Title: A Comparative Analysis of the Fibulin Protein Family
Abstract: Fibulins are a family of five extracellular matrix proteins characterized by tandem arrays of epidermal growth factor-like domains and a C-terminal fibulin-type module. They are widely distributed and often associated with vasculature and elastic tissues. In this study, we expressed the three more recently identified family members, fibulin-3, fibulin-4, and fibulin-5, as recombinant proteins in mammalian cells. The purified proteins showed short rod structures of ∼20 nm with a globule at one end, after rotary shadowing and electron microscopy. Two forms of mouse fibulin-3 were purified, and the O-glycan profiles of the larger form were characterized. Polyclonal antibodies raised against the purified proteins did not show any cross-reactivity with other family members and were used to assess the levels and localization of the fibulins in mouse tissues. Their binding interactions, cell adhesive properties, and tissue localization were analyzed in parallel with the previously characterized fibulin-1 and -2. Binding to tropoelastin was strong for fibulin-2 and -5, moderate for fibulin-4 and -1, and relatively weak for fibulin-3. Fibulin-4, but not fibulin-3 and -5, exhibited distinct interactions with collagen IV and nidogen-2 and moderate binding to the endostatin domain from collagen XV. Cell adhesive activities were not observed for all fibulins, except mouse fibulin-2, with various cell lines tested. All five fibulins were found in perichondrium and various regions of the lungs. Immunoelectron microscopy localized fibulin-4 and -5 to fibrillin microfibrils at distinct locations. Our studies suggest there are unique and redundant functions shared by these structurally related proteins. Fibulins are a family of five extracellular matrix proteins characterized by tandem arrays of epidermal growth factor-like domains and a C-terminal fibulin-type module. They are widely distributed and often associated with vasculature and elastic tissues. In this study, we expressed the three more recently identified family members, fibulin-3, fibulin-4, and fibulin-5, as recombinant proteins in mammalian cells. The purified proteins showed short rod structures of ∼20 nm with a globule at one end, after rotary shadowing and electron microscopy. Two forms of mouse fibulin-3 were purified, and the O-glycan profiles of the larger form were characterized. Polyclonal antibodies raised against the purified proteins did not show any cross-reactivity with other family members and were used to assess the levels and localization of the fibulins in mouse tissues. Their binding interactions, cell adhesive properties, and tissue localization were analyzed in parallel with the previously characterized fibulin-1 and -2. Binding to tropoelastin was strong for fibulin-2 and -5, moderate for fibulin-4 and -1, and relatively weak for fibulin-3. Fibulin-4, but not fibulin-3 and -5, exhibited distinct interactions with collagen IV and nidogen-2 and moderate binding to the endostatin domain from collagen XV. Cell adhesive activities were not observed for all fibulins, except mouse fibulin-2, with various cell lines tested. All five fibulins were found in perichondrium and various regions of the lungs. Immunoelectron microscopy localized fibulin-4 and -5 to fibrillin microfibrils at distinct locations. Our studies suggest there are unique and redundant functions shared by these structurally related proteins. Fibulins are a family of extracellular glycoproteins with distinctive features of a fibulin-type C-terminal domain preceded by tandem calcium-binding (cb) 4The abbreviations used are: cb, calcium binding; EGF, epidermal growth factor; NEM, N-ethylmaleimide; TBS, Tris-buffered saline; PMSF, phenylmethylsulfonyl fluoride; ESI-MS, electrospray ionization-mass spectrometry; MALDI, matrix-assisted laser desorption ionization; TIMP, tissue inhibitor of metalloproteinase; E, embryonic day. epidermal growth factor (EGF)-like modules (1Argraves W.S. Greene L.M. Cooley M.A. Gallagher W.M. EMBO Rep. 2003; 4: 1127-1131Crossref PubMed Scopus (257) Google Scholar, 2Timpl R. Sasaki T. Kostka G. Chu M.L. Nat. Rev. Mol. Cell. Biol. 2003; 4: 479-489Crossref PubMed Scopus (396) Google Scholar, 3Chu M.L. Tsuda T. Birth Defects Res. Part C. Embryo. Today. 2004; 72: 25-36Crossref PubMed Scopus (68) Google Scholar). The five-member family can be further classified into two subgroups. Fibulin-1 and -2, the first subgroup, are substantially larger than the other three members of the family because of the presence of an extra domain with three anaphylatoxin modules and higher numbers of cbEGF modules (see Fig. 1). Fibuin-1 at 90–100 kDa has variable C-terminal domains. Two major splice variants, fibulin-1C and -1D, are present in approximately equal amounts in most tissues of all animal species studied to date. Fibulin-2 at 200 kDa is the largest of all the fibulins, because it possesses an additional N-terminal domain of ∼400 amino acids not found in other fibulins. Members of the second subgroup, fibulin-3, -4, and -5, are similarly small in size (50–60 kDa) and highly homologous to one another in modular structure. They consist of a modified cbEGF domain at the N terminus followed by five tandem cbEGF modules and the fibulin-type C-terminal region (Fig. 1). Fibulin-1 and -2, the first subgroup, have been characterized extensively and shown to display distinct yet overlapping molecular interactions and expression patterns. Both proteins are localized in basement membranes, elastic fibers, and other connective tissue structures (4Pan T.C. Sasaki T. Zhang R.Z. Fassler R. Timpl R. Chu M.L. J. Cell Biol. 1993; 123: 1269-1277Crossref PubMed Scopus (148) Google Scholar, 5Roark 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 (165) Google Scholar, 6Reinhardt D.P. Sasaki T. Dzamba B.J. Keene D.R. Chu M.L. Gohring W. Timpl R. Sakai L.Y. J. Biol. Chem. 1996; 271: 19489-19496Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 7Zhang H.Y. Timpl R. Sasaki T. Chu M.L. Ekblom P. Dev. Dyn. 1996; 205: 348-364Crossref PubMed Scopus (87) Google Scholar). Fibulin-1 also is a plasma protein (8Argraves W.S. Tran H. Burgess W.H. Dickerson K. J. Cell Biol. 1990; 111: 3155-3164Crossref PubMed Scopus (186) Google Scholar, 9Kluge M. Mann K. Dziadek M. Timpl R. Eur. J. Biochem. 1990; 193: 651-659Crossref PubMed Scopus (62) Google Scholar), and its expression can be detected very early during embryonic development in most basement membranes (10Spence S.G. Argraves W.S. Walters L. Hungerford J.E. Little C.D. Dev. Biol. 1992; 151: 473-484Crossref PubMed Scopus (80) Google Scholar). Fibulin-2 expression initiates later during embryonic development and is distributed in a more restricted manner compared with fibulin-1. Notably, these two fibulins are both prominently expressed in the endocardial cushion tissue, great vessels, and developing cartilages during embryogenesis and remain abundant in the cardiac valves and blood vessel walls in the postnatal stage (7Zhang H.Y. Timpl R. Sasaki T. Chu M.L. Ekblom P. Dev. Dyn. 1996; 205: 348-364Crossref PubMed Scopus (87) Google Scholar, 10Spence S.G. Argraves W.S. Walters L. Hungerford J.E. Little C.D. Dev. Biol. 1992; 151: 473-484Crossref PubMed Scopus (80) Google Scholar, 11Tsuda T. Wang H. Timpl R. Chu M.L. Dev. Dyn. 2001; 222: 89-100Crossref PubMed Scopus (64) Google Scholar, 12Zhang H.Y. Chu M.L. Pan T.C. Sasaki T. Timpl R. Ekblom P. Dev. Biol. 1995; 167: 18-26Crossref PubMed Scopus (69) Google Scholar). They both are able to bind fibronectin, proteoglycans, tropoelastin, and various elastic fiber and basement membrane proteins (2Timpl R. Sasaki T. Kostka G. Chu M.L. Nat. Rev. Mol. Cell. Biol. 2003; 4: 479-489Crossref PubMed Scopus (396) Google Scholar), thereby participating in diverse extracellular supramolecular structures. The three smaller fibulins have been named using various acronyms before were classified as a family. Fibulin-3 is also known as S1–5 and EFEMP1, fibulin-4 as MBP1 and EFEMP2, and fibulin-5 as EVEC and DANCE. Fibulin-3 was initially identified as a gene highly up-regulated in senescent and Werner syndrome fibroblasts (13Lecka-Czernik B. Lumpkin C.K.J. Goldstein S. Mol. Cell. Biol. 1995; 15: 120-128Crossref PubMed Scopus (99) Google Scholar). Genetic linkage and molecular analysis has associated a missense mutation (R345W) in fibulin-3 with heritable macular degenerative disorders, and the protein has been shown to be localized in Bruch's membrane of the retina (14Blackburn J. Tarttelin E.E. Gregory-Evans C.Y. Moosajee M. Gregory-Evans K. Invest. Ophthalmol. Vis. Sci. 2003; 44: 4613-4621Crossref PubMed Scopus (33) Google Scholar, 15Stone 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 (407) Google Scholar). Fibulin-4 was identified through its sequence homology to fibulins-1, -2, and -3 and independently as a protein interacting with a mutant form of the tumor suppressor protein p53 (16Gallagher W.M. Argentini M. Sierra V. Bracco L. Debussche L. Conseiller E. Oncogene. 1999; 18: 3608-3616Crossref PubMed Scopus (52) Google Scholar, 17Giltay R. Timpl R. Kostka G. Matrix Biol. 1999; 18: 469-480Crossref PubMed Scopus (128) Google Scholar). The interaction with an intracellular protein may be explained by the presence of an alternative fibulin-4 transcript lacking the signal peptide coding sequences (18Gallagher W.M. Greene L.M. Ryan M.P. Sierra V. Berger A. Laurent-Puig P. Conseiller E. FEBS Lett. 2001; 489: 59-66Crossref PubMed Scopus (63) Google Scholar). Fibulin-5 was first characterized as a gene strongly expressed in large blood vessels during embryonic development and highly up-regulated upon vascular injury (19Kowal R.C. Richardson J.A. Miano J.M. Olson E.N. Circ. Res. 1999; 84: 1166-1176Crossref PubMed Scopus (105) Google Scholar, 20Nakamura 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). Through targeted ablation of genes in mice, both fibulin-4 and -5 have been shown to play essential roles in the assembly of elastic fibers during development, and both proteins bind tropoelastin in vitro (21Nakamura 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 (537) Google Scholar, 22Yanagisawa H. Davis E.C. Starcher B.C. Ouchi T. Yanagisawa M. Richardson J.A. Olson E.N. Nature. 2002; 415: 168-171Crossref PubMed Scopus (509) Google Scholar, 23McLaughlin 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 (175) Google Scholar). Mutations in fibulin-4 and -5 in humans lead to the cutis laxa syndrome, characterized by loose skin, emphysematous lungs, and tortuous blood vessels resulting from paucity and fragmentation of elastic fibers (24Loeys 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 (247) Google Scholar, 25Markova 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 (121) Google Scholar, 26Hucthagowder 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 (231) Google Scholar). Additionally, amino acid substitutions in fibulin-5 have been implicated in age-related macular degeneration (27Stone 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 (281) Google Scholar). Despite the involvement of the three fibulins of the second subgroup in human diseases, relatively little is known about their biochemical properties, molecular interactions, and expression patterns. In this study, we have prepared recombinant fibulin-3, -4, and -5 in their native forms without peptide tags in mammalian cells and raised specific antibodies. We performed a comprehensive biochemical analysis of these three fibulins in parallel with the previously characterized first subgroup. Our studies show that members of the fibulin protein family have unique and partially overlapping binding interactions and expression patterns, suggesting that they serve both distinct and redundant functions. Sources of Proteins—Laminin-1, collagen IV, and perlecan were purified from the mouse Engelbreth-Holm-Swarm tumor (28Timpl R. Paulsson M. Dziadek M. Fujiwara S. Methods Enzymol. 1987; 145: 363-391Crossref PubMed Scopus (86) Google Scholar). Mouse nidogen-1 (29Fox J.W. Mayer U. Nischt R. Aumailley M. Reinhardt D. Wiedemann H. Mann K. Timpl R. Krieg T. Engel J. EMBO J. 1991; 10: 3137-3146Crossref PubMed Scopus (386) Google Scholar), mouse nidogen-2 (30Salmivirta K. Talts J.F. Olsson M. Sasaki T. Timpl R. Ekblom P. Exp. Cell Res. 2002; 279: 188-201Crossref PubMed Scopus (90) Google Scholar), mouse fibulin-1 (31Sasaki T. Kostka G. Gohring W. Wiedemann H. Mann K. Chu M.L. Timpl R. J. Mol. Biol. 1995; 245: 241-250Crossref PubMed Scopus (92) Google Scholar), and mouse fibulin-2 (4Pan T.C. Sasaki T. Zhang R.Z. Fassler R. Timpl R. Chu M.L. J. Cell Biol. 1993; 123: 1269-1277Crossref PubMed Scopus (148) Google Scholar) and endostatins derived from collagens XV and XVIII (32Sasaki T. Larsson H. Tisi D. Claesson-Welsh L. Hohenester E. Timpl R. J. Mol. Biol. 2000; 301: 1179-1190Crossref PubMed Scopus (197) Google Scholar) were obtained as recombinant products as described previously. Human plasma fibronectin (Behringwerke) and vitronectin were purified by chromatography on heparin-Sepharose (33Yatohgo T. Izumi M. Kashiwagi H. Hayashi M. Cell Struct. Funct. 1988; 13: 281-292Crossref PubMed Scopus (490) Google Scholar). Recombinant human tropoelastin was kindly provided by Dr. Joel Rosenbloom (University of Pennsylvania). Integrin αVβ3 was purified by affinity chromatography from human placenta (34Pfaff M. Gohring W. Brown J.C. Timpl R. Eur. J. Biochem. 1994; 225: 975-984Crossref PubMed Scopus (86) Google Scholar). Expression Vectors and Recombinant Protein Production—To obtain a full-length cDNA encoding mouse fibulin-3, total RNA isolated from mouse lungs was amplified by reverse transcription-PCR using primers 5′-GTCAGCTAGCAGAGAATCACGATGTTG (forward) and 5′-GTCACTCGAGCTAAAATGAAAATGGCCCC (reverse). IMAGE cDNA clones for mouse fibulin-4 (3980048), mouse fibulin-5 (3482574), and human fibulin-5 (4693953) were used as templates to PCR amplify the full-length cDNAs. The primers for mouse fibulin-4 were 5′-GTCAGCTAGCCTCAGGATGCTCCC (forward) and 5′-GTCACTCGAGTCAGAAGGTATAGGCTCCC (reverse), and for mouse fibulin-5 were 5′-GTCAGCTAGCGCATCTTGGATATGCCAGG (forward) and 5′-GCCACTCGAGTCAGAACGGATACTGCGAC (reverse). The 5′ primers contained an NheI site, and the 3′ primers had an XhoI site following the stop codons. The resulting full-length cDNAs were inserted into the NheI and XhoI sites of the episomal expression vector pCEP-Pu (35Kohfeldt E. Maurer P. Vannahme C. Timpl R. FEBS Lett. 1997; 414: 557-561Crossref PubMed Scopus (205) Google Scholar). For human fibulin-5, the cDNA was amplified using primers 5′-GTCAGCTAGCACAGGCACAGTGCACG (forward) and 5′-GTCACTCGAGTCAGAATGGGTACTGCGAC (reverse), and the cDNA fragment flanked by NheI and XhoI sites was inserted into a pCEP-Pu expression vector containing the BM-40 signal peptide (35Kohfeldt E. Maurer P. Vannahme C. Timpl R. FEBS Lett. 1997; 414: 557-561Crossref PubMed Scopus (205) Google Scholar). Mouse MAGP-1 and MAGP-2 were also prepared recombinantly. IMAGE cDNA clones for MAGP-1 (6516202) and MAGP-2 (5325744) were used as templates, and the 5′ primers contained an NheI site and the 3′ primers contained an XhoI site for cloning into pCEP-Pu vector as described above. The sequences of all PCR products were confirmed by cycle sequencing using a BigDye Terminator Cycle Sequencing Ready Kit (ABI). These expression constructs were transfected into human embryonic kidney 293-EBNA cells, and serum-free media were collected as described (35Kohfeldt E. Maurer P. Vannahme C. Timpl R. FEBS Lett. 1997; 414: 557-561Crossref PubMed Scopus (205) Google Scholar). Purification and Characterization of Recombinant Proteins—For purification of mouse fibulin-3, mouse fibulin-5, and human fibulin-5, conditioned media were passed over a DEAE-cellulose column equilibrated in 0.05 m Tris-HCl, pH 8.6, and eluted with a linear 0–0.5 m NaCl gradient. Mouse fibulin-3 was eluted at 0.2–0.3 m NaCl, whereas mouse and human fibulin-5 eluted at 0.25–0.35 m NaCl. They were next purified on a Superose 12 column (HR16/50) equilibrated in 0.3 m NaCl/0.05 m Tris-HCl, pH 8.0, and then on a Mono Q (HR5/5) column equilibrated in 2 m urea/0.02 m Tris-HCl, pH 8.0. Two different forms of fibulin-3 were purified, and they were eluted at 0.2–0.23 m NaCl and 0.23–0.3 m NaCl from the Mono Q column. Both mouse and human fibulin-5 were eluted at 0.3–0.35 m NaCl from the Mono Q column. The purification of mouse fibulin-4 followed the same protocol except that the DEAE-cellulose, and Superose 12 columns were run in the presence of 2 m urea. Fibulin-4 was eluted at 0.3–0.34 m NaCl from the Mono Q column. All purified proteins were dialyzed against 0.2 m ammonium bicarbonate. Purification of MAGP-1 and MAGP-2 were performed basically by the same method as described above for the fibulins. From DEAE-cellulose, MAGP-1 was eluted at 0.3–0.4 m NaCl and MAGP-2 at 0.2–0.3 m NaCl. For the Superose 12 column, the buffers used were either 0.3 m NaCl/0.05 m Tris-HCl, pH 8.0, or 2 m urea/0.05 m Tris-HCl, pH 8.0. Both proteins were eluted through most of the fractions, indicating oligomerization of the proteins. The separation was not improved in the presence of 2 m urea. The monomer fractions were pooled and used for binding assays. Protein concentrations were determined on a Biotronik LC 3000 amino acid analyzer after hydrolysis with 6 m HCl for 16 h at 110 °C. N-terminal sequencing by Edman degradation was performed on a Procise sequencer (Applied Biosystems) following the manufacturer's instructions. Briefly, fibulins were separated by SDS-PAGE under reducing conditions and blotted onto polyvinylidene difluoride membranes. The stained bands were cut out, incubated with pyroglutamate aminopeptidase (TaKaRa Bio), and then sequenced. Electron microscopy of rotary shadowed proteins was carried out using standard protocols (36Engel J. Methods Enzymol. 1994; 245: 469-488Crossref PubMed Scopus (52) Google Scholar). Laser Light Scattering and Analytical Ultracentrifugation—The molecular weights of the recombinant fibulin-4 and -5 were determined by quasielastic laser light scattering (Dawn Eos, Wyatt Technology Corp.). The samples were chromatographed using a Superose 12 HR10/30 column equilibrated in 0.25 m NaCl/20 mm HEPES (pH 7.0), and the intensity of scattered light was simultaneously detected at 18 different angles. The absorbance and refractive index were also measured with inline detectors. The molecular weight of the samples was then calculated using Astra software provided with the instrument. For sedimentation equilibrium, measurements were done in double sector cells on a Beckman XLA analytical ultracentrifuge. The temperature of the runs was either 4 or 20 °C, and the speed was 12,000 rpm. The concentration was monitored at 230 nm as a function of the redial distance, and the data were analyzed by non-linear least squares fitting (Scientist, Micromath, St. Louis, MO). Treatment with Glycosidases—5 μg of lyophilized fibulins was dissolved in 18 μl of 20 mm phosphate buffer (pH 7.2) containing 1 mm EDTA, 0.5 mm N-ethylmaleimide (NEM) and 1 mm Pefabloc (Roche Applied Science) and incubated with neuraminidase (Roche Applied Science) and O-glycosidase (Roche Applied Science) in the presence or absence of N-glycosidase F (Roche Applied Science) at 37 °C for 24 h. N-Glycosidase F treatment was also done after denaturation of proteins by heating at 95 °C for 5 min in the presence of 1% SDS and 1% β-mercaptoethanol. Reductive β-Elimination and Permethylation of Glycan Alditols—For structural studies the glycans were liberated by reductive β-elimination according to a protocol applicable to microscale samples (37Schulz B.L. Packer N.H. Karlsson N.G. Anal. Chem. 2002; 74: 6088-6097Crossref PubMed Scopus (173) Google Scholar). The O-glycoprotein (10–30 μg) was dried in a 0.5-ml Eppendorf vial and treated with freshly prepared 0.5 m NaBH4 in 50 mm NaOH (20 μl) overnight at 50 °C. The sample was desalted with a 50-μl aliquot of Dowex 50Wx8(H+). To remove boric acid, 50-μl aliquots of 1% acetic acid in methanol were added to the dry sample (5×) and evaporated under nitrogen at 40 °C. To the dry sample 50 μl of anhydrous Me2SO was added followed by the same volume of base (NaOH/Me2SO), and the mixture was incubated for 30 min at room temperature with occasional shaking. Finally, an aliquot of 25 μl of methyl iodide was pipetted to the frozen reaction mixture followed by incubation for 30 min at room temperature. After neutralization with dilute acetic acid, the methylated glycans were extracted with chloroform-water. The chloroform phase was dried under nitrogen, and the glycans were solubilized in methanol. Analysis of O-Glycans by Mass Spectrometry—Matrix-assisted laser desorption ionization (MALDI) mass spectrometry was performed on a Bruker Reflex IV instrument (Bruker Daltonics, Bremen, Germany). The methylated glycan samples (∼500 pmol per μl) contained in methanol were applied to the stainless steel target by mixing a 0.5-μl aliquot with 1.0 μl of matrix (saturated solution of 2,5-dihydroxybenzoic acid in acetonitrile/0.1% trifluoroacetic acid, 1:2). Analyses were performed by positive ion detection in the reflectron mode as described previously (38Engelmann K. Kinlough C.L. Muller S. Razawi H. Baldus S.E. Hughey R.P. Hanisch F.G. Glycobiology. 2005; 15: 1111-1124Crossref PubMed Scopus (52) Google Scholar). Electrospray ionization (ESI) mass spectrometry data were acquired on a Q-T of two-quadrupole-time of flight mass spectrometer (Waters, Eschborn, Germany) equipped with a Z spray source. Mass spectrometry was performed in the positive ion mode using previously described conditions (38Engelmann K. Kinlough C.L. Muller S. Razawi H. Baldus S.E. Hughey R.P. Hanisch F.G. Glycobiology. 2005; 15: 1111-1124Crossref PubMed Scopus (52) Google Scholar). Collision energies varied in accordance with the type of molecular ion (M+Na: 50–75 V; M+H: 15–30 V). Site Determination of O-Glycosylation—Three independent strategies were applied to identify O-glycosylated peptides after proteolytic digestion. A first aliquot was partially deglycosylated by successive exoglycosidase treatment with sialidase (clostridium perfringens, New England Biolabs, 100 milliunits, pH 6.0, 24 h), β-galactosaminidase (bovine kidney, Glyko, sodium citrate, pH 6.0, 24 h), and β-hexosaminidase (Glyko, 100 milliunits, pH 6.0, 24 h), followed by reduction and alkylation according to standard protocols. The sample was successively digested with trypsin and V8 protease, and the peptide fragments were isolated by solid-phase extraction on a Pep-Clean C18 Spin column (Pierce). In a second approach, the protein was desialylated by mild acid treatment (0.1 m aqueous trifluoroacetic acid, 80 °C, 1 h), followed by trypsin/V8 digestion of the reduced and alkylated sample. In a third approach, the sample was reduced and alkylated prior to trypsin and V8 treatment, but the peptides and glycopeptides were analyzed without cleavage of the sugars by ESI-q-trap mass spectrometry (see below). MALDI-MS was performed under conditions described above. Nanoflow LC with online ESI-MS was performed on a Q-T of 2 quadrupole-time-of-flight mass spectrometer (Waters, Manchester, UK) equipped with a Z spray source. Samples were introduced using the Ultimate nano-LC system (LC-Packings, Amsterdam, Netherlands) equipped with the Famos autosampler and the Switchos column switching module. The column setup comprised a 0.3 × 1-mm trap column and a 0.075 × 150-mm analytical column, both packed with 3-μm PepMap C18 (LC-Packings). The analytical column flow rate was ∼200 nl/min, resulting from a 1:1000 split of the 200 μl/min flow delivered by the system pump. The samples were eluted onto the analytical column by using a gradient of acetonitrile in 0.1% formic acid over 30 min. Binding Assays—Protein ligands immobilized onto plastic wells were incubated with various concentrations of soluble ligands, and binding was detected with specific antisera against the soluble ligands following methods described previously (39Aumailley M. Wiedemann H. Mann K. Timpl R. Eur. J. Biochem. 1989; 184: 241-248Crossref PubMed Scopus (183) Google Scholar). The binding reactions were carried out in a buffer containing 0.15 m NaCl/0.05 m Tris-HCl (pH7.4) (TBS) supplemented with 2 mm CaCl2. For integrin αVβ3 binding assays, wells were coated with purified integrin dissolved in TBS containing 2 mm MgCl2, and the binding buffer used was TBS containing 1 mm MnCl2, 1 mm MgCl2, 0.1 mm CaCl2, and 0.01% Tween 20. Cell Attachment Assays—Adhesion of established cell lines and human umbilical cord endothelial cells to ligand-coated plastic wells was carried out for 30–60 min and detected colorimetrically according to a previously described protocol (40Aumailley M. Timpl R. Sonnenberg A. Exp. Cell Res. 1990; 188: 55-60Crossref PubMed Scopus (143) Google Scholar). Immunological Assays—Immunization of rabbits, affinity purification of antibodies, enzyme-linked immunosorbent assay titration, and radioimmunoassays were carried out using established protocols (41Timpl R. Methods Enzymol. 1982; 82: 472-498Crossref PubMed Scopus (135) Google Scholar). Fibroblasts obtained from 14.5-day mouse embryos were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Confluent cells grown on a 10-cm dish were used to obtain serum-free culture medium (5 ml), and the cell layers were extracted consecutively with three different buffers (1 ml/each) as described previously (42Sasaki T. Wiedemann H. Matzner M. Chu M.L. Timpl R. J. Cell Sci. 1996; 109: 2895-2904Crossref PubMed Google Scholar). These included a detergent extract with TBS containing 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 2 mm phenylmethylsulfonyl fluoride (PMSF) and 0.5 mm NEM; an EDTA extract with TBS containing 10 mm EDTA, 2 mm PMSF, and 0.5 mm NEM; and a urea extract with TBS containing 6 m urea, 2 mm PMSF, and 0.5 mm NEM. Tissues from 5- to 7-week-old mice were homogenized (200 mg of tissue/ml) in TBS containing 10 mm EDTA, 2 mm PMSF, and 0.5 mm NEM. The homogenates were centrifuged, and the supernatants were collected (EDTA extracts). The residual materials were extracted with the same buffer containing in addition 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS. The homogenates were centrifuged to collect the supernatants (detergent extracts). The precipitates were further extracted with 6 m guanidine hydrochloride/50 mm Tris-HCl (pH 7.5) at 4 °C overnight, and then centrifuged to collect the supernatant (guanidine extracts). Protein contents of the extracts were determined with BCA assay (Pierce). Cell and tissue extracts were separated by 5–20% SDS-PAGE gels and electroblotted onto Immobilon-P membrane (Millipore, Bedford, MA) followed by incubation with antibodies. The bound antibodies were detected with ECL Western blotting reagents (Amersham Biosciences). Immunohistochemistry—Mouse embryos obtained by mating of C57BL/6 mice (Jackson Laboratories) were harvested at day 15 of gestation (E15), fixed in 4% paraformaldehyde overnight, and cryosectioned at a thickness of 7–8 μm. Frozen sagittal sections of E14 mouse embryos were obtained from Zyagen (San Diego, CA). Affinity-purified antibodies of fibulins were used at a concentration of 5 μg/ml for immunohistochemistry. Tissue sections were incubated with primary antibodies overnight, and immunoreactivity was revealed by Cy-3-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch). Microscopic examination was performed with a Zeiss Axioskop epifluorescence microscope, and images were obtained with a Toshiba 3CCD camera. Immunoelectron Microscopy—Newborn mouse perichondrium was labeled using en bloc diffusion of primary antibodies (43Sakai L.Y. Keene D.R. Methods Enzymol. 1994; 245: 29-52Crossref PubMed Scopus (80) Google Scholar) followed by secondary anti-rabbit IgG conjugated with 5 nm gold particles. Expression and Purification of Recombinant Fibulins—Mouse fibulin-3 and fibulin-4, and both mouse and human fibulin-5 were prepared in recombinant forms using human embryonic kidney 293 cells transfected with the expression constructs. The recombinant fibulins were secreted into culture media at a concentration of 1–2 μg/ml except for mouse fibulin-4, which was produced at 5–10 μg/ml. All fibulins could be purified by similar chromatographic steps as described under “Experimental Procedures.” They appeared homogenous by SDS-gel electrophoresis (Fig. 2A) and by N-terminal amino acid sequencing. The molecular masses determined by SDS-PAGE under reducing conditions were 80 and 63 kDa for mouse fibulin-3 (see below), 61 kDa for mouse fibulin-4, 66 kDa for mouse fibulin-5, and 64 kDa for human fibulin-5 (Fig. 2A). A distinct increase in elect