Latent Transforming Growth Factor β-binding Protein 1 Interacts with Fibrillin and Is a Microfibril-associated Protein
2003; Elsevier BV; Volume: 278; Issue: 4 Linguagem: Inglês
10.1074/jbc.m209256200
ISSN1083-351X
AutoresZenzo Isogai, Robert N. Ono, Shin Ushiro, Douglas R. Keene, Yan Chen, Roberta Mazzieri, Noé L. Charbonneau, Dieter P. Reinhardt, Daniel B. Rifkin, Lynn Y. Sakai,
Tópico(s)Protease and Inhibitor Mechanisms
ResumoLatent transforming growth factor β-binding protein 1 (LTBP-1) targets latent complexes of transforming growth factor β to the extracellular matrix, where the latent cytokine is subsequently activated by several different mechanisms. Fibrillins are extracellular matrix macromolecules whose primary function is architectural: fibrillins assemble into ultrastructurally distinct microfibrils that are ubiquitous in the connective tissue space. LTBPs and fibrillins are highly homologous molecules, and colocalization in the matrix of cultured cells has been reported. To address whether LTBP-1 functions architecturally like fibrillins, microfibrils were extracted from tissues and analyzed immunochemically. In addition, binding studies were conducted to determine whether LTBP-1 interacts with fibrillins. LTBP-1 was not detected in extracted beaded-string microfibrils, suggesting that LTBP-1 is not an integral structural component of microfibrils. However, binding studies demonstrated interactions between LTBP-1 and fibrillins. The binding site was within three domains of the LTBP-1 C terminus, and in fibrillin-1 the site was defined within four domains near the N terminus. Immunolocalization data were consistent with the hypothesis that LTBP-1 is a fibrillin-associated protein present in certain tissues but not in others. In tissues where LTBP-1 is not expressed, LTBP-4 may substitute for LTBP-1, because the C-terminal end of LTBP-4 binds equally well to fibrillin. A model depicting the relationship between LTBP-1 and fibrillin microfibrils is proposed. Latent transforming growth factor β-binding protein 1 (LTBP-1) targets latent complexes of transforming growth factor β to the extracellular matrix, where the latent cytokine is subsequently activated by several different mechanisms. Fibrillins are extracellular matrix macromolecules whose primary function is architectural: fibrillins assemble into ultrastructurally distinct microfibrils that are ubiquitous in the connective tissue space. LTBPs and fibrillins are highly homologous molecules, and colocalization in the matrix of cultured cells has been reported. To address whether LTBP-1 functions architecturally like fibrillins, microfibrils were extracted from tissues and analyzed immunochemically. In addition, binding studies were conducted to determine whether LTBP-1 interacts with fibrillins. LTBP-1 was not detected in extracted beaded-string microfibrils, suggesting that LTBP-1 is not an integral structural component of microfibrils. However, binding studies demonstrated interactions between LTBP-1 and fibrillins. The binding site was within three domains of the LTBP-1 C terminus, and in fibrillin-1 the site was defined within four domains near the N terminus. Immunolocalization data were consistent with the hypothesis that LTBP-1 is a fibrillin-associated protein present in certain tissues but not in others. In tissues where LTBP-1 is not expressed, LTBP-4 may substitute for LTBP-1, because the C-terminal end of LTBP-4 binds equally well to fibrillin. A model depicting the relationship between LTBP-1 and fibrillin microfibrils is proposed. latent transforming growth factor β-binding protein latency associated peptide transforming growth factor β enzyme-linked immunosorbent assay Tris-buffered saline monoclonal antibody The fibrillins and latent transforming growth factor β-binding proteins (LTBPs)1 are members of a family of homologous molecules. The fibrillins and LTBPs contain multiple calcium-binding epidermal growth factor-like modules interspersed by a domain module (the 8-Cys or TB module), so far found only in these two proteins. Fibrillin-1 (1Sakai L.Y. Keene D.R. Engvall E. J. Cell Biol. 1986; 103: 2499-2509Crossref PubMed Scopus (926) Google Scholar, 2Maslen C.L. Corson G.M. Maddox B.K. Glanville R.W. Sakai L.Y. Nature. 1991; 352: 334-337Crossref PubMed Scopus (295) Google Scholar, 3Corson G.M. Chalberg S.C. Dietz H.C. Charbonneau N.L. Sakai L.Y. Genomics. 1993; 17: 476-484Crossref PubMed Scopus (230) Google Scholar, 4Pereira L. D'Alessio M. Ramirez F. Lynch J.R. Sykes B. Pangilinan T. Bonadio J. Hum. Mol. Genet. 1993; 2: 961-968Crossref PubMed Scopus (258) Google Scholar) and fibrillin-2 (5Lee B. Godfrey M. Vitale E. Hori H. Mattei M.G. Sarfarazi M. Tsipouras P. Ramirez F. Hollister D.W. Nature. 1991; 352: 330-334Crossref PubMed Scopus (589) Google Scholar, 6Zhang H. Apfelroth S.D., Hu, W. Davis E.C. Sanguineti C. Bonadio J. Mecham R.P. Ramirez F. J. Cell Biol. 1994; 124: 855-863Crossref PubMed Scopus (321) Google Scholar) share a highly similar overall structure. Both molecules are of equivalent size (∼350 kDa) and domain organization. In contrast, LTBP-1 (7Miyazono K. Hellman U. Wernstedt C. Heldin C.H. J. Biol. Chem. 1988; 263: 6407-6415Abstract Full Text PDF PubMed Google Scholar, 8Kanzaki T. Olofsson A Morén A. Wernstedt C. Hellman U. Miyazono K. Claesson-Welsh L. Heldin C.H. Cell. 1990; 61: 1051-1061Abstract Full Text PDF PubMed Scopus (380) Google Scholar), LTBP-2 (9Morén A. Olofsson A. Stenman G. Sahlin P. Kanzaki T. Claesson-Welsh L. ten Dijke P. Miyazono K. Heldin C.H. J. Biol. Chem. 1994; 269: 32469-32478Abstract Full Text PDF PubMed Google Scholar), LTBP-3 (10Yin W. Smiley E. Germiller J. Mecham R.P. Florer J.B. Wenstrup R.J. Bonadio J. J. Biol. Chem. 1995; 270: 10147-10160Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar), and LTBP-4 (11Giltay R. Kostka G. Timpl R. FEBS Lett. 1997; 411: 164-168Crossref PubMed Scopus (64) Google Scholar, 12Saharinen J. Taipale J. Monni O. Keski-Oja J. J. Biol. Chem. 1998; 273: 18459-18469Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar) are each smaller than the fibrillins and variable in size. Extensive immunolocalization data combined with structural analyses of the fibrillin-1 monomer and fibrillin-containing microfibrils (1Sakai L.Y. Keene D.R. Engvall E. J. Cell Biol. 1986; 103: 2499-2509Crossref PubMed Scopus (926) Google Scholar,13Sakai L.Y. Keene D.R. Glanville R.W. Bächinger H.P. J. Biol. Chem. 1991; 266: 14763-14770Abstract Full Text PDF PubMed Google Scholar, 14Keene D.R. Maddox B.K. Kuo H.J. Sakai L.Y. Glanville R.W. J. Histochem. Cytochem. 1991; 39: 441-449Crossref PubMed Scopus (184) Google Scholar, 15Reinhardt D.P. Keene D.R. Corson G.M. Pöschl E. Bächinger H.P. Gambee J.E. Sakai L.Y. J. Mol. Biol. 1996; 258: 104-116Crossref PubMed Scopus (210) Google Scholar) have established that fibrillin-1 is a major structural component of connective tissue microfibrils. In addition, genetic evidence in humans (16Hollister D.W. Godfrey M. Sakai L.Y. Pyeritz R.E. N. Engl. J. Med. 1990; 323: 152-159Crossref PubMed Scopus (313) Google Scholar, 17Dietz H.C. Cutting G.R. Pyeritz R.E. Maslen C.L. Sakai L.Y. Corson G.M. Puffenberger E.G. Hamosh A. Nanthakumar E.J. Curristin S.M. Stetten G. Meyers D.A. Francomano C.A. Nature. 1991; 352: 337-339Crossref PubMed Scopus (1676) Google Scholar) and mice (18Pereira L. Andrikopoulos K. Tian J. Lee S.Y. Keene D.R. Ono R.N. Reinhardt D.P. Sakai L.Y. Jensen-Biery N. Bunton T. Dietz H.C. Ramirez F. Nat. Genet. 1997; 17: 218-222Crossref PubMed Scopus (323) Google Scholar, 19Pereira L. Lee S.Y. Gayraud B. Andrikopoulos K. Shapiro S.D. Bunton T. Biery N.J. Dietz H.C. Sakai L.Y. Ramirez F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3819-3823Crossref PubMed Scopus (423) Google Scholar) has confirmed that fibrillin-1 performs a significant role in the maintenance of microfibrils and elastic fibers. Fibrillin-2, whose structure is predicted to be highly similar to fibrillin-1, has also been immunolocalized to microfibrils (20Zhang H., Hu, W. Ramirez F. J. Cell Biol. 1995; 129: 1165-1176Crossref PubMed Scopus (265) Google Scholar). However, in contrast to fibrillin-1, the contribution of fibrillin-2 to microfibril structure is temporally and spatially restricted. In situ hybridization studies in mice indicated that expression of the fbn2 gene is most prominent in the early developing fetus (20Zhang H., Hu, W. Ramirez F. J. Cell Biol. 1995; 129: 1165-1176Crossref PubMed Scopus (265) Google Scholar). Genetic evidence in humans (5Lee B. Godfrey M. Vitale E. Hori H. Mattei M.G. Sarfarazi M. Tsipouras P. Ramirez F. Hollister D.W. Nature. 1991; 352: 330-334Crossref PubMed Scopus (589) Google Scholar, 21Putnam E.A. Zhang H. Ramirez F. Milewicz D.M. Nat. Genet. 1995; 11: 456-458Crossref PubMed Scopus (230) Google Scholar) suggests that fibrillin-2 plays a more restricted role in the maintenance of microfibrils and elastic fibers in postnatal connective tissues. Recent immunolocalization studies demonstrate a ubiquitous early distribution of fibrillin-2 in fetal tissues followed by a restricted distribution in postnatal tissues (22Charbonneau, N. L., Dzamba, B. J., Ono, R. N., Keene, D. R., Reinhardt, D. P., and Sakai, L. Y. (November 11, 2002) J. Biol. Chem. 10.1074/jbc.M209201200Google Scholar). Mice produced by gene targeting experiments recapitulate the features (contractures of large and small joints) of the human disease congenital contractural arachnodactyly caused by mutations in fibrillin-2 (23Arteaga-Solis E. Gayraud B. Lee S.Y. Shum L. Sakai L.Y. Ramirez F. J. Cell Biol. 2001; 154: 275-281Crossref PubMed Scopus (181) Google Scholar). In addition, fibrillin-2 null mice revealed an unexpected role for fibrillin-2 in limb patterning, because the mutant animals display syndactyly (23Arteaga-Solis E. Gayraud B. Lee S.Y. Shum L. Sakai L.Y. Ramirez F. J. Cell Biol. 2001; 154: 275-281Crossref PubMed Scopus (181) Google Scholar). LTBP-1 forms a complex with latent TGF-β and targets it to the extracellular matrix (7Miyazono K. Hellman U. Wernstedt C. Heldin C.H. J. Biol. Chem. 1988; 263: 6407-6415Abstract Full Text PDF PubMed Google Scholar). Latent TGF-β consists of the mature growth factor plus the TGF-β propeptide, also known as the latency associated peptide (LAP). LAP binds to TGF-β by noncovalent interactions, and the association of LAP with TGF-β prevents the growth factor from binding to its receptor. During the secretory process, CR3, the second 8-Cys module, in LTBP-1 becomes disulfide-linked to the latency associated propeptide (LAP) of TGF-β (24Saharinen J. Taipale J. Keski-Oja J. EMBO J. 1996; 15: 245-253Crossref PubMed Scopus (190) Google Scholar, 25Gleizes P.E. Beavis R.C. Mazzieri R. Shen B. Rifkin D.B. J. Biol. Chem. 1996; 271: 29891-29896Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). The processed TGF-β remains noncovalently bound within this complex of LAP and LTBP. Studies of 8-Cys modules from fibrillin-1 indicate that the 8 conserved cysteine residues in this module form 4 intrachain disulfide bonds (15Reinhardt D.P. Keene D.R. Corson G.M. Pöschl E. Bächinger H.P. Gambee J.E. Sakai L.Y. J. Mol. Biol. 1996; 258: 104-116Crossref PubMed Scopus (210) Google Scholar). The solution structure of one 8-Cys module from fibrillin-1 has been determined (26Yuan X. Downing A.K. Knott V. Handford P.A. EMBO J. 1997; 16: 6659-6666Crossref PubMed Google Scholar), indicating the position of disulfide bonds. The structure of the 8-Cys module in LTBP-1, which is disulfide-linked and complexed with latent TGF-β, is not yet known. However, based upon the conformation of the fibrillin 8-Cys motif, binding of LTBP-1 to LAP has been proposed to occur through Cys4 and Cys7 or Cys2 and Cys6 (26Yuan X. Downing A.K. Knott V. Handford P.A. EMBO J. 1997; 16: 6659-6666Crossref PubMed Google Scholar). LTBP-3 and LTBP-4, but not LTBP-2, also interact with LAP (27Saharinen J. Keski-Oja J. Mol. Biol. Cell. 2000; 11: 2691-2704Crossref PubMed Scopus (228) Google Scholar). LTBP-1 becomes immobilized into the extracellular matrix of tissue culture cells in a covalent manner involving tissue transglutaminase-mediated cross-linking of a region in the N-terminal sequence of LTBP to an undefined matrix protein (28Nunes I. Gleizes P.E. Metz C.N. Rifkin D.B. J. Cell Biol. 1997; 136: 1151-1163Crossref PubMed Scopus (351) Google Scholar). In tissue culture, LTBP-1 colocalizes with both fibronectin and fibrillin-1 (29Taipale J. Saharinen J. Hedman K. Keski-Oja J. J. Histochem. Cytochem. 1996; 44: 875-889Crossref PubMed Scopus (196) Google Scholar,30Dallas S.L. Keene D.R. Bruder S.P. Saharinen J. Sakai L.Y. Mundy G.R. Bonewald L.F. J. Bone Miner. Res. 2000; 15: 68-81Crossref PubMed Scopus (133) Google Scholar). LTBP-1 has been immunolocalized to fibrillin-containing microfibrils in the skin (31Raghunath M. Unsold C. Kubitscheck U. Bruckner-Tuderman L. Peters R. Meuli M. J. Invest. Dermatol. 1998; 111: 559-564Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) and bone (30Dallas S.L. Keene D.R. Bruder S.P. Saharinen J. Sakai L.Y. Mundy G.R. Bonewald L.F. J. Bone Miner. Res. 2000; 15: 68-81Crossref PubMed Scopus (133) Google Scholar, 32Dallas S.L. Miyazono K. Skerry T.M. Mundy G.R. Bonewald L.F. J. Cell Biol. 1995; 131: 539-549Crossref PubMed Scopus (238) Google Scholar) and to microfibrillar structures in the heart (33Nakajima Y. Miyazono K. Kato M. Takase M. Yamagishi T. Nakamura H. J. Cell Biol. 1997; 136: 193-204Crossref PubMed Scopus (107) Google Scholar). LTBP-2 has also been immunolocalized to fibrillin-containing microfibrils (34Gibson M.A. Hatzinikolas G. Davis E.C. Baker E. Sutherland G.R. Mecham R.P. Mol. Cell. Biol. 1995; 15: 6932-6942Crossref PubMed Google Scholar) and exogenous LTBP-2 can be incorporated into the extracellular matrix by fibroblasts (35Hyytiäinen M. Taipale J. Heldin C.H. Keski-Oja J. J. Biol. Chem. 1998; 273: 20669-20676Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Studies of mRNA have indicated that LTBPs are differentially expressed in various tissues. However, surveys of the distribution of LTBP-1 protein in tissues have not been conducted. Moreover, the relationship between LTBPs and fibrillins within microfibril structures is not understood. The investigations presented here were undertaken to determine whether LTBP-1 is present in extracted fibrillin microfibrils, whether LTBP-1 and fibrillin bind to each other, and if so, which regions of the two proteins interact. Recombinant human fibrillin polypeptides used in these investigations have been previously described and characterized (15Reinhardt D.P. Keene D.R. Corson G.M. Pöschl E. Bächinger H.P. Gambee J.E. Sakai L.Y. J. Mol. Biol. 1996; 258: 104-116Crossref PubMed Scopus (210) Google Scholar, 22Charbonneau, N. L., Dzamba, B. J., Ono, R. N., Keene, D. R., Reinhardt, D. P., and Sakai, L. Y. (November 11, 2002) J. Biol. Chem. 10.1074/jbc.M209201200Google Scholar, 36Reinhardt 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: 19489-19496Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 37Reinhardt D.P. Gambee J.E. Ono R.N. Bächinger H.P. Sakai L.Y. J. Biol. Chem. 2000; 275: 2205-2210Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 38Keene 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: 1069-1082Crossref PubMed Scopus (72) Google Scholar). These are depicted schematically in Fig. 1 B. All recombinant fibrillin polypeptides were expressed using 293 human embryonic kidney cells. Full-length recombinant human LTBP-1 was expressed in insect cells as previously described (25Gleizes P.E. Beavis R.C. Mazzieri R. Shen B. Rifkin D.B. J. Biol. Chem. 1996; 271: 29891-29896Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). To make the expression construct rL1N, coding for Asn21 to Ile629 of LTBP-1, clone pACUW-51 was amplified with sense primer LTBP-1-1S (5′-CTGCTAGCAAACCACACTGGCCGCATCAAG-3′) introducing aNheI restriction site at the 5′ end, and antisense primer LTBP-1-1AS (5′-CTCGAGTCAATGATGATGATGATGATGTATGCAGTTAGTACCCTCCTC-3′), introducing the sequence for 6 histidine residues, a stop codon, and aXhoI restriction site at the 3′ end. The resultingNheI-XhoI fragment was subcloned into the expression vector pCEP/γ2III4, which contains the sequence for the BM40/SPARC signal peptide (39Mayer U. Pöschl E. Gerecke D.R. Wagman D.W. Burgeson R.E. Timpl R. FEBS Lett. 1995; 365: 129-132Crossref PubMed Scopus (68) Google Scholar). To make the expression construct rL1M, coding for Asp588 to Phe1139 of LTBP-1, template was amplified with LTBP-1-2S (5′-CTGCTAGCAGATATTGATGAGTGTACTCAGCAGGTC-3′) and LTBP-1-2AS (5′-CTCTCGAGTCAATGATGATGATGATGATGAAAGCACTGCAGTTTCACAGG-3′). For rL1C, coding for Asp1097 to Glu1394, the primer set LTBP-1-3S, (5′-CTGCTAGCAGATGCAGATGAATGCCTACTTTTTG-3′) and LTBP-1-3AS (5′-CTCTCGAGTCAATGATGATGATGATGATGCTCCAGGTCACTACTGTCTTTCTC-3′) was used. rL1K, coding for Arg1181 to Glu1394, was amplified with LTBP-1-29S (5′-AGCTGCTAGCACGACCGGCTGAGTCAAACGAAC-3′) and LTBP-1-3AS. The correct in-frame insertion of all constructs and the sequence of PCR amplified products were confirmed by sequence analysis using a DNA sequencer (Applied Biosystems 373A). The cDNA that was used to obtain expression construct rL4K (Ser1301-Ala1587 of LTBP-4) was derived from normal human dermal fibroblast RNA by reverse transcriptase-PCR. PCR amplification of the cDNA used sense primer rL4K-S (5′-GATCGCTAGCATCCAACGAGAGCCAGAGCC-3′) and antisense primer rL4K-AS (5′-GAGTCTCGAGCTCAGTGATGGTGATGGTGATGGGCCCGGGGCCGTGCGG-3′), which introduced, respectively, a 5′ NheI restriction site and a 3′ sequence for 6 histidine residues, a stop codon, and anXhoI site. An 894-bpNheI-XhoI-restricted insert was subcloned into the expression vector pCEP4/γ2III4. The entire insert of the resulting construct, designated pCEPSP-rL4K, was then verified by DNA sequencing. Mouse LTBP-3 specific sense primer L3-4F (5′-CCAGAAGGAGAGTCTGTGGC-3′) and antisense primer L3-4R (5′-TGTGGGCACTTGTGACACTT-3′) were designed based on the published sequence (10Yin W. Smiley E. Germiller J. Mecham R.P. Florer J.B. Wenstrup R.J. Bonadio J. J. Biol. Chem. 1995; 270: 10147-10160Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar) and used in reverse transcriptase-PCR to amplify a fragment of mouse LTBP-3 cDNA (nucleotide 460–902, with the A in the translation start codon ATG designated as +1) from RNA extracted from 2T3 mouse osteoblast cells. This fragment was used as a probe for screening of a mouse heart cDNA library (Clontech). Several full-length LTBP-3 cDNA clones were isolated and cloned as EcoRI-EcoRI fragments into pBluescript SK (Stratagene) vector. Several errors were found in the original sequence by analyzing these cDNA clones. 2Y. Chen, unpublished result. To make the expression vector rL3K, encoding the region from the beginning of CR4 to the COOH terminus, a fragment of the cDNA was amplified by PCR using sense primer L3CR4C-S (5′-CGGCTAGCCCCAAAGAGACGTGAAGTG-3′) and antisense primer L3CR4CAS (5′-CCGCTCGAGTCAGTGGTGGTGGTGGTGG-CGGCGGCGCTGAGGCAC-3′), introducing an NheI site at the 5′ end and the sequence for 6 histidine residues, a stop codon, and an XhoI site at the 3′ end. The NheI-XhoI fragment was subcloned into the expression vector pCEP4/γ2III4. The correct orientation of the insert and the sequence of the PCR amplified fragment were verified by DNA sequencing. Schematic representations of the LTBP constructs used for these studies are shown in Fig. 1 A. For stable episomal expression, 293 EBNA cells (Invitrogen) were transfected with the expression plasmids by a calcium phosphate precipitation method as described previously (40Chen C. Okayama H. Mol. Cell. Biol. 1987; 7: 2745-2752Crossref PubMed Scopus (5128) Google Scholar). Purification of the recombinant peptides was accomplished using chelating chromatography (HiTrap chelating, AmershamBiosciences) (15Reinhardt D.P. Keene D.R. Corson G.M. Pöschl E. Bächinger H.P. Gambee J.E. Sakai L.Y. J. Mol. Biol. 1996; 258: 104-116Crossref PubMed Scopus (210) Google Scholar) followed by molecular sieve chromatography, using Superose 6 (Amersham Biosciences) in 50 mm Tris-HCl, pH 7.5, 0.15 m NaCl (TBS) for rL1M and rL1C or 50 mm Tris-HCl, pH 7.5, 1 m NaCl for rL1N. Each of the expressed polypeptides was secreted into the medium resulting in yields of more than 0.5 μg/ml. N-terminal sequence analysis of the purified peptides using Edman degradation and amino acid analysis confirmed the expected polypeptide sequence and also demonstrated the purity of the peptides. SDS-PAGE analysis under nonreducing and reducing conditions revealed that the LTBP-1, LTBP-3, and LTBP-4 recombinant polypeptides were secreted as monomers from 293 cells (Fig. 2). Mouse monoclonal antibodies 201 and 69 to fibrillin-1 have been characterized previously (1Sakai L.Y. Keene D.R. Engvall E. J. Cell Biol. 1986; 103: 2499-2509Crossref PubMed Scopus (926) Google Scholar, 13Sakai L.Y. Keene D.R. Glanville R.W. Bächinger H.P. J. Biol. Chem. 1991; 266: 14763-14770Abstract Full Text PDF PubMed Google Scholar, 15Reinhardt D.P. Keene D.R. Corson G.M. Pöschl E. Bächinger H.P. Gambee J.E. Sakai L.Y. J. Mol. Biol. 1996; 258: 104-116Crossref PubMed Scopus (210) Google Scholar,38Keene 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: 1069-1082Crossref PubMed Scopus (72) Google Scholar). Polyclonal anti-fibrillin-1 9543 was also characterized (18Pereira L. Andrikopoulos K. Tian J. Lee S.Y. Keene D.R. Ono R.N. Reinhardt D.P. Sakai L.Y. Jensen-Biery N. Bunton T. Dietz H.C. Ramirez F. Nat. Genet. 1997; 17: 218-222Crossref PubMed Scopus (323) Google Scholar, 22Charbonneau, N. L., Dzamba, B. J., Ono, R. N., Keene, D. R., Reinhardt, D. P., and Sakai, L. Y. (November 11, 2002) J. Biol. Chem. 10.1074/jbc.M209201200Google Scholar). Monoclonal antibodies, 75G, and 42E, were generated using full-length LTBP-1 expressed by Sf9 insect cells (25Gleizes P.E. Beavis R.C. Mazzieri R. Shen B. Rifkin D.B. J. Biol. Chem. 1996; 271: 29891-29896Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Mouse monoclonal antibody 246 against the TGF-β1 propeptide, known as the LAP, was purchased from R & D Systems (Minneapolis, MN), and rabbit polyclonal antibody 39 against human LTBP-1 was purchased from BD Pharmingen. The specificity of monoclonal antibodies was determined by ELISA, as described (41Sakai L.Y. Engvall E. Hollister D.W. Burgeson R.E. Am. J. Pathol. 1982; 108: 310-318PubMed Google Scholar). Recombinant fibrillin-1 subdomains, rF11 and rF6, and LTBP-1 subdomains, rL1N, rL1M, and rL1C, were used to coat microtiter plates at 10 μg/ml. The antibodies were diluted in TBS. For Western blot analysis, serum-free conditioned medium from normal skin fibroblasts was collected for 48 h (13Sakai L.Y. Keene D.R. Glanville R.W. Bächinger H.P. J. Biol. Chem. 1991; 266: 14763-14770Abstract Full Text PDF PubMed Google Scholar). Proteins in the medium were precipitated, subjected to 7.5% SDS-PAGE, and analyzed by immunoblotting as previously described (42Isogai Z. Aspberg A. Keene D.R. Ono R.N. Reinhardt D.P. Sakai L.Y. J. Biol. Chem. 2002; 277: 4565-4572Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Extracts of normal adult human skin, bovine calf tendon, and human fetal membranes were prepared as follows. Nonexposed human skin (∼1 g) was obtained as excess tissue from a skin grafting procedure. Bovine tendon (∼1.5 g) was dissected from an 80-cm crown to rump fetal calf (∼245 days gestation, almost full-term) obtained from a local slaughterhouse. A 40-ml suspension of fetal membranes, washed and homogenized as described (42Isogai Z. Aspberg A. Keene D.R. Ono R.N. Reinhardt D.P. Sakai L.Y. J. Biol. Chem. 2002; 277: 4565-4572Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar), was also extracted as follows. Tissue samples were minced, and the pellets were washed briefly with 50 mm Tris-HCl, pH 7.5, containing 10 mmCaCl2, and 1 mm phenylmethylsulfonyl fluoride. The buffer was removed after centrifugation, and the pellet was extracted in 6 m guanidine hydrochloride, 50 mmTris-HCl, pH 7.5, containing 1 mm phenylmethylsulfonyl fluoride for 72 h at 4 °C with vigorous shaking. The supernatant was collected after centrifugation, and the pellet was extracted in the same buffer for 48 h followed by centrifugation and another 24 h of extraction. The supernatants were pooled and concentrated to 5 ml using an Amicon concentrator (cut-offM r = 30,000). Sieve chromatography under dissociative conditions was performed using a Sepharose CL-2B (AmershamBiosciences) molecular sizing column (90-ml total volume), equilibrated in 4 m guanidine HCl, 50 mm Tris-HCl, pH 7.5, at a flow rate of 0.1 ml/min. The fractions were collected every 2 ml. Protein concentrations were determined using a BCA protein assay kit (Pierce) with bovine serum albumin as the standard. Dot blot analysis was performed using 2.5 μl of spotted fractions and either polyclonal antibody 39 or monoclonal antibodies 69, 75G, and 42E, as described (42Isogai Z. Aspberg A. Keene D.R. Ono R.N. Reinhardt D.P. Sakai L.Y. J. Biol. Chem. 2002; 277: 4565-4572Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Western blot analysis was performed using two combined consecutive fractions. Guanidine hydrochloride was eliminated by ethanol precipitation as described (43Oike Y. Kimata K. Shinomura T. Nakazawa K. Suzuki S. Biochem. J. 1980; 191: 193-207Crossref PubMed Scopus (217) Google Scholar). Microfibrils were also isolated from tissues using crude collagenase (Sigma) digestion. Procedures used were the same as those we have previously detailed (42Isogai Z. Aspberg A. Keene D.R. Ono R.N. Reinhardt D.P. Sakai L.Y. J. Biol. Chem. 2002; 277: 4565-4572Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Light and electron microscopic immunohistochemical procedures were the same as those we have previously described (44Sakai L.Y. Keene D.R. Methods Enzymol. 1994; 245: 29-52Crossref PubMed Scopus (80) Google Scholar). Tissues were frozen in hexanes for light and confocal microscopy. Fluorescein isothiocyanate-conjugated rabbit anti-mouse IgG (Sigma) was used for immunofluorescence microscopy, using 8-μm sections. For confocal microscopy, 25-μm sections were incubated with primary antibodies, followed by Alexa Fluor 488 goat anti-mouse IgG or Alexa Fluor 594 goat anti-rabbit IgG (Molecular Probes, Eugene, OR). Stained sections were viewed with a Leica TCS SP2 confocal microscope and merged images were generated using Leica software. For electron microscopic immunolocalization, fresh tissue blocks were first incubated with dilutions of primary antibody, followed by a gold-conjugated second antibody, and then embedded and prepared for electron microscopy. Interactions between LTBP and fibrillin were investigated by solid phase ELISA binding or blot overlay assays. For ELISA binding assays, multiwell plates were coated with purified LTBP-1 peptides (50 nm, 100 μl/well) in 15 mmNa2CO3 and 35 mmNaHCO3, pH 9.2, at 4 °C overnight. Coated wells were blocked with 5% nonfat dry milk in TBS at room temperature for 1 h. Recombinant fibrillin-1 polypeptides were serially diluted 1:2 in 2% milk, TBS, containing 2 mm CaCl2 or 5 mm EDTA, and incubated in the wells for 3 h. Monoclonal antibodies against soluble ligands were diluted in 2% milk, TBS and used to detect the bound ligands, after a final incubation with enzyme-conjugated secondary antibodies. Color reaction of the enzyme immunoassay was achieved using p-nitrophenyl phosphate (Sigma tablets) or 1 mg/ml 5-aminosalicylic acid. Absorbance was determined at 405 nm using a Titertek Multiskan. For blot overlay assays, serum-free conditioned media was collected from High FiveTM cells that were transfected with recombinant LTBP-1. 1 ml of media was precipitated using trichloroacetic acid, resolved by SDS-PAGE, and transferred to a nitrocellulose membrane. After blocking with 5% nonfat milk in TBS at room temperature for 1 h, the membrane was incubated with recombinant fibrillin peptides (50 μg/ml or 1 μm) in 2% nonfat milk in TBS at 4 °C overnight or room temperature for 3 h. Monoclonal antibodies diluted in 2% milk, TBS were used to detect the bound ligands, after a final incubation with enzyme-conjugated secondary antibodies. The blots were developed by color reaction using 4-chloro-1-naphthol (Bio-Rad). Although immunolocalization studies of LTBP-1 have been performed using cultured cells and tissues, surveys of tissue distribution have not been published. In addition, most published results have relied upon polyclonal antiserum 39. To immunolocalize LTBP-1 with greater confidence and to establish tissue distribution patterns, monoclonal antibodies were generated using purified full-length recombinant human LTBP-1 expressed in insect cells. Two monoclonal antibodies (mAb 75G and mAb 42E), which immunoblotted and immunoprecipitated LTBP-1 produced by insect cells (data not shown), were selected for further characterization. Epitopes for these antibodies were mapped by immunoblotting using four LTBP-1 recombinant polypeptides (Fig. 1 A) expressed in 293 human embryonic kidney cells. MAb 75G recognized an epitope in rL1M, but did not bind to rL1N or rL1C (data not shown). mAb 42E bound to a site close to the C-terminal region in rL1C, not in rL1K (data not shown). In addition, mAb 75G and mAb 42E recognized authentic LTBP-1 present in the medium of cultured human fibroblasts (NSF lane, in Fig. 7). 75G and 42E displayed no reactivity with authentic fibrillin in fibroblast-conditioned medium, nor with rF11 and rF6, the two recombinant halves of fibrillin-1 (data not shown). When tested using a panel of human tissues, mAb 75G and mAb 42E yielded similar immunohistochemical results. In tissues such as tendon, perichondrium, and blood vessels, the staining patterns for LTBP-1 and fibrillin-1 were apparently identical. In tendon and perichondrium, long fluorescent fibrils were evident a
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