Pemphigus Foliaceus Sera Recognize an N-Terminal Fragment of Bovine Desmoglein 1

Pemphigus Foliaceus Sera Recognize an N-Terminal Fragment of Bovine Desmoglein 1 Monica Olague-Alcala,*t George J. Giudice,*t and Luis A. Diaz*t Departments of'Dermatology and tBiochemistry, Medical College of Wisconsin; and :|:Veterans Administration Medical Center, Milwaukee, Wisconsin, U.S.A.

It has previously been demonstrated that sera from endemic and nonendemic pemphigus foliaceus patients recognize three immunoreactive fragments of 80, 62, and 45 kilodaltons (kD) from extracts of the envelope fraction of human and bovine epidermis. These polypeptides are also immunoprecipitated by approximately 50% of pemphigus vulgaris sera, but are unreactive with sera from buUous pemphigoid patients or normal controls. The 80-kD antigen has been shown to be a glycoprotein with N-linked oligosaccharides. Complete removal of the carbohydrate moieties produced a 76-kD polypeptide that continued to react with pemphigus foliaceus autoantibodies in a Ca"'~'"-dependent manner. To further characterize this antigen/antibody system, the 80-kD pemphigus foliaceus antigen solubilized from a bovine epidermal envelope extract was purified by affinity chromatography using a pemphigus foliaceus patient's immunoglobulin (Ig)G immobilized on agarose. After elution with 0.2 M

I

diopathic pemphigus foliaceus (PF) and its endemic form Fogo Selvagem (FS) share clinical, histologic, and immunologic features [1,2], i.e., superficial skin blisters due to subcorneal acantholysis and the presence of pathogenic squamous epithelia-specific antoantibodies. Immunofluorescence (IF) techniques have been used to reveal the presence of autoantibodies bound in vivo to lesional epidermis and circulating in the sera of these patients. FS autoantibodies are predominantly of the immunoglobulin (Ig)G4 subclass [3] and the circulating antibody titers determined by indirect IF on normal skin correlate with the activity level and extent of the disease [4]. Using immunoelectron microscopy it has been shown recently that PF autoantibodies bind predominantly to the extracellular region of the epidermal desmosome [5]. Passive transfer of total IgG or the IgG4 fraction of FS sera as well as of Fab' fragments of FS IgG into neonatal BALB/c mice induced a cutaneous blistering disorder that closely mimicked the human disease [3,6,7]. Stanley and co-workers demonstrated that homogenization of human epidermis in nonionic detergents released two distinct antigenic complexes recognized by PF and pemr)higus vulgaris (PV) sera using immunoprecipitation techniques [8]. The PF complex Manuscript received September 27, 1993; accepted for publication January 14, 1994. Reprint requests to: Dr. Luis A. Diaz, Department of Dermatology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI, 53226. Abbreviations: Dsgl, desmoglein 1; Dsg3, desmoglein 3 (PV antigen); FS, Fogo Selvagem; PF, pemphigus foliaceus; PV, pemphigus vulgaris; PVDF, polyvinilidene difluoride.

0022-202X/94/$07.00

glycine/HCl, pH 2.8, 5 mM ethylene diaminetetraacetic acid, the polypeptide was mixed with a small amount of '^^I-labeled 80-kD antigen, added as a tracer, fractionated by sodium dodecylsulfate-polyacrylamide gel electrophoresis, and electrotransferred onto a polyvinylidene difluoride membrane. The 80-kD band detected by amido black staining and autoradiography was excised and characterized by amino acid sequence analysis. The resulting sequence, EXIKFAAAXREGEXNSKRNPIA, matched perfectly with the N-terminal 22 amino acids of the mature form of bovine desmoglein 1. These findings demonstrate that the 80-kD bovine autoantibody-reactive polypeptide is the glycosylated ectodomain of desmoglein 1, which may contain epitopes recognized by pathogenic autoantibodies. Key words: desmosomes/autoimmunity/cadherins/desmogleins. f Invest Dermatol 102:882-885, 1994

was shown to be a heterodimer of 260 kilodaltons (kD) made up of desmoglein-1 (Dsgl) (165 kD) and plakoglobin (85 kD) [8]. PV sera immunoprecipitated a 210-kD complex consisting of a 130-kD protein (PV antigen, recently named desmoglein 3 [Dsg3] [9]) and plakoglobin [8]. In these studies the PV complex was exclusively immunoprecipitated by PV sera, whereas the PF complex was recognized fjy all PF sera and by approximately 60% of PV sera tested. Immunoadsorption studies of either PV or PF sera with these antigenic fractions have not been performed due to tbe limited amounts of antigen present in the epidermal extracts. The cDNAs encoding human Dsg3 [10] and the human and bovine homologues of Dsgl [11-15] have been isolated and sequenced. Analysis of the deduced amino acid sequences have revealed that both Dsgl and Dsg3 belong to the cadherin family of cell adhesion molecules [10-15]. Other members of the cadberin family are known to mediate cell-cell adhesion in a Ca''~'"-dependent manner [16]. In an attempt to disclose the antigenic sites recognized by FS and PV sera on Dsgl and Dsg3, recombinant proteins containing the ectodomain segments of these proteins have been studied by immunoblot analysis [17,18]. Further, PV autoantibodies against the N-terminal domain of Dsg3 have been affinity purified and tested for pathogenicity using the passive transfer neonatal mouse model [18]. The purified PV autoantibodies produced limited microscopic suprabasilar acantholysis but no visible epidermal blisters in the injected animals [18]. Our laboratory has taken an independent approach to the characterization of epitopes that may be relevant in the pathogenesis of PF, FS, and PV. It was previously reported that a major pool of immunoreactive PF antigen(s) is highly insoluble and remains bound to the "epidermal envelope fraction" during extraction procedures

Copyright © 1994 by The Society for Investigative Dermatology, Inc.

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r j 9 _ 2 1 ] , Immunoreactive fragments of 80, 62, and 45 kD were released from this fraction by proteolysis [19] and repeated sonicad o n [20,21]. These studies also showed that the 80-kD band is a glycopeptide representing the major antigenic component recognized by PF autoantibodies using immunoprecipitation procedures. T h e immunoprecipitation reaction was Ca'*"'" dependent, denaturation sensitive, and unaltered by removal of the glycan from the glycopeptide by peptide N glycosidase F [21], I n this paper we report the isolation and characterization of the 8 0 - k D PF autoantibody-reactive glycopeptide from bovine muzzle epidermis, Amino acid sequence analysis of this antigen revealed the sequence EXIKFAAAXREGEXNSKRNPIA, which matched perfectly with the N-terminal 22 amino acids of the mature form of bovine Dsgl [13-15], It is postulated that the 80-kD epidermal fragment of bovine Dsg 1, which is recognized by all PF and FS sera tested, may bear pathogenically relevant epitopes, MATERIALS AND METHODS Sources of Sera As an initial step in isolating and characterizing the 80-kD PF antigen, we wished to confirm the specificity and reproducibility of the immunoprecipitation procedure in demonstrating this antigen. Therefore, we conducted immunoprecipitation using the sera of five FS patients and the sera of five normal human donors (NHS). The sera of patients and controls were tested for IgG anti-epidermal autoantibodies by indirect IF procedures reported elsewhere [22], using cryosections of normal human skin and fluorescein isothioeyanate (FITC)-conjugated goat antihuman IgG as a secondary antibody. The sera of FS patients and NHS controls were also tested by immunoblot against sodium dodecylsulfate (SDS) extracts of human epidermis, as reported elsewhere [20,21], Extraction of Bovine Epidermal Antigens Keratome-separated epidermis from freshly removed cow snouts kindly donated hy a local meat processing company was extracted following procedures reported in previous publications [20,21], Briefly, the epidermis was homogenized twice in 10 mM tris-buffered saline, pH 7,6, containing 5 mM CaCl2 (TBS/ Ca"^) and 1% Nonidet P-40 (NP-40) at 4°C in the presence ofthe following protease inhibitors: 10//g/ml pepstatin, antipain, chymostatin, leupeptin, a n d 1 mM phenylmethylsulfonyl fluoride (PMSF), The insoluble pellet was then extracted with a cold solution of 1,5 M KCl containing 5 tnM CaCl2 and the above-mentioned protease inhibitors. The insoluble pellet was resuspended in a solution containing 0.04 mg/ml of trypsin (Sigma, St. Louis, MO; from bovine pancreas type III) in TBS/Ca''^ and digested for 3 h at r o o m temperature (RT) under continuous stirring. Digestion was stopped hy t h e addition of 2 mM PMSF. The digest was centrifuged (15,000 X g for 15 min) and the trypsin-resistant pellet, also called "envelope fraction," was resuspended in cold TES/Ca"*"*", The mixture was again treated with 1 mM

PEMPHIGUS FOLIACEUS ANTIGEN

883

PMSF and the protease inhibitors listed above. This insoluble epidermal fraction resistant to trypsin was sonicated three times at 4°C (30 sec, pulsed at 25% efficiency and setting 4 [Sonicator W-385, Heat SystemsUltrasonics Inc., Farmingdale, NY]), The released soluble antigens were separated from the insoluble fraction by centrifugation at 15,000 X g during 15 min and treated with protease inhibitors. The potency ofthe antigenic activity in these fractions was titrated by immunoadsorption (blocking the indirect IF staining given by a dilution of a known FS serum) as previously described [19-21]; samples were aliquoted and stored frozen at — 70 ° C prior to use. Radiolabeling of Bovine Epidermal Antigens and Immunoprecipitation Techniques Bovine epidermal extracts were labeled with Na'^^I following the chloramine T method as previously described [20,21], Approximately 1 X 10' cpm of radiolabeled bovine epidermal extract were mixed with the patient's or control sera in immunoprecipitation buffer: 0.1 M tris-buffered saline, pH 7.6, containing 5 mM CaClj, 1% bovine serum albumin (BSA), and 1% Triton X-100. After a 1-h incubation at RT, 5 mg of fixed S. aureus cells (Sigma) in immunoprecipitation buffer were added and incubated for another hour. The cells were pelleted by centrifugation at 16,000 X g and washed with immunoprecipitation buffer containing no BSA. The immune complexes bound to 5. aureus were extracted with 0.2 M glycine/HCl pH 2.8, containing 5 mM ethylenediamine tetraacetic acid (EDTA). SDS was added to the eluted samples to bring the final concentration to 0.1% [20,21]. The pellet was redissolved on 0.1% SDS in TBS buffer without Ca"*^ and analyzed by one-dimensional 10% S D S polyacrylamide gel eiectrophoresis (PAGE) [23], The gel slabs were stained with 0.2% Coomassie blue and dried before exposure of x-ray film XAR-5 (Kodak). The molecular weight of radiolabeled bands was determined by comparison with protein standards (BioRad). Purification of PF Antigen by Affinity Chromatography In preparation for affinity chromatography, IgG from the serum of one ofthe abovementioned FS patient was bound to protein A immobilized on an agarose matrix (Sigma), following established procedures [24], Briefly, 64 ml of a 1:4 dilution of this well-characterized FS serum were incubated with 5 ml of protein A-agarose in 50 mM sodium borate buffer, pH 8.2, during 1 h at RT. The initial incubation step was followed by a wash with 20 vol ofthe same buffer. The gel/protein complex was then washed in 1 vol of 0.2 M triethanolamine, pH 8.2, suspended in 4 vol of dimethyl pimelidate (6.6 mg/ml) in triethanolamine buffer and incubated for another hour at RT. Finally, the gel was washed extensively and the remaining active sites were blocked with 0,1 M ethanolamine, pH 8.2. Before use, the gel was washed with 1 M NaCI followed by 0.2 M glycine/HCl pH 2.8, and suspended in TBS/Ca'*"^ buffer. Leakage of bound IgG was tested by extracting the crosslinked protein/gel with 1% SDS at 100° C and by analyzing the released products by SDS-PAGE. The optimal binding capacity of the immunoadsorbent gel was determined by adsorbing PF antigen(s) from freshly prepared batches of bovine epidermal extracts (known to have antigenic activity by immunoadsorption and immunoprecipitation). The bound bovine PF antigen(s) was eluted with 0,2 M glycine, 5 mM EDTA/HCl, pH 2,8, To increase the concentration of the proteins, the eluted fraction was lyophilized and then dissolved in a small volume of 0,1% SDS in TBS, '"Mabeled 80-kD PF antigen (approximately 1000 cpm) was added to this sample as a tracer prior to analysis by onedimensional 10% SDS-PAGE in the presence of reducing agents. The electrophoresed proteins were electrotransferred overnight to polyvinylidene difluoride (PVDF) membranes (Millipore) using 0.25 M ethylenemorpholine (Sigma) in 10% methanol/formic acid, pH 8,3, as the transfer medium. The membranes were stained with 0.1% amido black and exposed to x-ray film for autoradiographic analysis. Amino Acid Sequence Analysis of Bovine PF Antigen Amino acid sequence analysis was carried out at the Protein/Nucleic Acid Shared Facility at the Medical College of Wisconsin. The electroblotted 80-kD protein (stained by amido black and shown to co-localize with the radiolabeled hand) was excised and subjected to sequence analysis on a Beckman/Porton protein sequencer, model LF 3000 G. The sequencer used standard Edman degradation chemistry [25]. Phenylthiohydantoin-amino acids were detected on an integrated analyzer by high-pressure liquid chromatography using Beckman System Gold software (Beckman, FuUerton, CA),

Figure 1. Immunoadsorption of the indirect IF staining produced by FS autoantibodies on human epidermis by an extract of bovine epidermal "envelope fraction," A) Typical staining ofthe epidermis produced by FS autoantibodies present in a 1:80 dilution of a positive FS serum (indirect IF titer = 1:1280), B) The same FS serum previously immunoabsorbed with hovine PF antigen(s). Bar, 25 microns

RESULTS

Extraction of PF Antigen from Bovine Muzzle Epidermis The PF antigen(s) contained in the insoluble trypsin-resistant pellet of bovine epidermis was solubilized by repeated sonication. As shown in Fig 1, pre-incubation of an FS serum with the extract completely blocked the indirect IF staining produced by FS autoan-

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OLAGUE-ALCALA ET AL

97 •

97

66 • 43

66

43

10

Figure 2. Immunoprecipitates of "'I-labeled bovine PF antigen(s) produced by FS sera (lanes 1-5) and NHS (lanes 6-10), analyzed by SDS 10%-PAGE and autoradiography. The positions of the MW markers of 97, 66, and 43 kD are shown by diamonds and the three bands precipitated by FS sera are indicated by arrows. The 80-kD PF antigen is prominent and recognized hy all FS sera and none of the NHS. The 62- and 45-kD immunoreactive bands exhibit variable levels of intensity.

31

tibodies on human epidermis. A volume of the bovine epidermal extract containing approximately 200 /ig of total protein was capable of abolishing the epidermal staining produced by 1 ml of a 1:80 dilution of an FS serum that had an indirect IF titer of 1:1280. As seen in Fig 2, all five FS sera immunoprecipitated three specific bands of 80, 62, and 45 kD from >"I-labeled bovine epidermal extracts (lanes 1-5), whereas the five NHS showed negative results (lanes 6-10). The 80-kD band was more intense than the 62-kD and 45-kD bands. An FS serum that reacted with the 80-, 62-, and 45-kD antigens by immunoprecipitation but that did not react with Dsgl by immunoblotting was used throughout this investigation for analytical and preparative purposes.

Figure 3. Analysis of the affinity-purified 80-kD pemphigus foliaceus (PF) antigen. The PF antigen eluted from the FS IgG/protein A/agarose gel was mixed with '^^I-labeled 80-kD PF antigen, added as a tracer, fractionated by SDS 10%-PAGE, and electrotransferred onto a PVDF membrane. Auto-radiogram (A) of the amido black-stained blot (B). The positions of the MW standards are shown to the right of the panel. The affinity-purified PF antigen stained with amido black co-migrated precisely with the radiolabeled 80-kD antigen. This PF antigen was excised and characterized by amino acid sequence analysis.

Purification of the 80-kD Bovine PF Antigen by Affinity

DISCUSSION

Chromatography A 5-ml volume of FS IgG-protein A-agarose gel (approximately 32 mg of FS IgG per ml of gel) was sufficient to remove all the antigenic activity from a 50-ml volume of bovine epidermal extract (approximately 200 mg of total protein), obtained from 40 g of keratomed muzzle epidermis. The unbound epidermal fraction no longer blocked reactivity of FS sera with human epidermis when assayed by indirect IF. The unconcentrated immunoreactive peptides that were eluted from the immunoadsorbent matrix showed no absorbance at 280 nm. However, several amido black-stained proteins were detected when concentrated samples were fractionated by SDS-PAGE and electrotransferred onto PVDF membranes. The most prominent of these eluted bands were polypeptides of 80, 59, and 25 kD. Amido black staining and autoradiographic analysis of the blots demonstrated that the position of the major 80-kD polypeptide aligned precisely with the '^^I-labeled 80-kD PF-reactive antigen, which was added as a tracer. These results are clearly shown in Fig 3. Protein bands that were stained by amido black without a corresponding radiolabeled band may represent albumin, and heavy and light chains of IgG that had leaked from the immunoadsorbent gel.

Amino Acid Sequence of the 80-kD Bovine PF Antigen Matches Perfectly with the N-Terminus of Bovine Dsgl The results of the amino acid sequence analysis performed on the electroblotted 80-kD PF antigen are presented in Table I. This polypeptide sequenced at 5.2 pmol with an average repetitive yield of 86.1%. Analysis of the excised 80-kD band revealed no other contaminant sequences. The sequence of the PF antigen of 80 kD, which included 19 unambiguous residues and three indeterminate residues, matched perfectly with the sequence of the Nterminus of the mature form of Dsgl [13]. The alignment of these two amino acid sequences is also presented in Table I.

Calvanico et al [20], using human epidermis, and Olague-Alcala et al [21], using bovine muzzle epidermis, found that the PF antigen(s) bound to the trypsin-resistant epidermal envelope fraction could he released by repeated sonication. Three antigenic fragments of 80, 62, and 45 kD were recognized by all FS sera tested by immunoprecipitation using buffers containing Ca"*^. It was also noted in these studies that immunoprecipitation procedures carried out with freshly prepared '^^I-labeled epidermal fractions yielded a prominent 80-kD band and few or no 62-kD and 45-kD bands. Moreover, trypsin-treated '^^I-labeled epidermal extracts [21] or extracts that had been stored at 4 ° C (without protease inhibitors) for several days yielded a faint 80-kD band with more intense 62- and 45-kD bands. These findings suggested that the 80-kD peptide represented the major PF antigen present in these epidermal extracts. The 62- and 45-kD bands were thought to be degradation products of the 80-kD fragment. In the present investigation we have further characterized the 80-kD PF antigen from bovine epidermal extracts. This antigenic polypeptide was purified by immunoaffinity chromatography using the IgG fraction from an FS patient serum known to possess high titers of PF autoantibodies. The eluted 80-kD antigen was mixed with a trace amount of *^^I-labeled 80-kD protein, fractionated by SDS-PAGE, and electrotransferred onto a PVDF membrane. Unlabeled and radiolabeled 80-kD PF antigen co-migrated as a single band by amido black staining and autoradiography. Most significantly, the amino acid sequence of the 80-kD band exhibited identity with the N-terminal 22 amino acids of bovine Dsgl as reported by Koch e( a/ [13]. The sequence analysis of this band revealed no contaminants. Dsgl is a desmosomal glycoprotein that exhibits a surfaceexposed domain embedded in the desmosomal core, a transmembrane stretch, and an intracellular segment that is probably relevant

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Table I.

Comparison of the Amino Acid Sequence of the 80-kD PF Antigen and Bovine Dsg 1 9

80-kD band pmol-bkg Dsg I

a E 5.20 E W

885

l K F A A A f l 2.63 2.09 3.03 2.59 1.45 0.50 I K F A A A C

10

11

12

13

R E G E f 1.74 1.31 0.65 0.63 R E G E D

14 l

15

16

17

18

19

20

21

22

N S K R N P I A 0.58 0.18 0.13 0.48 0.40 0.10 0.067 0.087 N S K R N P I A

' A m i n o acid residues at positions 2, 9, and 14 could not be identified.

in membrane/cytoskeleton interactions [12,26]. The extracellular domain of Dsgl, like that of other members ofthe cadherin family of cell adhesion molecules, is predicted to be involved in homophilic interactions with Dsgl molecules of neighboring cells, thus mediating, in the presence of Ca"*^ ions, cell-cell adhesion [26-29]. T h e epitopes that are bound by pathogenic FS and PV autoantibodies on the ectodomain of Dsgl and Dsg3 remain undisclosed. It is clear, however, that keratinocyte detachment, which is a feature of b o t h PF and PV, may be precipitated by simple binding of PF and pV autoantibodies to these extracellular epitopes. This, in turn, could impair the adhesive functions of these cell adhesion molecules. This hypothesis was suggested by early in vitro studies [30] and by passive transfer of Fab' fragments from FS IgG into neonatal BALB/c mice, which induces intra-epidermal blisters due to keratinocyte detachment in these animals [7]. In summary, the 80-kD PF antigen represents a fragment of the extracellular domain of Dsgl that is bound by every PF sera tested by immunoprecipitation in our laboratory, and by approximately 50% of the PV sera tested by similar methods. Consequently, the epitopes located on the 80-kD PF antigen may be distinctively recognized by certain autoantibodies present in the sera of these patients or commonly recognized by other populations of crossreactive autoantibodies. It is anticipated that future studies aimed at characterizing relevant pathogenic epitopes on the Dsgl ectodomain will be facilitated by the availability of the bovine epidermal 80-kD PF antigen. This work was supported in part by U.S. Public Health Service Grants RO1AR32S99, R37-AR3208i (LAD), R01-HD24434 (LAD, sub-contractor), and R29 AR40410 (CJG)from the National Institutes of Health, anda VAMerit Grant (LAD) awarded by the Veterans Administration Cental Ojffice. We gratefully acknowledge the assistance of Drs. N. Dahms and S. Twining (Biochemistry Department, Medical College of Wisconsin) in matters related with the carbohydrate analysis of the pemphigus foliaceus antigen and the support ofDr. L. Mende-Mueller (Protein and Nucleic Acid Facility, Cancer Center ofMCW) in the amino acid sequence determination of the antigen. Dr. M. Steinberg, Princeton University, provided expert advice in the area of cell adhesion molecules. Superb technical assistance was provided by A.F. Taylor, P.M. Wilson, and S.f. Swartz.

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Anbalt GJ: Brazilian pempbigus foliaceus autoantibodies are pathogenic to BALB/c mice by passive asLnsfei.J Invest Dermatol 85:538-541, 1985 Rock B, Labib RS, Diaz LA: Monovalent Fab' immunoglobulin fragments from endemic pemphigus foliaceus autoantibodies reproduce tbe human disease in neonatal BALB/c mice.J Clin Invest 85:296-299, 1990 Korman NJ, Eyre RW, Klaus-Kovtun V, Stanley JR: Demonstration of an adhering-junction molecule (piakoglobin) in the autoantigens of pemphigus foliaceus and pemphigus vulgaris. N Engl J Med 321:631-635, 1989 Buxton RS, Cowin P, Franke W W , Garrod DR, Green KJ, King IA, Koch PJ, Magee AI, Rees DA, Stanley JR, Steinberg MS: Nomenclature ofthe desmosomal cadherins.J Cell Biol 121:481 - 4 8 3 . 1993 10. Amagai M, Klaus-Kovtun V. Stanley JR: Autoantibodies against a novel epithelial cadherin in pemphigus vulgaris, a disease of cell adhesion. Cell 67:869 - 877, 1991 11. Wheeler GN, Parker AE, Thomas CL, Ataliotis P, Poynter D. Amemann J, Rutman AJ, Pidsley SC. Watt FM, Rees DA. Buxton RS, Magee AI: Desmosomal glycoprotein DGI. a component of intercellular desmosome junctions, is related to the cadberin family of cell adhesion molecules. Proc Natl Acad Sci USA 88:4796-4800, 1991 12. Nilles LA, Parry DAD, Powers EE, Angst BD, Wagner RM, Green KG: Structural analysis and expression of buman desmoglein: a cadherin-like component of the desmosome.yCc//Sri 99:809-821. 1991 13. Koch PJ, Walsh MJ, Schmelz M, Goldscbmidt MD, Zimbelmann R, Franke W W : Identification of desmoglein, a constitutive desmosomal glycoprotein. as a member of the cadherin family of cell adhesion molecules. EurJ Cell Biol 53:1-12,1990 14. Goodwin L, Hill JE, Raynor K, R.iSZi L, Manabe M, Cowin P: Desmoglein shows extensive homology to the cadberin family of cell adhesion molecules. Biochem Biophys Res Commun 173:1224-1230. 1990 15. Koch PJ. Goldschmidt MD, Walsh MJ, Zimbelmann R, Franke W W : Complete amino acid sequence of the epidermal desmoglein precursor polypeptide and identification ofa second type of desmoglein gene. EurJ Cell Biol 55:200 - 208. 16. Geiger B, Ayalon O: Cadherins. Annu Ra/ Cell Biol 8:307-332, 1992 17. Allen EM, Giudice GJ, Diaz LA: Subclass reactivity of pemphigus foliaceus autoantibodies witb recombinant buman desmoglein. / Invest Dermatol 100:685691, 1993 18. Amagai M, Karpati S, Prussick R, Klaus-Kovtun V, Stanley JR: Autoantibodies against the amino-terminal cadherin-like binding domain of pemphigus vulgaris antigen are pathogenic./ Clin Invest 90:919-926, 1992 19. Labib RS, Camargo S, Futamura S, Martins CR, Rock B, Anhalt GJ, Diaz LA: Pemphigus foliaceus antigen: characterization ofa keratinocyte envelope associated pool and preparation of a soluble immunoreactive fragment. J Invest Dermatol 93:272-279, 1989 20. Calvanico NJ, Martins CR, Diaz LA: Characterization of pemphigus foliaceus antigen from buman epidermis./Jiimr Dermatol 96:815-821, 1991 21. Olague-Alcala M, Diaz LA: The epitopes on bovine pemphigus foliaceus antigen are calcium-dependent and located on the peptide backbone of this glycoprotein. Chron Dermatol 2:189-209. 1993 22. Matis WL, Anhalt GJ, Diaz LA, Rivitti EA, Martins CR, Berger RS: Calcium enhances tbe sensitivity of immunofluorescence for pemphigus antibodies. J Invest Dermatol 89:302-304, 1987 23. Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685, 1970 24. Hermanson GT, Mallia AK, Smith PK: Immobilized Affinity Ligand Techniques. Academic Press, Inc. San Diego, CA: Harcourt Brace Jovanovich, 1992, pp 224-226 25. Walker JM: The Dansyl-Edman Method for Peptide Sequencing. Methods in Molecular Biology 1: Proteins. The Humana Press Inc., Clifton, IL, 1984, pp 213-219 26. Schmelz M, Duden R, Cowin P, Franke W W : A constitutive transmembrane glyeoprotein of Mr 165000 (desmoglein) in epidermal and non-epidermal desmosomes. II. Immunolocalization and microinjection studies. EurJ Cell Biol 42:184-199, 1986 27. Koch PJ, Goldschmidt MD, Zimbelmann R. Troyanovsky R, Franke W W : Complexity and expression patterns of the desmosomal cadherins. Proc Natl Acad Sci USA 89:353-357. 1992 28. Buxton RS, Magee AI: Structure and interactions of desmosomal and other cadherins. Cell Biol 3:157-167, 1992 29. Garrod DR: Cell to cell and cell to matrix adhesion. BrMeii7306:703-705,1993 30. Diaz LA, Marcelo CL: Pemphigus and pemphigoid antigens in cultured epidermal cells. Br/Dermaw/98:631-637, 1978