Covalent and Non-covalent Interactions of βig-h3 with Collagen VI

Abstract

Transforming growth factor-␤ induced gene-h3 (␤ig-h3) 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 ␤ig-h3. When the fraction was analyzed under reducing conditions, collagen VI ␣ chains and ␤ig-h3 were the only species evident. The results indicated that ␤ig-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 ␤ig-h3 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 ␤ig-h3 was found to bind both native and pepsin-treated collagen VI but not individual pepsin-collagen VI ␣ chains. Blocking experiments indicated that the major in vitro ␤ig-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 K d was measured as 1.63 ؋ 10Rotary shadowing of immunogold-labeled complexes of recombinant ␤ig-h3 and pepsin-collagen VI indicated that the in vitro ␤ig-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 ␤ig-h3, although the possibility that both interactions use the same binding region is discussed. Overall the study supports the concept that ␤ig-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-␤-inducible gene h3 (␤ig-h3) 1 was first cloned from A549 lung adenocarcinoma cells that had been stimulated with transforming growth factor-␤1 (1, 2). ␤ig-h3 is also known variously as MP78/70 (3, 4), RGD-CAP (5), and keratoepithelin (6). ␤ig-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 (7-12). ␤ig-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 ␤ig-h3 molecule appears to undergo partial processing at the carboxylterminal end to yield a 68 -70 kDa isoform (2). ␤ig-h3 has been shown to bind in vitro to a number of other matrix components including fibro...

βig-h3 Interacts Directly with Biglycan and Decorin, Promotes Collagen VI Aggregation, and Participates in Ternary Complexing with These Macromolecules

“…The binding site is located close to the amino-terminal end of the collagen VI molecule. Additional binding assays have demonstrated that ␤ig-h3 binds in vitro to collagen VI, but in a non-covalent manner (32). The ␤ig-h3 attachment site on collagen VI appears to be close to those documented for the small leucinerich proteoglycans (SLRP), decorin and biglycan, and matrilins (33)(34)(35).…”

“…Human r␤ig-h3 and pepsin-treated collagen VI were prepared and purified as described previously (32,37). Purified r␤ig-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.…”

“…The non-solubilized material was then extracted extensively over 48 h with 0.6 M KCl, containing inhibitors (5 ϫ 10 volumes) to solubilize cytoskeletal proteins. The residue was rinsed with collagenase buffer (32) 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 ϭ 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.…”

“…Mutations in collagen VI genes (COL6A1, COL6A2, and COL6A3) have recently been linked to the muscle wasting diseases, Bethlem myopathy and Ullrich dystrophy (30,31). To define the relationship of ␤ig-h3 with collagen VI we have isolated collagen VI microfibrils from collagenase-treated nuchal ligament and demonstrated that ␤ig-h3 is covalently attached to collagen VI at regular intervals along at least some of the microfibrils (32). The binding site is located close to the amino-terminal end of the collagen VI molecule.…”

WOMEN IN CANCER THEMATIC REVIEW: Ovarian cancer–peritoneal cell interactions promote extracellular matrix processing

“…Effects of TGFBI on adhesion are mediated through interactions with various integrins including α1β1, α3β1, αvβ3 and αvβ5 (LeBaron et al 1995, Ohno et al 1999, Bae et al 2002, Jeong & Kim 2004, Kim & Kim 2008) via the different integrin-binding motifs. TGFBI also functions as a linker protein and connects many matrix proteins including collagens (type I, II and IV), fibronectin (Billings et al 2002) and proteoglycans (biglycan and decorin) with each other (Gibson et al 1997, Billings et al 2002, Hanssen et al 2003, Reinboth et al 2006.…”