nonoxidative pentose phosphate pathway. The sensitivity coefficients (also called "control strengths" or "flux-control coefficients") calculated from the computer simulations were 0.74 and 0.03 for transketolase and transaldolase, respectively.

Transketolase from human leukocytes. Isolation, properties and induction of polyclonal antibodies.

Transketolase has been purified for the first time from human leukocytes, according to a new procedure which consists of three conventional steps. The enzyme was finally detached from CM-cellulose by specific elution with a D-xylulose-5-phosphate/D-ribose-5-phosphate mixture and the isolated product exhibited a specific activity of about 10 units/mg protein at 37 degrees C. Transketolase preparations are contamination-free, except for a slight residual activity of phosphohexose isomerase. Kinetic constants for D-xylulose 5-phosphate and D-ribose 5-phosphate were found to be 0.19 mM and 0.63 mM, respectively. Pure transketolase migrates on SDS/PAGE as a single band, with a molecular mass of about 66 kDa. The isoelectrophoretic heterogeneity of transketolase was assessed either by activity staining or immunovisualization with anti-transketolase antisera, previously induced in rabbits. These techniques yielded two practically overlapping patterns consisting of 6-8 distinct bands within a pI range of 6.5-8.5. Both pure and crude transketolase preparations showed a similar heterogeneous profile, thus confirming the stability of the enzyme throughout purification. The occurrence of multiple enzyme forms in fresh human white cells has also been established by the analysis of transketolase in isolated populations of either lymphocytes or polymorphonuclear leukocytes, from individual healthy subjects.

Western blotting assay of transketolase concentration in human hemolysates.

Using a rabbit anti-human transketolase antiserum and Western blotting we can determine nanogram amounts of transketolase in human hemolysates quantitatively. Transketolase concentration in 18 apparently healthy subjects was 55.7 +/- 12.1 micrograms/g Hb (mean +/- SD). Transketolase concentration correlated positively with the enzyme activity both with and without in vitro addition of thiamin pyrophosphate. However, the former had a closer correlation (r = 0.8418, P less than 0.001) than the latter (r = 0.6703, P less than 0.01). A heavy drinker with an extremely low transketolase activity had proportionally low concentration to the activity. These results indicate that transketolase in hemolysates, whether it is holoenzyme or apoenzyme activated in vitro, has an identical specific activity among all subjects studied and that the reduced activity of transketolase in alcoholics is due to the reduced content of the enzyme protein. This method is applicable to study the dynamics and the abnormality of apotransketolase in human hemolysates.

Purification and characterization of, and preparation of an antibody to, transketolase from human red blood cells.

Transketolase (sedoheptulose-7-phosphate: D-glyceraldehyde-3-phosphate glycolaldehydetransferase, EC 2.2.1.1) was purified 16 000-fold from human red blood cells, using DEAE-Sephadex A-50, Sephadex G-150, FPLC on Mono P, and Sephadex G-100. The purified enzyme migrated as a single protein band on SDS-polyacrylamide gel electrophoresis. The FPLC step resolved transketolase into three peaks, designated I, II and III. From results of re-FPLC on Mono P, SDS-polyacrylamide gel electrophoresis, gel filtration, catalytic studies, amino acid analysis and immunological studies, it was concluded that I, II and III were originally the same protein, modified during storage and purification. Transketolase had a subunit (Mr 70 000) and appeared to be composed of two identical subunits. 1 mol of subunit contained 0.9 mol of thiamine pyrophosphate. The pH optimum of the reaction lay within the range 7.6-8.0, and the Km values were determined to be 1.5 X 10(-4) M for xylulose 5-phosphate and 4.0 X 10(-4) M for ribose 5-phosphate. Hg2+ and p-chloromercuribenzoate inhibited the enzyme reaction, and the inhibition of the latter disappeared upon the addition of cysteine. Thiamine and its phosphate esters did not, but cysteine (1 X 10(-2) M) and ethanol (10% and 1% v/v) did activate the enzyme reaction. Antibody prepared to II bound all forms of transketolase in the hemolysate, but inhibited the reaction only about 20%.