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• W2074001940 abstract "Mutations in fibrillin-1 lead to Marfan syndrome and some related genetic disorders. Many of the more than 600 mutations currently known in fibrillin-1 eliminate or introduce cysteine residues in epidermal growth factor-like modules. Here we report structural and functional consequences of three selected cysteine mutations (R627C, C750G, and C926R) in fibrillin-1. The mutations have been analyzed by means of recombinant polypeptides produced in mammalian expression systems. The mRNA levels for the mutation constructs were similar to wild-type levels. All three mutated polypeptides were secreted by embryonic kidney cells (293) into the culture medium. Purification was readily feasible for mutants R627C and C750G, but not for C926R, which restricted the availability of this mutant polypeptide to selected analyses. The overall folds of the mutant polypeptides were indistinguishable from the wild-type as judged by the ultrastructural shape, CD analysis, and reactivity with a specific antibody sensitive for intact disulfide bonds. Subtle structural changes caused by R627C and C750G, however, were monitored by proteolysis and heat denaturation experiments. These changes occurred in the vicinity of the mutations either as short range effects (R627C) or both short and long range effects (C750G). Enhanced proteolytic susceptibility was observed for R627C and C750G to a variety of proteases. These results expand and further strengthen the concept that proteolytic degradation of mutated fibrillin-1 might be an important potential mechanism in the pathogenesis of Marfan syndrome and other disorders caused by mutations in fibrillin-1. Mutations in fibrillin-1 lead to Marfan syndrome and some related genetic disorders. Many of the more than 600 mutations currently known in fibrillin-1 eliminate or introduce cysteine residues in epidermal growth factor-like modules. Here we report structural and functional consequences of three selected cysteine mutations (R627C, C750G, and C926R) in fibrillin-1. The mutations have been analyzed by means of recombinant polypeptides produced in mammalian expression systems. The mRNA levels for the mutation constructs were similar to wild-type levels. All three mutated polypeptides were secreted by embryonic kidney cells (293) into the culture medium. Purification was readily feasible for mutants R627C and C750G, but not for C926R, which restricted the availability of this mutant polypeptide to selected analyses. The overall folds of the mutant polypeptides were indistinguishable from the wild-type as judged by the ultrastructural shape, CD analysis, and reactivity with a specific antibody sensitive for intact disulfide bonds. Subtle structural changes caused by R627C and C750G, however, were monitored by proteolysis and heat denaturation experiments. These changes occurred in the vicinity of the mutations either as short range effects (R627C) or both short and long range effects (C750G). Enhanced proteolytic susceptibility was observed for R627C and C750G to a variety of proteases. These results expand and further strengthen the concept that proteolytic degradation of mutated fibrillin-1 might be an important potential mechanism in the pathogenesis of Marfan syndrome and other disorders caused by mutations in fibrillin-1. Fibrillins are major components of a class of 10-12-nm extracellular microfibrils, which occur either in association with elastin or in elastin-free bundles (1Sakai L.Y. Keene D.R. Engvall E. J. Cell Biol. 1986; 103: 2499-2509Google Scholar, 2Zhang 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). The highly homologous fibrillin family consists of three members, fibrillin-1, -2, and -3, which are encoded by different genes (2Zhang 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, 3Maslen C.L. Corson G.M. Maddox B.K. Glanville R.W. Sakai L.Y. Nature. 1991; 352: 334-337Google Scholar, 4Lee 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, 5Corson G.M. Chalberg S.C. Dietz H.C. Charbonneau N.L. Sakai L.Y. Genomics. 1993; 17: 476-484Google Scholar, 6Pereira L. D'Alessio M. Ramirez F. Lynch J.R. Sykes B. Pangilinan T. Bonadio J. Hum. Mol. Genet. 1993; 2: 961-968Google Scholar, 7Corson G.M. Charbonneau N.L. Keene D.R. Sakai L.Y. Genomics. 2004; 83: 461-472Google Scholar). Like many other extracellular glycoproteins, fibrillins are composed of individual modules in a mosaic fashion (Fig. 1A). Most of these modules are rich in cysteine residues, accounting for the high overall content of cysteine residues in fibrillins (12-13%). Multiple tandem arrays of epidermal growth factor-like (EGF) 1The abbreviations used are: EGF, epidermal growth factor-like module; cbEGF, calcium-binding epidermal growth factor-like module; 8-Cys/TB, 8-cysteine-containing modules; mAb, monoclonal antibody; MFS, Marfan syndrome; TBS, Tris-buffered saline; CCA, congenital contractural arachnodactyly. 1The abbreviations used are: EGF, epidermal growth factor-like module; cbEGF, calcium-binding epidermal growth factor-like module; 8-Cys/TB, 8-cysteine-containing modules; mAb, monoclonal antibody; MFS, Marfan syndrome; TBS, Tris-buffered saline; CCA, congenital contractural arachnodactyly. domains constitute the majority of the fibrillin molecules. There are 46-47 EGF modules in each fibrillin, 42-43 of which are associated with calcium binding. Each EGF module contains six highly conserved cysteine residues, which form disulfide bonds in a 1-3, 2-4, 5-6 arrangement, generating an anti-parallel β-pleated sheet conformation (8Campbell I.D. Bork P. Curr. Opin. Struct. Biol. 1993; 3: 385-392Google Scholar, 9Downing A.K. Knott V. Werner J.M. Cardy C.M. Campbell I.D. Handford P.A. Cell. 1996; 85: 597-605Google Scholar). It has been shown that calcium binding to calcium-binding EGF (cbEGF) modules in fibrillin-1 protects the molecules against proteolytic degradation by a variety of proteases (10Reinhardt D.P. Ono R.N. Sakai L.Y. J. Biol. Chem. 1997; 272: 1231-1236Google Scholar) and that calcium plays a crucial role in stabilizing tandem repeats of cbEGF modules in fibrillin-1 (9Downing A.K. Knott V. Werner J.M. Cardy C.M. Campbell I.D. Handford P.A. Cell. 1996; 85: 597-605Google Scholar, 11Reinhardt D.P. Mechling D.E. Boswell B.A. Keene D.R. Sakai L.Y. Bächinger H.P. J. Biol. Chem. 1997; 272: 7368-7373Google Scholar). Other prominent modules in fibrillins are 8-cysteine-containing structures typically referred to as 8-Cys/TB modules and hybrid modules. Both types of modules are exclusively found in fibrillins and latent transforming growth factor-β-binding proteins (for a review, see Ref. 12Saharinen J. Hyytiäinen M. Taipale J. Keski-Oja J. Cytokine Growth Factor Rev. 1999; 10: 99-117Google Scholar). Mutations in fibrillins lead to various connective tissue disorders such as the Marfan syndrome (MFS; MIM 154700) and some other related disorders of connective tissue commonly referred to as type-1 fibrillinopathies caused by mutations in the gene for fibrillin-1 (FBN1), and congenital contractural arachnodactyly (CCA; MIM 121050) caused by mutations in the gene for fibrillin-2 (FBN2) (for a review, see Ref. 13Milewicz D.M. Urban Z. Boyd C. Matrix Biol. 2000; 19: 471-480Google Scholar). Most of the known mutations in FBN1 (95%) result in various forms of MFS (14Collod-Beroud G. Le Bourdelles S. Ades L. Ala-Kokko L. Booms P. Boxer M. Child A. Comeglio P. De Paepe A. Hyland J.C. Holman K. Kaitila I. Loeys B. Matyas G. Nuytinck L. Peltonen L. Rantamaki T. Robinson P. Steinmann B. Junien C. Beroud C. Boileau C. Hum. Mutat. 2003; 22: 199-208Google Scholar). MFS is an autosomal dominant disorder of connective tissue with an estimated prevalence of about 1 in 5000 individuals (15Pyeritz R.E. Annu. Rev. Med. 2000; 51: 481-510Google Scholar). MFS is characterized by highly variable clinical manifestations including aortic dilation and dissection, ectopia lentis, dolichostenomelia, arachnodactyly, scoliosis, pectus deformities, and other musculoskeletal abnormalities (15Pyeritz R.E. Annu. Rev. Med. 2000; 51: 481-510Google Scholar). A dominant negative model for the pathogenesis of MFS has been proposed in which the product of the mutant allele interferes with polymerization of fibrillin into microfibrils or destabilizes the microfibrils after incorporation, and the severity of the disease is dependent on the mutant protein level (16Dietz H.C. McIntosh I. Sakai L.Y. Corson G.M. Chalberg S.C. Pyeritz R.E. Francomano C.A. Genomics. 1993; 17: 468-475Google Scholar). In addition, other mechanisms must play a role, since some premature termination mutations that cause selective degradation of the mutant mRNA can result in severe disease (17Hewett D. Lynch J. Child A. Firth H. Sykes B. Am. J. Hum. Genet. 1994; 55: 447-452Google Scholar, 18Halliday D. Hutchinson S. Kettle S. Firth H. Wordsworth P. Handford P.A. Hum. Genet. 1999; 105: 587-597Google Scholar, 19Montgomery R.A. Geraghty M.T. Bull E. Gelb B.D. Johnson M. McIntosh I. Francomano C.A. Dietz H.C. Am. J. Hum. Genet. 1998; 63: 1703-1711Google Scholar). Independent of the involved mechanism, the common pathway of various types of mutations appears to involve a reduction in the amount of functional microfibrils in the extracellular matrix (20Robinson P.N. Booms P. Cell. Mol. Life Sci. 2001; 58: 1698-1707Google Scholar). The majority of the more than 600 mutations in FBN1 currently known are point mutations (72.4%); the rest are frame-shifts caused by deletions and insertions (16.1%) and splice site mutations (11.5%) (data set of the FBN1 online data base (available on the World Wide Web at www.umd.be) as of April 2004; see Ref. 14Collod-Beroud G. Le Bourdelles S. Ades L. Ala-Kokko L. Booms P. Boxer M. Child A. Comeglio P. De Paepe A. Hyland J.C. Holman K. Kaitila I. Loeys B. Matyas G. Nuytinck L. Peltonen L. Rantamaki T. Robinson P. Steinmann B. Junien C. Beroud C. Boileau C. Hum. Mutat. 2003; 22: 199-208Google Scholar). Missense point mutations substituting (28.0%) or generating new (6.7%) cysteine residues represent the largest group, whereby the majority of cysteine-substituting (23.1%) or cysteine-generating (3.8%) mutations occur in cbEGF modules. Another prominent group (8%) are missense mutations that affect amino acid residues in cbEGF modules involved in calcium binding. For both groups, it is predicted that the mutations locally abolish or reduce calcium binding either directly by changing residues involved in calcium binding or indirectly by changing the structure of the mutated cbEGF module. It has been shown in vitro that a number of mutations affecting calcium binding residues render fibrillin-1 more susceptible to proteolysis, providing a potential mechanistic explanation for dominant negative effects (21Ashworth J.L. Murphy G. Rock M.J. Sherratt M.J. Shapiro S.D. Shuttleworth C.A. Kielty C.M. Biochem. J. 1999; 340: 171-181Google Scholar, 22Reinhardt D.P. Ono R.N. Notbohm H. Müller P.K. Bächinger H.P. Sakai L.Y. J. Biol. Chem. 2000; 275: 12339-12345Google Scholar, 23McGettrick A.J. Knott V. Willis A. Handford P.A. Hum. Mol. Genet. 2000; 9: 1987-1994Google Scholar). For some of these mutations, it has been shown that the enhanced susceptibility is caused by subtle structural changes in the region of the mutation (22Reinhardt D.P. Ono R.N. Notbohm H. Müller P.K. Bächinger H.P. Sakai L.Y. J. Biol. Chem. 2000; 275: 12339-12345Google Scholar). For the much larger group of cysteine mutations, however, very little information is available on their structural and functional consequences. Based on quantitative pulse-chase analyses of a number of cysteine mutations, normal synthesis and stability were observed for the majority of mutations analyzed accompanied with delayed secretion in about 55% (24Schrijver I. Liu W. Brenn T. Furthmayr H. Francke U. Am. J. Hum. Genet. 1999; 65: 1007-1020Google Scholar). In another study, two cysteine mutations studied in a recombinant system were retained and accumulated in the cells (25Whiteman P. Handford P.A. Hum. Mol. Genet. 2003; 12: 727-737Google Scholar). Enhanced protease susceptibility induced by a cysteine mutation have been shown in another study (26Booms P. Tiecke F. Rosenberg T. Hagemeier C. Robinson P.N. Hum. Genet. 2000; 107: 216-224Google Scholar). Here we demonstrate that typical cysteine mutations in cbEGF modules, which result in the classical form of Marfan syndrome, render recombinantly expressed polypeptides susceptible to proteolysis. Structural analyses reveal minor changes introduced by the mutations. These results extend and strengthen the concept that enhanced proteolytic degradation of mutated fibrillin-1 may be the causative mechanism for at least certain groups of mutations leading to type-1 fibrillinopathies. Expression Constructs—The construction of an episomal plasmid pCEPSP-rF45 to express wild-type human fibrillin-1 fragment rF45 (Asp451-Lys1027) as well as the cloning plasmid pBS-rF45 was described in detail previously (22Reinhardt D.P. Ono R.N. Notbohm H. Müller P.K. Bächinger H.P. Sakai L.Y. J. Biol. Chem. 2000; 275: 12339-12345Google Scholar). To introduce the mutation C1879T leading to amino acid substitution R627C (27Hayward C. Rae A.L. Porteous M.E. Logie L.J. Brock D.J. Hum. Mol. Genet. 1994; 3: 373-375Google Scholar), site-directed mutagenesis was performed with plasmid pBS-rF45 and complementary primer pairs 5′-GATCTGCATGAATGGGTGTT GCGTCAACACTG-3′ and 5′-CAGTGTTGACGCAACACCCATTCATGCAGATC-3′ using the QuikChange™ procedure as instructed by the supplier (Stratagene). Mutations in the oligonucleotides are in boldface type and underlined. The resulting plasmid was restricted with NheI-NotI, and the 1745-bp fragment was subcloned into the NheI-NotI-restricted pCEPSP-rF45, resulting in pCEPSP-rF45-C1879T. To introduce the mutation T2248G, leading to amino acid substitution C750G (27Hayward C. Rae A.L. Porteous M.E. Logie L.J. Brock D.J. Hum. Mol. Genet. 1994; 3: 373-375Google Scholar), an analogous procedure was employed using primer pairs 5′-GGGACCTATAAATGTATAGGCAATTCAGGATATGAAGTGG-3′ and 5′-CCACTTCATATCCTGAATTGCCTATACATTTATAGGTCCC-3′. To produce the mutation T2776C resulting in amino acid substitution C926R (28Nijbroek G. Sood S. McIntosh I. Francomano C.A. Bull E. Pereira L. Ramirez F. Pyeritz R.E. Dietz H.C. Am. J. Hum. Genet. 1995; 57: 8-21Google Scholar), template HFBN23 was used to amplify a 346-bp fragment with sense primer 5′-CCTTGCATTAATGGAGTCTGC-3′ and antisense primer 5′-TAGTGTTAACACGCAGGCCATTTTTACACACTCC-3′ by polymerase chain reaction. A 267 bp BamHI-HpaI fragment was excised from the product and subcloned into the BamHI-HpaI-restricted pBS-rF45 plasmid. A 1745-bp NheI-NotI fragment was then subcloned into NheI-NotI-restricted pCEPSP-rF45, resulting in pCEPSP-rF45-T2776C. Correct insertions of all mutations as well as the absence of new mutations introduced by DNA amplification were verified by DNA sequencing. All mutation constructs code for a protein with the sequence Ala-Pro-Leu-Ala-Asp451-Lys1027 including the individual mutation and without an additional histidine tag. The Flp-In system was used to generate stable recombinant cell clones with exactly one copy of the expression plasmid incorporated at a predefined locus of the cellular genome (Invitrogen). All mutation constructs in the pCEPSP plasmids were restricted with NheI-NotI, and the 1745-bp fragments were subcloned into NheI-NotI-restricted pcDNA5-FRT plasmid (Invitrogen), which had been modified by adding the sequence encoding the BM40 signal peptide. The sequences of the proteins expressed were identical to what is described above for the pCEPSP expression plasmids. To facilitate epitope mapping, several new expression plasmids for fragments of human fibrillin-1 have been generated. All of the following expression constructs are designed with a His6 tag at the C-terminal end to facilitate protein purification. To assemble an expression plasmid coding for Ser19-Gly714 of human fibrillin-1, plasmid pDNSP-rF16 (29Jensen S.A. Reinhardt D.P. Gibson M.A. Weiss A.S. J. Biol. Chem. 2001; 276: 39661-39666Google Scholar) was cut with EcoRI-NotI, and the 7516-bp fragment was religated using two complementary oligonucleotides 5′-AATTCAGCGGAATATCAGGCACTCTGCAGCAGTGGGCATCACCATCACCATCACTAATAGTGC-3′ and 5′-GGCCGCACTATTAGTGATGGTGATGGTGATGCCCACTGCTGCAGAGTGCCTGATATTCCGCTG-3′ as linkers. The resulting plasmid pDNSP-rF1F codes for a recombinant polypeptide (rF1F) with the sequence Ala-Pro-Leu-Ala-Ser19-Gly714-His6. An expression plasmid coding for Asp613-Leu951 was generated by amplification of template HFBN23-29 (30Reinhardt 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) with primers 5′-CGTAGCTAGCAGACATTAACGAGTGTGAAACCC-3′ and 5′-ACCGCTCGAGCTATTAGTGATGGTGATGGTGATGAAGACAGATCCTTCCTGTGGC-3′. The product was restricted with NheI-XhoI (1048 bp) and subcloned into pDNSP-rF16 (29Jensen S.A. Reinhardt D.P. Gibson M.A. Weiss A.S. J. Biol. Chem. 2001; 276: 39661-39666Google Scholar) restricted with the same enzymes. The resulting plasmid codes for a recombinant protein (rF51) with the sequence Ala-Pro-Leu-Ala-Asp613-Leu951-His6. To prepare an expression plasmid coding for Asp723-Leu951, HFBN23-29 (30Reinhardt 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) was amplified with primers 5′-CGTAGCTAGCTGATATAAATGAATGTGCACTAGATCC-3′ and 5′-ACCGCTCGAGCTATTAGTGATGGTGATGGTGATGAAGACAGATCCTTCCTGTGGC-3′. The resulting product was cut with NheI-XhoI (730 bp) and subcloned into the NheI-XhoI-restricted pcDNA5-FRT plasmid (Invitrogen) including a sequence for the BM40 signal peptide. The resulting expression plasmid codes for the recombinant polypeptide rF1A with the sequence Ala-Pro-Leu-Ala-Asp723-Leu951-His6. To assemble an expression plasmid coding for Asp952-Lys1027, template pCEPSP-rF18 (30Reinhardt 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) was amplified by polymerase chain reaction with primers 5′-CGTAGCTAGCCGATATCCGCCTGGAAACCTGC-3′ and 5′-ATAGTTTAGCGGCCGCTAGTGATGGTGATGGTGATGTTTGAAGAAAGGCTTTCCATTTG-3′. A 256-bp fragment (NheI-NotI) was cut from the product and subcloned into the NheI-NotI-restricted pCEPSP-rF18, resulting in pCEPSP-rF35. This expression plasmid codes for recombinant polypeptide rF35 with the sequence Ala-Pro-Leu-Ala-Asp952-Lys1027-His6. The relative position of all expressed polypeptides in comparison with full-length fibrillin-1 is shown in Fig. 1A. Transfection of Cells and Culture Conditions—293-EBNA cells (Invitrogen) were used to generate episomal recombinant cell clones with the pCEPSP plasmids, Flp-In-293 cells (Invitrogen) were used to generate stable recombinant cell clones with the pcDNA5-FRT plasmids, and 293 cells (American Type Culture Collection) were used for the production of stable clones with the pDNSP-based plasmids. All cell types were grown in Dulbecco's modified Eagle's medium supplemented with 2 mm glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, and 10% fetal calf serum in a 5% CO2 atmosphere at 37 °C. For 293-EBNA and 293 cells, calcium phosphate transfection was performed as described in detail (31Chen C. Okayama H. Mol. Cell. Biol. 1987; 7: 2745-2752Google Scholar). For Flp-In-293 cells, cotransfection with a plasmid encoding the Flp recombinase (pOG44) was performed as instructed by the supplier (Invitrogen). Selection of recombinant clones was started after 24 h with 0.25 mg/ml hygromycin (293-EBNA, Flp-In-293) or 0.25 mg/ml G418 (293). Single colonies resistant to the antibiotics were picked and propagated. Positive clones were identified by analysis of conditioned medium by Western blotting and SDS-gel electrophoresis using standard protocols. mRNA Synthesis and Secretion of Recombinant Proteins—Nontransfected and transfected Flp-In-293 cell clones were grown to confluence in 75-cm2 flasks and used for RNA purification and secretion analysis. After isolation of total RNA with Trizol following the supplier's instructions (Invitrogen), the mRNA was reverse transcribed into cDNA with Superscript II and oligo(dT) primer according to the manufacturer's protocol (Invitrogen). Polymerase chain reaction was performed using Taq polymerase (Roche Applied Science) and primer pair 5′-AAGTGTCAGTGTCCCAGTGG-3′ and 5′-TAGAAGGCACAGTCGAGGC-3′, which produced a 350-bp fragment specific for the rF45 constructs. A standard control (glyceraldehyde-3-phosphate dehydrogenase) was included, which resulted in a 1173-bp DNA fragment. The intensity of the DNA fragments was visualized by standard agarose gel electrophoresis and ethidium bromide staining. The conditions for the DNA amplifications (rF45 constructs and control) were optimized for quantification with respect to the cycle number and the amount of cDNA used as template. For this goal, the intensities of the stained DNA bands were correlated with a wide range of cycle numbers and the amount of cDNA used in the amplification procedure. The final parameters for quantification were chosen from linear ranges and were 0.5 μl of template cDNA solution for both reactions, 26 cycles for the recombinant constructs, and 22 cycles for the control. To test the secretion of the recombinant polypeptides, confluent cell layers were washed twice with phosphate-buffered saline and incubated with 10 ml of serum-free Dulbecco's modified Eagle's medium, which was harvested after 48 h. Equal amounts (1 ml) of secreted proteins, precipitated from the serum-free medium with 10% (w/v) trichloroacetic acid, were analyzed by standard Western blotting techniques using monoclonal antibody (mAb) 201 (∼4 μg/ml), which is specific for authentic fibrillin-1 and only reacts with nonreduced material (1Sakai L.Y. Keene D.R. Engvall E. J. Cell Biol. 1986; 103: 2499-2509Google Scholar, 22Reinhardt D.P. Ono R.N. Notbohm H. Müller P.K. Bächinger H.P. Sakai L.Y. J. Biol. Chem. 2000; 275: 12339-12345Google Scholar). Production and Purification of Recombinant Polypeptides—For large scale production of rF45-wt, rF45-R627C, rF45-C750G, rF45-C926R, and the histidine tag-containing recombinant polypeptides rF35, rF51, rF1A, and rF1F, cells were grown in triple layer flasks (500 cm2; Nalge Nunc International) to confluence, washed twice with 20 mm HEPES, 150 mm NaCl, pH 7.4, or alternatively with serum-free Dulbecco's modified Eagle's medium and incubated with 60-70 ml of serum-free Dulbecco's modified Eagle's medium for 48 h. The conditioned medium was centrifuged for 15 min at 4 °C (5000 × g) to remove cells, supplemented with phenylmethylsulfonyl fluoride (Fluka) to a final concentration of 0.1 mm, and frozen at -20 °C. About 2-3 liters of serum-free medium were collected and concentrated by ultrafiltration (10-30-kDa cut-off) to 40-50 ml. For the recombinant polypeptides without a histidine tag (rF45 wild-type and mutation constructs), the concentrated medium was dialyzed against 20 mm Tris-HCl, pH 8.6. The dialyzed medium was passed over an anion exchange column (1 ml of HiTrap Q; Amersham Biosciences) equilibrated in the same buffer, and bound proteins were eluted with a gradient of 25 mm NaCl/ml (0-400 mm NaCl total). Fractions containing the recombinant polypeptides were pooled and concentrated by ultrafiltration (10-kDa cut-off) to ∼1.5 ml. Fractions of ∼0.5 ml were passed over a Superose 12 gel filtration column (24 ml; Amersham Biosciences) equilibrated in 50 mm Tris-HCl, pH 7.4, 150 mm NaCl (TBS). Fractions containing the protein of interest were pooled and stored at -80 °C. Purification of recombinant polypeptides with a His6 tag (rF35, rF51, rF1A, and rF1F) was performed by chelating chromatography as described in detail previously for other recombinant polypeptides with minor modifications (32Lin G. Tiedemann K. Vollbrandt T. Peters H. Bätge B. Brinckmann J. Reinhardt D.P. J. Biol. Chem. 2002; 277: 50795-50804Google Scholar). Assessment of the purity and visualization of the purified material was performed by standard SDS gel electrophoresis and Coomassie Blue staining. Epitope Mapping—Overlapping recombinant fragments of the N-terminal part of fibrillin-1 were used to localize the epitope of mAb 201 by standard Western blotting as described in detail (33Keene D.R. Jordan C.D. Reinhardt D.P. Ridgway C.C. Ono R.N. Corson G.M. Fairhurst M. Sussman M.D. Memoli V.A. Sakai L.Y. J. Histochem. Cytochem. 1997; 45: 1069-1082Google Scholar). The purified recombinant fragments (5 μg each) were subjected to SDS-gel electrophoresis under nonreducing conditions, transferred to nitrocellulose membrane, and incubated with ∼4 μg/ml mAb 201. To correlate bands recognized by mAb 201 with the position of the purified proteins, a control was included with Coomassie Blue-stained purified polypeptides after SDS gel electrophoresis under reducing and nonreducing conditions. Degradation Experiments and N-terminal Sequence Analysis—Recombinant polypeptides rF45-wt, rF45-R627C, and rF45-C750G (1.0 mg/ml in TBS) were supplemented with 5 mm CaCl2 and incubated for 10 min at room temperature. After removal of a control, enzymes were added at a concentration of 1:20 (w/w) for plasmin (EC 3.4.21.7; Roche Applied Science) or 1:100 (w/w) for trypsin (EC 3.4.21.4; Sigma) treated with tosylphenylalanyl chloromethyl ketone and α-chymotrypsin (EC 3.4.21.1; Sigma) treated with Nα-p-tosyl-l-lysine chloromethyl ketone for incubation periods of 60 min (plasmin) or 10 min (trypsin and chymotrypsin). The reaction was stopped by adding 2-fold concentrated reducing SDS sample buffer to equal aliquots of the samples and heating at 95 °C for 3 min. Degradation products were separated by SDS gel electrophoresis (7.5-12% gradient gels) and visualized by Coomassie Blue staining. For N-terminal sequence analysis, the degradation products were blotted on polyvinylidene difluoride membrane (Immobilon-P (Milli-pore) or Pro Blott (Applied Biosystems)), stained with Coomassie Blue, excised, and analyzed on a protein sequencer (Applied Biosystems model 494). Circular Dichroism Spectroscopy—The purified recombinant polypeptides rF45-wt, rF45-R627C, and rF45-C750G (0.5 mg/ml in TBS) were analyzed either in the presence of 5 mm CaCl2 or 0.25 mm EDTA from 200 to 260 nm in a quartz cuvette at 20 °C on a Jasco J-715 instrument. Heat denaturation of the purified recombinant polypeptides was determined by measuring the circular dichroism at 220 nm at increasing temperatures (20-100 °C in steps of 0.4 °C/min). The signal at 20 °C was set to 0% denaturation, and the signal at 100 °C was set to 100% denaturation. Low Angle Rotary Shadowing Electron Microscopy—Purified polypeptides rF45-wt, rF45-R627C, rF45-C750G, and rF45-C926R (∼0.25 mg/ml) were supplemented with 5 mm CaCl2 and dialyzed against H2O. The samples were mixed with equal volumes of glycerol to a final concentration of 50% (v/v) glycerol, sprayed onto freshly cleaved mica, and dried under vacuum (Edwards Auto 306). Rotary shadowing was performed as described in detail previously (32Lin G. Tiedemann K. Vollbrandt T. Peters H. Bätge B. Brinckmann J. Reinhardt D.P. J. Biol. Chem. 2002; 277: 50795-50804Google Scholar). Replicates were examined at 100 kV in a transmission electron microscope (Zeiss TEM 109). In this study, we have investigated structural and functional consequences of cysteine mutations in fibrillin-1, leading to the classical form of MFS. Missense mutations substituting or eliminating cysteine residues in cbEGF modules represent the largest group of mutations in fibrillin-1 (see Introduction and Ref. 14Collod-Beroud G. Le Bourdelles S. Ades L. Ala-Kokko L. Booms P. Boxer M. Child A. Comeglio P. De Paepe A. Hyland J.C. Holman K. Kaitila I. Loeys B. Matyas G. Nuytinck L. Peltonen L. Rantamaki T. Robinson P. Steinmann B. Junien C. Beroud C. Boileau C. Hum. Mutat. 2003; 22: 199-208Google Scholar). The mutations in the FBN1 gene analyzed in this study were C1879T and T2248G, resulting in an additional cysteine residue (R627C) in cbEGF module 6 or a cysteine substitution at the C5 position of cbEGF module 7 (C750G), respectively (27Hayward C. Rae A.L. Porteous M.E. Logie L.J. Brock D.J. Hum. Mol. Genet. 1994; 3: 373-375Google Scholar). The third mutation analyzed was T2776C, leading to a cysteine substitution at the C3 position of cbEGF module 10 (C926R) (28Nijbroek G. Sood S. McIntosh I. Francomano C.A. Bull E. Pereira L. Ramirez F. Pyeritz R.E. Dietz H.C. Am. J. Hum. Genet. 1995; 57: 8-21Google Scholar). The mutations were introduced by site-directed mutagenesis into the expression vector for the previously described fibrillin-1 wild-type construct rF45-wt, spanning the fourth generic non-calcium-binding EGF-like module to the third 8-Cys/TB module (Fig. 1A) (22Reinhardt D.P. Ono R.N. Notbohm H. Müller P.K. Bächinger H.P. Sakai L.Y. J. Biol. Chem. 2000; 275: 12339-12345Google Scholar). The relative position of the cysteine mutations within a typical cbEGF module is shown in Fig. 1B. In order to analyze and compare mRNA expression and protein secretion from cells, stable expression clones were generated with the Flp-In system (Invitrogen). With this system, exactly one copy of the expression plasmid is incorporated into a predefined locus in the genome of the cells, eliminating differences of the copy number and the locus between different recombinant cell clones. Analysis of the mRNA by polymerase chain reaction after reverse transcription into cDNA demonstrated that all recombinant clones expressed the mutant mRNA at similar levels as compared with the wild type (Fig. 2A). Control analyses of mRNA isolated from nontra" @default.
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• W2074001940 title "Consequences of Cysteine Mutations in Calcium-binding Epidermal Growth Factor Modules of Fibrillin-1" @default.
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