˛ ˛

Journal of Cellular Biochemistry 104:27–37 (2008)

Characterization of the Antibodies to p62 Nucleoporinin Primary Biliary Cirrhosis Using HumanRecombinant Antigen

Jozefa Wesierska-Gadek,1* Anna Klima,1 Carmen Ranftler,1 Oxana Komina,1 John Hanover,2

Pietro Invernizzi,3 and Edward Penner4

1Cell Cycle Regulation Group, Division: Institute of Cancer Research, Department of Medicine I,Medical University of Vienna, Vienna, Austria2National Institute of Health, Bethesda, Maryland3Division of Internal Medicine and Liver Unit, San Paolo Hospital School of Medicine,University of Milan, Milan, Italy4Department of Medicine III, Medical University of Vienna, Vienna, Austria

Abstract Reactivity of sera from patients with primary biliary cirrhosis (PBC) with a 60 kDa component of nuclearpore complexes (NPCs), purified by affinity chromatography on wheat-germ agglutinin (WGA)-Sepharose, was previouslydetected. Recently, clinical significance of the anti-NPC antibodies in PBC became evident. In the light of recent reports,indicating the correlation of the anti-NPC antibodies with severity and progression of the disease, the characterization ofthe reactive antigens is becoming essential in the clinical management of patients with PBC. Since accurate autoantibodydetection represents one of the fundamental requirements for a reliable testing, we have generated a human recombinantp62 protein and validated an immunoprecipitation assay for the detection of anti-p62. We also demonstrated that thegenerated human recombinant p62 nucleoporin was modified by N-acetylglucosamine residues. More than 50% of testedPBC sera precipitated 35S-radioactively labeled p62 recombinant nucleoporin and 40% recognized this recombinantantigen by immunoblotting. We compared the reactivity of PBC sera with rat and human nucleoporin. The incidence ofanti-p62 nucleoporin positive PBC sera increased by 15% when human recombinant antigen was used. The titer ofautoantibodies in p62-positive PBC samples strongly varied. Preadsorption of the PBC sera with p62 recombinant proteincompletely abolished their reactivity with the antigen. In conclusion, this study unequivocally proves that autoantibodiesreacting with the 60 kDa component of NPCs target p62 nucleoporin and, more importantly, provide a better antigensource for future evaluations of the clinical role of anti-p62 in PBC. J. Cell. Biochem. 104: 27–37, 2008. �2007Wiley-Liss, Inc.

Key words: ANA; autoimmunity; nuclear pore complexes; p62 nucleoporin; gp210 glycoprotein; nuclear envelope;human recombinant p62 nucleoporin

Patients with primary biliary cirrhosis (PBC)[Talwalkar and Lindor, 2003] generate a varietyof autoantibodies, which are primarily directedagainst mitochondrial antigens [Walker et al.,1965; Van der Water et al., 1989]. However, inabout 50% of patients there are also antibodiesdirected against nuclear components [ANA,anti-nuclear antibodies; for review, see Refe-rence Invernizzi et al., 2005]. A number ofnuclear structures have been recognized asspecific targets of ANA in PBC. These includeSp100 and promyelocytic leukemia proteins,major components of nuclear bodies, whichgenerate a nuclear dot pattern in indirectimmunofluorescence (IIF) and constituents ofthe nuclear pore complexes (NPCs) that havebeen specifically associated with a perinuclear

� 2007 Wiley-Liss, Inc.

Abbreviations used: AMA, anti-mitochondrial antibodies;ANA, anti-nuclear antibodies; ELISA, enzyme-linkedimmunosorbent assay; GlcNAc, N-acetylglucosamine;kDa; kilodalton; LBR, lamin binding receptor; NPC,nuclear pore complex; PBC, primary biliary cirrhosis; Sf9,Spodoptera frugiperda; Tpr, translocated promoter region;WGA, wheat-germ agglutinin.

*Correspondence to: Jozefa Wesierska-Gadek, Cell CycleRegulation Group, Division: Institute of Cancer Research,Department of Medicine I, Medical University of Vienna,Borschkegasse 8 a, A-1090 Vienna, Austria.E-mail: jozefa.gadek-wesierski@meduniwien.ac.at

Received 27 July 2007; Accepted 7 September 2007

DOI 10.1002/jcb.21595

staining pattern [Lozano et al., 1988; Lassouedet al., 1990; Courvalin et al., 1990a; Wesierska-Gadek et al., 1995, 1996a,b; Miyachi et al., 1996,2003; Invernizzi et al., 2005]. PBC-specificANAs are associated with more severe diseaseand seem to have a prognostic role in the disease[Invernizzi et al., 2001, 2004; Wesierska-Gadeket al., 2006; Nakamura et al., 2007].

The frequency of the PBC sera staining thenuclear periphery strongly differs betweenreports and is in the range from 29% to 53%[Lozano et al., 1988; Lassoued et al., 1990;Courvalin et al., 1990a; Wesierska-Gadek et al.,1995, 1996a,b, 2006; Miyachi et al., 1996, 2003;Invernizzi et al., 2001, 2005]. The observeddivergence is not surprising, considering the factthat in previous studies antigens isolatedfrom different tissues (cervical carcinoma cells[Wesierska-Gadek et al., 1996a,b] vs. liver [Miya-chi et al., 1996]) originating from different species(human [Wesierska-Gadek et al., 1996a,b] vs. rat[Miyachi et al., 1996]) were used for testing. Anumber of studies revealed that two majorcomponents of the NPC are targeted by PBC sera[Courvalin et al., 1990a; Lassoued et al., 1990;Wesierska-Gadek et al., 1995, 1996a,b; Miyachiet al., 1996]. First, gp210 protein, a mannose-substituted integral component of NPCs, wasidentified as a major target [Courvalin et al.,1990a; Lassoued et al., 1990; Wesierska-Gadeket al., 1995]. A few years later the reactivity ofPBC sera with a 60 kDa component of NPCs hasbeen reported by our group [Wesierska-Gadeket al., 1996a] and then confirmed by otherlaboratories [Miyachi et al., 1996, 2003]. Thefact that the PBC sera also reacted with a60 kDa constituent of human NPCs purifiedby affinity chromatography on wheat-germagglutinin (WGA)-Sepharose indicated that thep62 nucleoporin is the reactive autoantigen[Wesierska-Gadek et al., 1996a].

To unequivocally prove the identity of thereactive antigen, we generated and charac-terized human recombinant p62 protein baculo-virally expressed in insect cells. Moreover, weadditionally used rat recombinant antigen andcompared by immunoprecipitation the reactivityof PBC sera with human and rat 35S-methioninelabeled p62 nucleoporin. In the attempt tovalidate a novel detection method and tobetter define the anti-p62 reactivity of patientswith PBC, we also developed an immunopreci-pitation (IP) assay using this new recombinantprotein.

Approximately half of PBC sera stronglyreacted with human p62 recombinant nucleo-porin and preadsorption experiments with His-tagged p62 recombinant protein completelyabolished their reactivity with the antigen.These results unequivocally evidence thatthe p62 nucleoporin is the target for thepreviously described autoantibodies recogniz-ing the 60 kDa component of NPC.

MATERIALS AND METHODS

Antibodies

Monoclonal anti-nucleoporin p62 antibodies(clone 53) from Transduction Laboratories(Lexington, KY) and anti-polyhistidine (cloneHIS-1) from Sigma–Aldrich Co. (St. Louis, MO)were used.

Plasmids

We used following plasmids encoding ratp62 nucleoporin (pETp62) and encoding ratgp210.

Human Sera

Twenty non-selected sera of patients dia-gnosed with PBC were collected. The diagnosisof PBC was based on internationally acceptedcriteria [Kaplan and Gershwin, 2005]. Patientswith serum positivity for hepatitis B surfaceantigen (HbsAg) and hepatitis C virus anti-bodies (anti-HCV) were not included in thepresent study. Additionally, 10 sera from nor-mal healthy blood donors and from patientswith diagnosed autoimmune hepatitis wereused as controls.

Indirect Immunofluorescence forSerum Autoantibody Analysis

Serum anti-mitochondrial antibodies (AMA),ANA, and anti-smooth muscle antibodies(SMA) were determined blindly by an unbiasedobserver by IIF as previously described[Invernizzi et al., 2001; Wesierska-Gadeket al., 2006]. Briefly, sera were tested for thepresence of AMA on rodent tissue preparations(rat kidney sections, normal< 1:40) and ANA onhuman Hep-2 cells (Kallestad, Houston, TX)and HeLaS3 cells. HeLaS3 cells grown onslides were fixed with ice-cold methanol/ace-tone-mixture (3:2) for 20 min. After PBS wash-ing and saturation with 5% BSA, specimenwere incubated overnight with PBC seradiluted to a final concentration of 1:500.

˛ ˛28 Wesierska-Gadek et al.

Thereafter, preparations were washed withPBS and incubated with secondary anti-bodies coupled to Cy2. Immunostained prepa-rations were inspected under fluorescencemicroscopy using an FITC filter. Sera wereconsidered positive for ANA at a dilutionof �1:40. A fluorescein-conjugated antibodydirected against human immunoglobulins (Ig)A, G, and M (Sigma ImmunoChemicals,St. Louis, MO) diluted to a final concentrationsof 1:100 were used as secondary antibody.Positivity and patterns were evaluated usingfluorescence microscopy (Orthoplan, Leitz,Wetzlar, Germany).

Cloning of Human p62 Nucleoporin

Human p62 cDNA generated and sequencedfrom a HeLa cDNA library in Dr. Hurt’slab [Carmo-Fonseca et al., 1991] was clonedinto a baculovirus recombination vector (pBlue-BacHis2B; Invitrogen Life Technologies, SanDiego, CA). A poly-His-tag sequence was intro-duced in the NH2-terminus of human recombi-nant p62 protein to facilitate its one-steppurification by Ni-affinity chromatography. Toensure that newly generated human recom-binant p62 protein possesses a proper primarystructure, the p62 nucleporin was sequenced.

Transfection of Insect Cells

For infection, medium containing the virusharboring the pBlueBacHis2B vector with a p62insert was used. After 3 days the baculovirusstarted to lyse the Spodoptera frugiperda (Sf9)insect cells and 1 week after transfection thesupernatant including the newly formed virusparticles could be saved for subsequent infec-tion of cells [Wesierska-Gadek et al., 2003,2005]. The optimal time for maximal expres-sion of the recombinant p62 protein wasdetermined. For selection, serial dilutions ofthe virus suspension (e.g., 1,000–100,000-fold)were put into petri dishes (10 cm diameter)containing 107 logarithmically growing Sf9cells. After 1 h the medium was removed andthe cells were covered with 6 ml of low meltingagarose containing X-Gal. Starting 3 days later,the petri dishes were inspected for the appear-ance of blue plaques, generated by X-Galmetabolized by the protein expressed by thereconstituted lacZ gene. Blue plaques contain-ing the recombinant virus were cut out and putinto insect cell medium to elute the virusparticles. This was then used for subsequent

infections of Sf9 insect cells. The human p62protein was in vivo labeled after exposure ofinsect cells for 15 h in methionine-deprivedmedium supplemented with 35S-methionine.

In Vitro Transcription/Translation Reaction

To generate 35S-labeled p62 and gp210 re-combinant proteins, an in vitro transcription/translation reaction was performed using therabbit reticulocyte Kit (Promega Corporation,Madison, WI), a cDNA coding for gp210 andp62 as well as 35S-labeled methionine (NewEngland Nuclear, Perkin-Elmer, Waltham,MA) [Wesierska-Gadek et al., 2007b]. Theidentity of the radioactively labeled recombi-nant p62 proteins was checked by immunopre-cipitation using monoclonal anti-p62 antibodies(Transduction Laboratories) and by sequentialanalysis of the immune complexes.

Immunoprecipitation

35S-labeled recombinant p62 protein was usedfor immuno-precipitation using human sera. Forcontrol, monoclonal anti-p62 antibodies (clone53) from Transduction Laboratories were used.Immunoprecipitation was performed accordingto the previously described stringent protocol[Wesierska-Gadek et al., 1995, 1996a, 2007a].The immune complexes were isolated by affinitychromatography on Protein G-Sepharose beads(Amersham Biosciences, Little Chalfont, UK).Protein-G-Sepharose bound immune complexeswere eluted twice with 40 ml of non-reduced SDSsample buffer and analyzed on 10% SDS-gels.Proteins separated on gels were transferredelectrophoretically onto the membranes. Theamount of precipitated 35S-labeled p62 proteinwas determined by liquid scintillation countingof the aliquots (3 ml) of both eluates and addi-tionally by autoradiography after exposure ofthe dried membranes to BMR film for theappropriate time (Eastman Kodak Co., Roches-ter, NY). As a control, 35S-labeled unrelatedhuman recombinant proteins (e.g., p53 protein)were used for immunoprecipitation.

Preparation of Cell Extracts

PBS-washed cells were lysed in RIPAbuffer [50 mM Tris–HCl (pH 7.4), 500 mMNaCl, 1% Nonidet-P40, 0.5% Na-deoxycholate,0.1% SDS, 0.05% NaN3, 1 mM phenylmethyl-sulfonylfluoride (PMSF)] for 20 min at þ48C[Wesierska-Gadek et al., 2005, 2007b]. Foraffinity purification of baculovirally expressed

Anti-p62 Nucleoporin Autoantibodies in PBC 29

proteins, insect cells were lysed directly innative binding buffer provided in the X-presspurification Kit (Invitrogen Life Technologies),and cell structures were destroyed by repeatedfreeze/thaw procedure and sonication [Wesier-ska-Gadek et al., 2003, 2005]. The cell suspen-sion was spun off. Clear supernatant was usedfor further analysis. The protein concentrationof cell extracts was determined by the DC assay(Bio-Rad Laboratories, Richmond, CA) withbovine serum albumin as standard.

Purification of His-Tagged Recombinantp62 Protein by Affinity Chromatography

on Ni-Agarose

Cell lysates prepared in native bindingbuffer (pH 7.8) were loaded on pre-equilibratedNi-agarose beads ProBond (Invitrogen LifeTechnologies) and purification of p62 recom-binant protein was performed according tothe manufacturer’s protocol [Wesierska-Gadeket al., 2003, 2005]. Recombinant p62 proteinwas eluted from the Ni-beads with the nativeelution buffer at pH 4.0 and immediatelyneutralized.

Isolation of Nuclear Pore Complexes(NPCs) From Human HeLaS3 Cells

The nuclear pore complex-lamina fraction(NPC-LF) was isolated from cultivated humanHeLaS3 cells according to the method of Dwyerand Blobel [1976]. Cells arrested in a stationaryphase were used for isolation of nuclei. Theircell cycle status was determined by measure-ment of the propidium iodide stained cellsin FACS. HeLaS3 cells were harvested andwashed in PBS as previously described in detail[Wesierska-Gadek et al., 1995, 1996a]. To avoida proteolytic degradation of antigens during allisolation steps PMSF and Pefabloc were addedto a final concentration of 1 mM and 50 mM,respectively. Briefly, PBS washed cells weresuspended in ice-cold low salt buffer (RSBbuffer), swollen and after stepwise addition ofdetergents (NP-40 and freshly prepared sodiumdeoxycholate) they were homogenized. Aftercentrifugation through sucrose cushion nucleiwere pelleted. Then isolated nuclei weredigested with DNase I and then chromatinwas extracted sequentially with the non-ionicdetergent Triton X-100 and high salt buffer. Theremaining insoluble structure pelleted after thelast centrifugation step represents the NPC-LF.

Electrophoretic Separation of Proteinsand Immunoblotting

Proteins of the NPC-LF fraction and proteinsisolated by affinity chromatography dissolvedin SDS-sample buffer were separated bySDS–PAGE on 10% slab gels and then theywere electrophoretically transferred onto aPVDF membrane (Amersham International,Little Chalfont, UK) [Wesierska-Gadek et al.,1995, 1996a, 2007a,b]. The efficiency of thetransfer as well as protein loading of proteinswas confirmed by Ponceau S staining. Blotswere incubated with human sera or with anti-p62 specific antibodies (clone 53) and theimmune complexes were detected autor-adiographically using appropriate peroxidase-conjugated secondary antibodies and theenhanced chemiluminescent detection reagentECLþ (Amersham International). The radio-actively labeled proteins were detected byautoradiography after exposure of the blots toBMR film (Eastman Kodak Co.).

Preadsorption Experiments

To examine the specificity of anti-p62 anti-bodies, an excess of cold p62 recombinantprotein (free or immobilized on Ni-beads) wasincubated with human PBC sera prior to theaddition of 35S-recombinant p62 protein or priorto the immunoblotting. After incubation for 1 hat RT, the immune complexes as well asfree recombinant p62 protein were removedby Ni-agarose. The supernatant samples re-presenting preadsorbed sera were used forimmunoprecipitation and for immunoblotting.The effect of the excess of cold recombinantp62 on the amount of precipitated 35S-p62protein was evaluated [Wesierska-Gadeket al., 1995, 1996a, 2006; Invernizzi et al., 2001].

RESULTS

PBC Sera React Primarily WithTwo Components of NPC

Incubation of human PBC sera with HeLapreparations generated different staining pat-tern (Table I). Some PBC sera displayedhomogenous nuclear (Fig. 1A) or nuclear dot(not shown) staining pattern by means of IIF.Moreover, four PBC sera stained the nuclearperiphery of HeLaS3 cells indicating that theantibody in PBC sera targets the components ofthe nuclear envelope (Fig. 1A). As depicted in

˛ ˛30 Wesierska-Gadek et al.

Figure 1A, two different PBC sera generated arim-like staining pattern. Unfortunately, mostPBC sera are heterogeneous and have anti-bodies with different specificities, for example,AMAs. For these reasons, overlapping stainingpatterns may occur and it is difficult to exactlydistinguish which subcellular structures arestained by the reactive autoantibodies [Inver-

nizzi et al., 2005]. Testing of PBC sera withproteins of isolated NPCs revealed a strongreactivity with two major autoantigens: with aprotein at 200 kDa and with a protein atapproximately 60 kDa (Fig. 1B, Table I). SomePBC sera additionally reacted with a few minorbands. However, the intensity of the minorbands was weak and it cannot be excluded that

TABLE I. Reactivity of Human Sera With The Recombinant p62 Nucleoporin

PatientsNo. ofsera

IIF WB IP

AMA ANA

NPCsHuman

p62Rat

gp210 Rat p62Human

p62p200 p60

PBC 20 19 7 5 7 8 2 11 14AIH 10 0 n.d. n.d. n.d. 0 0 0 0Healthy controls 10 0 0 0 0 0 0 0 0

n.d., not determined.

Fig. 1. Reactivity of PBC sera with NPCs. A: Immunostaining ofHeLaS3 cells by ANA-positive PBC sera. Fixed preparations ofHeLaS3 cells were incubated overnight with PBC sera diluted to afinal concentration of 1:500. Thereafter, they were washedwith PBS and incubated with secondary antibodies coupledto Cy2. Immunostained preparations were inspected underfluorescence microscopy using a FITC filter. The reactivity of aserum generating homogeneous nuclear staining (serum H) andtwo sera displaying a rim-like nuclear pattern (serum A and G)was shown. B: Detection of reactivity of PBC sera with proteins ofNPCs using Western blotting. Proteins of isolated NPCs were

resolved on 8% SDS-gel and electrophoretically transferred ontoPVDF membrane. For control of equal protein loading and ofproper electrotransfer, proteins immobilized on the membranewere detected by Ponceau S staining (lower panel). Blot stripswere incubated with distinct PBC sera (A–D and E–M) at a finaldilution of 1:500. After incubation with secondary antibodieslinked to HRP, immune complexes were detected by chemo-luminescence using ECLþ reagent. Marker proteins were loadedbetween lanes D and E. Arrows indicate the position of the majorautoantigens. [Color figure can be viewed in the online issue,which is available at www.interscience.wiley.com.]

Anti-p62 Nucleoporin Autoantibodies in PBC 31

the observed weak reactivity is attributable tounspecific cross-reactivity. Serum E was reac-tive with a protein at about 70 kDa, a positioncharacteristic for lamin A.

The 200 kDa component of NPCs reactingwith PBC sera (Fig. 1B, lane A,B) was identifieda couple of years ago. The glycoprotein gp210,an integral component of NPCs was evidenc-ed as the PBC-specific autoantigen. Unlikethe 200 kDa protein, the 60 kDa antigenrecognized by a number of PBC patients sharessome features, for example, modification byN-acetylglucosamine (GlcNAc) residues withother related constituents of NPC and itsidentity assumed so far by different biochemicalapproaches has to be proved by the reactivitywith the recombinant antigen.

Characterization of the HumanRecombinant p62 Protein

In the first step we verified the overexpressionof poly-His tagged human p62 recombinantprotein. Proteins solubilized during lysis ofcontrol and p62 baculovirus infected Sf9 insectcells were separated on SDS slab gels. Proteinswere Coomassie blue stained or were immobi-lized on a membrane. The comparison of thestaining pattern revealed the presence of aprominent protein band at approximately60 kDa solely in samples obtained from insectcells infected with baculovirus encoding p62,but not from cell lysate obtained from controlcells (Fig. 2A, left panel). The identity of theoverexpressed recombinant human protein wasexamined by immunoblotting using specificmonoclonal anti-p62 nucleoporin antibodiesand antibodies directed against the poly-His-tag sequence. As shown in Figure 2A (rightpanel), monoclonal anti-p62 reacted stronglywith a 62 kDa protein only in a sample obtainedfrom Sf9 cells infected with baculovirus. Anti-bodies targeting poly-His sequence generated asimilar staining pattern (not shown). Monoclo-nal anti-p62 antibodies additionally stained thenucleoporin band in the nuclei sample isolatedfrom human HeLaS3 cells (Fig. 2A, right panel).Moreover, the human recombinant His-taggedp62 was purified by one-step Ni-affinity chro-matography under native conditions. Sf9 cellextract loaded on Ni-Sepharose at slightly basicpH was eluted from the beads at acidic pH andimmediately neutralized (not shown). Finally,the glycosylation status of the human recombi-nant p62 nucleoporin was proved by two

independent methods, that is, WGA-affinitychromatography (Fig. 2B) and overlay withWGA-lectin (not shown); both methods evi-denced that human recombinant p62 nucleo-porin generated in insect cells was glycosylatedby GlcNAc residues. Considering the fact thatunder physiological conditions p62 nucleoporinis modified by GlcNAc residues, the modifica-tion could be of importance for its reactivity.

Characterization of the Recombinant Ratp62 Protein Generated by In VitroTranscription/Translation Reaction

To assess the glycosylation status of therecombinant rat p62 nucleoporin generatedby in vitro transcription/translation reaction(Fig. 2C, upper panel), 35S-labeled protein waspurified on WGA-affinity chromatography. Theanalysis of the fractions stepwise eluted fromWGA-Sepharose (eluates 1–4) (Fig. 2C, lowerpanel) revealed that also recombinant antigenin vitro synthesized in the rabbit reticulocytesystem was post-translationally modified.

PBC Sera Precipitate 35S-Labeled p62Recombinant Protein

In the next step we performed immunopreci-pitation assays using 35S-labeled p62 recom-binant protein. We used rat p62 proteinradioactively labeled during an in vitro tran-scription/translation reaction and humanp62 protein labeled in vivo in Sf9 cells. Wetested 20 non-selected PBC sera. As shown inFigure 3 and Table I, more than 50% of non-selected PBC sera strongly precipitated 35S-labeled rat p62 nucleoporin. Noteworthy, a fewsera that did not recognize rat p62 proteinprecipitated human antigen (Table I). Forcontrol, 35S-labeled p62 nucleoporin was pre-cipitated by monoclonal anti-p62 antibodies(Fig. 3). The amount of 35S-labeled p62 proteinmeasured in both eluates was summed up andcompared with the amount of radioactivitylabeled protein precipitated by monoclonalanti-p62 antibodies. Moreover, to exclude thepossibility that the p62 seroreactivity was over-estimated by immunoprecipitation, rat gp210protein 35S-methionine labeled with com-parable specific activity was used in parallelassays. Only 2 out of 20 PBC sera precipi-tated recombinant gp210 protein (Table I)[Wesierska-Gadek et al., 2007a]. These dataare in concordance with our recent observations[Wesierska-Gadek et al., 2007a]. Finally, the

˛ ˛32 Wesierska-Gadek et al.

human recombinant 35S-methionine labeledp62 nucleoporin that was precipitated by PBCsera (Fig. 3C, upper panel) was additionallydetected by immunoblotting using anti-p62-antibodies (Fig. 3C, lower panel) and anti-His antibodies (not shown). As depicted inFigure 3C, p62 nucleoporin positive signals

were very strong thereby evidencing that dis-tinct PBC sera precipitated high amounts of therecombinant antigen. Interstingly, two PBCsera (serum No. 4 and 13) that precipitated ratp62 displayed very weak, if any, reactivity withthe 35S-labeled human p62 nucleoporin andwere evaluated as human p62 negative.

Fig. 2. Characterization of human recombinant p62 protein.A: Cell lysates obtained from control Sf9 cells and cells infectedwith a baculovirus encoding human p62 nucleoporin (Sf9þhp62) were separated on 10% SDS slab gels. One gel wasCoomassie blue stained (left panel) and the second was blottedonto the PVDF membrane (right panel). p62 protein was detectedby immunoblotting using anti-p62 monoclonal antibody (clone53) at a final dilution of 1:2,000. Position of molecular weightprotein markers (M) is indicated by arrows. Sample of nucleiproteins isolated from HeLaS3 cells was additionally loaded. Toavoid overstaining, lysate from Sf9 cells expressing lower h p62

levels was loaded for immunoblotting. B: Human recombinantp62 nucleoporin was affinity purified on WGA-Sepharose. p62nucleoporin bound to WGA-Sepharose was eluted with puffercontaining N-acetylglucosamine. The majority of the recombi-nant protein was detected by immunoblotting in the first eluate.C: 35S-labeled rat p62 protein generated by in vitro transcription/translation reaction was precipitated by a monoclonal anti-p62(upper panel) or purified by affinity chromatography on WGA-Sepharose (lower panel) antibody and analyzed on 10%SDS-gels. [Color figure can be viewed in the online issue, whichis available at www.interscience.wiley.com.]

Anti-p62 Nucleoporin Autoantibodies in PBC 33

PBC Sera Recognize Human Recombinantp62 Protein by Immunoblotting

PBC sera reactive with 35S-labeled nucleo-porin p62 were further tested by immunoblot-ting. A lysate of Sf9 cells generating humanHis-tagged p62 nucleoporin and proteins ofNPCs isolated from HeLaS3 cells separatedon 10% SDS-slab gels were immobilized on themembrane. As negative control a Sf9 celllysate obtained from non-infected cells wasloaded. As shown in Figure 4, PBC sera react-ed with human recombinant p62 protein andreacted with the antigen in the NPC samples

isolated from HeLaS3 cells. However, theresults of the immunoblotting experimentsrequire an additional explanation. SerumNo. 15 that precipitated huge amounts of35S-labeled p62 nucleoporin, heavily stainedthe His-tagged p62 band. It also reacted withthe samples isolated from HeLaS3 cells. How-ever, to resolve the stained p62 band, this blotwas exposed very shortly. Similarly, the blotprobed with monoclonal anti-nucleoporin anti-bodies was also exposed very shortly and thep62 band in the sample of NPCs was notvisible. In contrast, serum No. 20 displayed amoderate reactivity with the denaturated

Fig. 3. PBC sera precipitate 35S-labeled recombinant p62protein. A: Analysis of the precipitated 35S-labeled human p62protein by autoradiography. An aliquot of the sample used forimmunoprecipitation (input) was loaded. B: Semi-quantitativeevaluation of the amount of precipitated 35S-labeled ratp62 protein. The amount of precipitated 35S-labeled p62 proteinwas determined by liquid scintillation counting of the aliquots(3 ml) of both eluates. Total activity of the eluate 1 and eluate2 was summed up. As a positive control 35S-labeled p62 protein

precipitated by a monoclonal anti-p62 antibody was included.Two PBC sera previously tested as reactive with the WGA-purified 60 kDa component of NPC (designated anti-NPCþ), twoAIH sera and one serum from normal healthy control wereadditionally included. C: Human recombinant p62 nucleoporinwas expressed and labeled with 35S-methionine in Sf9 cells.Radioactively labeled p62 protein precipitated by human sera orby monoclonal anti-p62 antibody was detected by autoradio-graphy (upper panel) and by immunoblotting (lower panel).

˛ ˛34 Wesierska-Gadek et al.

antigen. It generated a positive signal with therecombinant p62 protein but not with thesample of NPCs. The frequency of p62 positivePBC sera estimated by Western blotting isshown in Table I. It becomes obvious thatmuch more PBC sera react with the nativehuman recombinant p62 nucleoporin by im-munoprecipitation than by immunoblotting.

Preadsorption of p62 Reactive PBC SeraWith an Excess of Recombinant p62 Protein

Abolishes Their Reactivity

To prove the specificity of the anti-p62 positivePBC sera, preadsorption experiments wereperformed. As shown in Figure 5, the preadsorp-tion of PBC sera with human recombinantp62 protein abolished their reactivity with35S-labeled antigen. However, preadsorptionwith an excess of BSA or human normal serumdid not affect the amounts of the precipitatedradioactive antigen.

DISCUSSION

Approximately 30% of patients with PBCdevelop autoantibodies against proteins ofthe nuclear envelope [Invernizzi et al., 2005].These sera frequently display a rim-like nuclearstaining pattern. These autoantibodies aredirected against different nuclear proteins suchas lamin B receptor (lamin binding receptor,LBR) [Courvalin et al., 1990b; Lin et al., 1996],nuclear lamins [Wesierska-Gadek et al., 1988],translocated promoter region (Tpr) [Ou et al.,2004], LAP1 and two proteins [Miyachi et al.,2003; Invernizzi et al., 2005] and components ofNPCs [Lassoued et al., 1990; Courvalin et al.,

1990a; Wesierska-Gadek et al., 1995, 1996a,b;Miyachi et al., 2003]. The incidence of autoanti-bodies directed against proteins of the NPC isrelatively high and these antibodies seem tobe specific for this entity. In particular,the gp210 glycoprotein [Lassoued et al., 1990;Courvalin et al., 1990a; Wesierska-Gadek et al.,1995, 1996b], the integral protein of the nuclearpores [Wozniak et al., 1989; Greber et al.,1990], and a protein of approximately 60 kDarepresent the major autoantigens [Wesierska-Gadek et al., 1996a]. The autoantibodies direct-ed against gp210 occur in about 30% of PBC sera[Lassoued et al., 1990; Courvalin et al., 1990a;Wesierska-Gadek et al., 1995, 1996a,b] and thecarbohydrate moieties are an essential partof their antigenicity [Wesierska-Gadek et al.,1995, 1996a,b]. The antibodies against the60 kDa component of NPC were detected inapproximately 14–32% of PBC patients. Sincethe reactive autoantigen at 60 kDa was isolatedfrom human cells by affinity chromatography onWGA, it has been assumed that p62 nucleoporinrepresents the autoantigen [Wesierska-Gadek

Fig. 4. Reactivity of human PBC sera with p62 nucleoporin.Proteins of nuclei and of NPCs isolated from HeLaS3 cells as wellas lysate of control and p62 baculovirus infected Sf9 cells wereloaded on 10% SDS-gels and blotted onto membrane. Blots wereincubated with distinct PBC sera at a final dilution of 1:500 orwith monoclonal antibodies against p62 at a final dilution of1:2,000. Due to extremely strong reactivity of the monoclonalantibody with the sample of Sf9 cells overexpressing p62, the blotwas exposed only 1 s. After the short exposure p62 signal in NPCswas nicely detectable (without a strong background).

Fig. 5. Preadsorption of PBC sera abolished their reactivitywith 35S-labeled p62 recombinant protein. A: Detection of35S-labeled recombinant p62 protein precipitated directly byindicated PBC sera or after sera preadsorption with p62recombinant protein by autoradiography. 35S-labeled recombi-nant p62 protein used for immunoprecipitation (designated asinput p62) was loaded as a positive control. B: Quantification of35S-labeled recombinant p62 protein precipitated by non- andpreadsorbed PBC sera. [Color figure can be viewed in the onlineissue, which is available at www.interscience.wiley.com.]

Anti-p62 Nucleoporin Autoantibodies in PBC 35

et al., 1996a]. Moreover, by immunoblottingafter two-dimensional gel electrophoresis PBCsera reacted with 2–3 spots exhibiting pI valuessimilar to that of p62 nucleoporin [Wesierska-Gadek et al., 1996a].

p62 nucleoporin belongs to a distinct subset ofNPC proteins substituted by O-linked GlcNAc[Davis and Blobel, 1987]. This heterogeneousgroup of proteins, ranging in molecular weightfrom 45 to 200 kDa is glycosylated at serine andthreonine with 10–15 GlcNAc residues permolecule [Davis and Blobel, 1987; Starr andHanover, 1990; Starr et al., 1990]. The GlcNAcresidues covalently linked to nucleoporins playan essential role in the regulation of transportthrough NPCs. However, it should be stressedthat by WGA lectin binding not only the GlcNAcmodified p62 was separated from its unmodifiedcounterparts, but also other GlcNAc-linkedNPC components of this protein family wereisolated. Since nucleoporins possess a similarmolecular mass, the testing of PBC sera with arecombinant p62 protein became indispensable.Insect cells and lytic baculoviruses providea well-established method for high-level ex-pression of functional full-length mammalianproteins. In addition, recombinant proteinsexpressed in insect cells are post-translation-ally modified in a similar manner to thatcatalyzed in mammalian cells.

Using the recombinant p62 nucleoporin, wedetermined the specificity of the autoantibodiesby two independent approaches: immunopreci-pitation and immunoblotting. PBC sera pre-viously tested as reactive with the affinitypurified NPCs 62 kDa protein precipitatedrecombinant p62 protein and recognized itby immunoblotting, thereby evidencing thatthese sera are directed against p62 nucleoporin[Wesierska-Gadek et al., 1996a]. More than50% of 20 tested PBC sera were reactivewith the recombinant p62 nucleoporin. Consi-dering the fact that some previously publishedstudies on the reactivity of PBC sera withp62 nucleoporin were performed with the anti-gen purified from rat liver [Miyachi et al.,1996], we decided to examine the reactivityof our PBC sera with both recombinant p62proteins: human and rat. The analysis of pre-cipitated immune complexes revealed higherincidence of anti-p62 reactivity using humanantigen. The incidence of anti-p62 reactivityincreased by 15% as compared with the ratcounterpart.

Preadsorption of anti-p62 positive PBCsera with an excess of recombinant humanp62 protein abolished their reactivity with35S-labeled p62 protein and with the p62 proteinband in the sample of NPCs, thereby sub-stantiating the high specificity of the autoanti-bodies. The incidence of anti-p62 nucleoporinpositive sera assessed by immunoblottingis much lower (approximately 40%) and isin concordance with our previous results[Wesierska-Gadek et al., 1996a]. The nucleo-porin p62 antibodies seem to be characteristicfor PBC, although they were also reportedin 13% of Sjogrens’s syndrome [Miyachi et al.,2003; Enarson et al., 2004], and in rare cases ofmixed connective tissue disease [Kraemeret al., 2003; Enarson et al., 2004]. The observeddivergence in the number of anti-p62-positivePBC sera determined by immunoprecipitationand by immunoblotting may reflect the dif-ference in their immunoreactivity depending onthe status of the autoantigen. Whereas duringimmunoprecipitation autoantibodies interactwith a native antigen, for Western blottingdenaturated antigen immobilized on the mem-brane reacts with antibodies.

Evaluation of the clinical significance of ANAin PBC markedly progressed in recent yearsindicating [Invernizzi et al., 2001, 2004; Miya-chi et al., 2003; Wesierska-Gadek et al., 2006]that, in contrast to AMA, ANA in general, andPBC-specific anti-NPC in particular, correlatewith the severity of disease and may be ofimportant prognostic value [Invernizzi et al.,2001, 2004; Wesierska-Gadek et al., 2006]. Wegenerated in the present study a recombinantp62 nucleoporin antigen source that will beuseful in future serum testing of patients withPBC. Based on this data further multi-centerstudies based on a large collection of PBC serahave to be performed to elucidate the clinicalsignificance of the anti-p62 specific reactivities.

ACKNOWLEDGMENTS

The authors thank Dr. E.C. Hurt as well asDrs. M.E Gershwin and H.J. Worman for a kindgift of p62 and gp210 plasmids, respectively.

REFERENCES

Carmo-Fonseca M, Kern H, Hurt EC. 1991. Human nucleo-porin p62 and the essential yeast nuclear pore proteinNSP1 show sequence homology and a similar domainorganization. Eur J Cell Biol 55:17–30.

˛ ˛36 Wesierska-Gadek et al.

Courvalin J-C, Lassoued K, Bartnik E, Blobel G, WozniakRW. 1990a. The 210-kDa nuclear envelope polypeptiderecognized by human autoantibodies in primary biliarycirrhosis is the major glycoprotein of the nuclear pore.J Clin Inv 86:279–285.

Courvalin J-C, Lassoued K, Worman HJ, et al. 1990b.Identification and characterization of autoantibodiesagainst the nuclear envelope lamin B receptor frompatients with primary biliary cirrhosis. J Exp Med 172:961–967.

Davis LI, Blobel G. 1987. Nuclear pore complex contains afamily of glycoproteins that includes p62: Glycosylationthrough a previously unidentified cellular pathway. ProcNatl Acad Sci U S A 84:7552–7556.

Dwyer N, Blobel G. 1976. A modified procedure for theisolation of a pore-complex-lamina fraction from rat livernuclei. J Cell Biol 70:581–591.

Enarson P, Rattner JB, Ou Y, et al. 2004. Autoantigens ofthe nuclear pore complex. J Mol Med 82:423–433.

Greber UF, Senior A, Gerace L. 1990. A major glycoproteinof the nuclear pore complex is a membrane-spanningpolypeptide with a large lumenal domain and a smallcytoplasmic tail. EMBO J 9:1495–1502.

Invernizzi P, Podda M, Battezzati PM, et al. 2001.Autoantibodies against nuclear pore complexes areassociated with more active and severe liver disease inprimary biliary cirrhosis. J Hepatol 34:366–372.

Invernizzi P, Wesierska-Gadek J, Battezzati PM, et al.2004. Prognostic value of autoantibodies against proteinsof nuclear pore complexes (anti-NPCs) in earlyprimary biliary cirrhosis (PBC). J Hepatol 40:159–160(abstract).

Invernizzi P, Selmi C, Ranftler C, Podda M, Wesierska-Gadek J. 2005. Antinuclear antibodies in primary biliarycirrhosis. Semin Liver Dis 25:298–310.

Kaplan MM, Gershwin ME. 2005. Primary biliary cirrho-sis. N Engl J Med 353:1261–1273.

Kraemer DM, Kraus MR, Kneitz C, et al. 2003. Nucleoporinp62 antibodies in a case of mixed connective tissuedisease. Clin Diagn Lab Immunol 10:329–331.

Lassoued K, Brenard R, Degos F, Courvalin JC, et al. 1990.Antinuclear antibodies directed to a 200 kDa polypeptideof the nuclear envelope in primary biliary cirrhosis. Aclinical and immunological study of a series of 150patients with primary biliary cirrhosis. Gastroenterology99:181–186.

Lin F, Noyer CM, Ye Q, et al. 1996. Autoantibodies frompatients with primary biliary cirrhosis recognize a regionwithin the nucleoplasmic domain of inner nuclearmembrane protein LBR. Hepatology 23:57–61.

Lozano F, Pares A, Borsche L, et al. 1988. Autoantibodiesagainst nuclear envelope-associated proteins in primarybiliary cirrhosis. Hepatology 8:930–938.

Miyachi K, Shibata M, Onozuka Y, et al. 1996. Primarybiliary cirrhosis sera recognize not only gp210 but alsoproteins of the p62 complex bearing N-acetylglucosamineresidues from rat liver nuclear envelope. Mol Biol Rep23:227–234.

Miyachi K, Hankins RW, Matsushima H, et al. 2003. Profileand clinical significance of anti-nuclear envelope anti-bodies found in patients with primary biliary cirrhosis: Amulticenter study. J Autoimmun 20:247–254.

Nakamura M, Kondo H, Mori T, et al. 2007. Anti-gp210 andanti-centromere antibodies are different risk factors forthe progression of primary biliary cirrhosis. Hepatology45:118–127.

Ou Y, Enarson P, Barr SG, et al. 2004. The nuclear porecomplex protein Tpr is a common autoantigen in serathat demonstrate nuclear envelope staining by indirectimmunofluorescence. Clin Exp Immunol 136:379–387.

Starr CM, Hanover JA. 1990. Glycosylation of nuclear poreprotein p62. J Biol Chem 265:6868–6873.

Starr CM, D’Onofrio M, Park MK, Hanover JA. 1990.Primary sequence and heterologous expression of nuclearpore glycoprotein p62. J Cell Biol 110:1861–1871.

Talwalkar JA, Lindor KDA. 2003. Primary biliary cirrhosis.Lancet 362:53–61.

Van der Water J, Cooper A, Surh CD, et al. 1989. Detectionof autoantibodies to recombinant mitochondrial proteinsin patients with primary biliary cirrhosis. N Engl J Med320:1377–1380.

Walker JG, Doniach D, Roitt IM, Sherlock S. 1965.Serological tests in diagnosis of primary biliary cirrhosis.Lancet 39:827–831.

Wesierska-Gadek J, Penner E, Hitchman E, et al. 1988.Antibodies to nuclear lamins in autoimmune liverdisease. Clin Immunol Immunopathol 49:107–115.

Wesierska-Gadek J, Hohenauer H, Hitchman E, Penner E.1995. Autoantibodies from patients with primary biliarycirrhosis preferentially react with the amino-terminaldomain of nuclear pore complex glycoprotein gp210.J Exp Med 182:1159–1162.

Wesierska-Gadek J, Hohenauer H, Hitchman E, Penner E.1996a. Autoantibodies against nucleoporin p62 consti-tute a novel marker of primary biliary cirrhosis. Gastro-enterology 110:840–847.

Wesierska-Gadek J, Hohenauer H, Hitchman E, Penner E.1996b. Anti-gp210 antibodies in sera of patients withprimary biliary cirrhosis. Identification of a 64 kDafragment of gp210 as a major epitope. Hum AntibodiesHybridomas 7:167–174.

Wesierska-Gadek J, Wojciechowski J, Schmid G. 2003.Central and carboxy-terminal regions of human p53protein are essential for interaction and complex for-mation with PARP-1. J Cell Biochem 89:220–232.

Wesierska-Gadek J, Wojciechowski J, Schmid G. 2005.Advantage of baculovirus expression system. Acta Bio-chim Pol 52:713–719.

Wesierska-Gadek J, Penner E, Battezzati PM, et al. 2006.Correlation of initial autoantibody profile and clinicaloutcome in primary biliary cirrhosis. Hepatology 43:1135–1144.

Wesierska-Gadek J, Klima A, Komina O, Ranftler C,Invernizzi P, Penner E. 2007a. Characterization ofautoantibodies against components of the nuclear porecomplexes. High frequency of anti-p62 nucleoporin anti-bodies. Annals N Y Acad Sci 1109: (in press).

Wesierska-Gadek J, Schmitz ML, Ranftler C. 2007b.Roscovitine-activated HIP2 kinase induces phosphoryla-tion of wt p53 at Ser-46 in human MCF-7 breast cancercells. J Cell Biochem 100:865–874.

Wozniak RW, Bartnik E, Blobel G. 1989. Primary structureanalysis of an integral membrane glycoprotein of thenuclear pore. J Cell Biol 108:2083–2092.

Anti-p62 Nucleoporin Autoantibodies in PBC 37