Characterisation of the structural and functional correlation between GAD65 and GAD67

2017-05-18T02:32:44Z (GMT) by Langendorf, Christopher Grant
γ-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian central nervous system (CNS) and is critical for normal motor function, neurogenesis, neural plasticity, cognition and learning. GABA is produced by two different isoforms of an enzyme, the pyridoxal-5’-phosphate (PLP) dependent enzyme, glutamic acid decarboxylase (GAD). The 65kDa and 67kDa isoforms of GAD, (GAD65 and GAD67), share ~70 % sequence identity and function synchronously to produce and regulate the levels of GABA. GAD67 is constitutively active and is found the majority of the time in the PLP-bound (active, holoGAD) form, where it produces basal levels of GABA. GAD65 is transiently active and is largely found in the PLP-unbound (inactive; apoGAD) form. However, GAD65 can be switched on by PLP in response to extra demand for GABA. In contrast to GAD67, PLP-bound active holoGAD65 readily auto-inactivates to form the inactive PLP-unbound apoGAD65. The cycle of interconversion between apoGAD and holoGAD GAD65 is critical for the proper regulation of GABA homeostasis. Despite their high sequence identity, GAD65 but not GAD67 is an auto-antigen in Type 1 diabetes (T1D). GAD65 autoantibodies can be detected up to 10 years before clinical onset. Autoantibodies to GAD65 have been epitope mapped onto the surface of GAD65, with the most significant reactive region being within the flexible C-terminal domain. Autoantibodies to GAD67 are predicted to be a cross-reactive population of anti-GAD65 antibodies. Perturbations in the levels of GABA are implicated in many disorders in humans such as, epilepsy, Parkinson’s disease, Schizophrenia and post-traumatic stress disorder (PTSD). Treatments for some of the aforementioned diseases centre on chemically increasing GABA levels in the body. To date most of the known molecular components of the inhibitory neurotransmitter system except GAD have been targeted by chemical therapeutics. However, current GABA therapeutics that block GABA degradation and transportation have significant side effects, as they act indiscriminately throughout the body. To reduce the likelihood of side effects an approach to positively modulate GAD activity, which is activated when and where the body requires GABA, would be better. Currently no drug exists that enhances GAD activity in vivo. There are a number of technical challenges that hamper the search for GAD-targeted therapeutics. Firstly, there is no high-throughput approach to measure GAD activity; secondly, detailed understanding of the function of structural motifs within GAD during normal catalytic activity and auto-inactivation remain unclear. The first aim of this study was to develop a high throughput screening method that could be used to screen sizable libraries of compounds (>100,000) against GAD activity. Current methods (all of which rely on trapping radiolabelled product) are not appropriate for use in a high-throughput screen, therefore a new method for measuring GAD activity needed to be established. Herein is presented the development of a three-enzyme colorimetric assay that utilizes two reporter enzymes coupled to the activity of GAD to measure real-time production of GABA by GAD. The assay is suitable for both high-throughput drug screening and has been used to screen 120,000 compounds and GAD steady state enzyme kinetics. Here, this assay is further used to kinetically characterize wild-type and mutant enzymes. These data reveal that structural flexibility aids substrate binding, while structural stability increases product turnover. Further, the impact of catalytic and regulatory residues is also determined. The structural mechanisms involved in GAD auto-inactivation are key to understanding GAD65 regulation. Structural comparisons between the two GAD isoforms revealed that a key part of the catalytic machinery (the “catalytic loop”) is located on a loop that is mobile in GAD65. In contrast, the corresponding region in GAD67 is stable. Additionally the C-terminal domain of GAD65, which packs against the catalytic loop, showed increased flexibility when compared to GAD67. Accordingly, it is suggested that mobility of the catalytic loop and C-terminal domain results in enhanced auto-inactivation of GAD65 in comparison to GAD67. This study aims to further test the idea that the catalytic loop and C-terminal domain are important for auto-inactivation through interchanging both regions between GAD65 and GAD67 and measuring the rates of auto-inactivation. These mutational data are further supported through crystal structure determination. The results of this study demonstrate that a mutant GAD65 can be constructed that no longer auto-inactivates. Further, the study revealed that GAD67 could be mutated to a form that auto-inactivates at a rate comparable to that of GAD65. Together, these data permit a comprehensive description of the mechanism of GAD65 auto-inactivation. Finally, the idea that GAD67 reactivity occurs through GAD65-autoantibody cross reactivity was tested. To achieve this, the cross-reactivity of the C-terminal domain to autoantibodies was tested by producing C-terminal domain swap GAD chimaeras. The results reveal that autoantibodies from T1D binds to sequences both within and outside the C-terminal domain. These data suggest that binding to GAD67 is not a result of cross reactivity to GAD65.