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. Little C.D. Dev. Biol. PubMed Scopus Google Scholar). Fibulin-2 expression during embryonic development and is distributed in a more with these two fibulins are expressed in the and during and in the and in the H.Y. Timpl R. Sasaki T. Chu M.L. Ekblom P. Dev. Dyn. 1996; 205: 348-364Crossref PubMed Scopus (87) Google Scholar, S.G. Argraves W.S. Little C.D. Dev. Biol. PubMed Scopus Google Scholar, T. H. Timpl R. Chu M.L. Dev. Dyn. PubMed Scopus Google Scholar, H.Y. Chu M.L. T.C. Sasaki T. Timpl R. Ekblom P. Dev. Biol. 1995; PubMed Scopus Google Scholar). They are to and various elastic and basement proteins R. Sasaki T. Kostka G. Chu M.L. Nat. Rev. Mol. Cell. Biol. 2003; 4: 479-489Crossref PubMed Scopus (396) Google Scholar), in extracellular The three fibulins have been various were classified as a is also as and fibulin-4 as and and as and was identified as a highly in and S. Mol. Cell. Biol. 1995; PubMed Scopus Google Scholar). and molecular has associated a in fibulin-3 with and the protein has been shown to be localized in of the J. M. K. 2003; PubMed Scopus Google Scholar, K. P. G. Nat. PubMed Scopus Google Scholar). was identified its to -2, and and as a protein with a form of the protein W.M. M. PubMed Scopus Google Scholar, R. Timpl R. Kostka G. Biol. PubMed Scopus Google Scholar). The with an protein be by the presence of an fibulin-4 the W.M. Greene L.M. P. PubMed Scopus Google Scholar). was first characterized as a expressed in during embryonic development and highly Res. PubMed Scopus Google Scholar, T. P. M. K. K. R. S. T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of in fibulin-4 and -5 have been shown to in the of elastic during and proteins tropoelastin in T. S. K. K. T. PubMed Scopus Google Scholar, H. T. M. PubMed Scopus Google Scholar, M. R. T. L.Y. Mol. Cell. Biol. PubMed Scopus Google Scholar). in fibulin-4 and -5 in to the characterized by and from and of elastic G. P. S. Mol. PubMed Scopus Google Scholar, Sasaki T. Kostka G. Timpl R. J. Chu M.L. J. 2003; 72: Full Text Full Text PDF PubMed Scopus Google Scholar, L.Y. J. Full Text Full Text PDF PubMed Scopus Google Scholar). amino in have been in J. 2004; PubMed Scopus Google Scholar). the of the three fibulins of the second in relatively is properties, molecular interactions, and expression patterns. In this study, we have recombinant fibulin-3, -4, and -5 in forms in mammalian and raised a of these three fibulins in parallel with the previously characterized first Our studies show members of the protein family have unique and overlapping binding interactions and expression distinct and redundant of collagen and were purified from the mouse R. M. Dziadek M. S. PubMed Scopus Google Scholar). R. M. H. Mann K. Timpl R. T. J. EMBO J. PubMed Scopus Google Scholar), mouse nidogen-2 K. M. Sasaki T. Timpl R. Ekblom P. Cell Res. PubMed Scopus Google Scholar), mouse fibulin-1 T. Kostka G. Gohring W. H. Mann K. Chu M.L. Timpl R. J. Mol. Biol. 1995; PubMed Scopus 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 and from and T. H. Timpl R. J. Mol. Biol. PubMed Scopus Google were as recombinant as plasma and were purified by T. M. H. M. Cell PubMed Scopus Google Scholar). tropoelastin was by of was purified by from M. Gohring W. Timpl R. Eur. J. Biochem. PubMed Scopus Google Scholar). and a mouse fibulin-3, from mouse was by and for mouse fibulin-4 mouse and were used as to the The for mouse fibulin-4 were and and for mouse were and The an and the an the The were into the and of the expression P. C. Timpl R. PubMed Scopus Google Scholar). fibulin-5, the was and and the by and was into a expression the P. C. Timpl R. PubMed Scopus Google Scholar). and were also for and were used as and the an and the an for into as The of all were by a expression were into embryonic and were as P. C. Timpl R. PubMed Scopus Google Scholar). and of of mouse fibulin-3, mouse fibulin-5, and fibulin-5, were a in and with a fibulin-3 was at mouse and at They were purified a in and a in Two forms of fibulin-3 were purified, and were at and from the Both mouse and were at from the The of mouse fibulin-4 followed the except the and were in the presence of was at from the All purified proteins were against of and were by the as for the fibulins. was at and at the the used were Both proteins were most of the of the proteins. The was not in the presence of The were and used for binding were a amino after with for at N-terminal by was a the fibulins were by and The were with and microscopy of rotary proteins was J. PubMed Scopus Google Scholar). and molecular of the recombinant fibulin-4 and -5 were by laser The were a in and the of was detected at The and were also with The molecular of the was with the were in a The of the was and the was The was at nm as a of the and the were analyzed by with of fibulins was in of and and with and in the presence of at for was also after of proteins by at for in the presence of and and of studies the were by to a to Chem. PubMed Scopus Google Scholar). The was in a and with in at The was with a of of in were to the and at the of was followed by the of and the was for at with an of of was to the followed by for at with the were with The was and the were in of by laser desorption was a IV The in were to the by a with of matrix of in were by in the as previously K. S. H. PubMed Scopus Google Scholar). were a of of with a was in the previously K. S. H. PubMed Scopus Google Scholar). in with the of molecular of were to after first was by with and followed by and to The was with and and the were by a In a second the protein was by followed by of the and In a the was and to and but the and were analyzed of the by (see was with was a of with a were the with the and the module. The a and a with The was from a of the 200 by the The were the by a of in Binding were with various of and binding was detected with against the previously M. H. Mann K. Timpl R. Eur. J. Biochem. PubMed Scopus Google Scholar). The binding were in a with binding were with purified in and the binding used was and Cell of cell lines and to was for and detected to a previously M. Timpl R. Cell Res. 1990; PubMed Scopus Google Scholar). of of and were R. PubMed Scopus Google Scholar). from mouse were in modified with a were used to and the cell were with three as previously T. H. M. Chu M.L. Timpl R. J. Cell 1996; PubMed Google Scholar). a with phenylmethylsulfonyl and an with PMSF, and and a with PMSF, and from to were of in PMSF, and The were and the were The were with the in The were to the The were further with at and to the of the were with Cell and tissue were by and followed by with The antibodies were detected with by of were at of in and at a of of mouse were from antibodies of fibulins were used at a of for were with antibodies and was by was with a and were with a Immunoelectron mouse perichondrium was of antibodies L.Y. Keene D.R. PubMed Scopus Google followed by with nm and of fibulin-3 and fibulin-4, and mouse and were in recombinant forms embryonic with the expression The recombinant fibulins were into at a of except for mouse fibulin-4, was at All fibulins be purified by as They by (Fig. and by N-terminal amino The molecular by were and kDa for mouse fibulin-3 (see kDa for mouse fibulin-4, kDa for mouse fibulin-5, and kDa for (Fig. distinct in for all fibulins was observed not the of The N-terminal of the recombinant mouse fibulin-3, -4, and -5 were found to be the at the N of these proteins are was by to for fibulin-3 and for fibulin-4 as from the the to any amino for mouse with the N-terminal of was expressed the the a N-terminal after R. Mann K. K. M. Timpl R. EMBO J. 1993; PubMed Scopus Google Scholar). of the recombinant the N-terminal of Two forms of mouse fibulin-3 by kDa were purified (Fig. and the N-terminal amino there are two and five in mouse fibulin-3, the these two forms from was by with and (Fig. with and but not the size of the kDa these the size of the The the size of the two forms was at in to and the mouse fibulin-3 were by and analyzed as by and The a of molecular at and were detected at and The structures of the were by the in the 1). The at was as a of two at and at The structures were identified as the and the profiles of fibulin-3 expressed in in a to the for five the N-terminal domains of the protein be and of the and be by as because were found in in the were not found in any of the as as be as a to a of in Fig. the protein was by lines in Fig. and amino not in any of the were and (Fig. be the C-terminal and were not but in and were not found as in any of the in Fig. of of recombinant fibulin-3, -4, and -5 after rotary shadowing short of with a domain at one (Fig. The domain of fibulin-4 than of and in with the structures of the fibulins (Fig. the domains the cbEGF In to the shown in Fig. were also detected by electron microscopy. is with studies of recombinant fibulin-4 by amounts of in to R. Timpl R. Kostka G. Biol. PubMed Scopus Google Scholar). recombinant mouse fibulin-4 and -5 were analyzed by molecular with of laser and at The two fibulins as and the molecular of fibulin-4 and -5 were and of the protein the with laser recombinant mouse fibulin-4 and -5 not form Binding to binding were with all five fibulins as in binding The binding to was for all five fibulins, the fibulin-1 and T. Gohring W. J. Timpl R. PubMed Scopus Google Scholar), fibulin-4 M. R. T. L.Y. Mol. Cell. Biol. PubMed Scopus Google Scholar), and T. S. K. K. T. PubMed Scopus Google Scholar, H. T. M. PubMed Scopus Google Scholar). The for binding were to be nm for fibulin-2 and fibulin-5, nm for fibulin-4, and nm for fibulin-1C (Fig. Binding of fibulin-3 to tropoelastin was because the of binding was not at a of binding of any to and two was In to fibulin-1 and -2, -4, and -5 did not show any binding to and (Fig. and not interactions with collagen IV and nidogen-2 were found for fibulin-4 and -2, a binding at nm (Fig. and Fibulin-1 and to the C-terminal from and T. H. Timpl R. J. Mol. Biol. PubMed Scopus Google Scholar). also exhibited a moderate binding to collagen endostatin and a weak with collagen to were observed for fibulin-3 and -5, did not levels at (Fig. and Cell and cell adhesive of all fibulins were in parallel various cell lines and cells. mouse fibulin-2 was to by cell as previously M. Sasaki T. K. Chu M.L. Timpl R. Cell Res. 1995; PubMed Scopus Google not the in binding purified was used as the (Fig. The showed a strong binding to at moderate binding to mouse fibulin-2 as M. Sasaki T. K. Chu M.L. Timpl R. Cell Res. 1995; PubMed Scopus Google Scholar), but binding to and mouse not The binding of was after and the the the binding of the was with were raised against mouse fibulin-3, -4, and -5 and purified by The purified antibodies were highly with fibulins (Fig. the antibodies be to the in of Fibulins in and of fibulin-3, -4, and -5 the and three cell of embryonic mouse was analyzed by (Fig. The is to the the and consist of proteins are in the extracellular was found in amounts in the and not in the three cell in the amounts used for the not was detected in the and in all three cell was found in the three cell but was not detected in of the amounts of fibulin-4 and -5 were observed in as with the a calcium for matrix from were with and the were analyzed by (Fig. fibulin-3 and -4, all of the proteins from the and of proteins were by the The of these fibulins in the tissue were by with the other the of was detected in the first two from and of the protein was present in the the of in these its into the is in to the major fibulin-3 a and a protein were found in and the was than of the recombinant The the presence of fibulin-3 in the tissues. The tissue of fibulins in and were by The was at for fibulin-3, for fibulin-4, and for were as the of the amounts in most of the of fibulins were with in all tissues and -5 were substantially more than the other three fibulins. All of the five fibulins were in tissues in elastic fibers, and tissue were not was detected at higher in and and fibulin-1 in and of fibulins in of mouse tissues by in a of antibodies against the fibulins were used to of mouse at and of All five fibulins were found in the perichondrium of the in the (Fig. Fibulin-1 was and expressed in the basement of tissues the the expression of the other fibulins in these regions was more to the basement In the embryonic (Fig. fibulin-1 was localized in the basement of all and and and expressed the Fibulin-2 expression was to and was from all and was localized in the and basement of the but not Fibulin-4, was and expressed in the but the expression in the basement of the was not as distinct as is expressed in the and in the basement and of the it is present in the and perichondrium was used to fibulin-4 and -5 at the The of fibulin-4 was found in the microfibrils (Fig. and was detected at the and microfibrils (Fig. Members of the second of the fibulin-3, -4, and -5, have modular (Fig. but cbEGF modules in and amino acids the and of the modified cbEGF modules in fibulin-3, -4, and -5, Their are and the is fibulin-3 and microscopy of the recombinant fibulin-3, -4, and -5 a short rod with a globule at one as for with tandem modules (Fig. Our these three fibulins as fibulin-2 forms and fibulin-1 to into T. Kostka G. Gohring W. H. Mann K. Chu M.L. Timpl R. J. Mol. Biol. 1995; PubMed Scopus Google Scholar, K. Tran H. Godyna S. Argraves W.S. J. Biol. Chem. Full Text PDF PubMed Google Scholar, T. Mann K. H. Gohring W. J. Chu M.L. Timpl R. EMBO J. PubMed Scopus Google Scholar). The molecular from the are kDa for fibulin-3, kDa for fibulin-4, kDa for mouse fibulin-5, and kDa for has two five are for fibulin-3, for fibulin-4, three for mouse fibulin-5, and one for The molecular of the recombinant proteins by (Fig. are larger than the these three fibulins are to have with and also have and are to be in and a for of in has been J. J. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Nat. Rev. Mol. Cell. Biol. 2003; 4: PubMed Scopus Google Scholar, 1993; PubMed Scopus Google Scholar). the for 1993; PubMed Scopus Google is not present in all fibulins. The and of the and recombinant fibulin-3 proteins the first for the presence of fibulin-3 in of the from fibulin-3 by an O-glycan to of proteins and previously analyzed Chem. PubMed Scopus Google Scholar, M. S. D.P. M. P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). these proteins were also expressed in the cell as the recombinant fibulins, the of O-glycan structures is the protein but more by the of The of can the in size of fibulin-3 after (Fig. because this the The the two forms of fibulin-3 found by be by the of but this is often the for splice in N-terminal have previously been for fibulin-3, and the largest and are expressed in amounts at the protein S. Mol. Cell. Biol. 1995; PubMed Scopus Google Scholar). The and forms of mouse fibulin-3 have the N-terminal and to be the splice in a fibulin-3 with a size to the kDa form also be detected in tissues (Fig. In identified a fibulin-3 in to the splice a fibulin-3 functions are by the further and interactions, with are in matrix and of Our studies the second of fibulins have more binding than the first in not with and most basement proteins a binding than fibulin-3 and -5, because it is of with collagen and collagen endostatin in a to is a for all five fibulins, and the binding from strong for fibulin-2 and -5, moderate for fibulin-1 and -4, to weak for fibulin-3. these all five fibulins are localized in tissues in elastic fibers, as and The suggest all fibulins to elastic fibulin-4 and -5 have been found to be for elastic T. S. K. K. T. PubMed Scopus Google Scholar, H. T. M. PubMed Scopus Google Scholar, M. R. T. L.Y. Mol. Cell. Biol. PubMed Scopus Google Scholar). of fibulin-1 fibulin-2 in not have elastic G. R. W. K. Timpl R. Fassler R. 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In we have the of all members of the family in showed of the five fibulins distinct binding and tissue there are in these fibulins, and the in the are higher members the than subgroups. members in the first with elastic and basement in the second to elastic All fibulins are expressed in elastic but tissues are distinct and also Our studies suggest unique and redundant functions shared by these structurally related proteins and the of the family are for the of and Mann for for of modified cbEGF of fibulin-5, and Sakai for of the