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• W2021002011 abstract "α-Helical coiled coils, frequent protein oligomerization motifs, are commonly observed in vital proteins. Here, using collagen XVII as an example, we provide evidence for a novel function of coiled coils in the regulation of ectodomain shedding. Transmembrane collagen XVII, an epithelial cell surface receptor, mediates dermal-epidermal adhesion in the skin, and its dysfunction is linked to human skin blistering diseases. The ectodomain of this collagen is constitutively shed from the cell surface by proteinases of a disintegrin and metalloprotease family; however, the mechanisms regulating shedding remain elusive. Here, we used site-specific mutagenesis to target the coiled-coil heptad repeats within the juxtamembranous, extracellular noncollagenous 16th A (NC16A) domain of collagen XVII. This resulted in a substantial increase of ectodomain shedding, which was not mediated by disintegrin and metalloproteases. Instead, conformational changes induced by the mutation(s) unmasked a furin recognition sequence that was used for cleavage. This study shows that apart from their functions in protein oligomerization, coiled coils can also act as regulators of ectodomain shedding depending on the biological context. α-Helical coiled coils, frequent protein oligomerization motifs, are commonly observed in vital proteins. Here, using collagen XVII as an example, we provide evidence for a novel function of coiled coils in the regulation of ectodomain shedding. Transmembrane collagen XVII, an epithelial cell surface receptor, mediates dermal-epidermal adhesion in the skin, and its dysfunction is linked to human skin blistering diseases. The ectodomain of this collagen is constitutively shed from the cell surface by proteinases of a disintegrin and metalloprotease family; however, the mechanisms regulating shedding remain elusive. Here, we used site-specific mutagenesis to target the coiled-coil heptad repeats within the juxtamembranous, extracellular noncollagenous 16th A (NC16A) domain of collagen XVII. This resulted in a substantial increase of ectodomain shedding, which was not mediated by disintegrin and metalloproteases. Instead, conformational changes induced by the mutation(s) unmasked a furin recognition sequence that was used for cleavage. This study shows that apart from their functions in protein oligomerization, coiled coils can also act as regulators of ectodomain shedding depending on the biological context. α-Helical coiled coils represent the most frequent protein oligomerization motifs in nature, and they are often found in vital proteins (1Grigoryan G. Keating A.E. Structural specificity in coiled-coil interactions.Curr. Opin. Struct. Biol. 2008; 18: 477-483Crossref PubMed Scopus (219) Google Scholar, 2Mason J.M. Arndt K.M. Coiled-coil domains. Stability, specificity, and biological implications.ChemBioChem. 2004; 5: 170-176Crossref PubMed Scopus (542) Google Scholar, 3Kammerer R.A. α-Helical coiled-coil oligomerization domains in extracellular proteins.Matrix Biol. 1997; 15: 555-565Crossref PubMed Scopus (67) Google Scholar, 4Burkhard P. Stetefeld J. Strelkov S.V. Coiled coils. A highly versatile protein folding motif.Trends Cell Biol. 2001; 11: 82-88Abstract Full Text Full Text PDF PubMed Scopus (844) Google Scholar). Indeed, the prediction is that 3–5% of all protein residues form coiled coils (5Wolf E. Kim P.S. Berger B. MultiCoil. A program for predicting two- and three-stranded coiled coils.Protein Sci. 1997; 6: 1179-1189Crossref PubMed Scopus (649) Google Scholar). These are generally characterized by heptad repeat sequences, -abcdefg-, within which the first a and the fourth d positions are commonly occupied by hydrophobic amino acids (1Grigoryan G. Keating A.E. Structural specificity in coiled-coil interactions.Curr. Opin. Struct. Biol. 2008; 18: 477-483Crossref PubMed Scopus (219) Google Scholar, 2Mason J.M. Arndt K.M. Coiled-coil domains. Stability, specificity, and biological implications.ChemBioChem. 2004; 5: 170-176Crossref PubMed Scopus (542) Google Scholar, 3Kammerer R.A. α-Helical coiled-coil oligomerization domains in extracellular proteins.Matrix Biol. 1997; 15: 555-565Crossref PubMed Scopus (67) Google Scholar, 4Burkhard P. Stetefeld J. Strelkov S.V. Coiled coils. A highly versatile protein folding motif.Trends Cell Biol. 2001; 11: 82-88Abstract Full Text Full Text PDF PubMed Scopus (844) Google Scholar). Particularly, leucine is typically found at d position, where it plays a role as a “leucine zipper” for oligomerization (6Landschulz W.H. Johnson P.F. McKnight S.L. The leucine zipper. A hypothetical structure common to a new class of DNA-binding proteins.Science. 1988; 240: 1759-1764Crossref PubMed Scopus (2534) Google Scholar). In extracellular matrix proteins, which guide cell functions in most tissues, mostly three-stranded coiled-coil structures mediate oligomerization and thus provide functional advantages such as multivalency, enhanced binding strength, or combined functions of different domains for the protein (7Engel J. Role of oligomerization domains in thrombospondins and other extracellular matrix proteins.Int. J. Biochem. Cell Biol. 2004; 36: 997-1004Crossref PubMed Scopus (16) Google Scholar). Coiled coils are found in most, if not all, members of the collagen superfamily (8McAlinden A. Smith T.A. Sandell L.J. Ficheux D. Parry D.A. Hulmes D.J. α-Helical coiled-coil oligomerization domains are almost ubiquitous in the collagen superfamily.J. Biol. Chem. 2003; 278: 42200-42207Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). In fibrillar collagens, they are usually located in the C-terminal propeptides and function to initiate trimerization and therefore triple-helix folding (8McAlinden A. Smith T.A. Sandell L.J. Ficheux D. Parry D.A. Hulmes D.J. α-Helical coiled-coil oligomerization domains are almost ubiquitous in the collagen superfamily.J. Biol. Chem. 2003; 278: 42200-42207Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). In contrast, in cell surface-associated transmembrane collagens, the N-terminal noncollagenous domains harbor the coiled-coil sequences (8McAlinden A. Smith T.A. Sandell L.J. Ficheux D. Parry D.A. Hulmes D.J. α-Helical coiled-coil oligomerization domains are almost ubiquitous in the collagen superfamily.J. Biol. Chem. 2003; 278: 42200-42207Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 9Snellman A. Tuomisto A. Koski A. Latvanlehto A. Pihlajaniemi T. The role of disulfide bonds and α-helical coiled coils in the biosynthesis of type XIII collagen and other collagenous transmembrane proteins.J. Biol. Chem. 2007; 282: 14898-14905Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar), consistent with the fact that in these collagens the triple helix formation and ectodomain folding proceed from the N to C terminus (8McAlinden A. Smith T.A. Sandell L.J. Ficheux D. Parry D.A. Hulmes D.J. α-Helical coiled-coil oligomerization domains are almost ubiquitous in the collagen superfamily.J. Biol. Chem. 2003; 278: 42200-42207Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 9Snellman A. Tuomisto A. Koski A. Latvanlehto A. Pihlajaniemi T. The role of disulfide bonds and α-helical coiled coils in the biosynthesis of type XIII collagen and other collagenous transmembrane proteins.J. Biol. Chem. 2007; 282: 14898-14905Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). Thus far, the major, if not sole, function of coiled coils in collagens has been believed to be trimerization prior to triple helix formation. In this study, using collagen XVII as an example, we provide evidence for a novel function of coiled coils in transmembrane collagens, i.e. in the control of ectodomain shedding. Ectodomain shedding, the release of extracellular domains of transmembrane proteins, is one form of proteolytic maturation of functional proteins. Proteolytic processing of proteins is a common and crucial event in biology, underlined by the fact that more than 2% of mammalian genes encode proteases (10Overall C.M. Blobel C.P. In search of partners. Linking extracellular proteases to substrates.Nat. Rev. Mol. Cell Biol. 2007; 8: 245-257Crossref PubMed Scopus (284) Google Scholar). Ectodomain shedding is mainly catalyzed by proteinases of the “a disintegrin and metalloprotease” (ADAM) 3The abbreviations used are: ADAMa disintegrin and metalloproteaseM-β-CDmethyl-β-cyclodextrinDHSN-hydroxysuccinimideDecdecanoylCMKchloromethyl ketone. family (11Reiss K. Saftig P. The “a disintegrin and metalloprotease” (ADAM) family of sheddases. Physiological and cellular functions.Semin. Cell Dev. Biol. 2009; 20: 126-137Crossref PubMed Scopus (327) Google Scholar), and it is involved in a variety of essential functions mediated by TNF-α, amyloid precursor protein, Notch1, epidermal growth factor receptor ligands (11Reiss K. Saftig P. The “a disintegrin and metalloprotease” (ADAM) family of sheddases. Physiological and cellular functions.Semin. Cell Dev. Biol. 2009; 20: 126-137Crossref PubMed Scopus (327) Google Scholar, 12Huovila A.P. Turner A.J. Pelto-Huikko M. Kärkkäinen I. Ortiz R.M. Shedding light on ADAM metalloproteinases.Trends Biochem. Sci. 2005; 30: 413-422Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar), or transmembrane collagens (13Franzke C.W. Bruckner P. Bruckner-Tuderman L. Collagenous transmembrane proteins. Recent insights into biology and pathology.J. Biol. Chem. 2005; 280: 4005-4008Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 14Franzke C.W. Tasanen K. Schumann H. Bruckner-Tuderman L. Collagenous transmembrane proteins. Collagen XVII as a prototype.Matrix Biol. 2003; 22: 299-309Crossref PubMed Scopus (74) Google Scholar), for example. a disintegrin and metalloprotease methyl-β-cyclodextrin N-hydroxysuccinimide decanoyl chloromethyl ketone. Collagen XVII is an epithelial cell surface receptor in the skin. Its vital role in dermal-epidermal adhesion and cell migration is indirectly demonstrated by the fact that its dysfunction in genetic and acquired human diseases results in skin blistering (13Franzke C.W. Bruckner P. Bruckner-Tuderman L. Collagenous transmembrane proteins. Recent insights into biology and pathology.J. Biol. Chem. 2005; 280: 4005-4008Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 14Franzke C.W. Tasanen K. Schumann H. Bruckner-Tuderman L. Collagenous transmembrane proteins. Collagen XVII as a prototype.Matrix Biol. 2003; 22: 299-309Crossref PubMed Scopus (74) Google Scholar). Collagen XVII is a type II transmembrane protein with an intracytoplasmic N terminus and an extracellular collagenous C terminus (13Franzke C.W. Bruckner P. Bruckner-Tuderman L. Collagenous transmembrane proteins. Recent insights into biology and pathology.J. Biol. Chem. 2005; 280: 4005-4008Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 14Franzke C.W. Tasanen K. Schumann H. Bruckner-Tuderman L. Collagenous transmembrane proteins. Collagen XVII as a prototype.Matrix Biol. 2003; 22: 299-309Crossref PubMed Scopus (74) Google Scholar). The ectodomain can be proteolytically released (shed) from the cell surface both in vitro (15Franzke C.W. Tasanen K. Schäcke H. Zhou Z. Tryggvason K. Mauch C. Zigrino P. Sunnarborg S. Lee D.C. Fahrenholz F. Bruckner-Tuderman L. Transmembrane collagen XVII, an epithelial adhesion protein, is shed from the cell surface by ADAMs.EMBO J. 2002; 21: 5026-5035Crossref PubMed Scopus (188) Google Scholar) and in vivo (16Nishie W. Lamer S. Schlosser A. Licarete E. Franzke C.W. Hofmann S.C. Jackow J. Sitaru C. Bruckner-Tuderman L. Ectodomain shedding generates neoepitopes on collagen XVII, the major autoantigen for bullous pemphigoid.J. Immunol. 2010; 185: 4938-4947Crossref PubMed Scopus (52) Google Scholar, 17Hirako Y. Yoshino K. Zillikens D. Owaribe K. Extracellular cleavage of bullous pemphigoid antigen 180/type XVII collagen and its involvement in hemidesmosomal disassembly.J. Biochem. 2003; 133: 197-206Crossref PubMed Scopus (22) Google Scholar) to yield a shorter collagenous triple-helical molecule. The cleavage occurs at different sites within the juxtamembranous NC16A domain (16Nishie W. Lamer S. Schlosser A. Licarete E. Franzke C.W. Hofmann S.C. Jackow J. Sitaru C. Bruckner-Tuderman L. Ectodomain shedding generates neoepitopes on collagen XVII, the major autoantigen for bullous pemphigoid.J. Immunol. 2010; 185: 4938-4947Crossref PubMed Scopus (52) Google Scholar, 18Hirako Y. Nishizawa Y. Sitaru C. Opitz A. Marcus K. Meyer H.E. Butt E. Owaribe K. Zillikens D. The 97-kDa (LABD97) and 120-kDa (LAD-1) fragments of bullous pemphigoid antigen 180/type XVII collagen have different N termini.J. Invest. Dermatol. 2003; 121: 1554-1556Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Under physiological conditions, ADAM9, -10, and -17 appear to be the major sheddases (19Franzke C.W. Bruckner-Tuderman L. Blobel C.P. Shedding of collagen XVII/BP180 in skin depends on both ADAM10 and ADAM9.J. Biol. Chem. 2009; 284: 23386-23396Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar), but involvement of neutrophil elastase and serine proteinases has been suggested in pathological settings such as bullous pemphigoid, an autoimmune blistering disease (16Nishie W. Lamer S. Schlosser A. Licarete E. Franzke C.W. Hofmann S.C. Jackow J. Sitaru C. Bruckner-Tuderman L. Ectodomain shedding generates neoepitopes on collagen XVII, the major autoantigen for bullous pemphigoid.J. Immunol. 2010; 185: 4938-4947Crossref PubMed Scopus (52) Google Scholar, 20Hofmann S.C. Voith U. Schönau V. Sorokin L. Bruckner-Tuderman L. Franzke C.W. Plasmin plays a role in the in vitro generation of the linear IgA dermatosis antigen LADB97.J. Invest. Dermatol. 2009; 129: 1730-1739Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 21Lin L. Betsuyaku T. Heimbach L. Li N. Rubenstein D. Shapiro S.D. An L. Giudice G.J. Diaz L.A. Senior R.M. Liu Z. Neutrophil elastase cleaves the murine hemidesmosomal protein BP180/type XVII collagen and generates degradation products that modulate experimental bullous pemphigoid.Matrix Biol. 2012; 31: 38-44Crossref PubMed Scopus (50) Google Scholar). The full spectrum of biological functions of collagen XVII ectodomain shedding remains uncertain, but binding to laminin-332 and association with migration and differentiation of keratinocytes seem clear (13Franzke C.W. Bruckner P. Bruckner-Tuderman L. Collagenous transmembrane proteins. Recent insights into biology and pathology.J. Biol. Chem. 2005; 280: 4005-4008Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 14Franzke C.W. Tasanen K. Schumann H. Bruckner-Tuderman L. Collagenous transmembrane proteins. Collagen XVII as a prototype.Matrix Biol. 2003; 22: 299-309Crossref PubMed Scopus (74) Google Scholar). Consistent with this notion, we have shown that migrating keratinocytes constitutively shed and leave the collagenous ectodomain of collagen XVII in the extracellular matrix (22Tasanen K. Tunggal L. Chometon G. Bruckner-Tuderman L. Aumailley M. Keratinocytes from patients lacking collagen XVII display a migratory phenotype.Am. J. Pathol. 2004; 164: 2027-2038Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 23Nishie W. Kiritsi D. Nyström A. Hofmann S.C. Bruckner-Tuderman L. Dynamic interactions of epidermal collagen XVII with the extracellular matrix. Laminin 332 as a major binding partner.Am. J. Pathol. 2011; 179: 829-837Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). However, the regulation mechanisms of collagen XVII ectodomain shedding are still unclear. The first cue of functional association of coiled coils and ectodomain shedding in transmembrane collagens comes from the fact that the coiled-coil heptad repeats are located within the juxtamembranous noncollagenous domains adjacent to the cell surface, which also harbor the sheddase recognition and cleavage sites (8McAlinden A. Smith T.A. Sandell L.J. Ficheux D. Parry D.A. Hulmes D.J. α-Helical coiled-coil oligomerization domains are almost ubiquitous in the collagen superfamily.J. Biol. Chem. 2003; 278: 42200-42207Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 9Snellman A. Tuomisto A. Koski A. Latvanlehto A. Pihlajaniemi T. The role of disulfide bonds and α-helical coiled coils in the biosynthesis of type XIII collagen and other collagenous transmembrane proteins.J. Biol. Chem. 2007; 282: 14898-14905Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 24Balding S.D. Diaz L.A. Giudice G.J. A recombinant form of the human BP180 ectodomain forms a collagen-like homotrimeric complex.Biochemistry. 1997; 36: 8821-8830Crossref PubMed Scopus (61) Google Scholar). Interestingly, in collagen XVII, the physiological cleavage sites are located 8–11 amino acid residues C-terminally from the coiled coils within the NC16A domain (16Nishie W. Lamer S. Schlosser A. Licarete E. Franzke C.W. Hofmann S.C. Jackow J. Sitaru C. Bruckner-Tuderman L. Ectodomain shedding generates neoepitopes on collagen XVII, the major autoantigen for bullous pemphigoid.J. Immunol. 2010; 185: 4938-4947Crossref PubMed Scopus (52) Google Scholar), indicating the coiled coils are not included in the shed ectodomain. In addition, it has been reported that the recombinant collagenous COL15 domain of collagen XVII can form a trimeric structure without the NC16A domain (25Van den Bergh F. Fu C.L. Olague-Marchan M. Giudice G.J. The NC16A domain of collagen XVII plays a role in triple helix assembly and stability.Biochem. Biophys. Res. Commun. 2006; 350: 1032-1037Crossref PubMed Scopus (13) Google Scholar), suggesting that trimerization of collagen XVII may not always require coiled-coil repeats. These findings led us to investigate the biological functions of coiled coils in the NC16A domain of collagen XVII. We targeted the coiled-coil heptad repeats in the NC16A domain using site-directed mutagenesis and uncovered a novel and essential role of the coiled coils within the NC16A domain in the regulation of collagen XVII ectodomain shedding. The candidate regions for coiled coils on human collagen XVII (NM_000494) were assessed using the COILS (version 2.2) and Paircoil2 (26McDonnell A.V. Jiang T. Keating A.E. Berger B. Paircoil2. Improved prediction of coiled coils from sequence.Bioinformatics. 2006; 22: 356-358Crossref PubMed Scopus (326) Google Scholar) programs. To determine essential residues for coiled-coil regions, each leucine was changed into a proline as described previously (9Snellman A. Tuomisto A. Koski A. Latvanlehto A. Pihlajaniemi T. The role of disulfide bonds and α-helical coiled coils in the biosynthesis of type XIII collagen and other collagenous transmembrane proteins.J. Biol. Chem. 2007; 282: 14898-14905Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). Human collagen XVII cDNA (a kind gift from Dr. L. Borradori) was introduced into the NotI site of pcDNA5/FRT (Invitrogen) (16Nishie W. Lamer S. Schlosser A. Licarete E. Franzke C.W. Hofmann S.C. Jackow J. Sitaru C. Bruckner-Tuderman L. Ectodomain shedding generates neoepitopes on collagen XVII, the major autoantigen for bullous pemphigoid.J. Immunol. 2010; 185: 4938-4947Crossref PubMed Scopus (52) Google Scholar). This construct was designated as COL17. For site-directed mutagenesis of the coiled-coil sequence, two different 1187-bp fragments spanning the EcoRV and ClaI DNA restriction sites, which contain substitution mutations of either Leu495 or both Leu502 and Leu495 into Pro, were chemically produced and cloned into pUC57 (GenScript) (Fig. 1B). EcoRV- and ClaI-digested DNA fragments were then inserted into the COL17-pcDNA5/FRT vector. These constructs were designated as L495P and L495P/L502P. The Flp-In-293 cell line was grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum, 2 mm l-glutamine, and 1 mm sodium pyruvate (all Invitrogen). To establish stably expressing cell lines, pcDNA5/FRT plasmid with wild type and mutant collagen XVII cDNA, as well as the empty vector as a control, were co-transfected with pOG44 into the Flp-In-293 host cells by Lipofectamine 2000 (Invitrogen). Stably expressing cells were selected under 200 μg/ml hygromycin B (Invitrogen), as described previously (16Nishie W. Lamer S. Schlosser A. Licarete E. Franzke C.W. Hofmann S.C. Jackow J. Sitaru C. Bruckner-Tuderman L. Ectodomain shedding generates neoepitopes on collagen XVII, the major autoantigen for bullous pemphigoid.J. Immunol. 2010; 185: 4938-4947Crossref PubMed Scopus (52) Google Scholar, 23Nishie W. Kiritsi D. Nyström A. Hofmann S.C. Bruckner-Tuderman L. Dynamic interactions of epidermal collagen XVII with the extracellular matrix. Laminin 332 as a major binding partner.Am. J. Pathol. 2011; 179: 829-837Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Subconfluent stably transfected Flp-In-293 cells were incubated in serum-free DMEM for 24 h prior to harvesting. Freshly prepared ascorbic acid was added to the culture medium in a final concentration of 50 μg/ml to allow prolyl and lysyl hydroxylation of collagen and proper triple helix formation (15Franzke C.W. Tasanen K. Schäcke H. Zhou Z. Tryggvason K. Mauch C. Zigrino P. Sunnarborg S. Lee D.C. Fahrenholz F. Bruckner-Tuderman L. Transmembrane collagen XVII, an epithelial adhesion protein, is shed from the cell surface by ADAMs.EMBO J. 2002; 21: 5026-5035Crossref PubMed Scopus (188) Google Scholar). Then the cells were lysed for 30 min on ice in a buffer containing 1% Nonidet P-40, 0.1 m NaCl, 25 mm Tris-HCl, pH 7.4, 10 mm EDTA, and 1 mm Pefabloc (15Franzke C.W. Tasanen K. Schäcke H. Zhou Z. Tryggvason K. Mauch C. Zigrino P. Sunnarborg S. Lee D.C. Fahrenholz F. Bruckner-Tuderman L. Transmembrane collagen XVII, an epithelial adhesion protein, is shed from the cell surface by ADAMs.EMBO J. 2002; 21: 5026-5035Crossref PubMed Scopus (188) Google Scholar). The medium proteins were concentrated either with ethanol precipitation (15Franzke C.W. Tasanen K. Schäcke H. Zhou Z. Tryggvason K. Mauch C. Zigrino P. Sunnarborg S. Lee D.C. Fahrenholz F. Bruckner-Tuderman L. Transmembrane collagen XVII, an epithelial adhesion protein, is shed from the cell surface by ADAMs.EMBO J. 2002; 21: 5026-5035Crossref PubMed Scopus (188) Google Scholar) or by Amicon ultrafiltration cassette (30 kDa, Millipore) before further processing. For characterization of responsible sheddases and transportation of cellular proteins, protease inhibitors, including 10 μm Marimastat (British Biotech), 1 mm Pefabloc SC (Roche Applied Science), 1:100 diluted protease inhibitor mixture P8340 (Sigma), 0.1 mm membrane permeable furin inhibitor Dec-RVKR-CMK (344930, Calbiochem), 8 μm of membrane impermeable furin inhibitor α1-antitrypsin Portland, or 50 ng/ml macrocyclic lactone brefeldin A (Cell Signaling) were added into the medium as described previously (27Veit G. Zimina E.P. Franzke C.W. Kutsch S. Siebolds U. Gordon M.K. Bruckner-Tuderman L. Koch M. Shedding of collagen XXIII is mediated by furin and depends on the plasma membrane microenvironment.J. Biol. Chem. 2007; 282: 27424-27435Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Isolation of proteins from the cell surface was carried out using EZ-Link Sulfo-NHS-SS-Biotin (21328, Thermo Scientific) as described elsewhere (15Franzke C.W. Tasanen K. Schäcke H. Zhou Z. Tryggvason K. Mauch C. Zigrino P. Sunnarborg S. Lee D.C. Fahrenholz F. Bruckner-Tuderman L. Transmembrane collagen XVII, an epithelial adhesion protein, is shed from the cell surface by ADAMs.EMBO J. 2002; 21: 5026-5035Crossref PubMed Scopus (188) Google Scholar, 27Veit G. Zimina E.P. Franzke C.W. Kutsch S. Siebolds U. Gordon M.K. Bruckner-Tuderman L. Koch M. Shedding of collagen XXIII is mediated by furin and depends on the plasma membrane microenvironment.J. Biol. Chem. 2007; 282: 27424-27435Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Concentrated cell culture medium and cell lysate were mixed with Laemmli's sample buffer, and the samples, with or without boiling for 5 min, were separated on SDS-PAGE using 4–13% gradient or 7% uniform gels, followed by transfer onto a nitrocellulose membrane. After blocking the membranes with 2% skimmed milk in Tris-buffered saline and incubation with primary antibodies in the same buffer overnight at 4 °C, HRP-conjugated secondary antibodies were reacted for 1 h at room temperature. The signals were visualized by ECL-Plus (GE Healthcare). The following antibodies were employed for immunoblotting: mouse monoclonal antibody NC16A-1 and NC16A-3 directed to distinct epitopes within the NC16A domain of collagen XVII (20Hofmann S.C. Voith U. Schönau V. Sorokin L. Bruckner-Tuderman L. Franzke C.W. Plasmin plays a role in the in vitro generation of the linear IgA dermatosis antigen LADB97.J. Invest. Dermatol. 2009; 129: 1730-1739Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) were used in 1:50 (NC16A-1) and 1:2,000 dilutions (NC16A-3). For detection of the intracytoplasmic domain of collagen XVII, the polyclonal goat antibody N18 (Santa Cruz Biotechnology) was diluted 1:500. A new rabbit polyclonal antibody (Ab-SILP) was produced using the synthetic peptide NH2-SILPYGDS, as described previously (16Nishie W. Lamer S. Schlosser A. Licarete E. Franzke C.W. Hofmann S.C. Jackow J. Sitaru C. Bruckner-Tuderman L. Ectodomain shedding generates neoepitopes on collagen XVII, the major autoantigen for bullous pemphigoid.J. Immunol. 2010; 185: 4938-4947Crossref PubMed Scopus (52) Google Scholar, 23Nishie W. Kiritsi D. Nyström A. Hofmann S.C. Bruckner-Tuderman L. Dynamic interactions of epidermal collagen XVII with the extracellular matrix. Laminin 332 as a major binding partner.Am. J. Pathol. 2011; 179: 829-837Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Purification of the Ab-SILP was performed using SulfoLink coupling resin (20404, Thermo Scientific) according to the manufacturer's instructions, and the final concentration of 1 μg/ml of the antibody was used. The β-tubulin antibody Ab6046 (Abcam) in a 1:2,000 dilution was employed as an internal loading control for immunoblotting. Total RNA was extracted from subconfluent transformed Flp-In-293 cells with RNeasy Mini kit (Invitrogen), followed by RT-PCR using SuperScript III reverse transcriptase (Invitrogen), according to the manufacturer's instructions. Total cDNA was then amplified with primers F14 (5′-tacaatgagctggctgtgag-3′) and B14 (5′-cggcttgacagcaatacttc-3′) to yield a 374-bp COL17A1 cDNA fragment spanning nucleotides 4117–4491. The primers GAP-F (5′-tcatctctgccccctctgct-3′) and GAP-B (5′-cgacgcctgcttcaccacct-3′) were used for amplification of GAPDH cDNA (NM_002046.3) as a control. Subconfluent cells were washed twice with PBS and then treated with 5–20 mm methyl-β-cyclodextrin (M-β-CD, Sigma) in serum-free DMEM for 60 min (28Zimina E.P. Bruckner-Tuderman L. Franzke C.W. Shedding of collagen XVII ectodomain depends on plasma membrane microenvironment.J. Biol. Chem. 2005; 280: 34019-34024Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). The medium proteins were concentrated as described above. Precipitated medium proteins were boiled for 5 min with Laemmli's sample buffer, followed by SDS-PAGE. To acetylate all free N termini, a fraction of the medium proteins was treated with 40 mm sulfo-acetate NHS (Thermo Fisher Scientific) before SDS-PAGE, as described (16Nishie W. Lamer S. Schlosser A. Licarete E. Franzke C.W. Hofmann S.C. Jackow J. Sitaru C. Bruckner-Tuderman L. Ectodomain shedding generates neoepitopes on collagen XVII, the major autoantigen for bullous pemphigoid.J. Immunol. 2010; 185: 4938-4947Crossref PubMed Scopus (52) Google Scholar). After the SDS-PAGE, the Coomassie Blue-stained 120-kDa band corresponding to the monomer form of the ectodomain was cut out the gel and subjected to mass spectrometry (MS) analysis (Japan Bio Services), as reported previously (16Nishie W. Lamer S. Schlosser A. Licarete E. Franzke C.W. Hofmann S.C. Jackow J. Sitaru C. Bruckner-Tuderman L. Ectodomain shedding generates neoepitopes on collagen XVII, the major autoantigen for bullous pemphigoid.J. Immunol. 2010; 185: 4938-4947Crossref PubMed Scopus (52) Google Scholar). In silico prediction by COILS (29Lupas A. Van Dyke M. Stock J. Predicting coiled from protein sequences.Science. 1991; 252: 1162-1164Crossref PubMed Scopus (3466) Google Scholar) indicated that the stretch Val492–Ile505 in the juxtamembranous NC16A domain of collagen XVII contained two heptad repeats of potential coiled coils (Fig. 1A). The amino acid sequence of this region in collagen XVII is highly conserved among different species (Fig. 1A), suggesting that it has a vital function. When one or both leucines at the d position, Leu495 and/or Leu502, were changed into proline(s), the probability scores predicting a coiled-coil structure calculated by COILS were significantly reduced from 1.0 to 0.4 and 0, respectively (window; amino acids 14, Fig. 1, B and C). Accordingly, the calculated probability value for the prediction of coiled coils in this region by Paircoil2 was significant (p = 0.057) for wild type COL17, whereas those of the mutants L495P and L495P/L502P were both decreased (p = 0.33 and 0.68, respectively). To address the role of coiled coils within the NC16A domain, stably transformed Flp-In-293 cells expressing wild type (COL17) and mutant (L495P/L502P or L495P) collagen XVII were produced. Isogenic expression of genes encoding these constructs was confirmed by RT-PCR (Fig. 2A). For recombinant protein expression, to ensure proper folding of collagen XVII, ascorbic acid was added to the cell culture medium. It is a cofactor for collagen hydroxylases and necessary for proper triple helix formation (15Franzke C.W. Tasanen K." @default.
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• W2021002011 title "Coiled Coils Ensure the Physiological Ectodomain Shedding of Collagen XVII" @default.
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