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W2047152078 abstract "Fibrillin-containing microfibrils in elastic and nonelastic extracellular matrices play important structural and functional roles in various tissues, including blood vessels, lung, skin, and bone. Microfibrils are supramolecular aggregates of several protein and nonprotein components. Recently, a large region in the N-terminal portion of fibrillin-1 was characterized as a multifunctional protein interaction site, including binding sites for fibulin-2 and -5 among others. Using a panel of recombinant fibrillin-1 swapped domain and deletion fragments, we demonstrate here that the conserved first hybrid domain in fibrillin-1 is essential for binding to fibulin-2, -4, and -5. Fibulin-3 and various isoforms of fibulin-1 did not interact with fibrillin-1. Although the first hybrid domain in fibrillin-1 is located in close vicinity to the self-assembly epitope, binding of fibulin-2, -4, and -5 did not interfere with self-assembly. However, these fibulins can associate with microfibrils at various levels of maturity. Formation of ternary complexes between fibrillin-1, fibulins, and tropoelastin demonstrated that fibulin-2 and -5 but much less fibulin-4, are able to act as molecular adaptors between fibrillin-1 and tropoelastin. Fibrillin-containing microfibrils in elastic and nonelastic extracellular matrices play important structural and functional roles in various tissues, including blood vessels, lung, skin, and bone. Microfibrils are supramolecular aggregates of several protein and nonprotein components. Recently, a large region in the N-terminal portion of fibrillin-1 was characterized as a multifunctional protein interaction site, including binding sites for fibulin-2 and -5 among others. Using a panel of recombinant fibrillin-1 swapped domain and deletion fragments, we demonstrate here that the conserved first hybrid domain in fibrillin-1 is essential for binding to fibulin-2, -4, and -5. Fibulin-3 and various isoforms of fibulin-1 did not interact with fibrillin-1. Although the first hybrid domain in fibrillin-1 is located in close vicinity to the self-assembly epitope, binding of fibulin-2, -4, and -5 did not interfere with self-assembly. However, these fibulins can associate with microfibrils at various levels of maturity. Formation of ternary complexes between fibrillin-1, fibulins, and tropoelastin demonstrated that fibulin-2 and -5 but much less fibulin-4, are able to act as molecular adaptors between fibrillin-1 and tropoelastin. The microfibril/elastic fiber system provides tissues, such as lung, blood vessels, and skin, with elastic properties. Microfibrils with a diameter of 10–12 nm are typically located on the outer surface of elastic fibers and are thought to play an essential role in elastogenesis (1Mecham R.P. Davis E. Yurchenco P.D. Extracellular Matrix Assembly and Structure. Academic Press, Inc., New York1994: 281-314Crossref Google Scholar). Whereas elastic fibers are always associated with microfibrils, microfibrils themselves can occur in the absence of elastin in certain tissues such as ocular ciliary zonules, the kidney, or in close proximity to various basement membranes. The microfibril/elastic fiber system is a multicomponent assembly in the extracellular matrix, and for both, the microfibrils and the elastic fibers, a number of constituents have been described (for a review, see Ref 2Kielty C.M. Sherratt M.J. Shuttleworth C.A. J. Cell Sci. 2002; 115: 2817-2828Crossref PubMed Google Scholar). For most of the associated molecules, the exact relationship in terms of physical interaction with microfibrils and/or elastic fibers, and their functional relevance is not clear. The best described components of the microfibrils are a family of proteins consisting of three highly homologous members, fibrillin-1, -2, and -3 (3Sakai L.Y. Keene D.R. Engvall E. J. Cell Biol. 1986; 103: 2499-2509Crossref PubMed Scopus (909) Google Scholar, 4Zhang H. Apfelroth S.D. Hu W. Davis E.C. Sanguineti C. Bonadio J. Mecham R.P. Ramirez F. J. Cell Biol. 1994; 124: 855-863Crossref PubMed Scopus (321) Google Scholar, 5Maslen C.L. Corson G.M. Maddox B.K. Glanville R.W. Sakai L.Y. Nature. 1991; 352: 334-337Crossref PubMed Scopus (284) Google Scholar, 6Lee B. Godfrey M. Vitale E. Hori H. Mattei M.G. Sarfarazi M. Tsipouras P. Ramirez F. Hollister D.W. Nature. 1991; 352: 330-334Crossref PubMed Scopus (572) Google Scholar, 7Corson G.M. Chalberg S.C. Dietz H.C. Charbonneau N.L. Sakai L.Y. Genomics. 1993; 17: 476-484Crossref PubMed Scopus (227) Google Scholar, 8Pereira L. D'Alessio M. Ramirez F. Lynch J.R. Sykes B. Pangilinan T. Bonadio J. Hum. Mol. Genet. 1993; 2: 961-968Crossref PubMed Scopus (256) Google Scholar, 9Nagase T. Nakayama M. Nakajima D. Kikuno R. Ohara O. DNA. Res. 2001; 8: 85-95Crossref PubMed Scopus (121) Google Scholar). Fibrillins, like many other extracellular glycoproteins, are characterized by a number of tandemly arranged domains. The most prominent domain is an epidermal growth factor-like domain (EGF), 2The abbreviations used are: EGF, epidermal growth factor-like domain; cbEGF, calcium-binding EGF; TB, transforming growth factor β-binding protein domain; TBS, Tris-buffered saline. which occurs 46–47 times in fibrillins. These domains are stabilized by three intramolecular disulfide bonds, and the majority (42–43 domains) contain a consensus sequence for calcium binding (cbEGF) (10Handford P.A. Mayhew M. Baron M. Winship P.R. Campbell I.D. Brownlee G.G. Nature. 1991; 351: 164-167Crossref PubMed Scopus (246) Google Scholar, 11Downing A.K. Knott V. Werner J.M. Cardy C.M. Campbell I.D. Handford P.A. Cell. 1996; 85: 597-605Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar, 12Lee S.S. Knott V. Jovanovic J. Harlos K. Grimes J.M. Choulier L. Mardon H.J. Stuart D.I. Handford P.A. Structure (Camb.). 2004; 12: 717-729Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). The tandemly arranged EGF and cbEGF domains are interspersed by two other types of domains, the transforming growth factor β-binding protein (TB) or 8-Cys domains and the hybrid domains. The seven TB/8-Cys domains are characterized by four intramolecular disulfide bonds, and a similar arrangement is predicted for the two hybrid domains, although no structural data are available for this domain (12Lee S.S. Knott V. Jovanovic J. Harlos K. Grimes J.M. Choulier L. Mardon H.J. Stuart D.I. Handford P.A. Structure (Camb.). 2004; 12: 717-729Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 13Yuan X. Downing A.K. Knott V. Handford P.A. EMBO J. 1997; 16: 6659-6666Crossref PubMed Google Scholar). Sequence data have shown that the first hybrid domain in all fibrillins contains nine cysteine residues as compared with eight in the second hybrid domain. This 9-cysteine pattern is highly conserved in all species, ranging from invertebrates to humans. Previously, we have determined that cysteine 204 in human fibrillin-1 and cysteine 233 in human fibrillin-2 is unpaired and available for intermolecular disulfide bonding, which is essential for initial fibrillin assembly (14Reinhardt D.P. Gambee J.E. Ono R.N. Bächinger H.P. Sakai L.Y. J. Biol. Chem. 2000; 275: 2205-2210Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Genetic mutations in fibrillins cause a number of related connective tissue disorders, including Marfan syndrome (fibrillin-1), Beals-Hecht syndrome (fibrillin-2), Weill-Marchesani syndrome (fibrillin-1 and potentially fibrillin-3), and others (for a review, see Ref. 15Robinson P. Arteaga-Solis E. Baldock C. Collod-Beroud G. Booms P. De Paepe A. Dietz H.C. Guo G. Handford P.A. Judge D.P. Kielty C.M. Loeys B. Milewicz D.M. Ney A. Ramirez F. Reinhardt D.P. Tiedemann K. Whiteman P. Godfrey M. J. Med. Genet. 2006; 43: 769-787Crossref PubMed Scopus (315) Google Scholar). The clinical symptoms that characterize these connective tissue disorders exemplify the important roles for fibrillins in development and homeostasis of the cardiovascular, skeletal, and ocular systems. More specifically, for the cardiovascular system, it has been demonstrated by murine gene deletion experiments that fibrillin-1 and -2 are important for proper elastogenesis (16Carta L. Pereira L. Arteaga-Solis E. Lee-Arteaga S.Y. Lenart B. Starcher B. Merkel C.A. Sukoyan M. Kerkis A. Hazeki N. Keene D.R. Sakai L.Y. Ramirez F. J. Biol. Chem. 2006; 281: 8016-8023Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). Fibulins constitute another family of extracellular matrix proteins that contain clusters of various domains (for a review, see Refs. 17Chu M.L. Tsuda T. Birth Defects Res. C. Embryo Today. 2004; 72: 25-36Crossref PubMed Scopus (68) Google Scholar, 18Argraves W.S. Greene L.M. Cooley M.A. Gallagher W.M. EMBO Rep. 2003; 4: 1127-1131Crossref PubMed Scopus (254) Google Scholar, 19Timpl R. Sasaki T. Kostka G. Chu M.L. Nat. Rev. Mol. Cell. Biol. 2003; 4: 479-489Crossref PubMed Scopus (385) Google Scholar). They are characterized by common homologous C-terminal domains of 120–140 amino acid residues preceded by an array of cbEGF domains. Fibulin-1 and -2 are larger in size as compared with other fibulins, with anaphylatoxin domains N-terminal of the tandem cbEGF stretches (20Argraves W.S. Tran H. Burgess W.H. Dickerson K. J. Cell Biol. 1990; 111: 3155-3164Crossref PubMed Scopus (182) Google Scholar, 21Pan T.C. Sasaki T. Zhang R.Z. Fässler R. Timpl R. Chu M.L. J. Cell Biol. 1993; 123: 1269-1277Crossref PubMed Scopus (148) Google Scholar). Fibulin-2 contains an additional large N-terminal domain, which is absent from all other fibulins. A second subgroup is formed by the relatively small (∼50–70 kDa) fibulin-3, -4, and -5 (22Lecka-Czernik B. Lumpkin Jr., C.K. 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Genet. 2000; 106: 66-72Crossref PubMed Scopus (25) Google Scholar), which each contain five cbEGF domains preceded by a variably modified cbEGF domain at the N terminus. In respect to the relationship of fibulins with the microfibril/elastic fiber system, it has been shown that several fibulins are associated with this system. Immunohistochemical experiments at the light and electron microscopic level revealed that (i) fibulin-1 is associated with the amorphous elastin core of elastic fibers (29Roark E.F. Keene D.R. Haudenschild C.C. Godyna S. Little C.D. Argraves W.S. J. Histochem. Cytochem. 1995; 43: 401-411Crossref PubMed Scopus (164) Google Scholar, 30Sasaki T. Gohring W. Miosge N. Abrams W.R. Rosenbloom J. Timpl R. FEBS Lett. 1999; 460: 280-284Crossref PubMed Scopus (68) Google Scholar), (ii) fibulin-2 is colocalized with microfibrils at the interface with elastic fibers as well as with microfibrils in the absence of elastin (31Reinhardt D.P. Sasaki T. Dzamba B.J. Keene D.R. Chu M.L. Göhring W. Timpl R. Sakai L.Y. J. Biol. Chem. 1996; 271: 19489-19496Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 32Raghunath M. Tschödrich-Rotter M. Sasaki T. Meuli M. Chu M.L. Timpl R. J. Invest. Dermatol. 1999; 112: 97-101Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar), and (iii) fibulin-5 is found on the internal aortic elastic lamina and elastic fibers produced by dermal fibroblasts in vivo and in vitro (33Yanagisawa H. Davis E.C. Starcher B.C. Ouchi T. Yanagisawa M. Richardson J.A. Olson E.N. Nature. 2002; 415: 168-171Crossref PubMed Scopus (501) Google Scholar, 34Nakamura T. Lozano P.R. Ikeda Y. Iwanaga Y. Hinek A. Minamisawa S. Cheng C.F. Kobuke K. Dalton N. Takada Y. Tashiro K. Ross J.J. Honjo T. Chien K.R. Nature. 2002; 415: 171-175Crossref PubMed Scopus (531) Google Scholar, 35Zheng Q. Choi J. Rouleau L. Leask R.L. Richardson J.A. Davis E.C. Yanagisawa H. J. Invest. Dermatol. 2006; (in press)Google Scholar). Additional evidence for a close functional relationship of fibulins with components of the microfibril/elastic fiber system comes from in vitro protein-protein interaction studies. It has been demonstrated that fibulin-1, -2, -4, and -5 can interact with tropoelastin (30Sasaki T. Gohring W. Miosge N. Abrams W.R. Rosenbloom J. Timpl R. FEBS Lett. 1999; 460: 280-284Crossref PubMed Scopus (68) Google Scholar, 33Yanagisawa H. Davis E.C. Starcher B.C. Ouchi T. Yanagisawa M. Richardson J.A. Olson E.N. Nature. 2002; 415: 168-171Crossref PubMed Scopus (501) Google Scholar, 36McLaughlin P.J. Chen Q. Horiguchi M. Starcher B.C. Stanton J.B. Broekelmann T.J. Marmorstein A.D. McKay B. Mecham R. Nakamura T. Marmorstein L.Y. Mol. Cell. Biol. 2006; 26: 1700-1709Crossref PubMed Scopus (174) Google Scholar, 37Freeman L.J. Lomas A. Hodson N. Sherratt M.J. Mellody K.T. Weiss A.S. Shuttleworth A. Kielty C.M. Biochem. J. 2005; 388: 1-5Crossref PubMed Scopus (85) Google Scholar). In addition, fibulin-2 and -5 were shown to interact with fibrillin-1, and the binding epitopes were mapped to a relatively large N-terminal region of fibrillin-1 (31Reinhardt D.P. Sasaki T. Dzamba B.J. Keene D.R. Chu M.L. Göhring W. Timpl R. Sakai L.Y. J. Biol. Chem. 1996; 271: 19489-19496Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 37Freeman L.J. Lomas A. Hodson N. Sherratt M.J. Mellody K.T. Weiss A.S. Shuttleworth A. Kielty C.M. Biochem. J. 2005; 388: 1-5Crossref PubMed Scopus (85) Google Scholar). Gene targeting experiments in mice have shown that fibulin-4 and -5 are both essential for the biogenesis of elastic fibers (33Yanagisawa H. Davis E.C. Starcher B.C. Ouchi T. Yanagisawa M. Richardson J.A. Olson E.N. Nature. 2002; 415: 168-171Crossref PubMed Scopus (501) Google Scholar, 34Nakamura T. Lozano P.R. Ikeda Y. Iwanaga Y. Hinek A. Minamisawa S. Cheng C.F. Kobuke K. Dalton N. Takada Y. Tashiro K. Ross J.J. Honjo T. Chien K.R. Nature. 2002; 415: 171-175Crossref PubMed Scopus (531) Google Scholar, 36McLaughlin P.J. Chen Q. Horiguchi M. Starcher B.C. Stanton J.B. Broekelmann T.J. Marmorstein A.D. McKay B. Mecham R. Nakamura T. Marmorstein L.Y. Mol. Cell. Biol. 2006; 26: 1700-1709Crossref PubMed Scopus (174) Google Scholar). Mutations in some of the fibulins lead to a number of human genetic disorders, including Malattia Leventinese and Doyne honeycomb retinal dystrophy (fibulin-3), age-related macular degeneration (fibulin-5), and cutis laxa (fibulin-4 and -5) (38Stone E.M. Lotery A.J. Munier F.L. Heon E. Piguet B. Guymer R.H. Vandenburgh K. Cousin P. Nishimura D. Swiderski R.E. Silvestri G. Mackey D.A. Hageman G.S. Bird A.C. Sheffield V.C. Schorderet D.F. Nat. Genet. 1999; 22: 199-202Crossref PubMed Scopus (393) Google Scholar, 39Stone E.M. Braun T.A. Russell S.R. Kuehn M.H. Lotery A.J. Moore P.A. Eastman C.G. Casavant T.L. Sheffield V.C. N. Engl. J. Med. 2004; 351: 346-353Crossref PubMed Scopus (280) Google Scholar, 40Loeys B. Van Maldergem L. Mortier G. Coucke P. Gerniers S. Naeyaert J.M. De Paepe A. Hum. Mol. Genet. 2002; 11: 2113-2118Crossref PubMed Scopus (244) Google Scholar, 41Markova D. Zou Y. Ringpfeil F. Sasaki T. Kostka G. Timpl R. Uitto J. Chu M.L. Am. J. Hum. Genet. 2003; 72: 998-1004Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 42Hucthagowder V. Sausgruber N. Kim K.H. Angle B. Marmorstein L.Y. Urban Z. Am. J. Hum. Genet. 2006; 78: 1075-1080Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). Here, we have characterized the molecular interactions of fibulins with fibrillin-1. Fibulin-2, -4, and -5 show similar binding to the N-terminal region of fibrillin-1, whereas other fibulins did not interact. A panel of swapped domain constructs established that the first hybrid domain in fibrillin-1 is essential but not sufficient for binding to fibulin-2, -4, and -5. Whereas these fibulins are able to associate with microfibrils, they do not interfere with the ability of fibrillin-1 to self-interact. However, fibulin-2 and -5 can promote ternary complexes between fibrillin-1 and tropoelastin, indicating that they act as molecular adaptors between microfibrils and elastic fibers. Recombinant Proteins—In order to map fibulin binding sites on fibrillin-1, wild-type and swapped domain recombinant fibrillin-1 fragments were generated. Production and characterization of recombinant fibrillin-1 fragments rF6H and rF16 (43Jensen S.A. Reinhardt D.P. Gibson M.A. Weiss A.S. J. Biol. Chem. 2001; 276: 39661-39666Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar) and rF51 and rF1F (44Vollbrandt T. Tiedemann K. El-Hallous E. Lin G. Brinckmann J. John H. Bätge B. Notbohm H. Reinhardt D.P. J. Biol. Chem. 2004; 279: 32924-32931Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) have been described in detail previously. The generation of N- and C-terminal deletion constructs rF16N and rF16NEH will be described elsewhere. 3E. El-Hallous, D. Hubmacher, H. Notbohm, and D. P. Reinhardt, manuscript in preparation. The plasmids HFBN23, HFBN25, pBS-HFBN8-1-4, and pcDFRTSP-rF1A used as templates in PCRs have been described in detail in other studies (7Corson G.M. Chalberg S.C. Dietz H.C. Charbonneau N.L. Sakai L.Y. Genomics. 1993; 17: 476-484Crossref PubMed Scopus (227) Google Scholar, 44Vollbrandt T. Tiedemann K. El-Hallous E. Lin G. Brinckmann J. John H. Bätge B. Notbohm H. Reinhardt D.P. J. Biol. Chem. 2004; 279: 32924-32931Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 45Reinhardt D.P. Keene D.R. Corson G.M. Pöschl E. Bächinger H.P. Gambee J.E. Sakai L.Y. J. Mol. Biol. 1996; 258: 104-116Crossref PubMed Scopus (208) Google Scholar). All fibrillin-1 expression plasmids were designed with a sequence for a signal peptide from the BM40 protein to ensure secretion into the culture medium and a sequence for a C-terminal hexahistidine tag to facilitate protein purification. Due to the cloning strategy, the recombinant proteins are expressed with an additional Ala-Pro-Leu-Ala sequence at their N terminus. All expression plasmids described below were analyzed and verified for correct insertion and orientation by DNA sequencing (Agowa, Berlin, Germany). The recombinant proteins expressed by the expression plasmids are schematically shown in Fig. 1. Fragment rF1F is a wild-type fibrillin-1 fragment spanning the region between the N terminus and the second TB/8-Cys domain (Ser19–Gly714) (44Vollbrandt T. Tiedemann K. El-Hallous E. Lin G. Brinckmann J. John H. Bätge B. Notbohm H. Reinhardt D.P. J. Biol. Chem. 2004; 279: 32924-32931Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). The plasmid for rF1G has been designed to express the identical region compared with rF1F but with swapped domains cbEGF 8 and 9 replacing cbEGF 1 and 2. For the cloning strategy, it was necessary to modify the polylinker region of the cloning plasmid pBluescript II SK+ (Stratagene) to introduce additional ClaI and AgeI restriction sites. For this goal, the pBluescript plasmid was restricted with XhoI and SacI, and the 2870-bp fragment was religated with complementary oligonucleotides pBS3-S (5′-TCGAGTATCGATTGACGTCTACCGGTGAGCT-3′) and pBS3-AS (5′-CACCGGTAGACGTCAATCGATAC-3′), resulting in a plasmid termed pBS3. A 923 bp ClaI-AgeI fragment from pDNSP-rF1F (44Vollbrandt T. Tiedemann K. El-Hallous E. Lin G. Brinckmann J. John H. Bätge B. Notbohm H. Reinhardt D.P. J. Biol. Chem. 2004; 279: 32924-32931Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) was ligated with the ClaI-AgeI-restricted pBS3 plasmid to yield plasmid pBS3-rF1F. To obtain the sequence for domains cbEGF 8 and 9 of fibrillin-1 and to introduce additional BtgI and NsiI cloning sites, template HFBN23 (7Corson G.M. Chalberg S.C. Dietz H.C. Charbonneau N.L. Sakai L.Y. Genomics. 1993; 17: 476-484Crossref PubMed Scopus (227) Google Scholar) was amplified by PCR using oligonucleotides rF1G-S (5′-ATTACCGTGGCTTCATTCCAAATATCCGCACGGGACTTGTCAAGATATTAATGAATGTGTACTGAACAG-3′) and rF1G-AS (5′-CTGAATGCATATGGTTTTTGTTGGATCCAAAGTAC-3′), resulting in a 293-bp product. The amplified DNA was ligated with the pCR4Blunt-TOPO vector (Invitrogen), and the 283-bp BtgI-NsiI fragment isolated form this plasmid was subcloned into the BtgI-NsiI-restricted pBS3-rF1F, resulting in plasmid pBS3-rF1G. Finally, the 917-bp ClaI-AgeI fragment from pBS3-rF1G was ligated into the ClaI-AgeI-restricted plasmid pDNSP-rF1F. The resulting expression plasmid was named pDNSP-rF1G. The fibrillin-1 construct rF1H is identical to rF1F except that the first hybrid domain of fibrillin-1 was replaced by the second hybrid domain. To generate the expression plasmid for this construct, template pcDFRTSP-rF1A (44Vollbrandt T. Tiedemann K. El-Hallous E. Lin G. Brinckmann J. John H. Bätge B. Notbohm H. Reinhardt D.P. J. Biol. Chem. 2004; 279: 32924-32931Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) was amplified using oligonuceotides rF1H-S (5′-ATGTGCATGCACTTACGGATTTACTGGACCCCAGTGTATAGAAACCATCAAGGGCACTTGC-3′) and rF1H-AS (5′-AGAGCCCGGGGATGGCCTGGCATTCATCCACATCTTCACATTGTGTTCCTTTAATTCTTGAG-3′) resulting in a 263-bp product, which was subcloned into the pCR4Blunt-TOPO vector. This plasmid was restricted with SphI and XmaI, and the 245-bp fragment was ligated with the SphI-XmaI-restricted pBS3-rF1F, resulting in plasmid pBS-rF1H. A 911-bp ClaI-AgeI fragment isolated from pBS-rF1H was then ligated into the ClaI-AgeI-restricted pDNSP-rF1F, and the new expression plasmid was termed pDNSP-rF1H. The rF16H construct is identical to wild-type rF16 coding for the region in fibrillin-1 between the N terminus and cbEGF22 (43Jensen S.A. Reinhardt D.P. Gibson M.A. Weiss A.S. J. Biol. Chem. 2001; 276: 39661-39666Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar), except that the first hybrid domain was replaced with the second hybrid domain. A 1355-bp NheI-AgeI fragment from plasmid pDNSP-rF1H was subcloned into the NheI-AgeI restricted pDNSP-rF16 (43Jensen S.A. Reinhardt D.P. Gibson M.A. Weiss A.S. J. Biol. Chem. 2001; 276: 39661-39666Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar), and the new plasmid was designated pDNSP-rF16H. The wild-type fragment rF51 (44Vollbrandt T. Tiedemann K. El-Hallous E. Lin G. Brinckmann J. John H. Bätge B. Notbohm H. Reinhardt D.P. J. Biol. Chem. 2004; 279: 32924-32931Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) was modified to replace the second hybrid domain with the first hybrid domain (rF51H). Template pDNSP-rF1F (44Vollbrandt T. Tiedemann K. El-Hallous E. Lin G. Brinckmann J. John H. Bätge B. Notbohm H. Reinhardt D.P. J. Biol. Chem. 2004; 279: 32924-32931Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) was amplified using oligonucleotides rF51H-S 5′-CTTTGGATCCAACAAAAACCATCTGCATAAGAGATTACAGGACAGGCC-3′) and rF51H-AS (5′-GTGTTAACACACAGGCCATTTTTACACACTCCTGGGAACACTTCACATTCATCTATATCTTGACAAGCTCCCGTGCGG-3′), resulting in a 289-bp product, which was ligated with the pCR-Blunt II-TOPO plasmid (Invitrogen). The 279-bp BamHI-HpaI-restricted fragment from this plasmid was ligated into the BamHI-HpaI-restricted pDNSP-rF51, resulting in the expression plasmid pDNSP-rF51H. Generation of stable recombinant cell clones using human embryonic kidney cells 293, production of recombinant medium, and purification of the histidine-tagged proteins by chelating chromatography was performed as described previously for other fibrillin-1 and -2 fragments with minor modifications (46Lin G. Tiedemann K. Vollbrandt T. Peters H. Bätge B. Brinckmann J. Reinhardt D.P. J. Biol. Chem. 2002; 277: 50795-50804Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Recombinant fibulin-1C, -1D, -2, and -4 were prepared as described previously (21Pan T.C. Sasaki T. Zhang R.Z. Fässler R. Timpl R. Chu M.L. J. Cell Biol. 1993; 123: 1269-1277Crossref PubMed Scopus (148) Google Scholar, 25Giltay R. Timpl R. Kostka G. Matrix Biol. 1999; 18: 469-480Crossref PubMed Scopus (126) Google Scholar, 47Sasaki T. Kostka G. Göhring W. Wiedemann H. Mann K. Chu M.L. Timpl R. J. Mol. Biol. 1995; 245: 241-250Crossref PubMed Scopus (93) Google Scholar, 48Sasaki T. Göhring W. Pan T.C. Chu M.L. Timpl R. J. Mol. Biol. 1995; 254: 892-899Crossref PubMed Scopus (108) Google Scholar). Expression and characterization of fibulin-3 and -5 will be described in detail elsewhere. 4N. Kobayashi, G. Kostka, J. H. O. Garbe, D. R. Keene, H. P. Bächinger, F.-G. Hanisch, D. Markova, T. Tsuda, R. Timpl, M.-L. Chu, and T. Sasaki, submitted for publication. Briefly, cDNA coding for each fibulin was inserted into the pCEP-Pu or pCEP-Pu/AC7 vector, and 293-EBNA cells (Invitrogen) were transfected by standard methods. Fibulins were purified from serum-free culture medium as described previously with minor modifications (25Giltay R. Timpl R. Kostka G. Matrix Biol. 1999; 18: 469-480Crossref PubMed Scopus (126) Google Scholar). Recombinant tropoelastin was kindly provided by Dr. Anthony S. Weiss (49Martin S.L. Vrhovski B. Weiss A.S. Gene. 1995; 154: 159-166Crossref PubMed Scopus (168) Google Scholar). Solid Phase Microwell Assays—For protein-protein interaction assays, multiwell plates (Maxisorp, 96 wells; Nalge Nunc International) were coated with purified proteins (10–20 μg/ml; 50–100 μl/well) in 50 mm Tris-HCl, 150 mm NaCl, pH 7.4 (TBS), at 4 °C overnight. All subsequent steps were performed at 20 °C. Nonspecific binding sites were blocked for 1–2 h with 100 μl of TBS containing 2 mm CaCl2 and 5% (w/v) nonfat milk (binding buffer). The wells were washed three times with TBS, including 2 mm CaCl2 and 0.05% (v/v) Tween 20 (washing buffer). The coated proteins were typically incubated with either a single or with a duplicate serial dilution of the soluble ligands starting at 100–150 μg/ml for 2 h. In some cases, a second protein (fibulins or fibrillin-1 fragments) was added at constant concentrations in order to either test inhibitory effects of fibrillin self-assembly or enhancing effects on the fibrillin-1-tropoelastin interactions. After ligand incubation, the wells were washed three times with washing buffer and incubated for 2 h with 100 μl of the primary antibodies against the respective soluble ligands (diluted 1:500–1:1000 in binding buffer). After washing, the wells were incubated with 100 μlof horseradish peroxidase-conjugated secondary goat anti-rabbit antibodies (1:800 diluted in binding buffer) for 1.5 h. Color development was performed with 1 mg/ml 5-aminosalicylic acid in 20 mm phosphate buffer, pH 6.8, including 0.045% (v/v) H2O2 (100 μl/well) for 3–5 min and stopped by adding 100 μlof 2 m NaOH to each well. Color yields were determined at 490 nm using a Microplate EL310 autoreader (Bio-Tek Instruments). All solid phase interaction assays were repeated 3–7 times, resulting in similar binding profiles each time. Nonspecific binding of the soluble ligands to either the blocking reagents or the plastic surface was subtracted from binding profiles. Blot Overlay Assay—Extraction of authentic fibulin-2 followed an established procedure (31Reinhardt D.P. Sasaki T. Dzamba B.J. Keene D.R. Chu M.L. Göhring W. Timpl R. Sakai L.Y. J. Biol. Chem. 1996; 271: 19489-19496Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). Briefly, confluent layers of human skin fibroblasts were first washed two times with TBS, including protease inhibitors (2 mm phenylmethylsulfonyl fluoride, 5 mm N-ethylmaleimide), and the extracellular layer was then extracted with 0.1 ml/cm2 TBS, including protease inhibitors and 10 mm EDTA for 10 min at 20 °C. Proteins in 1-ml aliquots of the EDTA extracts were precipitated with 10% (w/v) trichloroacetic acid, dissolved in nonreducing SDS sample buffer, and separated by SDS gel electrophoresis (5% (w/v) acrylamide). The proteins were transferred onto nitrocellulose membrane (Bio-Rad) in 10 mm sodium borate, pH 9.2, at a constant current of 0.4 A for 45 min at 4 °C. All of the following incubations were performed at 20 °C. The membranes were first incubated for 1 h with TBS containing 5% (w/v) nonfat milk to block nonspecific binding sites and then with 100 μg/ml recombinant fibrillin-1 fragments as soluble ligands for3 h in TBS containing 5% (w/v) nonfat milk and 2 mm CaCl2 (binding buffer). Incubation with binding buffer alone served as a negative control. After washing three times with TBS, including 0.05% (v/v) Tween 20 and 2 mm CaCl2, the membranes were incubated for 2 h with monoclonal antibody 26 (∼5 μg/ml in binding buffer) against the soluble ligands. The membranes were incubated for 1.5 h with horseradish peroxidase-conjugated goat-anti-mouse antibodies (1:800 diluted in binding buffer). The color was developed in TBS, 17% methanol, including 0.02% (v/v) H2O2 and 0.5 mg/ml 4-chloro-1-naphthol (Bio-Rad). Antibodies—The following polyclonal and monoclonal antibodies have been generated and characterized previously. Polyclonal antisera anti-rF16 and anti-rF6H were raised in rabbits against the recombinant N- and C-terminal halves of human fibrillin-1, respectively (50Tiedemann K. Bätge B. Müller P.K. Reinhardt D.P. J. Biol. Chem. 2001; 276: 36035-36042Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 51Tiedemann K. Sasaki" @default.
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W2047152078 date "2007-03-01" @default.
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W2047152078 title "Fibrillin-1 Interactions with Fibulins Depend on the First Hybrid Domain and Provide an Adaptor Function to Tropoelastin" @default.
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