Calcium Stabilizes Fibrillin-1 against Proteolytic Degradation

The calcium-binding epidermal growth factor (cbEGF)-like domain is a structural motif that is present in many matrix proteins throughout the animal kingdom from invertebrates to mammals. This module has been demonstrated to bind calcium in the micromolar range. However, little is known about the functional consequences of calcium binding to proteins that contain this structural element.We used fibrillin-1, an extracellular matrix protein consisting of ∼60% cbEGF-like motifs, as a model system to study stabilizing effects of calcium in protease degradation assays. Authentic human fibrillin-1 and recombinant human fibrillin-1 subdomains, spanning the whole molecule, showed significantly slower proteolytic degradation in the presence of CaCl2 than in the presence of EDTA, demonstrating that calcium stabilizes the structure of fibrillin-1 and protects the molecule against proteolytic degradation.Information about cleavage sites protected by calcium was obtained with a new recombinant subdomain, rF17 (Asp952-Val1527), comprising the longest stretch of cbEGF-like motifs in the center of the fibrillin-1 molecule. The most sensitive sites for trypsin and endoproteinase Glu-C were observed in cbEGF-like motifs 11 (Met1034 and Asn1046), 12 (Ser1103), and 17 (Thr1318). Since most of the currently known mutations in fibrillin-1 are found within cbEGF-like motifs and are predicted to disrupt calcium binding, we suggest that these mutations render fibrillin-1 more susceptible to proteolytic cleavage, and this might be one of the reasons why these mutations result in Marfan's syndrome. The calcium-binding epidermal growth factor (cbEGF)-like domain is a structural motif that is present in many matrix proteins throughout the animal kingdom from invertebrates to mammals. This module has been demonstrated to bind calcium in the micromolar range. However, little is known about the functional consequences of calcium binding to proteins that contain this structural element. We used fibrillin-1, an extracellular matrix protein consisting of ∼60% cbEGF-like motifs, as a model system to study stabilizing effects of calcium in protease degradation assays. Authentic human fibrillin-1 and recombinant human fibrillin-1 subdomains, spanning the whole molecule, showed significantly slower proteolytic degradation in the presence of CaCl2 than in the presence of EDTA, demonstrating that calcium stabilizes the structure of fibrillin-1 and protects the molecule against proteolytic degradation. Information about cleavage sites protected by calcium was obtained with a new recombinant subdomain, rF17 (Asp952-Val1527), comprising the longest stretch of cbEGF-like motifs in the center of the fibrillin-1 molecule. The most sensitive sites for trypsin and endoproteinase Glu-C were observed in cbEGF-like motifs 11 (Met1034 and Asn1046), 12 (Ser1103), and 17 (Thr1318). Since most of the currently known mutations in fibrillin-1 are found within cbEGF-like motifs and are predicted to disrupt calcium binding, we suggest that these mutations render fibrillin-1 more susceptible to proteolytic cleavage, and this might be one of the reasons why these mutations result in Marfan's syndrome. INTRODUCTIONA structural motif found in the epidermal growth factor (EGF) 1The abbreviations used are: EGFepidermal growth factorcbEGFcalcium-binding epidermal growth factormAbmonoclonal antibodyTBSTris-buffered saline. is widely distributed throughout the animal kingdom as a subdomain in a variety of extracellular proteins. These proteins include functionally diverse members such as the fibrillin and fibulin families (1Lee B. Godfrey M. Vitale E. Hori H. Mattei M.G. Sarfarazi M. Tsipouras P. Ramirez F. Hollister D.W. Nature. 1991; 352: 330-334Google Scholar, 2Maslen C.L. Corson G.M. Maddox B.K. Glanville R.W. Sakai L.Y. Nature. 1991; 352: 334-337Google Scholar, 3Corson G.M. Chalberg S.C. Dietz H.C. Charbonneau N.L. Sakai L.Y. Genomics. 1993; 17: 476-484Google Scholar, 4Pereira L. D'Alessio M. Ramirez F. Lynch J.R. Sykes B. Pangilinan T. Bonadio J. Hum. Mol. Genet. 1993; 2: 961-968Google Scholar, 5Zhang H. Apfelroth S.D. Hu W. Davis E.C. Sanguineti C. Bonadio J. Mecham R.P. Ramirez F. J. Cell Biol. 1994; 124: 855-863Google Scholar, 6Argraves W.S. Tran H. Burgess W.H. Dickerson K. J. Cell Biol. 1990; 111: 3155-3164Google Scholar, 7Pan T.-C. Kluge M. Zhang R.-Z. Mayer U. Timpl R. Chu M.-L. Eur. J. Biochem. 1993; 215: 733-740Google Scholar, 8Pan T.-C. Sasaki T. Zhang R.-Z. Fässler R. Timpl R. Chu M.-L. J. Cell Biol. 1993; 123: 1269-1277Google Scholar, 9Zhang R.-Z. Pan T.-C. Zhang Z.-Y. Mattei M.-G. Timpl R. Chu M.-L. Genomics. 1994; 22: 425-430Google Scholar), nidogen/entactin (10Durkin M.E. Chakravarti S. Bartos B.B. Liu S.H. Friedman R.L. Chung A.E. J. Cell Biol. 1988; 107: 2749-2756Google Scholar, 11Mann K. Deutzmann R. Aumailley M. Timpl R. Raimondi L. Yamada Y. Pan T.-C. Conway D. Chu M.-L. EMBO J. 1989; 8: 65-72Google Scholar), blood coagulation factors (reviewed in 12Stenflo J. Blood. 1991; 78: 1637-1651Google Scholar) and anticoagulants (13Lundwall Å. Dackowski W. Cohen E. Shaffer M. Mahr A. Dahlbäck B. Stenflo J. Wydro R. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 6716-6720Google Scholar), and proteins for specification of cell fate (14Wharton K.A. Johansen K.M. Xu T. Artavanis-Tsakonas S. Cell. 1985; 43: 567-581Google Scholar, 15Yochem J. Weston K. Greenwald I. Nature. 1988; 335: 547-550Google Scholar, 16Coffman C. Harris W. Kintner C. Science. 1990; 249: 1438-1441Google Scholar, 17Ellisen L.W. Bird J. West D.C. Soreng A.L. Reynolds T.C. Smith S.D. Sklar J. Cell. 1991; 66: 649-661Google Scholar). A characteristic pattern of amino acid residues ((D/N)X(D/N)(Q/E)Xn(D*/N*)Xm(Y/F); residues with an asterisk may be β-hydroxylated) has been identified in a subset of EGF-like motifs that is responsible for calcium binding. Calcium binding to this type of motif has been demonstrated with blood clotting factors IX and X (18Persson E. Selander M. Linse S. Drakenberg T. Öhlin A.-K. Stenflo J. J. Biol. Chem. 1989; 264: 16897-16904Google Scholar, 19Handford P.A. Baron M. Mayhew M. Willis A. Beesley T. Brownlee G.G. Campbell I.D. EMBO J. 1990; 9: 475-480Google Scholar), with anticoagulants Protein C (20Öhlin A.-K. Linse S. Stenflo J. J. Biol. Chem. 1988; 263: 7411-7417Google Scholar) and Protein S (21Dahlbäck B. Hildebrand B. Linse S. J. Biol. Chem. 1990; 265: 18481-18489Google Scholar), and the microfibrillar protein fibrillin-1 (22Glanville R.W. Qian R.-Q. McClure D.W. Maslen C.L. J. Biol. Chem. 1994; 269: 26630-26634Google Scholar, 23Handford P. Downing A.K. Rao Z. Hewett D.R. Sykes B.C. Kielty C.M. J. Biol. Chem. 1995; 270: 6751-6756Google Scholar, 24Reinhardt D.P. Keene D.R. Corson G.M. Pöschl E. Bächinger H.P. Gambee J.E. Sakai L.Y. J. Mol. Biol. 1996; 258: 104-116Google Scholar).Little is known about the functional significance of these motifs and the consequences of calcium binding on the properties of proteins containing these motifs. It has been suggested that calcium binding to cbEGF-like motifs mediates protein-protein interactions. For example, mutations of amino acid residues crucial for calcium binding affect the clotting activity of factor IX (25Rees D.J.G. Jones I.M. Handford P.A. Walter S.J. Esnouf M.P. Smith K.J. Brownlee G.G. EMBO J. 1988; 7: 2053-2061Google Scholar). The interaction of Protein S with complement C4b-binding protein is mediated by this type of module (26Dahlbäck B. Frohm B. Nelsestuen G. J. Biol. Chem. 1990; 265: 16082-16087Google Scholar), and the interaction of the Drosophila transmembrane protein Notch with Delta and Serrate depends on two tandemly repeated cbEGF-like motifs (27Rebay I. Fleming R.J. Fehon R.G. Cherbas L. Cherbas P. Artavanis-Tsakonas S. Cell. 1991; 67: 687-699Google Scholar). Possibly, cbEGF-like motifs are also involved in mediating the calcium-dependent interaction of fibrillin-1 with fibulin-2 (28Reinhardt 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: 19489-19496Google Scholar).The fibrillin family consists of two members, fibrillin-1 and fibrillin-2, both of which are integral constituents of the 10-12-nm diameter extracellular matrix microfibrils (5Zhang H. Apfelroth S.D. Hu W. Davis E.C. Sanguineti C. Bonadio J. Mecham R.P. Ramirez F. J. Cell Biol. 1994; 124: 855-863Google Scholar, 29Sakai L.Y. Keene D.R. Engvall E. J. Cell Biol. 1986; 103: 2499-2509Google Scholar). Both proteins consist of repetitive motifs: 47 of them are EGF-like repeats, and 43 have the consensus sequence for calcium binding. The cbEGF-like repeats are dispersed over both entire molecules in nine stretches of 1-12 tandemly repeated motifs.Mutations in fibrillin-1 result in Marfan's syndrome, a connective tissue disorder affecting the cardiovascular, skeletal, and ocular systems (reviewed in 30Dietz H.C. Pyeritz R.E. Hum. Mol. Genet. 1995; 4: 1799-1809Google Scholar). On the other hand, mutations in fibrillin-2 give rise to congenital contractural arachnodactyly, a disorder characterized primarily by joint contractures and other skeletal features (31Putnam E.A. Zhang H. Ramirez F. Milewicz D.M. Nat. Genet. 1995; 11: Scholar). of the more than known mutations in fibrillin-1 and the two mutations in fibrillin-2 in cbEGF-like repeats and are predicted to calcium binding, an of the cbEGF-like repeats in these this we the of calcium binding on the of fibrillin-1 in protease degradation assays. These that calcium stabilizes fibrillin-1 against proteolytic degradation. This may be a functional for cbEGF-like motifs in other proteins. We that mutations in fibrillin-1, and in other that calcium binding to cbEGF-like motifs render the molecules more susceptible to proteolytic degradation. degradation of the molecules to the of the were to study the functional of calcium binding to cbEGF-like motifs. Since the a family of extracellular matrix contain A.K. C.M. Campbell I.D. Handford P.A. Cell. 1996; Scholar) cbEGF-like repeats in we used fibrillin-1 as a protein for fibrillin-1 from cell were with for the was from the cell by fibrillin-1 was from the by on which is for R. C. D. D. P. C. C. R. G. M. M. M. D. A. and L. Y. for of fibrillin-1 with trypsin in the presence of CaCl2 in significantly slower degradation with demonstrating that calcium stabilizes fibrillin-1 and protects the molecule against an of fibrillin-1 was in the presence of On the other hand, a showed degradation in the presence of calcium as with which is to the stabilizing of calcium on trypsin G. J. J. Mol. Biol. 1995; Scholar). the observed in fibrillin-1 degradation in the presence of calcium and may be the slower degradation of in the presence of calcium that the fibrillin-1 molecule L.Y. Keene D.R. Glanville R.W. Bächinger H.P. J. Biol. Chem. 1991; Scholar) is more susceptible to degradation than the we demonstrated structural and functional properties of of fibrillin-1 D.P. Keene D.R. Corson G.M. Pöschl E. Bächinger H.P. Gambee J.E. Sakai L.Y. J. Mol. Biol. 1996; 258: 104-116Google Scholar, 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: 19489-19496Google Scholar). the we used recombinant and which the whole fibrillin-1 molecule, for protease degradation with with endoproteinase and and were and protected against in the presence of calcium the were of in the presence of EDTA, in the presence of the These that of amino acid residues and within the fibrillin-1 molecule are more susceptible to proteolytic in the of with as showed for and in the presence of For endoproteinase degradation were in the presence of of calcium on the protease of recombinant of of recombinant and were in the presence of CaCl2 with trypsin endoproteinase Glu-C from an of for the as of the was as in the of The were by and The of proteins are This was repeated with and these sequence about protease cleavage which are protected by we the longest stretch of 12 tandemly repeated cbEGF-like motifs by the motif in fibrillin-1 for calcium assays. This recombinant subdomain was by human in of degradation and amino acid of rF17 were as from the The on was slower than of rF17 with to by that of the sites present in rF17 are by with and other recombinant fibrillin-1 were D.P. Keene D.R. Corson G.M. Pöschl E. Bächinger H.P. Gambee J.E. Sakai L.Y. J. Mol. Biol. 1996; 258: 104-116Google Scholar). of rF17 demonstrated to in fibrillin from cell L.Y. Keene D.R. Glanville R.W. Bächinger H.P. J. Biol. Chem. 1991; of recombinant subdomain rF17 as with of subdomain rF17 with trypsin endoproteinase Glu-C in the presence of CaCl2 demonstrated the of calcium to fibrillin-1 of rF17 in one to rF17 These that most of the cleavage sites are within and cbEGF-like repeats, which are by of proteolytic cleavage sites that are protected by subdomain rF17 in a and in was for with trypsin for with endoproteinase Glu-C in the presence of CaCl2 were by and with The were repeated with with these a the degradation were to and were by degradation. of the degradation and the of proteins are The of the sites identified are demonstrated in protein obtained a with trypsin endoproteinase Glu-C in the presence of were by degradation which are protected by were in cbEGF-like 11 (Met1034 and Asn1046), 12 (Ser1103), and 17 and in the of the motif and from the and and and of cbEGF-like motif These are to the calcium-binding M. M. E. Stenflo J. Drakenberg T. J. Biol. Chem. Scholar, Z. Handford P. Mayhew M. Brownlee G.G. D. Cell. 1995; Scholar). The other sites from the and of cbEGF-like 12 and 17 from the the motif and cbEGF-like 11 These are from the calcium-binding of sites cbEGF-like repeats of cbEGF-like repeats and 17 are amino acid is by a and is identified by the The of degradation obtained with trypsin and with endoproteinase Glu-C are binding to fibrillin is and has been demonstrated with fibrillin from cell G.M. Chalberg S.C. Dietz H.C. Charbonneau N.L. Sakai L.Y. Genomics. 1993; 17: 476-484Google Scholar), with of microfibrils (22Glanville R.W. Qian R.-Q. McClure D.W. Maslen C.L. J. Biol. Chem. 1994; 269: 26630-26634Google Scholar), with recombinant of fibrillin-1 D.P. Keene D.R. Corson G.M. Pöschl E. Bächinger H.P. Gambee J.E. Sakai L.Y. J. Mol. Biol. 1996; 258: 104-116Google Scholar, Downing C.M. Handford P. J. Mol. Biol. 1996; Scholar), and with P. Downing A.K. Rao Z. Hewett D.R. Sykes B.C. Kielty C.M. J. Biol. Chem. 1995; 270: 6751-6756Google Scholar, Chem. Biol. 1995; 2: Scholar). little is known about the of calcium to the of the has been demonstrated that calcium mediates protein-protein interaction (28Reinhardt 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: 19489-19496Google Scholar) and that calcium a in of a fibrillin M. Kielty C.M. K. A. L. B. Biochem. J. 1994; Scholar) and in of fibrillin molecules and microfibrils Downing C.M. Handford P. J. Mol. Biol. 1996; Scholar, A.K. C.M. Campbell I.D. Handford P.A. Cell. 1996; Scholar, C.M. 1993; this we stabilizing effects of calcium on fibrillin-1 in protease degradation We found that fibrillin-1 as as recombinant that the of fibrillin-1 are significantly more susceptible to proteolytic degradation in the of These that calcium stabilizes fibrillin-1 against by of fibrillin and the was C.M. 1994; Scholar). The of calcium to protease of fibrillin and was that are protected by we a new recombinant subdomain of fibrillin-1 that the longest stretch of cbEGF-like motifs in the molecule and the motif the of this subdomain by an to in fibrillin from cell L.Y. Keene D.R. Glanville R.W. Bächinger H.P. J. Biol. Chem. 1991; Scholar). The of degradation demonstrated that sites that are protected by calcium are in the and and and of cbEGF-like motif It was that the structural calcium binding is to the of a cbEGF-like of factor X M. M. E. Stenflo J. Drakenberg T. J. Biol. Chem. Scholar) factor IX Z. Handford P. Mayhew M. Brownlee G.G. D. Cell. 1995; Scholar). sites and are this in cbEGF-like motif and proteolytic degradation of calcium be by a structural and by degradation were observed in the and of cbEGF-like motifs 12 and 17 and and in the the motif with cbEGF-like motif However, structural in these calcium binding have been observed in cbEGF-like motifs M. M. E. Stenflo J. Drakenberg T. J. Biol. Chem. Scholar, Z. Handford P. Mayhew M. Brownlee G.G. D. Cell. 1995; Scholar), and stabilizing for the calcium in this in a of repeats repeats and A.K. C.M. Campbell I.D. Handford P.A. Cell. 1996; Scholar). suggested by Chem. Biol. 1995; 2: Scholar), sites for calcium binding be by an from the of the cbEGF-like stabilizing the and of this However, is in the of cbEGF-like repeats 12 and of for these sites may suggest that tandemly repeated cbEGF-like repeats of are by calcium in a the stabilizing of calcium binding on the of the to be suggest that the of calcium against proteolytic degradation of cbEGF-like repeats may be a of calcium binding to this type of as as in other proteins containing these repeats, this of stretches functional against proteolytic For example, the in consisting of EGF-like repeats, two of which are of the calcium-binding be by is the binding of the Drosophila transmembrane protein Notch motifs 11 and to Delta and Serrate (27Rebay I. Fleming R.J. Fehon R.G. Cherbas L. Cherbas P. Artavanis-Tsakonas S. Cell. 1991; 67: 687-699Google Scholar), calcium binding, in to mediating the protein-protein also the binding domain against proteolytic in this study are of for the of in which mutations in cbEGF-like motifs are known and predicted to disrupt calcium binding. These include Marfan's H.C. Pyeritz R.E. Hum. Mol. Genet. 1995; 4: 1799-1809Google Scholar), F. M. R. M. A. Brownlee G.G. 1996; Scholar), and Protein S S. D. M. M. C. P. B. P. P. M. Blood. 1995; Scholar). Marfan's syndrome, most of the over mutations known (reviewed in 30Dietz H.C. Pyeritz R.E. Hum. Mol. Genet. 1995; 4: 1799-1809Google Scholar) in cbEGF-like motifs and are predicted to disrupt calcium binding by in amino acid residues for calcium binding by in an of with demonstrated that a affecting the predicted to be D.R. Lynch J.R. Smith R. Sykes B.C. Hum. Mol. Genet. 1993; 2: Scholar) in cbEGF-like motif of fibrillin-1 calcium binding by P. Downing A.K. Rao Z. Hewett D.R. Sykes B.C. Kielty C.M. J. Biol. Chem. 1995; 270: 6751-6756Google Scholar). We suggest that mutations that affect calcium binding to cbEGF-like motifs render the molecule more susceptible to This affect the of fibrillin on as molecules as fibrillin molecules be are which in to a of microfibrils in fibrillin molecules are a may be for the of fibrillin microfibrils have been the of the cbEGF-like motifs the P. Downing A.K. Rao Z. Hewett D.R. Sykes B.C. Kielty C.M. J. Biol. Chem. 1995; 270: 6751-6756Google Scholar, 24Reinhardt D.P. Keene D.R. Corson G.M. Pöschl E. Bächinger H.P. Gambee J.E. Sakai L.Y. J. Mol. Biol. 1996; 258: 104-116Google Scholar, A.K. C.M. Campbell I.D. Handford P.A. Cell. 1996; Scholar). Calcium binding to these cbEGF-like repeats has been suggested to the two cbEGF-like Downing C.M. Handford P. J. Mol. Biol. 1996; Scholar, A.K. C.M. Campbell I.D. Handford P.A. Cell. 1996; Scholar) and to a in of the fibrillin molecules within microfibrils Z. Handford P. Mayhew M. Brownlee G.G. D. Cell. 1995; Scholar, C.M. 1993; Scholar). from a cell that was from an the in P. Downing A.K. Rao Z. Hewett D.R. Sykes B.C. Kielty C.M. J. Biol. Chem. 1995; 270: 6751-6756Google Scholar), to the of the of microfibrils from with C.M. 1993; Scholar). These with suggest that mutations that calcium binding may result in a of the of the microfibrils and of fibrillin molecules in the the are by demonstrating that within and to a motifs. Marfan's microfibrils are more susceptible to is that the microfibrils with degradation sites be Marfan's microfibrils of fibrillin-1 and sites for degradation. degradation these sites might protein binding the this might to a of the which the of Marfan's syndrome. we are this with recombinant mutations in cbEGF-like motifs.Mutations in a of fibrillin-1 motif and cbEGF-like have been suggested to result in Marfan's K. L. L. Sakai L. L. Nat. Genet. 1994; Scholar), a of the within the of fibrillin in cell from with Marfan's and A. M. J. Genet. Scholar, M. M. J. C.L. A. M. J. K. P. Y. M. A. U. B. J. 1995; Scholar, M. C. J. J. K. A. Godfrey M. Hum. Mol. Genet. 1995; 4: Scholar). This is in to observed in cell obtained from Marfan's the of the fibrillin in These to the that the is for E.A. M. Milewicz D.M. J. Genet. 1996; Scholar). of sites observed in study are in the of the molecule. This may be sensitive to proteolytic degradation mutations Marfan's calcium binding to this It is that fibrillin molecules to the for in the of the microfibrils might be to the of calcium degradation within the one of the observed sensitive sites is in the the motif with the cbEGF-like motif and is within a proteolytic degradation to this the microfibrils to This the and microfibrils observed in with Marfan's cell and the of Marfan's syndrome. INTRODUCTIONA structural motif found in the epidermal growth factor (EGF) 1The abbreviations used are: EGFepidermal growth factorcbEGFcalcium-binding epidermal growth factormAbmonoclonal antibodyTBSTris-buffered saline. is widely distributed throughout the animal kingdom as a subdomain in a variety of extracellular proteins. These proteins include functionally diverse members such as the fibrillin and fibulin families (1Lee B. Godfrey M. Vitale E. Hori H. Mattei M.G. Sarfarazi M. Tsipouras P. Ramirez F. Hollister D.W. Nature. 1991; 352: 330-334Google Scholar, 2Maslen C.L. Corson G.M. Maddox B.K. Glanville R.W. Sakai L.Y. Nature. 1991; 352: 334-337Google Scholar, 3Corson G.M. Chalberg S.C. Dietz H.C. Charbonneau N.L. Sakai L.Y. Genomics. 1993; 17: 476-484Google Scholar, 4Pereira L. D'Alessio M. Ramirez F. Lynch J.R. Sykes B. Pangilinan T. Bonadio J. Hum. Mol. Genet. 1993; 2: 961-968Google Scholar, 5Zhang H. Apfelroth S.D. Hu W. Davis E.C. Sanguineti C. Bonadio J. Mecham R.P. Ramirez F. J. Cell Biol. 1994; 124: 855-863Google Scholar, 6Argraves W.S. Tran H. Burgess W.H. Dickerson K. J. Cell Biol. 1990; 111: 3155-3164Google Scholar, 7Pan T.-C. Kluge M. Zhang R.-Z. Mayer U. Timpl R. Chu M.-L. Eur. J. Biochem. 1993; 215: 733-740Google Scholar, 8Pan T.-C. Sasaki T. Zhang R.-Z. Fässler R. Timpl R. Chu M.-L. J. Cell Biol. 1993; 123: 1269-1277Google Scholar, 9Zhang R.-Z. Pan T.-C. Zhang Z.-Y. Mattei M.-G. Timpl R. Chu M.-L. Genomics. 1994; 22: 425-430Google Scholar), nidogen/entactin (10Durkin M.E. Chakravarti S. Bartos B.B. Liu S.H. Friedman R.L. Chung A.E. J. Cell Biol. 1988; 107: 2749-2756Google Scholar, 11Mann K. Deutzmann R. Aumailley M. Timpl R. Raimondi L. Yamada Y. Pan T.-C. Conway D. Chu M.-L. EMBO J. 1989; 8: 65-72Google Scholar), blood coagulation factors (reviewed in 12Stenflo J. Blood. 1991; 78: 1637-1651Google Scholar) and anticoagulants (13Lundwall Å. Dackowski W. Cohen E. Shaffer M. Mahr A. Dahlbäck B. Stenflo J. Wydro R. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 6716-6720Google Scholar), and proteins for specification of cell fate (14Wharton K.A. Johansen K.M. Xu T. Artavanis-Tsakonas S. Cell. 1985; 43: 567-581Google Scholar, 15Yochem J. Weston K. Greenwald I. Nature. 1988; 335: 547-550Google Scholar, 16Coffman C. Harris W. Kintner C. Science. 1990; 249: 1438-1441Google Scholar, 17Ellisen L.W. Bird J. West D.C. Soreng A.L. Reynolds T.C. Smith S.D. Sklar J. Cell. 1991; 66: 649-661Google Scholar). A characteristic pattern of amino acid residues ((D/N)X(D/N)(Q/E)Xn(D*/N*)Xm(Y/F); residues with an asterisk may be β-hydroxylated) has been identified in a subset of EGF-like motifs that is responsible for calcium binding. Calcium binding to this type of motif has been demonstrated with blood clotting factors IX and X (18Persson E. Selander M. Linse S. Drakenberg T. Öhlin A.-K. Stenflo J. J. Biol. Chem. 1989; 264: 16897-16904Google Scholar, 19Handford P.A. Baron M. Mayhew M. Willis A. Beesley T. Brownlee G.G. Campbell I.D. EMBO J. 1990; 9: 475-480Google Scholar), with anticoagulants Protein C (20Öhlin A.-K. Linse S. Stenflo J. J. Biol. Chem. 1988; 263: 7411-7417Google Scholar) and Protein S (21Dahlbäck B. Hildebrand B. Linse S. J. Biol. Chem. 1990; 265: 18481-18489Google Scholar), and the microfibrillar protein fibrillin-1 (22Glanville R.W. Qian R.-Q. McClure D.W. Maslen C.L. J. Biol. Chem. 1994; 269: 26630-26634Google Scholar, 23Handford P. Downing A.K. Rao Z. Hewett D.R. Sykes B.C. Kielty C.M. J. Biol. Chem. 1995; 270: 6751-6756Google Scholar, 24Reinhardt D.P. Keene D.R. Corson G.M. Pöschl E. Bächinger H.P. Gambee J.E. Sakai L.Y. J. Mol. Biol. 1996; 258: 104-116Google Scholar).Little is known about the functional significance of these motifs and the consequences of calcium binding on the properties of proteins containing these motifs. It has been suggested that calcium binding to cbEGF-like motifs mediates protein-protein interactions. For example, mutations of amino acid residues crucial for calcium binding affect the clotting activity of factor IX (25Rees D.J.G. Jones I.M. Handford P.A. Walter S.J. Esnouf M.P. Smith K.J. Brownlee G.G. EMBO J. 1988; 7: 2053-2061Google Scholar). The interaction of Protein S with complement C4b-binding protein is mediated by this type of module (26Dahlbäck B. Frohm B. Nelsestuen G. J. Biol. Chem. 1990; 265: 16082-16087Google Scholar), and the interaction of the Drosophila transmembrane protein Notch with Delta and Serrate depends on two tandemly repeated cbEGF-like motifs (27Rebay I. Fleming R.J. Fehon R.G. Cherbas L. Cherbas P. Artavanis-Tsakonas S. Cell. 1991; 67: 687-699Google Scholar). Possibly, cbEGF-like motifs are also involved in mediating the calcium-dependent interaction of fibrillin-1 with fibulin-2 (28Reinhardt 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: 19489-19496Google Scholar).The fibrillin family consists of two members, fibrillin-1 and fibrillin-2, both of which are integral constituents of the 10-12-nm diameter extracellular matrix microfibrils (5Zhang H. Apfelroth S.D. Hu W. Davis E.C. Sanguineti C. Bonadio J. Mecham R.P. Ramirez F. J. Cell Biol. 1994; 124: 855-863Google Scholar, 29Sakai L.Y. Keene D.R. Engvall E. J. Cell Biol. 1986; 103: 2499-2509Google Scholar). Both proteins consist of repetitive motifs: 47 of them are EGF-like repeats, and 43 have the consensus sequence for calcium binding. The cbEGF-like repeats are dispersed over both entire molecules in nine stretches of 1-12 tandemly repeated motifs.Mutations in fibrillin-1 result in Marfan's syndrome, a connective tissue disorder affecting the cardiovascular, skeletal, and ocular systems (reviewed in 30Dietz H.C. Pyeritz R.E. Hum. Mol. Genet. 1995; 4: 1799-1809Google Scholar). On the other hand, mutations in fibrillin-2 give rise to congenital contractural arachnodactyly, a disorder characterized primarily by joint contractures and other skeletal features (31Putnam E.A. Zhang H. Ramirez F. Milewicz D.M. Nat. Genet. 1995; 11: Scholar). of the more than known mutations in fibrillin-1 and the two mutations in fibrillin-2 in cbEGF-like repeats and are predicted to calcium binding, an of the cbEGF-like repeats in these this we the of calcium binding on the of fibrillin-1 in protease degradation assays. These that calcium stabilizes fibrillin-1 against proteolytic degradation. This may be a functional for cbEGF-like motifs in other proteins. We that mutations in fibrillin-1, and in other that calcium binding to cbEGF-like motifs render the molecules more susceptible to proteolytic degradation. degradation of the molecules to the of the