Covalent and non-covalent interactions of b ig-h3 with collagen VI - big-h3 is covalently attached to the amino-terminal region of collagen VI in tissue microfibrils

Abstract

Transforming growth factor-β induced gene-h3 (β igh3) 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 αchains and β ig-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 β 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 β 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 Kd was measured as 1.63 x 10–8 M. Rotary shadowing of immunogold-labeled complexes of recombinant β ig-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.