The immortalized human keratinocyte cell line HaCaT (a kind gift from P. Boukamp, German Cancer Research Center (DKFZ), Heidelberg, Germany) was grown in DMEM (Invitrogen Corp.) supplemented with 50 U/ml penicillin-G, 50 μg streptomycin, and 10% FCS (Biochrom) in a humidified atmosphere (95% air, 5% CO2) at 37°C. The cultures were used for all experiments when grown to confluent monolayers, and Dsg1 expression was detected by dot blot analysis (Figure ) as well as immunostaining (day 7 after plating) (see Figure A). For dot blot analysis, cells from a T25 tissue flask were harvested in 150 μl of HBSS following incubation for 20 minutes in PBS consisting of 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, and 1.5 mM KH2PO4, pH 7.4) containing 1% EGTA (Roth), followed by 10 minutes of incubation in EGTA/trypsin (0.025%/0.05% final concentration each). Dot blotting was performed as described below.
HaCaT cells were grown on cover slips to confluence as described above (7 days) and incubated with PF-IgGs or control IgGs for 24 hours at 37°C. After incubation with PF-IgG, culture medium was removed and monolayers fixed for 10 minutes at room temperature (RT) with 2% formaldehyde (freshly prepared from paraformaldehyde) in PBS. Afterwards, monolayers were treated with 0.1% Triton X-100 in PBS for 5 minutes at RT. After rinsing with PBS at RT, HaCaT cells were preincubated for 30 minutes with 10% normal goat serum (NGS) and 1% BSA at RT and incubated for 16 hours at 4°C with mouse monoclonal antibodies directed against the ectodomain of human Dsg1 (clone p124; Progen Industries Ltd.) or Dsg3 (Zytomed) (dilution 1:100 in PBS each). After several rinses with PBS (3 times for 5 minutes each time), monolayers were incubated for 60 minutes at RT with Cy3-labeled goat anti-mouse IgGs (Dianova). For visualization of F-actin, Alexa-phalloidin (MoBiTec; diluted 1:60 in PBS) was used (incubation for 1 hour at RT). Cells incubated with antibodies or Alexa-phalloidin were rinsed with PBS (3 times for 5 minutes each time). Cover slips were mounted on glass slides with 60% glycerol in PBS containing 1.5% n-propyl gallate (Serva) as antifading compound. For visualization of Dsg1, Dsg3, and plakoglobin in association with bound Dsg1-coated beads, Dynabeads could not be used because of strong autofluorescence. Therefore, immunolocalization studies were performed with latex sulphate beads (Interfacial Dynamics Corp.). Bead solution (125 μl; 5 μl packed beads) was washed twice in 2-morpholino-ethane sulfonic acid (MES) (25 mM, pH 6.0) buffer by centrifugation and resuspension at 3000 g for 10 minutes. Beads were resuspended in 500 μl MES including 2.5 μg Dsg1-Fc and incubated overnight at RT under permanent slow overhead rotation. After centrifugation at 3000 g for 10 minutes, beads were washed 3 times in 1 ml PBS containing 0.1% albumine. Beads were stored for up to 10 days under permanent overhead rotation at 4°C. After incubation of HaCaT with Dsg1-coated microbeads for 30 minutes, culture medium was removed and monolayers were treated as described above. Monolayers were examined using an LSM 510 (Zeiss). Images were processed using Adobe Photoshop 7.0 software.
Purification of PF-IgG.
Sera from 2 PF patients whose diagnosis was confirmed clinically, histologically, and serologically and from a volunteer without any skin disease (control) were used for the present study. Subjects provided informed consent to the Department of Dermatology, University of Lübeck for the research studies. IgG fractions were purified by affinity chromatography using protein A agarose (Oncogene), eluted by citrate buffer (25 mM, pH 2.4), and immediately dialyzed against HBSS. The purity of isolated IgGs was checked by Coomassie blue staining of 7.5% SDS-PAGE. Activities against Dsg1 of IgGs purified from PF/control sera and purchased mouse monoclonal antibody directed against Dsg1 were adapted by dot blot immunodetection analysis of recombinant Dsg1-Fc (0.1–1 μg per dot) transferred to nitrocellulose using mouse monoclonal Dsg1 antibody followed by HRP-labeled goat anti-mouse IgGs (Dianova) as secondary antibody and the enhanced chemiluminescence technique (Amersham Biosciences) or biotinylated PF-IgGs and biotinylated control IgGs followed by HRP-labeled streptavidin (Dianova). For biotinylation, 300 μg of PF-IgGs was incubated twice for 2 hours at RT with 20 μg/μl N-hydroxy-succimide–biotin (NHS-Biotin) (Pierce Biotechnology Inc.), and afterwards reaction was stopped using saturated glycin in PBS followed by dialysis against PBS overnight at 4°C. Dot blotting of HaCaT cells was performed as indicated above using the monoclonal antibodies directed against Dsg1 and Dsg3, respectively.
Preparation of Fab fragments.
PF-IgGs were dialyzed overnight against buffer A (0.1 M Tris, pH 8.0, 2 mM EDTA) at 4°C. Papain (Sigma-Aldrich) was incubated in buffer A containing dithiothreitol (DTE) for 15 minutes at 37°C for activation and added at 1% final concentration to PF-IgG fractions for a 2-hour incubation at 37°C. Afterwards, reaction was stopped by incubation for 1 hour at 37°C in 2 mM iodacetamid (35
). Preparations of Fab fragments were analyzed by Coomassie blue staining of 7.5% SDS-PAGE and by Western blotting using HRP-labeled goat anti-FC IgGs or goat anti-Fab IgGs (Dianova; 1:600 each) followed by HRP-labeled rabbit anti-goat IgGs as outlined above. Capability of Fab fragments to bind to recombinant Dsg1 was demonstrated by dot blot analysis.
The transfer vector pEVmodPFIg, containing the sequence of the entire extracellular domain (EC 1–5) of Dsg1 and the constant region of human IgG 1 (Fc), was a kind gift from Masayuki Amagai (Keio University School of Medicine, Tokyo, Japan) (4
). For expression in eucaryotic cells, the Dsg1-Fc sequence was excised from pEVmodPFIg by digestion with BglII
and ligated with similarly cut pEGFP-N3 mammalian expression vector (BD Biosciences — Clontech). Dsg1-Fc and VE-cadherin were expressed by stably transfected CHO cells and purified from culture supernatants by affinity chromatography using protein A agarose (Oncogene). The protein was eluted by citrate buffer (25 mM, pH 2.4) and immediately dialyzed against HBSS for 16 hours at 4°C and stored in aliquots at –80°C. Protein content was determined using the Bradford method, and purity was checked by Coomassie blue staining of 10% SDS-PAGE. Western blotting and immunodetection were performed as described above using mouse monoclonal antibody against Dsg1 as well as goat anti-human IgGs directed against the Fc-portion of the fusion protein. Both antibodies detected the recombinant Dsg1-Fc fusion protein as a single band migrating slightly above the calculated molecular weight (not shown).
Coating of polystyrene beads.
After vortexing, 10 μl solution of protein A–coated superparamagnetic polystyrene microbeads (Dynabeads, diameter 2.8 μm; Dynal) containing 2 × 109 beads/ml were washed 3 times using 100 μl of buffer A (100 mM sodium phosphate buffer, pH 8.1). Washing was performed by immobilization of beads for 1 minute in a magnetic tube holder (MPC-E-1; Dynal Biotech) and reuptake in the corresponding buffer. Washed beads were suspended in 100 μl of 100 mM sodium phosphate buffer, pH 8.1, in HBSS containing 10 μg of Dsg1-Fc or of the Fc part of human IgGs (Dianova) and allowed to react for 16 hours at 4°C under permanent slow overhead rotation to avoid aggregation. After washing 3 times for 5 minutes each time in 100 μl of buffer A and 3 times for 5 minutes each time in buffer B (100 mM sodium borate, pH 9.0), beads were incubated for 45 minutes at RT in 100 μl buffer B containing 0.54 mg dimethyl pimelimidate dihydrochloride (DMP) (Pierce Biotechnology Inc.) to covalently cross-link protein A and bound Fc parts. After washing 2 times for 5 minutes each time in buffer C (100 μl 0.2 M ethanolamine, pH 8.0), beads were incubated in buffer C for 2 hours at RT. Finally, beads were washed 3 times for 5 minutes each time in HBSS and stored in HBSS at 4°C for up to 8 days under permanent slow overhead rotation to avoid aggregation of beads. The concentration of beads in these stocks was about 1.6 × 108 beads/ml.
PF-IgGs were added to 1 ml of DMEM (35 μg/ml final concentration) and incubated overnight at 4°C with Dynabeads coated with 15 μg recombinant Dsg1-Fc or VE-cadherin–Fc as described above. To saturate potential free protein A binding sites, beads were postincubated with 200 μl of human control IgGs (0.3 μg/ml) for 1 hour at RT. Following immunoabsorption, beads were separated using a magnetic tube holder, and DMEM containing the IgG fractions was applied to HaCaT monolayers.
The setup was used as described previously (17
). The home-built laser tweezer setup consisted of an Nd:Yag laser (1064 nm), the beam of which was expanded to fill the back aperture at a high NA-objective (100 × 1.3 oil; Zeiss), coupled through the epi-illumination port of an Axiovert 135 microscope (Zeiss) and reflected to the objective by a dicroic mirror (FT 510; Zeiss). Through all experiments, the laser intensity was 42 mW in the focal plane. Coated beads (10 μl of stock solution) were suspended in 200 μl of culture medium and allowed to interact with HaCaT monolayers for 30 minutes at 37°C before measuring the number of bound beads (control values). Beads were considered tightly bound when resisting laser displacement at the 42-mW setting. For every condition, 100 beads were counted. Afterwards, control and PF-IgG fractions (35 μg/ml) were applied for 30 minutes, and the number of bound beads was counted again. Percentage of beads resisting laser displacement under various experimental conditions was normalized to control values. For preincubation experiments, either HaCaT cells or Dsg1-coated beads were incubated with PF-IgGs (35 μg/ml, 30 minutes) and rinsed twice in HBSS before beads were allowed to settle on HaCaT cells for 30 minutes. Negative controls were performed using Dsg1-coated beads incubated on the surface of HaCaT cells in the presence of 5 mM EGTA (Roth) or in the presence of monoclonal mouse IgG antibody (1:50 in HBSS) directed against Dsg-1 (clone p124; Progen Industries Ltd.).
Scanning electron microscopy.
HaCaT monolayers with adhering Dsg1-coated Dynabeads were fixed overnight with 6.5% glutaraldehyde in HBSS. After dehydration with graded acetone series, critical point drying, and sputtercoating with palladium-gold (CPD 030, BAL-TEC), cells were examined with a JSM-840 scanning electron microscope (JEOL).
Transmission electron microscopy.
HaCaT monolayers with adhering Dsg1-coated Dynabeads were fixed in 2.5% glutaraldehyde containing 0.01% ruthenium red in 0.1 M sodium cacodylate, pH 7.35, for 1 hour at 4°C. After rinsing in 0.1 M cacodylate (3 times for 5 minutes each time), cells were postfixed in 2% OSO4 in 0.1 M cacodylate for 1 hour at RT. Cells were rinsed again in cacodylate and dehydrated in ascending concentrations of methanol (25%, 50%, 70%, 80%, 95% [twice], 100% [twice]; 10 minutes each time). Following incubation in 70% methanol, monolayers were placed for 1 hour in saturated uranyl acetate in 70% methanol for 1 hour in the dark. After dehydration, cells were equilibrated in propylene oxide (2 times for 10 minutes each time) and embedded in Epon 812, semithin sections (1 μM) were stained with toluidine blue, and ultrathin sections were contrasted with uranyl acetate and lead citrate and examined with a LEO AB 912 electron microscope.
Atomic force microscopy measurements.
Dsg1-Dsg1 interactions were characterized by force-distance measurements of Dsg1 coupled via flexible linkers to the tip and substrate of a Bioscope AFM driven by a Nanoscope III controller (Digital Instruments Inc.). Dsg1 was linked covalently to the Si3N4 tip of the cantilever (Park Scientific Ltd.) and freshly cleaved mica plates (Wacker) using PEG spacers containing an amino-reactive cross-linker group (NHS ester) at 1 end and a thiol-reactive group (2-[pyridyldithio] propionate) at the other, as previously described in detail (21
). The NHS group served to link PEG to free amino acid groups at both the Si3N4 tip and the SiOH plate introduced by treatment of the tip and plate with 2-aminoethanol HCl (Sigma-Aldrich). Binding events were measured in HBSS by force-distance cycles at amplitudes of 300 nm and at frequencies ranging from 1–2 Hz. Force-distance cycles were performed either at constant lateral positions or with lateral shifts of 1 nm/s. Force-distance cycles were analyzed as described previously in detail (15
Differences in bead adhesion or single molecule transinteraction between different protocols were assessed using 2-tailed Student’s t test. Values throughout are expressed as mean ± SEM. Statistical significance is assumed at P < 0.05.