Covalent and Non-covalent Interactions of βig-h3 with Collagen VI
2003; Elsevier BV; Volume: 278; Issue: 27 Linguagem: Inglês
10.1074/jbc.m303455200
ISSN1083-351X
AutoresEric Hanssen, Betty Reinboth, Mark Gibson,
Tópico(s)Cell Adhesion Molecules Research
ResumoTransforming growth factor-β induced gene-h3 (βigh3) was found to co-purify with collagen VI microfibrils, extracted from developing fetal ligament, after equilibrium density gradient centrifugation under both nondenaturing and denaturing conditions. Analysis of the collagen VI fraction from the non-denaturing gradient by gel electrophoresis under non-reducing conditions revealed the present of a single high molecular weight band that immunostained for both collagen VI and βigh3. When the fraction was analyzed under reducing conditions, collagen VI α chains and βig-h3 were the only species evident. The results indicated that βig-h3 is associated with collagen VI in tissues by reducible covalent bonding, presumably disulfide bridges. Rotary shadowing and immunogold staining of the collagen VI microfibrils and isolated tetramers indicated that βigh3 was specifically and periodically associated with the double-beaded region of many of the microfibrils and that this covalent binding site was located in or near the amino-terminal globular domain of the collagen VI molecule. Using solid phase and co-immunoprecipitation assays, recombinant βig-h3 was found to bind both native and pepsin-treated collagen VI but not individual pepsin-collagen VI α chains. Blocking experiments indicated that the major in vitro βig-h3 binding site was located in the pepsin-resistant region of collagen VI. In contrast to the tissue situation, the in vitro interaction had the characteristics of a reversible non-covalent interaction, and the Kd was measured as 1.63 × 10–8m. Rotary shadowing of immunogold-labeled complexes of recombinant βig-h3 and pepsin-collagen VI indicated that the in vitro βig-h3 binding site was located close to the amino-terminal end of the collagen VI triple helix. The evidence indicates that collagen VI may contain distinct covalent and non-covalent binding sites for βigh3, although the possibility that both interactions use the same binding region is discussed. Overall the study supports the concept that βig-h3 is extensively associated with collagen VI in some tissues and that it plays an important modulating role in collagen VI microfibril function. Transforming growth factor-β induced gene-h3 (βigh3) was found to co-purify with collagen VI microfibrils, extracted from developing fetal ligament, after equilibrium density gradient centrifugation under both nondenaturing and denaturing conditions. Analysis of the collagen VI fraction from the non-denaturing gradient by gel electrophoresis under non-reducing conditions revealed the present of a single high molecular weight band that immunostained for both collagen VI and βigh3. When the fraction was analyzed under reducing conditions, collagen VI α chains and βig-h3 were the only species evident. The results indicated that βig-h3 is associated with collagen VI in tissues by reducible covalent bonding, presumably disulfide bridges. Rotary shadowing and immunogold staining of the collagen VI microfibrils and isolated tetramers indicated that βigh3 was specifically and periodically associated with the double-beaded region of many of the microfibrils and that this covalent binding site was located in or near the amino-terminal globular domain of the collagen VI molecule. Using solid phase and co-immunoprecipitation assays, recombinant βig-h3 was found to bind both native and pepsin-treated collagen VI but not individual pepsin-collagen VI α chains. Blocking experiments indicated that the major in vitro βig-h3 binding site was located in the pepsin-resistant region of collagen VI. In contrast to the tissue situation, the in vitro interaction had the characteristics of a reversible non-covalent interaction, and the Kd was measured as 1.63 × 10–8m. Rotary shadowing of immunogold-labeled complexes of recombinant βig-h3 and pepsin-collagen VI indicated that the in vitro βig-h3 binding site was located close to the amino-terminal end of the collagen VI triple helix. The evidence indicates that collagen VI may contain distinct covalent and non-covalent binding sites for βigh3, although the possibility that both interactions use the same binding region is discussed. Overall the study supports the concept that βig-h3 is extensively associated with collagen VI in some tissues and that it plays an important modulating role in collagen VI microfibril function. Transforming growth factor-β-inducible gene h3 (βig-h3) 1The abbreviations used are: βig-h3, transforming growth factor-β-inducible gene-h3; rβig-h3, recombinant βig-h3; BSA, bovine serum albumin; GdnHCl, guanidine hydrochloride; TBS, Tris-buffered saline. 1The abbreviations used are: βig-h3, transforming growth factor-β-inducible gene-h3; rβig-h3, recombinant βig-h3; BSA, bovine serum albumin; GdnHCl, guanidine hydrochloride; TBS, Tris-buffered saline. was first cloned from A549 lung adenocarcinoma cells that had been stimulated with transforming growth factor-β1 (1Skonier J. Neubauer M. Madisen L. Bennett K. Plowman G.D. Purchio A.F. DNA Cell Biol. 1992; 11: 511-522Google Scholar, 2Skonier 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: 571-584Google Scholar). βig-h3 is also known variously as MP78/70 (3Gibson M.A. Kumaratilake J.S. Cleary E.G. J. Biol. Chem. 1989; 264: 4590-4598Google Scholar, 4Gibson M.A. Hatzinikolas G. Kumaratilake J.S. Sandberg L.B. Nichol J.K. Sutherland G.R. Cleary E.G. J. Biol. Chem. 1996; 271: 1096-1103Google Scholar), RGD-CAP (5Hashimoto 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: 303-314Google Scholar), and keratoepithelin (6Munier F.L. Korvatska E. Djemai A. Le Paslier D. Zografos L. Pescia G. Schorderet D.F. Nat. Genet. 1997; 15: 247-251Google Scholar). βig-h3 has been established as a extracellular matrix protein in a wide variety of tissues including developing nuchal ligament, aorta, lung, and kidney and mature cornea, skin, bladder, and bone (7Gibson M.A. Kumaratilake J.S. Cleary E.G. J. Histochem. Cytochem. 1997; 45: 1683-1696Google Scholar, 8Escribano J. Hernando N. Ghosh S. Crabb J. Coca-Prados M. J. Cell. Physiol. 1994; 160: 511-521Google Scholar, 9LeBaron R.G. Bezverkov K.I. Zimber M.P. Pavelec R. Skonier J. Purchio A.F. J. Invest. Dermatol. 1995; 104: 844-849Google Scholar, 10Hirano K. Klintworth G.K. Zhan Q. Bennett K. Cintron C. Curr. Eye Res. 1996; 15: 965-972Google Scholar, 11Billings 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: 261-273Google Scholar, 12Kitahama S. Gibson M.A. Hatzinikolas G. Hay S. Kuliwaba J.L. Evdokiou A. Atkins G.J. Findlay D.M. Bone (NY). 2000; 27: 61-67Google Scholar). βig-h3 is a 76–78-kDa protein containing 4 repeat regions, 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 (2Skonier 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: 571-584Google Scholar). βig-h3 has been shown to bind in vitro to a number of other matrix components including fibronectin, laminin, and several collagen types (13Billings 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: 28003-28009Google Scholar, 14Kim 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: 656-661Google 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 (15Kim J.E. Kim S.J. Lee B.H. Park R.W. Kim K.S. Kim I.S. J. Biol. Chem. 2000; 275: 30907-30915Google Scholar, 16Bae 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: 940-948Google Scholar), α1β1 (17Ohno 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: 196-205Google Scholar), or αVβ5 (18Kim 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: 46159-46165Google Scholar). The precise functions of βig-h3 are unknown, but it has been proposed that it may act as a cell adhesion molecule (18Kim 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: 46159-46165Google Scholar) and as a bifunctional linker protein interconnecting different matrix molecules to each other and to cells (7Gibson M.A. Kumaratilake J.S. Cleary E.G. J. Histochem. Cytochem. 1997; 45: 1683-1696Google Scholar, 11Billings 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: 261-273Google Scholar). Recent evidence suggests that βig-h3 may be important in endothelial cell-matrix interactions during vascular remodeling and angio-genesis (19Aitkenhead M. Wang S.J. Nakatsu M.N. Mestas J. Heard C. Hughes C.C. Microvasc. Res. 2002; 63: 159-171Google Scholar) and that the protein functions as a negative regulator of mineralization during cartilage differentiation and osteogenesis (20Ohno S. Doi T. Tsutsumi S. Okada Y. Yoneno K. Kato Y. Tanne K. Biochim. Biophys. Acta. 2002; 1572: 114-122Google Scholar, 21Kim 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: 169-178Google Scholar). Mutations in the human βig-h3 gene have been linked to several autosomal dominant corneal dystrophies (6Munier F.L. Korvatska E. Djemai A. Le Paslier D. Zografos L. Pescia G. Schorderet D.F. Nat. Genet. 1997; 15: 247-251Google Scholar) characterized by severe visual impairment resulting from the progressive accumulation of βig-h3-containing protein deposits in the corneal matrix (22Streeten B.W. Qi Y. Klintworth G.K. Eagle R.C.,Jr. Strauss J.A. Bennett K. Arch. Ophthalmol. 1999; 117: 67-75Google Scholar). 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 (7Gibson M.A. Kumaratilake J.S. Cleary E.G. J. Histochem. Cytochem. 1997; 45: 1683-1696Google Scholar). Collagen VI is present in the extracellular matrix of a wide range of tissues, usually as fine microfibrils 3–10 nm in diameter that exhibit a characteristic, double-beaded period of about 100 nm (23Timpl R. Chu M-L. Yurchenko P.D. Birk D. Mecham R.P. Extracellular Matrix Assembly and Structure. Academic Press, Inc., New York1994: 207-242Google Scholar). In some tissues collagen VI appears to form additional structures including thicker cross-banded fibrils and hexagonal networks (24Bruns R.R. Press W. Engvall E. Timpl R. Gross J. J. Cell Biol. 1986; 103: 393-404Google Scholar, 25Reale E. Groos S. Luciano L. Eckardt C. Eckardt U. Matrix Biol. 2001; 20: 37-51Google Scholar). Collagen VI consists of monomers containing three distinct polypeptides, α1(VI), α2(VI), and α3(VI), which form a triple helical collagenous central domain flanked by globular amino- and carboxyl-terminal domains. The monomers aggregate intracellularly, first into anti-parallel dimers with a 30-nm stagger and then into tetramers stabilized by intermolecular disulfide bonds before their secretion into the extracellular matrix (26Engvall E. Hessle H. Klier G. J. Cell Biol. 1986; 102: 703-710Google Scholar). Ultrastructurally, the tetramers are symmetrical with double beads at each end of an extended central rod-like region. The outer beads represent the amino-terminal globular domains, and the inner beads correspond to the carboxyl-terminal globular domains (27Kuo H.J. Keene D.R. Glanville R.W. Biochemistry. 1989; 28: 3757-3762Google Scholar). Extracellularly, the amino-terminal domains of the tetramers overlap end to end to form the collagen VI microfibrils (24Bruns R.R. Press W. Engvall E. Timpl R. Gross J. J. Cell Biol. 1986; 103: 393-404Google Scholar, 28Furthmayr H. Wiedemann H. Timpl R. Odermatt E. Engel J. Biochem. J. 1983; 211: 303-311Google Scholar). The microfibrils are resistant to digestion with bacterial collagenase (29Gibson M.A. Cleary E.G. Biochem. Biophys. Res. Commun. 1982; 105: 1288-1295Google Scholar, 30Kielty C.M. Hanssen E. Shuttleworth C.A. Anal. Biochem. 1998; 255: 108-111Google Scholar), and treatment with pepsin removes the bulk of the globular domains from the tetramers but does not completely dissociate the microfibrils (28Furthmayr H. Wiedemann H. Timpl R. Odermatt E. Engel J. Biochem. J. 1983; 211: 303-311Google Scholar). However, the microfibrils can readily be dissociated into tetrameric subunits by treatment with a chaotropic agent (27Kuo H.J. Keene D.R. Glanville R.W. Biochemistry. 1989; 28: 3757-3762Google Scholar) or low pH (31Spissinger T. Engel J. Matrix Biol. 1995; 14: 499-505Google Scholar), indicating that the subunits are not covalently linked together within the microfibrils. The precise functions of collagen VI are unclear, but the protein is considered to be important for tissue architecture, interconnecting structural components of the matrix with each other and with cells (23Timpl R. Chu M-L. Yurchenko P.D. Birk D. Mecham R.P. Extracellular Matrix Assembly and Structure. Academic Press, Inc., New York1994: 207-242Google Scholar). Mutations in collagen VI genes have recently been linked to the muscle-wasting disease, Bethlem myopathy (32Jobsis 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: 113-115Google Scholar). In the present study 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. The binding site is located close to the amino-terminal end of the collagen VI molecule. Additional binding assays showed that rβig-h3 binds in vitro to collagen VI but in a non-covalent manner. The results indicate that direct βig-h3/collagen VI interactions occur in vivo, suggesting that they are likely to be important for the normal development and morphology of a range of tissues including cornea. Moreover these interactions appear to involve two distinct mechanisms of binding. Materials—Pepsin-treated collagen VI was prepared as described previously (33Gibson M.A. Cleary E.G. J. Biol. Chem. 1985; 260: 11149-11159Google Scholar). Individual α chains were prepared from the pepsin-collagen VI by high performance liquid chromatography. Briefly, reduced and alkylated pepsin-collagen VI was chromatographed on a Protein Pak DEAE-5PW column (Waters) in 50 mm Tris buffer, pH 8.4, containing 6 m urea using a linear gradient of 0–0.2 m NaCl gradient (50 ml). The polypeptides eluted in the order pepsin-α3 (VI), pepsin-α2 (VI), and pepsin-α1 (VI) (data not shown). The polypeptides were dialyzed into TBS before use in the binding assay. Affinity-purified polyclonal antibodies to pepsin-treated collagen VI and to βig-h3 peptide TQLYTDRTEKLRPEMEG have been described previously (7Gibson M.A. Kumaratilake J.S. Cleary E.G. J. Histochem. Cytochem. 1997; 45: 1683-1696Google Scholar). Highly purified bacterial collagenase (type VII) and hyaluronidase from Streptomyces hyalurolyticus were purchased respectively from Sigma and Seikagaku Corp. (Tokyo, Japan). Isolation of Collagen VI Microfibrils—Collagen VI microfibrils were purified using a method based on that of Kielty et al. (30Kielty C.M. Hanssen E. Shuttleworth C.A. Anal. Biochem. 1998; 255: 108-111Google Scholar). Briefly, nuchal ligament tissue (9 g) from a bovine fetus of 200 days of gestation was finely diced with a razor blade, rinsed several times with TBS, and resuspended in 18 ml of collagenase digestion buffer (50 mm Tris-HCl, pH 7.4, containing 0.4 m NaCl, 10 mm CaCl2, 2 mmN-ethylmaleimide, and 1 mm phenylmethylsulfonyl fluoride). The suspension was incubated with 1.8 mg of collagenase (specific activity = 1980 units/mg) for 8hat37 °C then centrifuged at 10,000 × g for 20 min. The supernatant was chromatographed in 3 batches on a column of Sepharose CL-2B (1.6 × 60 cm) equilibrated in Tris/NaCl buffer (composition as above but lacking CaCl2). Flow rate was 30 ml/h, and 3-ml fractions were collected. Fractions containing βig-h3 and collagen VI were identified by direct dot blotting on nylon membranes with specific antibodies. The void volume peaks from the 3 runs were combined and treated with 5 units of hyaluronidase for 24 h at 4 °C. Samples of this digest were then analyzed by CsCl density gradient centrifugation in Tris/NaCl buffer (native conditions) or in Tris/NaCl buffer containing 4 m GdnHCl (denaturing conditions). Each sample was adjusted to a density of 1.30 g/ml with CsCl and then ultracentrifuged in a 70.1 Ti head (Beckman) at 30,000 rpm (62,000 × g) at 15 °C for 72 h. Each gradient was divided into 15 fractions, and those containing βig-h3 and collagen VI were identified by dot blotting. Those fractions containing collagen VI microfibrils were analyzed by rotary shadowing (see below in "Experimental Procedures") and by SDS-PAGE and immunoblotting as described previously (34Gibson M.A. Hughes J.L. Fanning J.C. Cleary E.G. J. Biol. Chem. 1986; 26: 111429-111436Google Scholar) except that PVDF-Plus membranes (Osmonics, Westborough, MA) were used. In some instances, samples were pretreated with 10 mm cysteine followed by 40 mm iodoacetamide using a previously described method (33Gibson M.A. Cleary E.G. J. Biol. Chem. 1985; 260: 11149-11159Google Scholar). Rotary Shadowing and Immunogold Labeling—Samples (5 μl) from selected the CsCl gradient fractions were applied for 10 s to Formvarcoated nickel grids either directly or after adjustment to 4 m GdnHCl to depolymerize the collagen VI microfibrils into tetramer subunits. The grids were then rinsed with 3 changes of TBS and incubated with a 1:20 dilution of anti-(βig-h3 peptide) antibody (or a control antibody) in TBS plus Tween (0.05%) for 30 min. Grids were then rinsed again with TBS and incubated further with anti-(rabbit IgG) antibody conjugated to 10-nm gold particles (Auroprobe EM GAR G10, Amersham Biosciences) following the manufacturer's instructions. After further rinsing with TBS, the grids were dried, and rotary shadowed with platinum at an angle of 8° under a vacuum of 2 × 10–6 torr in a Cressington CFE50 Freeze Etch Unit. Each grid was observed using a Philips CM100 transmission electron microscope at 80 kV. Expression and Purification of rβig-h3—Total RNA was purified as described previously from cultured osteoblast-like cells derived from a trabecular bone biopsy (12Kitahama S. Gibson M.A. Hatzinikolas G. Hay S. Kuliwaba J.L. Evdokiou A. Atkins G.J. Findlay D.M. Bone (NY). 2000; 27: 61-67Google Scholar). First strand cDNA was synthesized using total RNA (1 μg), random hexamer primers (50 ng), and Superscript II reverse transcriptase (200 units) (Invitrogen) following the manufacturer's instructions. The βig-h3 cDNA was then amplified by PCR using forward primer 5′-CCCGCTCCATGGCGCTCTTCGTG-3′, reverse primer 5′-GCCGTGGTGCATTCCTCCTGTAGT-3′, and high fidelity Pfx DNA polymerase (Invitrogen). PCR was conducted as described previously for 28 cycles with an annealing temperature of 60 °C (12Kitahama S. Gibson M.A. Hatzinikolas G. Hay S. Kuliwaba J.L. Evdokiou A. Atkins G.J. Findlay D.M. Bone (NY). 2000; 27: 61-67Google Scholar). The PCR product (2092 bp) was purified by gel electrophoresis, A-tailed by incubation with dATP, and platinum Taq DNA polymerase (Invitrogen) at 70 °C for 30 min, and cloned into pGEM T-easy vector (Promega, Madison, WI) following the manufacturer's instructions. An authentic βig-h3 cDNA clone was selected, and a sequence encoding a His6 tag was inserted into the construct using site-directed mutagenesis with a complementary pair of primers (5′-GGGTCCCGCCAAGCATCACCACCATCACCATTCGCCCTACCAGCTGGTG-3′ and 5′-CACCAGCTGGTAGGGCGAATGGTGATGGTGGTGATGCTTGGCGGGACCC-3′) and the QuikChange kit (Stratagene, La Jolla, CA) following the manufacturer's instructions. Again an authentic cDNA clone was selected, excised with NotI from the T-easy vector, and subcloned into the NotI site of the mammalian expression vector, pCEP4 (Invitrogen). Several clones were fully sequenced to check for errors, and the selected construct (pCEP/BH18) was transfected into 293-EBNA cells using the LipofectAMINE 2000 system (Invitrogen) following the manufacturer's instructions. Stably transfected cells were established in Dulbecco's modified Eagle's medium containing fetal calf serum (10%), penicillin, streptomycin, and nonessential amino acids and selected with hygromycin (50 μg/ml). Recombinant βig-h3 was harvested from the conditioned medium of confluent cells that had been incubated for 4 days in the above medium without serum. The medium was adjusted to binding conditions using an 8× stock of binding buffer (10 mm phosphate, pH 7.60, containing 0.5 m NaCl and 10 mm imidazole) and loaded onto a nickel-agarose column (chelating-Sepharose fast flow, Amersham Biosciences). The column was washed with 10 volumes of binding buffer, and rβig-h3 was then eluted using the same buffer containing 500 mm imidazole. The quality and yield of rβig-h3 was determined by SDS-PAGE and immunoblotting using anti-(penta-His) antibody (Qiagen, Valencia, CA) and anti-(βig-h3 peptide) antibody. Radiolabeling of rβig-h3—Recombinant βig-h3 was biosynthetically radiolabeled by incubating the transfected 293-EBNA cells in serum-free Dulbecco's modified Eagle's medium depleted in cysteine and methionine for 3 days with [35S]methionine and [35S]cysteine (Tran35S-label, ICN, Costa Mesa, CA) at 100 μCi/ml. The 35S-labeled rβig-h3 was then purified from the medium as described above. Binding Assays—Solid phase binding assays were conducted as described previously (35Finnis M.L. Gibson M.A. J. Biol. Chem. 1997; 272: 22817-22823Google Scholar). Briefly, microtiter plates (Immuno Maxisorp modules; Nunc, Roskilde, Denmark) were coated with collagen VI at 4 °C for 18 h. Control wells were coated with BSA at the same concentration. After rinsing with TBS the wells were blocked in 3% nonfat dried milk in TBS for 1 h. After further rinsing, the wells were incubated either with unlabeled rβig-h3 or with 35S-labeled rβig-h3 for 3 h at 37 °C. See Figs. 6 and 5, respectively, for details. The wells were extensively rinsed with 0.05% Tween 20 in TBS. Binding of unlabeled rβig-h3 was measured using anti-βig-h3 antibody and a peroxide enzyme-linked immunosorbent assay technique (35Finnis M.L. Gibson M.A. J. Biol. Chem. 1997; 272: 22817-22823Google Scholar), and that of 35S-labeled rβig-h3 was determined by liquid scintillation counting. In some binding experiments, samples of rβig-h3 were pretreated with 40 mm iodoacetamide, 10 mm cysteine, or cysteine followed by iodoacetamide using a previously described method (33Gibson M.A. Cleary E.G. J. Biol. Chem. 1985; 260: 11149-11159Google Scholar) and then dialyzed into TBS before use in the binding assay. The dissociation constant (Kd) for the interaction of βig-h3 with collagen VI was calculated from non-linear regression analysis of the specific binding curve using the Prism program version 3 (GraphPad software, San Diego, CA).Fig. 5Recombinant βig-h3 interacts with triple-helical collagen VI but not individual α chains.Panel A shows solid phase binding assays. Purified proteins (500 ng/well) were coated onto microtiter plates. After blocking, 7 × 103 dpm of 35S-labeled rβig-h3 (specific activity 3.5 × 105 dpm/μg) was added, and the wells were incubated at 37 °C for 3 h. After washing, binding was measured by liquid scintillation counting. Column 1, native collagen VI microfibrils; column 2, pepsin-collagen VI; column 3, pepsin-α 1 (VI); column 4, pepsin-α 2 (VI); column 5, pepsin-α 3 (VI); column 6, BSA. Panel B shows that in the solid phase assay, the binding of rβig-h3 is proportional to the amount of pepsin-collagen VI on the well. Pepsin-VI collagen was coated onto microtiter wells in serial dilution and incubated with 35S-labeled rβig-h3 as described in panel A. Panel C shows co-immunoprecipitation experiments. 35S-Labeled rβig-h3 (1 × 104 dpm) was incubated with 1 μg of pepsin-collagen VI, individual α chains, or BSA for 4 h at 37 °C. Collagen VI species were then recovered by the addition of anti-collagen VI antibody followed by protein A-Sepharose. Co-immunoprecipitated 35S-labeled rβig-h3 was measured by liquid scintillation counting. Column 1, pepsin-collagen VI; column 2, pepsin-α 1 (VI); column 3, pepsin-α 2 (VI); column 4, pepsin-α 3 (VI); column 5, BSA control. In all panels, the means ± S.D. of triplicate determinations are shown.View Large Image Figure ViewerDownload (PPT) Co-immunoprecipitation assays were performed following a previously described method (36Reinboth B. Hanssen E. Cleary E.G. Gibson M.A. J. Biol. Chem. 2002; 277: 3950-3957Google Scholar) using affinity-purified collagen VI antibody (1 μl) followed by protein A-Sepharose (20 μl). See Fig. 5 for details. Co-immunoprecipitated 35S-labeled rβig-h3 was measured by liquid scintillation counting. For rotary shadowing analysis of binding interactions, pepsin-collagen VI (0.04 nmol) and rβig-h3 (0.02 nmol) were incubated together for 1 h in TBS, coated onto grids, and immunogold-labeled for βig-h3 as described above. For a negative control, pepsin-collagen VI was incubated without rβig-h3. βig-h3 Co-purifies with Collagen VI Microfibrils—A collagenase-digested extract of nuchal ligament tissue was analyzed by gel permeation chromatography on Sepharose CL-2B (Fig. 1A). Dot blotting revealed that most of the collagen VI was present in the Vo peak (peak 1), consistent with the molecule being present as microfibrillar aggregates. Interestingly, a significant signal for βig-h3 was also detected in the Vo peak. To isolate collagen VI microfibrils, the Vo peak was treated with hyaluronidase and analyzed by CsCl equilibrium density gradient centrifugation. The gradient was selected so that collagen VI banded in the middle of the gradient with a buoyant density of about 1.3 g/ml, whereas fibrillin-containing microfibrils migrated to the bottom of the tube and other proteins occupied the fractions at the top of the gradient (Fig. 1B). Dot blotting showed that the βig-h3 in the sample co-distributed precisely with the collagen VI microfibrils under non-denaturing conditions (NaCl) and under denaturing conditions (4 m GdnHCl) even though the buoyant density of the denatured collagen VI had decreased slightly. Rotary shadowing of fraction 7 from the non-denatured gradient revealed that type VI collagen microfibrils were the only discernable structures present (data not shown). βig-h3 Is Covalently Bound to Collagen VI Tetramers in Nuchal Ligament Tissue—SDS-PAGE analysis of fraction 7 under reducing conditions on a 12% gel revealed several bands around 200 kDa, a strongly staining band at 140 kDa, and a band at 66 kDa (Fig. 2A, lane 1). No other protein bands were detected on the gel. Immunoblot analysis revealed that the 66-kDa band was βig-h3 (Fig. 2A, lane 2) and that the 200- and 140-kDa bands were collagen VI polypeptides corresponding, respectively, to isoforms of the α3 (VI) chain and a mixture of the α1 (VI) and α2 (VI) chains (Fig. 2A, lane 3). When the sample was analyzed under non-reducing conditions no bands were detected on 12% gels (data not shown). This finding indicated that both βig-h3 and collagen VI were present as high molecular weight aggregates. However, when the sample was analyzed on a 3–5% gradient gel under non-reducing conditions, a single slowly migrating band was detected (Fig. 2B, lane 2) similar in size to the tetrameric form of collagen VI (apparent Mr ∼2,000,000). The band stained with antibodies to βig-h3 (Fig. 2B, lane 4) and collagen VI (Fig. 2B, lane 6), suggesting that the two proteins were present in the same complex. When analyzed under reducing conditions the complex was disrupted, and bands corresponding to βig-h3 and collagen VI were again the only bands detected in the sample (Fig. 2B, lanes 1, 3, and 5). Colorimetric analysis of the Coomassie Blue staining of the bands indicated an approximate molecular ratio of one molecule of βig-h3 to two collagen VI monomers assuming that both protein exhibit similar uptake of stain. In a further experiment an aliquot of fraction 7 was treated with a mild reducing agent, 10 mm cysteine, followed by the alkylating agent iodoacetamide to irreversibly break intermolecular but not intramolecular disulfide bonds. When analyzed by SDS-PAGE on composite agarose-polyacrylamide gels under non-reducing conditions, bands corresponding to collagen VI tetramers, dimers, and monomers were obtained (Fig. 2C, lane 1), which immunoblotted with anti-(collagen VI) antibodies (Fig. 2C, lane 3). However, immunoblotting with anti-βig-h3 antibodies stained only the βig-h3 polypeptide (migrating at the dye front on gels of this porosity) and not the collagen VI bands, indicating that the covalent bonds between βig-h3 and collagen VI had been completely disrupted by the cysteine treatment (Fig. 2C, lane 2). Overa
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