Revisiting the mystery of fibronectin multimers: the fibronectin matrix is composed of fibronectin dimers cross-linked by non-covalent bonds.
Fibronectin (FN) matrix fibrils have long been thought to be formed by disulfide-bonded FN multimers, although there is no direct evidence that they are covalently linked with each other. To understand the biochemical properties of these fibrils, we extracted a crude FN matrix from FN-YPet transfected 3T3 cell culture using 0.2% deoxycholate and DNase. The insoluble extracted matrix preserved fibrillar structures and a major portion of the extracted proteins migrated as FN monomers on an SDS gel under reducing conditions. Under non-reducing conditions, some FN molecules appeared to be trapped at the top of the stacking gel. We tested this by mixing fluorescently labeled FN dimers with the extracted matrix just before loading on an SDS gel, and found that most of them were trapped with the extracted proteins at the top of the stacking gel. These results suggested that some components of the extracted matrix plugged the stacking gel and FN dimers were trapped with them. Rotary shadowing electron microscopy showed that the extracted matrix had some fibers that resembled fibrillin microfibrils. Peptide mass fingerprinting confirmed the presence of fibrillin in the extracted matrix. Fibrillin is known to form disulfide-bonded multimers and it is likely to be one of the components that plug the stacking gel and trap FN molecules in this system. The phenomenon by which FN molecules appear to migrate as multimers on SDS gels is thus an artifact rising from the presence of other large components in the extract. We conclude that FN matrix fibrils are made of FN dimers that are further cross-linked by non-covalent protein-protein bonds.
Ohashi
Tomoo
T
Department of Cell Biology, Box 3709, Duke University Medical Center, Durham, NC 27710, USA. t.ohashi@cellbio.duke.edu
Erickson
Harold P
HP
D000818:Animals; D055598:Chemical Phenomena; D003902:Detergents / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005109:Extracellular Matrix / Q000737:chemistry* / Q000648:ultrastructure; D000071837:Fibrillins; D005353:Fibronectins / Q000737:chemistry* / Q000302:isolation & purification; D051379:Mice; D008840:Microfilament Proteins / Q000737:chemistry; D008854:Microscopy, Electron; D041681:NIH 3T3 Cells; D055503:Protein Multimerization
D003902:Detergents; D000071837:Fibrillins; D005353:Fibronectins; D008840:Microfilament Proteins