Motifs and structural fold of the cofactor binding site of human glutamate decarboxylase

Qu, Kunbin; Lawrence, Charles E.

doi:10.1002/pro.5560070503

Cited by 34 publications

(24 citation statements)

References 38 publications

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“…These studies, and a statistical analysis of alignment data (15) indicated that the active site of GAD is in a fold formed by the interaction of two GAD monomers and that the interaction does not depend on disulfide bonds, but is instead due to some other interaction between the C-terminal domains of the two interacting monomers. This information is in accord with previous findings that GAD must be a dimer to be active and that it can consist of homodimers of either isoform, or as heterodimers formed from monomers of the two types (16).…”

Section: Introductionmentioning

confidence: 94%

Glutamate Decarboxylase: Loss of N-terminal Segment Does Not Affect Homodimerization and Determination of the Oxidation State of Cysteine Residues

2005

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Glutamate decarboxylase (GAD) produces GABA, the main inhibitory neurotransmitter in adult mammalian brain. The physical characteristics of GAD were studied using mass spectrometry and partial protein digests. The N-termini of the two main isoforms, GAD65 and GAD67, were processed by removal of the initial methionine residues and acetylation of the penultimate alanines. Native recombinant GAD65 and GAD67 exist as homodimers that can be dissociated with non-reducing methods, indicating that homodimerization does not involve intermolecular disulfide bonds. Truncation of the N-terminal segment with trypsin digestion did not affect homodimerization but increased activity by decreasing the Km of GAD67 and increasing the Vmax of both isoforms. Of the 15 cysteines in GAD65, the six found in the N-terminal segment can form disulfide bonds and of the 13 cysteines in GAD67, cysteines 32 and 38 can form a disulfide bond. The in vitro formation of disulfide bonds in the N-termini, and the removal of the termini with relatively low amounts of trypsin, indicate that the N-terminal segments of GAD65 and GAD67 are exposed and flexible. The formation of a disulfide bridge between cysteines 30 and 45 of GAD65 suggests that alteration of normal redox conditions could affect GAD targeting.

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Section: Introductionmentioning

confidence: 94%

Glutamate Decarboxylase: Loss of N-terminal Segment Does Not Affect Homodimerization and Determination of the Oxidation State of Cysteine Residues

2005

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“…The sequence of the Ab-binding peptides identifies amino acids critical for binding. The second is sequence alignment and secondary structure prediction of PLP-dependent decarboxylases and aminotransferases, which have shown that the PLP binding domain of ornithine decarboxylase (OrnDC) and aspartate aminotransferase from bacteria share a protein fold that is common to other PLPdependant proteins, including GAD65 (25,26). This has enabled the derivation of structural models of the GAD PLP binding domain (27), and such models can be used to formulate specific hypotheses regarding epitope structure.…”

Section: G Lutamic Acid Decarboxylase (Gad)mentioning

confidence: 99%

Conformational Epitopes on the Diabetes Autoantigen GAD65 Identified by Peptide Phage Display and Molecular Modeling

Myers

Davies

Tong

et al. 2000

The Journal of Immunology

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The major diabetes autoantigen, glutamic acid decarboxylase (GAD65), contains a region of sequence similarity, including six identical residues PEVKEK, to the P2C protein of coxsackie B virus, suggesting that cross-reactivity between coxsackie B virus and GAD65 can initiate autoimmune diabetes. We used the human islet cell mAbs MICA3 and MICA4 to identify the Ab epitopes of GAD65 by screening phage-displayed random peptide libraries. The identified peptide sequences could be mapped to a homology model of the pyridoxal phosphate (PLP) binding domain of GAD65. For MICA3, a surface loop containing the sequence PEVKEK and two adjacent exposed helixes were identified in the PLP binding domain as well as a region of the C terminus of GAD65 that has previously been identified as critical for MICA3 binding. To confirm that the loop containing the PEVKEK sequence contributes to the MICA3 epitope, this loop was deleted by mutagenesis. This reduced binding of MICA3 by 70%. Peptide sequences selected using MICA4 were rich in basic or hydroxyl-containing amino acids, and the surface of the GAD65 PLP-binding domain surrounding Lys358, which is known to be critical for MICA4 binding, was likewise rich in these amino acids. Also, the two phage most reactive with MICA4 encoded the motif VALxG, and the reverse of this sequence, LAV, was located in this same region. Thus, we have defined the MICA3 and MICA4 epitopes on GAD65 using the combination of phage display, molecular modeling, and mutagenesis and have provided compelling evidence for the involvement of the PEVKEK loop in the MICA3 epitope.

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“…Homo-and Heterodimerization of GAD65 Does Not Involve the NH 2 -terminal Membrane-anchoring Region-Molecular modeling studies have suggested that the middle domain of each subunit is involved in GAD65 dimerization (16,17). Since there are no proteins of known structure that share sequence homology with GAD65 in the first 100 amino acids at the NH 2 terminus of GAD65 (16), a structure prediction is not available for this region.…”

Section: Targeting To Nerve Terminals and Membrane Anchoringmentioning

confidence: 99%

“…The results presented here show that the GAD65⌬1-101 mutant is exclusively detected as a dimer in nondenaturing gels and therefore provide the first evidence that the NH 2 -terminal region of GAD65 is not important for homodimerization. Thus, the predicted interactions of several residues in the middle domain of one subunit with PLP in the active site of the second subunit may represent the main bonds between the subunits (17).…”

Section: Targeting To Nerve Terminals and Membrane Anchoringmentioning

confidence: 99%

“…In this model, the two GAD65 subunits are joined in the middle region (amino acids 201-461) and related by a P 21 symmetry. In a separate analysis, several residues in the middle domain were predicted to reach into the cofactor-binding site of the other subunit and interact directly with the phosphate group of PLP and with residues in the other subunit of the enzyme (17). Thus, the middle domains of GAD65 and GAD67 seem to be responsible for the basic homodimeric structure of the enzyme.…”

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confidence: 99%

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The Hydrophilic Isoform of Glutamate Decarboxylase, GAD67, Is Targeted to Membranes and Nerve Terminals Independent of Dimerization with the Hydrophobic Membrane-anchored Isoform, GAD65

Kanaani¹,

Lissin²,

Kash³

et al. 1999

Journal of Biological Chemistry

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GAD67, the larger isoform of the ␥-aminobutyric acidsynthesizing enzyme glutamic acid decarboxylase, is a hydrophilic soluble molecule, postulated to localize at nerve terminals and membrane compartments by heterodimerization with the smaller membrane-anchored isoform GAD65. We here show that the dimerization region in GAD65 is distinct from the NH 2 -terminal membrane-anchoring region and that a membrane anchoring GAD65 subunit can indeed target a soluble subunit to membrane compartments by dimerization. However, only a fraction of membrane-bound GAD67 is engaged in a heterodimer with GAD65 in rat brain. Furthermore, in GAD65؊/؊ mouse brain, GAD67, which no longer partitions into the Triton X-114 detergent phase, still anchors to membranes at similar levels as in wild-type mice. Similarly, in primary cultures of neurons derived from GAD65؊/؊ mice, GAD67 is targeted to nerve terminals, where it co-localizes with the synaptic vesicle marker SV2. Thus, axonal targeting and membrane anchoring is an intrinsic property of GAD67 and does not require GAD65. The results suggest that three distinct moieties of glutamate decarboxylase localize to membrane compartments, an amphiphilic GAD65 homodimer, an amphiphilic GAD65/67 heterodimer, tethered to membranes via the GAD65 subunit, and a hydrophilic GAD67 homodimer, which associates with membranes by a distinct mechanism.Glutamic acid decarboxylase (GAD) 1 (EC 4.1.1.15) is the key enzyme in the synthesis of ␥-aminobutyric acid (GABA) (1). GAD is expressed at comparable levels in GABA-ergic neurons in the central nervous system and in the insulin-producing ␤ cells in the islets of Langerhans (2, 3). GABA has been established as the major inhibitory neurotransmitter in the central nervous system, whereas its role in islet cell function remains elusive (4).Mammalian species express two isoforms of GAD designated GAD65 and GAD67 in accordance with their relative molecular masses in kDa (3, 5, 6). GAD65 and GAD67 are highly conserved in evolution. Although the two proteins differ substantially in the first 95 NH 2 -terminal amino acids, they share a significant homology in the remaining part of the molecule, which contains the catalytic portion of the enzyme (ϳ78% identity, ϳ95% similarity). GAD65 and GAD67 show significant differences in their levels of expression in different brain regions and steady state saturation with the co-enzyme pyridoxal 5Ј-phosphate (PLP) (1). More than half of GAD in rat brain is present as the PLP-free apoenzyme (7, 8), and GAD65 constitutes the majority of this reservoir (9 -11). GAD67 constitutes a small fraction of the apo-GAD reservoir in the brain (11) and is predominantly found as a holoenzyme tightly associated with PLP (9 -11). GAD65, but not GAD67, is a major target of autoimmune responses directed against pancreatic ␤ cells in type 1 diabetes (12, 13) and toward GABA-ergic neurons in a rare disease of the central nervous system, stiff-man syndrome (14).Most biochemical studies on the quaternary structure of purified GAD are consistent wit...

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Motifs and structural fold of the cofactor binding site of human glutamate decarboxylase

Cited by 34 publications

References 38 publications

Glutamate Decarboxylase: Loss of N-terminal Segment Does Not Affect Homodimerization and Determination of the Oxidation State of Cysteine Residues

Glutamate Decarboxylase: Loss of N-terminal Segment Does Not Affect Homodimerization and Determination of the Oxidation State of Cysteine Residues

Conformational Epitopes on the Diabetes Autoantigen GAD65 Identified by Peptide Phage Display and Molecular Modeling

The Hydrophilic Isoform of Glutamate Decarboxylase, GAD67, Is Targeted to Membranes and Nerve Terminals Independent of Dimerization with the Hydrophobic Membrane-anchored Isoform, GAD65

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