Differential Regulation of Elastic Fiber Formation by Fibulin-4 and -5
2009; Elsevier BV; Volume: 284; Issue: 36 Linguagem: Inglês
10.1074/jbc.m109.019364
ISSN1083-351X
AutoresRawshan Choudhury, Amanda McGovern, Caroline Ridley, Stuart A. Cain, Andrew K. Baldwin, Ming-Chuan Wang, Chun Guo, Mironov Aa, Zoe Drymoussi, Dorothy Trump, Adrian Shuttleworth, Clair Baldock, Cay M. Kielty,
Tópico(s)Dermatological and Skeletal Disorders
ResumoFibulin-4 and -5 are extracellular glycoproteins with essential non-compensatory roles in elastic fiber assembly. We have determined how they interact with tropoelastin, lysyl oxidase, and fibrillin-1, thereby revealing how they differentially regulate assembly. Strong binding between fibulin-4 and lysyl oxidase enhanced the interaction of fibulin-4 with tropoelastin, forming ternary complexes that may direct elastin cross-linking. In contrast, fibulin-5 did not bind lysyl oxidase strongly but bound tropoelastin in terminal and central regions and could concurrently bind fibulin-4. Both fibulins differentially bound N-terminal fibrillin-1, which strongly inhibited their binding to lysyl oxidase and tropoelastin. Knockdown experiments revealed that fibulin-5 controlled elastin deposition on microfibrils, although fibulin-4 can also bind fibrillin-1. These experiments provide a molecular account of the distinct roles of fibulin-4 and -5 in elastic fiber assembly and how they act in concert to chaperone cross-linked elastin onto microfibrils. Fibulin-4 and -5 are extracellular glycoproteins with essential non-compensatory roles in elastic fiber assembly. We have determined how they interact with tropoelastin, lysyl oxidase, and fibrillin-1, thereby revealing how they differentially regulate assembly. Strong binding between fibulin-4 and lysyl oxidase enhanced the interaction of fibulin-4 with tropoelastin, forming ternary complexes that may direct elastin cross-linking. In contrast, fibulin-5 did not bind lysyl oxidase strongly but bound tropoelastin in terminal and central regions and could concurrently bind fibulin-4. Both fibulins differentially bound N-terminal fibrillin-1, which strongly inhibited their binding to lysyl oxidase and tropoelastin. Knockdown experiments revealed that fibulin-5 controlled elastin deposition on microfibrils, although fibulin-4 can also bind fibrillin-1. These experiments provide a molecular account of the distinct roles of fibulin-4 and -5 in elastic fiber assembly and how they act in concert to chaperone cross-linked elastin onto microfibrils. Fibulins are a family of extracellular glycoproteins containing contiguous calcium-binding epidermal growth factor-like domains (cbEGFs) 3The abbreviations used are: cbEGFcalcium-binding epidermal growth factor-like domainFCC-terminal fibulinLOXlysyl oxidaseMALLSmultiangle laser light scatteringsiRNAsmall interfering RNAshRNAshort hairpin RNA. and a characteristic C-terminal fibulin (FC) domain (1.Chu M.L. Tsuda T. Birth Defects Res. C Embryo Today. 2004; 72: 25-36Crossref PubMed Scopus (68) Google Scholar, 2.de Vega S. Iwamoto T. Yamada Y. Cell Mol. Life Sci. 2009; 66: 1890-1902Crossref PubMed Scopus (203) Google Scholar, 3.Timpl R. Sasaki T. Kostka G. Chu M.L. Nat. Rev. Mol. Cell Biol. 2003; 4: 479-489Crossref PubMed Scopus (385) Google Scholar). Recent studies have revealed that fibulin-4 and -5 are both essential for elastic fiber formation (4.Kobayashi N. Kostka G. Garbe J.H. Keene D.R. Bächinger H.P. Hanisch F.G. Markova D. Tsuda T. Timpl R. Chu M.L. Sasaki T. J. Biol. Chem. 2007; 282: 11805-11816Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 5.McLaughlin 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, 6.Nakamura T. Lozano P.R. Ikeda Y. Iwanaga Y. Hinek A. Minamisawa S. Cheng C.F. Kobuke K. Dalton N. Takada Y. Tashiro K. Ross Jr., J. Honjo T. Chien K.R. Nature. 2002; 415: 171-175Crossref PubMed Scopus (531) Google Scholar, 7.Yanagisawa 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). Fibulin-4 is widely expressed from early embryogenesis and is necessary for normal vascular, lung, and skin development, since mice that lack fibulin-4 do not form elastic fibers and die perinatally (5.McLaughlin 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). Furthermore, mice with reduced fibulin-4 expression develop aneurysms (8.Hanada K. Vermeij M. Garinis G.A. de Waard M.C. Kunen M.G. Myers L. Maas A. Duncker D.J. Meijers C. Dietz H.C. Kanaar R. Essers J. Circ. Res. 2007; 100: 738-746Crossref PubMed Scopus (135) Google Scholar). Fibulin-5 is abundant in the aorta and large arteries during embryogenesis and following vascular injury (9.Nakamura T. Ruiz-Lozano P. Lindner V. Yabe D. Taniwaki M. Furukawa Y. Kobuke K. Tashiro K. Lu Z. Andon N.L. Schaub R. Matsumori A. Sasayama S. Chien K.R. Honjo T. J. Biol. Chem. 1999; 274: 22476-22483Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 10.Kowal R.C. Richardson J.A. Miano J.M. Olson E.N. Circ. Res. 1999; 84: 1166-1176Crossref PubMed Scopus (105) Google Scholar). Lack of fibulin-5 causes a less severe phenotype, with viable homozygous mice, but the elastic fibers in skin, lungs, and aorta are irregular and fragmented (6.Nakamura T. Lozano P.R. Ikeda Y. Iwanaga Y. Hinek A. Minamisawa S. Cheng C.F. Kobuke K. Dalton N. Takada Y. Tashiro K. Ross Jr., J. Honjo T. Chien K.R. Nature. 2002; 415: 171-175Crossref PubMed Scopus (531) Google Scholar, 7.Yanagisawa 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), and there is altered vascular remodeling (11.Spencer J.A. Hacker S.L. Davis E.C. Mecham R.P. Knutsen R.H. Li D.Y. Gerard R.D. Richardson J.A. Olson E.N. Yanagisawa H. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 2946-2951Crossref PubMed Scopus (96) Google Scholar). These mice models also highlight that fibulin-4 and -5 have non-compensatory roles in elastic fiber formation. Mutations in both molecules can cause cutis laxa, a heritable disorder associated with elastic fiber degeneration leading to sagging skin, vascular tortuosity, and emphysematous lungs (12.Dasouki M. Markova D. Garola R. Sasaki T. Charbonneau N.L. Sakai L.Y. Chu M.L. Am. J. Hum. Genet. A. 2007; 143A: 2635-2641Crossref PubMed Scopus (100) Google Scholar, 13.Hu Q. Loeys B.L. Coucke P.J. De Paepe A. Mecham R.P. Choi J. Davis E.C. Urban Z. Hum. Mol. Genet. 2006; 15: 3379-3386Crossref PubMed Scopus (77) Google Scholar, 14.Hu Q. Reymond J.L. Pinel N. Zabot M.T. Urban Z. J. Invest. Dermatol. 2006; 126: 283-290Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 15.Hucthagowder 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). A third isoform, fibulin-3, may play a minor role in elastic fiber formation, since its deficiency disrupts elastic fibers in Bruch's membrane of the eye (16.Fu L. Garland D. Yang Z. Shukla D. Rajendran A. Pearson E. Stone E.M. Zhang K. Pierce E.A. Hum. Mol. Genet. 2007; 16: 2411-2422Crossref PubMed Scopus (107) Google Scholar) and vaginal tissues (17.Rahn D.D. Acevedo J.F. Roshanravan S. Keller P.W. Davis E.C. Marmorstein L.Y. Word R.A. Am. J. Pathol. 2009; 174: 206-215Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). calcium-binding epidermal growth factor-like domain C-terminal fibulin lysyl oxidase multiangle laser light scattering small interfering RNA short hairpin RNA. Elastic fiber formation is a complex multistep process (18.Kielty C.M. Expert Rev. Mol. Med. 2006; 8: 1-23Crossref PubMed Scopus (215) Google Scholar, 19.Wagenseil J.E. Mecham R.P. Birth Defects Res. C Embryo Today. 2007; 81: 229-240Crossref PubMed Scopus (300) Google Scholar, 20.Sato F. Wachi H. Ishida M. Nonaka R. Onoue S. Urban Z. Starcher B.C. Seyama Y. J. Mol. Biol. 2007; 369: 841-851Crossref PubMed Scopus (61) Google Scholar). Initial pericellular microassembly of tropoelastin, which may involve the 67-kDa elastin-binding protein receptor, generates elastin globules that are stabilized by desmosine cross-links catalyzed mainly by lysyl oxidase (LOX) but also by LOXL1 (LOX-like 1). These globules are deposited on a fibrillin microfibril template, where they coalesce and undergo further cross-linking to form the elastin core of mature fibers. The ability of fibulin-4 and -5 to bind tropoelastin and fibrillin-1, the major structural component of microfibrils, supports a model in which these fibulins direct elastin deposition on microfibrils (4.Kobayashi N. Kostka G. Garbe J.H. Keene D.R. Bächinger H.P. Hanisch F.G. Markova D. Tsuda T. Timpl R. Chu M.L. Sasaki T. J. Biol. Chem. 2007; 282: 11805-11816Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 5.McLaughlin 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, 6.Nakamura T. Lozano P.R. Ikeda Y. Iwanaga Y. Hinek A. Minamisawa S. Cheng C.F. Kobuke K. Dalton N. Takada Y. Tashiro K. Ross Jr., J. Honjo T. Chien K.R. Nature. 2002; 415: 171-175Crossref PubMed Scopus (531) Google Scholar, 7.Yanagisawa 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, 21.El-Hallous E. Sasaki T. Hubmacher D. Getie M. Tiedemann K. Brinckmann J. Bätge B. Davis E.C. Reinhardt D.P. J. Biol. Chem. 2007; 282: 8935-8946Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 22.Freeman 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, 23.Hirai M. Ohbayashi T. Horiguchi M. Okawa K. Hagiwara A. Chien K.R. Kita T. Nakamura T. J. Cell Biol. 2007; 176: 1061-1071Crossref PubMed Scopus (141) Google Scholar, 24.Wachi H. Nonaka R. Sato F. Shibata-Sato K. Ishida M. Iketani S. Maeda I. Okamoto K. Urban Z. Onoue S. Seyama Y. J. Biochem. 2008; 143: 633-639Crossref PubMed Scopus (29) Google Scholar, 25.Zheng Q. Davis E.C. Richardson J.A. Starcher B.C. Li T. Gerard R.D. Yanagisawa H. Mol. Cell Biol. 2007; 27: 1083-1095Crossref PubMed Scopus (55) Google Scholar). This model does not delineate the unique molecular contributions of fibulin-4 and -5 to elastic fiber formation, but some molecular differences have emerged. Tropoelastin was bound more strongly by fibulin-5 than by fibulin-4, whereas fibulin-5 was at the microfibril-elastin interface, but perichondrial fibulin-4 localized mainly to microfibrils (4.Kobayashi N. Kostka G. Garbe J.H. Keene D.R. Bächinger H.P. Hanisch F.G. Markova D. Tsuda T. Timpl R. Chu M.L. Sasaki T. J. Biol. Chem. 2007; 282: 11805-11816Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). Fibulin-4 null mice offer tantalizing clues to how fibulin-4 contributes to elastic fiber formation (5.McLaughlin 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). They had dramatically reduced (94%) desmosine cross-links despite no change in elastin or LOX expression levels, and electron-dense rodlike structures were prominent within elastin aggregates. Morphologically similar structures seen after chemically inhibiting LOX were previously identified as glycosaminoglycans, which can bind charged free ϵ-amino groups on lysines in tropoelastin (26.Fornieri C. Baccarani-Contri M. Quaglino Jr., D. Pasquali-Ronchetti I. J. Cell Biol. 1987; 105: 1463-1469Crossref PubMed Scopus (73) Google Scholar). However, fibulin-4+/− mice showed ∼20% increase in desmosine (5.McLaughlin 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). LOX-null mice have phenotypic features similar to those of fibulin-4 null mice, dying perinatally with 60% reduced desmosine cross-links and major abnormalities in vascular and other elastic tissues (27.Hornstra I.K. Birge S. Starcher B. Bailey A.J. Mecham R.P. Shapiro S.D. J. Biol. Chem. 2003; 278: 14387-14393Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar, 28.Mäki J.M. Sormunen R. Lippo S. Kaarteenaho-Wiik R. Soininen R. Myllyharju J. Am. J. Pathol. 2005; 167: 927-936Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). In contrast, LOXL1-null mice are viable but have reduced desmosine (29.Liu X. Zhao Y. Gao J. Pawlyk B. Starcher B. Spencer J.A. Yanagisawa H. Zuo J. Li T. Nat. Genet. 2004; 36: 178-182Crossref PubMed Scopus (525) Google Scholar), whereas fibulin-5 null mice have a 16% reduction in desmosine cross-links and survive well into adulthood (7.Yanagisawa 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). Detection of the LOXL1 pro-domain in fibulin-5 null mice skin but not wild-type skin implicates fibulin-5 in activation of LOXL1 (30.Choi J. Bergdahl A. Zheng Q. Starcher B. Yanagisawa H. Davis E.C. Matrix Biol. 2009; 28: 211-220Crossref PubMed Scopus (50) Google Scholar). We and others have shown that fibrillin-1 and the microfibrillar protein MAGP-1 can both directly bind tropoelastin (31.Clarke A.W. Wise S.G. Cain S.A. Kielty C.M. Weiss A.S. Biochemistry. 2005; 44: 10271-10281Crossref PubMed Scopus (56) Google Scholar, 32.Jensen 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, 33.Rock 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, 34.Trask T.M. Trask B.C. Ritty T.M. Abrams W.R. Rosenbloom J. Mecham R.P. J. Biol. Chem. 2000; 275: 24400-24406Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). However, the fibulin-null mice show that the fibrillin-1 interaction with tropoelastin is insufficient to support elastic fiber formation in vivo. Fibulin-5 has been reported to facilitate tropoelastin binding to the N-terminal half of fibrillin-1 (21.El-Hallous E. Sasaki T. Hubmacher D. Getie M. Tiedemann K. Brinckmann J. Bätge B. Davis E.C. Reinhardt D.P. J. Biol. Chem. 2007; 282: 8935-8946Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). A study of elastin polypeptide self-assembly through coacervation and maturation phases showed that, although the N-terminal half of fibrillin-1 increased maturation velocity and droplet clustering, fibulin-4 and -5 both slowed maturation and limited globule growth (35.Cirulis J.T. Bellingham C.M. Davis E.C. Hubmacher D. Reinhardt D.P. Mecham R.P. Keeley F.W. Biochemistry. 2008; 47: 12601-12613Crossref PubMed Scopus (65) Google Scholar). These studies imply that fibulins and fibrillin-1 act together to regulate elastin accretion on microfibrils. To gain further insights into the contributions of fibulin-4 and -5 to elastic fiber formation, we have delineated how they interact with tropoelastin, LOX, and fibrillin-1. Novel findings are that fibulin-4 directly binds LOX, and this interaction enhances fibulin-4 binding to tropoelastin, thus forming a ternary complex that may be critical for elastin cross-linking. Fibulin-5 can concurrently bind fibulin-4 and tropoelastin, but the interaction of both fibulins with fibrillin-1 strongly inhibits their binding to tropoelastin. These interactions indicate the molecular basis of how fibulins act as chaperones for deposition of elastin onto microfibrils. Our study thus provides a molecular account of the differential roles of fibulins-4 and -5 in elastic fiber formation. All human elastic fiber proteins were expressed with an N-terminal His6 tag, using the mammalian expression vector pCEP-His and 293-EBNA cells, and purified using nickel affinity chromatography as described (22.Freeman 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, 33.Rock 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, 36.Lomas A.C. Mellody K.T. Freeman L.J. Bax D.V. Shuttleworth C.A. Kielty C.M. Biochem. J. 2007; 405: 417-428Crossref PubMed Scopus (68) Google Scholar, 37.Baldock C. Siegler V. Bax D.V. Cain S.A. Mellody K.T. Marson A. Haston J.L. Berry R. Wang M.C. Grossmann J.G. Roessle M. Kielty C.M. Wess T.J. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 11922-11927Crossref PubMed Scopus (49) Google Scholar, 38.Cain S.A. Baldock C. Gallagher J. Morgan A. Bax D.V. Weiss A.S. Shuttleworth C.A. Kielty C.M. J. Biol. Chem. 2005; 280: 30526-30537Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 39.Cain S.A. Baldwin A.K. Mahalingam Y. Raynal B. Jowitt T.A. Shuttleworth C.A. Couchman J.R. Kielty C.M. J. Biol. Chem. 2008; 283: 27017-27027Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 40.Marson A. Rock M.J. Cain S.A. Freeman L.J. Morgan A. Mellody K. Shuttleworth C.A. Baldock C. Kielty C.M. J. Biol. Chem. 2005; 280: 5013-5021Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 41.Mellody K.T. Freeman L.J. Baldock C. Jowitt T.A. Siegler V. Raynal B.D. Cain S.A. Wess T.J. Shuttleworth C.A. Kielty C.M. J. Biol. Chem. 2006; 281: 31854-31862Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar) (Fig. 1 and supplemental Fig. S1). Full-length human fibulin-4 (residues 26–418) was designated F4 (Fig. 1A). A truncated version of full-length fibulin-4 (designated tF4) was expressed by one culture of transfected cells; analysis by mass spectrometry revealed the absence of the C-terminal sequence (53 residues, comprising the FC domain and 6 residues of the preceding linker region) (supplemental Fig. S2A). We also expressed the N-terminal four domains of fibulin-4 (residues 26–217; designated nF4) and the central cbEGFs 2–6 (residues 98–303; designated eF4). The C-terminal four domains did not express efficiently. Full-length human fibulin-5 (residues 42–448) was designated F5 (Fig. 1A). Full-length fibulin-5 incorporating two cutis laxa mutations, C217R and S227P (F5C217R and F5S227P) (13.Hu Q. Loeys B.L. Coucke P.J. De Paepe A. Mecham R.P. Choi J. Davis E.C. Urban Z. Hum. Mol. Genet. 2006; 15: 3379-3386Crossref PubMed Scopus (77) Google Scholar), were expressed together with five overlapping domain pairs of fibulin-5 (residues 42–167 (F5-E1+2), 127–206 (F5-E2+3), 207–287 (F5-E4+5), 247–333 (F5-E5+6), and 288–448 (F5-E6FC)) (Fig. 1A). A sixth domain pair (F5-E3+4) did not express efficiently. Human fibrillin-1 fragments generated for this study were PF1, PF2, PF4, PF5, PF7, PF8, PF9, PF11, PF12, and PF13 (33.Rock 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, 37.Baldock C. Siegler V. Bax D.V. Cain S.A. Mellody K.T. Marson A. Haston J.L. Berry R. Wang M.C. Grossmann J.G. Roessle M. Kielty C.M. Wess T.J. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 11922-11927Crossref PubMed Scopus (49) Google Scholar, 38.Cain S.A. Baldock C. Gallagher J. Morgan A. Bax D.V. Weiss A.S. Shuttleworth C.A. Kielty C.M. J. Biol. Chem. 2005; 280: 30526-30537Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 39.Cain S.A. Baldwin A.K. Mahalingam Y. Raynal B. Jowitt T.A. Shuttleworth C.A. Couchman J.R. Kielty C.M. J. Biol. Chem. 2008; 283: 27017-27027Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 40.Marson A. Rock M.J. Cain S.A. Freeman L.J. Morgan A. Mellody K. Shuttleworth C.A. Baldock C. Kielty C.M. J. Biol. Chem. 2005; 280: 5013-5021Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 41.Mellody K.T. Freeman L.J. Baldock C. Jowitt T.A. Siegler V. Raynal B.D. Cain S.A. Wess T.J. Shuttleworth C.A. Kielty C.M. J. Biol. Chem. 2006; 281: 31854-31862Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar) (Fig. 1B). We also generated PF1 variants with the Marfan mutation R62C, T101A, or S115C. In addition, we used our panel of N-terminal deletion mutants: PF1 encoded by exons 1–11 (residues 31–489), Ex1–11 encoded by residues 45–489), Ex3–11 encoded by exons 3–11 (residues 81–489), Ex4–11 encoded by exons 4–11 (residues 115–489), Ex5–11 encoded by exons 5–11 (residues 147–489), Ex6–11 encoded by exons 6–11 (residues 179–489), Ex7–11 encoded by exons 7–11 (residues 246–489), and a three-domain fragment Ex5–7 encoded by exons 5–7 (residues 147–287) (39.Cain S.A. Baldwin A.K. Mahalingam Y. Raynal B. Jowitt T.A. Shuttleworth C.A. Couchman J.R. Kielty C.M. J. Biol. Chem. 2008; 283: 27017-27027Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Cells transfected with full-length fibrillin-1 expressed a large fragment comprising the N-terminal two-thirds of the molecule (250 kDa; designated tFib-1) (Fig. 1B and supplemental Fig. S1B); mass spectrometry indicated that the cleavage site was after the domain encoded by exon 43 (supplemental Fig. S2B). Human full-length LOX (Fig. 1C) was expressed and purified, as outlined above (supplementary Fig. S1C). Recombinant human tropoelastin lacking domain 26A was a generous gift from Dr. A. S. Weiss (Sydney, Australia) (42.Martin S.L. Vrhovski B. Weiss A.S. Gene. 1995; 154: 159-166Crossref PubMed Scopus (168) Google Scholar, 43.Toonkool P. Jensen S.A. Maxwell A.L. Weiss A.S. J. Biol. Chem. 2001; 276: 44575-44580Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). After nickel affinity chromatography, monomeric recombinant proteins were isolated by Superdex S200 10/300 GL size fractionation (GE Healthcare) in 10 mm Hepes-buffered saline, pH 7.4, containing 150 mm NaCl (HBS), using an AKTA purifier system. cDNA sequencing, SDS-PAGE, Western blotting with anti-His antibody (Sigma), and mass spectrometry confirmed the correct products (Fig. 1 and supplemental Figs. S1 and S2; data not shown). All molecules with NX(S/T) motifs were N-glycosylated, as determined by treatment with peptide:N-glycanase F (New England Biolabs). In all cases, the monomer fractions from S200 chromatography were equilibrated in HBS containing 0.5 mm CaCl2 (HBS+Ca) for subsequent binding assays. Monomers of fibulin-4 (F4, nF4, and tF4) and fibulin-5 (F5, F5S227P, and domain pairs F5-E1+2, F5-E2+3, F5-E4+5, F5-E5+6, and F5-E6FC) that had been isolated by Superdex 200 gel filtration in HBS, were analyzed by multiangle laser light scattering (MALLS) in HBS or HBS+Ca. Samples eluting from the column passed through a Wyatt EOS 18-angle light scattering detector fitted with a 688-nm laser and an Optilab r-EX refractometer. The solute molecular mass was determined using in-line MALLS attached to quasielastic light scattering and a differential refractometer (Wyatt Technology Corp.) (Table 1 and supplemental Fig. S3). The monomers had molecular masses that corresponded well with SDS-PAGE analysis (see supplemental Fig. S1).TABLE 1MALLS analysis of fibulin-4 and -5Molecule (buffer)MALLS-measured molecular massSDS-PAGE-measured molecular massCalculated molecular masskDakDakDaF4 (HBS) monomer525249nF4 (HBS) monomer304027F4 (HBS+Ca) "dimer"94F4 (HBS+Ca) monomer56nF4 (HBS+Ca) "dimer"60nF4 (HBS+Ca) monomer31F5 (HBS) monomer515350F5-S227P (HBS) monomer52F5-E1+2 (HBS) monomer202514F5-E2+3 (HBS) monomer13159F5-E4+5 (HBS) monomer13159F5-E5+6 (HBS) monomer161810F5-E6FC (HBS) monomer243019 Open table in a new tab LOX monomers were isolated on a Superdex 200 gel filtration column in 10 mm Tris, pH 7.4, with 150 mm NaCl containing 1 mm CaCl2 (TBS+Ca). MALLS analysis revealed that LOX was monomeric with a molecular mass of 47.5 kDa, compared with 50 kDa by SDS-PAGE (supplemental Fig. S1). Analysis of LOX by circular dichroism revealed 5% α-helix, 31% β-strand, and 13% β-turn. Analysis of LOX by analytical ultracentrifugation revealed a molecular mass of 48.9 kDa, measured S20,w of 2.2, calculated hydrodynamic radius of 5.41, and frictional ratio of 2.25. Purified F4 monomers in 10 mm Tris, pH 7.4, containing 500 mm NaCl (TBS) were adsorbed onto glow-discharged carbon-coated 400-mesh copper grids, negatively stained with 2% uranyl acetate, and analyzed by single particle averaging, as described (Fig. 2) (37.Baldock C. Siegler V. Bax D.V. Cain S.A. Mellody K.T. Marson A. Haston J.L. Berry R. Wang M.C. Grossmann J.G. Roessle M. Kielty C.M. Wess T.J. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 11922-11927Crossref PubMed Scopus (49) Google Scholar). Briefly, samples were observed at ×52,000 magnification using an FEI Tecnai 12 transmission electron microscope operating at 120 kV with a LaB6 filament, and images were recorded by a CCD camera (TVIPS Tem Cam, 2048 × 2048 resolution) using low dose (<10e−/Å2) at defocus values of 2 μm. The pixel size was 1.9 Å. Images were analyzed using electron microscopy analysis (EMAN) (44.Ludtke S.J. Baldwin P.R. Chiu W. J. Struct. Biol. 1999; 128: 82-97Crossref PubMed Scopus (2110) Google Scholar) for all data processing. The data set contained 3538 particles, which were aligned and classified by reference-free methods. A preliminary three-dimensional structure was calculated by averaging classes that represented distinct views of the sample. Fourier common lines were applied to determine the relative orientation of these classes. The preliminary structure was subsequently refined by iterative (eight times) projection matching. Each refinement was examined by the convergence of the Fourier shell correlation coefficient. Final resolution of the F4 reconstruction was ∼20 Å using the Fourier shell correlation = 0.5 criterion. For kinetic binding studies by surface plasmon resonance, a BIAcore biosensor was used (BIAcore 3000; GE Healthcare). Tropoelastin or LOX was immobilized onto CM5 sensor chips by amine coupling using 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide hydrochloride, N-hydroxysuccinimide, and ethanolamine-HCl, as described (33.Rock 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, 39.Cain S.A. Baldwin A.K. Mahalingam Y. Raynal B. Jowitt T.A. Shuttleworth C.A. Couchman J.R. Kielty C.M. J. Biol. Chem. 2008; 283: 27017-27027Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). All binding experiments were performed in HBS+Ca. LOX, fibulin-4, or fibulin-5 was injected at concentration ranges of 2–10 μg/ml for F4 or 0.4–10 μg/ml for F5 or LOX for 3–5 min at a flow rate of 30 μl/min. Curves were fitted using a 1:1 Langmuir association/dissociation model (BIAevaluation 4.1; GE Healthcare). This model was found to fit the tropoelastin-fibulin-5 and LOX-fibulin-4 data, with low χ2 values. χ2 values are a standard statistical measure of the closeness of fit (mean square of the signal noise). Each binding interaction was performed at least twice, with one concentration in duplicate each time. Some BIAcore experiments investigated potential inhibitory effects of a given elastic fiber protein on binding of a second protein to immobilized tropoelastin or LOX. Kinetic analysis was performed using ligand protein (F5 at 100 nm; F4 at 200 nm; LOX at 200 nm) preincubated with a 0.01 nm to 3 μm concentration of the potential inhibitor protein or with a non-binding control fibrillin-1 fragment PF12, in HBS+Ca, as described (38.Cain S.A. Baldock C. Gallagher J. Morgan A. Bax D.V. Weiss A.S. Shuttleworth C.A. Kielty C.M. J. Biol. Chem. 2005; 280: 30526-30537Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Inhibition curves were plotted using the response value of each normalized curve at the end of the association period. Because fibrillin-1 and the two fibulins when immobilized to BIAcore CM5 chips do not interact sufficiently with other proteins and to investigate further the interactions between LOX and tropoelastin and between fibrillin-1 and LOX, solid phase assays were utilized, as described (22.Freeman 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, 33.Rock 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, 39.Cain S.A. Baldwin A.K. Mahalingam Y. Raynal B. Jowitt T.A. Shuttleworth C.A. Couchman J.R. Kielty C.M. J. Biol. Chem. 2008; 283: 27017-27027Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Soluble ligands were biotinylated using sulfosuccinimidyl 2-(biotinamido)-ethyl-1,3-dithiopropionate (Pierce). Flat-bottomed microtiter plates were coated with tropoelastin, LOX, fibulin-4 or -5, or N-terminal fibrillin-1 fragments, at 100 nm in 10 mm Tris, pH 7.4, containing 150 mm NaCl and 1 mm CaCl2 (TBS+Ca) buffer, overnight at 4 °C. Nonspecific binding sites were blocked with TBS+Ca containing 5% bovine serum albumin at room temperature for at least 2 h. The plates were washed three times with TBS+Ca, 0.1% bovine serum albumin and incubated with 100 nm biotinylated ligand. Control wells with only soluble biotinylated ligand were included in all experiments. After a further three washes, plates were incubated with a 1:500 dilution of extravidin peroxidase conjugate at room t
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