Isoform identification and partial characterisation of Glutaminase in CD4+ T lymphocytes
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The proliferating phenotype is highly glycolytic and reliant on sufficient supply and synthesis of biosynthetic intermediaries to meet the demands of rapid cell replication. Glutamine contributes to this up-regulated synthetic machinery by providing critical anaplerotic carbon to the TCA cycle; nitrogen for nucleoside production; and, through its rapid conversion to lactate, the reducing agent NADPH necessary to maintain the increase in anabolic activity. Glutaminase represents a key step in this catabolism of glutamine and has been shown to be up-regulated in many different proliferating tissues. It presents a target for the possible suppression of undesired proliferation in over-reactive immunity. The expression of the three Glutaminase isoforms (KGA, LGA & GAC) where partially characterized using Western Blots in mature naïve and proliferating human CD4+ T lymphocytes after TCR-CD3/CD28 activation. Two Glutaminase inhibitors, BPTES and compound 968, were evaluated through 3H-Thymidine incorporation assays and mass spectrometry analysis of glutamine flux. In addition, the effects of glutamine removal from proliferating populations of human CD4+ T lymphocytes were also analysed Our results showed glutamine is absolutely necessary for optimal CD4+ T lymphocyte proliferation and that the Glutaminase isoform GAC is up-regulated in proliferating human CD4+ T lymphocytes. Its inhibition leads to attenuation in proliferative activity.