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• W2042173676 abstract "Type VII collagen, the major component of anchoring fibrils, consists of a central collagenous triple-helical domain flanked by two noncollagenous domains, NC1 and NC2. The NC2 domain has been implicated in catalyzing the antiparallel dimer formation of type VII procollagen. In this study, we produced the entire 161 amino acids of the NC2 domain plus 186 amino acids of adjacent collagenous domain (NC2/COL) and purified large quantities of the recombinant NC2/COL protein. Recombinant NC2/COL readily formed disulfide-bonded hexamers, each representing one antiparallel dimer of collagen VII. Removal of the collagenous helical domain from NC2/COL by collagenase digestion abolished the antiparallel dimer formation. Using site-directed mutagenesis, we found that mutation of either cysteine 2802 or cysteine 2804 alone within the NC2 domain blocked antiparallel dimer formation. In contrast, a single cysteine mutation, 2634, within the collagenous helical domain had no effect. A generated methionine to lysine substitution, M2798K, that is associated with recessive dystrophic epidermolysis bullosa, was unable to form antiparallel dimers. Furthermore, autoantibodies from epidermolysis bullosa acquisita patients also reacted with NC2/COL. We conclude that NC2 and its adjacent collagenous segment mediate antiparallel dimer formation of collagen VII. Epidermolysis bullosa acquisita autoantibodies bound to this domain may destabilize anchoring fibrils by interfering with antiparallel dimer assembly leading to epidermal-dermal disadherence. Type VII collagen, the major component of anchoring fibrils, consists of a central collagenous triple-helical domain flanked by two noncollagenous domains, NC1 and NC2. The NC2 domain has been implicated in catalyzing the antiparallel dimer formation of type VII procollagen. In this study, we produced the entire 161 amino acids of the NC2 domain plus 186 amino acids of adjacent collagenous domain (NC2/COL) and purified large quantities of the recombinant NC2/COL protein. Recombinant NC2/COL readily formed disulfide-bonded hexamers, each representing one antiparallel dimer of collagen VII. Removal of the collagenous helical domain from NC2/COL by collagenase digestion abolished the antiparallel dimer formation. Using site-directed mutagenesis, we found that mutation of either cysteine 2802 or cysteine 2804 alone within the NC2 domain blocked antiparallel dimer formation. In contrast, a single cysteine mutation, 2634, within the collagenous helical domain had no effect. A generated methionine to lysine substitution, M2798K, that is associated with recessive dystrophic epidermolysis bullosa, was unable to form antiparallel dimers. Furthermore, autoantibodies from epidermolysis bullosa acquisita patients also reacted with NC2/COL. We conclude that NC2 and its adjacent collagenous segment mediate antiparallel dimer formation of collagen VII. Epidermolysis bullosa acquisita autoantibodies bound to this domain may destabilize anchoring fibrils by interfering with antiparallel dimer assembly leading to epidermal-dermal disadherence. dystrophic epidermolysis bullosa NH2-terminal noncollagenous domain of type VII collagen COOH-terminal noncollagenous domain of type VII collagen epidermolysis bullosa acquisita polyacrylamide gel electrophoresis enzyme-linked immunosorbent assay bullous systemic lupus erythematosus cytomegalovirus Type VII collagen, a genetically distinct member of the collagen family, is found within the basement membrane zone beneath stratified squamous epithelium (1Uitto J. Chung-Honet L.C. Christiano A.M. Exp. Dermatol. 1992; 1: 2-11Crossref PubMed Scopus (62) Google Scholar, 2Burgeson R.E. J. Invest. Dermatol. 1993; 101: 252-255Abstract Full Text PDF PubMed Google Scholar). Type VII collagen is a major component of anchoring fibrils, attachment structures within the basement membrane between the epidermis and dermis of human skin (3Keene D.R. Sakai L.Y. Lundstrum G.P. Morris N.P. Burgeson R.E. J. Cell Biol. 1987; 104: 611-621Crossref PubMed Scopus (290) Google Scholar, 4Lunstrum G.P. Sakai L.Y. Keene D.R. Morris N.P. Burgeson R.E. J. Biol. Chem. 1986; 261: 9042-9048Abstract Full Text PDF PubMed Google Scholar). In inherited forms of dystrophic epidermolysis bullosa (DEB),1 anchoring fibrils are diminutive and/or reduced in number (5Uitto J. Christiano A.M. Semin. Dermatol. 1993; 12: 191-201PubMed Google Scholar, 6Tidman M.J. Eady R.A.J. J. Invest. Dermatol. 1985; 84: 373-377Abstract Full Text PDF Scopus (197) Google Scholar, 7Lin, A. N., and Carter, D. M. (eds) (1992) Epidermolysis Bullosa: Basic and Clinical Aspects, Springer-Verlag, New YorkGoogle Scholar). In addition to inherited DEB, epidermolysis bullosa acquisita (EBA) is an acquired autoimmune form of epidermolysis bullosa. EBA is characterized by circulating and tissue-bound IgG autoantibodies to type VII collagen (8Woodley D.T. Gammon W.R. O'Keefe E.J. Inman A.O. Queen L.L. Gammon W.R. N. Engl. J. Med. 1984; 310: 1007-1013Crossref PubMed Scopus (435) Google Scholar, 9Woodley D.T. Burgeson R.E. Lunstrum G.P. Bruckner-Tuderman L. Reese M.J. Briggaman R.A. J. Clin. Invest. 1988; 81: 683-687Crossref PubMed Scopus (269) Google Scholar). Like DEB, ultrastructural studies have demonstrated a dramatic paucity of anchoring fibrils in EBA skin (10Nieboer C. Boorsma D.M. Woerdeman M.J. Kalsbeck G.L. Br. J. Dermatol. 1980; 102: 383-392Crossref PubMed Scopus (169) Google Scholar). These observations suggest that type VII collagen plays an important role in maintaining epidermal-dermal adherence. Type VII collagen has been cloned, and a genetic linkage has been established between inherited DEB and mutations in the gene that encodes for type VII collagen, COL7A1 (11Parente M.G. Chung L.C. Ryynanen J. Woodley D.T. Wynn K.C. Bauer E.A. Mattei M-G. Chu M-L. Uitto J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6931-6935Crossref PubMed Scopus (171) Google Scholar, 12Christiano A.M. Greenspan D.S. Lee S. Uitto J. J. Biol. Chem. 1994; 269: 20256-20262Abstract Full Text PDF PubMed Google Scholar, 13Uitto J. Bauer E.A. Moshell A.N. J. Invest. Dermatol. 1992; 38: 391-395Crossref Scopus (23) Google Scholar, 14Hovnanian A. Duquesnoy P. Blanchet-Bardon C. Knowlton R.G. Amselm S. Lathrop M. Dubertret L. Uitto J. Goosens M. J. Clin. Invest. 1992; 90: 1032-1036Crossref PubMed Scopus (127) Google Scholar). There have been over 100 distinct COL7A1 gene mutations identified in patients with DEB, and these mutations have occurred within NC1, NC2, and the helical domain (15Christiano A.M. Uitto J. Curr. Opin. Dermatol. 1996; 3: 225-232Google Scholar, 16Uitto J. Hovnanian A. Christiano A.M. Proc. Assoc. Am. Phys. 1995; 107: 245-252PubMed Google Scholar). Type VII collagen is composed of three identical α chains, each consisting of a 145-kDa central collagenous triple-helical segment, flanked by a large 145-kDa amino-terminal non-collagenous domain (NC1), and a smaller 34-kDa carboxyl-terminal non-collagenous domain (NC2) (4Lunstrum G.P. Sakai L.Y. Keene D.R. Morris N.P. Burgeson R.E. J. Biol. Chem. 1986; 261: 9042-9048Abstract Full Text PDF PubMed Google Scholar,17Lunstrum G.P. Kuo H.-J. Rosenbaum L.M. Keene D.R. Glanville R.W. Sakai L.Y. Burgeson R.E. J. Biol. Chem... 1987; 262: 13706-13712Google Scholar). In the extracellular space, individual type VII collagen molecules form antiparallel tail-to-tail dimers stabilized by disulfide bonding through a small carboxyl-terminal overlap (NC2), and a portion of the NC2 domain is proteolytically removed (18Morris N.P. Keene D.R. Glanville R.W. Bentz H. Burgeson R.E. J. Biol. Chem. 1986; 261: 5638-5644Abstract Full Text PDF PubMed Google Scholar, 19Bruckner-Tuderman L. Nilssen O. Zimmermann D.R. Dours-Zimmermann M.T. Ulrike-Kalinke D. Gedde-Dahl T. Jan-Olof W. J. Cell Biol. 1995; 131: 551-559Crossref PubMed Scopus (128) Google Scholar). The antiparallel dimers then aggregate laterally in a nonstaggered manner to form anchoring fibrils (4Lunstrum G.P. Sakai L.Y. Keene D.R. Morris N.P. Burgeson R.E. J. Biol. Chem. 1986; 261: 9042-9048Abstract Full Text PDF PubMed Google Scholar). NC2 consists of 161 amino acids. The NC2 domain has a segment that is homologous with the Kunitz-type protease inhibitor (12Christiano A.M. Greenspan D.S. Lee S. Uitto J. J. Biol. Chem. 1994; 269: 20256-20262Abstract Full Text PDF PubMed Google Scholar). It also contains four potential sites for phosphorylation by casein kinase I and II (12Christiano A.M. Greenspan D.S. Lee S. Uitto J. J. Biol. Chem. 1994; 269: 20256-20262Abstract Full Text PDF PubMed Google Scholar). The overall conservation between mouse and human NC2 domain sequences is 90% at the amino acid level. The relatively high degree of conservation of this segment is highlighted by the presence of 67-amino acid residues spanning the junction of the collagenous and NC2 domains which is 100% conserved between hamster, mouse, and human sequences (20Greenspan D.S. Human Mol. Genet. 1993; 2: 273-278Crossref PubMed Scopus (43) Google Scholar). It has been proposed that this region contains the cleavage site for proteolytic removal of the NC2 domain. This region also contains a pair of cysteines in amino acid positions 2802 and 2804 which are likely to be involved in the disulfide-bond linkages that stabilize antiparallel dimers. In addition, there is a conserved single cysteine residue in amino acid position 2634 within the adjacent collagenous domain which may be involved in interchain disulfide bonding (20Greenspan D.S. Human Mol. Genet. 1993; 2: 273-278Crossref PubMed Scopus (43) Google Scholar). The NC2 domain of type VII collagen has been implicated in initiating the triple-helical assembly of type VII collagen and catalyzing the antiparallel dimer formation of procollagen (2Burgeson R.E. J. Invest. Dermatol. 1993; 101: 252-255Abstract Full Text PDF PubMed Google Scholar). However, the exact function of the NC2 domain is not known. This is at least partly due to the extremely low quantities of procollagen VII in skin and cell culture. Furthermore, there has been a very limited amount of NC2 available because of its susceptibility to nonspecific proteolytic degradation during biochemical purification. Therefore, there has been great difficulty obtaining sufficient quantities of pure NC2. The major antigenic epitopes for autoantibodies in patients with EBA and bullous systemic lupus erythematosus (BSLE) reside within the NC1 domain of type VII collagen (21Lapiere J.C. Woodley D.T. Parente M.G. Iwasaki T. Wynn K.C. Christiano A.M. Uitto J. J. Clin. Invest. 1993; 92: 1831-1839Crossref PubMed Scopus (174) Google Scholar, 22Gammon W.R. Abernethy M.L. Padilla K.M. Prisayanh P.S. Cook M.E. Wright J. Briggaman R.A. Hunt III, S.W. J. Invest. Dermatol. 1992; 99: 691-696Abstract Full Text PDF PubMed Google Scholar, 23Tanaka T. Furukawa F. Imamura S. J. Invest. Dermatol. 1994; 102: 706-709Abstract Full Text PDF PubMed Google Scholar). However, recently a novel EBA subgroup with a milder clinical presentation was defined in children with tissue bound and circulating autoantibodies targeting only the triple-helical central domain of type VII collagen (24Tanaka H. Yamamoto A.I. Hashimoto T. Hiramoto K. Harada T. Kawachi Y. Shimizu H. Tanaka T. Kishiyama K. Hopfner B. Takahashi H. Lizuka H. Bruckner-Tuderman L. Lab. Invest. 1997; 77: 623-632PubMed Google Scholar). These data suggest that EBA may exhibit a wider, more heterogeneous spectrum of autoantibody reactivities than previously assumed. In this study, we prepared a cDNA construct for the NC2 domain plus its immediately adjacent collagenous domain (COL). This construct was used in an eukaryotic expression system to produce large quantities of purified NC2/COL, a 50-kDa carboxyl terminus of the type VII collagen α-chain. We demonstrate herein that purified NC2/COL readily forms disulfide-bonded hexameric aggregates, each representing one anti-parallel dimer of collagen VII. We also demonstrate that the adjacent COL domain is essential for the formation of antiparallel dimers. Using site-directed mutagenesis, we found that mutating either cysteine 2802 or cysteine 2804 within the NC2 domain inhibits antiparallel dimer formation. In contrast, a single cysteine mutation, cysteine 2634, within the collagenous domain had no effect. Furthermore, a methione to lysine substitution mutation, M2798K, within the NC2 domain also caused an inability to form antiparallel dimers. Interestingly, this mutation has been reported to be associated with recessive DEB. The eukaryotic expression vector pRC/CMV (Invitrogen, San Diego, CA) which contains the cytomegalovirus (CMV) promoter and enhancer was used to express a chimeric cDNA (Fig. 1). The cDNA contains a 16-amino acid signal peptide sequence derived from human type VII collagen, a 186-amino acid COL domain, and the entire 161-amino acid NC2 domain. To generate the expression vector, a 1.8-kilobase pair of COL and NC2 cDNA fragment was released from pGEX-PCR10 byBamHI/EcoRI digestion (21Lapiere J.C. Woodley D.T. Parente M.G. Iwasaki T. Wynn K.C. Christiano A.M. Uitto J. J. Clin. Invest. 1993; 92: 1831-1839Crossref PubMed Scopus (174) Google Scholar) and made blunt-ended by DNA polymerase I (Klenow fragment) following a standard protocol. This fragment was then ligated to the KasI-digested and blunt-ended pRC/CMV to generate a construct designated CMV/NC2*. Then, a cDNA fragment containing signal peptide, COL domain, and NC2 domain was released from CMV/NC2* by NruI/MscI digestion and ligated into the NruI/XbaI (blunt-ended) pRC/CMV vector, resulting in the final expression construct as shown in Fig. 1. We designated the protein product of this construct NC2/COL. All the constructs were verified by DNA sequencing. The expression vector was used to transfect a human embryonic kidney cell line 293 (ATCC, Rockville, MD) using Lipofectin (Life Technologies, Inc., Gaithersburg, MD), and stable clones were selected using 500 μg of G418/ml as previously described (25Chen M. Marinkovich M.P. Veis A. Cai X.Y. Rao C.N. O'Toole E.A. Woodley D.T. J. Biol. Chem. 1997; 272: 14516-14522Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). For Northern blot analysis, total RNA was extracted using guanidine isothiocyanate-CsCl2 density gradient centrifugation as previously described (26Wang S.Y. Gudas L.J. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 5880-5884Crossref PubMed Scopus (114) Google Scholar). The total RNA was size fractionated and transferred to a nylon membrane (GeneScreen Plus, DuPont, Boston, MA) and hybridized to a 32P-labeled (random-primer labeling kit, BMB, Indianapolis, IN) 1.4-kilobase pair human type VII collagen cDNA probe containing NC2/COL domain. Site-directed mutagenesis was performed on the NC2/COL chimeric cDNA in the pRC/CMV vector using a commercial kit (QuikChangeTM site-directed mutagenesis kit, Stratagene Inc., La Jolla, CA) according to the manufacturer's instructions. Briefly, a pair of complementary primers with 39 bases was designed, and mutation to change cysteine to serine or methione to lysine was placed in the middle of the primers. A double cysteine mutant was created by changing cysteine at a given site to serine and using this mutant DNA as a template in the next round of mutagenesis. Parental cDNA inserted in pRC/CMV was amplified usingPyrococcus furiosus DNA polymerase with these primers for 12 cycles in a DNA thermal cycler (PerkinElmer Life Sciences). After digestion of the parental DNA with DpnI, the amplified DNA with nucleotide substitution incorporated was transformed intoEscherichia coli (XL1-Blue). The mutations were confirmed by automated DNA sequencing. For Western blot analysis, clonal cell lines resistant to G418 were grown to confluence, the medium was changed to serum-free medium, and the cultures were maintained for an additional 24 h. The media were collected, concentrated 10-fold by Centricon-30 (Amicon, Beverly, MA), and subjected to 10% SDS-PAGE. Proteins were then electrotransferred onto a nitrocellulose membrane. The presence of recombinant NC2/COL was detected with a rabbit polyclonal antibody prepared from our laboratory against the NC2/COL domain of type VII collagen followed by a horseradish peroxidase-conjugated goat anti-rabbit IgG and enhanced chemiluminescence detection reagent (Amersham Corp.) (27Chen M. O'Toole E.A. Muellenhoff M. Medina E. Kasahara N. Woodley D.T. J. Biol. Chem. 2000; 275: 24429-24435Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). For large-scale purification of recombinant NC2/COL, serum-free media were equilibrated to 5 mm EDTA, 50 μmphenylmethylsulfonyl fluoride, and 50 μm N-ethylmaleimide, and precipitated with 400 mg/ml ammonium sulfate at 4 °C overnight with stirring. Precipitated proteins were collected by centrifuging at 13,000 rpm for 1 h, resuspended and dialyzed in buffer A containing 500 mm NaCl, 25 mm Tris-HCl, pH 7.8, and 1 mm EDTA and passed over a Q-Sepharose column (Amersham Pharmacia Biotech, Piscataway, NJ) equilibrated in the same buffer. Elution was then carried out with a linear gradient from 0.5 to 1.0 m NaCl of appropriate volume size. The proteins that eluted between 0.8 and 1 mNaCl were pooled and further purified on a Superdex 200 (HR 10/30, Pharmacia) equilibrate in 25 mm Tris-HCl, pH 7.8, 1 mm EDTA, and 200 mm NaCl. The inhibitory activity of recombinant NC2/COL for various proteases was examined. We tested whether NC2/COL could inhibit selected proteases from degrading recombinant NC1 domain of type VII collagen. The recombinant NC1 (200 μg/ml) as substrate was dissolved in 50 mm Tris-HCl, pH 7.8, 100 mm NaCl, and 2 mm CaCl2 as described previously (28Mayer U. Poschl E. Nischt R. Specks U. Pan T.C. Chu M.-L. Timpl R. Eur. J. Biochem. 1994; 225: 573-580Crossref PubMed Scopus (41) Google Scholar). The proteases (2 μg/ml) were incubated with inhibitors in 10- or 50-fold excess (by molar) for 1 h at 37 °C prior to the addition of recombinant NC1. Digestions were allowed to incubate for 4 h at 37 °C and then analyzed by SDS-PAGE. Purified recombinant NC2/COL (5 μg) was dialyzed against 50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 10 mm CaCl2, and 2 mm N-ethylmaleimide and then subjected to 10 units of bacterial collagenase (clostridiopeptidase A, type III, Sigma) digestion at 37 °C for 1 h. For testing IgG reactivity with NC2/COL, EBA and BSLE sera were used. The diagnosis of EBA was based on clinical findings, characteristic histology, demonstration of linear IgG deposits at the dermal-epidermal junction by direct immunofluorescence, and demonstration of IgG deposits at the dermal floor of the patients skin when dermal-epidermal junction was fractured though the lamina lucida by treatment with 1 m NaCl (29Gammon W.R. Kowalewski C. Chorzelski T.P. Kumar V. Briggaman R.A. Beutner E.H. J Am. Acad. Dermatol. 1990; 22: 664-679Abstract Full Text PDF PubMed Scopus (160) Google Scholar, 30Woodley D.T. Sauder D. Talley M.J. Silver M. Grotendorst G. Qwarnstrom E. J. Invest. Dermatol. 1983; 81: 149-153Abstract Full Text PDF PubMed Scopus (150) Google Scholar). We showed previously that these sera contained autoantibodies against the NC1 domain of type VII collagen (31Chen M. Chan L.S. Cai X.Y. O'Toole E.A. Sample J.C. Woodley D.T. J. Invest. Dermatol. 1996; 108: 68-72Abstract Full Text PDF Scopus (137) Google Scholar). ELISA was performed as described previously except that purified recombinant NC2/COL (0.15 μg/per well) was used as antigen to coat 96-well microtiter plates (Immulon-4, Dynatch Laboratory Inc., Alexandria, VA) (31Chen M. Chan L.S. Cai X.Y. O'Toole E.A. Sample J.C. Woodley D.T. J. Invest. Dermatol. 1996; 108: 68-72Abstract Full Text PDF Scopus (137) Google Scholar). Purified recombinant NC2/COL protein (100 ng/well) was subjected to a 10% SDS-PAGE, then electrotransferred to a nitrocellulose membrane. Cut strips of the nitrocellulose were blocked for 1 h at room temperature with 5% nonfat dry milk in 50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 0.1% Tween 20 (TTBS). After washing with TTBS buffer, the strips were incubated for 1 h at room temperature with individual patient antisera or control antisera diluted in TTBS with 5% nonfat dry milk (1:200). The strips were then washed as before with TTBS three times. The immunoreactivity was detected with a horseradish peroxidase-conjugated goat anti-human IgG (Organon Teknika-Cappel, Durham, NC) diluted in TTBS with 5% nonfat dry milk (1:5,000) for 30 min at room temperature and enhanced chemiluminescence detection reagent (ECL, Amersham Pharmacia Biotech). To determine directly if the purified recombinant NC2/COL could form hexameric structures (antiparallel dimers), the purified protein was subjected to rotary shadowing electron microscopy by an established protocol (18Morris N.P. Keene D.R. Glanville R.W. Bentz H. Burgeson R.E. J. Biol. Chem. 1986; 261: 5638-5644Abstract Full Text PDF PubMed Google Scholar). Briefly, 10 μg/ml of the purified NC2/COL protein was dialyzed against 0.2m ammonium bicarbonate, pH 7.8, and then sprayed onto freshly cleaved mica. The mica was then transferred to a Balzers BAE 250 evaporator and coated while rotating with a mixture of Pt-C at an angle of 6°. The surface replica was floated free from the mica, then transferred to grids in preparation for examination using a Philips 410 transmission electron microscope. A potent eukaryotic expression vector pRC/CMV was used to express the entire 161 amino acids of the NC2 domain plus 186 amino acids of the adjacent collagenous domain of type VII collagen attached to the type VII collagen signal peptide (Fig.1 A). The signal peptide sequence from human type VII collagen was used because efficient expression and secretion was obtained for the recombinant NC1 domain using the same expression vector (25Chen M. Marinkovich M.P. Veis A. Cai X.Y. Rao C.N. O'Toole E.A. Woodley D.T. J. Biol. Chem. 1997; 272: 14516-14522Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). The expression vector NC2/COL was initially examined by transient transfection of human 293 embryonic kidney cells. Western blot analysis with a polyclonal antibody against the NC2/COL domain of type VII collagen detected the expression of a 50-kDa doublet in both serum-free medium and cellular extracts of transfected 293 cells (Fig.1 B, lanes 2 and 4). These bands were absent in the control, vector alone transfected 293 cells (Fig. 1B,lanes 1 and 3). The parental 293 cells did not produce endogenous type VII collagen. Stable cell clones were then established by G418 selection. Northern hybridization of the NC2/COL stably transfected 293 cell clones demonstrated abundant amounts of a 1.5-kilobase pair exogenous mRNA which corresponds in size to that of the cDNA insert (Fig.1C, lane 2). No hybridization, even after longer exposure, was observed with parental 293 cell RNA, indicating that there is very little or no production of endogenous type VII collagen in 293 cells (lane 1). The recombinant NC2/COL was purified from serum-free culture medium from a high expressing 293 cell clone by a two-step procedure. An ammonium sulfate precipitate of the conditioned medium proteins (Fig.2, lane 1) was passed over a Q-Sepharose column. Most of the NC2/COL proteins eluted from the column in high salt fractions (0.8–1 m NaCl) (lane 2). Further purification was achieved using molecular sieve chromatography on a Superdex-200 (lane 3). The final purified yields were 2–5 mg/liter of culture medium. Previous studies suggested that dimerization is catalyzed by binding of the NC2 domain to a specific region of the triple helix (2Burgeson R.E. J. Invest. Dermatol. 1993; 101: 252-255Abstract Full Text PDF PubMed Google Scholar). We assessed the ability of recombinant NC2/COL to assemble into disulfide-bonded oligomers. Fig. 3 shows that prior to reduction, the recombinant NC2/COL migrated as a disulfide-bonded 300-kDa hexamer, equivalent to one antiparallel dimer (that is, a molecular dimer) (lane 3). Reduction effectively dissociated the aggregate into a 50-kDa monomer of NC2/COL (lane 1). These results indicate that the recombinant NC2/COL is capable of forming a hexameric complex such as those that comprise antiparallel dimers consisting of six α-chains of type VII collagen. To examine whether the adjacent COL domain is required for the hexamer formation, we subjected purified recombinant NC2/COL to collagenase digestion. As also shown in Fig. 3, digestion of NC2/COL by bacterial collagenase converted the 50-kDa NC2/COL into a 32-kDa fragment under reducing conditions (lane 2). This apparent molecule mass corresponds to that expected for the NC2 domain of type VII collagen. Under nonreducing conditions, the 32-kDa NC2 runs as a 64-kDa dimer (D) and a 100-kDa trimer (T), but not as a hexameric complex of 192 kDa (lane 4). The NC2 domain is capable of forming trimers, but not hexamers. Therefore, the 186-amino acid collagenous sequences adjacent to NC2 are essential for the formation of hexamers and the initiation of antiparallel dimers. To determine more directly if NC2/COL formed hexameric structures akin to antiparallel dimers, we subjected purified recombinant protein to rotary shadowing. Rotary-shadowed images of the recombinant NC2/COL showed an extended overlapping rod COL domain with two distinct small globular folding NC2 domains at both ends (Fig.4 A). Within the field of rotary-shadowed structures, some of the molecules were separated along their length and appeared to be held together at their globular ends (Fig. 4 B). The rotary-shadowed molecules measured ∼60 nm within the overlapping COL domains, similar to the 60-nm lengths estimated from intact native type VII collagen dimers (18Morris N.P. Keene D.R. Glanville R.W. Bentz H. Burgeson R.E. J. Biol. Chem. 1986; 261: 5638-5644Abstract Full Text PDF PubMed Google Scholar). To further identify the cysteine residues involved in the formation of the disulfide linkages to stabilize the antiparallel type VII collagen dimers, we generated four site-directed Cys-Ser mutant constructs and transfected them into 293 cells. As shown in the immunoblot analysis in Fig.5 A, transfection with C2634S, C2802S, C2804S, and C2802S/C2804S constructs resulted in the secretion of the 50-kDa NC2/COL at a similar level as wild type (comparelanes 2–5 to 1). Under nonreducing conditions, the protein product of two single cysteine mutations within the NC2 domain (C2802S and C2804S) runs as a 50-kDa monomer, a 100-kDa dimer, and a 150-kDa trimer (lanes 8 and 9). A double cysteine mutation within the NC2 domain (C2802S/C2804S) runs predominately as a 50-kDa monomer, but some 100-kDa dimers are also observed (lane 10). In contrast, a single cysteine mutation within the collagenous domain (C2634S) runs as a 300-kDa hexamer similar to the wild type (lanes 6 and 7). Thus, both cysteine residues at amino acid 2802 and 2804 within the NC2 domain are vital for hexamer (antiparallel dimer) formation. Previous studies have identified a homozygous methionine to lysine missense mutation at the amino acid residue 2798 in a patient with recessive DEB (32Christiano A.M. Greenspan D.S. Hoffman G.G. Zhang X. Tamai Y. Lin A.N. Dietz H.C. Hovnanian A. Uitto J. Nat. Genet. 1993; 4: 62-66Crossref PubMed Scopus (176) Google Scholar). To investigate the molecular defect underlying this mutation, site-directed mutagenesis was used to generate the same mutation in the NC2/COL construct. As shown in the immunoblot analysis in Fig. 5 B, transfection of the M2798K construct into 293 cells resulted in the secretion of a 50-kDa NC2/COL at a similar level as the wild type (compare lanes 1 and2). Under nonreducing conditions, the M2789K mutation protein product runs mostly as a 50-kDa monomer and a 150-kDa trimer (lane 4). However, the mutation protein product was incapable of forming hexameric complexes. These data indicate that a methione to lysine mutation within the NC2 domain impairs the ability of NC2/COL to form antiparallel dimers. Sequence analysis of the NC2 domain reveals the presence of a segment with homology to Kunitz-type protease inhibitors (12Christiano A.M. Greenspan D.S. Lee S. Uitto J. J. Biol. Chem. 1994; 269: 20256-20262Abstract Full Text PDF PubMed Google Scholar). However, it is unclear whether NC2 functions as a protease inhibitor. To examine this issue, trypsin, chymotrypsin, and several other proteases with a trypsin-like specificity or other specificities were incubated with the NC1 domain of type VII collagen with and without the presence of purified recombinant NC2/COL added at 10- or 50-fold excess. Recombinant NC1 domain of type VII collagen was selected as the protein substrate simply because of its large size (145 kDa) and the presence of many protease cleavage sites. As shown in Fig.6, degradation of NC1 by either trypsin or chymotrypsin was completely protected by a 50-fold excess of aprotinin (lanes 2 and 6). However, no protective effect could be observed with recombinant NC2/COL added at 10-fold excess (lanes 3 and 7) or 50-fold excess (lanes 4 and 8). The same lack of inhibitory activity of NC2/COL was also found for thrombin and plasmin (data not shown). Previously, we and others have identified the 4 immunodominant epitopes within the NC1 domain of type VII collagen for EBA and BSLE autoantibodies (21Lapiere J.C. Woodley D.T. Parente M.G. Iwasaki T. Wynn K.C. Christiano A.M. Uitto J. J. Clin. Invest. 1993; 92: 1831-1839Crossref PubMed Scopus (174) Google Scholar, 22Gammon W.R. Abernethy M.L. Padilla K.M. Prisayanh P.S. Cook M.E. Wright J. Briggaman R.A. Hunt III, S.W. J. Invest. Dermatol. 1992; 99: 691-696Abstract Full Text PDF PubMed Google Scholar, 23Tanaka T. Furukawa F. Imamura S. J. Invest. Dermatol. 1994; 102: 706-709Abstract Full Text PDF PubMed Google Scholar). To investigate whether NC2/COL contained any antigenic epitopes for EBA and BSLE autoantibodies, recom" @default.
• W2042173676 created "2016-06-24" @default.
• W2042173676 creator A5000398282 @default.
• W2042173676 creator A5007855326 @default.
• W2042173676 creator A5027358797 @default.
• W2042173676 creator A5040445442 @default.
• W2042173676 creator A5079997154 @default.
• W2042173676 date "2001-06-01" @default.
• W2042173676 modified "2023-10-18" @default.
• W2042173676 title "The Carboxyl Terminus of Type VII Collagen Mediates Antiparallel Dimer Formation and Constitutes a New Antigenic Epitope for Epidermolysis Bullosa Acquisita Autoantibodies" @default.
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