Aromatic - L - amino - acid decarboxylase , a pyridoxal phosphate - dependent enzyme , is a ( 8 - cell autoantigen FREDRIK RORSMAN

Different autoantigens are thought to be involved in the pathogenesis of insulin-dependent diabetes mellitus, and they may account for the variation in the clinical presentation of the disease. Sera from patients with autoimmune polyendocrine syndrome type I contain autoantibodies against the 13-cell proteins glutamate decarboxylase and an unrelated 51-kDa antigen. By screening of an expression library derived from rat insulinoma cells, we have identified the 51-kDa protein as aromatic-L-amino-acid decarboxylase (EC 4.1.1.28). In addition to the previously published fulllength cDNA, forms coding for a truncated and an alternatively spliced version were identified. Aromatic L-amino acid decarboxylase catalyzes the decarboxylation of L-5-hydroxytryptophan to serotonin and that of L-3,4-dihydroxyphenylalanine to dopamine. Interestingly, pyridoxal phosphate is the cofactor of both aromatic L-amino acid decarboxylase and glutamate decarboxylase. The biological significance of the neurotransmitters produced by the two enzymes in the 18 cells remains largely unknown. The cell-specific destruction of the insulin-producing pancreatic ,B cells in insulin-dependent diabetes mellitus (IDDM) is mediated by autoimmune mechanisms. Glutamate decarboxylase (GAD), an enzyme catalyzing the synthesis of y-aminobutyric acid (GABA) from glutamate, has been identified as a major autoantigen (1). Autoantibodies against this enzyme are present in up to 80% of the patients with IDDM (2, 3), and prediabetic individuals often exhibit reactivity against GAD before the appearance of any clinical manifestations of the disease (4). Patients with IDDM are prone to develop autoimmunity against other organs-e.g., Addison disease and Graves disease (5). In the rare disease autoimmune polyendocrine syndrome type I (APS I), an autoimmune attack against the parathyroid glands, the adrenal glands, and the gonads begins in childhood (6), whereas IDDM may occur at a higher age (5, 6). Nonendocrine symptoms-e.g., chronic mucocutaneous candidiasis, chronic active hepatitis, alopecia, vitiligo, and malabsorption-are also frequent manifestations of the disease (6). In the adrenal cortex (7) and gonads (8), cytochrome P450 side-chain cleavage enzyme-the catalyst of the rate-limiting step in the steroid synthesis-is the major autoantigen. GAD is recognized in the insulin-producing f3 cells by a majority of sera from APS I patients (9, 10). In addition, strong reactivity against a 51-kDa protein of previously unknown identity has been observed in all APS I sera examined (9). The 51-kDa protein has now been identified by immunoscreening of a cDNA library constructed from a rat insulinoma cell line.t MATERIALS AND METHODS Subjects and Sera. Sera were obtained from seven patients with diagnosed APS I. Clinical characteristics of six of these patients have been described previously (9). The seventh patient was a 17-year-old woman with candidiasis, hypoparathyroidism, Addison disease, gonadal insufficiency, and hypothyroidism. Normal goat serum was obtained from Dakopatts (Glostrup, Denmark), and a specific rabbit serum against aromatic-L-amino-acid decarboxylase (AADC, EC 4.1.1.28) (anti-AADC) was purchased from Biogenesis (Poole, Dorset, U.K.). Construction of RINm 5F cDNA Library. The LiCl/urea method (11) was used to prepare total cellular RNA from the rat insulinoma cell line RINm 5F. Cells were cultured in Ham's F12 medium (Nord Vacc, Skarholmen, Sweden) supplemented with nonessential amino acids and 10% fetal calf serum (FCS; Biochrom, Berlin). Poly(A)+ RNA was purified with Oligotex-dT (Qiagen, Chatsworth, CA). cDNA was synthesized and the library in the A ZAP II vector was constructed according to instructions of the supplier (Stratagene), except for size fractionation of the cDNA, which was performed on a continuous 5-20% potassium acetate gradient. Five milliliters of gradient was overlaid with 100 ,ul of 10mM Tris HCl and 1 mM ethylenediaminetetraacetic acid (EDTA), pH 7.4, containing the synthesized cDNA. After centrifugation for 3 hr at 50,000 rpm (230,000 x g) in an SW 50.1 rotor (Beckman) at 22°C, 0.5-ml fractions were collected and analyzed by electrophoresis in a 1.0% agarose gel. Fractions containing cDNAs larger than 1000 bp were pooled and ligated into A ZAP II. The completed library, containing 4.5 x 106 recombinant clones, was amplified once. Immunoscreening of the Library. We plated 5 x 105 plaqueforming units (pfu) of the amplified library on Escherichia coli XL-1 at a density of 350 pfu/cm2. After 3.5 hr of culture at 42°C, plates were overlaid with nitrocellulose filters (Hybond C, Amersham) previously soaked with 10 mM isopropyl f-Dthiogalactopyranoside (IPTG; Sigma) and cultured for another 3.5 hr at 37°C. The filters were carefully removed from the plates and washed in 15 ml of 20 mM Tris-HCl, pH 7.5/0.1% gelatin/0.05% Tween-20 (TBS-GT) three times for 5 min. Nonspecific protein binding was blocked with 15 ml of 1% gelatin in 20 mM Tris HCl, pH 7.5, for 1 hr, after which the filters were washed in TBS-GT as above. To reduce unspecific binding of the secondary antibody during color development, the filters were incubated with normal goat serum at a dilution of 1/1000 in TBS-GT for 30 min. After a third wash cycle, filters were incubated overnight with sera diluted 1/5000 in TBS-GT, from one of the patients. Filters were washed in 20 mM Tris HCI, pH 7.5, supplemented with 0.1% gelatin and then incubated with alkaline phosphatase-conjugated goat anti-human IgG for 1.5 hr prior to color development with a p-nitrobluetetrazolium chloride (NBT)/5-bromo-4-chloro-3Abbreviations: IDDM, insulin-dependent diabetes mellitus; GAD, glutamate decarboxylase; GABA, y-aminobutyric acid; APS I, autoimmune polyendocrine syndrome type I; AADC, aromatic-L-aminoacid decarboxylase; FCS, fetal calf serum. *To whom reprint requests should be addressed. tThe sequence reported in this paper has been deposited in the GenBank data base (accession no. U31884). 8626 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Medical Sciences: Rorsman et al. indoyl phosphate/toluidine (BCIP) system (Bio-Rad). Positive clones were rescreened until pure isolates were obtained. DNA Sequence Analysis. The nucleotide sequence of positive cDNA clones was determined by the dideoxynucleotide method (12) after subcloning into M13 derivatives. Sequence analysis was performed with the Applied Biosystems automatic DNA sequenator 373A, using T7 DNA polymerase (Sequenase) with either -21M13 dye primer (Applied Biosystems) or unlabeled synthesized internal primers (Scandinavian Gene Synthesis, Koping, Sweden) together with dye terminators (Applied Biosystems). Recombinant AADC. Clone 3.1 was ligated into the Not I and Kpn I sites of the pBK-CMV vector (Stratagene) for transient expression in COS cells. COS cells (2 x 106) were cultured to 80% confluence, harvested by trypsin treatment, washed twice in 10 ml of Dulbecco's modified Eagle's medium (DMEM; Biochrom) buffered with 10 mM Hepes, pH 7.4, and finally resuspended in 200 1.l of the same medium. Fifty micrograms of vector was added to the cells. After incubation on ice for 10 min, electroporation was performed with a Bio-Rad Genepulser at 200 V and 500 mF. The cells were incubated on ice for another 10 min and then seeded in DMEM containing 10% FCS. After 48 hr of culture, the cells were used in immunoprecipitation experiments. Isolation of Islets of Langerhans. Islets of Langerhans from Wistar-Furth rats were manually isolated after collagenase digestion (13) and cultured for 48 hr in RPMI 1640 medium (Biochrom) containing 11 mM glucose prior to metabolic labeling.