Glutaminase inhibition impairs CD8 T cell activation in STK11-/Lkb1-deficient lung cancer.

The tumor microenvironment (TME) contains a rich source of nutrients that sustains cell growth and facilitate tumor development. Glucose and glutamine in the TME are essential for the development and activation of effector T cells that exert antitumor function. Immunotherapy unleashes T cell antitumor function, and although many solid tumors respond well, a significant proportion of patients do not benefit. In patients with KRAS-mutant lung adenocarcinoma, KEAP1 and STK11/Lkb1 co-mutations are associated with impaired response to immunotherapy. To investigate the metabolic and immune microenvironment of KRAS-mutant lung adenocarcinoma, we generated murine models that reflect the KEAP1 and STK11/Lkb1 mutational landscape in these patients. Here, we show increased glutamate abundance in the Lkb1-deficient TME associated with CD8 T cell activation in response to anti-PD1. Combination treatment with the glutaminase inhibitor CB-839 inhibited clonal expansion and activation of CD8 T cells. Thus, glutaminase inhibition negatively impacts CD8 T cells activated by anti-PD1 immunotherapy.

The glutaminase (CgGLS-1) mediates anti-bacterial immunity by prompting cytokine synthesis and hemocyte apoptosis in Pacific oyster Crassostrea gigas.

Glutaminase, an amidohydrolase enzyme that hydrolyzes glutamine to glutamate, plays crucial roles in various immunomodulatory processes such as cell apoptosis, proliferation, migration, and secretion of cytokines. In the present study, a glutaminase homologue (designated as CgGLS-1) was identified from Pacific oyster Crassostrea gigas, whose open reading frame was of 1836 bp. CgGLS-1 exhibited high sequence identity with vertebrate kidney-type GLS, and closely clustered with their homologues from mollusc C. virginica. The enzyme activity of recombinant CgGLS-1 protein (rCgGLS-1) was estimated to be 1.705 U/mg. CgGLS-1 mRNA was constitutively expressed in all the tested tissues of oysters, with the highest expression level in hemocytes. CgGLS-1 mRNA expression in hemocytes was significantly up-regulated and peaked at 6 h (2.07-fold, p < 0.01) after lipopolysaccharide (LPS) stimulation. The CgGLS-1 protein was mainly distributed in the cytoplasm with a significant co-location with mitochondria in oyster hemocytes. The content of Glu in the oyster serum was significantly decreased after the inhibition of CgGLS-1 using specific inhibitor Bis-2- [5-(phenyl acetamido)-1,3,4-thiadiazol-2-yl] ethyl sulfide (BPTES), and the expression levels of CgmGluR6, CgAP-1, cytokines CgIL17-5 and CgTNF-1 were significantly decreased after BPTES and LPS stimulation. The transcripts of CgCaspase3 as well as the apoptosis index of hemocytes were also decreased. These results collectively suggest that CgGLS-1 is the enzyme to synthesize Glu in oyster, which can modulate anti-bacterial immunity by regulating the secretion of pro-inflammatory cytokines CgIL17-5 and CgTNF-1, as well as hemocyte apoptosis.

TNFα increases STAT3-mediated expression of glutaminase isoform KGA in cultured rat astrocytes.

Glutamate related excitotoxicity and excess of cerebral levels of tumor necrosis factor alpha (TNFα) are interrelated and well documented abnormalities noticed in many central nervous system diseases. Contribution of kidney type glutaminase (KGA) and shorter alternative splicing form (GAC) to glutamine degradation in astrocytes has been recently a matter of dispute and extensive study but the regulation of the GLS isoforms by inflammatory factors is still not well known. Here we show that treatment of cultured rat cortical astrocytes with pathophysiologically relevant (50 ng/ml) concentration of TNFα specifically increases the expression of KGA but not GAC and increases activity of GLS. No changes in the expression of either of two GLS isoforms were observed following treatment with other tested cytokines IL-1ß and IL-6. The TNFα mediated KGA expression was associated with increased phosphorylation of signal transducer and activator of transcription 3 (STAT3). Stimulatory effect of TNF-α on KGA expression was reduced by selective inhibition of (STAT3) but not by inhibition of STAT1 nor nuclear transcription factor kappa. Additionally, the role of miRNA in TNFα-induced expression of KGA in astrocytes was excluded, since the expression of miR-23a/b and miR-200c, potential regulators of KGA expression, was unchanged. This study documents increased KGA expression in the astrocytes under inflammatory stimulation, identifying TNFα as a cytokine mediating this response, and demonstrates the specific and selective involvement of STAT3.

Distinct Regulation of Th17 and Th1 Cell Differentiation by Glutaminase-Dependent Metabolism.

Activated T cells differentiate into functional subsets with distinct metabolic programs. Glutaminase (GLS) converts glutamine to glutamate to support the tricarboxylic acid cycle and redox and epigenetic reactions. Here, we identify a key role for GLS in T cell activation and specification. Though GLS deficiency diminished initial T cell activation and proliferation and impaired differentiation of Th17 cells, loss of GLS also increased Tbet to promote differentiation and effector function of CD4 Th1 and CD8 CTL cells. This was associated with altered chromatin accessibility and gene expression, including decreased PIK3IP1 in Th1 cells that sensitized to IL-2-mediated mTORC1 signaling. In vivo, GLS null T cells failed to drive Th17-inflammatory diseases, and Th1 cells had initially elevated function but exhausted over time. Transient GLS inhibition, however, led to increased Th1 and CTL T cell numbers. Glutamine metabolism thus has distinct roles to promote Th17 but constrain Th1 and CTL effector cell differentiation.

Understanding the roles of glutamine synthetase, glutaminase, and glutamate decarboxylase autoantibodies in imbalanced excitatory/inhibitory neurotransmission as etiological mechanisms of autism.

AIM: Autism is a heterogeneous neurological disorder that is characterized by impairments in communication and social interactions, repetitive behaviors, and sensory abnormalities. The etiology of autism remains unclear. Animal, genetic, and post-mortem studies suggest that an imbalance exists in the neuronal excitation and inhibition system in autism. The aim of this study was to determine whether alterations of the measured parameters in children with autism are significantly associated with the risk of a sensory dysfunction. METHODS: The glutamine synthetase (GS), kidney-type glutaminase (GLS1), and glutamic acid decarboxylase autoantibody levels were analyzed in 38 autistic children and 33 age- and sex-matched controls using enzyme-linked immunosorbent assays. RESULTS: The obtained data demonstrated significant alterations in glutamate and glutamine cycle enzymes, as represented by GS and GLS1, respectively. While the glutamic acid decarboxylase autoantibodies levels were remarkably increased, no significant difference was observed compared to the healthy control participants. CONCLUSION: The obtained data indicate that GS and GLS1 are promising indicators of a neuronal excitation and inhibition system imbalance and that combined measured parameters are good predictive biomarkers of autism.

Decrease by 50% of plasma IgA tissue transglutaminase antibody concentrations within 2 months after start of gluten-free diet in children with celiac disease used as a confirming diagnostic test.

BACKGROUND: Histological examination of small bowel biopsies is normally the gold standard for the diagnosis of celiac disease (CD). The objective of this study was to investigate whether the rate of decreases in elevated plasma IgA tissue transglutaminase antibody (IgA-tTG) and/or IgG deamidated gliadin peptides antibody (IgG - DGP) concentrations could be used as a confirming test for CD in children on a gluten-free diet (GFD) when biopsy was omitted in the diagnostic process. METHODS: In this retrospective study we compared children (≤18 years old) with a CD-confirming biopsy (n = 16) to children without a biopsy (n = 18). After initiation of GFD the antibody half-life (the time (T½) when the antibody concentration is 50% decreased) was determined in all children. RESULTS: Children with a biopsy (IgA-tTG, T½ = 1.9 months; IgG - DGP, T½ = 2.2 months) and children without a biopsy (IgA-tTG, T½ = 1.6 months; IgG - DGP, T½ = 2.7 months) had comparable T½ (mean) results (p < 0.05) supporting that all children had the CD diagnosis. CONCLUSIONS: When biopsy was omitted a rapid rate of decrease in CD antibody concentrations confirmed the CD diagnosis in children on GFD. The half-lives (T½) of IgA-tTG were less than 2 months in CD children.

Prevalence of coeliac disease in healthy blood donors: a study from north India.

BACKGROUND: Blood donor screening can help predict prevalence of coeliac disease in population. METHODS: Between December 2010 and June 2011, healthy blood donors were screened using anti-tissue glutaminase antibodies. Those positive underwent duodenoscopy. Their age, gender, body mass index and haemoglobin and histological changes were recorded. RESULTS: Of the 1610 blood donors screened, 1581 (98.2%) were males. The mean age of donors was 31.51 ± 9.66 years and the mean body mass index was 22.12 ± 4.24 kg/m(2). Nine (0.56%) men were seropositive. Endoscopic features included reduced fold height (9), scalloping (8), grooving (7) and mosaic mucosal pattern (3). Eight had Marsh IIIa changes whilst one had IIIb change. The prevalence of coeliac disease was 1:179 (0.56%, 95% confidence interval 1/366-1/91, 0.27-1.1%). None of the 9 patients had any symptoms. Their mean haemoglobin and body-mass index was similar to rest of the cohort. CONCLUSION: The prevalence of coeliac disease amongst apparently healthy blood donors was 1:179 (0.56%).

Malabsorption may contribute to malnutrition in the elderly.

Malnutrition, either actually malnourished or at risk, is present in 80% of the elderly population presenting to hospital for admission. Although many factors contribute to this situation, one yet to be explored is malabsorption. We therefore aimed to assess nutritional status as well as the prevalence of altered mucosal permeability and celiac disease among a group of elderly patients presenting for rehabilitation. Forty-eight subjects were recruited (16 females) with a mean age of 83.7 (SD 6.1), body mass index 21.8 kg/m(2) (SD 3.9), mini-nutritional assessment (MNA) 19.5 (SD 3.4). They had no current gastrointestinal symptoms and undertook an assessment of mucosal permeability using the dual sugar absorption test of lactulose (7.5 g) and rhamnose (1 g). Ten of the 48 subjects had increased mucosal permeability with an L:R ration ranging from 0.0860 to 7.706 (N 0.01-0.08). These subjects were all at risk or malnourished according to the MNA score and they had a significantly lower mean MNA score of 17.2 (SD 3.5) compared to normal absorbers with a mean of 19.5 (SD 3.4). Two of the subjects had positive tissue trans-glutaminase antibodies. The higher risk of potential malabsorption in this elderly population has significant implications both for nutritional supplementation and for drug absorption as well as being a possible major contributor to malnutrition.

Expression of recombinant human L-glutaminase in Escherichia coli: polyclonal antibodies production and immunological analysis of mouse tissues.

The first complete sequence of human L-glutaminase was deduced from breast cancer glutaminase cDNA cloned in our laboratory. This cDNA clone has now been engineered to synthesize both precursor and mature forms of the protein in Escherichia coli. Among several different plasmid constructions, the expression system based on phage T7 promoter (vector pET-3c) was found to be the most efficient for glutaminase overproduction. Upon induction, precursor glutaminase accounts for about 25% of total E. coli protein, whereas a lower amount (12%) was achieved for the putative mature protein. The optimal length of the translational spacer on the ribosome binding site was shown to be eight nucleotides. However, using this length of spacer, we were unable to obtain expression in the pQE vector, tagged with a 6x His sequence at the NH(2)-terminus, stressing the importance of the 5'-coding sequence in the expression efficiency. Although the precursor and mature recombinant forms of glutaminase were devoid of catalytic activity, the purified protein allowed us to obtain highly specific polyclonal antibodies, as shown by immunoblot analysis of mouse tissues. Furthermore, the antibodies were able to immunoprecipitate the in vitro translated enzyme using a reticulocyte lysate system; these antibodies might be a valuable tool for studies on L-glutaminase expression in mammalian tissues.

Ehrlich ascites tumor cells expressing anti-sense glutaminase mRNA lose their capacity to evade the mouse immune system.

Glutaminase (EC 3.5.1.2) is a key enzyme in rapidly proliferating cells. Using anti-sense technology, an Ehrlich ascites tumor cell line (0.28AS-2) with reduced glutaminase activity has been obtained. We investigated the in vivo growth characteristics of the 0.28AS-2 cells. When injected i.p. into normal Swiss albino mice, the 0.28AS-2 cells were unable to grow. On the contrary, when injected into nude mice, they developed into solid tumors. Mice inoculated with 0.28AS-2 cells kept immunologic memory and rejected a second inoculation with parental Ehrlich ascites tumor cells. Expression of both polymorphic epithelial mucin-1 (MUC-1) and the enzyme N-acetyl-alpha-D-galactosaminidase, proteins implicated in host immune system escape, were markedly diminished in 0.28AS-2 cells. Study of the immune system response in mice inoculated with 0.28AS-2 cells revealed an increase in splenic CD18 cells and the presence of a large number of activated F4/80+ macrophages in the ascites cavity. These features, not observed in mice inoculated with parental Ehrlich ascites tumor cells, indicate that a distinctive, strong immune response occurred in animals inoculated with 0.28AS-2 cells. Our results suggest that inhibition of glutaminase expression using anti-sense technology induces phenotypic changes in Ehrlich ascites tumor cells that allow the development of an effective anti-tumor immune response, which makes the cells unable to develop in vivo tumors.

Neurotransmission in the vestibular endorgans--glutamatergic transmission in the afferent synapses of hair cells.

In the sensory pathways the first synapse is that between hair cells and primary afferent neurons and its most likely neurotransmitter candidate has long been thought to be glutamate. A number of pharmacological and electrophysiological studies have lent credence to this theory (reviewed by Bledsoe et al. 1988, Bobbin 1979, Ehrenberger and Felix 1991, Puel et al. 1991; Puel 1995) as has recent neurochemical and immunocytochemical work (reviewed by Ottersen et al. 1998; Usami et al. 2000). These recent studies reveal that the afferent hair cell synapse resembles the central glutamate synapses in many ways. Of the proteins confirmed to be involved in signal transduction and transmitter metabolism at most central synapses, many are also seen in the afferent hair cell synapse, and have an analogous compartmentation. On the other hand, there are also important differences, especially those related to the molecular mechanisms that underlie transmitter release.

[Antibodies against tissue transglutaminase as a serological marker in dermatitis herpetiformis During]. / Antikörper gegen Gewebstransglutaminase als serologischer Marker bei Dermatitis herpetiformis Duhring.

Dermatitis herpetiformis is associated with celiac disease. IgA antibodies to endomysium are considered as sensitive and specific markers for celiac disease. Recently, tissue transglutaminase was identified as the antigen of anti-endomysium antibodies. Moreover, serum levels of IgA antibodies to tissue transglutaminase were found to correlate with titers of IgA antibodies to endomysium in patients with both celiac disease and dermatitis herpetiformis. These findings confirm the close pathogenic relation between the two diseases. The determination of serum levels of antibodies to tissue transglutaminase may be a tool that can be routinely used for the diagnosis of dermatitis herpetiformis in the future.

Characterization of glutaminase in the developing rat small intestine.

As a primary substrate in the small intestine, glutamine is a very important source of energy. Glutaminase (GA) is the enzyme involved in the deamination of glutamine to glutamate, which is utilized for energy production via the TCA cycle. Although the enzymatic activity of GA in the small intestine is known to undergo maturational changes, the tissue localization of the protein and its mRNA, the intracellular processing of this enzyme and levels of its mRNA in the small intestine at different maturational stages have not yet been described. In this study, using immuno-histochemical staining, we confirm previous studies using other techniques that suggested GA is localized in the epithelial layer of the rat small intestine. Some GA is also found in cells of the lamina propria and crypt epithelium. Using in situ hybridization studies, we have corroborated the presence of the protein in the epithelial cells of the villi by localizing the mRNA of this protein to the same layer and its precursor layer in the crypt region. An ontogenic analysis of GA mRNA and protein from rat small intestines, using RNA dot blots, gel blots and protein immunoblotting revealed differences in immunoreactive GA protein and mRNA during maturation. Immunoreactive GA and steady-state levels of GA mRNA increased around the 3rd wk of life, coincident with weaning and the endogenous glucocorticoid surge. Whether these findings have nutritional or pathophysiological implications remains speculative.

Tumor glutaminase purification.

Two alternative purification schemes to obtain the glutaminase from Ehrlich tumor cells in a highly purified form have been developed. One experimental approach is based on conventional and high-performance liquid chromatography fractionation techniques, yielding a 37-fold higher purification than has been previously reported. The method comprises: isolation of mitochondria, solubilization with Triton X-100, ion-exchange and hydroxyapatite chromatography, ammonium sulfate precipitation, and hydrophobic interaction chromatography. A second purification schedule has been optimized employing native polyacrylamide gel electrophoresis, in situ activity staining, and electroelution of the protein band. This approach resulted in a simple and rapid isolation of a 10-fold higher purified glutaminase than before, minimizing also the potential for proteolytic inactivation of the enzyme. The apparent molecular weight of the protein in native form was determined by gel filtration and sucrose density gradient ultracentrifugation. Polyclonal antibodies raised against Ehrlich glutaminase were immunopurified against the pig kidney enzyme. Immunoblot analyses employing these antibodies as well as anti-rat kidney glutaminase antibodies revealed the same pattern of bands seen with the purified enzyme.

The vitamin D3 hydroxylase-associated protein is a propionamide-metabolizing amidase enzyme.

Previously we isolated a novel protein that coimmunoprecipitates with the 1,25-dihydroxyvitamin D3-24R-hydroxylase and 25-hydroxyvitamin D3-1 alpha-hydroxylase. This kidney-specific protein found in the inner membrane of mitochondria is named the vitamin D3 hydroxylase-associated protein (VDHAP). To determine a putative function for this protein, an extensive computer search of the deduced amino acid sequence of VDHAP was performed. A BLAST homology search identified amino acid residues 133 through 321 in acetamidase from Aspergillus nidulans that exhibit 38% amino acid identify and 65% amino acid similarity to VDHAP. A protein consensus sequence dictionary, MOTIFS, identified an amidase consensus sequence in VDHAP. This sequence, G-G-S-S-G-G-E-G-A-L-I-A-G-G-G-S-L-L-G-I-G-S-D-V-A-G-S-I-R-L-P-S, in VDHAP is located between amino acids 223 and 254. Propionamide, acetamide, and acrylamide were identified as substrates for an amidase activity in soluble chicken kidney mitochondria. Propionamide is the best substrate with a Vmax of 16.7 nmol NH4+/min/mg protein and an apparent Km of 7.9 mM in soluble chicken kidney mitochondria. A VDHAP monoclonal antibody, IVC2G8, immunoprecipitates 78% of the total propionamidase activity in soluble chicken kidney mitochondria. These results suggest that VDHAP is a propionamidase enzyme in soluble chicken kidney mitochondria and a member of the amidase signature gene family.

Glutaminase immunoreactive neurons in the rat dorsal root ganglion contain calcitonin gene-related peptide (CGRP).

The co-localization of glutaminase and calcitonin gene-related peptide (CGRP) was examined with immunohistochemistry in the rat dorsal root ganglion (DRG). The majority of the DRG neurons were immunoreactive for glutaminase and all DRG neurons that contained CGRP also contained glutaminase. These results indicate that some DRG neurons release glutamate and CGRP from the same axon terminals in the spinal cord. Co-release of glutamate and CGRP from primary afferents may have multiple effects including fast and slow neurotransmission of sensory information in the spinal cord.

Paucity of glutaminase-immunoreactive nonpyramidal neurons in the rat cerebral cortex.

Glutaminase has been considered to be a synthesizing enzyme of transmitter glutamate in pyramidal neurons of the cerebral cortex. In the present study, an attempt was made to examine with a double immunofluorescence method whether or not nonpyramidal neurons of the cerebral cortex are immunoreactive for glutaminase. Glutaminase was stained with mouse anti-glutaminase IgM and FITC-labeled anti-[mouse IgM] antibody. In the same section, parvalbumin (PA), calbindin (CB), choline acetyltransferase (CAT), vasoactive intestinal polypeptide (VIP), corticotropin releasing factor (CRF), cholecystokinin (CCK), somatostatin (SS), or neuropeptide Y (NPY) was visualized as a marker for nonpyramidal neurons with an antibody to each substance, biotinylated secondary antibody and Texas Red-labeled avidin. Virtually no glutaminase immunoreactivity was seen in PA-, CB-, CAT-, VIP-, CRF-, CCK-, SS-, or NPY-immunoreactive neuronal perikarya in the neocortex and mesocortex (cingulate and retrosplenial cortices), although it was detected in a few PA-, CB-, VIP-, CCK-, SS-, or NPY-immunoreactive nonpyramidal neurons in the piriform, entorhinal, and hippocampal cortices. PA- and CB-positive neurons have been reported to constitute the major population of GABAergic neurons in the cerebral cortex. Thus, the present results, together with the previous reports, suggest that most GABAergic, cholinergic and peptidergic nonpyramidal neurons in the neo- and mesocortex do not contain glutaminase.

Mosaic distribution of phosphate-activated glutaminase-like immunoreactivity in the rat striatum.

The dorsal and ventral striatum of mammals has been known to be organized in a mosaic manner, referred to as "patches" and "matrix" of the caudatoputamen. The present study was primarily attempted in order to reveal the relationship of glutamatergic neuronal components to the mosaic organization in the rat striatum by using a monoclonal antibody to phosphate-activated glutaminase, a major synthetic enzyme of transmitter glutamate. Antibodies against glutamate decarboxylase and choline acetyltransferase were also used as the markers for GABAergic and cholinergic neuronal components, respectively. Glutaminase immunoreactivity was seen in a number of large- and a few medium-sized neurons in the caudatoputamen, nucleus accumbens and olfactory tubercle. The large neurons with glutaminase immunoreactivity were observed in the neuropil of the caudatoputamen and nucleus accumbens; glutaminase immunoreactivity was particularly marked in the neuropil of island-like patchy areas although it was seen throughout the neuropil of the nuclei. In the caudatoputamen, island-like areas with marked glutaminase immunoreactivity exhibited less marked choline acetyltransferase immunoreactivity than the surrounding background region, and were thus considered to correspond to the patches. The mosaic distribution of glutamate decarboxylase immunoreactivity in the caudatoputamen seemed identical with that of glutaminase immunoreactivity. However, in the nucleus accumbens, the mosaic pattern of neuropil labeling for glutaminase was neither consistent with that for glutamate decarboxylase nor that for choline acetyltransferase, suggesting the presence of non-GABAergic glutaminase-containing nerve terminals in the nucleus. In an attempt to clarify the origin of neuropil labeling for glutaminase in the striatum, lesions were made in the regions sending projection fibers to the caudatoputamen and nucleus accumbens. After placing lesions in the cerebral cortex, glutaminase immunoreactivity was decreased in neuropil of the caudatoputamen, but the mosaic pattern remained. Lesions which were placed in the intralaminar thalamic nuclei, amygdaloid body, globus pallidus or substantia nigra produced no substantial change in glutaminase immunoreactivity in the caudatoputamen and nucleus accumbens. After injection of kainic acid into the caudatoputamen or nucleus accumbens, glutaminase immunoreactivity in the neuropil of the affected regions was decreased to lose the mosaic pattern, indicating that neuronal components with glutaminase immunoreactivity in the neuropil of the patches were mainly of intrinsic origin. In summary, possible axon terminals containing glutaminase were observed with mosaic patterns in the caudatoputamen and nucleus accumbens, in which large cholinergic and medium-sized non-cholinergic neurons were immunoreactive for glutaminase. In the caudatoputamen, glutaminase immunoreactivity in neuropil was more marked in the patches than in the matrix.(ABSTRACT TRUNCATED AT 400 WORDS)

Enhancement of glutaminase-like immunoreactivity in rat brain by an irreversible inhibitor of the enzyme.

Changes of glutaminase immunoreactivity in rat brain were examined after intracranial injection of 6-diazo-5-oxo-L-norleucine (DON), an irreversible inhibitor of glutaminase. When 1 M DON was injected into the lateral ventricle, a half-lethal dose was 7.5-10 mumol. After intraventricular injection of 2-7.5 mumol DON, glutaminase immunoreactivity was dose dependently enhanced with the maximum enhancement 3-5 days after the injection. The enhanced glutaminase immunoreactivity was recognized by enlarged granular immunodeposits in both perikarya and neuropil in many regions, such as the hippocampus, thalamus, hypothalamus, periaqueductal gray, and some brain stem, cerebellar, and spinal cord regions. Intrathalamic injection of 0.2 mumol DON enhanced glutaminase immunoreactivity in many neuronal perikarya in the thalamus and in some perikarya in layer VI of the cerebral cortex. Intrastriatal injection of the same dose of DON enhanced glutaminase immunoreactivity in neuropil of the caudoputamen and in many neuronal perikarya of the intralaminar thalamic nuclei. These results suggested that DON induced a new massive synthesis of glutaminase in the affected neurons.

Monoclonal antibodies against bacterial glucosamine 6-phosphate synthase: production and use for structural studies.

Fifteen mouse x rat hybridoma cell lines producing rat monoclonal antibodies (MAbs) directed to Escherichia coli Glucosamine 6-P Synthase (GlmS) were established and characterized. Most of them (13/15) are IgG2a while 2 were typed as IgG1. Their Kaff ranged from 1.5 x 10(6) to 9.6 x 10(8) M-1 as determined by Beatty et al. (1). The epitopes recognized by these MAbs were assigned to one of the two catalytical domains of the enzyme (CT1 and CT2) as demonstrated both by ELISA and Western-blotting using purified GlmS proteolytic fragments. The binding of the MAbs on either the native or denatured forms of GlmS, CT1 and CT2 was further analyzed by competitive immunoassay and most of the MAbs were found to bind preferentially to the denatured proteins. The study of the antigenic topography of GlmS by competitive radioimmunoassay demonstrated the existence of at least 10 independent epitopes on GlmS, divided into three groups. The first one (3/15) includes MAbs whose binding was not inhibited by any of the other MAbs. The second group (9/15) is comprised of MAbs that exhibit reciprocal binding inhibitory activity while the third group includes MAbs (3/15) presenting asymmetric inhibitory activity. Finally, since most of the isolated antibodies (10/15) bind to the 27 kDa amino-terminal glutamine binding domain (CT2), the capacity of these MAb to interfere with the associated glutaminase activity was analyzed.