Differential structural properties and expression patterns suggest functional significance for multiple mouse desmoglein 1 isoforms

Differentiation (2004) 72:434–449

r International Society of Differentiation 2004

OR IGI N A L A R T IC L E

Donna Brennan . Ying Hu . Ana Kljuic . YooWon Choi . Sohaila Joubeh . Marisa Bashkin . James Wahl . Andrzej Fertala . Leena Pulkkinen . Jouni Uitto . Angela M. Christiano . Andrey Panteleyev . My˜ G. Mahoney

Differential structural properties and expression patterns suggest functional significance for multiple mouse desmoglein 1 isoforms

Received August 28, 2004; accepted in revised form September 25, 2004

Abstract The four isoforms of desmosomal cadherin desmogleins (Dsg1–4) are expressed in epithelial tissues in a differentiation-specific manner. Extensive sequencing of the human genome has revealed only one copy of the Dsg1 gene. However, we recently cloned two novel additional mouse Dsg1 genes, Dsg1-b and -g, which flank the original Dsg1-a on chromosome 18. Sequence conservation between the Dsg1 isoforms diverged significantly at exon 11, particularly in the region that encodes for the extracellular anchoring (EA) domains. Computational analysis revealed very low hydrophilic potential of the Dsg1-g EA compared with the corresponding sequences of Dsg1-a and -b, suggesting that the Dsg1-g EA domain may have a stronger affinity Donna Brennan
Ying Hu
YooWon Choi
Sohaila Joubeh
Marisa Bashkin
Andrzej Fertala
Leena Pulkkinen
. ) Jouni Uitto
My˜ G. Mahoney (* Department of Dermatology and Cutaneous Biology Thomas Jefferson University Philadelphia, PA, USA Tel: (215) 503-3240 Fax: (215) 503-5788 E-mail: my.mahoney@jefferson.edu Ana Kljuic
Angela M. Christiano
Andrey Panteleyev Department of Dermatology Columbia University New York, NY, USA James Wahl Department of Oral Biology University of Nebraska Omaha, NE, USA Angela M. Christiano Department of Genetics and Development Columbia University New York, NY, USA U.S. Copyright Clearance Center Code Statement:

to the cell membrane. We generated antibodies using synthetic peptides or recombinant proteins localized within the EA domains. These antibodies were tested for their specificity and were then used to demonstrate expression of Dsg1 isoforms in various tissues. In the epidermis, all Dsg1 isoforms were differentially expressed in the differentiating cell layers. In the hair follicle, all Dsg1 isoforms were present throughout the entire process of its development and cycling but the expression of Dsg1 isoforms is subject to significant hair cycle-dependent changes. Dsg1-b and -g, but not Dsg1-a, were detected in the sebaceous gland epithelium and the stratified epithelium of the stomach. Finally, Dsg1-a and Dsg1-b, but not Dsg1-g, are proteolytically cleaved by exfoliative toxin A. These results suggest that the developmental complexity of mouse tissues, including skin and hair, may play a significant role in the evolutionary driving force to maintain multiple Dsg1 genes in mouse. Key words cadherins
cell adhesion
desmoglein
desmosome
differentiation
hair follicle

Introduction Organization of cells into a complex three-dimensional structure that can withstand mechanical stress, such as the epidermis, requires dynamic assembly and disassembly of cell–cell contacts that are highly regulated by growth factor-mediated cell signaling pathways (for a review, see Jamora and Fuchs, 2002). Desmosomes are specialized epithelial cell junctions composed of electron dense disc-like structures and clear cytoplasmic plaques to which keratin intermediate filaments associate, as displayed by transmission electron microscopy. The integral transmembrane components of the desmosomes

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are the cadherins, i.e., desmogleins and desmocollins, which form cell–cell contacts through heterotypic interactions of the extracellular domains in a calcium-dependent manner (for review, see Cheng and Koch, 2004; Garrod et al., 2002a). The intracellular domains of desmogleins and desmocollins recruit and interact either directly or indirectly with several cytoplasmic plaque proteins, including plakoglobin, plakophilin, and desmoplakin. Desmoplakin serves the role of the linker that connects the desmosomal complex to the intermediate cytokeratin filaments providing a stable, yet dynamic, three-dimensional scaffolding network for cell– cell adhesion and structural integrity of cells and tissues. The desmosomal cadherins comprise of four distinct desmogleins (Dsg1, 2, 3, and 4) and three desmocollins (Dsc1, 2, and 3), which are differentially expressed reflecting the tissue-type specificity of the epithelial cells (Green and Gaudry, 2000; Garrod et al., 2002b). Dsg2 and Dsc2 are expressed in all desmosome-containing tissues, including simple epithelia (e.g., colon, intestine, and bladder), and in select non-epithelial tissues (e.g., myocardium). Similar to Dsg2, a wide tissue distribution of DSG4 mRNA has been detected in humans. Conversely, the expression of Dsc1, Dsg1, Dsc3, and Dsg3 is more restricted to complex stratified epithelia. In the epidermis, Dsg2 is expressed at extremely low, if not undetectable, levels in the basal cell layer (Koch et al., 1992; Scha¨fer et al., 1994). Dsg3 is expressed mainly in the basal and most immediate suprabasal cell layers, while Dsc1, Dsc3, Dsg1, and Dsg4 are expressed throughout all vital cell layers. Nevertheless, Dsc1 and Dsg1 are found in significantly higher abundance in the differentiated cell layers. The role of desmosomal cadherins, particularly the desmogleins, in maintaining cell–cell adhesion and tissue stability has been supported by observations that loss of cadherin function would result in loss of cell–cell adhesion and subsequently lead to skin fragility. In particular, ablation of the Dsg3 gene in mice results in fragility of the skin and oral mucous membranes (Koch et al., 1997; Pulkkinen et al., 2002). In addition to skin fragility, ablation of the Dsc1 gene in mice results in compromised epidermal barrier defect, acantholysis in the granular layer, and abnormal epithelial cell differentiation (Chidgey et al., 2001). In humans, the mutation in the DSG1 gene results in the dominantly inherited disorder, striate palmoplantar keratoderma, typified by hyperkeratosis in the palms and soles (Rickman et al., 1999; Hunt et al., 2001; Kljuic et al., 2003b). In addition, disturbing the normal expression pattern of a desmosomal protein can disrupt formation of normal functioning epidermis, as demonstrated in transgenic mice overexpressing Dsg3 in the superficial epidermis where it is normally not expressed (Elias et al., 2001). This overexpression results in disruption of epidermal barrier function by altering the permeability of the stratum corneum (Elias et al., 2001; Merritt et al., 2002). Furthermore, we recently cloned a

novel member of the desmoglein gene family, Dsg4, and demonstrated that mutations in the human DSG4 gene result in recessively inherited hypotrichosis (Kljuic et al., 2003a). A similar hair phenotype was observed in lanceolate mouse having a mutation in the Dsg4 gene. Disrupting the function of desmosomal cadherins, in particular Dsg1 and/or Dsg3, by pathogenic antibodies results in cell–cell disadhesion and causes a blistering of skin and mucous membranes, resulting in a group of disorders collectively known as pemphigus (for reviews, see Stanley, 2000; Amagai, 2003). In pemphigus foliaceus (PF), pathogenic autoantibodies against Dsg1 disrupt desmosome function and promote cell– cell disadhesion in the superficial epidermis (Mahoney et al., 1999). Passive transfers of purified PF immunoglobulins (Igs) into newborn mice produce blisters similar to those observed in PF patients (Anhalt et al., 1982). In staphylococcal scalded skin syndrome (SSSS), proteolytic cleavage of Dsg1 (but not of Dsg3) by exfoliative toxins (ETs), serine proteases produced by Staphylococcus aureus, results in skin blisters in the superficial epidermis (Amagai et al., 2000; Amagai et al., 2002; Hanakawa et al., 2002). Newborn mice treated with ETs develop skin blisters similar to those observed with passive transfer of PF Igs (Amagai et al., 2000; Amagai et al., 2002). These results attest to the critical role of desmosomal cadherins in cell adhesion, structural stability, and formation of a functional tissue. Recent cloning of three novel desmoglein genes has added significant diversity to the desmosomal cadherin family. First, we cloned and characterized the biological function of human and mouse Dsg4 (Kljuic et al., 2003a). Although extensive searches of the gene bank (BLAST and BLAT programs) resulted in no additional orthologous human DSG1 genes, we and others recently identified two mouse desmoglein genes, Dsg1-b and Dsg1-g, that share high homology to Dsg1, now designated as Dsg1-a (Pulkkinen et al., 2003). To date, the expression profiles of these Dsg1 isoforms have been based solely on RT-PCR experiments. This prompted us to generate antibodies specific against each Dsg1 isoform. In this report, we examined the expression pattern of Dsg1-a, -b, and -g in various complex stratified epithelia. We demonstrated that the Dsg1 isoforms possess different biophysical properties, are overexpressed in calcium treated keratinocytes, and are differentially expressed in various mouse epithelial tissues. Collectively, these results provide insights into the significance of having multiple Dsg1 genes during cell differentiation and tissue development.

Materials and methods Antibodies Anti-Dsg1-a antibodies 3113 and 3123 (1:1000, raised against a glutathione S-transferase (GST)-fusion protein with the Dsg1-a

436 extracellular anchoring (EA) domain) were kind gifts from Dr. John Stanley (University of Pennsylvania, Philadelphia, PA). The antibody DG3.10 (1:100; Research Diagnostics, Flanders, NJ) was raised against bovine muzzle desmosome and recognizes Dsg1 and Dsg2 (Koch et al., 1990). Anti-Dsg1 monoclonal antibody 18D4 was generated against the cytoplasmic domain (amino acids 664763) of human Dsg1 (Wahl, 2002). Anti-FLAG antibodies (1:200) were from Sigma (St. Louis, MO) or Stratagene (La Jolla, CA). FITC- or Texas Red-conjugated secondary antibodies (1:200) were from Molecular Probes (Eugene, OR), and horse-radish peroxidase (HRP)-conjugated secondary antibodies (1:5000) were from Jackson Labs (West Grove, PA). To raise antibodies against Dsg1-a and -g, we synthesized the peptides ‘‘N’’-FQGDPDETLETPLYG-‘‘C’’ and N’-TSTEKPVTLSITPNV-‘‘C’’ corresponding specifically to the EA domains of Dsg1-a and -g, respectively (Genemed Synthesis, Los Angeles, CA). The peptides were conjugated to keyhole limpet hemocyanin and used to immunize rabbits or chicken (Genemed Synthesis and CoCalico Biologicals, Reamstown, PA). We raised antibodies against Dsg1-b in rabbits using Dsg1-b-EA-GST protein as described below (CoCalico Biologicals). For antibody affinity purification, we crosslinked 1 mg of GST, Dsg1-a-EA-GST, Dsg1-b-EA-GST, or Dsg1-g-EA-GST proteins to 5 ml of Affigel-10 (Bio-Rad Labs, Hercules, CA) in 10 mM Mops (pH 7.5) and 1 mM CaCl2. Crude antisera were passed through GST columns to remove non-specific antibodies. The flow-through material was added to the Dsg1-a-EA-GST, Dsg1-b-EA-GST, or Dsg1-g-EA-GST columns and incubated for 2 hr to room temperature. Columns were then washed with PBS, 20 times column bed volume, and antibodies were eluted using IG elution buffer (Pierce, Rockford, IL).

denaturing solution containing 8 M urea, 20 mM Tris, pH 8.0, and 0.5 M NaCl. The GST fusion proteins were clarified by centrifugation at 15,000 rpm for 20 min at 41C and bound to glutathione Sepharose beads and eluted with glutathione elution buffer according to the manufacturer’s protocol (Amersham). The eluted proteins were dialyzed against PBS and concentration was determined by the Pierce protein assay. We also generated GST fusion proteins of the intracellular domain (ID) of Dsg1-a, -b, and -g. The primers used to generate these products were: Dsg1-a, forward 5 0 -GCC GAA TTC TGT GAT TGT GGA GGG GC-3 0 , and reverse, 5 0 -GCC GCG GCC GCC TTG CTA TAC TGT AC-3 0 ; Dsg1-b, forward, 5 0 -GCC GAA TTC TGT GAT TGT GGA GGG GC-3 0 and reverse, 5 0 -GCC GCG GCC GCC TTG CTA TAC TGT AC-3 0 ; Dsg1-g, forward, 5 0 -GCC GAA TTC TGT GAT TGT GGA GGG GC-3 0 and reverse, 5 0 GCC GCG GCC GCG GGG GGG ATG CTC AG-3 0 . PCR products were digested with EcoRI and NotI and ligated into pGEX-4 T-1 vector. GST fusion proteins were produced as described above.

Subcloning of the full-length expression construct for Dsg1-b

Immunohistochemistry and Western blotting analysis

We obtained Dsg1-a-FLAG in pcDNA3 as a kind gift from Dr. John Stanley. For Dsg1-b, we designed the following nucleotide primers: forward, 5 0 -GCA AGC TTG CAG CGA TGG ACT GGC ACT CC-3 0 ; and reverse, 5 0 -GCG GAT CCC TAC TTG TCA TCG TCG TCC TTG TAG TCC TTG CTA TAC TG-3 0 , to amplify both Dsg1-a and Dsg1-b cDNAs by PCR using total mouse keratinocyte cDNA as a template. The FLAG sequence (underlined above) encoding for the amino acids Asp–Tyr–Lys–Asp–Asp–Asp– Asp–Lys (DYKDDDDK) was added to the 3 0 nd of the Dsg1-a and Dsg1-b cDNAs. The PCR product was digested with HindIII and BamHI, and because Dsg1-a but not Dsg1-b, contains an internal BamHI restriction site, we were able to distinguish Dsg1-b from Dsg1-a after BamHI digestion. The full-length Dsg1-b cDNA was ligated into pCDNA3 at the BamHI and HindIII sites, which were also present in the primers (see above). All DNA products were analyzed by automated nucleotide sequencing (ABI, Piscataway, NJ) with specific primers and the Prism Ready Reaction DiDeoxy Terminator cycle sequencing system.

Tissues were fixed in 10% formalin (Sigma) and embedded in paraffin prior to sectioning (5 mm) as previously described in detail (Mahoney et al., 1999). Briefly, tissues were deparaffinized in xylene and gradually decreasing concentration of ethanol in water. Antigens were retrieved in antigen-retrieving medium (Signet, Dedham, MA) by the microwave method and digestion with trypsin (Sigma) or pepsin (Invitrogen, Carlsbad, CA). Primary and secondary antibodies were suspended in blocking buffer consisting of 1% BSA 0.1% Tx-100 in PBS. Nuclei were stained with DAPI (Sigma) prior to mounting for viewing. For immunostaining of transfected HaCaT or Cos-7 cells, the cells were fixed in methanol for 5 min at  201C and permeabilized in 50:50 methanol/acetone for 2 min at  201C. Nonspecific sites blocked for 30 min in blocking buffer (TBS10.1% Triton X-10011% BSA) and primary and secondary antibodies were incubated in the same buffer. Again, nuclei were stained with DAPI and cells were mounted for analysis under fluorescence microscope.

Cell culture HaCaT cells (from Dr. Norberto Fusenig, DKFZ, Heidelberg, Germany) were grown in W489 medium consisting of four parts of MCDB153 (Sigma) and one part L15 (Sigma) media supplemented with 2% fetal bovine serum (Mahoney et al., 2002b). Cos-7 cells were obtained from ATCC (Rockville, MD) and maintained in DMEM supplemented with 10% FBS. Primary mouse keratinocytes were cultured from newborn C57Bl/6 mouse skin and maintained in CnT-02 medium (CELLnTEC, Berne, Switzerland) as previously described (Caldelari et al., 2000).

ETA cleavage of Dsg1-a, -b, and -g GST-fusion proteins GST-fusion proteins were obtained by in-frame cloning the EA domains of Dsg1-a, -b, and -g cDNAs into pGEX-4 T-1 (Amersham, Piscataway, NJ). The primers were: For Dsg1-a and -b, forward 5 0 -AGG GAA TTC CAA GGC ACT TCT-3 0 , and reverse 5 0 AGG GTC GAC TCA GTG AAC GTT GTC-3 0 ; for Dsg1-g, forward 5 0 -GCG AAT TCT TTA CCT TTT GTG TA-3 0 and reverse 5 0 -GCG TCG ACT CCG ACG TTT GAC TG-3 0 . The PCR amplified products were digested with EcoRI and SalI and inserted in frame into pGEX-4T-1. GST fusion proteins were produced in DH5a and BL21 Escherichia coli cells after induction with isopropyl-beta-D-thiogalactoside (0.5 mM). Cells were suspended in an STE buffer (10 mM Tris, pH 8.0, 150 mM NaCl, 10 mM EDTA, 1% Triton X-100, 2% sarcosyl) with phenylmethylsulfonyl fluoride (PMSF, 1 mM). Because Dsg1-g-EA-GST proteins were not soluble in STE buffer, the bacteria pellet was resuspended in a mild

Newborn C57Bl/6 mice were injected subcutaneously in the back of the neck with ETA (10 mg in 50 m PBS, Toxin Technology, Sarasota, FL) or PBS alone (Amagai et al., 2000). The next day, gross blister formation was documented and skin tissues were processed for histology or lysed in Laemmli buffer, boiled for 10 min, and subjected to SDS-PAGE (Bio-Rad Labs) for Western blotting with Dsg1specific antibodies. In addition, mouse keratinocytes were treated with 1 mM calcium for 3 days and then treated with ETA (1 mg/ml) for 1–2 hr at 371C in the presence of 1 mM calcium. Cells were then washed in PBS and prepared for Western blotting analysis. Modeling software Computer analysis was performed using a modeling program (Sybyl 6.9; Tripos, Inc., St. Louis, MO) installed on an Octane computer station (Silicon Graphics, Inc., Mountain View, CA).

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Fig. 1 Desmosomal cadherin gene locus on mouse chromosome 18 and functional domains of Dsg1 isoforms. (A) Genomic structure of the desmosomal cadherin genes, including Dsg1-a, -b, and -g. Arrows indicate the direction of transcription, and sizes of the genes and intragenic regions are indicated in kb. (B) Putative domains based on the deduced amino acid sequence of Dsg1-a, Dsg1-b, and Dsg1-g polypeptides revealing truncated EA and RUD domains and the absence of the TD domain in Dsg1-g. Values shown are numbers of amino acids found within each domain. SP, signal peptide; EC, extracellular; EA, extracellular anchoring; TM, transmembrane; IA, intracellular anchoring; ICS, intracellular cadherin-typical segment; LD, intracellular linker domain; RUD, intracellular repeated unit domain; TD, terminal domain.

Results Characterization of mouse Dsg1-a, -b, and -g We recently reported the cloning of two novel mouse desmoglein genes (Dsg1-b and -g) sharing high homology with both mouse Dsg1-a (Mahoney et al., 2002a) and human DSG1 (Pulkkinen et al., 2003). The Dsg1-b gene is 32 kb downstream of the Dsg1-a gene and is

encoded by 15 exons spanning approximately 45 kb on mouse chromosome 18 (Fig. 1A and Table 1). The Dsg1g gene is 28 kb upstream of the Dsg1-a gene and is encoded by 15 exons spanning approximately 37 kb (Fig. 1A and Table 1). The full-length mouse Dsg1-b cDNA contains an open reading frame of 3180 bp encoding a precursor protein of 1060 amino acids, while the Dsg1-g cDNA contains an open reading frame of only 2733 bp encoding a precursor protein of 911 amino acids.

Table 1 Exon/intron sizes of mouse Dsg1-a, Dsg1-b, and Dsg1-g genes #

Dsg1-a Exon (bp)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1

0

48 (15 non-coding) 36 132 156 145 167 135 186 260 140 330 137 73 209 1017 (13 0 non-coding)

Kljuic and Christiano (2003).

Dsg1-g1

Dsg1-b Intron (bp) 16,812 652 54,023 1,597 2,532 1,450 291 2,606 304 1,324 2,092 752 674 1,179

Exon (bp) 0

48 (15 non-coding) 36 132 156 145 167 135 186 266 140 339 137 73 209 1017 (13 0 non-coding)

Intron (bp) 11,160 357 1,144 1,592 2,305 1,170 291 790 304 1,574 5,128 310 13,609 996

Exon (bp) 0

48 (15 non-coding) 36 132 156 145 167 135 186 269 140 186 128 73 209 723 (13 0 non-coding)

Intron (bp) 16,812 652 1,098 1,601 2,346 1,456 291 2,067 303 1,581 2,136 311 2,124 998

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Nucleotide sequence conservation between the three Dsg1 genes diverges significantly at exon 11; 79% between Dsg1-a and Dsg1-b, 37% between Dsg1-a and Dsg1-g, and 38% between Dsg1-b and Dsg1-g. The largest difference is localized to the region that encodes for the EA domain (Fig. 1B and Table 2). Overall, Dsg1-a shares 94% identity with Dsg1-b and 83% identity with Dsg1-g at the amino acid level. However, within the EA domain the percent identity drops to only 44% between Dsg1-a and Dsg1-b, 12% between Dsg1-a and Dsg1-g, and 4% between Dsg1-b and Dsg1-g (Table 2 and Fig. 2A). The EA domain of Dsg1-g is significantly shorter (28 amino acids) compared with those of Dsg1-a (49) and Dsg1-b (52) (Fig. 2A). In addition, peptide analysis of the EA domains revealed higher hydrophobicity of the Dsg1-g EA (Figs. 2B, 2C). The pI for Dsg1-g-EA was 5.16 as compared wih 3.77 and 3.86 for Dsg1-a-EA and Dsg1-b-EA, respectively. Since structural data are not available for homology comparison to known crystalderived models, we subjected the EA sequences to computer modeling. The XX, YY, ZZ regions of the Dsg1a, the Dsg1-b, and the Dsg1-g, respectively, were analyzed, and the electron density surface, which represents an isosurface of electron densities, was calculated. The electrostatic potential (EP; expressed as kcal/mole) and lipophilicity potential (LP) of the surfaces were then evaluated (Fig. 2C). Computational analysis revealed a very low hydrophilic potential of the Dsg1-g EA compared to the EA domains of Dsg1-a and -b (Figs. 2B,

Table 2 Amino acid sequence homology of mouse Dsg1-a, Dsg1-b, and Dsg1-g within different functional domains1 Domain2

Dsg1-a: Dsg1-b

Dsg1-a: Dsg1-g

Dsg1-b: Dsg1-g

SP EC1 EC2 EC3 EC4 EA TM IA ICS LD RUD TD

100 100 100 99 95 44 100 97 100 92 94 94

100 100 99 91 65 12 96 95 93 86 82

100 100 99 91 63 4 96 93 93 82 83

1

Values shown are % of identical amino acids. For the structural arrangement and relative sizes of the protein domains, see Fig. 1B. SP, signal peptide; EC, extracellular domains 1–4; EA, extracellular anchoring; TM, transmembrane; IA, intracellular anchoring; ICS, cadherin-typical intracellular segment; LD, intracellular linker domain; RUD, intracellular repeated unit domain; TD, terminal domain.

2

2C). These results suggest that the Dsg1-g EA domain may have a stronger affinity, compared with Dsg1-a and Dsg1-b, to the cell membrane. Indeed, while generating recombinant proteins of the Dsg1 EA domains (see below), we observed that the Dsg1-g-EA protein was insoluble in non-denaturing buffers and required

Fig. 2 Differential biophysical properties of the EA domains of Dsg1 isoforms. (A) Amino acid sequence alignment of the EA domains of Dsg1a, -b, and -g. Homologous amino acids are shown in bold. (B) Molecular analyses of lipophilicity potential (LP) and electrostatic potential (EP) of the Dsg1 EA domains orientated from N-to-C terminus. Color code panels indicate differences in the EP or LP: RED, highly positive EP; BLUE, highly negative EP; BROWN, highly hydrophobic; BLUE, highly hydrophilic. (C) Numeric values for the LP and EP for the Dsg1-a, -b, and -g EA fragments.

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8 M urea to solubilize the protein. Whether Dsg1-g possesses different physical properties with respect to the plasma membrane or the desmosomal complex requires further analysis. Nevertheless, the results obtained here demonstrate structural and physicochemical differences between the Dsg1 proteins.

Production of specific antibodies against Dsg1 proteins To distinguish the expression pattern of Dsg1-a, -b, and -g at the protein level, we produced specific antibodies against each protein. Based on the amino acid sequences, we raised antibodies against the EA domains. This region shows the least homology among the desmogleins (Table 2), and previous antibodies have been successfully raised against Dsg3 and Dsg1-a using this domain (Koch et al., 1997; Wu et al., 2000). We raised antibodies against peptides specific for Dsg1-a (Ab61 and Ab62; rabbits), Dsg1-b (Ab24 and Ab25; chicken), and Dsg1-g (Ab15 and Ab16; chicken). Although the antibodies Ab24 and Ab25 were effective in immunohistochemistry, we were unable to use them in Western blotting analysis. This prompted us to raise additional antibodies against bacterially produced recombinant GST fusion proteins of the Dsg1-b-EA domain (Ab497 and Ab498; rabbits). Dsg1-b-EA-GST proteins were N-crosslinked to Affigel-10 sepharose, and Ab498 was subjected to affinity purification. The affinity-purified (AP) antibody was named AP498. We also generated Dsg1-a-EA-GST recombinant fusion proteins and used them in affinity purification of antibody Ab61 (AP61). Affinity purification of antibody Ab15 was not necessary since the crude antibody gave clean Western blots and immunostainings. To assess the specificity of these antibodies, we produced Dsg1-a-EAGST, Dsg1-b-EA-GST, and Dsg1-g-EA-GST recombinant fusion proteins and performed Western blotting analysis (Fig. 3A). Results demonstrate that AP61 recognized Dsg1-a but not Dsg1-b or Dsg1-g, antibody AP498 recognized Dsg1-b but not Dsg1-a or Dsg1-g, and Ab15 recognized Dsg1-g but not Dsg1-a or Dsg1-b (Fig. 3A). In summary, we have generated antibodies against each Dsg1 isoform and have confirmed their specificities. We next examined the expression of Dsg1 isoforms at the protein level. Newborn mouse skin was extracted in lysis buffer containing 1% Triton X-100. Triton X-100soluble and -insoluble proteins were boiled in SDS-containing Laemmli buffer. Proteins were resolved by SDSPAGE and Western blotted with anti-Dsg1 specific antibodies. All Dsg1 antibodies, including the anti-Dsg1/ Dsg2 monoclonal antibody DG3.10, detected a protein of approximately 160 kDa in size (Fig. 3B). To determine whether the commercially available antibodies DG3.10 and 18D4 recognize all Dsg1 isoforms, we generated GST fusion proteins of the intracellular

domains of Dsg1-a, Dsg1-b, and Dsg1-g. Western blotting analyses demonstrate that both antibodies DG3.10 and 18D4 recognized all Dsg1 isoforms but not GST alone (Fig. 3C; middle and right panels). The same blot was re-probed with anti-GST (Fig. 3C, left panel). Collectively, these results demonstrate that we have obtained and characterized antibodies specific against each Dsg1 isoform and antibodies that recognize all Dsg1 isoforms.

Calcium induced up-regulation of Dsg1 isoforms in cultured cells and differential expression in stratified squamous epithelia We first examined the effect of calcium-induced cellular differentiation on the expression level of Dsg1 isoforms. Primary mouse keratinocytes were isolated from newborn mouse skin and were grown in a low-calciumcontaining medium. Cells were switched to a high-calcium (1 mM) medium for 3 days and then processed for immunohistochemistry using the Dsg1 isoform-specific antibodies. In response to the increase in calcium in the medium, mouse keratinocytes overexpressed the differentiation protein involucrin (not shown). Under lowcalcium conditions, mouse keratinocytes did not express any Dsg1 isoforms (Figs. 4A, 4C, 4E). We believe that the high background staining for Dsg1-a (Fig. 4A) and Dsg1-b (Fig. 4C) with our polyclonal antibodies was nonspecific because DG3.10, which recognized all Dsg1 isoforms, did not detect any Dsg1 expression in the mouse keratinocytes grown under low-calcium conditions (Fig. 4G). In response to high calcium, Dsg1-b and Dsg1-g were up-regulated and localized to cell–cell borders (Figs. 4D, 4F). The expression level of Dsg1-a (Fig. 4B) appeared to increase, but less distinct at the cell–cell border as compared with Dsg1-b or Dsg1-g (Figs. 4D, 4F). These results demonstrate that in cultured keratinocytes, the expression of the Dsg1 isoforms was mediated by cellular differentiation. We next examined the expression patterns of Dsg1 isoforms in normal mouse epithelia during embryonic development and in adult tissues. While Dsg3 was detected only in the basal cell layer, the Dsg1 isoforms were detected in the differentiated cell layers of the epidermis of day14 embryos (Fig. 5). Interestingly, Dsg1-g expression appeared to be restricted to the upper more differentiated layers of the epidermis (Figs. 6A–6F) compared with Dsg1-a and Dsg1-b. In the newborn mouse epidermis, Dsg1-a was expressed throughout all suprabasal cell layers (Figs. 6A, 6D). In addition, some staining was also observed in the border between the basal and most immediate suprabasal cells and between some basal cells. Only the basolateral surface that interfaces the basement membrane was negative for Dsg1a. Dsg1-b (Fig. 6B) and Dsg1-g (Fig. 6E) were expressed throughout the differentiating cell layers while

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Fig. 3 Testing of antibodies specific against Dsg1-a, -b, and -g. (A) Fusion proteins were generated with GST and the extracellular anchoring (EA) domains of Dsg1-a, Dsg1-b, and Dsg1-g. Western blotting analysis was performed with AP61 (anti Dsg1a), AP498 (anti-Dsg1-b), and Ab15 (anti-Dsg1-g) antibodies to demonstrate specificity for each Dsg1 protein. Staining with antiGST antibody demonstrates equal loading. (B) Newborn mouse skin was homogenized in buffer containing 1% Triton X-100. The Triton-soluble (S) and -insoluble (I) fractions were boiled in SDS-containing Laemmli buffer and proteins prepared

for Western blotting analysis. The panel of anti-Dsg1 antibodies recognized proteins of approximately160 kDa from the Triton-X100-insoluble fraction of mouse skin extracts. Similar results were observed with DG3.10 antibody (right panel). (C) Fusion proteins were generated with GST and the intracellular domain (ID) of Dsg1-a, Dsg1-b, and Dsg1-g. By immunoblotting, the antiDsg1 antibodies DG3.10 (middle panel) and 18D4 (right panel) recognized all three Dsg1 isoforms but not the GST alone. The membranes were re-probed with anti-GST antibodies (left panel).

completely absent from the basal cells. This pattern of expression was more pronounced in the epithelia of adult oral mucosa (Fig. 6G–6L) where the expression of both Dsg1-b and Dsg1-g was noted in the spinous cell layers and increased significantly toward the highly dif-

ferentiated cell layers (Figs. 6H, 6K). Similar to the epidermis, Dsg1-a was expressed throughout all cell layers of the oral mucosa (Figs. 6G, 6J). We note here that in all tissues examined, the expression pattern for Dsg1-b obtained with the chicken antibodies Ab24 and Ab25

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Fig. 4 Dsg1-b and -g are overexpressed in calcium-induced keratinocyte differentiation. Cultured mouse keratinocytes were grown to confluency in low calcium (o100 mm) and then switched to high calcium (1 mM) for 3 days. Cells were fixed and stained with Dsg1 isoform-specific and DG3.10 antibodies. No cell–cell

border staining was observed in cells grown under low-calcium condition. In response to high calcium, expression levels of Dsg1-b and Dsg1-g, and to a lesser extent Dsg1-a, were up-regulated. Antibody DG3.10, which recognizes all Dsg1 isoforms, also showed a significantly high level of staining in the calcium-treated cells.

and the rabbit antibody AP498 was identical. In summary, these results demonstrate that while all three Dsg1 isoforms are co-expressed in the suprabasal cell layers, unlike Dsgl-a, Dsg1-b and Dsg1-g were not expressed in the basal cell layers. In addition to the skin and oral mucosa, differential expression pattern of Dsg1 isoforms was observed in other mouse epithelia, including the sebaceous glands and stomach epithelium. In the sebaceous glands of adult mouse ears, Dsg1-a was completely absent (Fig. 7Ai, asterisks) while Dsg1-b was expressed in the outer epithelial lining (Fig. 7Aii) and Dsg1-g was expressed throughout, including the more differentiated cell layers (Fig. 7Aiv). The merged image also showed that Dsg1-a (green) was expressed further into the granular cell layers of the epidermis as compared with Dsg1-b (red) (Fig. 7Aiii). Dsg1-g and Dsg1-b expression co-localized to the spinous and granular cell layers; however, some cells expressed only Dsg1-g, not Dsg1-b (Fig. 7vi; as indicated by arrows). Dsg1-g was also absent in highly differentiated cells of the epidermis (Fig. 7iv). Similar to the sebaceous gland, in the stomach epithelium, Dsg1-a (Fig. 7Bi, DAPI shown in blue) was virtually absent when compared with Dsg1-b (Fig. 7Bii) and Dsg1-g (Fig. 7Biii). Some cells expressed both Dsg1-b (red) and Dsg1-g (green) while others expressed only Dsg1-g (Fig. 7Biv). We also examined the expression of Dsg1 isoforms in various mouse tissues using a commercially available mouse tissue panel slide (Imgenex, Sorrento Valley, CA). The results are summarized in Table 3. In the

pancreas, Dsg1-g antibody produced both cytoplasmic and cell–cell border staining. In the small intestine, Dsg1-g antibodies produced cytoplasmic staining in non-epithelial cells. In the seminal vesicle, Dsg1-g staining was observed in the apical surface of the epithelial lumen and in the cytoplasm of the glandular epithelium. In the testis, nuclear staining was observed in the spermatozoas for Dsg1-b and in spermatocytes for Dsg1-g. Dsg1-b was detected in the cytoplasm of the columnar cells of the epididymis. In the uterus, Dsg1-b antibody produced cytoplasmic staining in the glandular epithelium while Dsg1-g antibody produced staining on the apical surface as well as some cell–cell border staining. Finally, in the thymus, we observed intense staining for Dsg1-g, but not Dsg1-a or Dsg1-b, in what appeared to be thymic Hassals’ corpuscles and epithelial reticular cells. The results summarized in Table 3 demonstrate the complex differential expression of Dsg1 isoforms in various mouse tissues. Complex differentially regulated expression of Dsg1 isoforms in the hair follicle The hair follicle is a complex appendage of the epidermis compartmentalized into structures composed of keratinocytes distinguished by their state of differentiation. To further demonstrate the complexity of Dsg1 gene regulation, we compared the expression of Dsg1 isoforms in the hair follicles during development. Similar to the expression of Dsg1 in human anagen hair (Wu et al., 2003), Dsg1 isoforms were expressed in the

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Fig. 5 Differential expression of Dsg1 isoforms during embryonic development. Immunofluorescence staining of Dsg1-a (top left, red), Dsg1-b (top right, green), Dsg1-g (bottom left, green), and Dsg3 (lower right, red) in the epidermis of mouse embryo day 14. Unlike the expression of Dsg3, which was restricted mainly to the basal cell layer, Dsg1 isoforms were expressed only in the highly differentiated uppermost layers. Most lower suprabasal layers were negative for Dsg1 isoforms. Nuclear staining with DAPI is shown in blue.

outer root sheath (ORS) of the infundibulum (Fig. 8). Immediately below the sebaceous glands, expression Dsg1 isoforms became distinctly restricted to the most suprabasal cell layer of ORS. Yet further down the hair follicle, Dsg1 isoforms expression switched from the ORS to the inner root sheath (IRS) (Figs. 8A–8C). Interestingly, the transition from ORS to IRS appeared to start earlier in the hair follicle for Dsg1-a (Fig. 8A) and Dsg1-g (Fig. 8C) than Dsg1-b (Fig. 8B). However, Dsg1-b seemed to have a wider expression within the matrix and the precortex (Fig. 8B). Finally, the dermal papilla cells, which during anagen are on the move and are dispersed from one another, were negative for all Dsg1 isoforms (Figs. 8A–8C). In summary, when cells are undergoing transformation or remodeling, they do not express Dsg1s. Only stable structures express all three Dsg1 isoforms.

During telogen, Dsg1-b and Dsg1-g, but not Dsg1-a, were expressed throughout the entire hair follicle epithelium, higher in the cells with immediate contact with the club hair (Fig. 9). Dsg1-a staining was detected just in the cornified portion of club and in the uppermost layers of hair follicle infundibulum. Some faint expression of Dsg1-a was also observed in cells attached to the hair shaft, most likely representing the hair cuticle (Fig. 9A). Dsg1-b and Dsg1-g were seen not only in the cornified (belonging to hair shaft) portion of the club but also in the layer of epithelial cells immediate to the club, which are involved in club anchoring (Figs. 9B, 9C). In general, we observed higher level and more extensive expression of Dsg1-b and Dsg1-g, compared to Dsg1-a, in the telogen follicle. At this stage of hair development, follicular papilla cells are active, compressed, and establish tight connections. These cells "

Fig. 6 Suprabasilar expression of Dsg1 isoforms in the newborn mouse epidermis and oral mucosa. Immunofluorescence staining of Dsg1-a (A, D, G, and J), Dsg1-b (B and H), and Dsg1-g (E and K) in newborn skin (A)–(F) and oral mucosa (G)–(L). Dsg1-a was evenly distributed throughout all suprabasal cell layers and was also observed in the portion of basal cell border facing the first suprabasal layer and between neighboring basal cells (zone of

desmosomal junctions). The part of the basal cells facing the basement membrane (zone of hemidesmosomes) was totally negative. Dsg1-b and Dsg1-g were totally absent from the basal layer. Dsg1b staining was also reduced in the granular layer while Dsg1-g staining remained high. The dashed lines define the boundary of the epidermis and dermis and nuclear staining with DAPI is shown in blue.

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expressed low levels of Dsg1-b, but not Dsg1-a or Dsg1g. In summary, all three Dsg1 isoforms are present throughout the entire process of its development and cycling and are subject to significant changes during hair follicle development.

Dsg1-a and Dsg1-b, but not Dsg1-g, are proteolytic targets of ETA To determine the potential role of Dsg1 isoforms in cell–cell adhesion, we examined whether Dsg1-b or Dsg1-g, similar to Dsg1-a, are also targets of proteolytic cleavage by ETA. Previous studies have demonstrated that ETs cleave Dsg1-a after glutamic acid residue 381 located between extracellular domains 3 and 4 (EC3 and EC4; see Fig. 1B; (Hanakawa et al., 2002)). Dsg1-a and Dsg1-b, but not Dsg1-g, share identical consensus sequence surrounding the cleavage site (Fig. 10B), suggesting that Dsg1-b but not Dsg1-g would also be a target for proteolysis by ETs. To determine whether Dsg1-b and Dsg1-g are cleaved by ETs, we subjected newborn mice (n 5 2) to ETA treatment overnight according to protocols previously described (Hanakawa et al., 2002). The next day, gross blisters were observed in the ETA-treated animals but not in the PBS treated controls (not shown). Histology revealed extensive acantholysis in the superficial epidermis similar to PF patients. Proteins were subjected to Western blotting analysis using DG3.10- and anti-Dsg1-specific antibodies (Fig. 10A). The Dsg1/Dsg2 antibody DG3.10 detected a protein of approximately 160 kDa in the control skin. Since Dsg2 is virtually undetectable in mouse skin due to low level of expression, it is conceivable that essentially all DG3.10 antigens are representative of Dsg1 isoforms. In the ETA-treated skin lysate, DG3.10 recognized a weak band approximately 160 kDa and a strong degraded product at approximately 105 kDa. These results demonstrate that ETA did not cleave all Dsg1 proteins. Both the anti-Dsg1-a (AP61) and anti-Dsg1-b (AP498) antibodies detected the 160 kDa band in the controls and 105 kDa band in the ETA-treated samples, respectively. However, the anti-Dsg1-g (Ab15) antibody only detected the unprocessed 160 kDa protein in both control and ETA treated samples (Fig. 10A). These results demonstrate that

Dsg1-a and Dsg1-b, but not Dsg1-g, are proteolytic targets of ETA.

Discussion In this report, we have characterized biophysical properties and expression patterns of the mouse Dsg1 gene family: Dsg1-a, Dsg1-b, and Dsg1-g. We demonstrated that Dsg1 isoforms are differentially expressed in the skin; its appendages, including hair follicles and sebaceous glands; and other epithelial tissues. Collectively, these results suggest unique roles for Dsg1 isoforms in cell–cell adhesion during different stages of tissue development and that multiple isoforms of Dsg1 may be necessary for the development and proper function of mouse-stratified epithelia. At the gene level, mouse Dsg3 and Dsg4 genes are encoded by 16 exons while the Dsg2 and all three Dsg1 genes are encoded by only 15 exons. The missing exon in the Dsg1 gene may be the result of fusion between what would be designated as exons 11 and 12 in the Dsg3 gene (Ishikawa et al., 2000). This may be due to either a deletion of an intron in the Dsg1 genes or the addition of an intron in the Dsg3 gene. The core of the exon 11 in Dsg1 encodes the EA domain, the region most adjacent to the transmembrane domain. The amino acid sequences of EA domains, in comparison with the other domains of the polypeptide, diverge significantly between the three Dsg1s (Table 2). At the same time, the size of EA domains remained quite conserved in Dsg1-a and Dsg1-b (49 and 52 aa, respectively). However, the Dsg1-g EA domain (28 amino acids) is significantly truncated and possesses stronger hydrophobic properties compared with Dsg1-a and Dsg1-b (Fig. 1B and Table 2). The roles of the EA domain in the overall stability of the protein and the adhesiveness of the desmosomal junction remain untested at present. Complexity of Dsg1 expression The existing anti-Dsg1 antibodies, DG3.10 and 18D4, recognized all three mouse Dsg1 proteins, which necessitated the need to produce antibodies specific against each Dsg1 isoform. The cross-reactivity of our Dsg1 "

Fig. 7 Differential expression of Dsg1 isoforms in the sebaceous glands and stomach epithelium. (A) Immunofluorescence staining showing expression of Dsg1-b (middle panels) and Dsg1g (bottom left panel), but Dsg1-a (top left panel), in the sebaceous glands from adult mouse ears. Note that similar to newborn mouse epidermis (see Fig. 6), Dsg1-a was expressed throughout all cell layers while staining for Dsg1-b and Dsg1-g was restricted mainly to the suprabasilar cell layers. Dsg1-b staining declined in the granular layer. Merged images are shown to the right. Bracket in the upper right panel defines the area of the stratum corneum where Dsg1-a staining was present without Dsg1-b.

Arrows in the lower panels clearly demonstrate difference in Dsg1-b and Dsg1-g expression in the granular layer. In the sebaceous glands, staining for Dsg1-b and Dsg1-g, but not Dsg1-a, was detected. (B) Immunostaining for Dsg1-b and Dsg1-g, but not Dsg1-a, in the stratified epithelium of the stomach. Merged image of Dsg1-b and Dsg1-g staining is shown at the bottom right, demonstrating a higher level of Dsg1-g expression compared with Dsg1-b in the upper layers of the stomach epithelium. Nuclear staining with DAPI in blue is included to demonstrate the lack of Dsg1-a staining in the mouse stomach epithelium (top left panel).

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446 Table 3 Dsg1 expression in adult mouse tissues Organ

Dsg1-a

Dsg1-b

Dsg1-g

Skin Sebaceous gland Spleen Skeletal muscle Lung Heart Tongue Salivary gland Liver Pancreas Esophagus Stomach Small intestine Colon Kidney, cortex Kidney, medula Urinary bladder Seminal vesicle Testis Epididymis Uterus Ovary Thymus Cerebrum Pons Cerebellum

1      1                   

1 1     1    1        1 1 1     

1 1     1   1 1 1 1 1    1 1  1  1   

Panel of formalin fixed normal organs of adult mice (Imgenex) immunostained with specific antibodies against Dsg1-a (AP61), Dsg1-b (AP498), and Dsg1-g (Ab15).

antibodies to Dsg2, Dsg3, and Dsg4 was ruled out for the following reason. All Dsg1 isoforms are expressed in the superficial epidermis while Dsg2 and Dsg3 are expressed in the basal cell layer. None of our Dsg1-specific antibodies recognized Dsg4 recombinant proteins. Although overall RT-PCR and immuno staining results agreed that Dsg1-g was more widely distributed compared with Dsg1-a and Dsg1-b, the expression profiles of the Dsg1 isoforms at the protein level reported here were somewhat different to results previously observed by RT-PCR experiments (Kljuic and Christiano, 2003; Pulkkinen et al., 2003; Whittock, 2003). During embryonic development RT-PCR detected mRNA level of only Dsg1-a and Dsg1-b, but not Dsg1-g, by day 15; however, by immuno staining we detected all Dsg1 isoforms in the mouse embryonic epidermis, at day 14. This may be due to the low level of Dsg1-g mRNA relative to the total embryonic mRNA. This is supported by our observation that the expression of Dsg1-g was restricted mainly to the highly differentiated cell layers of the epidermis at this stage of development. In addition, unlike the published RT-PCR results, we did not detect Dsg1-g protein in the liver. Positive detection of Dsg1-g by RT-PCR may have resulted from one of several causes: cross-hybridization with another gene in the liver, RT-PCR being a more sensitive detection method than immuno staining, or the Dsg1-g mRNA was unstable.

Our expression studies demonstrated that the Dsg1 isoforms are differentially expressed in a variety of stratified and non-stratified postnatal epithelia. In the epidermis, all Dsg1 isoforms were detected but the expression of each was spatially distinct. Dsg1-a was detected throughout all cell layers and particularly high in the stratum corneum (Figs. 6 and 7). Dsg1-b and Dsg1g were detected mainly in the spinous and granular cell layers of the epidermis (Figs. 6 and 7). Interestingly, Dsg1-b and Dsg1-g, but not Dsg1-a, were expressed in the sebaceous glands and fore-stomach epithelium (Fig. 7). These results suggest that Dsg1-a may not be required for adhesion in these tissues, or the presence of Dsg1-a would hinder the function of these tissues, or that Dsg1-a plays a role in adhesion during cell cornification, which is present in the epidermis but not in the sebaceous glands or the stomach epithelium. Differential expression of Dsg1 isoforms was also observed in the hair follicle. Collectively the expression pattern of all Dsg1 isoforms observed in mouse anagen hair is similar to that previously observed in human hair follicle at the same stage of development (Wu et al., 2003). The expression of Dsg1 isoforms is a subject of significant hair cycle-dependent changes being up-regulated in stable, differentiating cellular compartments and declining significantly with the onset of proliferation/remodeling associated with elevated cell motility. During the resting stage of the telogen hair, we observed high expression of Dsg1-b and Dsg1-g in the cells surrounding the club hair and the IRS. Similar to previous results (Hanakawa et al., 2002; Wu et al., 2003), Dsg1-a was not detected in this region (Fig. 9A). Interestingly, Dsg3 null mice develop progressive hair loss presumed to be due to loss of adhesion between the keratinocytes surrounding the club and the ORS (Koch et al., 1998). This suggests that cell–cell adhesion by Dsg1-b and Dsg1-g is not sufficient to compensate for the loss of Dsg3. Interestingly, overexpression of Dsg1-a under the K14 promoter in cells surrounding the hair club in transgenic mice crossed with the Dsg3 null mice delays, although does not completely reverse, this hair loss phenotype (Hanakawa et al., 2002). Future experiments to determine the adhesive strength of each individual desmosomal protein within a desmosome would resolve these issues.

Significance of multiple Dsg1 genes in mouse Multiple Dsg1 isoforms could conceivably influence the observed skin phenotype in the mouse passive transfer model of pemphigus blistering diseases, such as PF and SSSS. In PF, the pathogenic antibodies target antigenic sites within the first four extracellular domains (EC1– EC4) of DSG1 (Amagai et al., 1992). These domains share significant homology between all three mouse Dsg1s (Table 2) and thus, it is highly likely that the PF

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Fig. 8 Comparison of Dsg1 expression in anagen follicles. Immunostaining for Dsg1-a (A), Dsg1-b (B), and Dsg1-g (C) in anagen hair follicles. All Dsg1 isoforms were expressed in the hair follicle infundibulum and inner root sheath (IRS) throughout the anagen hair. Hair shaft, matrix cells, and outer root sheath (ORS) were all negative. Some expression of Dsg1-b was also detected in the hair cuticle.

antibodies would target the same antigenic sites found in all Dsg1 isoforms. However, in SSSS, the serine proteases ETs cleave Dsg1-a at a glutamic acid localized

between EC3 and EC4 domains at position 381 (Amagai et al., 2000; Amagai et al., 2002; Hanakawa et al., 2002). Identical sequence homology between Dsg1-a

Fig. 9 Dsg1 expression in the telogen hair follicles. Immunostaining of Dsg1-a (A), Dsg1-b (B), and Dsg1-g (C) in the telogen hair follicles. All Dsg1 isoforms were present in the suprabasal cell layers of the hair follicle infundibulum. Dsg1-b and Dsg1-g, but not

Dsg1-a, were actively expressed in the sebaceous glands (). Below the sebaceous glands, expression of Dsg1-b and Dsg1-g was restricted to the keratinocytes adjacent to the club hair. Staining for Dsg1-a was virtually absent from the telogen club hair.

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Fig. 10 ETA cleaves Dsg1-a and Dsg1-b, but not Dsg1-g. Newborn C57Bl/6 mice were injected with ETA in PBS. The next day, total skin protein was extracted in Laemmli buffer and subjected to Western blotting analysis with DG3.10, AP61 (anti-Dsg1a), AP498 (anti-Dsg1-b), and Ab15 (anti-Dsg1-g) antibodies. DG3.10 recognized a band of approximately 160 kDa for Dsg1 s in the control mouse skin lysate (top arrow). After cleavage with ETA, most of the 160 kDa band was cleaved to a band approximately 105–110 kDa (bottom arrow); however, a residual

signal was detected at 160 kDa. The same blots were probed for Dsg1 isoform-specific antibodies and the results demonstrate that Dsg1-a and Dsg1-b, but not Dsg1-g, were cleaved by ETA. (B) Comparison of the amino acid sequence surrounding the putative ETA cleavage site between human Dsg1, canine Dsg1, bovine Dsg1, and mouse Dsg1s. All except Dsg1-g shared the same glutamic acid–glycine sequence for ETA cleavage. Summary of cleavage by ETA from in vivo or in vitro experiments is shown to the right.

and Dsg1-b surrounding this cleavage site and our results demonstrating that ETA cleaved Dsg1-a and Dsg1-b suggest that ETA cleaves Dsg1-a and Dsg1-b in vivo. Interestingly, Dsg1-g does not contain this ETconsensus cleavage sequence and in vivo experiments demonstrated that ETA did not cleave Dsg1-g. However, mice injected with ETA develop acantholysis in the superficial epidermis where both Dsg1-a and Dsg1-b were expressed. These two proteins were also cleaved by ETA, suggesting that Dsg1-a and Dsg1-b, but not Dsg1-g, may play a significant role in cell–cell adhesion. However, it is also possible that Dsg1-g may provide some resistance for the mouse against degradation by Staphylococcal proteases, but at a level not observable in experimental newborn mice injected with purified ETs. Through evolution there is selective pressure to eliminate functionally redundant genes, and simultaneously duplicate individual genes to enhance functional diversity and differential expression (Lynch and Conery, 2000). Two novel mouse desmoglein genes, Dsg1-b and Dsg1-g, were recently identified as sharing significant homology

to Dsg1-a (Kljuic and Christiano, 2003; Pulkkinen et al., 2003). The presence of multiple Dsg1 genes in mouse may attest to the evolutionary driving force to maintain these genes. As expected, these Dsg1 genes are differentially expressed in some tissues and co-expressed in others, suggesting that they may possess distinct, yet overlapping functions. Although the biological significance for multiple Dsgl isoforms requires further analysis, it is possible that their individual expression patterns collectively contribute to their overall biological function in diverse, complex tissues. Acknowledgments This work was supported by grants from the National Institutes of Health and the Dermatology Foundation. We would like to thank Dr. John Stanley for providing antibodies and constructs.

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