Proc. Nadl. Acad. Sci. USA Vol. 88, pp. 47%-4800, June 1991 Cell Biology Desmosomal glycoprotein DGI, a component of intercellular desmosome junctions, is related to the cadherin family of cell adhesion molecules (desmosome/desmoglein/ceD-cell junction) G. N. WHEELER*, A. E. PARKER*, C. L. THOMAS*, P. ATALIOTIS*, D. POYNTER*, J. ARNEMANN*, A. J. RUTMAN*, S. C. PIDSLEY*, F. M. WATTt, D. A. REES*, R. S. BUXTON*, AND A. I. MAGEE*t *National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom; and tImperial Cancer Research Fund, Lincoln's Inn Fields, London WC2A 3PX, United Kingdom Communicated by James Gowans, February 4, 1991 (received for review November 22, 1990) ABSTRACT Among the variety ofspecialized intercellular with the plaque or intermediate filaments. In contrast, the junctions, those of the adherens type have the most obvious cadherin family of proteins has been shown to codistribute association with cytoskeletal elements. This may be with the with actin bundles in the adherens type of cell junction (3). actin microfilanent system as in the zonula adherens or with intermediate ifiaments as in the macula adherens, or desmo- METHODS some. In the former case, it is clear that transmembrane glycoproteins of the cadherin family are important adhesive Generation and Screening ofAntibodies. Desmosomes were components of the molecular assembly. We now show for isolated from bovine muzzle epidermis (4). Rabbit anti-DGI desmosomes that a major glycoprotein component (desmo- serum was generated against DGI purified by SDS/PAGE somal glycoprotein DGI) has extensive homology with the and electroelution; its specificity was similar to the guinea pig cadherins, defining an extended fmily, but also has unique sera previously described (4). The serum specifically recog- features in its cytoplasmic domain that are likely to be relevant nized DGI on immunoblots of cow's nose desmosomes, to the association with intermediate rather than actin filaments. precipitated a 150-kDa protein from metabolically labeled A novel 282-residue extension contains repeats of --29 amino MDCK canine kidney epithelial cells, and stained MDCK acid residues predicted to have an antiparallel P-sheet struc- cells in a punctate cell junctional pattern by immunofluores- ture, followed by a glycine-rich sequence. As in the cadherins, cence microscopy (data not shown; ref. 4). Polyclonal anti- the extracellular domain contains possible Ca2+-binding se- bodies to DGI were affinity-purified on DGI immobilized on quences and a potential protease processing site. The cell Sepharose CL-4B (CNBr-Sepharose; Pharmacia). adhesion recognition region (His-Ala-Val) of the cadherins is A DGI-specific IgG mouse hybridoma (3D1) was generated modified to Arg-Ala-Leu. against bovine DGI. The antibody produced by this hybrid- oma recognized DGI on immunoblots of desmosomes, but The molecules that are involved in cell-cell adhesion are now did not immunoprecipitate well, and gave distinct punctate becoming known. Thus the Ca2l-dependent cadherin cell cell border staining on epithelial cell types, especially if the adhesion molecules (CAMs) have been distinguished from cells were unfixed (data not shown). Cell permeabilization the immunoglobulin-related proteins such as neural (N-) was required for staining, suggesting that the antibody rec- CAM (1). Little is known, however, of the molecules in- ognized intracellular epitopes (data not shown). volved in adhesion mediated by the desmosome, which is one For the preparation of antibodies to TrpE fusion proteins, of the major adhesive junctions of epithelial tissues with a a Sty I-Xho I fragment [base pairs (bp) 2218-3422] from clone characteristic and well-defined morphology (2). Two classes G4, encoding the intracellular part of the DGI molecule, of desmosomal proteins are candidates for a role in cell including the DGI-specific repeats, was subcloned in frame adhesion: (i) the glycosylated proteins DGII (Mr 120,000) and into the Xba I-Sal I restriction sites ofthe pATH2 expression DGIII (Mr 110,000), which have been shown to be related to vector (5). The TrpE fusion protein was expressed, separated one another by peptide mapping and immunological criteria; in a polyacrylamide gel, excised, and electroeluted. Rabbits and (it) another glycosylated protein, DGI (desmoglein; Mr were immunized and the resulting sera were preabsorbed 150,000) (2). These proteins are, like the cadherins, Ca2 - with Sepharose-bound proteins from Escherichia coli ex- binding transmembrane proteins, unlike other desmosomal pressing the pATH2 vector alone. Immunoblot analysis of proteins such as plakoglobin and the desmoplakins, which are fusion proteins expressed by putative DGI recombinants was present in an electron-dense plaque zone underneath the cell carried out according to Price et al. (6). membrane. These latter proteins may provide a link to the Peptide Sequencing. Bovine DGI purified by SDS/PAGE intermediate filaments with which desmosomes interact. and electroelution was dialyzed against 50 mM Mops buffer Here we provide detailed information on the structure of (pH 7) containing 0.05% SDS and was concentrated using an the desmosomal glycoprotein DGI, through the isolation and Amicon CX30 immersible concentrator. The protein was sequence analysis of cDNA clones.§ Our results show that digested with trypsin or Staphylococcus aureus V8 protease DGI is indeed related to the cadherin family of cell adhesion (Boehringer Mannheim) (1-5% enzyme/substrate weight ra- proteins, albeit more distantly than other members of the tio) overnight at 370C. Fragments were resolved by SDS/ family, and is thus implicated as having a role in adhesion. PAGE and transferred to Immobilon membrane (0.45 Eum; DGI differs from the cadherins in having a unique large Millipore). After staining with Coomassie brilliant blue, the cytoplasmic domain, which may be involved in interactions Abbreviation: CAM, cell adhesion molecule. The publication costs ofthis article were defrayed in part by page charge tTo whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" §The sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. X56654). 4796 Downloaded by guest on September 24, 2021 Cell Biology: Wheeler et al. Proc. Natl. Acad. Sci. USA 88 (1991) 4797 fragments were cut out and sequenced using an Applied 30 min at 65°C. They were then dried and exposed to Kodak Biosystems 477A sequenator with on-line phenylthiohydan- XAR-5 film. toin (PTH) analyzer model 120A. For Southern blotting, human genomic DNA was prepared Library Construction and Screening. A Agtll cDNA library from EDTA-treated blood of healthy male donors according (Clontech) prepared using mRNA from cultured human ke- to standard procedures (9). DNA (5 ,ug) was digested with ratinocytes was screened with affinity-purified anti-DGI an- various restriction enzymes, electrophoresed in 1% agarose tibodies (see above), essentially as detailed (7). The initial G1 gels, and transferred to Hybond-N membranes. Filters were clone was plaque-purified and shown to have an insert size of hybridized with various fragments of the DGI cDNA, 32p_ 1.1 kilobases (kb). To obtain clones covering more of the labeled by the random primer method (9). PCR-mediated coding sequence, we constructed a cDNA library in Agtll, amplification ofcDNA fragments was carried out in a volume using random hexanucleotide primers with 1 Ag of mRNA of 50 ,ud according to the supplier's recommendation (Cetus), isolated from human keratinocytes (Amersham cDNA syn- using cDNA clone pG4 as a source. thesis and cloning kit). This library and the poly(A)+ library DNASequencing. Phage A DNA was purifiedfromplate lysates were probed with a 32P-labeled G1 cDNA and yielded several (9), digested with EcoRI, separated in agarose gels, purified using overlapping clones. Geneclean (Bio 101, LaJolla, CA), and subcloned into M13 mplO Northern and Southern Analysis. For Northern blot anal- and pBluescript KS(+). The DNA sequence was determined by ysis, poly(A)+ RNA was isolated from cultured human epi- the dideoxynucleotide chain-termination method (10) using Se- dermal keratinocytes essentially by the guanidinium isothio- quenase (United States Biochemical). The initial G1 clone was cyanate method of Chirgwin et al. (8). Total RNA was sequenced by subcloning restriction fragments into M13. Sub- isolated from a myeloma cell line (JKAg8) that does not sequent clones were sequenced using appropriate oligodeoxy- express DGI. The RNA was electrophoresed in a 1% agarose nucleotides (synthesized with an Applied Biosystems synthe- gel containing 2.2 M formaldehyde and transferred to Hy- sizer) as primers. The whole ofclone G4 was sequenced in both bond-N by standard procedures (9). Hybridizations were directions. carried out for 16 hr at 480C with 32P-labeled probes in 1.5x SSC (0.225 M NaCI/0.0225 M trisodium citrate, pH 7.0) RESULTS 50%o 1% dried milk containing (vol/vol) formamide, (wt/vol) Isolation and Characterization of DGI cDNA. A human (Marvel, U.K.), 50 mM Tris Cl (pH 7.6), 3% (wt/vol) SDS, keratinocyte Agtll library was screened using affinity- 10% (wt/vol) dextran sulfate, and boiled salmon sperm DNA purified rabbit anti-DGI antibodies (see Methods). Screening at 25 u&g/ml. Filters were washed once with lx SSC/0.2% of -150,000 recombinants identified 3 positive clones. The SDS for 30 min and three times with 0.lx SSC/O.1% SDS for fusion proteins encoded by these recombinants were ana- lyzed by Western immunoblotting; the results were the same 13, i.: ',All '.' for all three clones. The fusion protein of A clone G1 showed I a molecular mass of -150 kDa (Fig. 1A, lane 1) when probed A with an anti-l3-galactosidase antibody; the lower bands were probably due to degradation. The fusion protein reacted 21)0 - A\[ A.- ki - _ *f: .:,.. C4. f, 0 fi! fo M,,.* 6 ': 68 - D LERlH C DPLGYGNVTVTESYTTSDTLKPSVHVHDNRPASNVWVTERWVGPI SGADLHGMLEMPDLRDGS 1 111111 LIYFNVTVTEWYT V8 peptide 1111111111i 1111111111 VVGPISGADLQGMLEMPDLRDGS 11 I 11 tryptic peptide YTSSGTL V8 peptide FIG.