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Structural biology of the GAD autoantigen
Autoimmunity Reviews 9 (2010) 148–152
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Autoimmunity Reviews j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a u t r ev
Structural biology of the GAD autoantigen☆ Gustavo Fenalti a, Ashley M. Buckle a,b,⁎ a b
Department of Biochemistry and Molecular Biology, Monash University, Clayton Campus, Melbourne, VIC 3800, Australia ARC Centre of Excellence in Structural and Functional Microbial Genomics, Monash University, Clayton Campus, Melbourne, VIC 3800, Australia
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Article history: Received 23 February 2009 Accepted 12 May 2009 Available online 22 May 2009 Keywords: Glutamic acid decarboxylase Autoantigen Autoepitope Antibody–antigen interactions Type 1 diabetes Antigenicity
a b s t r a c t For the past twenty years the type 1 diabetes autoantigen glutamic acid decarboxylase (65 kDa isoform; GAD65) has become a prototypic autoantigen, yielding a wealth of immunological and clinical insights. However for most of that period, much of the data could not be placed in a structural context, and relied upon modelling ‘guesswork’. The high-resolution crystal structure of GAD65, as well as that of its isoform GAD67, was determined in 2007, providing many insights into the molecular determinants of antigenicity, as well as an atomic positioning of the epitope-mapping data. Despite the two isoforms having the same fold and high sequence identity, it is intriguing that only the 65 kDa isoform functions as an autoantigen. The structures shed much light on this question, revealing striking differences in structure and mobility at the C-terminal domain of the isoforms, which agreed with remarkable accuracy with epitope-mapping data. Furthermore the structures provided an explanation of why two enzymes are required to catalyse the same reaction in mammals, and how this might be linked to their contrasting antigenicities. This review thus focuses on how the GAD system represents a unique testbed for understanding the relationships between molecular structure, function and antigenicity. © 2009 Elsevier B.V. All rights reserved.
Contents
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1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. The molecular structure of GAD65 . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. A structural rationalisation of epitope-mapping data . . . . . . . . . . . . . . . . . . 4. Structural differences between GAD65 and GAD67 dictate their contrasting antigenicities . 5. Functional differences between GAD isoforms: a function–antigenicity trade-off? . . . . . 6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Take-home messages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction The human neuroendocrine enzyme glutamic acid decarboxylase (GAD) catalyses the formation of the non-protein amino acid γaminobutyric acid (GABA), using L-glutamate as substrate and pyridoxal-5′-phosphate (PLP) as co-factor. GAD exists as two isoforms that are named according to their respective molecular weights, GAD65 and GAD67. Whereas most neurotransmitters are synthesized
☆ Grant support: We thank the CAPES Foundation, subordinated to the Ministry of Education, Brazil, for financial support of the PhD candidature of Gustavo Fenalti, and the National Health and Medical Research Council of Australia for funding and support. AMB is an NHMRC Senior Research Fellow. ⁎ Corresponding author. Department of Biochemistry and Molecular Biology, Monash University, Clayton Campus, Melbourne, VIC 3800, Australia. Tel.: +61 3 9902 0269; fax: +61 3 9905 4699. E-mail address: [email protected] (A.M. Buckle). 1568-9972/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.autrev.2009.05.003
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by a single enzyme, GABA's biosynthesis is catalysed by both GAD65 and GAD67, which function synchronously to produce and regulate physiological levels of GABA, the most abundant inhibitory neurotransmitter in the central nervous system (CNS) of higher vertebrates. Both isoforms share a high similarity and identity in their amino acid sequence, yet surprisingly, there are striking differences in their catalytic mechanisms of action; these include differences in interaction with the co-factor PLP, and enzyme kinetics for GABA synthesis [1]. Accordingly, while cytosolic GAD67 is more saturated with the cofactor PLP and constantly active to produce basal levels of GABA, the membrane associated GAD65, also abundant in the pancreatic islet βcells, exists mainly as autoinactivated apoenzyme (no co-factor bound) [1,2]. Apo-GAD65 can be reactivated by PLP to form holo-GAD65 (cofactor bound) and produce GABA when additional inhibitory neurotransmitter is required, for example in response to stress [3,4]. GAD65, but not GAD67, is a major autoantigen and autoantibodies to GAD65 are detected at high frequency in patients with newly-
G. Fenalti, A.M. Buckle / Autoimmunity Reviews 9 (2010) 148–152
diagnosed type 1 diabetes (T1D) (~ 80%), autoimmune polyendocrine syndrome (APS) type 1 and type 2, and more rarely in neurological disorders that include Stiff-Person syndrome (SPS) [5,6], Batten's disease [7], cerebellar ataxia [8] and certain forms of treatment resistant epilepsy [9]. The series of events responsible for initiation of these autoimmune responses are unknown, but in the case of T1D, the autoimmune process is associated with lymphocytic infiltration of the islets and destruction of pancreatic β-cells, and autoantibodies to several β-cell constituents including GAD65 [10,11]. Indeed, autoantibodies to GAD65 are considered an important diagnostic marker and are highly effective for predicting the development of T1D, or for distinguishing T1D from other hyperglicemic disorders (e.g. type 2 diabetes) [12]. In addition, there is evidence that the specificity of autoantibodies for particular epitopes on GAD65 may be a better indicator than the actual levels of anti-GAD65 in predicting impending or actual destruction of pancreatic islet β-cells [13]. Intriguingly, GAD67 is seldom independently autoantigenic and autoantibodies that react primarily with GAD65 occasionally crossreact with GAD67 [14], reflecting the contrasting autoantigenic potential of the GAD isoforms [15,16]. These antigenic differences are therefore interesting immunological phenomena, made more intriguing by the contrasting enzymatic functions of GAD isoforms. Most autoantibodies to GAD65 are reactive with the highly conserved PLP-binding and C-terminal domains where there is particularly high sequence identity between the two GAD isoforms, however, autoantibodies specifically to GAD67 are almost never detected. The finding that only GAD65 is autoantigenic in different diseases provides an excellent setting to study the humoral autoimmune response to this autoantigenic molecule. In addition, it is one of the few antigenic molecules for which numerous human monoclonal autoantibodies were derived [17–20], providing a unique model for the analysis of antibody diversity toward an immunodominant epitope. Further, the recent determination of the high-resolution crystal structures of both isoforms in combination with the epitope-mapping data allowed a full analysis of the B-cell autoreactivity to GAD. Here we review the structural features of GAD65 that appear to dictate its antigenicity, and in particular differentiate it from its non-antigenic isoform GAD67. 2. The molecular structure of GAD65 For the last twenty years in the absence of crystal structures, neither the antigenic differences between GAD isoforms nor the epitope-mapping data for GAD65 could be interpreted in a structural context. However in 2007 the crystal structures of both GAD65 and GAD67 were determined, offering precise interpretations of the contrasting enzymatic characteristics of the GAD isoforms as well as insights into the molecular mechanisms underlying their antigenic activities. Both GAD isoforms form obligate functional dimers (Fig. 1). The monomeric units comprise three domains—N-terminal, PLP-binding, and C-terminal, and the two active sites are located in the centre of the PLP-binding domain at the dimer interface [2]. Fig. 1 summarises the key elements of the structural immunology of GAD. A key finding of the crystal structural analysis was the contrasting architecture of the active sites of the isoforms. In GAD67 the active sites are covered by an extended catalytic loop, positioning a conserved catalytic tyrosine that is essential for continuous GABA synthesis [2]. In GAD65 however, there is high mobility in the same catalytic loop, permitting catalysis of a side reaction, loss of the PLP cofactor and subsequent enzyme auto-inactivation through apo-GAD65 formation [2]. This observation provided for the first time an elegant structural explanation for the requirement for two isoforms of GAD in mammals; the basal level of GABA is produced in a consistent and efficient manner by GAD67, whereas when extra GABA neurotransmitter is required for example in times of stress, inactive apo-GAD65 can be reactivated by supplying extra PLP co-factor [2].
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3. A structural rationalisation of epitope-mapping data The crystal structures for both GAD65 and GAD67 have provided a new opportunity to re-examine the location of epitope sites on GAD65, based on an “autoimmunological” panel of mAb to GAD65. The key finding was the observation of a major region of autoantigenic activity for GAD65 involving the C-terminal domain that differs strikingly between GAD65 and GAD67, but extending to parts of the PLP and N-terminal domains. Two mutually exclusive clusters of B-cell epitopes, cluster 1 epitope sites (ctc1) and cluster 2 epitope sites (ctc2), could be defined on opposing faces of the C-terminal domain according to the reactivity of mAb with particular mutants of GAD65, and competition studies using human rFab [21]. Further, four of five DRB1⁎0401-restricted epitopes on GAD65 formed a contiguous surface-exposed patch comprising parts of the two major B-cell epitope clusters, highlighting the close relationship of the T- and B-cell antigenic determinants on GAD65. 4. Structural differences between GAD65 and GAD67 dictate their contrasting antigenicities Overall, the structures of GAD65 and GAD67 show a high degree of similarity. A closer inspection, however, reveals three significant differences between the isoforms that provide important clues to their contrasting antigenicities. First, there are significant structural differences in the C-terminal domains encompassing the clusters of B-cell epitopes ctc1 and ctc2 (Fig. 1F). Second, GAD65 is strikingly more mobile than GAD67, particularly at the C-terminal domain and catalytic loop residues (Fig.1C and D). Indeed this is most acutely demonstrated by the absence of residues in the most exposed region of the C-terminal domain of GAD65, a key indicator of structural disorder (Fig. 1E and F). Third, the molecular surfaces of GAD65 and GAD67 show marked differences in their electrostatic charge distribution (Fig.1G and H). Critically, point mutations on GAD65 that affect mAb binding to the ctc1 region are located in the region of greater structural/surface characteristics and differences between GAD65 and GAD67. Taken together, these differences all correlate with the charged and flexible characteristics of other antigenic molecules [22–27]. Indeed, previous studies have suggested that antigen denaturation can focus an antibody response to flexible regions of the antigen [24]. This is consistent with suggestions that the autoantibody response in T1D first involves the C-terminal domain, with later spreading to the PLPand N-terminal domains [13,28]. 5. Functional differences between GAD isoforms: a function–antigenicity trade-off? The contrasting enzymatic properties of GAD isoforms may provide clues to their contrasting antigenicities. The mobility of the GAD65 catalytic loop is absolutely required for its particular enzymatic function. This requirement is satisfied, or at least facilitated, by the higher mobility and charge in the C-terminal domain of GAD65. This distinguishes it as an autoantigen and may therefore be an adventitious “by-product” of functional requirements for its particular enzymatic mechanism [2]. It is interesting that many potently autoantigenic molecules are important cellular enzymes, and that antigenicity often appears to reside at or near the catalytic site of the enzyme and that the same considerations might apply. Moreover most of the ensuing autoantibodies are enzyme inhibitory, examples including thyroid peroxidase in chronic thyroiditis [29], the cytochrome CYP450 2D6 in autoimmune hepatitis [30], the 2-oxoacid dehydrogenase complex enzymes, particularly PDC in primary biliary cirrhosis [31,32], and GAD65 in some instances [33]. The generality of an antigenicity–function relationship with other enzyme autoantigens is plausible given the demonstrated role of flexibility in catalysis [34–36], but awaits testing.
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6. Conclusions Whilst the crystal structural data rationalises much of the existing epitope-mapping data it also presents a new set of questions, and many gaps in knowledge still exist. The interplay between GAD function and antigenicity is currently being tested in our laboratory by structural and functional studies of engineered GAD65 molecules, as well as that of apo-GAD65. A more complete understanding of GAD65 epitopes will require the structural characterisation of GAD-antibody complexes, but it is noteworthy that to date only two structures of autoantibodies in complex with the entire antigen have been determined [37,38]. Nonetheless, the structural characterisation of the GAD system advances our current understanding of the structural basis of antigenicity, and will serve as a powerful benchmark for many years to come. Take-home messages • The 3D structures of GAD65 and GAD67 reveal key differences that correlate with known epitope regions in the antigenic isoform GAD65. • Immunodominant epitopes on GAD65 are highly mobile and charged, relative to the corresponding regions in the non-antigenic isoform GAD67. • The enzymatic function of GAD65 is regulated by in-built structural flexibility. • The inherent flexibility of GAD65, not present in GAD67, may be the cause of its increased B-cell antigenicity. • Other autoantigens that are enzymes may also exhibit a function– antigenicity trade-off, if their enzyme mechanisms rely on flexibility.
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Fig. 1. Summary of the structural immunology of GAD. Upper: Domain structure of GAD, with horizontal blue bars representing the GAD65 primary amino acid sequence, one for each of the mAbs observed to bind. Domain architecture with boundaries of N-, PLP and C-terminal domains are indicated by red lines and labeled. The epitope regions defined according to reactivity with chimeric GAD65/67 molecules are shown as colored boxes on the domain structure. Domains targeted by human mAbs are listed on the left-hand side and colored according to domain, to match domain structure key (shown below). Epitope-mapping data derived by Schwartz et al. [15] unless otherwise indicate (Powers et al. data [39] is indicated by “P”). Mutations that affect binding of mAbs to GAD65 are listed on the right hand side, in red. Lower: Structural and physico-chemical properties of GAD isoforms. (A) The epitope regions defined according to reactivity with chimeric GAD65/67 molecules are mapped on the crystal structure of dimeric GAD65 (monomer A colored dark green and monomer B light green) and each region is colored according to the boxes shown in the bar diagram above. (B) Mapping of single residues that affect binding of mAb to GAD65 onto a surface representation of its crystal structure (monomer A colored dark green and monomer B light green) showing their clustering in the C-terminal domain; Molecular surface of GAD65 (C) and GAD67 (D) colored according to flexibility, as measured by atomic temperature factors, showing that GAD65 is more flexible than GAD67, particularly in the C-terminal domain (blue (ordered) to red (flexible) gradient); (E) cartoon representation of the overall fold of dimeric GAD65 with the domains of monomer A colored light blue (N-terminal), dark blue (PLP) and light green (C-terminal), and monomer B blue (N-terminal), cyan (PLP) and green (C-terminal). Unstructured, highly mobile C-terminal residues are indicated by black dotted lines; (F) superposition of GAD65 and GAD67 structures indicating the regions of highest structural differences between the isoforms in the C-terminal domain (red ellipses); comparison of the surface electrostatic fields between GAD65 (G) and GAD67 (H) indicates striking differences (red = negative, blue = positive, with field strength proportional to surface volume—e.g., more highly charged regions appear inflated). Specifically, the C-terminal domain of GAD65 displays a strong negative electrostatic field, a characteristic absent in the C-terminal domain GAD67.
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Epigenetics in multiple sclerosis susceptibility: difference in transgenerational risk localizes to the major histocompatibility complex Major histocompatibility complex has been shown to play a weighty role in the susceptibility to develop multiple sclerosis (MS), especially when certain haplotypes contain the HLA-DRB1*1501 allele. However, some interesting epidemiological evidence has demonstrated that epigenetic factors might cause distortion in the transmission of the disease in aunt/uncle-niece/nephew (AUNN) pairs. That means that the frequency for the HLA-DRB1*1501 allele is different between the first and second generations of people affected by MS, suggesting that there are other epigenetic factors influencing such risk. A recent study performed by Chao MJ. Et al. (Hum Mol Genet. 2009;18:261-6) showed that the HLA-DRB1*1501 allele frequencies were significant different between the first and second generations affected. Affected aunts had a significantly lower HLA-DRB1*15 frequency compared with their affected nieces (Chi-square= 9.90, P = 0.0016), whereas the HLA-DRB1*15 frequency in affected males remains unaltered across the two generations (Chi-square= 0.23, P = 0.63). After comparing the transmissions for the HLA-DRB1*15 allele using a family-based transmission disequilibrium test approach in 1690 individuals from 350 affected sibling pair (ASP) families and 960 individuals from 187 AUNN families; they found that transmissions differed between the ASP and the AUNN families (Chi-square= 6.92; P = 0.0085). The risk carried by HLA-DRB1*15 was increased in families with affected second-degree relatives (AUNN: OR = 4.07) when compared with those consisting only first-degree relatives (ASP: OR = 2.17), establishing heterogeneity of risk among HLA-DRB1*15 haplotypes based on whether collateral parental relatives are affected. Such findings can be understood as the product of geneenvironment interactions among other unknown likely genetic interactions. These data also strongly suggest that the female-specific increasing risk of MS is mediated through these alleles or adjacent variation. The comparison of transmission of the same allele in vertically affected pedigrees (AUNN) to collinear sibling pairs (ASP) may provide a useful screen for putative epigenetic marks.
Autoantibodies against galectin-8: their specificity, association with lymphopenia in systemic lupus erythematosus and detection in rheumatoid arthritis and acute inflammation The role of autoantibodies in the pathogenesis of systemic lupus erythematosus (SLE) has not been completely defined. From more than a hundred autoantibodies described in SLE, relatively few have been associated with clinical manifestations. The glycan-binding proteins of the galectin family can modulate the immune system. Anti-galectin autoantibodies thus could have functional and/or pathogenic implications in inflammatory processes and autoimmunity. Massardo L et al. had previously reported function-blocking autoantibodies against galectin-8 (Gal-8) in SLE. Now Massardo L, et al. (Lupus 2009; 18:539-46) tested these autoantibodies against a series of other human galectins and demonstrated their specificity for Gal-8, being detectable in 23% of 78 SLE patients. Remarkably, they associated with lymphopenia (50% of 18 anti-Gal-8-positive versus 18% of 60 anti-Gal-8-negative cases, Fisher's Exact test two-tailed: P b 0.012). Lymphopenia is a common clinical manifestation in SLE, yet of unknown mechanism. In addition, six of eight patients with both lymphopenia and malar rash had antiGal-8 in their sera. Occurrence of these autoantibodies was not confined to SLE as we also found them in sera of patients with rheumatoid arthritis (16%) and septicemia (20%). This study establishes the occurrence of specific anti-Gal-8 autoantibodies in autoimmune rheumatic diseases and in acute inflammation, with an apparent association to a clinical subset patients with SLE.
Circulating endothelial cells and angiogenic proteins in patients with systemic lupus erythematosus The aim of this study was to assess absolute counts of different subpoulatins of circulating endothelial cells (CEC) in patients with active systemic lupus erythematosus (SLE). Robak E. et al (Lupus 2009; 18: 332-41). The authors investigated a potential correlation of CEC numbers with serum levels of angiogenic proteins as well as with clinical and laboratory symptoms of the disease. For the first time in SLE, CEC were enumerated directly, by the ‘single platform’ method. Resting (rCEC), activated (aCEC) and progenitor (pCEC) endothelial cells were identified in patients with SLE and healthy volunteers using four-colour flow cytometry. Serum concentrations of angiogenic proteins (vascular endothelial growth factor, placental growth factor (PIGF), soluble vascular cell adhesion molecule and endoglin) were evaluated by ELISA. The SLE activity was scored according to the Systemic Lupus Activity Measure system. The authors found that total CEC number in patients with SLE was significantly higher (median 14.2/microl) than in the control group (median 3.3/microl) (p b 0.0001). Absolute counts of aCEC, rCEC and pCEC (medians 4.9/microl, 6.8/microl and 2.3/microl, respectively) were also higher in patients with SLE than in healthy persons (medians 0.9/microl, 1.6/microl and 0.1/microl, respectively), with p b 0.0001 for all comparisons. There was no correlation between CEC or their subpopulations and SLE activity. Strong positive correlations were found between CEC, rCEC and pCEC number and serum levels of PIGF. Moreover, aCEC, rCEC and pCEC counts were significantly higher in SLE patients with leucopenia. In conclusion, our results show that absolute CEC counts and angiogenic proteins levels are elevated in patients with SLE as compared with healthy controls. These data may suggest that angiogenic process is involved in the pathogenesis of this disease.
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Autoimmunity Reviews j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a u t r ev
Structural biology of the GAD autoantigen☆ Gustavo Fenalti a, Ashley M. Buckle a,b,⁎ a b
Department of Biochemistry and Molecular Biology, Monash University, Clayton Campus, Melbourne, VIC 3800, Australia ARC Centre of Excellence in Structural and Functional Microbial Genomics, Monash University, Clayton Campus, Melbourne, VIC 3800, Australia
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Article history: Received 23 February 2009 Accepted 12 May 2009 Available online 22 May 2009 Keywords: Glutamic acid decarboxylase Autoantigen Autoepitope Antibody–antigen interactions Type 1 diabetes Antigenicity
a b s t r a c t For the past twenty years the type 1 diabetes autoantigen glutamic acid decarboxylase (65 kDa isoform; GAD65) has become a prototypic autoantigen, yielding a wealth of immunological and clinical insights. However for most of that period, much of the data could not be placed in a structural context, and relied upon modelling ‘guesswork’. The high-resolution crystal structure of GAD65, as well as that of its isoform GAD67, was determined in 2007, providing many insights into the molecular determinants of antigenicity, as well as an atomic positioning of the epitope-mapping data. Despite the two isoforms having the same fold and high sequence identity, it is intriguing that only the 65 kDa isoform functions as an autoantigen. The structures shed much light on this question, revealing striking differences in structure and mobility at the C-terminal domain of the isoforms, which agreed with remarkable accuracy with epitope-mapping data. Furthermore the structures provided an explanation of why two enzymes are required to catalyse the same reaction in mammals, and how this might be linked to their contrasting antigenicities. This review thus focuses on how the GAD system represents a unique testbed for understanding the relationships between molecular structure, function and antigenicity. © 2009 Elsevier B.V. All rights reserved.
Contents
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1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. The molecular structure of GAD65 . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. A structural rationalisation of epitope-mapping data . . . . . . . . . . . . . . . . . . 4. Structural differences between GAD65 and GAD67 dictate their contrasting antigenicities . 5. Functional differences between GAD isoforms: a function–antigenicity trade-off? . . . . . 6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Take-home messages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction The human neuroendocrine enzyme glutamic acid decarboxylase (GAD) catalyses the formation of the non-protein amino acid γaminobutyric acid (GABA), using L-glutamate as substrate and pyridoxal-5′-phosphate (PLP) as co-factor. GAD exists as two isoforms that are named according to their respective molecular weights, GAD65 and GAD67. Whereas most neurotransmitters are synthesized
☆ Grant support: We thank the CAPES Foundation, subordinated to the Ministry of Education, Brazil, for financial support of the PhD candidature of Gustavo Fenalti, and the National Health and Medical Research Council of Australia for funding and support. AMB is an NHMRC Senior Research Fellow. ⁎ Corresponding author. Department of Biochemistry and Molecular Biology, Monash University, Clayton Campus, Melbourne, VIC 3800, Australia. Tel.: +61 3 9902 0269; fax: +61 3 9905 4699. E-mail address: [email protected] (A.M. Buckle). 1568-9972/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.autrev.2009.05.003
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by a single enzyme, GABA's biosynthesis is catalysed by both GAD65 and GAD67, which function synchronously to produce and regulate physiological levels of GABA, the most abundant inhibitory neurotransmitter in the central nervous system (CNS) of higher vertebrates. Both isoforms share a high similarity and identity in their amino acid sequence, yet surprisingly, there are striking differences in their catalytic mechanisms of action; these include differences in interaction with the co-factor PLP, and enzyme kinetics for GABA synthesis [1]. Accordingly, while cytosolic GAD67 is more saturated with the cofactor PLP and constantly active to produce basal levels of GABA, the membrane associated GAD65, also abundant in the pancreatic islet βcells, exists mainly as autoinactivated apoenzyme (no co-factor bound) [1,2]. Apo-GAD65 can be reactivated by PLP to form holo-GAD65 (cofactor bound) and produce GABA when additional inhibitory neurotransmitter is required, for example in response to stress [3,4]. GAD65, but not GAD67, is a major autoantigen and autoantibodies to GAD65 are detected at high frequency in patients with newly-
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diagnosed type 1 diabetes (T1D) (~ 80%), autoimmune polyendocrine syndrome (APS) type 1 and type 2, and more rarely in neurological disorders that include Stiff-Person syndrome (SPS) [5,6], Batten's disease [7], cerebellar ataxia [8] and certain forms of treatment resistant epilepsy [9]. The series of events responsible for initiation of these autoimmune responses are unknown, but in the case of T1D, the autoimmune process is associated with lymphocytic infiltration of the islets and destruction of pancreatic β-cells, and autoantibodies to several β-cell constituents including GAD65 [10,11]. Indeed, autoantibodies to GAD65 are considered an important diagnostic marker and are highly effective for predicting the development of T1D, or for distinguishing T1D from other hyperglicemic disorders (e.g. type 2 diabetes) [12]. In addition, there is evidence that the specificity of autoantibodies for particular epitopes on GAD65 may be a better indicator than the actual levels of anti-GAD65 in predicting impending or actual destruction of pancreatic islet β-cells [13]. Intriguingly, GAD67 is seldom independently autoantigenic and autoantibodies that react primarily with GAD65 occasionally crossreact with GAD67 [14], reflecting the contrasting autoantigenic potential of the GAD isoforms [15,16]. These antigenic differences are therefore interesting immunological phenomena, made more intriguing by the contrasting enzymatic functions of GAD isoforms. Most autoantibodies to GAD65 are reactive with the highly conserved PLP-binding and C-terminal domains where there is particularly high sequence identity between the two GAD isoforms, however, autoantibodies specifically to GAD67 are almost never detected. The finding that only GAD65 is autoantigenic in different diseases provides an excellent setting to study the humoral autoimmune response to this autoantigenic molecule. In addition, it is one of the few antigenic molecules for which numerous human monoclonal autoantibodies were derived [17–20], providing a unique model for the analysis of antibody diversity toward an immunodominant epitope. Further, the recent determination of the high-resolution crystal structures of both isoforms in combination with the epitope-mapping data allowed a full analysis of the B-cell autoreactivity to GAD. Here we review the structural features of GAD65 that appear to dictate its antigenicity, and in particular differentiate it from its non-antigenic isoform GAD67. 2. The molecular structure of GAD65 For the last twenty years in the absence of crystal structures, neither the antigenic differences between GAD isoforms nor the epitope-mapping data for GAD65 could be interpreted in a structural context. However in 2007 the crystal structures of both GAD65 and GAD67 were determined, offering precise interpretations of the contrasting enzymatic characteristics of the GAD isoforms as well as insights into the molecular mechanisms underlying their antigenic activities. Both GAD isoforms form obligate functional dimers (Fig. 1). The monomeric units comprise three domains—N-terminal, PLP-binding, and C-terminal, and the two active sites are located in the centre of the PLP-binding domain at the dimer interface [2]. Fig. 1 summarises the key elements of the structural immunology of GAD. A key finding of the crystal structural analysis was the contrasting architecture of the active sites of the isoforms. In GAD67 the active sites are covered by an extended catalytic loop, positioning a conserved catalytic tyrosine that is essential for continuous GABA synthesis [2]. In GAD65 however, there is high mobility in the same catalytic loop, permitting catalysis of a side reaction, loss of the PLP cofactor and subsequent enzyme auto-inactivation through apo-GAD65 formation [2]. This observation provided for the first time an elegant structural explanation for the requirement for two isoforms of GAD in mammals; the basal level of GABA is produced in a consistent and efficient manner by GAD67, whereas when extra GABA neurotransmitter is required for example in times of stress, inactive apo-GAD65 can be reactivated by supplying extra PLP co-factor [2].
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3. A structural rationalisation of epitope-mapping data The crystal structures for both GAD65 and GAD67 have provided a new opportunity to re-examine the location of epitope sites on GAD65, based on an “autoimmunological” panel of mAb to GAD65. The key finding was the observation of a major region of autoantigenic activity for GAD65 involving the C-terminal domain that differs strikingly between GAD65 and GAD67, but extending to parts of the PLP and N-terminal domains. Two mutually exclusive clusters of B-cell epitopes, cluster 1 epitope sites (ctc1) and cluster 2 epitope sites (ctc2), could be defined on opposing faces of the C-terminal domain according to the reactivity of mAb with particular mutants of GAD65, and competition studies using human rFab [21]. Further, four of five DRB1⁎0401-restricted epitopes on GAD65 formed a contiguous surface-exposed patch comprising parts of the two major B-cell epitope clusters, highlighting the close relationship of the T- and B-cell antigenic determinants on GAD65. 4. Structural differences between GAD65 and GAD67 dictate their contrasting antigenicities Overall, the structures of GAD65 and GAD67 show a high degree of similarity. A closer inspection, however, reveals three significant differences between the isoforms that provide important clues to their contrasting antigenicities. First, there are significant structural differences in the C-terminal domains encompassing the clusters of B-cell epitopes ctc1 and ctc2 (Fig. 1F). Second, GAD65 is strikingly more mobile than GAD67, particularly at the C-terminal domain and catalytic loop residues (Fig.1C and D). Indeed this is most acutely demonstrated by the absence of residues in the most exposed region of the C-terminal domain of GAD65, a key indicator of structural disorder (Fig. 1E and F). Third, the molecular surfaces of GAD65 and GAD67 show marked differences in their electrostatic charge distribution (Fig.1G and H). Critically, point mutations on GAD65 that affect mAb binding to the ctc1 region are located in the region of greater structural/surface characteristics and differences between GAD65 and GAD67. Taken together, these differences all correlate with the charged and flexible characteristics of other antigenic molecules [22–27]. Indeed, previous studies have suggested that antigen denaturation can focus an antibody response to flexible regions of the antigen [24]. This is consistent with suggestions that the autoantibody response in T1D first involves the C-terminal domain, with later spreading to the PLPand N-terminal domains [13,28]. 5. Functional differences between GAD isoforms: a function–antigenicity trade-off? The contrasting enzymatic properties of GAD isoforms may provide clues to their contrasting antigenicities. The mobility of the GAD65 catalytic loop is absolutely required for its particular enzymatic function. This requirement is satisfied, or at least facilitated, by the higher mobility and charge in the C-terminal domain of GAD65. This distinguishes it as an autoantigen and may therefore be an adventitious “by-product” of functional requirements for its particular enzymatic mechanism [2]. It is interesting that many potently autoantigenic molecules are important cellular enzymes, and that antigenicity often appears to reside at or near the catalytic site of the enzyme and that the same considerations might apply. Moreover most of the ensuing autoantibodies are enzyme inhibitory, examples including thyroid peroxidase in chronic thyroiditis [29], the cytochrome CYP450 2D6 in autoimmune hepatitis [30], the 2-oxoacid dehydrogenase complex enzymes, particularly PDC in primary biliary cirrhosis [31,32], and GAD65 in some instances [33]. The generality of an antigenicity–function relationship with other enzyme autoantigens is plausible given the demonstrated role of flexibility in catalysis [34–36], but awaits testing.
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6. Conclusions Whilst the crystal structural data rationalises much of the existing epitope-mapping data it also presents a new set of questions, and many gaps in knowledge still exist. The interplay between GAD function and antigenicity is currently being tested in our laboratory by structural and functional studies of engineered GAD65 molecules, as well as that of apo-GAD65. A more complete understanding of GAD65 epitopes will require the structural characterisation of GAD-antibody complexes, but it is noteworthy that to date only two structures of autoantibodies in complex with the entire antigen have been determined [37,38]. Nonetheless, the structural characterisation of the GAD system advances our current understanding of the structural basis of antigenicity, and will serve as a powerful benchmark for many years to come. Take-home messages • The 3D structures of GAD65 and GAD67 reveal key differences that correlate with known epitope regions in the antigenic isoform GAD65. • Immunodominant epitopes on GAD65 are highly mobile and charged, relative to the corresponding regions in the non-antigenic isoform GAD67. • The enzymatic function of GAD65 is regulated by in-built structural flexibility. • The inherent flexibility of GAD65, not present in GAD67, may be the cause of its increased B-cell antigenicity. • Other autoantigens that are enzymes may also exhibit a function– antigenicity trade-off, if their enzyme mechanisms rely on flexibility.
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Fig. 1. Summary of the structural immunology of GAD. Upper: Domain structure of GAD, with horizontal blue bars representing the GAD65 primary amino acid sequence, one for each of the mAbs observed to bind. Domain architecture with boundaries of N-, PLP and C-terminal domains are indicated by red lines and labeled. The epitope regions defined according to reactivity with chimeric GAD65/67 molecules are shown as colored boxes on the domain structure. Domains targeted by human mAbs are listed on the left-hand side and colored according to domain, to match domain structure key (shown below). Epitope-mapping data derived by Schwartz et al. [15] unless otherwise indicate (Powers et al. data [39] is indicated by “P”). Mutations that affect binding of mAbs to GAD65 are listed on the right hand side, in red. Lower: Structural and physico-chemical properties of GAD isoforms. (A) The epitope regions defined according to reactivity with chimeric GAD65/67 molecules are mapped on the crystal structure of dimeric GAD65 (monomer A colored dark green and monomer B light green) and each region is colored according to the boxes shown in the bar diagram above. (B) Mapping of single residues that affect binding of mAb to GAD65 onto a surface representation of its crystal structure (monomer A colored dark green and monomer B light green) showing their clustering in the C-terminal domain; Molecular surface of GAD65 (C) and GAD67 (D) colored according to flexibility, as measured by atomic temperature factors, showing that GAD65 is more flexible than GAD67, particularly in the C-terminal domain (blue (ordered) to red (flexible) gradient); (E) cartoon representation of the overall fold of dimeric GAD65 with the domains of monomer A colored light blue (N-terminal), dark blue (PLP) and light green (C-terminal), and monomer B blue (N-terminal), cyan (PLP) and green (C-terminal). Unstructured, highly mobile C-terminal residues are indicated by black dotted lines; (F) superposition of GAD65 and GAD67 structures indicating the regions of highest structural differences between the isoforms in the C-terminal domain (red ellipses); comparison of the surface electrostatic fields between GAD65 (G) and GAD67 (H) indicates striking differences (red = negative, blue = positive, with field strength proportional to surface volume—e.g., more highly charged regions appear inflated). Specifically, the C-terminal domain of GAD65 displays a strong negative electrostatic field, a characteristic absent in the C-terminal domain GAD67.
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Epigenetics in multiple sclerosis susceptibility: difference in transgenerational risk localizes to the major histocompatibility complex Major histocompatibility complex has been shown to play a weighty role in the susceptibility to develop multiple sclerosis (MS), especially when certain haplotypes contain the HLA-DRB1*1501 allele. However, some interesting epidemiological evidence has demonstrated that epigenetic factors might cause distortion in the transmission of the disease in aunt/uncle-niece/nephew (AUNN) pairs. That means that the frequency for the HLA-DRB1*1501 allele is different between the first and second generations of people affected by MS, suggesting that there are other epigenetic factors influencing such risk. A recent study performed by Chao MJ. Et al. (Hum Mol Genet. 2009;18:261-6) showed that the HLA-DRB1*1501 allele frequencies were significant different between the first and second generations affected. Affected aunts had a significantly lower HLA-DRB1*15 frequency compared with their affected nieces (Chi-square= 9.90, P = 0.0016), whereas the HLA-DRB1*15 frequency in affected males remains unaltered across the two generations (Chi-square= 0.23, P = 0.63). After comparing the transmissions for the HLA-DRB1*15 allele using a family-based transmission disequilibrium test approach in 1690 individuals from 350 affected sibling pair (ASP) families and 960 individuals from 187 AUNN families; they found that transmissions differed between the ASP and the AUNN families (Chi-square= 6.92; P = 0.0085). The risk carried by HLA-DRB1*15 was increased in families with affected second-degree relatives (AUNN: OR = 4.07) when compared with those consisting only first-degree relatives (ASP: OR = 2.17), establishing heterogeneity of risk among HLA-DRB1*15 haplotypes based on whether collateral parental relatives are affected. Such findings can be understood as the product of geneenvironment interactions among other unknown likely genetic interactions. These data also strongly suggest that the female-specific increasing risk of MS is mediated through these alleles or adjacent variation. The comparison of transmission of the same allele in vertically affected pedigrees (AUNN) to collinear sibling pairs (ASP) may provide a useful screen for putative epigenetic marks.
Autoantibodies against galectin-8: their specificity, association with lymphopenia in systemic lupus erythematosus and detection in rheumatoid arthritis and acute inflammation The role of autoantibodies in the pathogenesis of systemic lupus erythematosus (SLE) has not been completely defined. From more than a hundred autoantibodies described in SLE, relatively few have been associated with clinical manifestations. The glycan-binding proteins of the galectin family can modulate the immune system. Anti-galectin autoantibodies thus could have functional and/or pathogenic implications in inflammatory processes and autoimmunity. Massardo L et al. had previously reported function-blocking autoantibodies against galectin-8 (Gal-8) in SLE. Now Massardo L, et al. (Lupus 2009; 18:539-46) tested these autoantibodies against a series of other human galectins and demonstrated their specificity for Gal-8, being detectable in 23% of 78 SLE patients. Remarkably, they associated with lymphopenia (50% of 18 anti-Gal-8-positive versus 18% of 60 anti-Gal-8-negative cases, Fisher's Exact test two-tailed: P b 0.012). Lymphopenia is a common clinical manifestation in SLE, yet of unknown mechanism. In addition, six of eight patients with both lymphopenia and malar rash had antiGal-8 in their sera. Occurrence of these autoantibodies was not confined to SLE as we also found them in sera of patients with rheumatoid arthritis (16%) and septicemia (20%). This study establishes the occurrence of specific anti-Gal-8 autoantibodies in autoimmune rheumatic diseases and in acute inflammation, with an apparent association to a clinical subset patients with SLE.
Circulating endothelial cells and angiogenic proteins in patients with systemic lupus erythematosus The aim of this study was to assess absolute counts of different subpoulatins of circulating endothelial cells (CEC) in patients with active systemic lupus erythematosus (SLE). Robak E. et al (Lupus 2009; 18: 332-41). The authors investigated a potential correlation of CEC numbers with serum levels of angiogenic proteins as well as with clinical and laboratory symptoms of the disease. For the first time in SLE, CEC were enumerated directly, by the ‘single platform’ method. Resting (rCEC), activated (aCEC) and progenitor (pCEC) endothelial cells were identified in patients with SLE and healthy volunteers using four-colour flow cytometry. Serum concentrations of angiogenic proteins (vascular endothelial growth factor, placental growth factor (PIGF), soluble vascular cell adhesion molecule and endoglin) were evaluated by ELISA. The SLE activity was scored according to the Systemic Lupus Activity Measure system. The authors found that total CEC number in patients with SLE was significantly higher (median 14.2/microl) than in the control group (median 3.3/microl) (p b 0.0001). Absolute counts of aCEC, rCEC and pCEC (medians 4.9/microl, 6.8/microl and 2.3/microl, respectively) were also higher in patients with SLE than in healthy persons (medians 0.9/microl, 1.6/microl and 0.1/microl, respectively), with p b 0.0001 for all comparisons. There was no correlation between CEC or their subpopulations and SLE activity. Strong positive correlations were found between CEC, rCEC and pCEC number and serum levels of PIGF. Moreover, aCEC, rCEC and pCEC counts were significantly higher in SLE patients with leucopenia. In conclusion, our results show that absolute CEC counts and angiogenic proteins levels are elevated in patients with SLE as compared with healthy controls. These data may suggest that angiogenic process is involved in the pathogenesis of this disease.