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• W2062173898 abstract "Recombinant human βig-h3 was found to bind 125I-labeled small leucine-rich proteoglycans (SLRPs), biglycan, and decorin, in co-immunoprecipitation experiments. In each instance the binding could be blocked by an excess of the unlabeled proteoglycan, confirming the specificity of the interaction. Scatchard analysis showed that biglycan bound βig-h3 more avidly than decorin with Kd values estimated as 5.88 × 10–8 and 1.02 × 10–7 m, respectively. In reciprocal blocking experiments both proteoglycans inhibited the others binding to βig-h3 indicating that they may share the same binding site or that the two binding sites are in close proximity on the βig-h3 molecule. Since βig-h3 and the SLRPs are known to be associated with the amino-terminal region of collagen VI in tissue microfibrils, the effects of including collagen VI in the incubations were investigated. Co-immunoprecipitation of 125I-labeled biglycan incubated with equimolar mixtures of βig-h3 and pepsin-collagen VI was increased 6-fold over βig-h3 alone and 3-fold over collagen VI alone. Similar increases were also observed for decorin. The findings indicate that βig-h3 participates in a ternary complex with collagen VI and SLRPs. Static light scattering techniques were used to show that βig-h3 rapidly forms very high molecular weight complexes with both native and pepsin-collagen VI, either alone or with the SLRPs. Indeed βig-h3 was shown to form a complex with collagen VI and biglycan, which appeared to be much more extensive than that formed by βig-h3 with collagen VI and decorin or those formed between the collagen and βig-h3, biglycan, or decorin alone. Biglycan core protein was shown to inhibit the extent of complexing of βig-h3 with native and pepsin-collagen VI suggesting that the glycosaminoglycan side chains of the proteoglycan were important for the formation of the large ternary complexes. Further studies showed that the direct interaction between βig-h3 and biglycan and between biglycan and collagen VI were also important for the formation of these complexes. The globular domains of collagen VI also appeared to have an influence on the interaction of the three components. Overall the results indicate that βig-h3 can differentially modulate the aggregation of collagen VI with biglycan and decorin. Thus this interplay is likely to be important in tissues such as cornea where such complexes are considered to occur. Recombinant human βig-h3 was found to bind 125I-labeled small leucine-rich proteoglycans (SLRPs), biglycan, and decorin, in co-immunoprecipitation experiments. In each instance the binding could be blocked by an excess of the unlabeled proteoglycan, confirming the specificity of the interaction. Scatchard analysis showed that biglycan bound βig-h3 more avidly than decorin with Kd values estimated as 5.88 × 10–8 and 1.02 × 10–7 m, respectively. In reciprocal blocking experiments both proteoglycans inhibited the others binding to βig-h3 indicating that they may share the same binding site or that the two binding sites are in close proximity on the βig-h3 molecule. Since βig-h3 and the SLRPs are known to be associated with the amino-terminal region of collagen VI in tissue microfibrils, the effects of including collagen VI in the incubations were investigated. Co-immunoprecipitation of 125I-labeled biglycan incubated with equimolar mixtures of βig-h3 and pepsin-collagen VI was increased 6-fold over βig-h3 alone and 3-fold over collagen VI alone. Similar increases were also observed for decorin. The findings indicate that βig-h3 participates in a ternary complex with collagen VI and SLRPs. Static light scattering techniques were used to show that βig-h3 rapidly forms very high molecular weight complexes with both native and pepsin-collagen VI, either alone or with the SLRPs. Indeed βig-h3 was shown to form a complex with collagen VI and biglycan, which appeared to be much more extensive than that formed by βig-h3 with collagen VI and decorin or those formed between the collagen and βig-h3, biglycan, or decorin alone. Biglycan core protein was shown to inhibit the extent of complexing of βig-h3 with native and pepsin-collagen VI suggesting that the glycosaminoglycan side chains of the proteoglycan were important for the formation of the large ternary complexes. Further studies showed that the direct interaction between βig-h3 and biglycan and between biglycan and collagen VI were also important for the formation of these complexes. The globular domains of collagen VI also appeared to have an influence on the interaction of the three components. Overall the results indicate that βig-h3 can differentially modulate the aggregation of collagen VI with biglycan and decorin. Thus this interplay is likely to be important in tissues such as cornea where such complexes are considered to occur. Transforming growth factor-β (TGF-β) 2The abbreviations used are: TGF-β, transforming growth factor β; βig-h3, transforming growth factor β-inducible gene-h3; BSA, bovine serum albumin; GAG, glycosaminoglycan; rβig-h3, recombinant βig-h3; SLRP, small leucine-rich repeat proteoglycan; TBS, Tris-buffered saline. -inducible gene-h3 (βig-h3) (also known variously as MP78/70 (1Gibson M.A. Kumaratilake J.S. Cleary E.G. J. Biol. Chem. 1989; 264: 4590-4598Abstract Full Text PDF PubMed Google Scholar, 2Gibson M.A. Hatzinikolas G. Kumaratilake J.S. Sandberg L.B. Nichol J.K. Sutherland G.R. Cleary E.G. J. Biol. Chem. 1996; 271: 1096-1103Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar), RGD-CAP (3Hashimoto K. Noshiro M. Ohno S. Kawamoto T. Satakeda H. Akagawa Y. Nakashima K. Okimura A. Ishida H. Okamoto T. Pan H. Shen M. Yan W. Kato Y. Biochim. Biophys. Acta. 1997; 1355: 303-314Crossref PubMed Scopus (150) Google Scholar), and keratoepithelin (4Munier F.L. Korvatska E. Djemai A. Le Paslier D. Zografos L. Pescia G. Schorderet D.F. Nat. Genet. 1997; 15: 247-251Crossref PubMed Scopus (530) Google Scholar)), is an extracellular matrix protein expressed in a wide variety of tissues including developing nuchal ligament, aorta, lung, kidney, and cartilage; and mature cornea, skin, bladder, and bone (5Gibson M.A. Kumaratilake J.S. Cleary E.G. J. Histochem. Cytochem. 1997; 45: 1683-1696Crossref PubMed Scopus (70) Google Scholar, 6Escribano J. Hernando N. Ghosh S. Crabb J. Coca-Prados M. J. Cell. Physiol. 1994; 160: 511-521Crossref PubMed Scopus (153) Google Scholar, 7LeBaron R.G. Bezverkov K.I. Zimber M.P. Pavelec R. Skonier J. Purchio A.F. J. Investig. Dermatol. 1995; 104: 844-849Abstract Full Text PDF PubMed Scopus (198) Google Scholar, 8Hirano K. Klintworth G.K. Zhan Q. Bennett K. Cintron C. Curr. Eye Res. 1996; 15: 965-972Crossref PubMed Scopus (59) Google Scholar, 9Billings P.C. Herrick D.J. Kucich U. Engelsberg B.N. Abrams W.R. Macarak E.J. Rosenbloom J. Howard P.S. J. Cell. Biochem. 2000; 79: 261-273Crossref PubMed Scopus (43) Google Scholar, 10Kitahama S. Gibson M.A. Hatzinikolas G. Hay S. Kuliwaba J.L. Evdokiou A. Atkins G.J. Findlay D.M. Bone. 2000; 27: 61-67Crossref PubMed Scopus (54) Google Scholar, 11Ferguson J.W. Mikesh M.F. Wheeler E.F. LeBaron R.G. Mech. Dev. 2003; 120: 851-856Crossref PubMed Scopus (58) Google Scholar). The nameβig-h3 stems from its identification and cloning as a major TGF-β-responsive gene in A549 lung adenocarcinoma cells (12Skonier J. Neubauer M. Madisen L. Bennett K. Plowman G.D. Purchio A.F. DNA Cell Biol. 1992; 11: 511-522Crossref PubMed Scopus (505) Google Scholar, 13Skonier J. Bennet K. Rothwell V. Kosowski S. Plowman G.D. Wallace P. Edelhoff S. Disteche C. Neubauer M. Marquardt H. Rodgers J. Purchio A.F. DNA Cell Biol. 1994; 13: 571-584Crossref PubMed Scopus (260) Google Scholar). βig-h3 protein is 76–78 kDa in size and contains four repeat domains, with homology to the insect protein fasciclin, and 11 cysteine residues most of which are clustered in a distinct amino-terminal region. The βig-h3 molecule appears to undergo partial processing at the carboxyl-terminal end to yield a 68–70-kDa isoform (13Skonier J. Bennet K. Rothwell V. Kosowski S. Plowman G.D. Wallace P. Edelhoff S. Disteche C. Neubauer M. Marquardt H. Rodgers J. Purchio A.F. DNA Cell Biol. 1994; 13: 571-584Crossref PubMed Scopus (260) Google Scholar). βig-h3 has been shown to bind in vitro to a number of other matrix components including fibronectin, laminin, and several collagen types (14Billings P.C. Whitbeck J.C. Adams C.S. Abrams W.R. Cohen A.J. Engelsberg B.N. Howard P.S. Rosenbloom J. J. Biol. Chem. 2002; 277: 28003-28009Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 15Kim J.E. Park R.W. Choi J.Y. Bae Y.C. Kim K.S. Joo C.K. Kim I.S. Investig. Ophthalmol. Vis. Sci. 2002; 43: 656-661PubMed Google Scholar). In addition, βig-h3 has multiple cell-adhesion motifs within the fasciclin-like domains that can mediate interactions with a variety of cell types via integrins α3β1 (16Kim J.E. Kim S.J. Lee B.H. Park R.W. Kim K.S. Kim I.S. J. Biol. Chem. 2000; 275: 30907-30915Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, 17Bae J.S. Lee S.H. Kim J.E. Choi J.Y. Park R.W. Park J-W. Park H.S. Sohn Y.S. Lee D.S. Lee E.B. Kim I.S. Biochem. Biophys. Res. Commun. 2002; 294: 940-948Crossref PubMed Scopus (132) Google Scholar), α1β1 (18Ohno S. Noshiro M. Makihira S. Kawamoto T. Shen M. Yan W. Kawashima-Ohya Y. Fujimoto K. Tanne K. Kato Y. Biochim. Biophys. Acta. 1999; 1451: 196-205Crossref PubMed Scopus (101) Google Scholar), or αVβ5 (19Kim J.E. Jeong H.W. Nam J.O. Lee B.H. Choi J.Y. Park R.W. Park J.Y. Kim I.S. J. Biol. Chem. 2002; 277: 46159-46165Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). The precise functions of βig-h3 are unknown but it has been proposed that it may act as a cell adhesion molecule (19Kim J.E. Jeong H.W. Nam J.O. Lee B.H. Choi J.Y. Park R.W. Park J.Y. Kim I.S. J. Biol. Chem. 2002; 277: 46159-46165Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar) and as a multifunctional linker protein interconnecting different matrix molecules to each other and to cells (5Gibson M.A. Kumaratilake J.S. Cleary E.G. J. Histochem. Cytochem. 1997; 45: 1683-1696Crossref PubMed Scopus (70) Google Scholar, 9Billings P.C. Herrick D.J. Kucich U. Engelsberg B.N. Abrams W.R. Macarak E.J. Rosenbloom J. Howard P.S. J. Cell. Biochem. 2000; 79: 261-273Crossref PubMed Scopus (43) Google Scholar). Recent evidence suggests that βig-h3 may be particularly important for skeletal muscle cell adhesion at the myotendinous junction (18Ohno S. Noshiro M. Makihira S. Kawamoto T. Shen M. Yan W. Kawashima-Ohya Y. Fujimoto K. Tanne K. Kato Y. Biochim. Biophys. Acta. 1999; 1451: 196-205Crossref PubMed Scopus (101) Google Scholar), and for the induction of keratinocyte differentiation (20Oh J.E. Kook J.K. Min B.M. J. Biol. Chem. 2005; 280: 21629-21637Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). The protein also appears to be involved in endothelial cell-matrix interactions during vascular remodeling and angiogenesis (21Aitkenhead M. Wang S.J. Nakatsu M.N. Mestas J. Heard C. Hughes C.C. Microvasc. Res. 2002; 63: 159-171Crossref PubMed Scopus (126) Google Scholar) and as a negative regulator of mineralization during cartilage differentiation and osteogenesis (22Ohno S. Doi T. Tsutsumi S. Okada Y. Yoneno K. Kato Y. Tanne K. Biochim. Biophys. Acta. 2002; 1572: 114-122Crossref PubMed Scopus (33) Google Scholar, 23Kim J.E. Kim E.H. Han E.H. Park R.W. Park I.H. Jun S.H. Kim J.C. Young M.F. Kim I.S. J. Cell. Biochem. 2000; 77: 169-178Crossref PubMed Scopus (110) Google Scholar, 24Thapa N. Kang K.B. Kim I.S. Bone. 2005; 36: 232-242Crossref PubMed Scopus (66) Google Scholar). Mutations in the human βig-h3 gene (TGFBI) have been linked to several autosomal dominant corneal dystrophies (4Munier F.L. Korvatska E. Djemai A. Le Paslier D. Zografos L. Pescia G. Schorderet D.F. Nat. Genet. 1997; 15: 247-251Crossref PubMed Scopus (530) Google Scholar) characterized by severe visual impairment resulting from the progressive accumulation of βig-h3-containing protein deposits in the corneal matrix (25Streeten B.W. Qi Y. Klintworth G.K. Eagle Jr., R.C. Strauss J.A. Bennett K. Arch. Ophthalmol. 1999; 117: 67-75Crossref PubMed Scopus (102) Google Scholar, 26Clout N.J. Hohenester E. Mol. Vis. 2003; 9: 440-448PubMed Google Scholar). To elucidate the function of βig-h3 within the extracellular matrix, studies in our laboratory have focused on the localization, molecular forms, and matrix interactions of βig-h3 within various tissues. Ultrastructural localization studies on developing tissues showed that, in most instances, βig-h3 was loosely associated with collagen fibers although, in developing kidney, labeling was also observed close to the tubular and capsular basement membranes. Double immunolabeling experiments with antibodies to βig-h3 and collagen VI indicated that much of the βig-h3 was associated with collagen VI microfibrils rather than the collagen fibers themselves (5Gibson M.A. Kumaratilake J.S. Cleary E.G. J. Histochem. Cytochem. 1997; 45: 1683-1696Crossref PubMed Scopus (70) Google Scholar). The collagen VI microfibrils, 3–10 nm in diameter, exhibit a characteristic, double-beaded period of about 100 nm (27Timpl R. Chu M-L. Extracellular Matrix Assembly and Structure.in: Yurchenko P.D. Birk D. Mecham R.P. Academic Press, New York1994: 207-242Crossref Google Scholar). In some tissues collagen VI appears to form additional structures including thicker cross-banded fibrils and hexagonal networks (28Bruns R.R. Press W. Engvall E. Timpl R. Gross J. J. Cell Biol. 1986; 103: 393-404Crossref PubMed Scopus (215) Google Scholar, 29Reale E. Groos S. Luciano L. Eckardt C. Eckardt U. Matrix Biol. 2001; 20: 37-51Crossref PubMed Scopus (18) Google Scholar). The precise functions of collagen VI are unclear but the protein is considered to be important for tissue architecture, interlinking structural components of the matrix, and for cell-matrix interactions (27Timpl R. Chu M-L. Extracellular Matrix Assembly and Structure.in: Yurchenko P.D. Birk D. Mecham R.P. Academic Press, New York1994: 207-242Crossref Google Scholar). Mutations in collagen VI genes (COL6A1, COL6A2, and COL6A3) have recently been linked to the muscle wasting diseases, Bethlem myopathy and Ullrich dystrophy (30Jobsis G.J. Keizers H. Vreijling J.P. de Visser M. Speer M.C. Wolterman R.A. Baas F. Bolhuis P.A. Nat. Genet. 1996; 14: 113-115Crossref PubMed Scopus (219) Google Scholar, 31Lampe A.K. Bushby K.M. J. Med. Genet. 2005; 42: 673-685Crossref PubMed Scopus (313) Google Scholar). To define the relationship of βig-h3 with collagen VI we have isolated collagen VI microfibrils from collagenase-treated nuchal ligament and demonstrated that βig-h3 is covalently attached to collagen VI at regular intervals along at least some of the microfibrils (32Hanssen E. Reinboth B. Gibson M.A. J. Biol. Chem. 2003; 278: 24334-24341Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). The binding site is located close to the amino-terminal end of the collagen VI molecule. Additional binding assays have demonstrated that βig-h3 binds in vitro to collagen VI, but in a non-covalent manner (32Hanssen E. Reinboth B. Gibson M.A. J. Biol. Chem. 2003; 278: 24334-24341Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). The βig-h3 attachment site on collagen VI appears to be close to those documented for the small leucine-rich proteoglycans (SLRP), decorin and biglycan, and matrilins (33Wiberg C. Hedbom E. Khairullina A. Lamande S.R. Oldberg A. Timpl R. Morgelin M. Heinegard D. J. Biol. Chem. 2001; 276: 18947-18952Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 34Wiberg C. Heinegard D. Wenglen C. Timpl R. Morgelin M. J. Biol. Chem. 2002; 277: 49120-49126Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 35Wiberg C. Klatt A.R. Wagener R. Paulsson M. Bateman J.F. Heinegard D. Morgelin M. J. Biol. Chem. 2003; 278: 37698-37704Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). SLRPs, in particular biglycan, have been shown to influence the aggregation and organization of collagen VI into networks in vitro, mimicking those found in some tissues, particularly cornea (28Bruns R.R. Press W. Engvall E. Timpl R. Gross J. J. Cell Biol. 1986; 103: 393-404Crossref PubMed Scopus (215) Google Scholar, 34Wiberg C. Heinegard D. Wenglen C. Timpl R. Morgelin M. J. Biol. Chem. 2002; 277: 49120-49126Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). In the present study we investigated the influence of rβig-h3 on collagen VI aggregation and its interactions with decorin and biglycan in vitro. We have shown that rβig-h3 directly binds to biglycan and decorin and in turn forms ternary complexes with collagen VI and these SLRPs. Furthermore, rβig-h3 promotes the rapid aggregation of collagen VI tetramers into very large assemblies in vitro and differentially influences the aggregation of the collagen with the two SLRPs. The findings indicate that βig-h3 is likely to be involved in the modulation of collagen VI-proteoglycan interactions during development of a range of tissues including cornea. It is possible that disruption of the normal interactions of βig-h3 with collagen VI and/or SLRPs may be important for the development of βig-h3-linked corneal dystrophies. Materials—Biglycan and decorin were purified from the nuchal ligaments of 230-day-old fetal calves as described previously (36Reinboth B. Hanssen E. Cleary E.G. Gibson M.A. J. Biol. Chem. 2002; 277: 3950-3957Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). Human rβig-h3 and pepsin-treated collagen VI were prepared and purified as described previously (32Hanssen E. Reinboth B. Gibson M.A. J. Biol. Chem. 2003; 278: 24334-24341Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 37Gibson M.A. Cleary E.G. J. Biol. Chem. 1985; 260: 11149-11159Abstract Full Text PDF PubMed Google Scholar). Purified rβig-h3 was stored at 4 °C in 50 mm Tris buffer, pH 7.4, containing 500 mm NaCl to prevent self-aggregation prior to aggregation experiments. Native collagen VI was purified from fetal bovine skeletal muscle. The frozen tissue was crushed and extracted at 4 °C for 18 h in 5 volumes of TBS, pH 7.4, containing proteinase inhibitors and 0.1% Nonidet P-40. The non-solubilized material was then extracted extensively over 48 h with 0.6 m KCl, containing inhibitors (5 × 10 volumes) to solubilize cytoskeletal proteins. The residue was rinsed with collagenase buffer (32Hanssen E. Reinboth B. Gibson M.A. J. Biol. Chem. 2003; 278: 24334-24341Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) and digested sequentially with highly purified bacterial collagenase (7500 units/ml) at 37 °C for 24 h and then hyaluronidase (200 units/ml) for a further 24 h. The supernatant was subjected at 4 °C to CsCl equilibrium density gradient centrifugation at 30,000 rpm (g = 193,000), with initial density of 1.325 g/ml, for 72 h in a fixed angle head (70.1 Ti) in a Beckman L7-55 ultracentrifuge. The fractions containing collagen VI microfibrils were pooled and purified further on a second CsCl gradient with initial density of 1.27 g/ml. Fractions containing collagen VI microfibrils were dialyzed into TBS, pH 7.4, containing 400 mm NaCl, and final purification was performed by fast protein liquid chromatography on a Superose 6 column (1.5 × 25 cm). Proteinase inhibitors were included throughout the purification process. Purified native and pepsin-collagen VI were depolymerized by dialysis into 100 mm sodium citrate buffer, pH 4.0 (34Wiberg C. Heinegard D. Wenglen C. Timpl R. Morgelin M. J. Biol. Chem. 2002; 277: 49120-49126Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Any remaining aggregates were removed from the tetramers by centrifugation at 15,000 × g for 10 min. Specific polyclonal rabbit antibodies to βig-h3 and type VI collagen have been described previously (32Hanssen E. Reinboth B. Gibson M.A. J. Biol. Chem. 2003; 278: 24334-24341Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 37Gibson M.A. Cleary E.G. J. Biol. Chem. 1985; 260: 11149-11159Abstract Full Text PDF PubMed Google Scholar). Anti-His5 antibody was purchased from Qiagen. Chondroitinase ABC was purchased from Seikagaku Corp. (Tokyo, Japan). Iodination of Biglycan and Decorin—Biglycan and decorin (100 μg) were radiolabeled with Na125I (Amersham Biosciences) using IODO-BEADS (Pierce). Each proteoglycan was reacted with an IODO-BEAD and 0.75 mCi of 125I for 5 min in 50 mm Tris buffer, pH 7.4, containing 0.3 m guanidinium chloride. Bound and free radiolabels were separated by gel filtration through Sephadex G-10. The specific activities of biglycan and decorin were both 3.4 × 106 dpm/μg of core protein unless stated otherwise. Core proteins of biglycan and decorin were prepared by digestion of the 125I-labeled proteoglycans with chondroitinase ABC as described previously (36Reinboth B. Hanssen E. Cleary E.G. Gibson M.A. J. Biol. Chem. 2002; 277: 3950-3957Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). Co-immunoprecipitation Assays—Co-immunoprecipitation assays were performed as described previously (35Wiberg C. Klatt A.R. Wagener R. Paulsson M. Bateman J.F. Heinegard D. Morgelin M. J. Biol. Chem. 2003; 278: 37698-37704Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). Briefly, the test protein (1 μg of rβig-h3 or 2 μg of collagen VI) was incubated with 7 × 104 dpm of 125I-labeled biglycan or decorin for 4 h at 37°C in 50 μl of TBS (10 mm Tris buffer, pH 7.4, containing 0.15 m NaCl and protease inhibitors, EDTA (2 mm), benzamidine (1 mm), ϵ-amino-n-caproic acid (1 mm), and phenylmethylsulfonyl fluoride (0.5 mm)) containing 2 μg of BSA. Control incubations contained no test protein. Specific rabbit polyclonal antiserum (10 μl) raised to the test protein was then added and incubation was continued for 18 h at 4 °C with gentle shaking. To ensure full recovery of the immunoprecipitate, protein A-Sepharose (30 μl) was added and incubation was continued for 1 h at room temperature with gentle shaking. The immunoprecipitate-protein A-Sepharose complex was recovered by centrifugation (3,000 × g for 10 min) and resuspended in 100 μl of TBS containing 0.05% Tween 20. The complex was washed by centrifugation three times through 200 μl of 1 m sucrose in TBS/Tween. The bound 125I-proteoglycan was eluted from the complex by resuspension in non-reduced electrophoresis buffer (30 μl) and heating to 100 °C for 2 min. Each sample was centrifuged to remove the Sepharose beads and then β-mercaptoethanol was added to a final concentration of 2%. The samples were reheated for 2 min to reduce disulfide bonds and then analyzed for 125I-labeled proteoglycan content by γ counting and SDS-PAGE/autoradiography as described previously (36Reinboth B. Hanssen E. Cleary E.G. Gibson M.A. J. Biol. Chem. 2002; 277: 3950-3957Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). Each co-immunoprecipitation was performed in triplicate. The protocol was slightly modified for estimation of Kd values, see Fig. 2 for details. Static Light Scattering Analysis of Collagen VI Aggregation—An argon laser (Lexel model 95, Lexel Laser Inc.) was operated at a wavelength of 488 nm with an output power of 300 milliwatts. Light was focused into a sample cell containing a solution of the test macromolecules. The sample cell was maintained at 15 °C by a temperature controller (Endocal model RTE-5DD, Neslab Instruments). The light scattered by the sample was detected at a scattering angle of 90°. Scattered light was detected by the single photon-counting photomultiplier tube (model EMI 9863B/350) with an aperture setting of 800 μm. The output pulses from the photomultiplier were passed through an amplifier discriminator to a digital correlator (model BI-2030AT, Brookhaven Instruments Inc.). The correlator was set to run continuously with a sample time of 1 μs and displayed the average count rate that was recorded every 30 s. Each experiment was commenced by mixing native or pepsin-collagen VI tetramers (120 nm) with rβig-h3 (120 nm) and/or a proteoglycan (biglycan or decorin (480 nm)) in TBS, directly in the cell. Analysis of Collagen VI-containing Aggregates by SDS-PAGE—Pepsin-treated and native collagen VI samples were incubated in TBS containing ovalbumin (120 nm) at 4 °C for 4 h with rβig-h3 only or with rβig-h3 plus biglycan or decorin at the concentrations indicated above. The samples were centrifuged at 18,000 × g for 20 min and the supernatants were removed. The pellets were rinsed with TBS and dissolved in electrophoresis buffer. The proteins in the supernatants were recovered by acetone precipitation and dissolved in electrophoresis buffer using previously described methods (37Gibson M.A. Cleary E.G. J. Biol. Chem. 1985; 260: 11149-11159Abstract Full Text PDF PubMed Google Scholar). The precipitated and non-precipitated protein fractions were analyzed by SDS-PAGE on 8% gels and Coomassie Blue staining. βig-h3 Binds to Biglycan and Decorin via Their Core Proteins—Co-immunoprecipitation experiments were performed using 125I-labeled biglycan and decorin to determine whether βig-h3 directly binds one or both of these proteoglycans. Both biglycan and decorin were found to specifically co-precipitate with rβig-h3 indicating molecular interactions with the protein (Fig. 1). When the binding experiments were repeated with isolated core proteins from the two proteoglycans, each core protein co-precipitated showing that the βig-h3 binding sites were contained within the core proteins and not in the GAG side chains. Gel and autoradiographic analysis of the co-precipitates confirmed that the precipitated radioactivity was associated with intact proteoglycans or, where appropriate, the isolated cores. Because the specific activities of the two proteoglycans and their cores were matched, comparison between the relative extent of binding could be made. Biglycan was precipitated to a greater extent than decorin suggesting that the former may have a higher affinity for βig-h3. In contrast, the two core proteins were precipitated to an equal extent suggesting that their affinities for βig-h3 were of similar magnitude. Because both cores were precipitated to a greater extent than the intact proteoglycans the results suggest that the interactions were inhibited to some degree by the GAG side chains. However, this could not explain the differences in the extent of βig-h3 binding between the intact proteoglycans, because biglycan has a greater glycosaminoglycan content with two GAG side chains compared with one in decorin. Further immunoprecipitation experiments were performed over a range of proteoglycan concentrations that allowed Kd values to be estimated for the interactions of βig-h3 with decorin and biglycan (Fig. 2). Scatchard analyses estimated the Kd values for βig-h3 binding to decorin and biglycan as 1.02 × 10–7 and 5.88 × 10–8 m, respectively. This result confirms that biglycan binds βig-h3 more strongly than decorin. Blocking experiments were performed to confirm the specificity of the interactions and to determine whether the two proteoglycans compete for binding to βig-h3 (Fig. 3, A and B). Preincubation of rβig-h3 with excess unlabeled biglycan completely blocked the subsequent binding of radiolabeled biglycan and decorin. Similarly unlabeled decorin blocked the binding of radiolabeled decorin and biglycan to the rβig-h3. The result indicates that biglycan and decorin either share the same binding site on βig-h3 or that the sites are in close proximity such that the binding of one proteoglycan prevents the subsequent binding of the other. The binding activities were also completely blocked by both biglycan and decorin core proteins, indicating that the GAG side chains of the proteoglycans did not contribute measurably to the interaction of the intact proteoglycans with βig-h3 (Fig. 3, C and D). βig-h3 Enhances the Binding of Biglycan and Decorin to Collagen VI—The co-immunoprecipitation experiments were extended to include collagen VI in its subunit tetrameric form (27Timpl R. Chu M-L. Extracellular Matrix Assembly and Structure.in: Yurchenko P.D. Birk D. Mecham R.P. Academic Press, New York1994: 207-242Crossref Google Scholar). The results are shown in Fig. 4. The co-immunoprecipitation of radiolabeled biglycan in the presence of equimolar mixtures of rβig-h3 and collagen VI was increased 6-fold over rβig-h3 alone (Fig. 4A) and 3-fold over collagen VI alone (Fig. 4B). Similarly co-immunoprecipitation of decorin with the rβig-h3/collagen VI mixture was increased 5-fold over rβig-h3 alone (Fig. 4C) and 3-fold over collagen VI alone (Fig. 4D). The amount of each proteoglycan precipitated with the rβig-h3/collagen VI mixture was similar when either anti-βig-h3 antibody and or anti-collagen VI antibody was used. Thus it appears that βig-h3 enhances the complexing of both proteoglycans to collagen VI and that it participates in a ternary complex in which it interacts directly with both the collagen VI and proteoglycan molecules. βig-h3 Rapidly Complexes with Collagen VI Tetramers in Vitro to Form Very Large Aggregates—Since βig-h3 was found to enhance the interaction of collagen VI with biglycan and decorin, its effect on collagen VI aggregation was investigated in the absence and presence of each proteoglycan. The process of collagen VI aggregation was measured by static light scattering experiments. The direct effect of βig-h3 on aggregation of collagen VI tetrameric subunits is shown in Fig. 5. βig-h3 was shown to cause the rapid aggregation of the pepsin-collagen VI tetramers into very high molecular weight complexes (Fig. 5A). As measured by the light scattering, the process appeared to be ess" @default.
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• W2062173898 title "βig-h3 Interacts Directly with Biglycan and Decorin, Promotes Collagen VI Aggregation, and Participates in Ternary Complexing with These Macromolecules" @default.
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