of April 11, 2018.This information is current as

Meningioma-Associated Antigen TXNDC16Secretion and Immunogenicity of the

Eckart MeeseandAndreas Keller, Michael Menger, Richard Zimmermann

Ruth Nickels, Elmar Krause, Martin Jung, Jens Rettig,Werner, Valentina Galata, Christina Backes, Katja Schmitt, Christian Harz, Nicole Ludwig, Sven Lang, Tamara V.

http://www.jimmunol.org/content/193/6/3146doi: 10.4049/jimmunol.1303098August 2014;

2014; 193:3146-3154; Prepublished online 13J Immunol 

MaterialSupplementary

8.DCSupplementalhttp://www.jimmunol.org/content/suppl/2014/08/12/jimmunol.130309

Referenceshttp://www.jimmunol.org/content/193/6/3146.full#ref-list-1

, 19 of which you can access for free at: cites 51 articlesThis article

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The Journal of Immunology

Secretion and Immunogenicity of the Meningioma-AssociatedAntigen TXNDC16

Christian Harz,* Nicole Ludwig,* Sven Lang,† Tamara V. Werner,* Valentina Galata,*,‡

Christina Backes,*,‡ Katja Schmitt,* Ruth Nickels,x Elmar Krause,{ Martin Jung,‖

Jens Rettig,{ Andreas Keller,‡ Michael Menger,x Richard Zimmermann,‖ and

Eckart Meese*

In a previous study, we identified thioredoxin domain containing 16 (TXNDC16) as a meningioma-associated Ag by protein macro-

array screening. Serological screening detected autoantibodies against TXNDC16 exclusively in meningioma patients’ sera and not

in sera of healthy controls. TXNDC16 was previously found to be an endoplasmic reticulum (ER)–luminal glycoprotein. In this

study, we show an additional ER-associated localization of TXNDC16 in the cytosol by in vitro synthesis, molecular mass shift

assay, and flow cytometry. We were able to show TXNDC16 secretion in different human cell lines due to masked and therefore

nonfunctional ER retrieval motif. A previously indicated exosomal TXNDC16 secretion could not be confirmed in HEK293 cells.

The secreted serum protein TXNDC16 is bound in circulating immune complexes, which were found both in meningioma and

healthy blood donor sera. Employing a customized array with 163 overlapping TXNDC16 peptides and measuring autoantibody

reactivity, we achieved discrimination of meningioma sera from healthy controls with an accuracy of 87.2% using a set of only five

immunogenic TXNDC16 epitopes. The Journal of Immunology, 2014, 193: 3146–3154.

With 35% of all primary brain tumors, meningiomas arethe most common intracranial neoplasias. They occurwith an average annual incidence of 7 in 100,000 and

are generally benign (1, 2). We previously showed that meningi-omas trigger a complex humoral immune response, suitable fora highly accurate minimally invasive detection by serologicalmethods (3–5). Serological screening of recombinantly expressedclones revealed several meningioma-associated Ags reacting withpatients’ sera. Tumor-associated Ags represent a class of bio-markers that are recognized by autoantibodies in tumor patients’sera. Although in general, normal proteins are not antigenic,deregulated expression, altered posttranslational modifications, or

subcellular localization often provoke loss of immune system’sself-tolerance (6–8).Thioredoxin domain containing 16 (TXNDC16) emerged as one

of the most important meningioma-associated Ags to discriminatemeningioma patients from healthy controls. TXNDC16 was ex-clusively seroreactive with meningioma patients’ sera (3, 4). It hasa molecular mass of ∼93 kDa (NP_065835.2), and the gene isubiquitously expressed in normal and in many cancer tissues (9).Mitochondrial localization as suggested by Barbe et al. (10) waslater disproved by Riemer et al. (11), who found TXNDC16 to bean endoplasmic reticulum (ER)–luminal glycoprotein. It containsa predicted N-terminal signal peptide of 27 aa that is supposed tobe necessary for cotranslational translocation into the ER. Thesignal peptide of the polypeptide chain is bound by signal rec-ognition particle (SRP) during translation. SRP subsequentlybinds to the SRP receptor at the ER membrane, and the ribosome–polypeptide–SRP complex is delivered to the Sec61 complex,where polypeptide elongation and translocation are sustained, andthe signal peptide is cleaved by signal peptidase (12).Riemer et al. (11) detected TXNDC16 in cell culture super-

natant, which they explained by either the lack of a C-terminalER retrieval motif or the artificial overexpression. In general,ER-luminal proteins possess C-terminal ER retrieval motifs,variants of the consensus sequence KDEL (13, 14). Humanproteins containing this motif bind to one of the three KDELreceptors (ERD21, ERD22, and ERD23) in cis-Golgi and areretrotranslocated into the ER via coat protein complex (COP)I–mediated vesicular transport (13–18). TXNDC16 contains theC-terminal KDEL variant DKEL with an additional 6 aa encodeddownstream.TXNDC16 was previously detected in human parotid gland

exosomes (19). Exosomes are small membrane vesicles witha diameter of 30–100 nm and show a characteristic cup-shapedmorphology as observed by electron microscopy (20, 21). Exo-somes derive from late endosomes/multivesicular bodies by in-vagination of the endosomal membrane and are released due to

*Department of Human Genetics, Medical School, Saarland University, 66421Homburg/Saar, Germany; †Buck Institute for Age Research, Novato, CA 94945;‡Chair for Clinical Bioinformatics, Medical School, Saarland University, 66421Homburg/Saar, Germany; xInstitute for Clinical and Experimental Surgery, MedicalSchool, Saarland University, 66421 Homburg/Saar, Germany; {Department of Physiol-ogy, Medical School, Saarland University, 66421 Homburg, Germany; and ‖MedicalBiochemistry and Molecular Biology, Medical School, Saarland University, 66421Homburg/Saar, Germany

Received for publication November 18, 2013. Accepted for publication July 16,2014.

This work was supported by the Homburger Forschungsforderungsprogramm 2009and European Union Grant ContraCancrum 2010.

The microarray data presented in this article have been submitted to the GeneExpression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under accession numberGSE58949.

Address correspondence and reprint requests to Prof. Eckart Meese, Departmentof Human Genetics, Medical School, Saarland University, Building 60, 66421Homburg/Saar, Germany. E-mail address: hgemee@uks.eu

The online version of this article contains supplemental material.

Abbreviations used in this article: CIC, circulating immune complex; COP, coat proteincomplex; DPBS, Dulbecco’s PBS; ER, endoplasmic reticulum; ERAD, ER-associateddegradation; HA, hemagglutinin; malPEG, maleimide–PEG; PBS-T, PBS-Tween; PEG,polyethylene glycol; PFA, paraformaldehyde; P/S, penicillin/streptomycin; PSS, DPBScontaining 0.1% (w/v) saponin; RM, rough microsome; RT, room temperature; SRP,signal recognition particle; TXNDC16, thioredoxin domain containing 16.

Copyright� 2014 by TheAmericanAssociation of Immunologists, Inc. 0022-1767/14/$16.00

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multivesicular bodies’ exocytic fusion with the plasma membrane.They represent an unconventional secretion pathway for cytosolicproteins (22–24).Protein secretion offers a potential target for humoral immune

response. Backes et al. (25) and Daniels et al. (26) showed anincreased seroreactivity of prostate cancer patients against thesecreted protein LEDGF/p75 in contrast to controls. Via in silicoanalyses, we recently calculated a significant enrichment of se-creted and extracellular Ags in autoimmune diseases. In our pre-vious study, TXNDC16 mRNA transcription was found upregulatedby up to three times in meningiomas of TXNDC16 seroreactivepatients (27).In this study, we identify an additional ER-associated cytosolic

localization of TXNDC16 and examined whether the potentialTXNDC16 signal peptide is essential for its translocation into theER as well as its ability to translocate GFP into the ER likewise. Weshow that also endogenous TXNDC16 is detectable at 100 kDa insupernatants of several human cell lines (e.g., benign and malign-ant meningioma cell lines). We addressed whether TXNDC16 isretained due to its C terminus or is secreted physiologically, maybevia an exosomal pathway in HEK293 cells.In addition, we demonstrate that TXNDC16 occurs as processed

serum protein with molecular masses of 55 and 70 kDa. The 55-kDa fragment of TXNDC16 was found in circulating immunecomplexes both in meningioma sera and healthy controls. We usedcustomized peptide arrays of 163 TXNDC16 peptides to identifyimmunogenic TXNDC16 epitopes and asked whether discrimi-nation of meningioma and control sera is possible with a specificsubset selection of all immunogenic epitopes.

Materials and MethodsPrimers and plasmids

Full-length TXNDC16 and GFP cDNAs were amplified from plasmidspCMV-Sport6-TXNDC16 (Open Biosystems) and pEGFP-N1 (Clontech),respectively. Vector pSG5 (Agilent Technologies) was used to express allvariants of TXNDC16 and GFP in HEK293 and HeLa cells.

Cell lines and transient transfection

For transfection, human cervix carcinoma cell line HeLa (DeutscheSammlung von Mikroorganismen und Zellkulturen), human embryonickidney cell line HEK293 (Deutsche Sammlung von Mikroorganismen undZellkulturen), the benign meningioma cell lines Ben-Men-1 and HBL-52,and the malignant meningioma cell line IOMM-Lee were used. Menin-gioma cell lines were kindly provided by Prof. ChristianMawrin (Otto-von-Guericke University of Magdeburg). Cells were cultured in DMEM (LifeTechnologies) supplemented with 10% (v/v) heat-inactivated FBS (Bio-chrom) and 1% (w/v) penicillin/streptomycin (P/S; Life Technologies) at37˚C and 5% CO2. HeLa cells were transiently transfected with the non-liposomal transfection reagent FuGENE HD (Roche) according to themanufacturer’s instructions. HEK293 cells were transfected using thecalcium phosphate method. In detail, 3 3 106 cells were seeded per 145-cm2 dish and cultured for 24 h. Cells were washed with Dulbecco’s PBS(DPBS) (Life Technologies) and covered with 40 ml freshly supplementedDMEM. A total of 50 mg plasmid DNA in a total volume of 4.7 ml sterile,deionized water was mixed with 600 ml CaCl2 and 4.7 ml sterile filtered23 HEPES-buffered saline (280 mM NaCl, 1.5 mM Na2HPO4, and 50 mMHEPES [pH 7.05]). Cells were incubated overnight with the transfectionreaction. Subsequently, medium was removed, and cells were cultured inFBS-free DMEM supplemented with P/S and harvested after 48 h.

Immunofluorescence

HeLa cells were seeded at 3 3 105 cells/well in six-well plates containingsterile coverslips. After transient transfection using FuGENE HD (Roche),cells were fixed with 4% (w/v) paraformaldehyde (PFA) in DPBS for20 min at 4˚C. Fixed cells were washed twice with DPBS and blocked for30 min with 1% (v/v) goat serum (Life Technologies) in DPBS containing0.1% (w/v) saponin (PSS). Cells were incubated with anti-hemagglutinin(HA) (H-9658; mouse monoclonal, clone HA-7; 1:500; Sigma-Aldrich)and anti-PDI (sc-20132; rabbit polyclonal, 1:400; Santa Cruz Biotech-

nology) for 1 h at room temperature (RT). After removal of Ab solution,cells were washed three times with PSS and subsequently incubated withone of the following secondary Abs, each diluted 1:2000 in PSS, for 1 h atRT: Alexa Fluor 488–conjugated goat anti-mouse IgG (H+L) (A11001;Invitrogen), Alexa Fluor 488–conjugated goat anti-rabbit IgG (H+L)(A11008; Invitrogen), or Alexa Fluor 594–conjugated goat anti-rabbit IgG(H+L) (A11012; Invitrogen). Finally, cells were washed three times withPSS, covered with 10 ml ProLong Gold Antifade (Invitrogen), and trans-ferred to glass slides. Immunostaining was imaged with a Zeiss Elyra(Zeiss) or an Olympus AX70 microscope (Olympus).

Exosomes isolation

For depletion of bovine exosomes, FBS was ultracentrifuged overnight at100,0003 g and 4˚C. HEK293 cells were cultured for 24 h in DMEM with10% (v/v) exosome-depleted FBS and P/S in three 175-cm2 flasks. Aftercalcium phosphate transfection, exosomes were released into FBS-freeDMEM. Medium was collected, and detached cells, dead cells, and celldebris were removed by sequential centrifugation at 300 3 g for 10 min,2000 3 g for 10 min, and 10,000 3 g for 30 min, respectively, at 4˚C each.Secreted exosomes were subsequently pelleted at 100,000 3 g for 1 h at 4˚C.The supernatant was retained for further protein precipitation. The exosome-containing pellet was resuspended in an appropriate volume of DPBS (atleast 100 ml), and the ultracentrifugation step was repeated. DPBS super-natant was discarded, and the exosomes were resuspended in 100 ml DPBS.

Circulating immune complex preparation

To remove any suspended particles, serum was first centrifuged at 10,000rpm for 5 min. Serum supernatant was mixed with equal volume of 7%polyethylene glycol (PEG 8000) in borate buffer (0.1 M boric acid, 0.5 mMdisodium tetraborate, and 75 mM NaCl [pH 8.4]). Circulating immunecomplex (CIC) precipitation was done at 4˚C overnight. CICs were pelletedat 13,000 rpm for 10 min, washed twice with 3.5% PEG, and resuspendedwith initial volume of 0.01 M PBS.

Cell lysis, protein precipitation, SDS-PAGE, and Westernblotting

Cells were trypsinized, diluted in DMEM, pelleted, and washed once withDPBS (300 3 g, 5 min each). Cells were resuspended in an appropriatevolume (200 ml per 1 3 107 cells) of cell lysis buffer (300 mM NaCl,50 mM Tris-HCl, pH 7.4, and 0.5% [v/v] Triton X-100) containing pro-tease inhibitors (1 tablet of complete EDTA-free protease inhibitor mixture[Roche] in 10 ml lysis buffer) and incubated 20 min on ice. Crudemembrane fraction was pelleted at 20,000 3 g and 4˚C for 20 min. Su-pernatant was collected for subsequent protein precipitation. Cell lysatesand exosomes harvested from cell-culture supernatant were incubated onice for 30 min in 10% (v/v) TCA, and precipitated proteins were pelleted at15,000 3 g for 15 min at 4˚C. The protein pellet was resuspended in300 ml ice-cold acetone and centrifuged at 20,000 3 g for 5 min at 4˚C.The air-dried protein pellet was dissolved in 100 ml DPBS with proteaseinhibitors (1 tablet of complete EDTA-free protease inhibitor mixture in10 ml DPBS). Protein concentration was determined by measuring theabsorbance at 280 nm in a NanoDrop 2000 (Thermo Fisher Scientific,Waltham, MA). Protein, CIC, and serum samples (10 mg protein/CIC foreach sample or 0.5% serum, respectively) were denatured in 43 RotiLoadsample buffer (Roth) for 5 min at 95˚C prior separation in a 10% SDSpolyacrylamide gel at 125 V for 2 to 3 h. Separated proteins were trans-ferred onto a polyvinylidene difluoride membrane (Amersham) by wetWestern blotting at 25 V for 2 to 3 h).

Immunodetection

Polyvinylidene difluoride membranes were blocked with 5% (w/v) BSA(Sigma-Aldrich) in PBS-T (PBS-Tween; 10 mM NaH2PO4, 130 mM NaCl[pH 7], and 0.2% [v/v] Tween 20) or TBST (20 mM Tris, 137 mM NaCl[pH 7.6], and 0.1% [v/v] Tween 20), depending on primary Ab, for 1 h atRT or overnight at 4˚C. The membranes were incubated with the follow-ing primary Abs for 1 h at RT or overnight at 4˚C: anti-TXNDC16(HPA002543; rabbit polyclonal, 1:500 in PBS-T; Sigma-Aldrich), anti-CD63 (HPA010088; rabbit polyclonal, 1:500 in PBS-T; Sigma-Aldrich),anti–b-actin (A1978; mouse monoclonal, clone AC-15, 1:1000 in PBS-T;Sigma-Aldrich), anti-myc (ab9106; rabbit polyclonal, 1:500 in TBST;Abcam), anti-PDI (sc-20132; rabbit polyclonal, 1:400 in PBS-T; Santa CruzBiotechnology), anti-GAPDH (sc-47724; mouse monoclonal, clone 0411,1:1000 in PBS-T; Santa Cruz Biotechnology), and anti-GFP (rabbit poly-clonal, 1:1000 in PBS-T; kindly provided by Prof. Richard Zimmermann,Medical School, Saarland University). The membranes were washed threetimes with PBS-T/TBST for 10 min at RT and subsequently incubated

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with HRP-conjugated goat anti-rabbit IgG (H + L) (1:30,000; Dianova) orHRP-conjugated sheep-anti-mouse IgG (H + L) (1:50,000; Dianova), eachdiluted in 5% (w/v) nonfat dry milk/PBS-T or TBST for 1 h at RT. Proteinswere detected using ECL Plus Western blotting Detection (GE Health-care).

Molecular weight shift

HeLa cells were seeded at 2 3 106 cells per 55-cm2 dish and transientlytransfected with FuGENE HD (Roche) according to the manufacturer’sinstructions. Molecular weight shift assay with maleimide-PEG (malPEG;5 kDa) (Sigma-Aldrich) was performed as described previously (28).Proteins were separated by SDS-PAGE and detected by Western blotting,as described above.

Flow cytometry

Confluently grown and untransfected HeLa cells were harvested from three75-cm2 flasks, and cell aggregates were separated with 10% FBS in DPBS.Cells were pelleted for 5 min at 300 3 g and RT, resuspended in 9 mlDPBS supplemented with 3% (w/v) BSA, and split into three aliquots. Thefirst aliquot was left unpermeabilized, the other aliquots were per-meabilized with 20 mM digitonin or 0.1% (v/v) Triton X-100, respectively,for 30 min at RT. Each of the three aliquots was split into five smalleraliquots. Unpermeabilized cells were washed twice with BSA/DPBS, andpermeabilized cells were washed twice with BSA/DPBS containing 2 mMdigitonin or 0.01% Triton X-100, respectively, and pelleted at 300 3 g for5 min at RT. Three pelleted cell aliquots were resuspended in 1 ml primaryAb solution (anti–a-tubulin, T9026; mouse monoclonal, clone DM-1A,1:1000; Sigma-Aldrich; anti-PDI, 1:500; and anti-TXNDC16, 1:500) andtwo pelleted cell aliquots in Ab-free BSA/DPBS. After incubation for 30min at RT, cells were washed three times by adding 2 ml BSA/DPBS andcentrifugation at 3003 g for 5 min. Cells were fixed with 2% (w/v) PFA inDPBS for 10 min at 37˚C, and the three washing steps were repeated. Cellswere incubated for 30 min with secondary Abs Alexa Fluor 488 goat-anti-rabbit and Alexa Fluor 488 goat anti-mouse (each 1:2000 in BSA/DPBS).As negative control, one aliquot previously not incubated with a primaryAb was incubated with both secondary Abs. Secondary Abs were removed,and cells were washed three times with BSA/DPBS. Cells were subse-quently kept in fixative (1% [w/v] PFA/DPBS) until FACScan (BD Bio-sciences) measurement. A total of 2000 cells was analyzed per run.

Protein transport and sequestration

Cotranslational protein transport experiments were performed by synthe-sizing precursor polypeptides in reticulocyte lysate (Promega) in thepresence of [35S]methionine (1000 Ci/mmol) (PerkinElmer). The in vitrosynthesis reaction was prepared with 1 mg plasmid DNA according tomanufacturer’s instructions and split into two aliquots (25 ml final vol-ume), in one of which 6% (v/v) pancreatic ER-derived rough microsomes(RMs) were added (29). The samples were incubated for 60 min at 30˚C.Each reaction was subsequently split into two aliquots and incubated withor without proteinase K (170 mg/ml) for 60 min at 0˚C. The reaction wasstopped by addition of PMSF (10 mM). Processed polypeptides wereseparated by SDS-PAGE and detected by phosphoimaging (Typhoon-Trioimaging system; GE Healthcare).

Patients’ sera, peptide arrays, and bioinformatics

Meningioma sera and sera from healthy controls were subsequently storedat 220˚C after blood withdrawal. CelluSpots peptide arrays were manu-factured by Intavis with 163 TXNDC16 spanning peptides spotted as tworeplicate subarrays. The 15-mer peptides overlapping by 10 aa were co-valently bound to cellulose membranes with their C termini (for peptidesequences, see Supplemental Table I). Arrays were incubated according tothe manufacturer’s instructions. Serum and secondary Ab (Cy5-conjugatedAffiniPure rabbit-anti-human IgA + IgG + IgM [H + L]; Dianova) wereboth diluted 1:1000 in 5% (w/v) nonfat dry milk/TBST (150 mM NaCl,50 mM Tris-HCl [pH 7.5], and 0.05% [v/v] Tween 20). Cy5 detection wasperformed with microarray scanner ScanArray Lite (PerkinElmer) at633 nm, and peptide intensity values were analyzed with ScanArray Ex-press (photomultiplier tube 40%, 20 mm). All peptides were classified intothree groups via their mean foreground intensity values (2 “strongly se-ropositive” if x$ quantile 0.95, 1 “weakly seropositive” if quantile 0.8, =x , quantile 0.95 and 0 “seronegative” if x , quantile 0.8) (SupplementalFig. 1). Each classified peptide value was multiplied with its correspondingduplicate on the same peptide array, therefore resulting in four final clas-sifying groups (4, strong seropositive; 2, moderate seropositive; 1, weakseropositive; and 0, seronegative). For the serum classification, linear sup-port vector machines with 20 repetitions of standard 10-fold cross-

validation were used as described previously (5). Unprocessed raw datacan be downloaded from National Center for Biotechnology InformationGene Expression Omnibus (www.ncbi.nlm.nih.gov/geo/index.cgi), acces-sion number GSE58949. The study was approved by the local ethicscommittee (approval numbers 44/05, 67/06, 68/06, and 42/07).

ResultsCytosolic association of TXNDC16 with the ER

Human TNXDC16 was previously found to be a soluble ER-luminal glycoprotein, cotranslationally translocated into the en-doplasmic reticulum due to its predicted N-terminal 27-aa signalpeptide (11). To confirm this subcellular localization, we per-formed immunostaining of HeLa cells transiently expressingTXNDC16 with a C-terminally added HA tag (TXNDC16-HA). Byconfocal microscopy, we detected a colocalization of TXNDC16-HA with the ER-luminal protein PDI (Fig. 1A). The N-terminalsignal peptide plays an essential role in the translocation ofTXNDC16 as a deletion variant of TXNDC16 lacking the first 27 aa(DSP-TXNDC16-HA), transiently expressed in HeLa cells causedan altered subcellular localization of the protein from ER-associatedto cytosolic (Fig. 1A).Next we examined the translocation of TXNDC16-HA into

canine pancreatic ER-derived RMs using a cell-free [35S]methi-onine labeling in vitro transcription and translation system. As anER-luminal protein, we expected TXNDC16 to be sequestered inthe RMs after protease digestion as it could be shown for the kL-chain of human Igs (Fig. 1B). In this study, only the unprocessed kL-chain precursor was accessible for proteinase K, but not thetranslocated and processed form. However, TXNDC16 did notyield similar results and was not sequestered despite RMs’ pres-ence.To exclude the possibility of insufficient translocation efficiency

into the RMs, we investigated the subcellular localization ofTXNDC16 by Western blotting. We used HeLa cells transientlyexpressing myc-TXNDC16-DKEL, an N-terminally myc-taggedversion of the protein with a 6-aa truncation at its C terminus.These amino acids flank a KDEL-like ER retrieval motif (DKEL),subsequently proven in this study to enrich the intracellular proteinyield of TXNDC16. The myc-tag was inserted downstream of thesignal peptide coding sequence between the two codons for aa 30and aa 31 to ensure a remaining N-terminal tag after signal peptidecleavage at the N terminus. The cells were treated with digitonin,which permeabilizes the plasma membrane but not the ERmembrane, and additionally incubated with malPEG, a membrane-impermeable cysteine modification reagent causing a molecularmass shift of 5 kDa per accessible endogenous cysteine. ER-luminal proteins are protected from malPEG binding due to thenonpermeabilized ER membrane. No molecular shift was ex-pected for myc-TXNDC16-DKEL because of its supposed ER-luminal localization. Yet after immunoblotting with myc Abs,we determined a molecular mass shift of myc-TXNDC16-DKELafter digitonin and malPEG treatment (Fig. 1C). As control, weperformed the similar treatment with HeLa cells transiently ex-pressing a cytosolic myc-tagged TXNDC16 variant without theN-terminal signal peptide (DSP-myc-TXNDC16). Both TXNDC16forms not only produced similar results but also their equal shiftsupported the ER-associated cytosolic localization of TXNDC16.An artificial influence of the TXNDC16 overexpression was ex-cluded by flow cytometry. Untransfected HeLa cells were per-meabilized with digitonin or Triton X-100, respectively, or re-mained unpermeabilized. For protein detection, a polyclonalTXNDC16 Ab recognizing a C-terminal epitope of the endoge-nous TXNDC16 was used. Although only few digitonin-treatedcells incubated with polyclonal Abs against the ER-luminal

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marker PDI yielded a fluorescence signal, .80% of these cellsfluoresced after incubation with the TXNDC16 Ab similarly tocells incubated with Abs against a-tubulin that served as a cyto-solic marker (Fig. 1D). As control, due to ER-membrane per-meabilization with Triton X-100, cells showed increasedfluorescence after incubation with PDI Ab. Nevertheless, it re-mains unclear how TXNDC16 is retrotranslocated into thecytosol. Despite its retrotranslocation into the cytosol, confocalmicroscopy did not show TXNDC16 as soluble cytosolic protein.Therefore, we propose that TXNDC16 is linked to the cytosolicER membrane surface through a currently unknown mechanism.

Nonexosomal secretion of TXNDC16

Riemer et al. (11) previously detected TXNDC16 in cell culturemedium of HEK293 cells and interpreted this finding as a satu-rated retention mechanism caused by a potentially artificialoverexpression. Hence we extracted proteins from culture medium

of various nontransfected cell lines including HeLa, HEK293, aswell as the benign meningioma cell lines Ben-Men-1 and HBL-52and the malignant meningioma cell line IOMM-Lee. After TCAprecipitation of the supernatant and SDS-PAGE, we performedimmunoblotting with a polyclonal Ab recognizing a C-terminalepitope of TXNDC16. TXNDC16 was detected in all supernatantfractions at different expression levels (Fig. 2A). We did not detectGAPDH in supernatant fractions for which we confirmed thepresence of TXNDC16. These data indicate that the TXNDC16that was found in supernatant fractions does not stem from thecytosol of dead cells. Notably, in comparison with HeLa andHEK293, virtually no TXNDC16 was detectable in all meningi-oma cell lysates, thus supporting a physiological secretion ofTXNDC16 in meningioma cells.Although TXNDC16 contains a KDEL-like ER retrieval motif

(DKEL), this sequence is followed by six additional amino acidsand therefore not directly located at the C terminus. Nevertheless,

FIGURE 1. ER-associated cytosolic localization of TXNDC16. (A) Immunostaining of HeLa cells transiently overexpressing TXNDC16-HA or DSP-

TXNDC16-HA, respectively. Cells were grown on coverslips in 35-mm dishes for 48 h after FuGENE HD transfection and fixed with 4% PFA. Confocal

microscopy shows colocalization of TXNDC16-HA (green) with the ER-luminal protein PDI (red). Signal peptide deletion causes cytosolic localization of

DSP-TXNDC16-HA. (B) [35S]methionine labeling in vitro transcription and translation system with plasmid-DNA was performed in presence of RMs.

Cotranslational transport assay with additional proteinase K digestion shows no sequestration of TXNDC16-HA in RM, in contrast to k L-chain of human

Igs. (C) Molecular mass shift of myc-TXNDC16-DKEL. HeLa cells were grown in 75-cm2 flasks and transiently transfected 24 h later. At 48 h after

transfection, cells were split into three parts and selectively permeabilized with either digitonin (plasma membrane permeabilization) or Triton X-100 or

remained unpermeabilized. Molecular weight shift was quenched by DTT. Total of 15 ml of each protein sample were loaded into separate wells of 10%

SDS-PAGE gel. Western blotting of myc, PDI, and b-actin was performed with same samples but different membranes. Cysteines of cytosolic proteins

(b-actin and DSP-myc-TXNDC16) are accessible for malPEG, whereas cysteines of ER-luminal proteins (PDI) are only bound by malPEG after Triton

X-100 treatment. Molecular mass shift of myc-TXNDC16-DKEL after digitonin treatment is shown, similar to the cytosolic variant DSP-myc-TXNDC16.

(D) Flow cytometry of untransfected and selectively permeabilized HeLa cells after indirect immunostaining. HeLa cells were grown on three 75-cm2 flasks

for 72 h. Total of 2000 events was analyzed per run. Most HeLa cells permeabilized with digitonin are fluorescent after incubation with primary Abs against

endogenous TXNDC16, similar to cells that were incubated with primary Abs against cytosolic a-tubulin. In contrast, only few cells are fluorescent after

incubation with primary Abs against ER-luminal PDI. 2nd Abs, HeLa cells only incubated with secondary Abs; without (w/o) Abs, HeLa cells incubated

with neither primary nor secondary Abs.

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we examined the potential function of the masked DKEL retrievalsequence by transient expression of different TXNDC16 variantsin HeLa cells. We generated an N-terminally myc-tagged andC-terminally truncated TXNDC16 form without the six flankingamino acids (myc-TXNDC16-DKEL) as well as an N-terminallymyc-tagged cytosolic form with wild-type C terminus (DSP-myc-TXNDC16). As control, we used N-terminally myc-taggedTXNDC16 with wild-type C terminus (myc-TXNDC16). AfterTCA precipitation of cell culture supernatant and immunoblottingwith primary myc-Ab, we observed marginal intracellular myc-TXNDC16 in contrast to very high protein levels in the superna-tant (Fig. 2B). Myc-TXNDC16-DKEL was not detected in thesupernatant but showed an increased intracellular yield, whichsupports the missing ER retrieval capacity of the masked DKELsequence. Interestingly, in addition to its cytosolic localization,a small amount of DSP-myc-TXNDC16 was present in the su-pernatant due to potential cell lysis.The nonfunctional ER retrieval motif was further examined

using several GFP fusion proteins. All GFP variants were fusedwith the TXNDC16 signal peptide at their N termini but differedat their C termini. We examined wild-type GFP C terminus(SP-TX-GFP), C-terminal fusion with DKEL (SP-TX-GFP-DKEL),

and C-terminal fusion with the C-terminal 10 TXNDC16 aa(SP-TX-GFP-TX-Cterm). We first investigated if TXNDC16 signalpeptide also facilitates GFP translocation into the ER. Confocalmicroscopy of transiently transfected HeLa cells shows thatSP-TX-GFP is colocalized with the ER-luminal protein PDI incontrast to wild-type GFP (Fig. 2C). After confirmation of theGFP translocation, we analyzed HEK293 cells transientlyexpressing the above-mentioned GFP variants. Wild-type GFPserved as control and was detected almost exclusively intracel-lularly, although a small amount was found in the supernatant,presumably an artifact of GFP overexpression as previouslydescribed by Tanudji et al. (30). SP-TX-GFP-TX-Cterm wasexclusively detected in supernatant, which was similar to SP-TX-GFP and proves that TXNDC16 C terminus does not haveany influence on ER retention (Fig. 2D). In contrast, SP-TX-GFP-DKEL with C-terminal ER retrieval motif DKEL wasnow detected intracellularly. Despite SP-TX-GFP-DKEL reten-tion, we observed small amounts of secreted protein in the su-pernatant probably caused by a saturated retention mechanism.We investigated if N-terminal myc-tagged TXNDC16 is secreted

via exosomes released by HEK293 cells. After ultracentrifuga-tion, exosomal proteins, TCA/acetone precipitated proteins from

FIGURE 2. Nonexosomal secretion of TXNDC16. (A) TXNDC16 is secreted by different human cell lines, including benign (Ben-Men-1, HBL-52) and

malignant (IOMM-Lee) meningioma cell lines. All cells were grown in 75-cm2 flasks with FBS-free DMEM for 72 h. In meningioma cell lines, endogenous

TXNDC16 could not be detected in cell lysates, in contrast to HEK293 and HeLa. (B) In HEK293 cells, TXNDC16 is not retained intracellularly by

a masked KDEL-like ER retrieval motif (DKEL). N-terminally myc-tagged TXNDC16 with wild-type C terminus (myc-TXNDC16) is found in supernatant

but marginally detected in cell pellet, whereas the C-terminally truncated TXNDC16 variant with C-terminal DKEL (myc-TXNDC16-DKEL) is retained

and not secreted. Cytosolic TXNDC16 (DSP-myc-TXNDC16) is also found in supernatant likely due to unconventional secretion. (C) Immunostaining of

HeLa cells transiently overexpressing GFP fusion protein with N-terminal TXNDC16 signal peptide (SP-TX-GFP) or wild-type GFP, respectively. Cells

were grown on coverslips in 35-mm dishes for 48 h after FuGENE HD transfection and fixed with 4% PFA. Overexpressed GFP fusion protein with N-

terminal TXNDC16 signal peptide (SP-TX-GFP) is directed to secretory pathway and enriched in Golgi apparatus (see arrow) in contrast to ubiquitous

localization of overexpressed wild-type GFP. (D) in HEK293 cells nonfunctional ER retention of TXNDC16 C terminus is shown by different secreted GFP

variants. Secreted GFP with wild-type C terminus (SP-TX-GFP) as well as secreted GFP with TXNDC16 C terminus (SP-TX-GFP-TX-Cterm) are not

retained intracellularly, in contrast to secreted GFP with C-terminal ER retrieval motif (SP-TX-GFP-DKEL). (E) N-terminally myc-tagged TXNDC16 is not

secreted via an exosomal secretory pathway in HEK293 cells. Exosomal fraction was determined by presence of tetraspanins CD63 (B, D, E). HEK293 cells

were grown in 145-cm2 dishes. Cells were transiently transfected with calcium phosphate method 24 h after seeding and cultured in FBS-free medium for

additional 48 h. Total of 5 mg of each protein sample was loaded into separate wells of a 10% SDS-PAGE gel. Western blotting was performed with same

samples but different membranes.

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exosome-free supernatant, and proteins from cell lysate wereseparated by SDS-PAGE. We used a polyclonal Ab against theexosomal marker CD63 (31, 32). Although the exosomal fractionwas verified by presence of CD63, we did not detect TXNDC16 inthe exosomal fraction of HEK293 cells (Fig. 2E). Still, thesefindings do not exclude an exosomal secretion of TXNDC16 inother cell lines or cell types. Nevertheless, TXNDC16 is a secretedprotein and therefore offers itself as an accessible target for im-mune response.

TXNDC16 in circulating immune complexes in the serum

Because TXNDC16 was found in the supernatant of meningiomacells and because autoantibodies against TXNDC16were detected insera of patients with meningioma, we set out to confirm the presenceof TXNDC16 in patients’ sera. In detail, we isolated sera frompatients diagnosed with meningioma and performed Western blot-ting. The above-mentioned polyclonal Ab that recognized a C-terminal epitope of the endogenous TXNDC16 detected two dis-tinct bands at ∼55 and 70 kDa (Fig. 3A), which likely result fromproteolysis of the 100-kDa protein found in the cell lysates (Fig. 2A,2B). Because TXNDC16 was also found in the supernatant ofHEK293 cells and because TXNDC16 was ubiquitously expressed(33), we also tested for the presence of TXNDC16 in sera of healthycontrols and found the same signals as for the meningioma samples.To analyze whether TXNDC16 immune complexes are present

in patient’s serum, we isolated CICs from patients’ sera using PEG

and performed Western blotting using the same polyclonal Ab forTXNDC16. As shown in Fig. 3B, the TXNDC16 Ab only detectedthe signal of 55 kDa in the CICs of meningioma sera. In addition,we isolated CICs from healthy donors and found the same signalas for the meningioma samples.

TXNDC16 epitopes achieve high classification accuracy ofmeningioma sera

Because TXNDC16 was discovered as a meningioma-associatedAg including upregulated mRNA expression in meningioma tis-sues of seroreactive patients, we aimed at identifying specificlinear TXNDC16 epitopes exclusively recognized by meningiomapatients’ autoantibodies (3, 4, 27). We used customized TXNDC16peptide arrays with 163 peptides (15-mers) as duplicates over-lapping in sequence and analyzed 24 meningioma sera and 19 seraof healthy controls. We could not determine any TXNDC16-specific epitopes neither recognized by all analyzed meningio-mas nor with control sera (Fig. 4A). Yet we computed a set of fiveimmunogenic epitopes out of the 163 peptides (feature selection)with which we could successfully discriminate both meningiomaand control serum groups with an accuracy of 87.2%, a specificityof 83.7%, and a sensitivity of 90% compared with an accuracy of53.8%, a specificity of 24.2%, and a sensitivity of 77.3% when allpeptides were used (Table I). When examining the set of fiveepitopes, only peptide G06 was exclusively seroreactive with 8 of24 meningioma sera, with 6 sera showing weak seroreactivity and2 sera showing moderate seroreactivity (Fig. 4B). Peptide B12 wasnearly exclusively seroreactive with meningioma sera, showingstrong seroreactivity in three cases and moderate and weakseroreactivity in one case each, but also showed a weak seror-eactivity with one control serum. Peptide sequences of the fivebest epitopes are summarized in Table II.

DiscussionIn this study, we demonstrated that in addition to its previouslyreported occurrence as a soluble ER-luminal glycoprotein, TXNDC16shows an ER-associated subcellular localization in the cytosol, asconfirmed by confocal microscopy, molecular mass shift, and flowcytometry. Although Riemer et al. (11) conducted alkali extractionof crude membranes to postulate soluble ER-luminal localizationof TXNDC16, these findings are not in conflict with our results.Yamazaki et al. (34) recently identified MIZ1 (Mizu-Kussei 1)expressed in Arabidopsis root cells to be associated with the cy-tosolic face of the ER, likely due to hydrophobic interactions,although MIZ1 was also detected in the soluble fraction aftersodium carbonate extraction. Until recently, N-glycosylation wasthought to be specific for proteins of the secretory pathway;however, N-glycolysed cytosolic proteins were described (35–37).For example, a cytosolic localization of the normally ER-luminalchaperone clusterin was previously shown, including apoptosis-inhibiting function (38).It remains unclear how TXNDC16 is retrotranslocated into the

cytosol and linked to the cytosolic face of the ER membrane. Anexplanation might be its interaction with the ER-luminal proteinER flavoprotein associated with degradation, a component ofER-associated degradation (ERAD) pathway that has been dem-onstrated recently (11, 39). Actually, ERAD operates as an in-tracellular quality control component by targeting incompletelytranslated or misfolded proteins for cytosolic degradation via theubiquitin–proteasome system (40–43). However, an additionalretrotranslocating capability of ERAD without subsequent sub-strate degradation is discussed in literature (44, 45).TXNDC16 contains the potential C-terminal ER retrieval

motif DKEL even though followed by additional 6 aa. Yet we

FIGURE 3. TXNDC16 serum and in CICs. Serum samples from patients

diagnosed with meningioma and sera of healthy blood donors were diluted

1:200 with PBS and separated by SDS-PAGE. Following Western blotting,

a polyclonal Ab that recognized a C-terminal epitope of the endogenous

TXNDC16 identifies two distinct fragments of TXNDC16 (55 and 70 kDa)

in meningioma and in control sera. The same Ab identifies a signal at ∼100kDa for the myc-TXNDC16-DKEL in protein lysate of transfected HeLa

cells. CICs were isolated from patients’ sera and sera of healthy blood

donors using PEG. Total of 10 mg of PEG precipitated CICs was separated

by SDS-PAGE. Following Western blotting, the Ab against TXNDC16

identifies a signal at 55 kDa in CICs of meningioma patients’ sera and in

the sera of healthy controls.

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demonstrated that TXNDC16 is not retained in the ER and thussecreted into cell culture medium. The absence of GAPDH insupernatant fractions that contained TXNDC16 indicates thatTXNDC16 in cell supernatant is not due to cell death. In addition,we tested for b-actin as a structural protein, which was alsoabsent in the supernatant. Furthermore, we found comparablyless TXNDC16 in the meningioma cell lysates than in the su-pernatant, providing further evidence that the presence ofTXNDC16 in the supernatant is not simply the result of lysedcells. However, we are aware that GAPDH in the supernatantmight have been actively secreted, maybe via exosomes, andthat b-actin as a structural protein is less likely to be found inthe supernatant (46, 47). Nevertheless, the present evidencepoints toward secretion as cause of the TXNDC16 presence inthe cell supernatant. Although TXNDC16 was discovered inhuman parotic gland exosomes in a proteomic study ofGonzales-Begne et al. (19), we could not confirm TXNDC16detection in HEK293 exosomes.It has to be elucidated whether TXNDC16 is secreted by the

classical ER/Golgi dependent or an unconventional secretorypathway. The comparison of the subcellular localization ofTXNDC16 (Fig. 1A) and the secreted GFP variant SP-TX-GFP(Fig. 2C) argues for the unconventional pathway. In contrast toTXNDC16, a prominent Golgi localization of SP-TX-GFP was

observed as expected for secreted proteins. There may be multiplepotential secretory mechanisms for TXNDC16 as previously de-scribed for proportein convertase 7, which was reported to besecreted via the COP II–mediated vesicular transport as well asvia Golgi-independent vesicular transport lacking COP II (48).The detection of TXNDC16 protein in patients’ serum is in

agreement with the presence of autoantibodies against TXNDC16.The detection of TXNDC16 protein in normal sera is in agree-ment with the finding of this protein in the supernatant of non-meningioma cells (HEK293 cells) and an ubiquitous expressionpattern (33). Apparently, the cytosolic 100-kDa TXNDC16 isprocessed into two C-terminal proteins of 55 and 70 kDa duringthe release into the serum. Notably, the Ab does not detect addi-tional signals next to the 55- and the 70-kDa signal in the sera andnext to the 100-kDa signal in the cell lysate. Only the 55-kDasignal detected in the serum is found in CICs, again both inpatients and in healthy donors. There are previous reports onimmunogenic proteins in CICs not only in tumor patients but alsoin normal controls (49, 50). Our data indicate that the potentialproteolysis especially into proteins of 55 kDa may be a necessaryprerequisite to elicit an autoantibody response against TXNDC16.However, the presence of TXNDC16 in the serum and specificallyin the CICs of healthy donors also shows that other mechanismsmust be present to explain our finding of frequent autoantibodies

Table I. Accuracy, specificity, and sensitivity for discrimination ofmeningioma sera from healthy control sera using all 163 TXNDC16peptides or using a subset of 5 immunogenic epitopes after featureselection, respectively

Features Accuracy Specificity Sensitivity

All 53.8% 24.2% 77.3%A12, B12, C08, D19, and

G0687.2% 83.7% 90%

Serum classification was performed by linear support vector machines with 20repetitions of standard 10-fold cross-validation.

Table II. Peptide sequence and localization of immunogenic epitopeswithin TXNDC16 protein

ImmunogenicEpitope Peptide Sequence

Position in TXNDC16(aa)

A12 PRTSVFLEELNEAVR 56–70B12 VMEAAFVYGTTYQFV 176–190C08 IVSQQATYEADRRTA 276–290D19 DWSDVCTKQNVTEFP 451–465G06 YDFLSMIDAATSQRG 746–760

FIGURE 4. Immunogenic TXNDC16 epitopes. (A) Heat map of all classified TXNDC16 peptides. TXNDC16 is represented from N-terminal (left panel)

to C-terminal (right panel) by 163 15-mer peptides. All peptide intensity values are classified corresponding to the Materials and Methods. No TXNDC16

specific epitopes were recognized by all of the analyzed meningioma sera or with any of the control sera. (B) Heat map of immunogenic TXNDC16

epitopes after feature selection, which allows discrimination of meningioma sera from healthy control sera with high accuracy. Epitope G06 is exclusively

moderately or weakly seroreactive with 2 or 7 out of 24 meningioma sera, respectively, but not with control sera. Epitope B12 is strongly seroreactive with

three meningioma sera, moderately seroreactive with one, and weakly seroreactive with one meningioma serum, but additionally shows weak seroreactivity

with one control serum.

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against recombinantly expressed TXNDC16 in serum of menin-gioma patients. To understand these mechanisms that explain theautoantibody response against this TXNDC16, it is necessary tohave a more profound understanding of the meningioma devel-opment. As stated .10 y ago, “a theory that explains the pro-duction of autoantibodies needs also to be a theory of etiology andpathogenesis of the diseases in which they occur” (51).A potential upregulated but not yet confirmed TXNDC16 se-

cretion in meningioma, caused by previously described TXNDC16mRNA upregulation, could favor a patient’s humoral immuneresponse. Therefore, it is necessary to investigate potential im-munogenic epitopes for tumor diagnosis, as demonstrated forcancer/testis Ag NY-ESO-1. Zeng et al. (52) identified a 40-aaprotein fragment of NY-ESO-1 recognized by autoantibodies ina wide range of patients’ sera suffering from different tumor en-tities. In this study, we determined one TXNDC16 epitope ex-clusively recognized by autoantibodies in one-third of all testedmeningioma sera and one epitope recognized in approximatelyone-fifth of meningioma sera but also in one control serum. Inaddition, we achieved highly accurate differentiation of menin-gioma sera from controls by peptide array with just a smallnumber of peptides of the meningioma-associated Ag TXNDC16.Still these findings need to be validated with an independent setof sera.

AcknowledgmentsWe thank Prof. Dr. Wolf-Ingo Steudel (Department of Neurosurgery,

Medical School, Saarland University) for providing meningioma sera

and Prof. Dr. Hermann Eichler (Department of Clinical Hemostaseology

and Transfusion Medicine, Medical School, Saarland University) for

providing healthy control sera. We also thank Prof. Dr. Christian Mawrin

(Otto-von-Guericke University of Magdeburg, Magdeburg, Germany)

for providing meningioma cell lines.

DisclosuresThe authors have no financial conflicts of interest.

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