The presence of autoantibodies to multiple-islet autoantigens confers high risk for the development of type 1 diabetes. Four major autoantigens are established (insulin, glutamate decarboxylase, IA2, and zinc transporter-8), but the molecular identity of a fifth, a 38-kDa membrane glycoprotein (Glima), is unknown. Glima antibodies have been detectable only by immunoprecipitation from extracts of radiolabeled islet or neuronal cells. We sought to identify Glima to enable efficient assay of these autoantibodies. Mouse brain and lung were shown to express Glima. Membrane glycoproteins from extracts of these organs were enriched by detergent phase separation, lectin affinity chromatography, and SDS-PAGE. Proteins were also immunoaffinity purified from brain extracts using autoantibodies from the sera of patients with diabetes before SDS-PAGE. Eluates from gel regions equivalent to 38 kDa were analyzed by liquid chromatography–tandem mass spectrometry for protein identification. Three proteins were detected in samples from the brain and lung extracts, and in the immunoaffinity-purified sample, but not in the negative control. Only tetraspanin-7, a multipass transmembrane glycoprotein with neuroendocrine expression, had physical characteristics expected of Glima. Tetraspanin-7 was confirmed as an autoantigen by demonstrating binding to autoantibodies in type 1 diabetes. We identify tetraspanin-7 as a target of autoimmunity in diabetes, allowing its exploitation for diabetes prediction and immunotherapy.
Introduction
Detection of circulating autoantibodies to pancreatic islets (1), and identification of their molecular targets (2), has allowed the development of high-throughput autoantibody assays for clinical diagnosis of type 1 diabetes and identification of individuals at risk for disease. Evidence from both animal studies and human trials (3,4) indicates that type 1 diabetes may be prevented in individuals at risk. Hence, a range of therapies to interfere with immune responses has proved to be effective in preventing disease development in animal models of diabetes (5) and in slowing the loss of β-cell function occurring in the months after disease diagnosis in humans (6–8). There is now a focus on the development of procedures to interfere specifically in immune responses that cause type 1 diabetes, requiring knowledge of the major targets of the autoimmune response. There is no single common autoimmune target, and individuals differ in the antigen specificity of the autoimmune responses that develop. Four major humoral autoantigens have been identified in type 1 diabetes by defining the specificity of autoantibodies in the disease: insulin (9), glutamate decarboxylase (10), IA2 (11), and zinc transporter-8 (12). Autoantibodies to a fifth major humoral autoantigen, a 38-kDa glycosylated membrane protein (Glima), have been detected in 19–38% of patients with type 1 diabetes, with significantly higher prevalence (up to 50%) in children (13–15). The molecular identity of Glima has for many years proved elusive, hampering the characterization of autoimmunity to the protein and the development of sensitive, specific autoantibody assays. Glima is expressed in pancreatic β-cell and neuronal cell lines; is hydrophobic; is heavily N-glycosylated, having affinity for the lectin wheat germ agglutinin; and has a core protein backbone of ∼22 kDa (13–15). The aim of this study was to take advantage of these known physical properties to prepare Glima-enriched extracts for the identification of the autoantigen by mass spectrometry.
Research Design and Methods
Patients
Serum samples were obtained from 40 patients with type 1 diabetes (12–26 years of age) within 6 months of diagnosis from clinics in West Yorkshire with informed consent for screening for high-titer Glima antibodies, from 94 additional patients (12–63 years of age) for assay verification, and from 52 individuals without diabetes as negative control subjects. Approval for the analysis of autoantibodies in sera from these individuals was obtained from the Yorkshire and the Humber–Bradford Leeds Research Ethics Committee.
Screen of Sera of Patients With Type 1 Diabetes for Glima Antibodies
Glima antibodies were detected by a modification of immunoprecipitation assays previously described (13–15) using the neuronal mouse cell line GT1.7 as a source of antigen. Endogenous proteins in GT1.7 cells were labeled by incubation in methionine-free DMEM medium containing 4 MBq/mL 35S-methionine for 7 h at 37°C. Cells were washed with HEPES buffer (10 mmol/L HEPES, pH 7.4, 150 mmol/L NaCl, 10 mmol/L benzamidine) and stored at −80°C. Frozen cell pellets were extracted in HEPES buffer containing 2% Triton X-100 for 2 h on ice and insoluble material removed by centrifugation at 15,000g for 15 min at 4°C. Membrane glycoproteins were isolated by incubating cell extracts with wheat germ agglutinin-agarose on ice for 30 min and, after washing in HEPES buffer containing 0.5 mmol/L methionine, 100 mg/L BSA, and 0.5% Triton X-100, were eluted in the same buffer containing 0.5 mol/L N-acetyl glucosamine. Aliquots (20 μL) of eluate containing 3 × 105 cpm radiolabeled protein were incubated with 5 μL of test sera for 18 h at 4°C, and immune complexes were captured on 5 μL of Protein A-Sepharose. Immunoprecipitated proteins were eluted in 15 μL of SDS-PAGE Loading Buffer (Novex; Life Technologies, Paisley, U.K.) with heating at 90°C for 5 min and were subjected to SDS-PAGE on 12% polyacrylamide gels. After electrophoresis, gels were incubated in 40% v/v methanol, 2.5% v/v acetic acid, and subsequently in Enlightning Autoradiographic Enhancer (PerkinElmer, Coventry, U.K.), each for 30 min. Gels were dried and contacted with X-ray film (BioMax MR film; Kodak, Watford, U.K.) for up to 2 weeks. After exposure, X-ray film was developed to detect radiolabeled proteins specifically immunoprecipitated by sera from patients with type 1 diabetes, with bands detected in the 38,000 Mr region, indicating positivity for Glima antibodies.
Tissue Expression Screen
To identify mouse organs containing the highest levels of Glima for use in antigen purification, competitive binding studies were performed using detergent extracts of organs as unlabeled competitors with 35S-methionine–labeled Glima for binding to Glima antibodies in serum from a high-titer Glima antibody–positive patient. Mouse kidney, brain, heart, liver, thyroid, muscle, salivary gland, thymus, pancreas, spleen, adrenal, pituitary, and lung were dissected, frozen in liquid nitrogen, and stored at −80°C before extraction. Tissues were homogenized in homogenization buffer (10 mmol/L HEPES, pH 7.4, 0.25 mol/L sucrose, 10 mmol/L benzamidine), and membrane fractions sedimented by centrifugation at 15,000g for 30 min at 4°C. Supernatants were removed and pellets extracted in 2% Triton X-100 extraction buffer for 2 h on ice. Extracts were centrifuged at 15,000g for 30 min at 4°C, and supernatants were collected. The protein concentrations of extracts were determined using the Pierce BCA Protein Assay Kit (ThermoFisher Scientific, Loughborough, U.K.).
For the competition assay, wheat germ agglutinin agarose eluates from extracts of 35S-methionine–labeled GT1.7 cells were prepared as described above. Aliquots (20 μL) of GT1.7 cell glycoproteins containing 3 × 105 cpm radiolabeled proteins were incubated for 18 h at 4°C with 5 μL of serum from patient 029 alone, or with 10 μL of detergent extracts containing 100 μg of extracted protein from each mouse tissue. Immune complexes were captured on Protein A-Sepharose and processed for SDS-PAGE and autoradiography.
Partial Purification of Glima From Mouse Brain and Lung
Mouse brain and lung, shown to express Glima in the tissue screen (see results), were homogenized in ice-cold homogenization buffer in a Dounce homogenizer, and cell debris was removed by centrifugation at 500g for 5 min at 4°C. A membrane fraction was prepared by centrifugation of the supernatant at 10,000g for 15 min at 4°C, and the pellet was washed and extracted in 2% Triton X-114 extraction buffer for 2 h at 4°C. Insoluble material was removed by centrifugation at 10,000g for 15 min at 4°C, and a detergent phase was prepared by heat-induced phase separation as previously described (13). Fractions were added to wheat germ agglutinin-agarose at a ratio of 100 μL lectin-agarose to 5 mg total protein and incubated overnight at 4°C with gentle mixing. The beads were washed twice with HEPES buffer containing 0.5% Triton X-100 and twice in NOG buffer (1% N-octyl-glucopyroside in HEPES buffer). Wheat germ agglutinin–binding proteins were eluted in 0.5 mol/L N-acetyl-glucosamine in NOG buffer.
Eluates were concentrated using a Pierce SDS-PAGE Sample Preparation Kit (Life Technologies), solubilized in SDS-PAGE Loading Buffer (Novex; Life Technologies) for 10 min at 60°C, and electrophoresed on 12% Bis-Tris gels in MOPS running buffer. Gels were stained with Brilliant Blue G-Colloidal Coomassie (Sigma-Aldrich, Poole, U.K.), and gel slices corresponding to the 38,000 Mr region were excised for mass spectrometry.
Immunoaffinity Purification With Glima Antibodies
For immunoaffinity purification, 250 μL of pooled sera from three patients with high levels of Glima antibodies was used with 250 μL of sera from three antibody-negative individuals as a negative control. Sera were incubated with Protein A-Sepharose (250 μL) for 1 h at room temperature with rolling and washed three times in borate buffer (100 mmol/L boric acid, pH 8.3). Antibodies were cross-linked to Protein A-Sepharose with 20 mmol/L dimethyl pimelimidate in borate buffer for 1 h. Unreacted sites were blocked with 20 mmol/L ethanolamine for 10 min and washed before use.
Triton X-114 detergent phase–purified amphiphilic proteins from mouse brains prepared as above were added to the Glima antibody–positive and Glima antibody–negative beads and incubated overnight at 4°C with mixing. Beads were washed with 0.5% Triton X-100 in HEPES buffer prior to elution in 2% SDS at 90°C for 10 min. The eluate was concentrated to 20 μL using the SDS-PAGE Sample Preparation Kit and was subjected to SDS-PAGE and Colloidal Coomassie gel staining as above, and gel slices in the 38,000 Mr region were excised for mass spectrometry.
In-Gel Trypsin Digestion and Mass Spectrometry of 38,000 Mr Proteins
Gel slices representing 38,000 Mr regions of all samples were processed using the Pierce In-Gel Tryptic Digestion Kit (ThermoFisher Scientific) according to the manufacturer instructions, and the trypsin-treated extracts were vacuum dried and stored at −20°C prior to mass spectrometry. Samples were reconstituted in 30 μL of 50 mmol/L ammonium bicarbonate for 30 min at room temperature and centrifuged at 15,000g for 15 min to remove insoluble material. Samples were transferred to autosampler tubes, and 10 μL of each was analyzed by liquid chromatography–tandem mass spectrometry (LC-MS/MS). Peptides were resolved by reversed-phase chromatography on a 75-μm C18 EASY column using a linear gradient of acetonitrile in 0.1% formic acid at a flow rate of 300 nL/min over 50 min on an EASY Nano LC system (ThermoFisher Scientific). The eluate was ionized by electrospray ionization using an Orbitrap Velos Pro mass spectrometer (ThermoFisher Scientific) operating under Xcalibur (version 2.2; ThermoFisher Scientific) and by precursor ions selected based on their intensity for sequencing by collision-induced fragmentation. The tandem mass spectrometry (MS/MS) analyses were conducted using collision energy profiles that were chosen based on the charge/mass ratio and the charge state of the peptide.
Tandem mass spectra were processed into peak lists using Proteome Discoverer (version 1.3; ThermoFisher Scientific). All MS/MS samples were analyzed using Mascot (version 2.2.06; Matrix Science, London, U.K.) searching the UniProt Mus musculus database, assuming digestion with trypsin. Mascot was searched with a fragment ion mass tolerance of 0.80 Da and a parent ion tolerance of 10.0 ppm, with oxidation of methionine and carbamidomethylation of cysteine as variable modifications. Each data set was analyzed with a reverse FASTA database acting as a decoy.
Scaffold (version 4.3.2; Proteome Software Inc., Portland, OR) was used to validate MS/MS-based peptide and protein identifications, which were assigned by Peptide Prophet algorithms (16,17) and accepted at >95.0% probability. The UniProt database was manually searched for the physical characteristics of the proteins identified, including molecular weight, tissue distribution, and glycosylation. Of those identified, only one, tetraspanin-7 (Tspan7), matched the known properties of Glima (see results) and was characterized further.
Immunohistochemistry
Tspan7 localization in rodent tissues was performed by immunohistochemistry. Sections of formalin-fixed, paraffin-embedded rat brain, pituitary gland, pancreas, adrenal gland, lung, muscle, heart, liver, kidney, spleen, and thymus were dewaxed, and subjected to epitope retrieval in a microwave pressure cooker in 10 mmol/L citric acid (pH 6.0) and 0.05% Tween 20. Endogenous peroxidase activity was inhibited with 0.3% H2O2, and nonspecific binding was blocked with 25% nonimmune swine serum in PBS. Primary antibody to Tspan7 (anti-TM4SF2, catalog #HPA003140; Sigma-Aldrich) was applied at 1:1,000 dilution and incubated overnight at 4°C. Antibody labeling was detected with the Envision Kit (Dako, Ely, U.K.) according to the manufacturer instructions, and sections were counterstained in Mayer’s Hematoxylin Solution (Sigma-Aldrich) and visualized by microscopy.
Cloning and Expression of Recombinant Tspan7
cDNA for the coding region of mouse Tspan7 was amplified by RT-PCR from the mouse islet cell line Min6 using primers (5′-GAATTCATGGCATCGAGGAGAATGG-3′ and 5′-AGATCTCACCATCTCATACTGATTGGC-3′) that introduce EcoR1 and BglII sites at the 5′ and 3′ ends, respectively, with the native stop codon removed to allow expression as a fusion protein with a COOH-terminal purification tag. The PCR product was cloned into the pFLAG-CTS expression vector for protein expression in Escherichia coli BL21 cells after induction with isopropyl β-D-1-thiogalactopyranoside. Expressed protein was extracted from cells with Hen Egg Lysozyme (1 mg/mL) in PBS containing 10 mmol/L benzamidine, 1 mmol/L phenylmethylsulfonyl fluoride for 30 min at room temperature, followed by incubation in Triton X-100 (0.1%) for 5 min and DNase (1 µg/mL) for 10 min. The lysate was centrifuged at 10,000g for 10 min at 4°C, and the supernatant was used in immunoprecipitation assays.
Tspan7 Binding to Autoantibodies in Type 1 Diabetes
Individual Glima antibody–positive and Glima antibody–negative human sera (15 µL) were incubated with Protein A-Sepharose (15 µL), and the Ig captured was cross-linked to beads with dimethyl pimelimidate (18). Bead-bound antibodies were incubated overnight at 4°C with Triton X-100 extracts of mouse brain or with lysates of E. coli expressing recombinant mouse Tspan7. Beads were washed three times in 0.5% Triton X-100 in HEPES buffer, and captured proteins were subjected to SDS-PAGE and Western blotting using rabbit anti-Tspan7 antibody (anti-TM4SF, catalog #HPA003140; Sigma-Aldrich) at 1:250 dilution overnight at 4°C. Immunoprecipitated Tspan7 was detected with goat anti-rabbit IgG-peroxidase (catalog #A0545; Sigma-Aldrich) and SuperSignal West Pico Chemiluminescent substrate (ThermoFisher Scientific).
Luminescent Immunoprecipitation Assay for Detection of Tspan7 Antibodies
The coding region of human Tspan7 was cloned into the pCMVTnT vector as a fusion with nanoluciferase (NanoLuc) (Promega, Southampton, U.K.) at the 3′ end. The construct was transfected into HEK 293 cells with ExpiFectamine (ThermoFisher Scientific) for transient expression of antigen. Transfected cells were extracted in either 2% Triton X-114 or passive lysis buffer (Promega), and insoluble material was removed by centrifugation. Triton X-114 extracts were subjected to phase separation as described above and analyzed for fusion protein by Western blotting with rabbit antibodies to NanoLuc (a gift from Promega) or Tspan7 (anti-TM4SF). Luciferase expression in the cell extracts was quantified by luminometry using Nano-Glo assay reagent (Promega), and aliquots of passive lysis buffer extracts containing 106 light units of antigen were incubated with 5 µL of serum at 4°C for 16 h prior to the capture of antibody complexes on Protein A-Sepharose. Mouse brain extracts (150 µg protein) or lysates of E. coli expressing Tspan7 (250 µg protein) were added to reactions as sources of Tspan7 to compete for antibody binding. Complexes were washed and luciferase activity immunoprecipitation determined by luminometry with Nano-Glo assay reagent.
Results
Selection of Glima Antibody–Positive Sera
To identify patients with high levels of Glima antibodies for immunoaffinity purification, sera from 40 patients with recent-onset type 1 diabetes were screened by immunoprecipitation using radiolabeled mouse GT1.7 cell extracts (Fig. 1). Intense diffuse 38,000 Mr bands, which are indicative of high levels of Glima antibodies, were detected for three patients (Fig. 1, patients 029, 037, and 110). These three sera were used for subsequent Glima characterization and purification. Weaker 38,000 Mr bands indicative of Glima antibody positivity were detected in an additional 11 patients (Fig. 1).
Autoradiogram showing a screen of serum samples from patients with type 1 diabetes for Glima antibodies by immunoprecipitation of the 38,000 Mr protein from extracts of GT1.7 cells with detection by SDS-PAGE and autoradiography. A normal control serum (-ve) and a patient with type 1 diabetes previously determined to be positive for Glima antibodies (SL) were included in the assay. The location of Glima on the autoradiograph is marked. An indication of whether the samples were determined to be negative (−) or positive (+) for Glima antibodies is shown under each lane of the figure.
Autoradiogram showing a screen of serum samples from patients with type 1 diabetes for Glima antibodies by immunoprecipitation of the 38,000 Mr protein from extracts of GT1.7 cells with detection by SDS-PAGE and autoradiography. A normal control serum (-ve) and a patient with type 1 diabetes previously determined to be positive for Glima antibodies (SL) were included in the assay. The location of Glima on the autoradiograph is marked. An indication of whether the samples were determined to be negative (−) or positive (+) for Glima antibodies is shown under each lane of the figure.
Tissue Specificity of Glima Expression
To identify large organs in which Glima is expressed at suitably high levels for antigen purification, competitive binding studies were performed in which detergent extracts of normal mouse tissues acted as unlabeled competitors with radiolabeled Glima from GT1.7 cell lysates for binding to antibodies in serum from patient 029, who was strongly Glima antibody positive. Extracts of brain, pituitary, and lung reduced the intensity of radiolabeled 38,000 Mr protein, which is indicative of Glima immunoreactivity in these tissues (Fig. 2).
Autoradiograph of tissue expression screen demonstrating competition for Glima antibody binding to Glima antibody–positive (Glima Ab +ve) serum sample from patient 029 by proteins in Triton X-100 detergent (100 μg) extracts of normal mouse tissues. Serum from an individual without diabetes was included as a negative control (-ve control). Reduced intensity of the 38,000 Mr band is indicative of Glima immunoreactivity in that tissue.
Autoradiograph of tissue expression screen demonstrating competition for Glima antibody binding to Glima antibody–positive (Glima Ab +ve) serum sample from patient 029 by proteins in Triton X-100 detergent (100 μg) extracts of normal mouse tissues. Serum from an individual without diabetes was included as a negative control (-ve control). Reduced intensity of the 38,000 Mr band is indicative of Glima immunoreactivity in that tissue.
Identification of Glima Candidate Proteins by Mass Spectrometry
Extracts enriched for glycosylated membrane proteins from both brain and lung were prepared using Triton X-114 phase separation of amphiphilic membrane proteins followed by wheat germ agglutinin affinity purification. Proteins migrating at 38,000 Mr by SDS-PAGE were trypsinized and analyzed by LC-MS/MS. A total of 65 candidates in brain and 25 in lung were identified, of which 20 were common to both samples (Supplementary Table 1). Glycosylated membrane proteins immunoprecipitated from brain extracts by antibodies in the high Glima antibody titer patients’ serum pool were also subjected to SDS-PAGE and LC-MS/MS analysis, and 3 of the 20 protein candidates common to brain and lung were present in the Glima antibody–positive sample, but not in the negative control (Supplementary Table 1). These were as follows: 1) cytoplasmic actin-1 (Actb), a ubiquitous nonglycosylated cytoskeletal protein with a predicted molecular weight of 42 kDa; 2) guanine nucleotide-binding protein G(i) subunit α-2 (Gnai2), a nonglycosylated membrane–associated 40-kDa protein with a wide tissue distribution; and 3) Tspan7, a hydrophobic four-transmembrane domain protein with a core molecular weight of 27.5 kDa, five putative N-glycosylation sites, and a neuroendocrine distribution. Tspan7 closely matched the known properties of Glima, and additional studies were performed to compare properties and validate Tspan7 as the autoantigen.
Localization of Tspan7 in Rat Tissues
The tissue distribution of Tspan7 was determined by immunohistochemistry for comparison with patterns of Glima expression in the competition experiments described above. Strong immunolabeling for Tspan7 was detected in the rat brain, in particular the cerebral cortex, hippocampus, cerebellum, striatum, and thalamus (Fig. 3A); in the pancreatic islets (Fig. 3B); in the anterior pituitary (Fig. 3C); and in epithelial cells lining the alveoli in the lung (Fig. 3D). These observations agree with the Glima immunoreactivity described above and previously (13,15). Weak Tspan7 immunolabeling was also found in cells of the adrenal gland (Fig. 3E). No evidence of Tspan7 expression was found in the exocrine pancreas (Fig. 3B), muscle, heart, liver, kidney, spleen, or thymus (data not shown).
Immunohistochemical analysis of Tspan7 expression in rat tissues. Sections of formalin-fixed, paraffin-embedded rat tissues were labeled with rabbit anti-serum to Tspan7 and labeling detected by a peroxidase/3,3-diaminobenzidine–based system, with positive labeling detected as brown staining under the microscope. Representative images of the labeling of tissue sections of brain (A), pancreas (B), pituitary (C), lung (D), and adrenal gland (E) are shown.
Immunohistochemical analysis of Tspan7 expression in rat tissues. Sections of formalin-fixed, paraffin-embedded rat tissues were labeled with rabbit anti-serum to Tspan7 and labeling detected by a peroxidase/3,3-diaminobenzidine–based system, with positive labeling detected as brown staining under the microscope. Representative images of the labeling of tissue sections of brain (A), pancreas (B), pituitary (C), lung (D), and adrenal gland (E) are shown.
Immunoprecipitation of Tspan7 by Antibodies in Type 1 Diabetes
To demonstrate that Tspan7 is a target for autoantibodies in type 1 diabetes, extracts of mouse brain and lysates of E. coli expressing recombinant mouse Tspan7 were subject to immunoprecipitation with Glima antibody–positive and Glima antibody–negative sera followed by Western blotting with a rabbit antibody to Tspan7. A 38,000 Mr band representing Tspan7 was selectively immunoprecipitated from mouse brain detergent extract by three Glima antibody–positive sera, but not by control samples. The bands detected comigrated with the brain lysate control (Fig. 4A). Antibodies in the sera of patients with type 1 diabetes also specifically immunoprecipitated Tspan7 from bacterial lysates containing recombinant protein (Fig. 4B). Here, the protein migrated at ∼22,000 Mr, which is consistent with a lack of glycosylation in bacteria (Fig. 4B). The results confirm Tspan7 as a target of autoantibodies in type 1 diabetes.
A: Tspan7 labeling of Western blots of mouse brain proteins immunoprecipitated by antibodies in sera from Glima antibody–negative (-ve) and Glima antibody–positive (+ve) patients with recent onset of type 1 diabetes. The migration of molecular weight markers (10−3 × Mr) on the gel and the localization of Tspan7-specific bands at 38,000 Mr are marked. B: Tspan7 labeling of Western blots of proteins from lysates of Tspan7 expressing E. coli immunoprecipitated by antibodies in sera from Glima antibody–negative and Glima antibody–positive patients with recent onset of type 1 diabetes. The localization of Tspan7-specific bands at ∼22,000 Mr is marked; the migration of molecular weight markers is as in A. In both panels A and B, IgG heavy chains (50,000 Mr), light chains (25,000 Mr), and cross-linked Ig (>100,000 Mr) from all serum samples were also detected on the blot as a consequence of cross-reactivity with the peroxidase-conjugated anti-rabbit detection antibody. C: Tspan7 was expressed as a fusion protein with NanoLuc, and Triton X-114 extracts of cells were subject to heat-induced phase separation. Detergent and aqueous phases were subject to SDS-PAGE and Western blotting with antibodies to NanoLuc or Tspan7. The migration of molecular weight markers are shown (10−3 × Mr). D: Detergent extracts of NanoLuc-tagged Tspan7 were immunoprecipitated with normal control sera (Controls) (n = 30), sera from Glima antibody (Ab)–positive patients with type 1 diabetes (T1D) (n = 15), and the sera of Glima antibody–negative patients with type 1 diabetes and luciferase activity associated with each immunoprecipitate determined by luminometry. Data are plotted as luciferase activity immunoprecipitated in kilo light units (kLU), and sample codes for control or individuals with diabetes with high levels of antibodies are shown. E: Samples from control individuals or Glima antibody–positive patients with type 1 diabetes were tested for competitive binding by natural or recombinant Tspan7 in the LIPS by performing the immunoprecipitations in the absence (black bars) or presence of 150 μg of mouse brain extract (white bars) or 250 μg of lysates of E. coli expressing recombinant Tspan7 (hatched bars). Assays were performed in triplicate. The addition of brain and E. coli lysate significantly blocked antibody binding for all samples (P < 0.0001; ANOVA with Dunnett correction for multiple comparisons), with the exception of control sample CH.
A: Tspan7 labeling of Western blots of mouse brain proteins immunoprecipitated by antibodies in sera from Glima antibody–negative (-ve) and Glima antibody–positive (+ve) patients with recent onset of type 1 diabetes. The migration of molecular weight markers (10−3 × Mr) on the gel and the localization of Tspan7-specific bands at 38,000 Mr are marked. B: Tspan7 labeling of Western blots of proteins from lysates of Tspan7 expressing E. coli immunoprecipitated by antibodies in sera from Glima antibody–negative and Glima antibody–positive patients with recent onset of type 1 diabetes. The localization of Tspan7-specific bands at ∼22,000 Mr is marked; the migration of molecular weight markers is as in A. In both panels A and B, IgG heavy chains (50,000 Mr), light chains (25,000 Mr), and cross-linked Ig (>100,000 Mr) from all serum samples were also detected on the blot as a consequence of cross-reactivity with the peroxidase-conjugated anti-rabbit detection antibody. C: Tspan7 was expressed as a fusion protein with NanoLuc, and Triton X-114 extracts of cells were subject to heat-induced phase separation. Detergent and aqueous phases were subject to SDS-PAGE and Western blotting with antibodies to NanoLuc or Tspan7. The migration of molecular weight markers are shown (10−3 × Mr). D: Detergent extracts of NanoLuc-tagged Tspan7 were immunoprecipitated with normal control sera (Controls) (n = 30), sera from Glima antibody (Ab)–positive patients with type 1 diabetes (T1D) (n = 15), and the sera of Glima antibody–negative patients with type 1 diabetes and luciferase activity associated with each immunoprecipitate determined by luminometry. Data are plotted as luciferase activity immunoprecipitated in kilo light units (kLU), and sample codes for control or individuals with diabetes with high levels of antibodies are shown. E: Samples from control individuals or Glima antibody–positive patients with type 1 diabetes were tested for competitive binding by natural or recombinant Tspan7 in the LIPS by performing the immunoprecipitations in the absence (black bars) or presence of 150 μg of mouse brain extract (white bars) or 250 μg of lysates of E. coli expressing recombinant Tspan7 (hatched bars). Assays were performed in triplicate. The addition of brain and E. coli lysate significantly blocked antibody binding for all samples (P < 0.0001; ANOVA with Dunnett correction for multiple comparisons), with the exception of control sample CH.
Analysis of Tspan7 Antibodies by Luminescence Immunoprecipitation Assay
Patients screened for Glima antibodies were analyzed for Tspan antibodies by immunoprecipitation of recombinant NanoLuc-tagged human Tspan7. Western blotting with rabbit polyclonal antibodies to both NanoLuc and Tspan7 detected diffuse 38,000 Mr bands (the expected size of the nonglycosylated fusion protein) as the dominant immunoreactivity in cells transfected with the construct, with additional bands at approximately 80,000 Mr (Fig. 4C). The 38,000 Mr protein partitioned into the detergent on temperature-induced phase separation in Triton X-114. Transfected cell extracts were used in immunoprecipitation studies with normal control sera or with sera from Glima antibody–positive and Glima antibody–negative patients with type 1 diabetes. All but one of the control subjects (control sample V015) (n = 52) had low levels of Tspan7 antibodies (Fig. 4D). Four patients with high levels of Glima antibodies (Fig. 1) also immunoprecipitated high luciferase activity in the Tspan7 antibody assay (Fig. 4D), and significantly higher levels of Tspan7 antibodies were found in Glima antibody–positive patients than Glima antibody–negative patients (P < 0.0001; Mann-Whitney U test). In competition assays, natural or recombinant Tspan7 in brain or E. coli extracts partially (control sample V015) or completely (Glima antibody–positive patients with type 1 diabetes) blocked antibody binding to the NanoLuc-Tspan7 construct (Fig. 4E). Control sample V015 did not bind Tspan7 from mouse brain extracts when tested in the Western blotting assay. A second set of 94 patients with recent onset of type 1 diabetes was also tested in the Tspan7 antibody assay. Using a cutoff of mean ±3 SDs of control subjects (omitting the outlier), 40 (43%) were positive for Tspan7 antibodies (Fig. 4D).
Discussion
Autoantibodies to Glima in type 1 diabetes were first reported in 1996 (13), but its molecular identity has since then remained unknown. We used mass spectrometry of Glima-enriched fractions of brain and lung to search for likely candidates for Glima. LC-MS/MS analysis identified 65 proteins in 38,000 Mr gel samples of amphiphilic membrane glycoproteins from brain and 25 proteins from lung, of which 20 were common to both (Supplementary Table 1). Additional LC-MS/MS analysis of immunoaffinity-purified proteins isolated from brain extracts using Protein A-Sepharose–coupled Igs from Glima antibody–positive sera (with similar preparations from Glima antibody–negative sera as a negative control) further narrowed down the potential candidates for the testing of auto-antigenicity. Only six proteins detected in the brain or lung extracts were also present in the immunoaffinity-purified sample but absent in the negative control. Of these, five (Actb, Gnai2, Sfxn5 [sideroflexin-5], Kctd12 [potassium channel tetramerization domain 12], and Tuba1b [tubulin α-1B chain]) had a considerably higher predicted molecular weight (>36 kDa) than expected for the nonglycosylated Glima protein (∼22 kDa) (15). Furthermore, Actb, Gnai2, and Tuba1a are ubiquitous cytoplasmic proteins lacking the amphiphilic characteristics expected of Glima. Sfxn5 and Kctd12 were not detected in the lung extract. Tspan7 was, consequently, the most promising candidate for Glima identified in the LS-MS/MS analyses.
Tspan7 is a member of the tetraspanin family, members of which share structural characteristics of four transmembrane domains, with one short (EC1) and one long (EC2) extracellular loop (19). Four of the putative N-glycosylation sites are contained within the EC2 domain with a further site located in EC1. The presence of multiple transmembrane domains and N-glycosylation sites within Tspan7 is consistent with the hydrophobic properties and heavy N-glycosylation previously reported for Glima (13,15). There are four amino acid differences between mouse and human Tspan7, all of which are located in the long extracellular loop. The tissue distribution of Tspan7 expression has not been widely investigated, but analysis of the transcriptional activity of the Tspan7 gene has shown restricted tissue distribution with high levels being detected in regions of the adult mouse brain and lung (20). In the pancreas, Tspan7 is found specifically in the islets of Langerhans (21). Functionally, tetraspanin family members are involved in mediating signal transduction events and have been noted to regulate cell development, activation, growth, and motility through the trafficking of other transmembrane proteins (22). Both the extracellular and intracellular domains are able to interact with other proteins, and a number of tetraspanins bind integrins, thereby forming links with the actin cytoskeleton (23). Mutations in the Tspan7 gene are associated with X-linked mental retardation and neuropsychiatric diseases, potentially as a result of impaired ability of the actin cytoskeleton to drive neurite outgrowth (24). The ability of tetraspanins to form complexes with other membrane and cytosolic proteins may explain the copurification of multiple protein fragments identified in the LC-MS/MS analysis.
Proteins immunoprecipitated by Glima antibody–positive sera from brain extracts, or from bacterial extracts containing recombinant Tspan7, also bound rabbit antibodies to Tspan7 by Western blotting, confirming Tspan7 as the target of the antibodies. Glima autoantibodies bound both the glycosylated natural 38,000 Mr Tspan7 in brain and the 22,000 Mr nonglycosylated form of the protein expressed in E. coli. A luminescence-based immunoprecipitation system (LIPS) (25) using NanoLuc-tagged human Tspan7 expressed in mammalian cells showed that patients with high levels of Glima autoantibodies were also strongly positive in the anti-Tspan7 LIPS. The relationship between Glima and Tspan autoreactivity is imperfect, which may in part be the consequence of difficulties in ascertaining whether or not diffuse Glima bands are present on autoradiographs of immunoprecipitation reactions (Fig. 1). Individuals without diabetes had low levels of Tspan7 antibodies, with the exception of one strongly positive control. Antibodies in this control serum bound poorly to natural Tspan7 from mouse brain extracts, suggesting that antibodies in this particular sample may bind epitopes not displayed on the natural protein. The SDS-PAGE gel migration of the fusion protein indicated that the majority of recombinant luciferase-tagged proteins were not subject to the heavy glycosylation found on the natural protein, which is indicative of incorrect membrane insertion, protein folding, or intracellular targeting of the fusion protein required for appropriate posttranslational modification. Incorrect folding or lack of glycosylation may reveal antibody epitopes not normally displayed on the natural Tspan7. Further optimization of Tspan7 expression should permit the development of high-throughput assays for the detection of diabetes-associated Tspan7 autoantibodies with high sensitivity and specificity.
Autoimmunity to major autoantigens in type 1 diabetes appears within the first 5 years of life in at-risk children (26), with individual immune responses developing sequentially rather than simultaneously (27). Autoimmunity in the disease is therefore progressive, with the order of appearance of autoimmune responses to individual antigens differing between individuals and diversification of the immune response being essential for disease progression; therefore, disease rarely develops in individuals in whom autoimmunity develops only to single autoantigens (28). The optimum strategy currently adopted for assessing disease risk is to screen individuals for the presence of autoantibodies to multiple islet autoantigens. The inclusion of Tspan7 antibodies in the screen may improve the sensitivity and specificity of disease prediction in large populations, and will provide a fuller description of the major autoimmune responses that are developing in that individual, which is necessary for guiding the selection of autoantigen-specific immunotherapeutic agents to prevent the disease.
Article Information
Acknowledgments. The authors thank Raymond Chung and Malcolm Ward of King’s College London Proteomics Facility for LC-MS/MS analyses.
Funding. This study was funded by a research grant from Diabetes UK (grant 11/0004297) and by a Society for Endocrinology Early Career Award to K.A.M. C.C.R. was supported by a PhD Studentship from King’s College London Graduate School. Research by C.B., D.L., V.L., and L.P. was conducted within the framework of the Italian Ministry of Research project “Ivascomar project, Cluster Tecnologico Nazionale Scienze della Vita ALISEI.”
Duality of Interest. No potential conflicts of interest relevant to this article were reported.
Author Contributions. K.A.M. designed the study, researched and analyzed data, and wrote the manuscript. C.C.R., A.R., C.B., D.L., V.L., L.P., D.M., and R.G.F. researched and analyzed data. M.R.C. designed the study, researched and analyzed data, and contributed to the writing of the manuscript. All authors reviewed and edited the manuscript and approved the final version for submission. M.R.C. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Prior Presentation. Parts of this study were presented in abstract form at the 75th Scientific Sessions of the American Diabetes Association, Boston, MA, 5–9 June 2015.