Abstract
Transforming growth factor-beta induced gene-h3 (betaig-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 betaig-h3. When the fraction was analyzed under reducing conditions, collagen VI alpha chains and betaig-h3 were the only species evident. The results indicated that betaig-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 betaig-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 betaig-h3 was found to bind both native and pepsin-treated collagen VI but not individual pepsin-collagen VI alpha chains. Blocking experiments indicated that the major in vitro betaig-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 betaig-h3 and pepsin-collagen VI indicated that the in vitro betaig-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 betaig-h3, although the possibility that both interactions use the same binding region is discussed. Overall the study supports the concept that betaig-h3 is extensively associated with collagen VI in some tissues and that it plays an important modulating role in collagen VI microfibril function.
Highlights
Subsequent immunoelectron microscopic studies in a range of tissues showed ig-h3 to be associated with collagen VI microfibrils rather than fibrillin-containing microfibrils [7], and this prompted the current investigation into the molecular basis of the association
In the present study ig-h3 was found to co-purify with collagen VI microfibrils, extracted from fetal nuchal ligament, on CsCl density gradients even under denaturing conditions
Analysis of the molecular composition of the microfibrils by SDS-PAGE and immunoblotting showed that ig-h3 and collagen VI co-migrated under non-reducing conditions as a single high molecular weight complex close in size to that predicted for the tetramer subunit of collagen VI
Summary
Materials—Pepsin-treated collagen VI was prepared as described previously [33]. Individual ␣ chains were prepared from the pepsincollagen VI by high performance liquid chromatography. Affinity-purified polyclonal antibodies to pepsin-treated collagen VI and to ig-h3 peptide TQLYTDRTEKLRPEMEG have been described previously [7]. Fractions containing ig-h3 and collagen VI were identified by direct dot blotting on nylon membranes with specific antibodies. Samples of rig-h3 were pretreated with 40 mM iodoacetamide, 10 mM cysteine, or cysteine followed by iodoacetamide using a previously described method [33] and dialyzed into TBS before use in the binding assay. Co-immunoprecipitation assays were performed following a previously described method [36] using affinity-purified collagen VI antibody (1 l) followed by protein A-Sepharose (20 l). For rotary shadowing analysis of binding interactions, pepsin-collagen VI (0.04 nmol) and rig-h3 (0.02 nmol) were incubated together for 1 h in TBS, coated onto grids, and immunogold-labeled for ig-h3 as described above.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
