Calcium-Dependent Conformation of Desmoglein 1 Is Required for its Cleavage by Exfoliative Toxin
2003; Elsevier BV; Volume: 121; Issue: 2 Linguagem: Inglês
10.1046/j.1523-1747.2003.12362.x
ISSN1523-1747
AutoresYasushi Hanakawa, Trevor Selwood, Denise K. Woo, Chenyan Lin, Norman M. Schechter, John R. Stanley,
Tópico(s)Biochemical and Structural Characterization
ResumoIn bullous impetigo, Staphylococcus aureus spreads under the stratum corneum of skin by elaboration of exfoliative toxin, which hydrolyzes only one peptide bond in a highly structured calcium-binding domain of desmoglein 1, resulting in loss of its function. We investigated the basis of this exquisite specificity. Exfoliative toxin cannot cleave desmoglein 1 pretreated at 56°C or higher or at low or high pH, suggesting that the proper conformation of desmoglein 1 is critical for its cleavage. Because cleavage occurs in an area of desmoglein 1 stabilized by calcium, we determined if the conformation necessary for cleavage is calcium-dependent. Depletion of calcium from desmoglein 1 completely inhibited its cleavage by exfoliative toxin, even after calcium was added back. A change in conformation of desmoglein 1 by calcium depletion was shown, with immunofluorescence and enzyme-linked immunoassay, by loss of binding of PF sera, which recognize conformational epitopes. This change in conformation was confirmed by tryptophan fluorometry and circular dichroism, and was irreversible with repletion of calcium. These data suggest that the specificity of exfoliative toxin cleavage of desmoglein 1 resides not only in simple amino acid sequences but also in its calcium-dependent conformation. In bullous impetigo, Staphylococcus aureus spreads under the stratum corneum of skin by elaboration of exfoliative toxin, which hydrolyzes only one peptide bond in a highly structured calcium-binding domain of desmoglein 1, resulting in loss of its function. We investigated the basis of this exquisite specificity. Exfoliative toxin cannot cleave desmoglein 1 pretreated at 56°C or higher or at low or high pH, suggesting that the proper conformation of desmoglein 1 is critical for its cleavage. Because cleavage occurs in an area of desmoglein 1 stabilized by calcium, we determined if the conformation necessary for cleavage is calcium-dependent. Depletion of calcium from desmoglein 1 completely inhibited its cleavage by exfoliative toxin, even after calcium was added back. A change in conformation of desmoglein 1 by calcium depletion was shown, with immunofluorescence and enzyme-linked immunoassay, by loss of binding of PF sera, which recognize conformational epitopes. This change in conformation was confirmed by tryptophan fluorometry and circular dichroism, and was irreversible with repletion of calcium. These data suggest that the specificity of exfoliative toxin cleavage of desmoglein 1 resides not only in simple amino acid sequences but also in its calcium-dependent conformation. exfoliative toxins Impetigo is the most common bacterial infection of children and 30% of these impetigo patients have bullous impetigo, which is caused by Staphylococcus aureus strains that produce exfoliative toxins (ETs) (Ladhani et al., 1999Ladhani S. Joannou C.L. Lochrie D.P. Evans R.W. Poston S.M. Clinical, microbial, and biochemical aspects of the exfoliative toxins causing staphylococcal scalded-skin syndrome.Clin Microbiol Rev. 1999; 12: 224-242Crossref PubMed Google Scholar). Staphylococcal scalded skin syndrome is a generalized form of bullous impetigo in which patients, usually infants or young children, develop bullae and erosions over a large area of the skin surface due to the systemic circulation of ETs from a localized source of infection. In the early 1970s, two serotypes of ETs (ETA and ETB), were shown to produce blisters in the superficial epidermis of neonatal mice by passive transfer. (Melish and Glasgow, 1970Melish M.E. Glasgow L.A. The staphylococcal scalded-skin syndrome. Development of an experimental model.N Engl J Med. 1970; 282: 1114-1119Crossref PubMed Scopus (318) Google Scholar,Melish and Glasgow, 1971Melish M.E. Glasgow L.A. 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Sasaki M. et al.Identification of the Staphylococcus aureus etd pathogenicity island which encodes a novel exfoliative toxin, ETD, and EDIN-B.Infect Immun. 2002; 70: 5835-5845Crossref PubMed Scopus (195) Google Scholar). Recently, it has been shown that these ETs are serine proteases that specifically target and cleave desmoglein (Dsg) 1 (Amagai et al., 2000Amagai M. Matsuyoshi N. Wang Z.H. Andl C. Stanley J.R. Toxin in bullous impetigo and staphylococcal scalded-skin syndrome targets desmoglein 1.Nature Med. 2000; 6: 1275-1277Crossref PubMed Scopus (384) Google Scholar,Amagai et al., 2002Amagai M. Yamaguchi T. Hanakawa Y. Nishifuji K. Sugai M. Stanley J.R. Staphylococcal exfoliative toxin B specifically cleaves desmoglein 1.J Invest Dermatol. 2002; 118: 845-850Crossref PubMed Scopus (150) Google Scholar;Yamaguchi et al., 2002Yamaguchi T. Nishifuji K. Sasaki M. et al.Identification of the Staphylococcus aureus etd pathogenicity island which encodes a novel exfoliative toxin, ETD, and EDIN-B.Infect Immun. 2002; 70: 5835-5845Crossref PubMed Scopus (195) Google Scholar). The exquisite specificity of this protease–substrate interaction is underscored by the finding of one unique cleavage site caused by all ETs in both mouse and human Dsg1, and the inability of ETs to cleave closely homologous proteins such as Dsg3 and E-cadherin (Amagai et al., 2000Amagai M. Matsuyoshi N. Wang Z.H. Andl C. Stanley J.R. Toxin in bullous impetigo and staphylococcal scalded-skin syndrome targets desmoglein 1.Nature Med. 2000; 6: 1275-1277Crossref PubMed Scopus (384) Google Scholar;Hanakawa et al., 2002Hanakawa Y. Schechter N. Lin C. et al.Molecular mechanisms of blister formation in bullous impetigo and staphylococcal scalded skin syndrome.J Clin Invest. 2002; 110: 53-60Crossref PubMed Scopus (162) Google Scholar). Dsg1 is a member of the cadherin supergene family (Angst et al., 2001Angst B.D. Marcozzi C. Magee A.I. The cadherin superfamily. Diversity in form and function.J Cell Sci. 2001; 114: 629-641Crossref PubMed Google Scholar). These cadherins characteristically contain extracellular repeating domains of about 100 amino acids in length that are highly conserved. Most cadherins are involved in calcium-dependent cell–cell adhesion, and calcium is thought to be important in maintaining their structure and function (Kemler et al., 1989Kemler R. Ozawa M. Ringwald M. Calcium-dependent cell adhesion molecules.Curr Opin Cell Biol. 1989; 1: 892-897Crossref PubMed Scopus (80) Google Scholar;Steinberg and McNutt, 1999Steinberg M.S. McNutt P.M. Cadherins and their connections: Adhesion junctions have broader functions.Curr Opin Cell Biol. 1999; 11: 554-560Crossref PubMed Scopus (248) Google Scholar). Dsg1 is a member of the so-called desmosomal cadherin family of molecules which includes desmogleins and desmocollins. These molecules are critical to the proper function of desmosomes that maintain tissue integrity in epithelial and other tissues (Green and Gaudry, 2000Green K.J. Gaudry C.A. Are desmosomes more than tethers for intermediate filaments?.Nature Rev Mol Cell Biol. 2000; 1: 208-216Crossref PubMed Scopus (321) Google Scholar). The importance of these molecules in maintaining adhesion in epithelial tissues has been demonstrated by loss of adhesion with anti-desmoglein and anti-desmocollin antibodies from patients with pemphigus, cell culture studies in which these desmosomal cadherins confer cell–cell adhesion, and loss of adhesion in mice with genetic deletions of genes encoding these molecules (Chitaev and Troyanovsky, 1997Chitaev N.A. Troyanovsky S.M. Direct Ca2+-dependent heterophilic interaction between desmosomal cadherins, desmoglein and desmocollin, contributes to cell–cell adhesion.J Cell Biol. 1997; 138: 193-201Crossref PubMed Scopus (196) Google Scholar;Hashimoto et al., 1997Hashimoto T. Kiyokawa C. Mori O. et al.Human desmocollin 1 (Dsc1) is an autoantigen for the subcorneal pustular dermatosis type of IgA pemphigus.J Invest Dermatol. 1997; 109: 127-131Crossref PubMed Scopus (191) Google Scholar;Koch et al., 1997aKoch P.J. Mahoney M.G. Ishikawa H. et al.Targeted disruption of the pemphigus vulgaris antigen (desmoglein 3) gene in mice causes loss of keratinocyte cell adhesion with a phenotype similar to pemphigus vulgaris.J Cell Biol. 1997; 137: 1091-1102Crossref PubMed Scopus (374) Google Scholar;Marcozzi et al., 1998Marcozzi C. Burdett I.D. Buxton R.S. Magee A.I. 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As with classical cadherins, calcium is critical to the function of desmosomal cadherins, and the amino acid sequence of desmosomal and classical cadherins is highly conserved at the calcium-binding sites (Garrod et al., 1996Garrod D. Chidgey M. North A. Desmosomes: Differentiation, development, dynamics and disease.Curr Opin Cell Biol. 1996; 8: 670-678Crossref PubMed Scopus (129) Google Scholar;Chitaev and Troyanovsky, 1997Chitaev N.A. Troyanovsky S.M. Direct Ca2+-dependent heterophilic interaction between desmosomal cadherins, desmoglein and desmocollin, contributes to cell–cell adhesion.J Cell Biol. 1997; 138: 193-201Crossref PubMed Scopus (196) Google Scholar;Wallis et al., 2000Wallis S. Lloyd S. Wise I. Ireland G. Fleming T.P. Garrod D. The alpha isoform of protein kinase C is involved in signaling the response of desmosomes to wounding in cultured epithelial cells.Mol Biol Cell. 2000; 11: 1077-1092Crossref PubMed Scopus (144) Google Scholar;Syed et al., 2002Syed S.E. Trinnaman B. Martin S. Major S. Hutchinson J. Magee A.I. Molecular interactions between desmosomal cadherins.Biochem J. 2002; 362: 317-327Crossref PubMed Scopus (94) Google Scholar). Therefore, like classical cadherins, calcium is presumed to maintain the structure and function of desmosomal cadherins. ETs have been shown to attack Dsg1 precisely at one of its calcium-binding domains. They hydrolyze the peptide bond just after the glutamic acid at amino acid position 381 (as counted from the initiating methionine of both mouse and human Dsg1), between extracellular domains 3 and 4. By homology to the recent crystal structure of C-cadherin this cleavage site is in the third of four calcium-binding sites each of which binds three calcium ions (Boggon et al., 2002Boggon T.J. Murray J. Chappuis-Flament S. Wong E. Gumbiner B.M. Shapiro L. C-cadherin ectodomain structure and implications for cell adhesion mechanisms.Science. 2002; 296: 1308-1313Crossref PubMed Scopus (544) Google Scholar). These sites are thought to orient rigidly each extracellular domain to the next to result in a rod-like structure of the entire extracellular domain. Thus ETs show exquisite specificity for a highly structured site in Dsg1. This led us to ask whether this specificity resides only in the primary amino acid sequence of Dsg1 or in its conformation, and, if the latter, whether ETs conformational specificity was calcium dependent. Recombinant wild-type ETA and ETB with a V5 and His tag on the carboxy-terminus (these tagged ETs will be abbreviated ETA and ETB) were purified on Ni-NTA columns (Qiagen, Valencia, California) according to the manufacturer's protocol, then dialyzed against phosphate-buffered saline (Hanakawa et al., 2002Hanakawa Y. Schechter N. Lin C. et al.Molecular mechanisms of blister formation in bullous impetigo and staphylococcal scalded skin syndrome.J Clin Invest. 2002; 110: 53-60Crossref PubMed Scopus (162) Google Scholar). Protein concentrations of ETA and ETB were estimated with a Protein Assay Kit (Bio-Rad Laboratories, Hercules, California). The entire extracellular domain of human recombinant Dsg1 with a his and E-tag (hDsg1E) on the carboxyl-terminus was produced as a secreted protein by baculovirus as previously described (Ishii et al., 1997Ishii K. Amagai M. Hall R.P. Hashimoto T. et al.Characterization of autoantibodies in pemphigus using antigen-specific enzyme-linked immunosorbent assays with baculovirus-expressed recombinant desmogleins.J Immunol. 1997; 159: 2010-2017PubMed Google Scholar). Approximately, 10 nM of hDsg1E in the culture supernatant of High Five (Ca2+ concentration of 6 mM) was treated with various conditions then used for cleavage analysis as follows: (1) hDsg1E was incubated at 4, 25, 56, and 80°C for 1 h, then incubated with 3 μM ETA at 37°C for 1 h. (2) hDsg1E was dialyzed against Tris-buffered saline (TBS) with 1 mM CaCl2 (TBS+Ca2+), then the pH was adjusted to 4.0 and 4.5 by adding 0.1 M acetic acid, pH 4.0 and 4.5, which was preadjusted with NaOH. pH was adjusted to 5.0 and 6.0 by adding 0.1 M MES (2-(N-Morpholino)ethanesulfonic acid) buffer, pH 5.0 and 6.0, which was preadjusted with NaOH. pH was adjusted to 7.4 with 0.1 M Tris of pH 7.4. pH was adjusted to 10 with 0.1 M NaOH. After incubation at room temperature for 15 min, the solutions were dialyzed against TBS+Ca, then incubated with 3 μM ETA at 37°C for 1 h. (3) hDsg1E in culture supernatant was dialyzed against TBS+Ca2+, then treated with 5 mM ethylenediamine tetraacetic acid (EDTA) at room temperature for 1 h, then again dialyzed against TBS+Ca2+. As a control, hDsg1E dialyzed against TBS+Ca2+ without pretreatment with EDTA was used. Dialyzed hDsg1E was then incubated with 3 μM ETA or, as controls, 3 μM porcine pancreas trypsin (Sigma, St Louis, Missouri) or 3 μM staphylococcus V8 protease (Roche Applied Science, Indianapolis, Indiana) at 37°C for 1 h. (4) hDsg1E was dialyzed against TBS without CaCl2 (TBSminusCa2+), TBS+Ca2+ or TBSminusCa2+ followed by TBS+Ca2+, then incubated with 3 μM ETA or ETB at 37°C for 1 h. In all the above four conditions, degradation of hDsg1E was assayed by western blotting with anti-E-tag antibodies. For fluorometry and circular dichroism analysis, hDsg1E was purified from culture supernatant with an anti-E-tag column (Amersham Biosciences, Piscataway, New Jersey) and eluted using 0.1 mg per mL E-tag peptide in TBS+Ca2+ and 0.1% octylglucoside (Anatrace, Maumee, Ohio). Eluant was dialyzed against TBS+Ca2+ and 0.1% octylglucoside for fluorometry, and water with 1 mM CaCl2 for circular dichroism analysis. The purified samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, then stained by Coomassie Brilliant Blue, which showed one major band (Figure 1). Protein concentrations of hDsg1E were estimated with a Protein Assay Kit (Bio-Rad Laboratories). Proteins in Laemmli sample buffer were separated by 4 to 20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Bio-Rad Laboratories or Invitrogen Life Sciences, Carlsbad, California), then transferred to nitrocellulose sheets (Transblot; Bio-Rad Laboratories). The sheets were incubated for 1 h in blocking buffer of 5% fat-free milk powder in phosphate-buffered saline. The E-tag antibody conjugated with horseradish peroxidase (Amersham Biosciences), diluted in blocking buffer, was applied for 1 h at room temperature. After four washes with 0.1% Tween 20 in phosphate-buffered saline, the signals were detected with chemiluminescence (ECL or ECL plus, Amersham Biosciences). A pemphigus foliaceus (PF) patient's serum (PF982) was diluted to 1:1280 with TBS+Ca2+ or with 2 nM hDsg1E, which was purified on an anti-E tag column and dialyzed against TBSminusCa2+ or TBS+Ca2+, then incubated overnight at 4°C. These diluted sera were used for indirect immunofluorescent staining of cryostat sections of normal human skin. Epidermal bound human IgG from the diluted pemphigus sera were detected with antihuman IgG conjugated with Alexa 590 (Molecular Probes, Eugene, Oregon). hDsg1E was purified with the anti-E-tag column, then dialyzed against TBS+Ca2+ (hDsg1E+Ca2+) or TBSminusCa2+ (hDsg1EminusCa2+). PF sera (n=8), normal human serum (n=2), or bullous pemphigoid serum (n=2) were diluted into various concentrations of hDsg1E+Ca2+ or hDsg1EminusCa2+ and incubated overnight at 4°C. For ELISA substrate, Ni-NTA HisSorb strips (Qiagen) were coated by incubation with 2 nM hDsg1E+Ca in TBS+Ca2+ with 1% bovine serum albumin, overnight at 4°C. Normal, bullous pemphigoid, or PF sera, adsorbed with hDsg1E+Ca2+ or hDsg1EminusCa2+, were used as the first antibody. Anti-human IgG conjugated with alkaline phosphatase (DAKO, Carpinteria, CA) was used as the second antibody. The plates were developed with AmpliQ kit (DAKO) according to the manufacturer's procedure and the OD at 490 nm was measured with an ELISA plate reader. Circular dichroism measurements were performed with an AVIV Model 202 spectrometer. Far-ultraviolet spectra from 200 nm to 250 nm were recorded of hDsg1E solution at 25°C in a quartz cell with a path length of 1 mm. The analysis was of hDsg1E at a concentration of 5 μM in water with 1 mM CaCl2, pH approximately 6.0. For the second spectrum EDTA was added to a final concentration of 5 mM. Finally, the third analysis was taken following further addition of excess Ca2+ (final concentration 10 mM CaCl2). Spectra were corrected by subtraction of each corresponding solution, and data were normalized and presented as mean molar residue ellipticity (deg cm2 per dmol) using the calculation of: [θ]=(CDvalue of protein−CD value of buffer)(10×conc inM×path length in cm×#residues) Three independent preparations of hDsg1E, two in water and one in phosphate-buffered saline, pH 7.4, showed similar spectra. Tryptophan fluorescence emission of hDsg1E was measured with a QM-C60 fluorescence spectrophotometer (Photon Technology, Lawrenceville, NJ) in the photon counting mode. The cell holder was maintained at 25°C. Excitation was at 295 nm, and intrinsic fluorescence emission spectra were recorded from 305 to 405 nm at a rate of 4 nm per s and at 1 nm intervals. Excitation and emission slits were set to give a 4 nm band pass, and the cuvette had a path length of 1 cm. Background spectra of buffer were subtracted from all hDsg1E spectra to correct for the Raman fluorescence. In EDTA experiments, spectra of the same hDsg1E were taken in the following sequence: (1) in TBS+Ca2+, (2) following addition of EDTA to a final concentration of 5 mM, then (3) following further addition of excess Ca2+ (to 10 mM CaCl2). In pH experiments, spectra of the same hDsg1E were taken in the following sequence: (1) in TBS+Ca2+, (2) following addition of 4×10–4 M HCl to yield a pH of 3.5, then (3) following further addition of 10 mM Tris pH 7.4 to adjust the pH to 7.0. Finally, spectra were taken at 1 and 13 min after hDsg1E was directly added to 8 M urea in TBS+Ca2+. To determine, in general, if the conformation might be important in Dsg1 cleavage by ET, we analyzed if heating of Dsg1 affects its proteolysis (Figure 2a). Recombinant Dsg1E (extracellular domain of human Dsg1 with an E-tag at the carboxy-terminus) produced by baculovirus was treated at 4, 25, 56, or 80°C for 1 h, then incubated with excess ETA at 37° for 1 h. hDsg1E and its degradation product were detected by western blotting with anti-E-tag antibody. hDsg1E pretreated at 4°, 25° or 37°C was cleaved by ETA to yield the characteristic 35 kDa degradation product, but ETA was not able to cleave hDsg1E heated at 56° or 80°C. In contrast, trypsin, a serine protease in the same family as ETA, was able cleave hDsg1E after pretreatment at all temperatures (data not shown). Trypsin and other related proteases in this family (the chymotrypsin family) are sequence-dependent proteases and can cleave denatured proteins. These results suggest that heat may irreversibly change the conformation of Dsg1, and ETA cleavage of Dsg1 may be dependent on conformation, not just primary amino acid sequence. We next determined whether changes in pH, which may change the conformation of proteins, could affect the susceptibility of Dsg1 to proteolysis by ETA. hDsg1E was treated at pH 4.0, 4.5, 5.0, 6.0, and 7.4 for 15 min at room temperature then dialyzed against TBS+Ca2+ (pH 7.4) before incubation with ETA. Subsequent western blotting with anti-E-tag antibody indicated hDsg1E treated at pH 4.5 and below lost its susceptibility to proteolysis by ETA (Figure 2b). hDsg1E treated with high pH (pH 10 with NaOH) was also not cleaved by ETA (data not shown). These data again indicate that Dsg1 susceptibility to cleavage by ETA is not simply specified by its primary amino acid sequence, but is likely conformational dependent. In addition, these findings imply that any changes induced in conformation of Dsg1 by pH are not directly reversible by reversing the pH back to 7.4, as confirmed below by tryptophan fluorescence spectroscopy (Figure 6). The Ca2+ binding in cadherins is mediated by acidic amino acids, which have negative charge at neutral pH (Ringwald et al., 1987Ringwald M. Schuh R. Vestweber D. et al.The structure of cell adhesion molecule uvomorulin. Insights into the molecular mechanism of Ca2+-dependent cell adhesion.EMBO J. 1987; 6: 3647-3653Crossref PubMed Scopus (226) Google Scholar;Kemler et al., 1989Kemler R. Ozawa M. Ringwald M. Calcium-dependent cell adhesion molecules.Curr Opin Cell Biol. 1989; 1: 892-897Crossref PubMed Scopus (80) Google Scholar;Ozawa et al., 1990Ozawa M. Engel J. Kemler R. Single amino acid substitutions in one Ca2+ binding site of uvomorulin abolish the adhesive function.Cell. 1990; 63: 1033-1038Abstract Full Text PDF PubMed Scopus (238) Google Scholar;Nagar et al., 1996Nagar B. Overduin M. Ikura M. Rini J.M. Structural basis of calcium-induced E-cadherin rigidification and dimerization.Nature. 1996; 380: 360-364Crossref PubMed Scopus (564) Google Scholar;Pertz et al., 1999Pertz O. 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As controls for ETA we used trypsin and staphylo-coccus V8 protease, which are well-characterized sequence-specific serine proteases in the same family (chymotrypsin family) as ETA. hDsg1E not treated with EDTA was cleaved by ETA to yield the characteristic carboxy-terminal 35 kDa degradation product. Trypsin and V8 protease also digested Dsg1, but, as expected, no degradation product was visible on western blotting, because the substrate is digested into small peptides or alternatively, it is possible that these proteases cleaved off the E-tag. EDTA did not block hDsg1E digestion by trypsin and V8 protease, but did inhibit cleavage by ETA, even after Ca2+ was added back. Similarly, ethyleneglycol-bis-(β-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA)-treated Dsg1 was no longer susceptible to degradation by ETA (data not shown). Finally, as with ETA, ETB could not cleave EDTA or EGTA-treated Dsg1 even after Ca2+ was added back (data not shown). We then determined whether dialysis alone, without a chelating reagent, could affect the susceptibility of Dsg1 to proteolysis by ETs (Figure 3b). When hDsg1E was dialyzed against TBSminusCa2+, it was not cleaved by ETA or ETB. When hDsg1E was TBSminusCa2+ and sequentially dialyzed against TBS+Ca2+, it was still mostly resistant to degradation by ETA and ETB, although a slight amount of cleavage was observed. hDsg1E dialyzed against TBS with 1 mM MgCl2 and TBS with 1 mM ZnCl2 was not cleaved by ETA and ETB (data not shown). These results indicate that the cleavage of Dsg1 by ET is dependent on a Ca2+–Dsg1 interaction, and once Ca2+ is removed, Dsg1 loss of susceptibility to cleavage is almost completely irreversible. These data, combined with the known Ca2+-dependent conformation of cadherins, suggest that depletion of Ca2+ cause at least partial denaturation of Dsg1 that is not totally reversible once Ca2+ is restored. Combined with the loss of susceptibility to proteolysis by ET after heat and pH changes, these data suggest that the exquisite specificity of ET for Dsg1 is dependent on the conformation of Dsg1 that is stabilized by Ca2+. To investigate Ca2+-dependent changes in conformation of Dsg1, we measured its reactivity with PF sera, as it has been shown that most antibodies from PF sera bind conformational epitopes on Dsg1 (Koulu et al., 1984Koulu L. Kusumi A. Steinberg M.S. Klaus Kovtun V. Stanley J.R. Human autoantibodies against a desmosomal core protein in pemphigus foliaceus.J Exp Med. 1984; 160: 1509-1518Crossref PubMed Scopus (182) Google Scholar;Stanley et al., 1986Stanley J.R. Koulu L. Klaus Kovtun V. Steinberg M.S. A monoclonal antibody to the desmosomal glycoprotein desmoglein I binds the same polypeptide as human autoantibodies in pemphigus foliaceus.J Immunol. 1986; 136: 1227-1230PubMed Google Scholar;Eyre and Stanley, 1987Eyre R.W. Stanley J.R. 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Pemphigus sera recognize conformationally sensitive epitopes in the amino-terminal region of desmoglein-1 (Dsg1).J Invest Dermatol. 1995; 105: 147-152Crossref PubMed Scopus (79) Google Scholar) (Figure 4a). IgG in PF sera bind the cell surface of keratinocytes throughout the epidermis as determined by indirect immunofluorescence. hDsg1E stabilized with Ca2+ was able to adsorb anti-keratinocyte antibodies from PF sera; however, strikingly, in contrast, hDsg1E dialyzed against TBSminusCa2+ did not adsorb most of these cell-surface reactive antibodies. To semiquantitate these findings, we performed ELISA assays on Dsg1 using PF sera adsorbed with hDsg1E+Ca2+ or Dsg1EminusCa2+ (Figure 4b). Normal human serum (n=2) and bullous pemphigoid serum (n=2) showed background reactivity to Dsg1 and pretreatment with Dsg1EminusCa2+ or Dsg1E+Ca2+ did not cause a significant change in their reactivity. On the other hand, PF sera adsorbed with hDsg1EminusCa2+ showed high reactivity with Dsg1 that was mostly, although not entirely in some sera, adsorbed with hDsg1E+Ca2+. These data show that Dsg1 undergoes a major conformational change when Ca2+ is removed, and that this change removes most of the epitopes that bind anti-Dsg1 autoantibodies in PF sera. To investigate the Ca2+-dependent change in conformation of Dsg1 and its potential reversibility more directly, we performed circular dichroism and tryptophan fluorometry, two techniques that measure parameters dependent on a molecule's conformation. In circular dichroism (Figure 5), the spectrum of hDsg1E in 1 mM CaCl2 showed a minimum of -8.35×103 deg cm2 per dmol at 215 nm. The shape of the spectrum suggested the predominance of β sheets, consistent with the crystal structure of the ectodomains of cadherins (Shapiro et al., 1995Shapiro L. Fannon A.M. 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