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• W1996020433 abstract "The interactions of type VI collagen have been investigated, using solid phase binding assays, with two components of the fibrillin-containing microfibrils, the elastin-binding protein, MAGP-1 and its structural relative MAGP-2. Both native and pepsin-treated forms of type VI collagen specifically bound to MAGP-1 but not to MAGP-2. Pepsin type VI collagen was shown to block the binding of MAGP-1 to native type VI collagen indicating that the major MAGP-1-binding site was in the triple-helical region of the molecule. MAGP-1 was found not to bind to collagens I, III, and V. Affinity blotting of pepsin-treated type VI collagen showed that MAGP-1 binding was specific for the collagenous domain of the α3(VI) chain. Decorin and biglycan were found not to inhibit the interaction of pepsin-treated type VI collagen with MAGP-1, indicating that its binding site on the collagen is not close to that for the proteoglycans. Reduction and alkylation of disulfide bonds in MAGP-1 did not destroy its type VI collagen-binding properties, indicating that the binding site was likely to be in the cysteine-free, N-terminal domain of MAGP-1. Interestingly, the interaction of MAGP-1 with type VI collagen was inhibited by tropoelastin, suggesting that the binding sites for tropoelastin and type VI collagen may be in the same domain of MAGP-1. A peptide, corresponding to amino acids 29–38 of MAGP-1, was found to inhibit the interactions of MAGP-1 with type VI collagen and tropoelastin. The results suggest that the peptide may contain the binding sequences for both type VI collagen and tropoelastin, and thus that these two proteins may share the same binding site on MAGP-1. The interactions of MAGP-1 with type VI collagen and tropoelastin were both determined to be of moderately high affinity, withK d values of 5.6 × 10−7m and 2.6 × 10−7m, respectively. The findings indicate that MAGP-1 may mediate a molecular interaction between type VI collagen microfibrils and fibrillin-containing microfibrils, structures which are often found in close proximity to each other in a wide range of extracellular matrices. The interactions of type VI collagen have been investigated, using solid phase binding assays, with two components of the fibrillin-containing microfibrils, the elastin-binding protein, MAGP-1 and its structural relative MAGP-2. Both native and pepsin-treated forms of type VI collagen specifically bound to MAGP-1 but not to MAGP-2. Pepsin type VI collagen was shown to block the binding of MAGP-1 to native type VI collagen indicating that the major MAGP-1-binding site was in the triple-helical region of the molecule. MAGP-1 was found not to bind to collagens I, III, and V. Affinity blotting of pepsin-treated type VI collagen showed that MAGP-1 binding was specific for the collagenous domain of the α3(VI) chain. Decorin and biglycan were found not to inhibit the interaction of pepsin-treated type VI collagen with MAGP-1, indicating that its binding site on the collagen is not close to that for the proteoglycans. Reduction and alkylation of disulfide bonds in MAGP-1 did not destroy its type VI collagen-binding properties, indicating that the binding site was likely to be in the cysteine-free, N-terminal domain of MAGP-1. Interestingly, the interaction of MAGP-1 with type VI collagen was inhibited by tropoelastin, suggesting that the binding sites for tropoelastin and type VI collagen may be in the same domain of MAGP-1. A peptide, corresponding to amino acids 29–38 of MAGP-1, was found to inhibit the interactions of MAGP-1 with type VI collagen and tropoelastin. The results suggest that the peptide may contain the binding sequences for both type VI collagen and tropoelastin, and thus that these two proteins may share the same binding site on MAGP-1. The interactions of MAGP-1 with type VI collagen and tropoelastin were both determined to be of moderately high affinity, withK d values of 5.6 × 10−7m and 2.6 × 10−7m, respectively. The findings indicate that MAGP-1 may mediate a molecular interaction between type VI collagen microfibrils and fibrillin-containing microfibrils, structures which are often found in close proximity to each other in a wide range of extracellular matrices. Two structurally distinct microfibrillar elements, type VI collagen microfibrils and fibrillin-containing microfibrils, are abundant constituents of the extracellular matrix in a wide range of tissues. Type VI collagen microfibrils (3–5 nm in diameter) are present as an extensive network in virtually all soft connective tissues, where they are found in loose association with collagen fibers and basement membranes, and near the surface of cells (1Gibson M.A. Cleary E.G. Collagen Relat. Res. 1983; 3: 469-488Crossref PubMed Scopus (24) Google Scholar, 2Keene D.R. Engvall E. Glanville R.W. J. Cell Biol. 1988; 107: 1995-2006Crossref PubMed Scopus (321) Google Scholar, 3Maier A. Mayne R. Am. J. Anat. 1987; 180: 226-236Crossref PubMed Scopus (30) Google Scholar, 4Rittig M. Lutjen-Drecoll E. Rauterberg J. Jander R. Mollenhauer J. Cell Tissue Res. 1990; 259: 305-312Crossref PubMed Scopus (38) Google Scholar, 5Zhu D. Kim Y. Steffes M.W. Groppoli T.J. Butkowski R.J. Mauer S.M. J. Histochem. Cytochem. 1994; 42: 577-584Crossref PubMed Scopus (30) Google Scholar, 6Timpl R. Engel J. Mayne R. Burgeson R.E. Structure and Function of Collagen Types. Academic Press, New York1987: 105-143Google Scholar, 7Timpl R. Chu M.-L. Yurchenko P.D. Birk D. Mecham R.P. Extracellular Matrix Assembly and Structure. Academic Press, New York1994: 207-242Crossref Google Scholar). These microfibrils appear to contain only type VI collagen, present as three distinct α chains, assembled into monomers consisting of a short, central triple-helical region and large globular N-terminal and C-terminal domains (8Chu M.-L. Mann K. Deutzmann R. Pribula-Conway D. Hsu-Chen C.-C. Bernard M.P. Timpl R. Eur. J. Biochem. 1987; 168: 309-317Crossref PubMed Scopus (99) Google Scholar, 9Chu M.-L. Conway D. Pan T. Baldwin C. Mann K. Deutzmann R. Timpl R. J. Biol. Chem. 1988; 263: 18601-18606Abstract Full Text PDF PubMed Google Scholar). The monomers form anti-parallel, disulfide-bonded dimers then tetramers by lateral association. The tetramers are arranged end to end to form the microfibril with a periodic interval of 100 nm (7Timpl R. Chu M.-L. Yurchenko P.D. Birk D. Mecham R.P. Extracellular Matrix Assembly and Structure. Academic Press, New York1994: 207-242Crossref Google Scholar, 10Furthmayr H. Wiedemann H. Timpl R. Odermatt E. Engel J. Biochem. J. 1983; 211: 303-311Crossref PubMed Scopus (207) Google Scholar). Fibrillin-containing microfibrils are found in association with elastin in elastic fibers, which are prevalent in tissues such as arteries, lung, skin, and elastic ligaments, and as elastin-free bundles in tissues such as kidney, ocular zonule, and spleen (11Cleary E.G. Gibson M.A. Int. Rev. Connect. Tissue Res. 1983; 10: 97-209Crossref PubMed Google Scholar, 12Cleary E.G. Gibson M.A. Comper W.D. The Structure and Function of Extracellular Matrix. 2. Gordon & Breach Science Publisher, New York1996: 95-140Google Scholar, 13Gibson M.A. Cleary E.G. Immunol. Cell Biol. 1987; 65: 345-356Crossref PubMed Scopus (51) Google Scholar, 14Henderson M. Polewski R. Fanning J.C. Gibson M.A. J. Histochem. Cytochem. 1996; 44: 1389-1397Crossref PubMed Google Scholar, 15Keene D.R. Maddox B.K. Kuo H.-J. Sakai L.Y. Glanville R.W. J. Histochem. Cytochem. 1991; 39: 441-449Crossref PubMed Scopus (182) Google Scholar, 16Kumaratilake J.S. Gibson M.A. Fanning J.C. Cleary E.G. Eur. J. Cell Biol. 1989; 50: 117-127PubMed Google Scholar, 17Sakai L.Y. Keene D.R. Engvall E. J. Cell Biol. 1986; 103: 2499-2509Crossref PubMed Scopus (889) Google Scholar). In contrast to type VI collagen microfibrils, fibrillin-containing microfibrils appear to be complex structures that may contain, or be closely associated with, a number of glycoproteins (17Sakai L.Y. Keene D.R. Engvall E. J. Cell Biol. 1986; 103: 2499-2509Crossref PubMed Scopus (889) Google Scholar, 18Abrams W.R. Ma R.-I. Kucich U. Bashir M.M. Decker S. Tsipouras P. McPherson J.D. Wasmuth J.J. Rosenbloom J. Genomics. 1995; 26: 47-54Crossref PubMed Scopus (42) Google Scholar, 19Corson G.M. Chalberg S.C. Dietz H.C. Charbonneau N.L. Sakai L.Y. Genomics. 1993; 17: 476-484Crossref PubMed Scopus (223) Google Scholar, 20Gibson M.A. Hughes J.L. Fanning J.C. Cleary E.G. J. Biol. Chem. 1986; 261: 11429-11436Abstract Full Text PDF PubMed Google Scholar, 21Gibson M.A. Kumaratilake J.S. Cleary E.G. J. Biol. Chem. 1989; 264: 4590-4598Abstract Full Text PDF PubMed Google Scholar, 22Gibson M.A. Sandberg L.B. Grosso L.E. Cleary E.G. J. Biol. Chem. 1991; 266: 7596-7601Abstract Full Text PDF PubMed Google Scholar, 23Gibson 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, 24Gibson M.A. Hatzinikolas G. Kumaratilake J.S. Sandberg L.B. Nicholl J.K. Sutherland G.R. Cleary E.G. J. Biol. Chem. 1996; 271: 1096-1103Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 25Sakai L.Y. Keene D.R. Glanville R.W. Bachinger H.P. J. Biol. Chem. 1991; 266: 14763-14770Abstract Full Text PDF PubMed Google Scholar, 26Pereira L. D'Alessio M. Ramirez F. Lynch J.R. Sykes B. Pangilinan T. Bonadio J. Hum. Mol. Genet. 1993; 2: 961-968Crossref PubMed Scopus (250) Google Scholar, 27Yeh H. Chow M. Abrams W.R. Fan J. Foster J. Mitchell H. Muenke M. Rosenbloom J. Genomics. 1994; 23: 443-449Crossref PubMed Scopus (28) Google Scholar, 28Zhao Z. Lee C.-C. Jiralerspong S. Juyal R.C. Lu F. Baldini A. Greenberg F. Caskey C.T. Patel P.I. Hum. Mol. Genet. 1995; 4: 589-597Crossref PubMed Scopus (98) Google Scholar, 29Zhang H. Apfelroth S.D. Hu W. Davis E.E. Sanguineti C. Bonadio J. Mecham R.P. Ramirez F. J. Cell Biol. 1994; 124: 855-863Crossref PubMed Scopus (318) Google Scholar, 30Zhang H. Hu W. Ramirez F. J. Cell Biol. 1995; 129: 1165-1176Crossref PubMed Scopus (255) Google Scholar) and a chondroitin sulfate proteoglycan (31Kielty C.M. Whittaker S.P. Shuttleworth C.A. FEBS Lett. 1996; 386: 169-173Crossref PubMed Scopus (52) Google Scholar). Their major structural components are considered to be the fibrillins, which are a two-member family of large (350 kDa), rodlike, cysteine-rich glycoproteins, named fibrillin-1 and fibrillin-2 (17Sakai L.Y. Keene D.R. Engvall E. J. Cell Biol. 1986; 103: 2499-2509Crossref PubMed Scopus (889) Google Scholar, 19Corson G.M. Chalberg S.C. Dietz H.C. Charbonneau N.L. Sakai L.Y. Genomics. 1993; 17: 476-484Crossref PubMed Scopus (223) Google Scholar, 25Sakai L.Y. Keene D.R. Glanville R.W. Bachinger H.P. J. Biol. Chem. 1991; 266: 14763-14770Abstract Full Text PDF PubMed Google Scholar, 26Pereira L. D'Alessio M. Ramirez F. Lynch J.R. Sykes B. Pangilinan T. Bonadio J. Hum. Mol. Genet. 1993; 2: 961-968Crossref PubMed Scopus (250) Google Scholar, 29Zhang H. Apfelroth S.D. Hu W. Davis E.E. Sanguineti C. Bonadio J. Mecham R.P. Ramirez F. J. Cell Biol. 1994; 124: 855-863Crossref PubMed Scopus (318) Google Scholar, 30Zhang H. Hu W. Ramirez F. J. Cell Biol. 1995; 129: 1165-1176Crossref PubMed Scopus (255) Google Scholar, 32Reinhardt D.P. Keene D.R. Corson G.M. Poschl E. Bachinger H.P. Gambee J.E. Sakai L.Y. J. Mol. Biol. 1996; 258: 104-116Crossref PubMed Scopus (205) Google Scholar). Parallel arrays of 6–8 fibrillin molecules appear to aggregate, end to end, to form the microfibrils (25Sakai L.Y. Keene D.R. Glanville R.W. Bachinger H.P. J. Biol. Chem. 1991; 266: 14763-14770Abstract Full Text PDF PubMed Google Scholar, 32Reinhardt D.P. Keene D.R. Corson G.M. Poschl E. Bachinger H.P. Gambee J.E. Sakai L.Y. J. Mol. Biol. 1996; 258: 104-116Crossref PubMed Scopus (205) Google Scholar). It is uncertain whether the two forms of fibrillin form distinct microfibrils or if they can co-exist within the same microfibril. The fibrillin-containing microfibrils have a characteristic beaded-filament structure, with a 50-nm periodicity, when viewed by the rotary shadowing technique (14Henderson M. Polewski R. Fanning J.C. Gibson M.A. J. Histochem. Cytochem. 1996; 44: 1389-1397Crossref PubMed Google Scholar, 15Keene D.R. Maddox B.K. Kuo H.-J. Sakai L.Y. Glanville R.W. J. Histochem. Cytochem. 1991; 39: 441-449Crossref PubMed Scopus (182) Google Scholar, 33Wallace R.N. Streeten B.W. Hanna R.B. Curr. Eye Res. 1991; 10: 99-109Crossref PubMed Scopus (47) Google Scholar). Current evidence suggests that the bundles of fibrillin molecules form the interbead regions, whereas the beads correspond to regions of head to tail interaction between adjacent bundles (25Sakai L.Y. Keene D.R. Glanville R.W. Bachinger H.P. J. Biol. Chem. 1991; 266: 14763-14770Abstract Full Text PDF PubMed Google Scholar, 32Reinhardt D.P. Keene D.R. Corson G.M. Poschl E. Bachinger H.P. Gambee J.E. Sakai L.Y. J. Mol. Biol. 1996; 258: 104-116Crossref PubMed Scopus (205) Google Scholar). Evidence suggests that the beads may contain other components including the small structurally related glycoproteins, MAGP-1 1The abbreviations used are: MAGP, microfibril-associated glycoprotein; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; BSA, bovine serum albumin.1The abbreviations used are: MAGP, microfibril-associated glycoprotein; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; BSA, bovine serum albumin. (31 kDa) and MAGP-2 (25 kDa) (14Henderson M. Polewski R. Fanning J.C. Gibson M.A. J. Histochem. Cytochem. 1996; 44: 1389-1397Crossref PubMed Google Scholar, 24Gibson M.A. Hatzinikolas G. Kumaratilake J.S. Sandberg L.B. Nicholl J.K. Sutherland G.R. Cleary E.G. J. Biol. Chem. 1996; 271: 1096-1103Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). MAGP-1 specifically co-distributes with fibrillin-1 in tissues and has been shown to be localized periodically, at 50-nm intervals, to the beads of the microfibrils (14Henderson M. Polewski R. Fanning J.C. Gibson M.A. J. Histochem. Cytochem. 1996; 44: 1389-1397Crossref PubMed Google Scholar). MAGP-1 is covalently associated with the microfibrils by disulfide bonding, and it may play a role in the stabilization of the head to tail interaction between fibrillin molecules (14Henderson M. Polewski R. Fanning J.C. Gibson M.A. J. Histochem. Cytochem. 1996; 44: 1389-1397Crossref PubMed Google Scholar, 20Gibson M.A. Hughes J.L. Fanning J.C. Cleary E.G. J. Biol. Chem. 1986; 261: 11429-11436Abstract Full Text PDF PubMed Google Scholar, 21Gibson M.A. Kumaratilake J.S. Cleary E.G. J. Biol. Chem. 1989; 264: 4590-4598Abstract Full Text PDF PubMed Google Scholar, 22Gibson M.A. Sandberg L.B. Grosso L.E. Cleary E.G. J. Biol. Chem. 1991; 266: 7596-7601Abstract Full Text PDF PubMed Google Scholar). Immunofluorescence staining experiments indicate that the MAGP-1 molecule is accessible on the surface of the microfibril (13Gibson M.A. Cleary E.G. Immunol. Cell Biol. 1987; 65: 345-356Crossref PubMed Scopus (51) Google Scholar), and in vitro binding assays have shown that MAGP-1 will bind to the elastin precursor, tropoelastin (34Brown-Augsburger P. Broekelmann T. Mecham L. Mercer R. Gibson M.A. Cleary E.G. Abrams W.R. Rosenbloom J. Mecham R.P. J. Biol. Chem. 1994; 269: 28443-28449Abstract Full Text PDF PubMed Google Scholar). These findings suggest that MAGP-1 may participate in the binding and alignment of tropoelastin onto the surface of the microfibrils during elastinogenesis. However, MAGP-1 is also present in microfibrils that do not develop into elastic fibers (16Kumaratilake J.S. Gibson M.A. Fanning J.C. Cleary E.G. Eur. J. Cell Biol. 1989; 50: 117-127PubMed Google Scholar), suggesting that the protein may also be involved in the interaction of the microfibrils with other structural elements of the extracellular matrix. MAGP-2 is also specifically disulfide-bonded to fibrillin-associated microfibrils but has a much more restricted tissue distribution than MAGP-1 (21Gibson M.A. Kumaratilake J.S. Cleary E.G. J. Biol. Chem. 1989; 264: 4590-4598Abstract Full Text PDF PubMed Google Scholar, 24Gibson M.A. Hatzinikolas G. Kumaratilake J.S. Sandberg L.B. Nicholl J.K. Sutherland G.R. Cleary E.G. J. Biol. Chem. 1996; 271: 1096-1103Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). MAGP-2 is associated with microfibrils in tissues, such as nuchal ligament, muscle, spleen, kidney mesangium, and the adventitia of arteries, but is essentially absent from tissues, such as ocular zonule, media of arteries, and the peritubular matrix of kidney. 2M. A. Gibson, J. S. Kumaratilake, and E. G. Cleary, manuscript in preparation.2M. A. Gibson, J. S. Kumaratilake, and E. G. Cleary, manuscript in preparation. It is possible that MAGP-2 is specifically associated with microfibrils containing only fibrillin-2. The close similarity between MAGP-1 and MAGP-2 is confined to a central region of 60 amino acids where there is precise alignment of 7 cysteine residues. The two glycoproteins are very diverse in the remainder of their structures, suggesting that they may have very distinct functions in microfibril biology (24Gibson M.A. Hatzinikolas G. Kumaratilake J.S. Sandberg L.B. Nicholl J.K. Sutherland G.R. Cleary E.G. J. Biol. Chem. 1996; 271: 1096-1103Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Ultrastructurally, fine microfibrils of type VI collagen are often found in close proximity to the thicker fibrillin-containing microfibrils in a wide range of tissues (2Keene D.R. Engvall E. Glanville R.W. J. Cell Biol. 1988; 107: 1995-2006Crossref PubMed Scopus (321) Google Scholar, 16Kumaratilake J.S. Gibson M.A. Fanning J.C. Cleary E.G. Eur. J. Cell Biol. 1989; 50: 117-127PubMed Google Scholar, 35Okada Y. Naka K. Minamoto T. Ueda Y. Oda Y. Nakanishi I. Timpl R. Lab. Invest. 1990; 63: 647-656PubMed Google Scholar, 36Wu X.X. Gordon R.E. Glanville R.W. Kuo H.J. Uson R.R. Rand J.H. Am. J. Pathol. 1996; 149: 283-291PubMed Google Scholar), suggestive of molecular interactions between the two structures. In the present study we have investigated the binding, in vitro, of the microfibrillar proteins MAGP-1 and MAGP-2 with native type VI collagen and the pepsin-resistant fragment of the collagen (pepsin type VI collagen), which corresponds mainly to the triple-helical region. The results indicate that MAGP-1, but not MAGP-2, specifically interacts with type VI collagen. The binding appears to be via a region close to the N terminus of MAGP-1 with a site in or close to the helical region of the α3(VI) chain. The finding provides biochemical evidence that fibrillin-containing microfibrils may indirectly link elastic fibers to collagen fibers via the interaction of MAGP-1 with type VI collagen microfibrils. MAGP-1, MAGP-2, and MP78/70 (βig-h3) were extracted from fetal nuchal ligaments using reductive saline treatment and purified by DEAE-cellulose and gel permeation chromatography as described previously (21Gibson M.A. Kumaratilake J.S. Cleary E.G. J. Biol. Chem. 1989; 264: 4590-4598Abstract Full Text PDF PubMed Google Scholar), except the proteins were not alkylated and buffers contained 25 mm dithiothreitol. Disulfide bonds were reformed by dialysis into 50 mm Tris buffer, pH 8.0, containing 0.3 m guanidinium chloride, 4 mmcysteine, and 2 mm cystine. Collagen types I, III, V, and VI (native and pepsin-treated), were prepared from fetal calf skin or nuchal ligaments as described previously (37Gibson M.A. Cleary E.G. J. Biol. Chem. 1985; 260: 11149-11159Abstract Full Text PDF PubMed Google Scholar). Tropoelastin was purified using the method of Brown et al. (38Brown P.L. Mecham L. Tisdal C. Mecham R.P Biochem. Biophys. Res. Commun. 1992; 186: 549-555Crossref PubMed Scopus (46) Google Scholar), and decorin and biglycan were prepared from the fetal calf by the method of Choiet al. (39Choi H.U. Johnson T.L. Pal S. Tang L.-H. Rosenberg L. Neame P.J. J. Biol. Chem. 1989; 264: 2876-2884Abstract Full Text PDF PubMed Google Scholar). Antibodies to MAGP-1 and MAGP-2 have been described previously (14Henderson M. Polewski R. Fanning J.C. Gibson M.A. J. Histochem. Cytochem. 1996; 44: 1389-1397Crossref PubMed Google Scholar, 24Gibson M.A. Hatzinikolas G. Kumaratilake J.S. Sandberg L.B. Nicholl J.K. Sutherland G.R. Cleary E.G. J. Biol. Chem. 1996; 271: 1096-1103Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). For some experiments MAGP-1 was radiolabeled with Na125I (Amersham, Sydney, Australia) using IODO-BEADs (Pierce). Briefly MAGP-1 (95 μg) was dialyzed into 0.1 m phosphate buffer, pH 7.0, containing 6 murea and reacted with a washed IODO-BEAD and 1 mCi of 125I for 15 min at room temperature. The radiolabeled protein was then dialyzed into 50 mm Tris buffer, pH 8.0, containing 0.3m guanidinium chloride and stored at −20 °C. For some experiments MAGP-1 was equilibrated in Tris buffer, pH 8.0, containing 4 m guanidinium chloride and 25 mmdithiothreitol. A portion was then alkylated with 55 mmiodoacetamide for 30 min in the dark. Alkylated and nonalkylated aliquots of MAGP-1 were then either directly diluted or dialyzed into 50 mm Tris buffer, pH 8.0, containing 0.3 mguanidinium chloride, 4 mm cysteine, and 2 mmcystine. The samples were then diluted further into PBS/milk for the solid phase binding assays (see below). Synthetic MAGP-1 peptides YPDHVQYTHY (peptide 29–38), VIPAPTLEPGTVET (peptide 74–87), and QSVAAACARSCGGC (peptide 170–183), corresponding to amino acids 29–38, 74–87, and 170–183, respectively, of the 183-amino acid sequence of bovine MAGP-1 (22Gibson M.A. Sandberg L.B. Grosso L.E. Cleary E.G. J. Biol. Chem. 1991; 266: 7596-7601Abstract Full Text PDF PubMed Google Scholar), were a kind gift from Dr. Robert Mecham, Washington University, St Louis, MO. Each peptide was dissolved at a concentration of 20 mg/ml in dimethyl formamide and stored at −70 °C under nitrogen. Plastic flat-bottomed microtiter plates (Nunc-Immuno Maxisorb modules) were coated with different collagen types (usually 1 μg in 200 μl of PBS) at 4 °C for 18 h. Control wells were coated with BSA. The wells were then rinsed with PBS, blocked for 30 min with 3% low-fat dried milk in PBS, and washed three times with PBS. MAGP-1 or MAGP-2, either125I-labeled or unlabeled (0–1 μg in 100 μl of PBS, 0.05% milk), was then added to wells in quadruplicate, and incubation was continued for 2 h at 37 °C. The wells were then washed three times with PBS, 0.05% Tween 20. Binding of125I-labeled protein to each well was measured directly by scintillation counting. Dissociation constants for interactions were determined using the method of Bidanset et al. (40Bidanset D.J. Guidry C. Rosenberg L.C. Choi H.U. Timpl R. Hook M. J. Biol. Chem. 1992; 267: 5250-5256Abstract Full Text PDF PubMed Google Scholar). Binding of unlabeled protein was measured using antibody detection with anti-MAGP-1 or anti-MAGP-2 antibodies (diluted 1:500 in PBS/Tween) followed by goat anti-rabbit IgG antibodies conjugated to horse radish peroxidase (1:8000 in PBS/Tween). o-Phenylenediamine was used for color development, which was stopped after 30 min by addition of 0.25 volume of 8 m H2SO4. Color was measured at 490 nm. Controls included omission of protein (MAGP-1 or MAGP-2) from the mobile phase (antibody-only controls). Blocking experiments were also conducted as described in the appropriate figure legends. These included (a) preincubation of MAGP-1 in the liquid phase with pepsin type VI collagen or tropoelastin (3 μg/μg of MAGP-1) for 2 h at 37 °C; (b) incubation of pepsin type VI collagen or BSA-coated wells with decorin, biglycan, MP78/70 or ovalbumin (1 μg/well) for 1 h at 37 °C, prior to the addition of 125I-labeled MAGP-1 in the liquid phase; and (c) addition of synthetic MAGP-1 peptides (25 μg/μg of MAGP-1) to 125I-labeled MAGP-1 in the liquid phase. Duplicate samples (1 μg) of pepsin type VI collagen and MAGP-1 were subjected to SDS-PAGE under reducing conditions on 10% gels and transferred to nylon membranes using previously described methods (21Gibson M.A. Kumaratilake J.S. Cleary E.G. J. Biol. Chem. 1989; 264: 4590-4598Abstract Full Text PDF PubMed Google Scholar, 37Gibson M.A. Cleary E.G. J. Biol. Chem. 1985; 260: 11149-11159Abstract Full Text PDF PubMed Google Scholar). The membranes were blocked with PBS containing 3% milk for 30 min. One membrane was incubated with MAGP-1 (8 μg/ml) in PBS, 0.05% milk for 18 h, the other was incubated without MAGP-1 as a control. The membranes were rinsed three times with PBS/Tween and incubated with anti-MAGP-1 monoclonal antibody 11B (as ascites diluted 1:500 in PBS/Tween containing 3% milk) for 18 h at 4 °C. After rinsing, the membranes were treated for 1 h with goat anti-mouse IgG antibodies conjugated to alkaline phosphatase (Bio-Rad) diluted 1:5000 in PBS, Tween, 3% milk. The membranes were rinsed again, and binding was detected using nitro blue tetrazolium and bromo-chloro-indolylphosphate substrates as described previously (22Gibson M.A. Sandberg L.B. Grosso L.E. Cleary E.G. J. Biol. Chem. 1991; 266: 7596-7601Abstract Full Text PDF PubMed Google Scholar). After photography, the membranes were counterstained for type VI collagen using polyclonal rabbit anti-(pepsin type VI collagen) antiserum, followed by goat anti-rabbit IgG antibody conjugated to horse radish peroxidase (Bio-Rad). Color was developed using 50 mm Tris buffer, pH 7.6, containing imidazole (10 mm), hydrogen peroxide (0.01%), and diaminobenzidine (600 μg/ml). MAGP-1 and MAGP-2 were tested for binding to native type VI collagen using a modified ELISA system (Fig. 1). MAGP-1 showed strong binding that directly correlated with the amount of type VI collagen coated to each well of the microtiter plate. In contrast, MAGP-2 showed no specific interaction with the collagen. To determine if the binding site(s) for MAGP-1 were present in the triple-helical and/or globular domains of type VI collagen, the assay was repeated using pepsin type VI collagen that corresponds to the triple-helical region of the molecule (Fig. 2). Strong binding of MAGP-1 was again observed, indicating that the pepsin-resistant region contained a major binding site. Preincubation of the MAGP-1 with pepsin type VI collagen in the mobile phase greatly reduced the binding to wells coated with pepsin type VI collagen, confirming that the molecular interaction was specific. Interestingly preincubation with pepsin type VI collagen also greatly reduced the binding of MAGP-1 to native type VI collagen. This confirmed that the major MAGP-1-binding site(s) on type VI collagen was in the pepsin-resistant domain.Figure 2MAGP-1 specifically binds to the pepsin-resistant domain of type VI collagen. Native type VI collagen (2 μg/well) and pepsin type VI collagen (0.5 μg/well) were coated onto microtiter plates. The wells were then incubated with MAGP-1 (0.6 μg/well) or with MAGP-1, which had been preabsorbed with pepsin type VI collagen (3 μg/μg of MAGP-1). Binding was measured using the peroxidase ELISA technique and color development at 490 nm. The binding of preabsorbed MAGP-1 is expressed as a percentage of the binding of untreated MAGP-1, measured by absorbance at 490 nm. Mean ± S.D. of quadruplicate determinations is shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Other collagen types were also tested to determine if the binding of MAGP-1 was specific for type VI collagen rather than for collagens in general (Fig. 3). Radiolabeled MAGP-1 was used in these experiments to avoid variations in the nonspecific binding of the anti-MAGP-1 antibodies to wells coated with different collagens. MAGP-1 binding to wells coated with collagen types I, III, or V was very low and was comparable to the levels found in control wells coated with BSA. The binding to type VI collagen was at least 2.5-fold higher than to the other collagen types, indicating that it was specific for this microfibrillar collagen. Separation of the three α chains of pepsin type VI collagen by reduction and alkylation under denaturing conditions did not eliminate the binding of MAGP-1 to the collagen, provided that the collagen was renatured by dialysis before coating onto the microtiter wells (not shown). This finding suggested that the MAGP-1-binding site(s) was not dependent on the presence of the triple-helical conformation and thus that it was likely to be present on one of the three distinct α chains of the collagen. To identify which chain was involved, the three α chains of pepsin type VI collagen were separated by SDS-PAGE under reducing conditions and affinity-blotted with MAGP-1 (Fig. 4). MAGP-1 was found to bind to one band of the pepsin type VI collagen (Fig. 4 A), which was not present in the duplicate blot incubated without MAGP-1 but with anti-MAGP-1 antibodies (Fig. 4 C). This result indicated that the band was identified by MAGP-1 and not directly by the antibody, thus confirming that the binding was specific. Counterstaining with anti-type VI collagen antibodies (Fig.4 B) showed that the band corresponded to the α3 chain of pepsin type VI collagen, indicating that the MAGP-1-binding site was present on this chain. To determine if the MAGP-1-binding site on type VI collagen could be blocked by decorin or biglycan, the MAGP-1-binding assay was repeated with type VI collagen-coated wells that had been preincubated with each of the above proteoglycans (Fig.5). Neither macromolecule was found to reduce the binding of MAGP-1 to type VI collagen, suggesting that the proteoglycans bind to a different region of the collagen. MP78/70 was also tested as a potential inhibitor, without effect. To determine if intact disul" @default.
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• W1996020433 title "Microfibril-associated Glycoprotein-1 (MAGP-1) Binds to the Pepsin-resistant Domain of the α3(VI) Chain of Type VI Collagen" @default.
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