Two Forms of Collagen XVII in Keratinocytes
1998; Elsevier BV; Volume: 273; Issue: 40 Linguagem: Inglês
10.1074/jbc.273.40.25937
ISSN1083-351X
AutoresHeike Schäcke, Hauke Schumann, Nadja Hammami-Hauasli, Michael Raghunath, Leena Bruckner‐Tuderman,
Tópico(s)Cell Adhesion Molecules Research
ResumoThe cDNA sequence of human collagen XVII predicts an unusual type II transmembrane protein, but a biochemical characterization of this structure has not been accomplished yet. Using domain-specific antibodies against recombinant collagen XVII fragments, we identified two molecular forms of the collagen in human skin and epithelial cells. Full-length collagen XVII appeared as a homotrimeric transmembrane molecule of three 180-kDa α1(XVII) chains. The globular intracellular domain was disulfide-linked, and theN-glycosylated extracellular domain of three 120-kDa polypeptides was triple-helical at physiological temperatures. A second, soluble form of collagen XVII in keratinocyte culture media was recognized with antibodies to the ectodomain, but not the endodomain. The soluble form exhibited molecular properties of the collagen XVII ectodomain: a triple-helical, N-glycosylated molecule of three 120-kDa polypeptides. Northern blot analysis with probes spanning either the distal 5′or the distal 3′ end of the collagen XVII cDNA revealed an identical 6-kb mRNA, suggesting that both the 180- and 120-kDa polypeptides were translated from the same mRNA, and that the 120-kDa polypeptide was generated post-translationally. In concert, keratinocytes harboring a homozygous nonsense mutation in theCOL17A1 gene synthesized neither the 180-kDa α1(XVII) chain nor the 120-kDa polypeptide. Finally, treatment of normal keratinocytes with a synthetic inhibitor of furin proprotein convertases, decanoyl-RVKR-chloromethyl ketone, prevented the generation of the 120-kDa polypeptide. These data strongly suggest that the soluble 120-kDa polypeptide represents a specifically cleaved ectodomain of collagen XVII, generated through furin-mediated proteolytic processing. Thus, collagen XVII is not only an unusual type II transmembrane collagen, but the first collagen with a specifically processed, soluble triple-helical ectodomain. The cDNA sequence of human collagen XVII predicts an unusual type II transmembrane protein, but a biochemical characterization of this structure has not been accomplished yet. Using domain-specific antibodies against recombinant collagen XVII fragments, we identified two molecular forms of the collagen in human skin and epithelial cells. Full-length collagen XVII appeared as a homotrimeric transmembrane molecule of three 180-kDa α1(XVII) chains. The globular intracellular domain was disulfide-linked, and theN-glycosylated extracellular domain of three 120-kDa polypeptides was triple-helical at physiological temperatures. A second, soluble form of collagen XVII in keratinocyte culture media was recognized with antibodies to the ectodomain, but not the endodomain. The soluble form exhibited molecular properties of the collagen XVII ectodomain: a triple-helical, N-glycosylated molecule of three 120-kDa polypeptides. Northern blot analysis with probes spanning either the distal 5′or the distal 3′ end of the collagen XVII cDNA revealed an identical 6-kb mRNA, suggesting that both the 180- and 120-kDa polypeptides were translated from the same mRNA, and that the 120-kDa polypeptide was generated post-translationally. In concert, keratinocytes harboring a homozygous nonsense mutation in theCOL17A1 gene synthesized neither the 180-kDa α1(XVII) chain nor the 120-kDa polypeptide. Finally, treatment of normal keratinocytes with a synthetic inhibitor of furin proprotein convertases, decanoyl-RVKR-chloromethyl ketone, prevented the generation of the 120-kDa polypeptide. These data strongly suggest that the soluble 120-kDa polypeptide represents a specifically cleaved ectodomain of collagen XVII, generated through furin-mediated proteolytic processing. Thus, collagen XVII is not only an unusual type II transmembrane collagen, but the first collagen with a specifically processed, soluble triple-helical ectodomain. polyacrylamide gel electrophoresis digoxigenin kilobase(s). Collagen XVII, also known as the 180-kDa bullous pemphigoid antigen or BP180, is a structural component of the hemidesmosomes in epithelial cells (1Nishizawa Y. Uematsu J. Owaribe K. J. Biochem. (Tokyo). 1993; 113: 493-501Crossref PubMed Scopus (137) Google Scholar). The cDNA sequence predicts a type II integral transmembrane protein of 1497 amino acids, with an NH2-terminal intracellular domain of 466 amino acids, a transmembrane domain of 23 residues and a COOH-terminal extracellular domain of 1008 amino acid residues (2Giudice G.J. Emery D.J. Diaz L.A. J. Invest. Dermatol. 1992; 99: 243-250Abstract Full Text PDF PubMed Scopus (486) Google Scholar). Because of 15 collagenous subdomains characterized by -Gly-X-Y- repeat sequences within the ectodomain, the molecule was designated collagen XVII (3Li K. Tamai K. Tan E.M.L. Uitto J. J. Biol. Chem. 1993; 268: 8825-8834Abstract Full Text PDF PubMed Google Scholar, 4Gatalica B. Pulkkinen L. Li K. Kuokkanen K. Ryynänen M. McGrath J. Uitto J. Am. J. Hum. Genet. 1997; 60: 352-365PubMed Google Scholar). Traditionally, collagens are defined as triple-helical proteins with -Gly-X-Y- repeat sequences and with a function as a structural protein of the extracellular matrix (5Prockop D.J. Kivirikko K.I. Annu. Rev. Biochem. 1995; 64: 403-434Crossref PubMed Scopus (1388) Google Scholar). Among the more than 20 homo- and heterotrimeric collagens, types XVII and XIII represent the only putative transmembrane collagens (for review, see Ref. 6Pihlajaniemi T. Rehn M. Progr. Nucleic Acids Res. Mol. Biol. 1995; 50: 225-262Crossref PubMed Scopus (78) Google Scholar). 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In concert with these findings, recombinant extracellular fragments of human collagen XVII expressed in COS-1 cells showed a high molecular mass form with an elongated conformation (8Balding S.D. Diaz L.A. Giudice G.J. Biochemistry. 1997; 36: 8821-8830Crossref PubMed Scopus (61) Google Scholar). Immunoelectron microscopy demonstrated that antibodies against recombinant ectodomain fragments labeled structures outside the keratinocyte plasma membrane along the skin basement membrane (9Masunaga T. Shimizu H. Yee C. Borradori L. Lazarova Z. Nishikawa T. Yancey K.B. J. Invest. Dermatol. 1997; 109: 200-206Abstract Full Text PDF PubMed Scopus (99) Google Scholar). The functions of collagen XVII are not known, but as a transmembrane component of the hemidesmosomes, it is likely to play a role in maintaining linkage between the intracellular and the extracellular structural elements and in anchoring the epithelia to the underlying basement membrane (10Hopkinson S.B. Baker S.E. Jones J.C.R. J. 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Invest. Dermatol, in pressGoogle Scholar). Despite a growing number of COL17A1 mutations, the genotype-phenotype correlations and the molecular mechanisms underlying the phenotypes have remained elusive as a result of insufficient information on the structure and functions of normal human collagen XVII. In the present study, we used domain-specific antibodies and biochemical analyses to characterize collagen XVII from normal human skin and epidermal keratinocytes. We show that the collagen occurs in two triple-helical forms, as a full-length transmembrane protein and as a soluble ectodomain, a specific proteolytic cleavage product of the full-length molecule. Recombinant fusion proteins corresponding to two different fragments of the human collagen XVII were generated using the bacterial pQE expression system (Qiagen, Hilden, Germany). The fusion proteins contained an NH2-terminal His tag for easy purification of the expression products from bacterial lysates. The fusion protein Col17-intra spanned amino acids 61–360 in the intracellular domain, whereas the fusion protein Col17-extra corresponded to amino acids 1292–1497 in the carboxyl terminus of the extracellular domain of collagen XVII (Fig. 1). The corresponding cDNAs were synthesized by First Strand cDNA Synthesis Kit (Amersham, Braunschweig, Germany) of mRNA from normal human keratinocytes isolated by the QuickPrep mRNA isolation kit (Pharmacia, Freiburg, Germany). The polymerase chain reaction amplification was performed with the sense primer 5′-ACGGATCCGCAGCGGCTACATAAACTC-3′ and the antisense primer 5′-GCGAAGCTTGCAGCTCCATCTCCTTCTT-3′for the fusion protein Col17-intra, and with the sense primer 5′-CGGGATCCAGCAGCTCCTCCTCACACA-3′ and the antisense primer 5′-GCGAAGCTTGAGACCTTGGACCTAAGTG-3′ for the fusion protein Col17-extra. To generate the expression clones, 5′ end primers contained a BamHI site and 3′ end primers contained aHindIII site for cloning into the vector pQE32 (Col17-intra) and pQE30 (Col17-extra). The correct orientation and ligation of both clones was verified by dideoxynucleotide sequence analysis. The fusion proteins were produced using the QIAexpress Type IV kit (Qiagen) according to the manufacturer′s instructions and purified with affinity chromatography on Ni2+-NTA-agarose. Immunization of rabbits with the fusion proteins was performed using standard procedures (Eurogentec, Ougreé, Belgium). Since no immune response was obtained for Col17-extra, chicken were immunized, and the antibodies were purified from egg yolks by filtration and precipitation with polyethylene glycol (29Gassmann M. Thommes P. Weiser T. Hubscher U. FASEB J. 1990; 4: 2528-2532Crossref PubMed Scopus (304) Google Scholar). Bullous pemphigoid patient autoantiserum IF 77/95 (Fig. 1) that was strongly reactive with the NC16a-domain (15Zillikens D. Pose P.A. Balding S.D. Liu Z. Olague-Marchan M. Diaz L.A. Giudice G.J. J. Invest. Dermatol. 1997; 109: 573-579Abstract Full Text PDF PubMed Scopus (276) Google Scholar), but not with other domains of collagen XVII, was also used for immunoblotting. Normal human keratinocytes were obtained by trypsinization of skin biopsy samples, the human keratinocyte cell line HaCaT was a generous gift of Dr. N. Fusenig, German Cancer Research Center (DKFZ), Heidelberg, Germany. All cells were cultured in serum-free, low calcium keratinocyte growth medium, supplemented with bovine pituitary extract and epidermal growth factor (KGM, Life Technologies, Inc.) as described previously (30König A. Bruckner-Tuderman L. J. Cell Biol. 1992; 117: 679-685Crossref PubMed Scopus (68) Google Scholar). Prior to extraction and immunoblotting experiments, the cells were grown in the presence of 50 μg/ml l-ascorbate for 48 h (26Schumann H. Hammami-Hauasli N. Pulkkinen L. Mauviel A. Küster W. Lüthi U. Owaribe K. Uitto J. Bruckner-Tuderman L. Am. J. Hum. Genet. 1997; 60: 1344-1353Abstract Full Text PDF PubMed Scopus (73) Google Scholar). For analysis of collagen XVII from cell cultures, proteins in the cell layer and the media were processed separately. The cell layers were extracted for 30 min on ice with 1 ml/75 cm2 of a neutral buffer containing 1% Nonidet P-40, 0.1 m NaCl, 25 mm Tris-HCl, pH 7.4, and 10 mm EDTA, 1 mm Pefabloc (Merck, Darmstadt, Germany), and when appropriate,14 μg/ml chymostatin, 7 μg/ml antipain, 7 μg/ml leupeptin, and 14 μg/ml pepstatin as proteinase inhibitors (31Sonnenberg A. Gehlsen K.R. Aumailley M. Timpl R. Exp. Cell Res. 1991; 197: 234-244Crossref PubMed Scopus (63) Google Scholar). The cell lysate was then scraped with a rubber policeman, and the extract was centrifuged at 14,000 ×g at 4 °C. The supernatant was used for further analyses. In some experiments, the above extraction was preceded by incubation of the cells with 0.5 m NaCl in 0.05 m Tris-HCl, pH 8.2, to release neutral salt soluble proteins. For analysis of medium proteins, proteinase inhibitors were added immediately after collecting the medium onto ice. After removing cellular debris by centrifugation 1000 rpm for 10 min, the proteins from 10 ml medium were precipitated with ammonium sulfate to 30% saturation for 4 h at 4 °C. After centrifugation at 15 000 × g for 60 min at 4 °C, the pellets were dissolved in 100 μl of a buffer containing 65 mm NaCl, 25 mm Tris-HCl, pH 7.4, 1 mm Pefabloc (Merck), and 1 mm EDTA (32Marinkovich M.P. Lunstrum G.P. Keene D.R. Burgeson R.E. J. Cell Biol. 1992; 119: 695-703Crossref PubMed Scopus (235) Google Scholar). Fifty to 100 μl of the cell extracts or the medium concentrate were used for the enzyme digestions and 10–30 μl for immunoblotting. For extraction of collagen XVII from the epidermis, normal human skin was subjected to artificial epidermolysis in a neutral buffer containing 20 mm EDTA and the above proteinase inhibitors at 4 °C overnight (33Meyer L.J. Taylor T.B. Kadunce D.P. Thuong-Ngyen V. Zone J.J. J. Invest. Dermatol. 1991; 96: 991-993Abstract Full Text PDF PubMed Google Scholar), and the epidermis and the dermis were mechanically separated. Both skin layers were extracted with 400 μl/cm2 of a buffer containing 8 m urea, 2% SDS, 0.05 m Tris-HCl, pH 6.8, and proteinase inhibitors for 2 min at 95 °C, followed by extensive dialysis against 0.8m urea, 2% SDS, 5% glycerol in 0.1 m Tris, pH 6.8, as described previously (30König A. Bruckner-Tuderman L. J. Cell Biol. 1992; 117: 679-685Crossref PubMed Scopus (68) Google Scholar). Five to 15 μl of the extract was used for immunoblotting; application of significantly more than 15 μl of the epidermis extract onto SDS-PAGE1 was not possible, because the high content of keratins in the extract caused protein overloading of the lanes. For immunoblotting, proteins were separated on SDS-PAGE using gels with either 7% polyacrylamide or 3–15% polyacrylamide gradients under non-reducing or reducing (1 mm dithiothreitol) conditions. The incubations with the first antibodies were overnight, and with the alkaline phosphatase-linked anti-rabbit, -chick, and -human second antibodies for 2 h. Semiconfluent keratinocytes or HaCaT cells were incubated with 50 μg/ml ascorbic acid for 48 h, washed extensively and incubated with 3 μg/mld-biotinoyl-ε-aminocaproic acidN-hydroxysuccinimide ester (Boehringer Mannheim, Mannheim, Germany) in 0.15 m NaCl, 0.05 m sodium borate, pH 8.0, for 15 min. The reaction was stopped with 1 mmNH4Cl. After extensive washing, the cells were extracted with the Nonidet P-40-containing extraction buffer as described above, followed by immunoprecipitation with domain-specific collagen XVII antibodies. Prior to immunoprecipitation, preclearing was achieved by incubating 30 μl of protein A-Sepharose (Pharmacia, Uppsala, Sweden) with 500 μl of biotinylated cell extract containing 0.1% SDS for 2 h at 4 °C. After centrifugation for 5 min at 300 × g, the supernatants were added to protein A-Sepharose/antibody complexes. These were prepared by adding 50 μl of rabbit antibody SA3485 to 30 μl of protein A-Sepharose in 500 μl of the above Nonidet P-40 buffer with 0.1% SDS (Nonidet P-40 buffer, 0.1% SDS). Alternatively, 50 μl of chicken antibody Col17ecto-1 and 5 μl of rabbit-anti-chicken-IgG (Dianova, Hamburg, Germany) were added to 30 μl of protein A-Sepharose in 500 μl of Nonidet P-40 buffer, 0.1% SDS. The complexes were rotated for 2 h at 4 °C and centrifuged at 300 × g for 5 min, and the antibody/protein-A-Sepharose pellets were washed three times with Nonidet P-40 buffer, 0.1% SDS. The supernatants of precleared cell extracts were added to these antibody complexes and rotated at 4 °C overnight. After extensive washing with Nonidet P-40 buffer, 0.1% SDS, the pellets were suspended in 0.8 m urea, 2% SDS, 5% glycerol, 0.1 m Tris, pH 6.8, heated for 5 min at 95 °C, and centrifuged for 5 min at 300 × g. The supernatants were loaded onto 7% SDS-PAGE and analyzed in blots using streptavidin-coupled alkaline phosphatase (Sigma, Deisenhofen, Germany) for detection. For assessment of the domain structure and stability of collagen XVII, cell and medium extracts were subjected to collagenase, pepsin, sequential pepsin/trypsin, orN-glycosidase F digestions. The incubation with 40 units/ml highly purified bacterial collagenase (Advanced Biofacturers Inc., Lynbrook, NY) was carried out in 50 μl of the cell extraction buffer containing 15 mm CaCl2 and 1 mmPefabloc (Merck) for 4 h at 37 °C (34Burgeson R.E. J. Invest. Dermatol. 1993; 101: 252-255Abstract Full Text PDF PubMed Google Scholar). For a limited pepsin digestion of collagen XVII, 100 μl of extracts were acidified by adding glacial acetic acid to a final concentration of 0.1m, and the samples were incubated with 1 μg/ml pepsin (Fluka, Deisenhofen, Germany) at 5 °C for 2–24 h (34Burgeson R.E. J. Invest. Dermatol. 1993; 101: 252-255Abstract Full Text PDF PubMed Google Scholar). After neutralization with unbuffered Tris, the samples were either directly precipitated with ethanol at −20 °C overnight or treated with 10 μg/ml trypsin (Sigma) at temperatures between 15 °C and 47 °C for 2 min (35Bruckner P. Prockop D.J. Anal. Biochem. 1981; 110: 360-368Crossref PubMed Scopus (231) Google Scholar, 36Raghunath M. Bruckner P. Steinmann B. J. Mol. Biol. 1994; 236: 940-949Crossref PubMed Scopus (116) Google Scholar). The reaction was stopped by adding soy bean trypsin inhibitor (Sigma) to a final concentration of 10 μg/ml (36Raghunath M. Bruckner P. Steinmann B. J. Mol. Biol. 1994; 236: 940-949Crossref PubMed Scopus (116) Google Scholar). For deglycosylation, 50 μl of the protein extract were treated with 10% β-mercaptoethanol for 10 min at 60 °C prior to digestion with 10 units/ml N-glycosidase F (Boehringer Mannheim, Mannheim, Germany) overnight at 37 °C. For Northern blotting, 1.5 μg of mRNA isolated from cultured cells with Oligotex Direct mRNA Minikit (Qiagen, Hildesheim, Germany) was separated on a 0,8% agarose gel containing formaldehyde, transferred onto a positively charged nylon membrane (Boehringer Mannheim) overnight, and immobilized by baking at 120 °C for 30 min. The membranes were pre-hybridized and then hybridized with digoxigenin (DIG)-labeled collagen XVII cDNA (4Gatalica B. Pulkkinen L. Li K. Kuokkanen K. Ryynänen M. McGrath J. Uitto J. Am. J. Hum. Genet. 1997; 60: 352-365PubMed Google Scholar) at 50 °C. The DIG labeling of the cDNA was performed with the DIG DNA labeling kit (Boehringer Mannheim, Mannhein, Germany) following the manufacturer′s instructions for randomly primed DNA labeling. After hybridization, the filters were washed to a final stringency of 0.1 × standard saline citrate, 0.1% sodium dodecyl sulfate at 65 °C. The cDNA-mRNA hybrids were detected with alkaline phosphatase-labeled anti-DIG-antibodies and visualized by chemiluminescence using CDP-StarTM substrate (Boehringer Mannheim). Normal keratinocytes or HaCaT cells were grown in the presence of 50 μg/ml ascorbic acid. As proteinase inhibitors, 1–5 mm EGTA, 0.1–1 mm Pefabloc (Merck), or 100 μm decanoyl-RVKR-chloromethyl ketone (Ref. 37Garten W. Hallenberger S. Ortmann D. Schäfer W. Vey M., H. Angliker H. Shaw E. Klenk H.D. Biochimie (Paris). 1994; 76: 217-225Crossref PubMed Scopus (140) Google Scholar; Bachem, Basel, Switzerland) were added. After a culture period of 20 h, the medium and cell layer were analyzed with immunoblotting as described above. Polyclonal antibodies raised against recombinant procaryotic fragments spanning the endodomain and the distal ectodomain of collagen XVII (Fig. 1) gave an intensive immune response in rabbits (antibody SA 3485) or in chicken (antibody Col17ecto-1). The antibodies showed no cross-reactivity or reaction with other hemidesmosomal components or basement membrane proteins, such as BP230, laminin 5, collagen IV, or collagen VII. In contrast, they specifically recognized collagen XVII extracted from skin or cultured cells in immunoblots and by immunoprecipitation. They did not work in immunofluorescence staining of skin cryosections or cultured cells. Collagen XVII could be extracted from human epidermis, but not dermis (Fig. 2 A) with a chaotropic buffer containing 8 m urea and 2% SDS as described previously (30König A. Bruckner-Tuderman L. J. Cell Biol. 1992; 117: 679-685Crossref PubMed Scopus (68) Google Scholar). The native conformation of collagen XVII was preserved by extraction of cultured keratinocytes with a neutral buffer containing 1% Nonidet P-40 as a detergent (31Sonnenberg A. Gehlsen K.R. Aumailley M. Timpl R. Exp. Cell Res. 1991; 197: 234-244Crossref PubMed Scopus (63) Google Scholar). Notably, the α1(XVII) chains from both sources showed similar migration on SDS-PAGE (Fig. 2 A, lanes 1 and 2), indicating that the tissue form of collagen XVII was similar to the cell form, and that no typical procollagen → collagen conversion (38Bruckner-Tuderman L. Nilssen Ö. Zimmermann D. Dours-Zimmermann M.-T. Kalinke U.D. Gedde-Dahl Jr., T. Winberg J.-O. J. Cell Biol. 1995; 131: 551-559Crossref PubMed Scopus (128) Google Scholar) occurred prior to deposition in the tissue. The detergent was necessary for solubilization of collagen XVII from the cells, since no collagen XVII was extractable with phosphate-buffered saline or 0.5 m NaCl, 0.1 m Tris, pH 7.4, or 0.1 m acetic acid (data not shown). The transmembrane location of collagen XVII was verified biochemically using keratinocyte cell surface biotinylation and subsequent immunoprecipitation with antibodies to the endo- and ectodomains. Specifically, semiconfluent keratinocytes were biotinylated under conditions that preserved cell integrity to allow for labeling of the putative extracellular domain only. The cells were then extracted with a neutral buffer containing 1% Nonidet P-40, collagen XVII was immunoprecipitated from the extract, and the precipitates were visualized with streptavidin as a marker. Both antibodies SA 3485 and Col17ecto-1 precipitated a biotinylated 180-kDa band (Fig. 2 B), demonstrating that the polypeptide recognized by them contained a biotinylated extracellular domain. For characterization of the domain structure, native collagen XVII was subjected to limited proteolytic digestions. Treatment with highly purified bacterial collagenase yielded a band of approximately 65 kDa on SDS-PAGE under reducing conditions (Fig. 3 A), corresponding to the fragment predicted from the cDNA sequence (2Giudice G.J. Emery D.J. Diaz L.A. J. Invest. Dermatol. 1992; 99: 243-250Abstract Full Text PDF PubMed Scopus (486) Google Scholar). Under non-reducing conditions, the collagenase-resistant fragment migrated with an apparent molecular mass of 170 kDa (Fig. 3 C), suggesting that the endodomain formed a disulfide-linked trimer. In concert with this observation, intact collagen XVII migrated as a large polymer under non-reducing conditions (Fig. 3 C). The collagenase-resistant bands were recognized by the endodomain antibody SA 3485, but not by the ectodomain antibodies. Limited pepsin and/or trypsin digestion was used to isolate the collagenous domain, since triple-helical structures resist proteolysis under conditions in which globular sequences are digested (35Bruckner P. Prockop D.J. Anal. Biochem. 1981; 110: 360-368Crossref PubMed Scopus (231) Google Scholar). Incubation of collagen XVII with 1 μg/ml pepsin at 5 °C for 2 h was sufficient to digest the endodomain. During an extended incubation, the digestion proceeded in a stepwise manner, yielding several intermediate products with apparent sizes of 140, 120, and 105 kDa, and a final pepsin-resistant fragment of about 90 kDa, both under reducing and non-reducing conditions (Fig. 3, B and C), demonstrating that the collagenous domain did not contain disulfide bonds. Limited trypsin digestion also resulted in a 90-kDa fragment (Fig. 3 B, lane 5), indicating that the collagenous domain was triple-helical. The NH2 terminus of the pepsin/trypsin-resistant fragments extends to the NC16a domain, since the antibody IF 77/95 recognized all intermediate fragments. In contrast, antibody Col17ecto-1 raised against the 205 most COOH-terminal amino acid residues of collagen XVII reacted only with the 140-, 120-, and 105-kDa fragments, but not with the 90-kDa fragment (Fig. 4,lane 9), indicating that an extended pepsin incubation or trypsin treatment eliminated the distal ectodomain (compare with Fig. 1). Deglycosylation withN-glycosidase F resulted in faster migration of the 180-kDa α1(XVII) chain on SDS-PAGE (Fig. 4, lanes 1 and 2). When collagenase digestion preceded deglycosylation, no difference in the molecular mass of the endodomain was noted (Fig. 4,lanes 3 and 4). In contrast, differences emerged when the extracellular domain was deglycosylated: shifts in migration of the 140- and 120-kDa, but not of the 105- and 90-kDa pepsin fragments were observed (Fig. 4, lanes 5–8). These findings are consistent withN-glycosylation of the -N-V-T- site in the distal COOH terminus (Asn-1421, numbered according to Giudice et al.;Ref. 2Giudice G.J. Emery D.J. Diaz L.A. J. Invest. Dermatol. 1992; 99: 243-250Abstract Full Text PDF PubMed Scopus (486) Google Scholar) of collagen XVII. This site lies within the segment recognized by the antibody Col17ecto-1, which is eliminated during extended pepsin digestion (Fig. 4, lane 9). The thermal stability of the extracellular domain of collagen XVII was assessed with trypsin or sequential pepsin/trypsin digestions as probes for the triple-helical conformation (35Bruckner P. Prockop D.J. Anal. Biochem. 1981; 110: 360-368Crossref PubMed Scopus (231) Google Scholar, 36Raghunath M. Bruckner P. Steinmann B. J. Mol. Biol. 1994; 236: 940-949Crossref PubMed Scopu
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