Proc. Natl. Acad. Sci. USAVol. 91, pp. 242-246, January 1994Biochemnstry

A membrane form of brain L-glutamate decarboxylase:Identification, isolation, and its relation toinsulin-dependent diabetes mellitus

(raminobutyric acid/neurotransmitter enzyme)

BRiTTo NATHAN*, JUN BAO*, CHE-CHANG Hsu*, PATRICIA AGUILAR*, ROSEMARY WU*, MINA YAROM*,CHAO-YING KUOt, AND JANG-YEN WU*:*Department of Physiology and Cell Biology, University of Kansas, Lawrence, KS 66045-2106; and tDepartment of Pediatrics, University of Tennessee,Memphis, TN 38163

Communicated by Shang Fa Yang, September 28, 1993 (received for review June 29, 1993)

ABSTRACT A membrane form of L-glutamate decarbox-ylase (GAD) was identified and purified to apparent homoge-neity from hog brain. The purified GAD was established as anintegral membrane protein by phase-partitioning assay,charge-shift electrophoresis, and chromatography on a hydro-phobic interaction column. This membrane GAD has a nativemolecular mass of 96 ± 5 kDa and is a homodimer of 48 ±3-kDa subunits. Immunoprecipitation and immunoblottingtests revealed the presence of antibodies against this membraneGAD in sera from patients with insulin-dependent diabetesmeilitus. Since this form of GAD appears to be an integralmembrane protein and is presumed to have extracellulardomains exposed, it seems reasonable to suggest that mem-brane GAD is more likely than soluble GAD to be involved inthe pathogenesis of insulin-dependent diabetes and relatedautoimmune disorders such as stiff-man syndrome.

y-Aminobutyric acid (GABA) is the major inhibitory neuro-transmitter in the mammalian central nervous system (1) andalso serves signaling (2) and trophic (3) roles in severalneuronal and non-neuronal tissues. The rate-limiting step inGABA biosynthesis is the decarboxylation of L-glutamate byL-glutamate decarboxylase (GAD; EC 4.1.1.15). GAD hasbeen implicated in several neuronal disorders, such as epilepsy(4), schizophrenia (5), and stiff-man syndrome (6), and hasbeen identified as the autoantigen in insulin-dependent diabe-tes mellitus (IDDM) (7) and stiff-man syndrome (8). Severalforms of soluble GAD (sGAD), notably GAD-65 and GAD-67(65 and 67 kDa, respectively), have been extensively studied,ranging from kinetic studies, to studies of regional and cellu-lar/subcellular distribution, to structural analysis (for review,see ref. 9). From sequence information, it is clear that none ofthe sGADs, including GAD-65 and GAD-67, contains a stretchof hydrophobic amino acids long enough to span the mem-brane (about 20 residues), a typical feature for integral mem-brane proteins, or contains the appropriate consensus se-quences for the attachment of GAD to membranes throughfatty acylation via esterification, N-myristoylation, or glypia-tion (9-11). Unlike sGAD, little is known with certainty aboutthe structure and function ofmembraneGAD (mGAD) despitethe fact that about 50%6 of the total GAD activity in the brainis attributable to mGAD (12, 13). GAD can interact withmembranes by ionic or hydrophobic mechanisms. It wasreported that GAD could become associated with membranesin the presence of Ca2+ (14). Covarrubias and Tapia (15, 16)showed that this Ca2+-induced binding of GAD to the mem-branes occurred predominantly with pyridoxal 5'-phosphate(PLP)-dependent GAD, suggesting that mGAD might be dif-

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ferent from sGAD in terms ofaffinity toward its cofactor, PLP.Martin and Martin (17) have shown that apoGAD has a strongaffinity for polyanions (e.g., hexasulfate) and hence is at-tracted to synaptic vesicles, whose cytoplasmic face is knownto be enriched in acidic phospholipids (10). However, Changand Gottlieb (13) have demonstrated that 60% of GAD ismembrane-bound and can be released only with detergent(0.2% Triton X-100) but not-with high salt (1 M NaCl or KCl),suggesting that the interaction between GAD and membranesis predominantly hydrophobic. Christgau et al. (18) reportedthat pancreatic (3 cells expressed two autoantigenic forms ofGAD, GAD-65 and GAD-64. GAD-65 is a soluble protein,whereas GAD-64 is firmly membrane anchored and can bereleased only by detergent, such as Triton X-100, but not byany of the agents known to release peripheral membraneproteins. Christgau et al. (19) have shown that the autoantigenGAD-65 (previously referred to as GAD-64 by the sameauthors) is anchored to membranes of microvesicles in 3-cellsby palmitoylation in the N-terminal domain. Reetz et aL (20)also reported colocalization ofGAD and GABA with synaptic-like microvesicles in (3 cells and (3cell lines. Recently Solim-ena et al. (21) reported association ofGAD-65 but not GAD-67with the Golgi complex of transfected Chinese hamster ovarycells. Further, the same authors showed that the association ofGAD-65 with the Golgi complex was mediated by the N-ter-minal region. Hence, although GAD-65 is not an integralprotein, it can be associated with membranes through post-translational modification. In regard to possible functions, ithas been proposed that mGAD may be involved in processingof memory and information and regulation of the centralnervous system (22, 23). However, no definite results havebeen reported yet to support the above propositions. Thus itis clear that the information regarding the structure andfunction ofmGAD is quite limited. Therefore, we decided toundertake the purification of mGAD from porcine brain tohomogeneity and to obtain more precise information about itsstructure, properties, and function. We present evidenceindicating the presence of at least three different forms ofmGAD, referred to as mGADI, -II, and -III. In addition, wedescribe the isolation, purification, and characterization ofmGADI, which appears to be an integral membrane proteinand is different from any other forms ofGAD that have beenreported (9). Evidence suggesting that mGADI may be in-volved in certain autoimmune disorders such as IDDM is alsoincluded. mGADII is also an integral membrane protein and isan autoantigen for IDDM, whereas mGADIII is a peripheralmembrane protein and its association with membranes is

Abbreviations: GABA, y-aminobutync acid; GAD, L-glutamate de-carboxylase; mGAD, membrane GAD; sGAD, soluble GAD; AET,S-(2-aminoethyl)isothiouronium bromide hydrobromide; PLP, pyri-doxal 5'-phosphate; IDDM, insulin-dependent diabetes mellitus.iTo whom reprint requests should be addressed.

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Ca2+-dependent. These aspects of studies and the isolation,purification, and characterization of mGADII and mGADIIIare described elsewhere (24, 25).

MATERIALS AND METHODSPreparation of Membrane Fraction. All operations were

carried out at 4°C. In a typical preparation, a blender wasused to make a 10% porcine brain homogenate in doublydistilled water containing 2 mM S-(2-aminoethyl)isothiouro-nium bromide hydrobromide (AET) and 0.4 mM PLP. Thehomogenate was centrifuged at 100,000 x g for 1 hr, and thepellet was rehomogenized and centrifuged as before. Thepellet after the second centrifugation is referred to as P2M.P2M from fresh rat pancreas was prepared in the same way.

Solubilization of Brain mGAD. A 25% P2M homogenatewas made in 0.05 M standard buffer (0.05 M KPi, pH 7.2/0.4mM PLP/2 mM AET/1 mM EDTA) containing 0.5% TritonX-100. The mixture was gently stirred for 1 hr at 4°C and thencentrifuged at 100,000 x g for 1 hr. The supernatant servedas the starting material for mGAD purification.Enzyme and Protein Assays. GAD was assayed by measur-

ing the formation of 14CO2 from L-[1-14C]glutamic acid (12).Protein was assayed with a Bio-Rad protein assay kit. Bovineserum albumin was used as a standard.

Purification of mGADI. All procedures were carried out at4°C, and all solutions contained standard buffer. SolubilizedmGAD (2400 mg of protein obtained from 90 g of brain) wasapplied to a Whatman DE-52 column (5.0 x 55 cm) equili-brated in 0.05 M standard buffer containing 0.5% TritonX-100. The column was washed with 1 bed volume ofequilibration buffer, followed by elution with 2 bed volumesand 1 bed volume of 0.1 M and 0.3 M standard bufferscontaining 0.5% Triton X-100. The flow rate was 80 ml/hr.GAD was separated into three peaks, referred to as mGADI,-II, and -III. Fractions with specific activity > 60o that ofthepeak fraction were pooled and concentrated by ultrafiltrationin an Amicon stirred cell with YM30 membranes. The con-centrated sample was applied to an LKB Ultrogel AcA 34column (2.7 x 100 cm) equilibrated in 0.05 M standard buffercontaining 0.25% Triton X-100. MGADI was eluted with thesame buffer at 30 ml/hr. Sample volume was <3% of the bedvolume. Fractions with GAD activity from the AcA 34column were pooled, concentrated as described, and dia-lyzed overnight in 0.01 M KPi, pH 7.2/0.2% Triton X-100.The dialyzed sample was applied to a hydroxylapatite column(2.75 x 35 cm) which had been equilibrated in 0.01 M KPi, pH7.2/0.1% Triton X-100. Washing with 2 bed volumes ofequilibration buffer was followed by elution with 5 bedvolumes of a linear gradient of 0.05-0.15 M KPi buffer (pH7.2) containing 0.1% Triton X-100 and then with 2 bedvolumes of 0.3 M KP1, pH 7.2/0.1% Triton X-100. The flowrate was 160 ml/hr. Fractions with GAD activity werepooled, concentrated, and applied to a Sephadex G-200column (2.7 x 40 cm) equilibrated in 0.01 M KPi buffer.Sample volume was 2% of the bed volume. mGADI waseluted from the column at 20 ml/hr with the same buffer.Fractions from the Sephadex G-200 column with specificactivity > 80% that of the peak fraction were pooled,concentrated, and further purified by nondenaturing gradientPAGE as described.

Electrophoresis and Immunoblotting. Concentrated mGADI(1 mg/ml, 0.4 ml) from Sephadex G-200 was applied to anondenaturing 5-25% polyacrylamide slab gel (0.75 mm thick)in a Tris/glycine buffer system. The mGADI was in samplebuffer containing 1 mM AET, 0.2 mM PLP, 0.5% 2-mercap-toethanol, 25 mM Tris (pH 8.4), 10% glycerol, and 192 mMglycine. Electrophoresis was at 800 V for 12 hr at 4°C withbuffer containing all constituents of the sample buffer except10%o glycerol. After electrophoresis, one gel strip (1 cm wide)

was sliced into 0.2-cm squares and assayed for GAD activityand the second gel strip was stained for protein withCoomassie blue. mGADI was extracted from the band con-taining the highest GAD activity by homogenizing the gel(-200 ,u/cm2 ofgel) in 0.1M KPi, pH 6.0/1mM AET/0.2mMPLP/1 mM EDTA. The supernatant after a brief centrifuga-tion was the source of purified mGADI. SDS/PAGE ofpurified mGADI was performed as described (26). Immuno-blotting tests were carried out in both nondenaturing andSDS/PAGE systems. mGADI from Sephadex G-200 wassubjected to SDS/PAGE immunoblotting using IDDM serum(1:1000 to 1:6000 dilution) as described (26), with some minormodifications. Briefly, blotting of proteins was carried out at4°C in anLKB 2005 transfer unit, and the tank buffer contained25 mM Tris Cl (pH 8.3), 0.192M glycine, 0.5% SDS (or 0.05%SDS in the nondenaturing system), and 20% methanol. Blot-ting time varied from 3-12 hr based on the number of gels.Immunodetection was carried out with the ECL Western blotsystem (Amersham).

Immunoprecipitation. All operations were performed at4°C. Partially purified (-50%) mGADI or sGAD in standardbuffer was incubated with 5 ,ul of normal human serum,preimmune rabbit serum, IDDM serum, or standard bufferfor 24 hr. After incubation, samples were centrifuged at10,000 x g for 3 min, and the resulting pellets were washedonce in standard buffer. The pellets from the second washand the two supernatant solutions were assayed for GADactivity. The activity in the two supernatants was combinedto obtain the total GAD activity in the supernatant.

Kinetic Studies. Characterization of mGADI and sGADwith respect to Km, Vma, thermal stability, substrate spec-ificity, pH optima, and sensitivity toward various inhibitorswas carried out as described (26).Hydrophobic Interaction Chromatography. Partially puri-

fied sGAD and mGADI samples in 0.5 M standard bufferwere loaded on a SPW phenyl-Sepharose HPLC column (7.5mm x 7.5 cm) equilibrated in 0.5 M standard buffer. Elutionwith 0.5 M standard buffer for 20 min was followed by elutionwith 0.01 M standard buffer for 20 min. The flow rate was 1ml/min.

Triton X-114 Phase-Partitioning Assay. Triton X-114 phasepartitioning was carried out as described (27), except thatboth the sample and the sucrose cushion contained 150 mMKP1 (pH 7.2), 2 mM AET, and 0.4 mM PLP instead of 10mMTris HCl (pH 7.4) and 150 mM NaCl. After phase partition-ing, aliquots of the separated phases were assayed for GADactivity and expressed as percentage of the total activity.

Charge-Shift Electrophoresis. Charge-shift electrophoresiswas carried out (28) with modifications. Vertical 1% agaroseelectrophoresis was carried out at 4°C in a Hoeffer SE 600unit. The buffer in the gel was 0.05 M glycine NaOH, pH8.4/0.1M NaCl/1 mM AET/0.2mM PLP/0.5% Triton X-100with or without 0.25% sodium deoxycholate. The runningbuffer was the same except that Triton X-100 was omitted.Samples were in the running buffer containing 0.02% bro-mophenol blue and a 4-fold detergent concentration over thatused in the gels. Samples were allowed to equilibrate over-night at 4°C prior to electrophoresis. Electrophoresis wascarried out at 5 V/cm until the tracking dye reached within1 cm of the bottom of the gel. After electrophoresis, lanesloaded with enzymes were sliced into 1-cm squares andassayed for GAD activity.

RESULTSSolubilization and Purification of mGADI. The solubilized

mGAD was separated by DE-52 column chromatography intothree distinct peaks of GAD activity, mGADI, -II, and -III(Fig. 1), which contributed about 32%, 38%, and 30% of thetotal mGAD activity, respectively. mGADI from DE-52 was

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~~~~~~~~~~~~~~2

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FIG. 1. DEAE-cellulose anion-exchange chromatography of sol-ubilized mGAD. Solubilized mGAD (12) was applied to a WhatmanDE-52 column (5.0 x 55 cm) equilibrated in 0.05 M standard buffercontaining 0.5% Triton X-100. The column was washed with 1 bedvolume of equilibration buffer (first arrow), and bound material waseluted with 2 bed volumes of 0.1 M and 1 bed volume of 0.1 and 0.3M (second and third arrows) standard buffers containing 0.5% TritonX-100. The flow rate was 80 ml/hr. About 25 ml was collected perfraction and an aliquot of 100 ,l4 was used for GAD assay (18). GADactivity was expressed as the amount of 14CO2 formed in 30 min (o),and the protein concentration was monitored at 280 nm (o). ThreeGAD activity peaks referred to as mGAD-I, -II, and -III wereconsistently obtained.

further purified by a combination of nondenaturing PAGEand column chromatographies (Table 1). mGADI was puri-fied to apparent homogeneity (-2000-fold) with a yield of1.3%. If corrected for loss of activity from inactivation duringthe lengthy purification procedure, the overall yield and foldpurification will be even higher.

Criteria of Purity. mGADI purified by nondenaturingPAGE migrated as a single protein band at 96 ± 5 kDa innondenaturing gradient PAGE (Fig. 2C). Further, the proteinband correlated well with GAD activity (Fig. 2A). WhenmGADI was analyzed by SDS/PAGE, a single protein bandcorresponding to 48 ± 3 kDa was obtained, suggesting thatmGADI is a homodimer of48-kDa subunits (Fig. 2C, lane 1).The single protein band obtained in both nondenaturingPAGE and SDS/PAGE clearly indicates the homogeneity ofthe purified mGADI.

Kinetic Studies. The purified mGADI has a Km of 6.54 mMfor glutamate, Vma, of 0.63 ,umol of CO2 per min per mg andpH optimum of 7-7.5. Incubation at 45°C for 45 min resulted

A

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FIG. 2. Analysis of mGADI on various gel electrophoresis andimmunoblotting systems. (A) GAD activity profile of mGADI innondenaturing gradient PAGE. (B) Protein profiles in nondenaturinggradient PAGE. Lanes: 1, mGADI from Sephadex G-200 column(Table 1); 2, purified mGADI; 3, immunoblot ofmGADI with IDDMserum; M, size markers (kDa). (C) SDS/PAGE protein profile ofpurified mGADI (lane 1) and immunoblotting ofmGADI with IDDMserum (lane 2). Lane M, size markers (kDa).

in 80%o loss of the enzyme activity. mGADI was inhibited byreagents known to inhibit sGAD (29) such as the carbonyl-trapping agent aminooxyacetic acid, 50% at 10 ,uM; thesulfhydryl reagentp-chloromercuribenzoate, 50%o at 100 ,uM;and the thiol compound 3-mercaptopropionic acid, 50% at 10,uM. mGADI showed no detectable decarboxylase activitywith L-[U-14C]aspartic acid or L-[1-_4C]cysteinesulfinic acidas substrate.These kinetic properties are similar to those of sGAD. For

instance, sGAD has a Km of 1.32 mM and pH optimumaround 7.0. Incubation of sGAD at 45°C for 45 min resultedin loss of 75% of the activity. Aminooxyacetic acid, p-chlo-romercuribenzoate, and 3-mercaptopropionate inhibited 50%of sGAD activity at 10, 100, and 10 uM, respectively.

Immunoprecipitation with Antibodies Against sGAD. Anti-bodies against sGAD purified from porcine brain (29) werefound to crossreact with all three forms of mGAD as shownby immunoprecipitation (Table 2).

Triton X-114 Phase Partitioning. Fifty-eight ± 10% ofmGADI and 3 ± 1% ofsGAD partitioned in the detergent-richphase.Hydrophobic Interaction Chromatography. mGADI was

eluted at low ionic strength (Fig. 3A), like many other integralmembrane proteins (30), whereas sGAD was eluted at highionic strength, as expected for a hydrophilic protein (Fig. 3B).

Charge-Shift Electrophoresis. The electrophoretic mobilityof mGADI treated with a mixture of nonionic detergent(Triton X-100) and anionic detergent (sodium deoxycholate)

Table 1. Purification of mGADI from 267 g of porcine brain

Activity, Protein, Specific activity, Yield, Purification,Sample units mg (units/mg) x 103 % fold

Brain homogenate 4.7 29,663 0.16 100P2M 2.5 19,417 0.13 53Solubilized P2M 1.3 7,115 0.19 29DE-52 0.20 3,628 0.06 4.32 1Ultrogel AcA 34 0.18 979 0.19 3.92 3Hydroxylapatite 0.14 88 1.69 3.13 28Sephadex G-200 0.12 40 2.97 2.5 50Nondenaturing gradient PAGE 0.06 0.48 125 1.3 2083One unit of activity forms 1 iLmol of product per minute at 37°C. The specific activity of mGADI after DE-52 was used

as reference for calculation of fold purification.

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Table 2. Immunoprecipitation test using 10, 50, and 100 Al ofantibodies against sGAD

GAD activity in

GAD sample immunoprecipitate, cpm(GAD activity in cpm) 10 ,ul 50 Al 100 ,u

sGAD (18,577 cpm) 165 206 6372mGADI (7884 cpm) 201 8666 8125mGADII (14,839 cpm) 116 7061 8185mGADIII (20,479 cpm) 23 9250 7993

No detectable GAD activity was found in the precipitate whenanti-sGAD serum was replaced with the same amount of preimmuneserum.

shifted anodally relative to the mobility of mGADI treatedwith nonionic detergent alone (Fig. 4A). No significant shiftin the electrophoretic mobility was observed when sGADwas treated under the same conditions as mGADI (Fig. 4B).

Immunoprecipitation and Immunoblotting with IDDM Se-rum. IDDM serum precipitated almost 100% of mGADIactivity (Table 3). In immunoblotting tests using IDDM serum(1:6000 dilution), single protein bands at 96 ± 3 and 48 ± 2 kDa(Fig. 2) were obtained in nondenaturing gradient PAGE andSDS/PAGE, respectively. Similar results were obtained withother IDDM sera except that more concentrated sera (1:1000dilution) were used. Further, the protein band in nondenatur-ing gradient PAGE correlated with GAD activity.mGAD Activity in the Pancreas. About40% ofGAD activity

in the rat pancreas is associated with membranes. Thespecific activity ofmGAD in the pancreas was as 5.1 x 10-4unit per g of tissue. One unit is defined as 1 A.mol of CO2formed per min.

DISCUSSIONThere are three major findings presented in this report. (i)mGAD is as important as sGAD in GABA biosynthesis, since>50% of the total GAD activity in the brain is contributed by

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FIG. 3. Hydrophobic interaction chromatography of mGADI (A)and sGAD (B). Purified sample (2 ml) was loaded on a SPWphenyl-Sepharose HPLC column equilibrated in 0.5 M standardbuffer. Elution with 0.5M standard buffer (first arrow) for 20 min wasfollowed by elution with 0.01 M standard buffer (second arrow) foranother 20 min. Fractions were collected at 1 ml/min, and an aliquotof 100 IlI was used for GAD assay. The GAD activity was expressedas the amount of 14CO2 formed in 30 min (s) and the proteinconcentration was monitored at 280 nm (o).

2

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0.02 4 6 8 10 12Distance from the origin, cm

FIG. 4. Charge-shift electrophoresis (22) of mGADI (A) andsGAD (B). GAD activity of enzyme treated with Triton X-100 alone(o) or Triton X-100 plus sodium deoxycholate (o) was expressed asthe amount of 14CO2 formed in 30 min.

mGAD (Table 1). (ii) We have purified and characterized aform of GAD, mGADI, which is a homodimer of 48-kDasubunits, whereas both mGADII and mGADIII are ho-modimers of 60-kDa subunits (24, 25). Since 2-mercaptoeth-anol was included in both the nondenaturing and denaturinggel systems, we conclude that the native mGADI moleculeconsists of two 48-kDa subunits held together not throughdisulfide linkage but through noncovalent interaction. Thus,in molecular structure mGADI is different from other formsof GAD (9). Furthermore, purified mGADI may be anintegral membrane protein. (a) In the phase-partitioningassay, -58% of mGADI partitioned in the detergent-richphase, like many other integral membrane proteins (27),whereas <3% of sGAD partitioned in the detergent-richphase. (b) In charge-shift electrophoresis, the mobility ofmGADI treated with a mixture of nonionic detergent andanionic detergent shifted anodally relative to the mobility ofmGADI treated with nonionic detergent alone (Fig. 4A),whereas no significant shift in electrophoretic mobility wasobserved with sGAD (Fig. 4B). (c) In hydrophobic interactionchromatography, mGADI (Fig. 3A) was eluted in low-ionic-strength buffer, like many other integral membrane proteins(30), whereas sGAD (Fig. 3B) was eluted in high-ionic-strength buffer. However, proof that mGADI is an integralmembrane protein has to wait until the primary structure ofmGAD is known. If mGAD is an integral membrane protein,one would expect to see that it contains a stretch of hydro-phobic amino acid sequence of >20 amino acids so that it canspan the membrane. Although mGADI differs greatly fromsGAD in hydrophobic properties as indicated by detergentpartitioning, charge-shift electrophoresis, and hydrophobicinteraction chromatography, they are similar in kinetic prop-

Table 3. Immunoprecipitation test of mGADI with IDDM serum

GAD activity, cpmSerum Supernatant Pellet

Normal human 7224 + 111 153 ± 36IDDM 197 ± 21 7848 ± 220None* 8621 ± 162 164 ± 17

GAD activity is the average from five different samples.*Controls containing standard GAD buffer without serum.

A

.0.5 M 0.01 M

10 20 30

11A Q 1

0= (-T n r)Mi

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erties such as Ki, V., pH profile, and sensitivity towardvarious GAD inhibitors as well as in immunologic properties(Table 2). (iii) mGADI is a major autoantigen in IDDM, sinceIDDM serum contains high-titer antibodies against mGADIas indicated in both immunoprecipitation tests (Table 3) andimmunoblotting tests using nondenaturing, as well as SDS-containing polyacrylamide gels (Fig. 2). We have also foundmGADII to be an autoantigen in IDDM (25). Even thoughsGAD has been suggested as the autoantigen in IDDM andstiff-man syndrome, a direct role ofsGAD in their pathogen-esis has been considered unlikely because (a) sGAD is notexposed to the extracellular milieu and, therefore, is notlikely to trigger production of antibodies (8) and (b) even ifsGAD is exposed by lysis of cells as in the case of somedegenerative neurological diseases, antibodies against sGADhave not been detected in these patients (31). Based on ourfinding that mGADI may be an integral membrane proteinwhich is likely to have exposed extracellular domains, to-gether with the observation that IDDM serum containsantibodies against this protein, it seems reasonable thatmGADI could play a crucial role in the pathogenesis ofIDDMand related disorders. If this hypothesis proved to be true, itmight be possible to develop specific diagnostic tests andtherapies for these disorders.

The word processing ofthe manuscript done by Jan Elder and JudyWiglesworth is much appreciated. This study was supported in partby Public Health Service Grant NS20978, National Science Foun-dation Grant BNS-8820581, and a grant from Marion Merrell DowFoundation.

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