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• W1966152952 abstract "We have defined the homotypic interactions of fibrillin-1 to obtain new insights into microfibril assembly. Dose-dependent saturable high affinity binding was demonstrated between N-terminal fragments, between furin processed C-terminal fragments, and between these N- and C-terminal fragments. The N terminus also interacted with a downstream fragment. A post-furin cleavage site C-terminal sequence also interacted with the N terminus, with itself and with the furin-processed fragment. No other homotypic fibrillin-1 interactions were detected. Some terminal homotypic interactions were inhibited by other terminal sequences, and were strongly calcium-dependent. Treatment of an N-terminal fragment with N-ethylmaleimide reduced homotypic binding. Microfibril-associated glycoprotein-1 inhibited N- to C-terminal interactions but not homotypic N-terminal interactions. These fibrillin-1 interactions are likely to regulate pericellular fibrillin-1 microfibril assembly. We have defined the homotypic interactions of fibrillin-1 to obtain new insights into microfibril assembly. Dose-dependent saturable high affinity binding was demonstrated between N-terminal fragments, between furin processed C-terminal fragments, and between these N- and C-terminal fragments. The N terminus also interacted with a downstream fragment. A post-furin cleavage site C-terminal sequence also interacted with the N terminus, with itself and with the furin-processed fragment. No other homotypic fibrillin-1 interactions were detected. Some terminal homotypic interactions were inhibited by other terminal sequences, and were strongly calcium-dependent. Treatment of an N-terminal fragment with N-ethylmaleimide reduced homotypic binding. Microfibril-associated glycoprotein-1 inhibited N- to C-terminal interactions but not homotypic N-terminal interactions. These fibrillin-1 interactions are likely to regulate pericellular fibrillin-1 microfibril assembly. Fibrillins are large multidomain glycoproteins (∼340 kDa) and the major structural components of a class of 10-12-nm extracellular matrix microfibrils that are widely distributed in connective tissues (1Sakai L.Y. Keene D.R. Engvall E. J. Cell Biol. 1986; 103: 2499-2509Crossref PubMed Scopus (909) Google Scholar, 2Handford P.A. Downing A.K. Reinhardt D.P. Sakai L.Y. Matrix Biol. 2000; 19: 457-470Crossref PubMed Scopus (114) Google Scholar, 3Kielty C.M. Baldock C. Lee D. Rock M.J. Ashworth J.L. Shuttleworth C.A. Philos. Trans. R. Soc. Lond. B. 2002; 357: 207-217Crossref PubMed Scopus (92) Google Scholar). In elastic tissues such as aorta, lung, and skin, they are associated with tropoelastin deposition during elastic fibrillogenesis, and form an outer mantle for mature elastic fibers (4Mecham R.P. Davis E.C. Yurchenco P.D. Birk D.E. Mecham R.P. Extracellular Matrix Assembly and Structure. Academic Press, New York1994: 281-314Google Scholar, 5Kielty C.M. Sherratt M.J. Shuttleworth C.A. J. Cell Sci. 2002; 115: 2817-2828Crossref PubMed Google Scholar). Microfibril arrays are also abundant in dynamic tissues that do not express elastin, such as the cilary zonules of the eye (6Ashworth J.L. Kielty C.M. McLeod D.M. Br. J. Ophthalmol. 2000; 84: 1312-1317Crossref PubMed Scopus (51) Google Scholar). Structural analyses of isolated fibrillin-rich microfibrils have revealed a complex 56-nm “beads-on-a-string” appearance, whereas the predicted length of a fibrillin monomer is ∼160 nm (7Wright D.W. Mayne R. J. Ultrastruct. Mol. Struct. Res. 1988; 100: 224-234Crossref PubMed Scopus (89) Google Scholar, 8Keene D.R. Maddox B.K. Kuo H.J. Sakai L.Y. Glanville R.W. J. Histochem. Cytochem. 1991; 39: 441-449Crossref PubMed Scopus (183) Google Scholar, 9Kielty C.M. Cummings C. Whittaker S.P. Shuttleworth C.A. Grant M.E. J. Cell Sci. 1991; 99: 797-807Crossref PubMed Google Scholar, 10Sherratt M.J. Holmes D.F. Shuttleworth C.A. Kielty C.M. Int. J. Biochem. Cell Biol. 1997; 29: 1063-1070Crossref PubMed Scopus (36) Google Scholar). Mutations in fibrillin-1 cause Marfan syndrome, a heritable disease associated with severe aortic, ocular, and skeletal defects because of defective elastic fibers (11Robinson P.N. Booms P. Cell Mol. Life Sci. 2001; 58: 1698-1707Crossref PubMed Google Scholar). There are three closely related fibrillin isoforms that have distinct, but overlapping, developmental, and adult tissue distributions (12Pereira 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, 13Zhang 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, 14Corson G.M. Charbonneau N.L. Keene D.R. Sakai L.Y. Genomics. 2004; 83: 461-472Crossref PubMed Scopus (143) Google Scholar). Fibrillin-1 contains 47 epidermal growth factor (EGF) 1The abbreviations used are: EGF, epidermal growth factor; MAGP-1, microfibril-associated glycoprotein-1; GST, glutathione S-transferase; BSA, bovine serum albumin; ABTS, 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid); TBS, Tris-buffered saline; NEM, N-ethylmaleimide; CP, C-terminal fibrillin-1 protein.-like domains, 43 of which are calcium binding (cbEGF)-like domains, seven 8-cysteine (TB) modules, two hybrid motifs with similarities to both cbEGF-like domains and TB motifs, and a proline-rich region that may act as a hinge region (12Pereira 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). In fibrillin-2, this sequence is glycine-rich (13Zhang 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), and in fibrillin-3 it is proline/glycine-rich (14Corson G.M. Charbonneau N.L. Keene D.R. Sakai L.Y. Genomics. 2004; 83: 461-472Crossref PubMed Scopus (143) Google Scholar). The linkage to Marfan syndrome, and its abundance in developing and adult tissues (11Robinson P.N. Booms P. Cell Mol. Life Sci. 2001; 58: 1698-1707Crossref PubMed Google Scholar, 15Quondamatteo F. Reinhardt D P. Charbonneau N.L. Pophal G. Sakai L.Y. Herken R. Matrix Biol. 2002; 21: 637-646Crossref PubMed Scopus (76) Google Scholar), confirm fibrillin-1 as the major fibrillin isoform in elastic fibers. C-terminal furin processing of fibrillin-1 is important for extracellular fibrillin-1 deposition (16Wallis D.D. Putnam E.A. Cretoiu J.S. Carmical S.G. Cao S.N. Thomas G. Milewicz D.M. J. Cell. Biochem. 2003; 90: 641-652Crossref PubMed Scopus (36) Google Scholar, 17Raghunath M. Putnam E.A. Ritty T. Hamstra D. Park E.S. Tschodrich-Rotter M. Peters R. Rehemtulla A. Milewicz D.M. J. Cell Sci. 1999; 112: 1093-1100Crossref PubMed Google Scholar). However, mass spectrometry analysis of isolated tissue microfibrils has shown that at least some unprocessed molecules are present in tissue microfibrils. 2Cain, S. A., Morgan, A., Sherratt, M. J., Ball, S. G., Shuttleworth, C. A., and Kielty, C. M. (2005) Mol. Cell. Proteomics, in press. The molecular mechanisms by which fibrillins assemble into mature microfibrils remain unresolved. Several models of fibrillin alignment in microfibrils have been proposed (19Reinhardt D.P. Keene D.R. Corson G.M. Pöschl E. Bächinger H.P. Gambee J.E. Sakai L.Y. J. Mol. Biol. 1996; 258: 104-114Crossref PubMed Scopus (208) Google Scholar, 20Baldock C. Koster A.J. Ziese U. Rock M.J. Sherratt M.J. Kadler K.E. Shuttleworth C.A. Kielty C.M. J. Cell Biol. 2001; 152: 1045-1056Crossref PubMed Scopus (122) Google Scholar, 21Lee 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). Head-to-tail alignment of fibrillin-1 molecules within microfibrils has been proposed on the basis of antibody localizations (19Reinhardt D.P. Keene D.R. Corson G.M. Pöschl E. Bächinger H.P. Gambee J.E. Sakai L.Y. J. Mol. Biol. 1996; 258: 104-114Crossref PubMed Scopus (208) Google Scholar, 20Baldock C. Koster A.J. Ziese U. Rock M.J. Sherratt M.J. Kadler K.E. Shuttleworth C.A. Kielty C.M. J. Cell Biol. 2001; 152: 1045-1056Crossref PubMed Scopus (122) Google Scholar). Complex intramolecular folding in 56-nm microfibrils was also demonstrated by interbead antibody epitope reversal (20Baldock C. Koster A.J. Ziese U. Rock M.J. Sherratt M.J. Kadler K.E. Shuttleworth C.A. Kielty C.M. J. Cell Biol. 2001; 152: 1045-1056Crossref PubMed Scopus (122) Google Scholar). A one-third staggered arrangement has also been suggested (21Lee 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). Lateral fibrillin-1 assembly is another critical, but unexplained, feature of microfibril assembly. Electron microscopy of isolated microfibrils, and mass mapping, suggest that there are eight molecules in cross-section (20Baldock C. Koster A.J. Ziese U. Rock M.J. Sherratt M.J. Kadler K.E. Shuttleworth C.A. Kielty C.M. J. Cell Biol. 2001; 152: 1045-1056Crossref PubMed Scopus (122) Google Scholar, 22Wallace R.N. Streeten B.W. Hanna R.B. Curr. Eye Res. 1991; 10: 99-109Crossref PubMed Scopus (48) Google Scholar, 23Davis E.C. Roth R.A. Heuser J.E. Mecham R.P. J. Struct. Biol. 2002; 139: 65-75Crossref PubMed Scopus (31) Google Scholar). Recombinantly expressed N-terminal regions of fibrillin-1 have a tendency to dimerize (24Ashworth J.L. Kelly V. Wilson R. Shuttleworth C.A. Kielty C.M. J. Cell Sci. 1999; 112: 3549-3558Crossref PubMed Google Scholar, 25Trask T.M. Ritty T.M. Broekelmann T. Tisdale C. Mecham R.P. Biochem. J. 1999; 340: 693-701Crossref PubMed Scopus (62) Google Scholar), whereas the N-terminal half of the molecule interacts with the C-terminal half (26Lin G.K. Tiedemann K. Vollbrandt T. Peters H. Batge B. Brinckmann J. Reinhardt D.P. J. Biol. Chem. 2002; 277: 50795-50804Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). An unpaired cysteine residue in the first hybrid domain may covalently link aligned fibrillin-1 molecules (27Reinhardt 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). In vitro studies of interactions between fibrillin-1 and other elastic fiber molecules have revealed that its N terminus is highly interactive, binding to microfibril-associated glycoprotein-1 (MAGP-1) (28Jensen 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, 29Rock M.J. Cain S.A. Freeman L.J. Morgan A. Mellody K. Marson A. Shuttleworth C.A. Weiss A.S. Kielty C.M. J. Biol. Chem. 2004; 279: 23748-23758Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar) and fibulin-2 (30Reinhardt 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). It is unclear whether any of these interactions are mutually exclusive. We have undertaken a detailed analysis of fibrillin-1 homotypic interactions, using recombinant fragments that span the entire coding region of human fibrillin-1, to define fibrillin-1 sequences that interact. The N terminus binds very strongly to itself and to an overlapping downstream sequence. The furin-processed C terminus and the proteolytically released C-terminal 20-kDa fragment both bind homotypically and tightly to the N terminus. No other homotypic fibrillin-1 interactions were detected. MAGP-1 inhibits the N- to C-terminal interaction but not the N- to N-terminal interaction. These interactions may regulate linear head-to-tail and lateral fibrillin-1 assembly. Recombinant Fibrillin-1 Fragments and Full-length MAGP-1—Recombinant human fibrillin-1 fragments PF1 encoded by exons 1-11 (residues 1-489), PF2 encoded by exons 9-17 (residues 330-722), PF3 encoded by exons 1-17 (residues 1-722), PF4 encoded by exons 1-8 (residues 1-329), PF5 encoded by exons 18-25 (residues 723-1069), PF7 encoded by exons 24-30 (residues 952-1279), PF8 encoded by exons 30-38 (residues 1238-1605), PF9 encoded by exons 37-43 (residues 1528-2166), PF10 encoded by exons 41-52 (residues 1688-2165), PF11 encoded by exons 37-52 (residues 1528-2165), PF12 encoded by exons 50-58 (residues 2055-2443), and PF13 encoded by exons 57-65 (residues 2402-2871) (12Pereira 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) were expressed and purified in milligram amounts using a mammalian episomal expression system and 293-EBNA cells as described previously (29Rock M.J. Cain S.A. Freeman L.J. Morgan A. Mellody K. Marson A. Shuttleworth C.A. Weiss A.S. Kielty C.M. J. Biol. Chem. 2004; 279: 23748-23758Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 31Bax D.V. Bernard S.E. Lomas A. Morgan A. Shuttleworth C.A. Humphries M.J. Kielty C.M. J. Biol. Chem. 2003; 278: 34605-34616Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar) (Fig. 1A). The pCEP-pu/AC7 vector (32Mayer U. Pöschl 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, 33Kohfeldt E. Maurer P. Vannahme C. Timpl R. FEBS Lett. 1997; 414: 557-561Crossref PubMed Scopus (203) Google Scholar) was modified by incorporation of an N-terminal His6 tag following the signal peptide to allow rapid fragment purification by nickel chromatography. The His6 tag could subsequently be removed by enterokinase. All fragments had their predicted monomeric mass, as judged by SDS-PAGE and Coomassie Blue staining and by Western blotting using a penta-His antibody (Qiagen) in the presence or absence of 10 mm dithiothreitol (29Rock M.J. Cain S.A. Freeman L.J. Morgan A. Mellody K. Marson A. Shuttleworth C.A. Weiss A.S. Kielty C.M. J. Biol. Chem. 2004; 279: 23748-23758Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 31Bax D.V. Bernard S.E. Lomas A. Morgan A. Shuttleworth C.A. Humphries M.J. Kielty C.M. J. Biol. Chem. 2003; 278: 34605-34616Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). They also bound calcium because electrophoretic shifts were apparent following EDTA treatment, confirming that the cbEGF-like domains are correctly folded (34Ashworth J.L. Sherratt M.J. Rock M.J. Murphy G. Shapiro S.D. Shuttleworth C.A. Kielty C.M. Biochem. J. 1999; 340: 171-181Crossref PubMed Scopus (220) Google Scholar). PF1, PF2, PF3, PF5, PF7, PF8, PF9, PF10, PF11, and PF12 fragments were N-glycosylated as predicted from the primary sequence. PF13 was furin processed on secretion that removed an N-glycosylated C-terminal post-furin cleavage fragment (Fig. 1B). For some experiments, PF13 was treated with purified furin (10 units) (Sigma). In other experiments, after addition of decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone (10 μm) (Bachem, UK), ∼25% PF13 remained unprocessed (Fig. 1B). Fibrillin-1 fragments expressed in a similar system are correctly folded (19Reinhardt D.P. Keene D.R. Corson G.M. Pöschl E. Bächinger H.P. Gambee J.E. Sakai L.Y. J. Mol. Biol. 1996; 258: 104-114Crossref PubMed Scopus (208) Google Scholar, 26Lin G.K. Tiedemann K. Vollbrandt T. Peters H. Batge B. Brinckmann J. Reinhardt D.P. J. Biol. Chem. 2002; 277: 50795-50804Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 27Reinhardt 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). Full-length human MAGP-1 was expressed in the same mammalian episomal expression system, as described previously (29Rock M.J. Cain S.A. Freeman L.J. Morgan A. Mellody K. Marson A. Shuttleworth C.A. Weiss A.S. Kielty C.M. J. Biol. Chem. 2004; 279: 23748-23758Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Recombinant Expression of Post-furin Cleavage Site Fibrillin-1 Fragment—Fibrillin-1 fragment CP encoded by exon 65 (the final 420 bp) was expressed using the pGEX4T-3 expression vector in Escherichia coli. Cells were treated with 1% Triton X-100, then sonicated, and lysates were collected after centrifugation (7,800 × g, 10 min) in the presence of 2 mm sodium azide and a mixture of protease inhibitors (protease inhibitor mixture, Sigma). The recombinant protein was purified as a fusion protein on a glutathione-Sepharose column (Amersham Biosciences). The column was first washed with phosphate-buffered saline, then the protein was eluted in 30 mm Tris/HCl, pH 8.0, containing 30 mm glutathione, in 1-ml fractions. Eluted fractions were then dialyzed into 0.1 m NaCl, 0.02 m Tris/HCl, pH 7.8, containing 0.001 m CaCl2, and analyzed by SDS-PAGE in reducing conditions (Fig. 1C), Western blot, and size exclusion chromatography using Superdex 200. Pure GST expressed in this E. coli system was a gift from Prof. M. J. Humphries (University of Manchester, Manchester, UK). Solid-phase Binding Assays—Solid-phase binding assays utilized recombinant human fibrillin-1 fragments PF1, PF2, PF3, PF4, PF5, PF7, PF8, PF9, PF10, PF11, PF12, PF13, CP, and the GST control. The soluble ligands were biotinylated and the binding assays were conducted at 37 °C. The block for these biotinylated ligands was bovine serum albumin (BSA). For biotinylation of soluble ligands, each fibrillin-1 fragment or full-length human MAGP-1 was rotated at room temperature for 30 min with an approximate 10-fold molar excess of 10 mg/ml solution of Immunopure sulfo-N-hydroxysuccinimide ester-biotin (Pierce) diluted in phosphate-buffered saline. Each mixture was then dialyzed against several changes of 0.02 m Tris/HCl, pH 7.8, containing 0.1 m NaCl and 0.001 m CaCl2 (TBS/CaCl2) to remove excess biotin. Flat-bottomed microtiter plates (ThermoLabsystems, Franklin, WI) were coated with N- or C-terminal fibrillin-1, each at 0.12 μm (100 μl) in TBS/CaCl2, overnight at 37 °C. The plates were then incubated with TBS/CaCl2 containing 10 mg/ml heat denatured filtered BSA for at least 3 h at room temperature to block nonspecific binding sites and then washed with TBS/CaCl2 containing 1 mg/ml heat-denatured filtered BSA (wash buffer) (3 × 200 μl). Initially, 5 μg/ml soluble fibrillin-1 protein fragments that together span the full-length molecule were screened for binding to each other or to MAGP-1 by incubating in TBS/CaCl2 for 3 h, or overnight at 37 °C. Subsequently, detailed binding assays of the interacting fibrillin-1 fragments were conducted, with addition of soluble biotinylated ligand at concentrations from 0 to 0.15 μm in TBS/CaCl2 for 3 h or overnight at 37 °C. Plates were washed (3 × 200 μl) before detection of bound fibrillin-1. Biotinylated ligands were quantified by incubating with 1:200 dilution of ExtraAvidin peroxidase conjugate (Sigma) in TBS/CaCl2 at room temperature for 10-15 min, and read at a wavelength of 405 nm. For both methods, wells were washed four times and the color developed using 40 mm 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) solution (Sigma) for ∼10-20 min at room temperature. Plates were read at a wavelength of 405 nm. All assays were performed in triplicate and repeated at least twice to confirm results. Calcium Dependence Binding Assays—Binding assays were performed as above, except that dilution buffers and washes were with or without calcium (0.001 m CaCl2). The wash buffer contained 1 mg/ml filtered, heat-denatured BSA in Tris-buffered saline (TBS). Solid-phase protein fragments at 0.12 μm were incubated for 1 h at 4 °C in the presence or absence of 10 mm EDTA, before adherence to the plate and overnight incubation at 37 °C. The wells were blocked with 10 mg/ml filtered, heat-denatured BSA for 3 h, and washed with wash buffer with or without calcium, depending on whether or not the solid phase had been pretreated with EDTA. Each soluble protein ligand was biotinylated and dialyzed, as described above. Each protein ligand was incubated at 4 °C in the presence or absence of 10 mm EDTA for 1 h, re-equilibrated in TBS with or without calcium, then added to the wells at (0.02 μm) and incubated overnight at 37 °C. Plates were washed three times with wash buffer with or without calcium, before detection of bound fibrillin-1. Biotinylated ligand was quantified by incubating with 1:200 dilution of ExtraAvidin peroxidase conjugate (Sigma) in TBS with or without CaCl2, for 10-20 min at room temperature. Wells were washed four times with wash buffer with or without calcium, and the color was developed following addition of 40 mm ABTS solution for 10-20 min at room temperature. Plates were read at a wavelength of 405 nm. All experiments were done in triplicate wells, and repeated at least twice to confirm results. Fibrillin-1 Inhibition Binding Assays—Inhibition binding assays were also conducted using both non-biotinylated and biotinylated soluble protein ligands. Flat bottomed microtiter plates were coated with N-terminal (PF4) or C-terminal (PF13) fragments at 0.12 μm in TBS/CaCl2 overnight at 37 °C. Nonspecific binding sites were blocked with TBS/CaCl2 containing 10 mg/ml BSA, at room temperature for at least 3 h. The plates were washed with wash buffer (3 × 200 μl) and incubated with 0.02 μm (or as specified) non-biotinylated fibrillin-1 fragments in TBS/CaCl2 overnight at 37 °C. The plates were washed again three times, then 0.02 μm (or as specified) of the second biotinylated protein in TBS/CaCl2 was added, overnight at 37 °C. Control wells were incubated overnight in TBS/CaCl2 with the first non-biotinylated soluble ligand omitted, prior to addition of the biotinylated second soluble ligand. After a further three washes, plates were incubated with 1:200 dilution of ExtraAvidin peroxidase conjugate at room temperature for 15 min. Bound protein was quantified after four more washes by the colorimetric assay described above, using ABTS solution for 10-20 min at room temperature. Plates were read at a wavelength of 405 nm. All experiments were done in triplicate wells, and repeated at least twice to confirm results. MAGP-1 Inhibition Binding Assays—Assays were performed as described above for competition assays involving only fibrillin-1 protein fragments. Flat bottomed microtiter plates were coated with N-terminal fibrillin-1 (0.12 μm) in TBS/CaCl2 overnight at 37 °C. Nonspecific binding sites were blocked as described. The plates were washed with wash buffer (3 × 200 μl) and incubated with increasing concentrations of non-biotinylated MAGP-1 in TBS/CaCl2 overnight at 37 °C. The plates were then washed with wash buffer (3 × 200 μl) and incubated with 0.03 μm biotinylated N- (PF4) or C- (PF13, CP) terminal fibrillin-1 protein fragments in TBS/CaCl2 overnight at 37 °C. The reaction was completed as described above. N-Ethylmaleimide Treatment—Solid-phase assays were performed, as described above, to investigate N-ethylmaleimide (NEM) capping of a free cysteine within the PF4 hybrid domain (27Reinhardt 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). PF4, at a concentration of 0.12 μm was preincubated at 4 °C with or without 2 mm NEM (molar excess) for 1 h. NEM-treated PF4 was allowed to adsorb to flat-bottomed microtiter plates, prior to solid-phase binding assays with soluble untreated PF4, as outlined above. Dissociation Constants for Fibrillin-1 Interactions—We previously used surface plasmon resonance with a BIAcore biosensor (BIAcore 3000, BIAcore AB, Sweden) to define the dissociation constants for fibrillin-1 interactions with MAGP-1 and tropoelastin (29Rock M.J. Cain S.A. Freeman L.J. Morgan A. Mellody K. Marson A. Shuttleworth C.A. Weiss A.S. Kielty C.M. J. Biol. Chem. 2004; 279: 23748-23758Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). In those experiments, MAGP-1 or tropoelastin was attached to CM5 chips, with fibrillin-1 fragments as soluble analytes. Here it was possible to bind the C-terminal fragment CP to CM5 BIAcore chips for analysis of this interaction with PF4. However, fibrillin-1 PF1, PF3, PF4, and PF13 fragments were found not to attach stably to either CM5 or streptavidin-coated chips, in contrast to a previous report of N- and C-terminal fibrillin-1 halves (26Lin G.K. Tiedemann K. Vollbrandt T. Peters H. Batge B. Brinckmann J. Reinhardt D.P. J. Biol. Chem. 2002; 277: 50795-50804Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Therefore, we derived kinetic data on fibrillin-1-fibrillin-1 interactions using solid-phase assays, percentage saturation was analyzed using GraphPad Prism software and Scatchard plots. We investigated fibrillin-1 homotypic interactions to gain new insights into how microfibrils assemble. To date, there has not been a comprehensive analysis of fibrillin-1 interactions, although it was reported that the N-terminal half of fibrillin-1 (encoded by exons 1-36) interacted with the C-terminal half of the molecule (encoded by exons 36-65) (26Lin G.K. Tiedemann K. Vollbrandt T. Peters H. Batge B. Brinckmann J. Reinhardt D.P. J. Biol. Chem. 2002; 277: 50795-50804Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). We have used shorter overlapping human fibrillin-1 fragments encompassing the entire sequence (Fig. 1A), and full-length human MAGP-1 in solid-phase binding and inhibition assays, and BIAcore analyses. The interactions show dose dependence and saturation kinetics. Fibrillin-1 N-terminal Interactions—Homotypic N-terminal fibrillin-1 interactions were studied using solid-phase binding assays. Immobilized PF4 (encoded by exons 1-8) interacted strongly with soluble PF4 (Figs. 2A and 3A), so a homotypic interaction site must be present within this N-terminal fragment. PF4 also interacted strongly with downstream fragment PF2 (encoded by exons 9-17) (Fig. 2A), but PF2 bound only weakly to itself (not shown). The longer N-terminal fragment PF1 also bound PF2 (not shown). Thus, N-terminal sequences can interact with PF2. This interaction could stabilize predicted folding at the proline-rich region.Fig. 2Solid-phase binding assays of soluble fibrillin-1 fragments to immobilized fibrillin-1 fragments. Fibrillin-1 fragments were coated to the plastic surfaces of multiwell plates, and then incubated with soluble biotinylated fibrillin fragments at increasing concentrations. Soluble fragments that bound to immobilized fibrillin-1 fragments were quantified by incubating with ExtraAvidin peroxidase conjugate, then color development using ABTS solution and detection at 405 nm. Where BSA curves are not shown, nonspecific binding has been subtracted. Results are shown as the mean ± S.E. of triplicate values. A, the fibrillin-1 N-terminal fragment PF4 was immobilized, then incubated with soluble biotinylated PF4 (♦) or soluble biotinylated PF2 (▪). B, the fibrillin-1 N-terminal fragment PF4 was immobilized, then incubated with soluble biotinylated CP (▪); also, CP was immobilized and then incubated with soluble biotinylated CP (♦). C, fibrillin-1 N-terminal fragment PF4 was immobilized, then incubated with soluble biotinylated CP (♦). D, fibrillin-1 C-terminal fragment CP (♦), purified GST (▪), or BSA (▴) were immobilized, then incubated with soluble biotinylated PF13.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 3Non-linear regression analysis of N- and C-terminal fibrillin-1. Fibrillin-1 fragments were coated to the plastic surfaces of multiwell plates, and then incubated with soluble biotinylated fibrillin fragments at increasing concentrations. Soluble fragments that bound to immobilized fibrillin-1 fragments were quantified by incubating with ExtraAvidin peroxidase, then color development using ABTS solution and detection at 405 nm. Nonspecific binding was subtracted. Results are shown as the mean ± S.E. of triplicate values. Saturation binding curves are shown, with calculated maximum response values for each concentration, plotted against concentration. Using non-linear regression, KD and Bmax values were also calculated using GraphPad Prism version 2.0 (see Table I). A, the fibrillin-1 N-terminal fragment PF4 was immobilized, then incubated with soluble biotinylated PF4. B, the fibrillin-1 C-terminal fragment PF13 was immobilized, then incubated with soluble biotinylated PF13. C, the fibrillin-1 N-terminal fragment PF4 was immobilized, then incubated with soluble biotinylated PF13. D, the fibrillin-1 C-terminal fragment PF13 was immobilized, then incubated with soluble biotinylated PF4.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Fibrillin-1 N-terminal Interactions with Central Domains— Using solid-phase assays, the ability of PF1 and PF2 to interact with central domains was investigated to determine whether there are any strong interactions that might be important in microfibril assembly or packing. Screening of fragments PF5, PF7, PF8, PF9, PF10, and PF12 (Fig. 1) showed no significant binding to PF1 or PF2, or to each other (not shown). Fibrillin-1 N- and C-terminal Interactions—We next investigated the ability of N- (PF4) and C-terminal (processed PF13) domains to interact, using solid-phase binding assays (Fig. 3, C and D). Such an interacti" @default.
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• W1966152952 title "Homotypic Fibrillin-1 Interactions in Microfibril Assembly" @default.
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