GABA production by glutamic acid decarboxylase is regulated by a dynamic catalytic loop

Fenalti, Gustavo; Law, Ruby H. P.; Buckle, Ashley M.; Langendorf, Christopher G.; Tuck, Kellie L.; Rosado, Carlos J.; Faux, Noel; Mahmood, Khalid; Hampe, Christiane S.; Banga, J. Paul; Wilce, Matthew Charles James; Schmidberger, J.W.; Rossjohn, Jamie; El‐Kabbani, Ossama; Pike, Robert N.; Smith, A. Ian; Mackay, Ian R.; Rowley, M.J.; Whisstock, James C.

doi:10.1038/nsmb1228

Cited by 209 publications

(216 citation statements)

References 39 publications

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“…The 2.3-Å crystal structures of GAD67 and GAD65 have been reported (5). Both isoforms adopt the same fold and formed obligate dimers from two monomeric units, each comprising the NH 2 -terminal, PLPbinding, and COOH-terminal domains ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Presently, unanswered questions include the precise location of epitope sites and critical binding residues for autoantibody recognition, the differing immune reactivity of the GAD65 versus the GAD67 isoform, and the failure of natural immune tolerance to GAD65 in the first place. These questions prompted the recent crystallographic structure determination of both GAD67 and GAD65 isoforms (5).…”

mentioning

confidence: 99%

“…Details of the purification, crystallization, and structural determination of human GAD65 and GAD67 have been published previously (5). We used for crystallization an NH 2 -terminally truncated form of each isoform (hereinafter referred to as GAD67 and GAD65) that lacked the first 89 and 83 residues, respectively; the NH 2 -terminal truncation facilitated purification because this region is hydrophobic and highly susceptible to proteolysis and did not affect enzymatic properties (5) nor reactivity with specific antisera (12)(13)(14).…”

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confidence: 99%

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COOH-Terminal Clustering of Autoantibody and T-Cell Determinants on the Structure of GAD65 Provide Insights Into the Molecular Basis of Autoreactivity

et al. 2008

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OBJECTIVE-To gain structural insights into the autoantigenic properties of GAD65 in type 1 diabetes, we analyzed experimental epitope mapping data in the context of the recently determined crystal structures of GAD65 and GAD67, to allow "molecular positioning" of epitope sites for B-and T-cell reactivity. RESEARCH DESIGN AND METHODS-Datawere assembled from analysis of reported effects of mutagenesis of GAD65 on its reactivity with a panel of 11 human monoclonal antibodies (mAbs), supplemented by use of recombinant Fab to crossinhibit reactivity with GAD65 by radioimmunoprecipitation of the same mAbs. RESULTS-The COOH-terminal region on GAD65 was the major autoantigenic site. B-cell epitopes were distributed within two separate clusters around different faces of the COOHterminal domain. Inclusion of epitope sites in the pyridoxal phosphate-and NH 2 -terminal domains was attributed to the juxtaposition of all three domains in the crystal structure. Epitope preferences of different mAbs to GAD65 aligned with different clinical expressions of type 1 diabetes. Epitopes for four of five known reactive T-cell sequences restricted by HLA DRB1*0401 were aligned to solvent-exposed regions of the GAD65 structure and colocalized within the two B-cell epitope clusters. The continuous COOH-terminal epitope region of GAD65 was structurally highly flexible and therefore differed markedly from the equivalent region of GAD67.CONCLUSIONS-Structural features could explain the differing antigenicity, and perhaps immunogenicity, of GAD65 versus GAD67. The proximity of B-and T-cell epitopes within the GAD65 structure suggests that antigen-antibody complexes may influence antigen processing by accessory cells and thereby T-cell reactivity. Diabetes 57:1293-1301, 2008 T ype 1 diabetes is characterized by autoimmune reactivity to islet cell antigens that include the 65-kDa isoform of GAD (GAD65). Of the two isoforms of GAD, GAD67 and GAD65, only GAD65 is autoantigenic, in diseases that include particularly type 1 diabetes (1,2). Importantly, the specificity of antibodies for particular epitopes on GAD65 rather than their actual levels may be a better indicator of impending or actual destruction of islet ␤-cells. Thus in genetically prone individuals, changes in the focus of autoantibody responses to epitopes of GAD65, i.e., intramolecular epitope shifts during the period of pre-diabetic insulitis, herald the onset of overt type 1 diabetes (3).B-cell/autoantibody epitopes on GAD65 engage conformational determinants that are widely distributed over the linear sequence of the three domains, NH 2 -terminal (amino acids 1-234), pyridoxal phosphate (PLP)-binding (235-442), and COOH-terminal (443-585) domains (4). Presently, unanswered questions include the precise location of epitope sites and critical binding residues for autoantibody recognition, the differing immune reactivity of the GAD65 versus the GAD67 isoform, and the failure of natural immune tolerance to GAD65 in the first place. These questions prompted the recent crystallographic s...

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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confidence: 99%

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COOH-Terminal Clustering of Autoantibody and T-Cell Determinants on the Structure of GAD65 Provide Insights Into the Molecular Basis of Autoreactivity

et al. 2008

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“…Although the two isoforms display a high similarity in their amino acid sequences, differences are found in their interaction with the co-factor pyridoxal-5ƍ-phosphate and in enzyme kinetics for GABA production (Battaglioli 2003). Whereas cytosolic GAD67 is found to be constitutively active and is responsible for basal levels of GABA, the inducible cytosolic GAD65 associated with the GABA vesicle membrane is mostly present without its co-factor as an autoinactivated apoenzyme (Fenalti 2007). Upon reactivation with its co-factor, apo-GAD65 becomes holo-GAD65 that can catalyze synthesis of GABA when additional neurotransmitter is needed (Kash 1997).…”

Section: Glutamic Acid Decarboxylasementioning

confidence: 99%

MS and clinically isolated syndromes: Shared specificity but diverging clonal patterns of virus‐specific IgG antibodies produced in vivo and by CSF B cells in vitro

Skorstad

Vandvik

Vartdal

et al. 2009

Euro J of Neurology

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Background: Intrathecal synthesis of oligoclonal IgG antibodies against measles virus (MeV), varicella zoster virus (VZV) and herpes simplex virus type‐1 (HSV‐1) is a characteristic feature multiple sclerosis (MS). Methods: We have used isoelectric focusing‐immunoblot to define the clonal patterns of IgG and of IgG antibodies to MeV, VZV and HSV‐1 in supernatants of in vitro cultures of peripheral blood lymphocytes (PBL) and cerebrospinal fluid (CSF) cells and in sera and CSF from three patients with MS and three patients with clinically isolated syndromes (CIS) suspective of demyelinating disease. Results: In vitro synthesis of IgG by PBL was not detected in any patient. In contrast, in vitro synthesis by CSF cells of oligoclonal IgG and oligoclonal IgG antibodies to one or two of the three viruses tested was observed in all six patients. The clonal patterns of the in vitro synthesized IgG and virus specific IgG differed to varying extent from those synthesized intrathecally in vivo. However, in each patient, the in vitro and in vivo intrathecally produced antibodies displayed specificity for the same viruses. The addition of B cell activating factor (BAFF) had no effect on the amounts or clonal patterns of either total IgG or virus‐specific IgG produced by CSF cells in vitro. Conclusion: Virus specific B cells capable of spontaneous IgG synthesis are clonally expanded in the CSF of patients with MS. The B‐cell repertoire in CSF samples is only partially representative of the intrathecal B‐cell repertoire.

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“…To define the core domain of hHDC, the amino-acid sequence (662 amino acids) was aligned with those of 2 group II decarboxylases, namely GAD (Fenalti et al, 2007) and AroDC (Burkhard et al, 2001), using the program ClustalW (http://www.ebi.ac.uk/clustalw/).…”

Section: Site-directed Mutagenesis Expression and Purificationmentioning

confidence: 99%

Purification, crystallization and preliminary X-ray analysis of human histidine decarboxylase

et al. 2012

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The core domain of a human histidine decarboxylase mutant was purified and cocrystallized with the inhibitor l-histidine methyl ester. Using synchrotron radiation, a data set was collected from a single crystal at 100 K to 1.8 Å resolution. The crystal belonged to space group C2, with unit-cell parameters a = 215.16, b = 112.72, c = 171.39 Å , = 110.3 . Molecular replacement was carried out using the structure of aromatic l-amino-acid decarboxylase as a search model. The crystal contained three dimers per asymmetric unit, with a Matthews coefficient (V M ) of 3.01 Å 3 Da À1 and an estimated solvent content of 59.1%.

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GABA production by glutamic acid decarboxylase is regulated by a dynamic catalytic loop

Cited by 209 publications

References 39 publications

COOH-Terminal Clustering of Autoantibody and T-Cell Determinants on the Structure of GAD65 Provide Insights Into the Molecular Basis of Autoreactivity

COOH-Terminal Clustering of Autoantibody and T-Cell Determinants on the Structure of GAD65 Provide Insights Into the Molecular Basis of Autoreactivity

MS and clinically isolated syndromes: Shared specificity but diverging clonal patterns of virus‐specific IgG antibodies produced in vivo and by CSF B cells in vitro

Purification, crystallization and preliminary X-ray analysis of human histidine decarboxylase

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