Glutamic Acid Decarboxylase in Insulin-Dependent Diabetes Mellitus Henry J. DeAizpurua and Leonard C. Harrison Burnet Clinical Research Unit, The Walter and Eliza Hall lnstitute of Medical Research, Royal Melbourne Hospital, Parkville 3050, Australia

I. INTRODUCTION Type 1or insulin-dependent diabetes mellitus (IDDM) is due to the selective destruction of pancreatic islet beta cells. Over the last two decades, evidence has accumulated which incriminates autoimmune mechanisms in the pathogenesis of IDDM.' Although the molecular mechanisms which underly beta-cell destruction are not fully understood, three major lines of evidence support the role of autoimmunity. First, a mononuclear islet-cell infiltrate or insulitis is observed in newly-diagnosed IDDM subjects and in animal second, there are circulatmodels of the ing islet-cell and insulin antibodies4 and isletreactive T cells5 before and at the onset of clinical disease; and third, immunosuppressive drugs retard loss of residual beta-cell function after clinical diagnosis.6 Beta-cell destruction probably evolves through a series of stages following an initial environmental insult, with progressive insulitis culminating in symptomatic insulin deficiency (reviewed in ref. 7). The most practical markers of beta-cell autoimmunity currently available are circulating antibodies against islet antigens. Antibodies to at least three islet antigens have been identified in IDDM sera: insulin: a putative glycolipid that accounts for islet-cell cytoplasmic antibody (ICA) reactivity on frozen sections of pancreas,' and a M , 64000 (64 kD) antigenlo which appears to be the y-aminobutyric acid (GABA)-synthesizing enzyme glutamic acid decarboxylase (GAD)." Autoimmunity to GAD has recently generated a great deal of excitement because of the expectation DiabetesiMetabolism Reviews, Vol. 8, No. 2, 13?-147

0 1992 by John Wiley & Sons, Ltd.


that GAD antibodies may represent a very early marker in subjects destined to develop to clinical IDDM. In this paper we will review research findings from our own and other laboratories and attempt to clarify the current state of knowledge of autoimmunity to GAD and its relevance to IDDM.


A. Initial Description and Prevalence of Immunoreactivity Baekkeskov et al.'O reported in 1982 that sera from 8 of 10 newly diagnosed children precipitated a protein of M , -64 000 (64 kD) from detergent lysates of biosynthetically labelled human islet cells. Subsequently, antibodies to a 64 kD islet antigen were detected in two animal models of spontaneous diabetes: the Bio-Breeding (BB) rat'* and the non-obese diabetic (NOD) m 0 ~ s . e . ' ~ In six BB/Hagedorn rats studied prospectively, antibodies to the 64 kD antigen (64 KAb) were present in five rats up to 9 weeks before the onset of IDDM. Three of the six rats had decreased levels of 64 KAb after the onset of diabetes. Interestingly, in the NOD mouse the 64 KAb were first detected at weaning, disappeared within weeks after diabetes onset, and were absent in older non-diabetic NOD mice, apparently independent of i n ~ u l i t i s . ' In ~,~ humans, ~ Baekkeskov et al.15 examined 14 ICA-positive first-degree relatives of IDDM subjects and reported that 11 had 64KAb that preceded the clinical onset of IDDM by several years and sometimes preceded


the detection of ICA, the implication being that immunity to the 64 kD antigen is an early event in the development of IDDM. As was the case in the animal s t ~ d i e s , ' ~64 , ~KAb ~ in humans15 diminished after the onset of IDDM. In a study of Swedish children diagnosed with IDDM,16 29 of 40 (73%) were found to have 64KAb. There was only a weak, although significant, correlation between titres of ICA and 64 KAb. Using foetal pig proislets, we found that 9/14 (64%) of pre-clinical IDDM subjects but only 12/33 (36%) of recent-onset, and 4/12 (33%) of established diabetic subjects, had 64 KAb17 (Figure 1).If the frequency of 64KAb decreases with the development of clinical disease, this could reflect loss of antigenic drive; alternatively, 64KAb may not be predictive of progression to IDDM. Differences in the reported frequencies of 64KAb within IDDM groups may also reflect the use of different substrates, as well as the

Figure 1. Autoradiograph of biosynthetically-labelled foetal pig proislet 64 kD protein (arrowed)immunoprecipitated with serum from an IDDM subject (lane 2).

Lane 1 = Healthy control serum. Immune precipitates were analysed by 10% SDS-PAGE under reducing condi lions.


subjectivity of evaluating a radioactive band on an autoradiogram.

B. Cellular and Subcellular Localization The 64 kD antigen is not species-specific but appears to be tissue-specific, i.e., restricted to the islets, and probably to the beta cells.'*.'" A number of beta cell lines, derived from insulinomas from several species, have consistently failed to show expression of the 64 kD antigen at levels detectable by immunoprecipitation of biosynthetically labelled beta cells. One important implication of these findings is that the 64 kD antigen may be associated with a component of the glucosecoupled insulin secretory pathway, which is defective in transformed beta cell lines. Indeed, a MT 38 000 fraction from insulin secretory granules has been shown to generate T cell lines from IDDM subjects20 and is similar in size to a proteolytic cleavage product of the 64 kD antigen.21 It should be noted that a 38 kD antigen is sometimes co-precipitated with the 64 kD antigen. The question pertaining to the subcellular location of the 64 kD antigen is still unclear. Studies using Triton X-114 extracts of fractionated rat islets showed that it was co-localized with the 5'-nucleotidase-rich plasma membrane fraction1R,22,23 and it was proposed, therefore, that it could be a surface target for antibodies in IDDM. The 64 kD antigen detected in rat islet preparations has been reported to possess amphiphilic proper tie^.^^ When rat islets were subjected to Triton X-114 phase partitioning, the majority of the 64 kD antigen was recovered in the aqueous phase and a minority in the hydrophobic detergent phase, suggesting a membrane location.23 However, the 64 kD antigen could not be precipitated from Triton X-100 extracts of purified, hand-picked human islets that had been surface-labelled with 1251, which argues against its surface e x p r e ~ s i o n When .~~ analysed under reducing conditions, the protein was reported to exist in varying proportions of disulphide-linked dimers and trimers.26 C. Biochemical and Structural Characterization The 64 kD antigen has been reported to exist as a and p components which have identical charge and differ only in molecular weight.24 Using two-dimensional electrophoretic analysis,


Baekkeskov et al.24 provided convincing evidence that the 64 kD a/P dimer was the only islet protein specifically recognized by antibodies in IDDM sera. Our studies (unpublished) have failed to confirm the existence of two components of the 64 kD antigen. Partial tryptic proteolysis of [35S]methioninelabelled 64 kD antigen immunoprecipitated from detergent extracts of rat islets produces a series of fragments of 50, 40, and 37 kD.Z1When IDDM sera were used to screen the proteolytic fragments, 81% recognized the 50 kD fragment and 77% the 40 and 37 kD fragments. Interestingly, some 64 KAb-negative sera contained antibodies that precipitated the latter fragments, implying the existence of cryptic epitopes. Overall, 93% of IDDM sera precipitated at least one of the three tryptic peptides. The 64kD antigen has not been quantitatively purified to homogeneity and therefore no direct sequence information is available. The 64kD antigen does not bind wheat germ agglutinin, which indicates that it is therefore not an N-acetylglucosamine or sialic acid-containing glycoprotein.2s

D. Regulation of 64 kD Antigen Expression Kampe et aLZ7 cultured rat islets in 5, 11, or 28 mM glucose for varying times and found that in 28 mM glucose the amount of 64 kD protein recovered by immunoprecipitation with IDDM sera was significantly greater (up to four-fold over basal). They concluded that the synthesis of the 64 kD antigen may be increased by hyperglycaemia. A corollary is that the "honeymoon" period which occurs frequently with correction of hyperglycaemia after clinical onset could result, at least in part, from a decrease in antigenicity of the beta cell. Using high-resolution twodimensional PAGE, a 65 kD protein from rat islets was reported to be upregulated at least 20-fold by increasing the glucose concentration of the labelling medium from 3 to 18 mM.28This protein, termed a glucospondin by the authors, has not yet been reported to be recognized by IDDM sera. Viral infections are possible triggering agents for IDDM, with Coxsackie virus being one of the prime candidates. Gerling and ChatterjeeZ9 reported that the expression of two islet proteins was significantly enhanced in islets 72 h after infection of mice with Coxsackie virus. The first was glyceraldehyde-3-phosphatedehydrogenase, thought to be involved in the regulation of


glucose-stimulated insulin r e l e a ~ e . ~The " second was a M, 64 kD protein that was precipitated with sera from IDDM subjects containing antibodies to the 64 kD antigen. Indeed, the difference in frequency of 64KAb in uninfected (11%)mice versus infected (89%) mice supports a role for Coxsackie infection in triggering IDDM. A great deal of information has accumulated about the effects of cytokines on the beta cell (for a review see ref. 31). An obvious question is whether cytokines, such as IFN-y and TNF that upregulate the expression of immune molecules on beta cells, also modulate the expression of beta-cell antigens. There are no data available which answer this question for the 64 kD antigen.

E. Identification as Glutamic Acid Decarboxylase The 64 kD antigen is now thought to be glutamic acid decarboxylase (GAD), the enzyme responsible for the synthesis of the inhibitory neurotransmitter y-aminobutyric acid (GABA).I ' The genesis of the studies which led to this conclusion was the finding by Solimena et n1.32,3-' of a high frequency of IDDM and other organspecific autoimmune diseases in stiff man syndrome (SMS), a rare neurological disease characterized by GAD antibodies (Figure 2). In addition, it had been shown that GAD was expressed not only in GABA-ergic neurones of the brain, but also in pancreatic beta cells (Figure 3).34 The hypothesis was therefore formulated that the target of 64 KAb in IDDM was GAD. The experimental evidence for the equivalence of the 64 kD antigen and GAD is strong but indirect.",35 The seminal observations' I delineated four considerations. First, a sheep antiserum against rat brain GAD (S3) and SMS sera immunoprecipitated a [3sSS]methionine-labelled 64 kD doublet from rat islets which, on the basis of electrophoretic mobility and cross-immunoprecipitation experiments, appeared to be the same species precipitated by antibodies in IDDM sera. Second, trypsin treatment of the 64 kD protein produced a 55 kD fragment that was recognized by both S3 and antibodies in three IDDM sera. Third, immunoprecipitation of rat brain homogenates with seven IDDM sera and rat islet lysates with one IDDM serum revealed a species of -64 kD that could be immunoblotted with 53. Finally, GAD enzymatic activity was precipitated from lysates of rat islets and rat brain extracts with one IDDM serum.



. .




plasmalemma GABA-transporter

From Solknena and De Camefi TINS : 14, 1991 Figure 2. Synthesis of GABA from glutamic acid by GAD in inhibitory neurones of the central nervous system. High-titre GAD antibodies have been reported in subjects with stiff man syndrome.


GAD in the Islet



Figure 3. Production and effects of GABA in pancreatic islet cells. Most pre-clinical IDDM subjects have GAD antibodies which are cross-reactive with brainderived GAD.

GAD catalyses the a-decarboxylation of Lglutamic acid to form GABA and CO, (Figure 4) and is probably the rate-limiting enzyme determining steady-state levels of GABA in the brain. The synthesis of GABA requires three enzymes: GAD; GABA-glutamic acid transaminase (GABA-T), which converts GABA to succinic semialdehyde in the absence of a-ketoglutarate; and an NAD-requiring succinic semialdehyde dehydrogenase, which completes the metabolism of GABA to succinic acid (Figure 4).’6 GAD requires potassium ions and, in some of the more primitive forms,37 also a high concentration (20-100 mM) of 9-mercaptoethanol for optimal activity in vitro. In addition, the activity of all forms of GAD requires the cofactor pyridoxal-5phosphate.37 Native GAD has been purified in active form from several brain sources including lobster,36 mouse,38 pig3’ and human.40 A summary of the properties of GAD prepared from these tissues is presented in Table I. There is broad agreement



thought to be mildly acidic and highly pHsensitive. At least three isoforms of pig porcine brain GAD39 and four of rat brain GAD4' have been reported which differ in their isoelectric points and kinetic properties (Table 11). Currently, it is not known whether similar isoforms of GAD can be purified from human brain or islets. The majority of GAD activity in the central nervous system (CNS) can be recovered as a water-soluble component of the cellular cytosol,","""' but a significant component remains membraneas~ociated.~~

Glutamic Acid Decarboxylase


HOOC-(CH2)2- CHNH2 COOH glutamic acid







+ P5P

B. Cellular and Subcellular Distribution

+ HOOC - (CHJ2 - C - COOH + NH, CI 1 a -ketoglutarate Cycle

Although GABA was discovered in mammalian brain in 1950,J2 the exact localization of GABA and GAD is still the subject of investigation. Initially thought to occur exclusively within the inhibitory neurones of the CNS, GAD is now known to occur in active form in non-neuronal tissues such as the kidney, liver, and adrenal gland.35,44,45 Both GABA and GAD activity have been demonstrated in rat pancreatic islets and in human insulinoma cells.46 More recently, GAD mRNA has been detected by Northern blot analysis in rat seminiferous epithelium and further localized to spermatocytes and sperm at id^.'^ Thus,

Figure 4. Synthetic pathway for the production of GABA from glutamic acid, catalysed by GAD. The cofactor pyridoxal-5-phosphate is essential for the activity of both GAD and GABA-T.

that the native enzyme consists of two disulphidelinked chains, probably identical, which upon reduction produce a single species of 44-72 kD depending on the source of the enzyme. GAD is

Table I. Biochemical Properties of Purified GAD Source of brain GAD Pig ~


Biochemical properties






Molecular weight Native Reduced


44 000

NR* 60 000

120000 60 000

NR 60 000

100 000 67 500 ? 5000

Isoelectric point






pH dependence






Amino acid composition






N- terminus K,, glutamate (mM) Pyridoxal-5-phosphate (FM)

NR 0.70


NR 0.17 0.18

NR 0.45 0.35

NR 1.24 0.76

A 1.28 0.13

Substrate specificity






Stability (days) (- 7OoC/50% glycerol)






* NR = Not reported, +Single letter amino acid code.



Table 11. Comparison of Rat Brain GAD 67 and GAD 65

Molecular weight Sequence homology OO / identical YO similar Enzymatic activity (fold increase over basal) Antigenicity with @-GAD67 (K2) a-GAD 65 (GAD-6) a-GAD (S3) Subcellular localization

GAD is clearly not strictly confined with regard to its cellular distribution. In evolutionary terms, brain GAD is a member of a family of pyridoxal phosphate-dependent decarboxylases which arose from a single gene duplication event and branched away from histidine d e c a r b ~ x y l a s e This . ~ ~ family of enzymes is highly conserved with a common pyridoxal phosphate binding site and 74% sequence homology between Drosophila and feline GAD.48 The complex cellular distribution of GAD in the CNS has been examined in detail.49The expression of GAD within the CNS is subject to environmental influences which modulate the overall patterns of GAD expression without altering the kinetic properties of the enzyme.50 Lesions in cerebeller Purkinje cells of neighbouring tissue with a neurotoxin (3-acetylpyridine) lead to an increase of u p to 50% in GAD mRNA and a ~ t i v i t y . ~This ' suggests that GAD in islets may be subject to upregulation following a local insult, be i t environmental or immunological in nature. Diverse treatments, for example with dopamine receptor antagonists or agonists in the rat52 and monocular deprivation in adult monkey,53 have been shown to cause significant changes in GAD levels.

C. Molecular Characterization Brain GAD mRNA is present very early in embryogenesis, as early as day 15 in the fetal rat. At this time, GAD abundance is about 50% of the adult level and by the first postnatal day GAD mRNA in the whole brain is almost at the adult

GAD 67 (GAD- 1)


66 600

65 400

GAD 65

65 (to GAD 65) 80 (to GAD 65)



Yes No Yes Cell bodies, dendrites

No Yes Yes Nerve endings

level. However the level of enzyme activity at birth is only 8% of its adult specific activity.54 On the basis of evidence that embryonic brain GAD included an exon not present in adult brain GAD, it has been proposed that early in brain development transcripts encoding a truncated form of GAD are expressed.55 This form cannot function as a decarboxylase because the stop codon in the embryonic exon occurs upstream of the binding site for pyridoxal phosphate. No such data are currently available for islet GAD. The existence of embryonic forms of GAD could have implications for the development of immune tolerance to GAD. The molecular cloning of GAD from mouse,5' rat,57feline,58and human59brain has only recently been reported. Comparison of the deduced amino acid sequences shows that GAD is highly conserved. Rat and feline GAD are 96% identical and the partial human brain GAD sequence also displays a high degree of homology with the equivalent region of feline brain GAD.5" The chromosomal locations of mouse DNA sequences homologous to a feline brain GAD cDNA clone are at two distinct loci: on chromosome 2 in a region of conserved homology with the long arm of human chromosome 2 and on chromosome 10.60 Both neurones and pancreatic islet cells contain at least two forms of immunoreactive GAD which differ in size, charge, and a n t i g e n i ~ i t y " " ' ' ~ ~ (and reviewed in ref. 64). These two forms, with molecular weights of -67 000 and -65 000, known as GAD 67 (GAD-1) and GAD 65 (GAD-2), are encoded by two distinct genes in rat brain, have


only about 65% nucleotide identity, and have different pyridoxal phosphate requirements and subcellular locations65 (Table 11). Data are also available for cloned rat islet GAD 67 which displays > 96% homology with mouse, cat, and human GAD 67 from the brain. Interestingly, GAD 65 cDNA from human islets shares no more than 63% identity with previously published brain GAD 67 ~ e q u e n c e s . ~GAD ~ , ~ ~65 and GAD 67 have also been localized to human chromosomes 2 and 10, respectively.68

D. Antigenicity-Relevance in IDDM The first reports indicating that GAD was an autoantigen in SMS appeared at a time when GAD was thought to be almost exclusively found in the inhibitory neurones of the CNS (reviewed in ref. 69). It is now known that GAD also occurs at levels comparable to those i n the brain in the islet (beta) cells and it is assumed that antibodies to GAD (GAD Ab) are synonymous with those to the 64 kD antigen.'l However, the relationship between GAD and the 64 kD antigen is not fully clarified and will be discussed below. Nevertheless, there is no doubt that GAD is an autoantigen in I D D M . ' ' T ~ ~We -~~ have found that the majority of ICA-positive first-degree relatives of IDDM subjects (designated as pre-clinical IDDM subjects) and approximately half of the newly diagnosed IDDM subjects have autoantibodies to human brain GAD.72 More recent studies using foetal pig brain GAD show that 21/47 (45%) of pre-clinical, 26/66 (41%) of recentonset, and 15/30 (50%) of established IDDM subjects tested had GAD Ab.73a In addition, in longitudinal studies, the level of GAD antibody did not change either before or after clinical diagnosis, suggesting that loss of antigenic drive due to beta-cell destruction is unlikely to be operating in these subjects. Persistence of GAD Ab in established IDDM may reflect the response to GAD outside the beta cell. Human brain GAD was purified to homogeneity by us by a series of chromatography steps ending in affinity chromatography with a GAD monoclonal antibody, and we found that only IDDM and SMS sera specifically immunoprecipitated the enzyme.72 Martino et aZ.,70 using similar assay systems and partially purified GAD from adult male Wistar rat brains, reported GAD antibodies in only 22% of recent-onset IDDM subjects. It should be noted that the frequency of GAD antibodies in the recent-onset IDDM subjects


was lower than that reported for 64 KAb.'fl,12O n the other hand, Rowley et using 1251-labelled affinity-purified adult pig brain GAD and an ion exchange resin to selectively bind glutamate from an enzymatic reaction mix, reported that the frequency of GAD Ab was 69% in recent-onset IDDM and 59% in established IDDM. No data were presented for pre-clinical subjects. Differences in the prevalence of GAD Ab probably reflect the combination of different substrates, assay methods, and subjects. In addition, allowance must be made for the fact that some GAD Ab inhibit GAD enzymatic At the DNA and protein level there are at least two distinct immunoreactive forms of GAD (see Sections 1II.A and 1II.B). Christgau et ~ 1 reported the existence of two antigenic forms of GAD from islets; the first was a 65 kD hydrophilic form with a PI of 6.9-7.1 which corresponded to GAD 67 from the brain, and the second (presumably equivalent to GAD 65) was a more abundant 64 kD amphiphilic form which exists in three distinct isoforms with regard to cellular compartmentalization and hydrophobicity. In pulse-chase experiments, GAD 65 had a shorter half-life than GAD 67; remained hydrophilic and soluble; and did not resolve into isomers. It should be noted that isomeric forms of brain GAD 65 have not yet been reported. It is widely agreed that beta-cell destruction in IDDM is T cell-mediated (reviewed in refs 1 and 31). The evidence for this relies heavily on the spontaneous rodent models of IDDM, the NOD mouse,75 and the BB rat,76 in which cell transfer studies have directly demonstrated the key role of T cells in mediating beta-cell destruction. Evidence for the role of T cells in human IDDM is less direct but includes migration inhibition of blood leucocytes in the presence of islet h ~ m o g e n a t e s ,cytotoxicity ~~ of peripheral blood mononuclear cells (PBMCs) from IDDM subjects against human insulinoma cells78and rat and human i ~ l e t s , inhibition ~ ~ , ~ ~ by PBMCs of insulin release from mouse islets,81 generation from PBMCs of CD4 T lymphocyte clones to human islet cells,62reactivity to human insulin by PBMCs from subjects with pre-clinical IDDM,83and reactivity to human islets and foetal pig proislets by PBMCs from subjects with pre-clinical and recent-onset IDDM.5*84Reactivity of T cells in the peripheral blood of pre-clinical IDDM subjects, in response to islets, is a marker of pre-clinical IDDM, equivalent to either high-titre ICA ( 2 40 JDF units) or 64 KAb.5





Which antigen(s) elicits T-cell reactivity in the primary stage of IDDM is a key question. GAD is a candidate. There have been no published reports showing that purified native GAD is a Tcell autoantigen. Studies from our laboratory17 demonstrate that -50% of pre-clinical subjects react to crude GAD extracts from purified foetal pig proislets and, importantly, when GAD activity was removed from the crude GAD extract by affinity chromatography, reactivity decreased by more than half. In a preliminary reports5 we also showed that peripheral blood T cells from some pre-clinical and newly diagnosed IDDM subjects reacted to recombinant GAD 67 expressed as a hexahistidine fusion protein. Atkinson et aLg6 then reported that recombinant GAD 67 stimulated T cells of 12 of 18 (66%) newly diagnosed and 5 of 9 (55%) pre-clinical IDDM subjects, but only 2 of 19 (11%) of ICA-negative relatives. These studies indicate that GAD is an important T-cell antigen. Diaz et al.R7inoculated E . coli GAD into the footpads of diabetes-resistant BB rats and after 10 days assayed the popliteal lymph node population for GAD-specific T cells. Four stable CD4-positive MHC-restricted T cell lines were isolated which proliferated selectively in response to E . coli GAD. It is not known whether any of these lines can respond to eukaryotic GAD forms. The other major T-cell antigen which has been described is a 38 kD fraction associated with the insulin secretory granule.20 At this stage, it appears that the 38 kD granule-associated antigen is distinct from the 37/40 kD tryptic cleavage fragments previously discussed which are derived from the 64 kD antigen.21 Interestingly, antibodies that recognize the 37/40 kD fragments appear to be associated with more rapid progression to clinical IDDM,88 in contrast to antibodies to a 50 kD tryptic fragment of the 64 kD antigen which also recognize GAD.88*89

E. Relationship to ICA Islet-cell antibodies remain the primary reference point for a serological diagnosis of IDDM and at high titre they are predictive for clinical lDDM.90Insulin autoantibodies (IAA) are diagnostic signposts for an important reason-insulin is the only known beta cell-specific antigen.91-96The combination of IAA and ICA appears to be a more reliable predictor for progression to clinical IDDM than ICA a l ~ n e . ~ ' - ~ ' ICA-positive first-degree relatives of people with IDDM are operationally defined as "pre-


clinical". In this group (defined by ICA), it is difficult to analyse independently antibodies to the 64 kD antigen or GAD.'7,72Although there is a high degree of concordance between ICA and 64 KAb,72 there are exceptions. ICA-positive sera may be 64KAb and GAD Ab-negative, or vice versa. There is disagreement on the extent of concordance between ICA and GAD Ab. Martino et aL7" report an invariable association, whereas Rowley et aL7' report only a weak association between these two antibodies in either recentonset or established IDDM subjects. It should not be forgotten that 90% of people developing IDDM do not have a first-degree relative with the disease.9s Is ICA explained by GAD autoantibodies? Evidence has recently been presented that the autoantigen of "restricted ICA" (reacting with human and rat but not mouse islets) is GAD."" First-degree relatives with restricted ICA and GAD antibodies appeared to have a lower risk of IDDM than relatives with the non-restricted form of ICA, the implication being that GAD antibodies are a poor marker for progression to diabetes. Our findings (unpublished) and those of othersLo" suggest that while monoclonal GAD antibodies stain the whole islet in an identical pattern to ICA showing no specificity for beta cells, it is not possible to remove ICA or decrease their titre by adsorption with recombinant forms of GAD.

IV. RELATIONSHIP BETWEEN GAD AND THE 64 kD ANTIGEN The identity of the 64 kD antigen had been long awaited and the report of Baekkeskov et al." which identified the 64 kD antigen as GAD was received with great interest. As discussed in Section ILE, four lines of evidence implicated GAD as the 64 kD antigen. That GAD is an autoantigen in IDDM is now beyond doubt, but just how relevant GAD is to the pathogenesis of IDDM is unclear. It is not beyond doubt that GAD accounts for all of the 64 kD antigen. At a biochemical level, comparison of the properties of GAD antigen and the 64 kD antigen reveals several disparities (Table 111). The 64 kD antigen is reported to exist as two components of 65 and 64 kD which display identical charge characteristics. The 64 kD form of the antigen has been reported to have two isoforms (a and p).24,74 It is important to note



Table 111. Comparison of the Properties of the 64 kD Islet Antigen and Native Human Brain GAD 64 kD



Molecular weight Isoelectric point pH dependence Tissue specificity

64 000-65 000

59 000-67 000






Brain, pancreas,

Cellular specificity


kidney, testis All inhibitory neurones, p-cell, spermatozytes

Subcellular location


Soluble cytosolic enzyme

* NR


Not reported.

distinct isoforms of GAD exist and that non-islet, that the chemical structure of the 64 kD antigen non-neuronal isoforms may lack determinants has not been defined directly and some variation exists in the descriptions of its i ~ o f o r m s . ~ ~ , ~ ~recognized by IDDM-associated antibodies. A strong positive correlation between antibodies to GAD has been the subject of research reports brain GAD and islet 64 kD antigen ( r = 0.91) was since 1950 and is therefore more extensively reported. Interestingly, GAD antibodies were only defined. In the brain, GAD may have at least four weakly associated with a 50 kD tryptic fragment of different isoforms,41 all of identical molecular islet 64 kD antigen and not associated with the weight but with different isoelectric points and 37 kD or 40 kD tryptic fragments. This final obserh y d r o p h ~ b i c i t i e sThe . ~ ~ M,of GAD under reducvation is difficult to reconcile with data2',Rs,8' ing conditions ranges between 59 and 67kD. showing that the 37 kD/40 kD tryptic fragments Monoclonal antibodies raised against purified are first 64 kD antigen-derived and second predicGAD from rat brain recognize a group of tive of progression to IDDM. The isoforms of polypeptides in the range 55-65 kD.43 Although GAD which may exist in the beta cell are yet to GAD enzymatic activity can be detected in isolated be fully defined. It may well be that the beta cell pancreatic islets, the 64 kD antigen has not been exhibits forms of GAD that are not found in the studied in whole brain (for technical reasons) and brain. Presumably, if an islet-specific isoform has not been reported in neuronal cell lines. If exists, it may be particularly relevant to IDDM. GAD represents the 64 kD islet antigen, the lack Recombinant GAD 65 of islet origin has been of data on the 64 kD antigen in neuronal cell lines reported67 and the authors note that despite the may simply reflect a lack of research effort. suggestion that the 64 kD antigen might be The 64 kD antigen was initially reported to be expressed on the cell ~ u r f a c e , ' ~ they , ~ ~ , could ~~ a membrane-bound protein which co-localized with 5'-nucleotidase to the plasma membrane.18,22,23 find no conventional leader sequence or transmembrane domain in the deduced GAD sequence. However, after its identification as GAD, the Further, a C-terminal hydrophobic extension 64 kD antigen was reported to contain both common to phosphatidylinositol membrane-anchmembrane-bound and soluble forms.74 By conored proteins was not present. In order to reconcile trast, GAD has consistently been described as the physical and cellular differences between predominantly a soluble cytosolic enzyme with 64 kD antigen and GAD, it may be necessary to only a small amount existing as a membraneawait the purification of native islet antigens. bound There is no evidence that GAD Gianani et ~ 7 1 proposed . ~ ~ that a subgroup of is normally present on the plasma membrane. older, predominantly female subjects with highChristie et al.35 have reported that GAD titre antibodies to GAD progress only slowly, if at enzymatic activity from the kidney, liver, adrenal all, to clinical IDDM. These subjects have the gland, and testes could not be precipitated by "restricted" form of ICA attributed to GAD Ab (see serum antibodies that precipitated brain and islet Section 1II.D). If GAD Ab are not predictive for forms of GAD. They suggested that antigenically



progression to clinical IDDM, how can they be equated with 64 KAb reported to be highly predictive of clinical d i ~ e a s e ? ' ~ Recent , ' ~ ~ evidence of Genovese et ~ 1 suggests . ~ that ~ GAD ~ antibody does not account for all ICA. Pre-incubation of IDDM ICA-positive sera with rat brain homogenate led to the total block of beta cell-specific ICA staining, while staining of the remainder of the islet was unaffected. The authors note, but do not explain, that GAD immunostaining of islets with GAD-6 monoclonal antibody is not restricted to beta cells. At the T-cell level, Atkinson et d 1 O 0 found Tcell proliferative responses to recombinant GAD 65 in 63% of ICA-positive relatives of IDDM subjects and 61% of newly diagnosed IDDM subjects. Kaufman et a1.'03 have reported that GAD Ab levels generally decline after IDDM onset, in contrast to their previous claim for 64 KAb.'O' It is not yet clear whether GAD Ab per se are predictive of progression to IDDM as r e p ~ r t e d ' ~ , ' for ~ ' 64 KAb. In an attempt to define the molecular identity of the 64 kD antigen, Kaufman et a1.'03 depleted solubilized [35S]methionine-labelled islet cells with GAD-6 monoclonal antibody (anti-GAD 65) and used recombinant GAD 65 to adsorb the 64 kD antigen. They claimed that the 64 kD antigen is immunologically (but not necessarily biochemically) indistinguishable from GAD 65. Islet cells contain both GAD 65 and GAD 67, and they found that antibodies to both were present in IDDM.Io3 Although they were able to detect antibodies to either GAD 65 or GAD 67 in most of the 23 individuals tested, no reactivity with the amino terminus of GAD 65 (which contains the 30% sequence dissimilarity with GAD 67) was demonstrated. It is difficult to reconcile the claim that GAD 65 accounts for the 64 kD antiged7 and that GAD 67 is not cross-reactive with the 64 kD islet antigenlo3 if antibodies against these two forms of GAD are directed to their conserved sequences.lo3This question needs resolution, and recognition should be given to the heterogeneity of the anti-GAD immune response in IDDM.

and react with GAD exist, there are no data that demonstrate the presence of cytolytic GAD Ab or T cells in islets in IDDM. The available data can be interpreted only in the context of the predictive value of anti-GAD autoimmunity. As discussed in Section III.D, the degree to which GAD antibodies in IDDM subjects are predictive of progression to clinical diabetes remains controversial. The s ~ g g e s t i o n ~ " that ~ ' ~ ) GAD ~ Ab may denote a group of subjects at low risk of IDDM seems puzzling. This might be explained by an anti-islet immune response mediated by CD4positive Th2 cells that produce IL-4, IL-5, and IL10 for high-titre antibody production rather than by CD4-positive Th, cells which produce IFNy and IL-2 for delayed-type hypersensitivity reactions,lo4 consistent with the notion that beta-cell destruction is T ~ell-mediated.'~The prediction would be that "pre-clinical" subjects at high risk for progression to clinical disease are those with high T-cell and low antibody responses to GAD. GAD antibodies in IDDM were initially reported as a result of their association with SMS, which is itself frequently associated with polyendocrine autoimmune disease, including IDDM.69 IDDM is a relatively common disease of younger persons, whereas SMS is a rare disease of mainly older persons. While IDDM is due to T cell-mediated beta-cell destruction, in SMS there does not appear to be evidence of neuronal d e s t r ~ c t i o nIf. ~GAD ~ is an autoantigen common to both IDDM and SMS, it must be asked why these two diseases are so different. Why don't IDDM subjects have clinical signs of SMS? Th, cells may predominate in SMS, driving a largely antibody-mediated disease. This raises more questions. For example, why would IDDM and SMS arise, respectively, as the outcome of Th, (cell-mediated) and Th, (antibody-mediated) CD4 T-cell responses? Could there be tissue-specific differences in antigen processing and presentation? Do the antibody and T-cell epitopes of GAD differ between IDDM and SMS?



A pathogenetic role for GAD in IDDM has not yet been established. Until new data appear, GAD antibodies and T cells can be considered markers for, but not mediators of, IDDM. Although antibodies and T cells which specifically recognize

If GAD is to be recognized as a primary target of beta-cell destruction, then some conceptual problems need to be resolved. First, the lack of tissue and cellular specificity of GAD is difficult to reconcile with the specific loss of beta cells in



2. Foulis AK, Liddle CN, Farquharson MA, and IDDM. Second, despite the high frequency of Richmond JA: The histopathology of the pancreas CAD Ab in operationally-defined pre-clinical in type 1 (insulin-dependent) diabetes mellitus: IDDM subjects, there is no evidence that GAD a 25-year review of deaths in patients under 20 Ab are independent markers of progression to years of age in the United Kingdom. Diabetologia 29~267-274, 1986. IDDM. Longitudinal studies which address this 3. Makino S, Kunimoto K, Muroaka Y, Mizushima question are yet to be reported. As in other organY, Katagiri K and Tochino Y: Breeding of a Nonspecific autoimmune diseases, GAD is but one of Obese Diabetic Strain of Mice. E x p . Anim. 29(1) several disease-specific autoantigens. It is likely 1-13, 1980. that the predictive value of antibodies to beta4. Gleichman H, and Bottazzo GF: Islet cell and insulin autoantibodies in diabetes. Itnniunol Today cell antigens will require that they be analysed 8:157-168, 1987. conjointly rather than individually. Finally, why 5. Harrison LC, Chu XS, DeAizpurua HJ, Graham should GAD, a soluble cytosolic enzyme not M, Honeyman MC, and Colman PG: Islet-reactive known to exist on surface membranes, be specifically T cells are a marker of pre-clinical insulintargeted by the immune system? Enzymes are also dependent diabetes. ] Clin Inuest 89:1161-1165, 1992. antigens in other autoimmune diseases, e.g. thyroid 6. Harrison LC, Colman P, Dean 8, Baxter R, and peroxidase in Hashimoto's thyroiditis,'05 H+/K+ Martin FIR: Increase in remission rate in newly ATPase in pernicious anaemia,*06 pyruvate diagnosed type 1 diabetic subjects treated with dehydrogenase in primary biliary c i r r h o ~ i s , ' ~ ~ azathioprine. Diabetes 34:1306-1308, 1985. steroid 17a-hydroxylase in Addison's disease,lo8 7. Campbell I, and Harrison L: Molecular pathology of type I diabetes. Mol B i d Med 7:299-309, 1990. and cytochrome P-450 db-1 in autoimmune 8. Rayner ML, Harrison LC, Campbell IL, and hepatitis type II.'09 Heyma P: Detection of insulin antibodies in Criteria are required to define a pathogenic newly-diagnosed type 1 diabetic children after role for autoantigens like GAD in IDDM. Ideally, "acid-stripping" of sera. Diabetes Res 6:l-4, 1987. antigen-specific T-cell responses should reflect the 9. Colman PG, Nayak RC, Campbell IL, and Eisenbarth GS: Binding of cytoplasmic islet cell disease process and antigen-specific strategies; for antibodies is blocked by human pancreatic example, tolerance induction should downregulate glycolipid extracts. Diabetes 37:645-652, 1988. antigen-specific T cells and retard the disease 10. Baekkeskov S, Nielsen JH, Marner B, Bilde T, process. The availability of recombinant GAD will Ludevigsson J, and Lernmark A: Autoantibodies facilitate the development of improved diagnostic in newly diagnosed diabetic children immunoprecipitate human pancreatic islet cell proteins. assays73and the evaluation of GAD autoimmunity Nature 298:167-169, 1982. in the pathogenesis of IDDM, in particular the 11. Baekkeskov S, Aanstoot S, Christgau A, Reetz A, identification of T-cell epitopes and the role of Solimena A, Cascalho M, Folli F, Richter-Olesen CAD-reactive T cells. Although GAD autoimmunH, and DeCamilIi P: Identification of the 64 k ity has heralded a new wave of research into the autoantigen in IDDM as the GABA-synthesizing enzyme GAD. Nature 347:151-156, 1990. molecular mechanisms of IDDM, we still have a S, Dyrberg T, and Lernmark A: 12. Baekkeskov considerable way to go to establish that it is more Autoantibodies to a 64-kilodalton islet cell protein than an epiphenomenon. precede the onset of spontaneous diabetes in the BB rat. Science 224:1348-1350, 1984. Acknowledgements Margaret Thompson assisted with the preparation of the manuscript. HJDeA is a Senior Research Officer supported by Diabetes Australia and LCH is a Senior Principal Research Fellow of the National Health and Medical Research Council of Australia. The research of the authors and their colleagues is supported by grants from AMKAID Pty. Ltd., Juvenile Diabetes Foundation International, and the Victorian Health Promotion Foundation.

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Glutamic acid decarboxylase in insulin-dependent diabetes mellitus.

Glutamic Acid Decarboxylase in Insulin-Dependent Diabetes Mellitus Henry J. DeAizpurua and Leonard C. Harrison Burnet Clinical Research Unit, The Walt...
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