Genetic Evidence for a Novel Human Desmosomal Cadherin, Desmoglein 4

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Neil V.Whittock1 and Christopher Bower2 1Institute of Biomedical and Clinical Science, Peninsula Medical School, Exeter, United Kingdom, 2Department of Dermatology, Royal Devon and Exeter Hospital, Exeter, United Kingdom

Desmosomes are essential adhesion structures in most bp that encodes a precursor protein of 1040 amino acids. epithelia that link the intermediate ¢lament network of The predicted mature protein comprises 991 amino one cell to its neighbor, thereby forming a strong bond. acids with a molecular weight of 107822 Da at pI 4.38. The molecular components of desmosomes belong to Human desmoglein 4 shares 41% identity with human the cadherin superfamily, the plakin family, and the ar- desmoglein 1, 37% with human desmoglein 2, and 50% madillo repeat protein family.The desmosomal cadher- with human desmoglein 3.Analysis of the exon/intron ins are calcium-dependent transmembrane adhesion organization of the human desmoglein 4 gene (DSG4) molecules and comprise the desmogleins and desmo- demonstrates that it is composed of 16 exons spanning collins.To date, three human desmoglein isoforms have approximately 37 kb of 18q12 and is situated between been characterized, namely desmogleins 1, 2, and 3 that DSG1 and DSG3.We have demonstrated using are expressed in a tissue- and di¡erentiation-speci¢c RT-PCR on multiple tissue cDNA samples that desmo- manner.Here we have identi¢ed and characterized, at glein 4 has very speci¢c tissue expression in salivary the genetic level, a novel human desmoglein cDNA gland, testis, prostate, and skin. Keywords: Desmosome/ sharing homology with desmogleins 1, 2, 3 and we gene organization /desmosomopathy/pemphigus. J Invest Der- name this desmoglein 4.The human desmoglein 4 matol120:523 ^530, 2003 cDNA (3.6 kb) contains an open reading frame of 3120

esmosomes are disc-like structures of 0.1^0.5 mmin dent class of single pass transmembrane cell adhesion molecules, diameter and are located principally in epithelial the cadherins (Nilles et al, 1991) and were subsequently named cells where they play a major role in cell-cell adhe- ‘‘desmosomal cadherins’’. sion as well as cell communication (Garrod, 1993; Similar to the classical cadherins, the desmosomal cadherins Kouklis et al, 1994; Garrod, 1996; Green and Jones, possess ¢ve homologous extracellular domains containing puta- D1996). Viewed under standard transmission electron microscopy tive calcium-binding sites, a single transmembrane spanning do- desmosomes present with a distinctive ultrastructure consisting main, and a carboxy-terminal cytoplasmic tail (Wheeler et al, of two major domains. The ¢rst, termed the extracellular core 1991). The cytoplasmic tails of the desmogleins and desmocollins domain or ‘‘desmoglea’’, is approximately 30 nm in width and is contain many motifs including an intracellular anchor domain separated by an electron-dense mid line region. This extracellular (IA), an intracellular cadherin-typical segment (ICS) and a major core domain consists of the extracellular heads of the desmoso- binding site that contributes to the binding of the desmosomal mal cadherins that provide cellular adhesion to neighboring cells plaque protein, plakoglobin (Schmidt et al, 1994). The desmocol- (North et al, 1999). The second domain consists of an electron- lins exist in ‘‘a’’and ‘‘b’’ isoforms, the shorter ‘‘b’’ isoform lacking dense cytoplasmic plaque that can be further separated into an the major binding site that eliminates its ability to bind plakoglo- outer dense plaque and an inner dense plaque, which are sepa- bin (Troyanovsky et al, 1993; Troyanovsky et al, 1994a; Troyanovs- rated from one another by an electron lucent zone. The cytoplas- ky et al, 1994b). In addition, molecules in the desmoglein micplaque is composed of the desmosomal plaque proteins subgroup contain a unique carboxy-terminal extension contain- desmoplakin, plakoglobin, the plakophilins and the amino ter- ing a ‘‘repeat unit domain’’ (RUD) that comprises a repeating minal domains of the intermediate ¢lament proteins. Early stu- motif of approximately 29 amino acids (Koch et al, 1990; Wheeler dies on desmosomes identi¢ed a group of glycoproteins and et al, 1991) that is predicted to form b-strands. In order to avoid evidence suggested that they mediated the speci¢c recognition confusion the nomenclature was simpli¢ed by renaming the hu- and adhesion functions of desmosomes (Cohen et al, 1983). With man proteins such that there were six desmocollins (i.e., 1a, 1b, 2a, the resulting isolation and characterization of these glycoproteins 2b, 3a, and 3b), and three desmogleins (i.e., 1, 2, and 3) (Buxton it was demonstrated that they were related to the calcium depen- et al,1993). The largest desmosomal cadherins are desmocollin 2 and desmoglein 2; they are the essential desmosomal cadherins common Manuscript received November 15, 2002; revised December 1, 2002; to all desmosome-possessing tissues, including simple epithelia, accepted for publication December 9, 2002 myocardium and many cell cultures (Koch et al, 1992; Theis et al, Reprint requests to: Dr. Neil V Whittock, Molecular Genetics Labora- tory, Old Pathology Building, Royal Devon and Exeter Hospital, Barrack 1993; Schmidt et al, 1994). In contrast, the epidermal isoforms Road, Exeter, United Kingdom, EX2 5DW. E-mail: nwhittoc@hgmp. desmocollins 1 and 3, and desmogleins 1 and 3 are restricted to mrc.ac.uk specialized epithelia, mostly strati¢ed squamous ones (Koch et al,

0022-202X/03/$15.00 . Copyright r 2003 by The Society for Investigative Dermatology, Inc.

523 524 WHITTOCK AND BOWER THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

1992; Schafer et al, 1994; Schmidt et al, 1994; Schafer et al,1996). an ABI 377 geneticanalyzer (Applied Biosystems) and analyzed using Within the epidermis the three desmocollin/glein isoforms are Sequence Navigator 1.0.1. software (Applied Biosystems). To determine expressed in a di¡erentiation-speci¢c manner, with desmocollin the expression of desmoglein 4 a multiple tissue RNA panel II (adult) 2 and desmoglein 2 being basal, desmoglein 3 and desmocollin (BD Clontech, Basingstoke, UK) was reverse transcribed and ampli¢ed as 3 being basal layer and ¢rst suprabasal layer, and desmoglein 1 described above. and desmocollin 1 being upper spinous and granular layer of epi- RACE PCR The 50 and 30 ends of the desmoglein 4 cDNA were dermis (Arnemann et al,1993;Kinget al, 1993; Theis et al, 1993; isolated from skin and testis total RNA using the Smart Race Schmidt et al,1994;Kinget al, 1995; Nuber et al,1995;Yueet al, ampli¢cation kit (BD Clontech) according to the manufacturer’s 1995; Adams et al, 1998; Denning et al, 1998). In addition to its role instructions. Primers used to amplify the 50 and 30 ends of desmoglein 4 in cell adhesion in deep strati¢ed squamous epithelia, desmoglein were EX1B RACE 50 -CTG AAG AAG AGC CAA TCC ATT CCT TTG GG-30, and 3UTRRACEA 50 -GCA TCC CTT GAT ACT GTC TAA 3 is also critical in anchoring the telogen hair to the outer root 0 sheath of the hair follicle and thus demonstrates that desmosomes CGA ATA GC-3 . RACE PCR was performed using the Advantage II PCR kit (BD Clontech) according to the manufacturer’s instructions, and are important in maintaining the normal structure and function PCR products were puri¢ed and sequenced as above. of the hair (Koch et al,1998). The human desmoglein genes comprise either 15 or 16 exons (Silos et al, 1996; Frank et al,2001;Huntet al, 2001) that are clus- RESULTS tered within a region of approximately 150 kb in the order 50 - DSG1-DSG3-DSG2-30 (Simrak et al, 1995). The human desmo- Sequence analysis reveals a novel human desmosomal collin genes comprise 17 exons (Whittock et al, 2000b; Cserhal- cadherin Analysis of the annotated human genome data mi-Friedman et al, 2001), where exon 16 is alternatively spliced, (http://genome.ucsc.edu) for chromosome 18 demonstrated giving rise to the ‘‘a’’ and ‘‘b’’ forms of the protein (Collins et al, several predicted genes in the 100 kb gap between DSG1 1991; Greenwood et al, 1997). However, the genomicorganization and DSG3 using various algorithms. These included of the desmocollin genes is closer to that of the classical cadherins ENST00000308128 (Ensembl), chr18.29.005.a and chr18.30.001.a than the desmogleins (Greenwood et al, 1997). The entire DSC/ (Twinscan), and NT_010966.11 (Genescan). All of these predicted DSG gene complex occupies approximately 700 kb on 18q12.1, genes demonstrated varying levels of homology with the known and the genes are arranged in the order cen-30 -DSC3-DSC2- desmoglein genes, DSG1-3. In addition, the predicted mRNA’s DSC1-50 -50 -DSG1-DSG3-DSG2-30tel, where the two gene clus- and proteins showed a high level of homology to desmogleins ters are transcribed outward from the interlocus region (Arnemann 1^3. However, it was clear based on exon size conservation with et al, 1991; King et al, 1993; Cowley et al,1997;Huntet al, 1999). other DSG genes and amino acid sequence that the gene Desmosome integrity is critical for normal cell-cell adhesion. prediction algorithms had incorrectly identi¢ed several exons This is clearly highlighted in a group of disorders whereby de- and most importantly had not correctly identi¢ed exon 1 and its fects occur within desmosomal proteins that lead to the loss of associated promoter. In order to locate exon 1, ¢ve regions this adhesion. The geneticdiseases of desmosomes cover a range containing high homology with the syntenic mouse region of disorders that can be inherited in autosomal dominant or re- were identi¢ed as HumMus.18.14374, 14379, 14381, 14382, and cessive modes and that can range from a¡ecting just palmoplantar 14383. Sequence analysis of HumMus.18.14374, 14379, 14382, and skin to those that a¡ect skin, hair and heart and can lead to death 14383 revealed no potential ¢rst exon; however, analysis of during the second or third decade of life. In addition, desmoso- HumMus.18.14381 revealed a region predicted to encode 16 mal proteins are targets for the autoimmune diseases pemphigus amino acid residues with high homology to desmoglein 1 vulgaris, pemphigus foliaceous and paraneoplastic pemphigus, as followed by a donor splice site. To determine whether these well as substrates in staphylococcal scalded skin syndrome (SSSS). predicted genes were indeed expressed in skin and to con¢rm Here we present the identi¢cation of an additional human des- the correct exons, overlapping RT-PCR was used on skin biopsy moglein which we term desmoglein 4 and demonstrate tissue total RNA from nonlesional skin. Sequence analysis of the speci¢c expression. Based on gene structure and protein identity desmoglein 4 cDNA indicated a transcript of at least 3.6 kb we show that desmoglein 4 is closely related to desmoglein 3. containing an open reading frame of 3120 bp (Fig 1). Exon/intron organization revealed that the new gene, DSG4, comprised 16 exons spanning approximately 37 kb of human chromosome 18 MATERIALS AND METHODS with the entire desmoglein gene complex spanning approximately 250 kb of 18q12 (Fig 2). The exons varied from Reverse transcription PCR (RT-PCR) and cDNA expression 36 bp (exon 2) to 765 bp coding (exon 16) in size, and the studies Total RNA was extracted from normal skin biopsy using the introns varied from 8176 bp (intron 1), to 153 bp (intron 13) in Perfect RNA kit (Eppendorf AG, Cambridge, UK) according to size (Tab l e 1). By gene structure alone DSG4 is more closely manufacturer’s instructions. RNA was reverse transcribed using random related to DSG3 as they both consist of 16 exons whereas DSG1 primers with the Thermo-script cDNA kit (Invitrogen Ltd, Paisley, UK) and DSG2 comprise 15 exons (Tab l e 2 ). In addition, the sizes according to the manufacturer’s instructions. Gene-speci¢c primers used of exons for DSG3andDSG4 are the same for exons 1, 2, 3, 4, 5, for overlapping RT-PCR of desmoglein 4 are available on request. Gene- 6, 8, 9, 10, 13 and 14, demonstrating a high level of conservation speci¢c intron crossing primers were designed for use in RT-PCR based cDNA expression studies to exclude ampli¢cation of genomic DNA. (Tab l e 2 ). Primers used for desmoglein 4 were cDNA4F (forward-situated in exon 4) 50 -GTA GGG ATT GAT CGA CCA CC-30 and cDNA7R (reverse- Analysis of the deduced amino acid sequence of human situated in exon 7) 50 -CTTGATTCTACAGTCACACTC-30 to yield a desmoglein 4 and comparison with the desmoglein 507-bp product. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) family The 3120 bp open reading frame of the human was ampli¢ed using the primers 50 -TGA AGG TCG GAG TCA ACG desmoglein 4 cDNA encodes a precursor protein of 1040 GAT TTG GT-30 (forward) and 50 -CAT GTG GGC CAT GAG GTC residues with a predicted molecular weight of 113812 Da and CAC CAC-30 (reverse). PCR was performed in a 50-mL reaction mixture isoelectric point of 4.42 (Fig 1). This precursor comprises a 21 containing 20 pmol forward and reverse primers, 10 nmol each of dTTP, amino acid signal sequence followed by a 28 amino acid dCTP and dATP, 7.6 nmol dGTP, 2.5 nmol deaza GTP, 2.5 mL DMSO, prosequence that includes a NXS/T glycosylation site and ends 50 mmol betaine, 10 mM Tris^HCl, 50 mM KCl, 1.5 mM MgCl2 and in a basicputative proteolysis sequenceRRQKR. Cleavage 1.0 U Amplitaq Gold Ta q polymerase (Applied Biosystems, Warrington, UK). After an initial denaturation of 951C for 12 min, 40 cycles were at this sequence would result in a mature desmoglein 4 performed of 951C for 1 min, 551C for 1 min, and 721C for 2 min, with a unglycosylated peptide of 991 amino acids with a molecular ¢nal extension step of 721C for 10 min. The PCR products were examined mass of 107822 Da and a pI of 4.38. The extracellular region of by 1.5% agarose gel electrophoresis, puri¢ed using spin columns (Qiagen, desmoglein 4 (578 amino acids), by homology with other Crawley, England) and directly sequenced using Big Dye terminators on desmogleins is divided into four domains of about equal size, VOL. 120, NO. 4 APRIL 2003 HUMAN DESMOGLEIN 4 525

Figure 2.Predicted gene and protein details of human desmoglein 4. (a) Schematic representation of the human desmoglein complex. DSG4 lies between DSG1andDSG3 with the entire desmoglein complex occu- pying approximately 250 kb (b) Genomicorganization of the human DSG4. Exons are represented by vertical boxes, introns by horizontal lines. The gene consists of 16 exons, spanning approximately 37 kb of genomic DNA. The size of the individual exons and introns are presented in Table 1. (c) The encoded protein, desmoglein 4, comprises a signal (S) and pre- protein (p) domain followed by 5 extracellular domains (EC1-4, EA), a transmembrane domain (TM), an intracellular anchor domain (IA), an intracellular cadherin-typical segment domain (ICS), a linker domain (LD), a repeat unit domain (RUD) containing two repeats, and a terminal domain (TD).

EC1 to EC4 that have homology with each other, followed by an extracellular anchor domain (EA).The highly conserved sequence HAVof typical cadherins, which is thought to be involved in cell adhesion, is represented in desmoglein 4 by the conservatively substituted sequence RAL. Other conserved sequences in the extracellular domains of desmoglein 4 and other desmogleins with potential function include putative calcium binding motifs (DXNDN and A/VXDXD). In addition, there are two further potential N-glycosylation sites (NXS/T) in the extracellular domain of desmoglein 4, in EC1 that is conserved in the equivalent positions of desmogleins 1^3, and in EA that is conserved only in desmoglein 3. The extracellular domain is followed by a 25 amino acid predicted transmembrane domain. The cytoplasmic domain of desmoglein 4 (388 amino acids) comprises an intracellular anchor domain (IA), an intracellular cadherin-typical segment (ICS), an intracellular linker domain (LD), a repeat unit domain (RUD) and a terminal domain (TD). The RUD domain contains one NVXVTE repeat and a highly related NVIYAE predicting two repeats as found in desmoglein 3. The overall protein identity of desmoglein 4 with human desmoglein 1 (Nilles et al, 1991;Wheeler et al, 1991) is 41%, with human desmoglein 2 (Koch et al, 1991; Arnemann et al, 1992) is 37%, and human desmoglein 3 (Amagai et al,1991)is50% (Tab l e 3)(Fig 3). We therefore conclude that desmoglein 4 is a member of the cadherin family and that it is more closely Figure1.Nucleotide and predicted amino acid sequence of human related to the desmogleins than the typical cadherins. desmoglein 4. Horizontal arrows under the amino acid sequence show Comparison of the individual regions for desmoglein 4 with the beginning of each domain. The putative signal sequence is double desmoglein 1, desmoglein 2 and desmoglein 3 demonstrates that striked. The presumed recognition site for proteolytic cleavage (RRQKR) desmoglein 4 is more identical to desmoglein 1 over the is single striked. The RAL sequence, which corresponds to the HAV se- extracellular domain whereas it is more identical with quence of typical cadherins is underlined and boxed. Putative calcium desmoglein 3 over the intracellular domain (Tab l e 3). binding sites (DXNDN and A/VXDXD) are bold and underlined. The transmembrane domain is underlined. The related NVXVTE repeats in RT-PCR on multiple tissue panel reveals tissue speci¢c the repeat unit domain are bold and double-underlined. Potential N-gly- expression To determine whether the desmoglein 4 transcript cosylation sites are boxed. The translation initiation codon (ATG) is at nu- is expressed in other human tissues in addition to skin we cleotide position 1, and the predicted translation termination codon (TAA) examined its tissue-speci¢c expression pro¢le using RT-PCR on is marked with an asterisk (n). EC-extracellular domain, EA- extracellular a cDNA panel derived from several adult tissues. The gene was anchor domain, TM-transmembrane domain, IA-intracellular anchor do- highly expressed in testis, prostate, and skin, but was less main, ICS-intracellular cadherin-typical segment domain, LD-linker do- abundant in salivary gland (Fig 4). No expression was detected main, RUD-repeat unit domain,TD-terminal domain. in thymus, uterus, skeletal muscle, thyroid, trachea, spleen, liver, 526 WHITTOCK AND BOWER THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Table1.Exon-Intron Boundaries of the Human Desmoglein 4 Gene, DSG4 Exon Exon size (bp) 50 splice donor sequence Intron size (bp) 30 splice donor sequence

1 Non coding þ 48 CTAATGgtaagt 8176 aaacagGTGGTG 2 36 GTTGAGgtaatg 1516 cttaagGTGAAG 3 132 GCCAAAgtaagt 1547 ttatagATTCGA 4 156 TTCTTGgtaagt 351 ccacagATCTAT 5 145 ATGCCAgtaagt 1637 ttctagATACAT 6 167 AGAGAGgtaaag 255 attaagCAACAC 7135 ACTTCAgtaagt 942 ttttagTACTCA 8 186 GTCAAGgtacag 6931 tttcagATGCTG 9272 TGTCAGgtactg 1337 aaacagATATAT 10 140 TAGATGgtaaga 2395 tttcagATGGCT 11 219 CAAATGgtaagt 2442 tttcagCTACCT 12 297 TGATTTgtaagt 3078 gggcagTGGCTC 13 140 GACAGGgtaagt 153 tttcagGATGTG 14 6 4 CCTCTGgtcagt 1422 tgtcagCTTTTT 15 218 TCGGAGgtaatg 1379 ttttagAAAGCG 16 765 þ non coding ND

Table 2.Comparison of the Deduced Exon Sizes for the Four Human Desmoglein Genes Exon DSG1 DSG2 DSG3 DSG4

1 Non coding þ 48 Non coding þ 45 Non coding þ 48 Non coding þ 48 236 36 36 36 3 132 135 132 132 4 156 162 156 156 5 145 145 145 145 6 167 167 167 167 7 135 138 129 135 8 186 186 186 186 9 260 266 272 272 10 14 0 143 14 0 14 0 11 282 228 225 219 12 134 228 261 297 13 70 122 140 140 14 209 333 64 64 15 1047 þ non coding 1020 þ non coding 284 218 16 612 þ non coding 765 þ non coding

Table 3.Identity of the Desmoglein 4 Protein with Desmogleins 1^3 Domain Dsg4 vs. Dsg1 Dsg4 vs. Dsg2 Dsg4 vs. Dsg3

E1 82 (93) 64 (81) 70 (85) E2 67 (82) 51 (67) 65 (81) E3 50 (76) 50 (76) 67 (81) E4 35 (59) 42 (60) 47 (72) EA N/S 31 (53) 34 (54) TM 52 (82) 64 (80) 75 (95) IA 40 (60) N/S 49 (62) ICS 53(70) 43(57) 58(72) LD N/S N/S N/S RUD 39(66) 30(48) 51(69) TD 46 (67) N/S N/S Entire protein 41 (56) 37 (53) 50 (66) Values are expressed as a percentage (%). Values in brackets are percentage similarity. N/S ^ no signi¢cant identity or similarity. lung, placenta, fetal brain, heart, adrenal gland, bone marrow, DISCUSSION cerebellum, whole brain, or kidney. Desmosomes are essential adhesion structures in most epithelia GenBank accession number Human desmoglein 4 genomic and consist of a speci¢c subset of proteins necessary to attach the DNA and desmoglein 4 cDNA sequences have been deposited intermediate ¢lament network of one cell to the intermediate in the GenBank sequence database under Accession Nos ¢lament network of a neighboring cell, thus forming a strong AY177663 and AY177664, respectively. connection. VOL. 120, NO. 4 APRIL 2003 HUMAN DESMOGLEIN 4 527

Figure 3.Multiple amino acid sequence alignment of human desmogleins 1^4. Horizontal arrows above the amino acid sequence show the beginning of each domain. Desmoglein 1 (Nilles et al, 1991), desmoglein 2 (Koch et al, 1991), and desmoglein 3 (Amagai et al, 1991) are from published sequences. Conserved amino acids are represented by asterisks (n) under the sequence. For abbreviations see Figure 1 legend.

The amino-terminal domains of both the desmocollins and domains of the desmosomal cadherins protrude into the outer desmogleins comprise the extracellular core domain of the dense plaque at varying distances due to their di¡erent lengths. desmosome (North et al, 1999). However, the carboxy-terminal The carboxy-terminal domains of the desmogleins reach at least 528 WHITTOCK AND BOWER THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

generalized skin blistering disease staphylococcal scalded skin dyndrome (SSSS) (Melish and Glasgow, 1970; Melish et al,1972; Amagai et al,2000;Amagaiet al, 2002). It therefore follows that mutations in other undiscovered desmosomal proteins may underlie inherited disorders or themselves be targets for autoimmune blistering disease and microbial attack. Using geneticanalysis we have identi¢ed a novel human desmoglein gene, desmoglein 4, sharing signi¢cant homology with all cadherins, but most markedly with desmoglein 3. Like all other members of the cadherin family, desmoglein 4 has a putative signal sequence and a well-conserved sequence of basic amino acids that presumably serves as a signal for cleavage to a mature protein, followed by ¢ve extracellular domains, of which Figure 4.Tissue speci¢c expression of human desmoglein 4. Intron EC1, EC2, EC3, and EC4 show variable homology with each crossing primers for desmoglein 4 and GAPDH (control ^ not shown) other. Like the typical cadherins, EA shows minimal or no signif- were used to amplify cDNA from a multiple adult human tissue panel icant homology with the other extracellular domains. Near the (BD Clontech). Speci¢c expression of desmoglein 4 was observed in sali- amino terminus of the mature protein, the area important for vary gland, testis, prostate, and skin. The negative control consisted of no homophilic binding in typical cadherins, desmoglein 4 shows cDNA. much greater identity to corresponding domains of desmoglein 1 than to those of desmogleins 2 and 3. Like desmoglein 1 and the inner edge of the outer dense plaque approximately 20 nm desmoglein 3, desmoglein 4 has an RAL site in EC1 that corre- from the plasma membrane and may even reach into the inner sponds to the conserved HAV site in an equivalent position in dense plaque. Within the desmosome the cytoplasmic tail of the typical cadherins. Desmoglein 4 also has several conserved puta- desmogleins bind the ‘arm’ repeat domain of plakoglobin (Troya- tive calcium binding domains with all cadherins as well as several novsky et al, 1993; Mathur et al, 1994; Troyanovsky et al, 1994a; conserved N-glycosylation sites with desmoglein 1 and desmo- Troyanovsky et al, 1994b). Speci¢cally,‘‘arm’’ repeats 1^3 and per- glein 3. Amongst the desmoglein genes, the desmoglein 4 gene haps repeat 4 are required for binding of desmoglein (Kapprell is most similar to desmoglein 3, as both comprise 16 exons. et al, 1988; Ozawa et al, 1995; Troyanovsky et al,1996;Wahlet al, Expression studies using RT-PCR on a human cDNA panel 1996; Witcher et al, 1996; Andl and Stanley, 2001).The stoichiome- demonstrated weak expression of desmoglein 4 in salivary gland. try of binding between the desmosomal cadherins and plakoglo- It has previously been shown using RNA protection assays that bin has been extensively studied. Initial reports indicated that the only desmoglein 2, and not desmoglein 1 or desmoglein 3, is cytoplasmic tail of desmocollins could bind one molecule of pla- expressed in the salivary gland (Schmidt et al,1994).Aswellas koglobin only, whereas the cytoplasmic tail of desmogleins could skin, the desmoglein 4 transcript is also highly expressed in testis bind 6^7 plakoglobin molecules (Kowalczyk et al, 1996; Witcher and prostate gland. Comparative data for the other desmoglein et al, 1996). A recent study, however, has demonstrated that the isoforms is only available for prostate gland, which shows that desmogleins bind no more than 2 plakoglobin molecules (Bannon only desmoglein 2 is expressed (Schmidt et al, 1994). The tissue et al, 2001). Indeed it was demonstrated that desmogleins 2 and 3 speci¢c transcript expression of desmoglein 4 in testis and pros- bound only one molecule of plakoglobin, where as the di¡erentia- tate has added signi¢cance due to the fact that loss of heterozyg- tion speci¢c isoform, desmoglein 1, bound 2 molecules of plako- osity (LOH) at 18q is found in both testicular and prostate globin. The authors suggest that the isoform speci¢c binding of cancers. The spectrum of genetic alterations in human cancer plakoglobin may exert control over processes related to dif- a¡ect speci¢c genes that play crucial roles in a varied range of ferentiation, junctional assembly and homeostasis in epithelia cellular processes such as cell adhesion, signal transduction, devel- (Bannon et al, 2001). opment, DNA-repair and di¡erentiation. Testicular germ cell The requirement for functionally intact desmosomes has been tumors (TGCT) are probably the most prevalent type of tumor demonstrated with the characterization of a signi¢cant group of among adolescent and young adult males. Suggestive linkage be- inherited disorders caused by mutations in the genes of the des- tween disease and geneticmarkers has been reported for several mosomal proteins. These disorders include the genetically hetero- chromosomes including loss of genetic material at 18q12-ter (al- geneous autosomal dominant condition, striate palmoplantar Jehani et al, 1995; Lothe et al, 1995; Ottesen et al, 1997; Looijenga keratoderma, that can be caused by mutations in the desmosomal et al, 2000; Skotheim et al, 2001). Prostaticcarcinoma is the most cadherin, desmoglein 1, the constitutive desmosomal plaque common form of male cancer found in the Western world and member desmoplakin, or the type II epidermal keratin, keratin 1 LOH has been found for at least two locations on 18q12-21 (Armstrong et al, 1999; Rickman et al, 1999; Whittock et al, 1999; (Bostwick et al, 1998; Verma et al, 1999; Padalecki et al, 2000; Chu Hunt et al,2001;Kljuicet al, 2003; Whittock et al, 2002a). Muta- et al,2001;Yinet al, 2001). The role of cell-cell adhesion molecules tions in the desmosomal plaque member plakophilin 1 can result in cancer is well understood as mutations in E-cadherin underlie in an autosomal recessive condition characterized by ectodermal di¡use gastric cancers and lobular breast cancers (Berx et al,1998), dysplasia and skin fragility (McGrath et al, 1997; McGrath et al, and in most cases, the mutations occur in combination with LOH 1999; Whittock et al, 2000a; Hamada et al, 2002), and more re- of the wild-type allele. Loss of heterozygosity has also been re- cently, mutations in plakoglobin and desmoplakin have been ported at 18q12 in esophageal carcinomas as well as cancer of the shown to underlie a group of autosomal recessive conditions in- head and neck (Papadimitrakopoulou et al, 1998; Karkera volving skin, hair and heart (McKoy et al, 2000; Norgett et al, et al, 1999; Pack et al, 1999). It is therefore possible that mutations 2000; Whittock et al, 2002b). in desmoglein 4 in combination with LOH may underlie some Indeed, the involvement of desmosomal proteins in human forms of testicular or prostate cancer. disease does not end with inherited disorders, as desmoglein 1, In summary, using geneticanalysis of human genomicdata we desmoglein 3, and desmoplakin are targets for the autoimmune have identi¢ed a novel member of the human desmoglein gene blistering diseases pemphigus foliaceous, pemphigus vulgaris, family that demonstrates highest homology to desmoglein 3, and paraneoplasticpemphigus (Stanley et al, 1984; Labib et al, and using RT-PCR demonstrate that the transcript and presum- 1990; Amagai et al, 1991; Oursler et al, 1992; Joly et al,1994; ably its protein is expressed only in speci¢c tissues. Further work Olague-Alcala et al, 1994; Emery et al,1995;Linet al, 1997a; Lin is now required to con¢rm desmoglein 4 expression at the pro- et al, 1997b; Allen and Camisa, 2000; Lin et al, 2000). The extra- tein level and to identify in which cell layers desmoglein 4 is ex- cellular domain of desmoglein 1 is also the target of exfoliative pressed. In addition, studies are required to determine whether toxins ETA and ETB from Staphylococcal aureus that cause the some cases of inherited disorders of desmosomes such as striate VOL. 120, NO. 4 APRIL 2003 HUMAN DESMOGLEIN 4 529 palmoplantar keratoderma are caused by mutations in desmogle- Denning MF, Guy SG, Ellerbroek SM, Norvell SM, Kowalczyk AP, Green KJ: The in 4, to see whether desmoglein 4 is a target antigen of autoanti- expression of desmoglein isoforms in cultured human keratinocytes is regu- lated by calcium, serum, and protein kinase C. Exp Cell Res 239:50^59, 1998 bodies in pemphigus vulgaris or pemphigus foliaceus, and to Emery DJ, Diaz LA, Fairley JA, Lopez A,Taylor AF, Giudice GJ: Pemphigus foliaceus determine whether the extracellular domain of desmoglein 4 is and pemphigus vulgaris autoantibodies react with the extracellular domain of cleaved by either or both exfoliative toxins A and B. desmoglein-1. J Invest Dermatol 104:323^328, 1995 Frank J, Cserhalmi-Friedman PB, Ahmad W, Panteleyev AA, Aita VM, Christiano AM: Characterization of the desmosomal cadherin gene family: Genomic organization of two desmoglein genes on human chromosome 18q12. Exp Der- ELECTRONIC-DATABASE INFORMATION matol 10:90^94, 2001 Garrod DR: Desmosomes and hemidesmosomes. Curr Opin Cell Biol 5:30^40, 1993 URLs for data in this article are as follows: Garrod DR: Desmosomes and hemidesmosomes: Epithelial development and di¡erentiation: The role of desmosomes. 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