Size distribution and molecular associations of plasma fibronectin and fibronectin crosslinked by transglutaminase 2.
Fibronectin (FN) is a ubiquitously expressed cell adhesion protein capable of assembling into large, extended fibrillar networks as part of an extracellular matrix (ECM) that regulates cell behavior. FN is a substrate for certain members of the transglutaminase family of protein-crosslinking enzymes-enzymes which can modify the ability of FN to support cell adhesion. In this study, we have analyzed the thermo-chemical stability of plasma FN in its noncrosslinked form, and after crosslinking by transglutaminase 2 (TG2), using dynamic light scattering. We report that FN is found in a generally globular (8.7 nm hydrodynamic radius), dimerized form in aqueous solutions, but unfolds into a linear arrangement at high ionic (1 M NaCl) and chaotropic (5 M urea) environments. FN conformation remained stable after multiple heating and cooling cycles ranging from 4 to 60 degrees C. Crosslinking of FN with TG2 formed large, multimeric complexes having high chemical stability in aqueous, high ionic and chaotropic environments, demonstrating that this covalent modification stabilizes FN. Given recent data that substrate (e.g. ECM) rigidity profoundly affects cell differentiation and behavior, we further studied how TG2 crosslinking affects the molecular rigidity of FN by obtaining atomic force microscopy nanoindentation measurements from untreated and crosslinked FN samples embedded in acrylamide gels. We demonstrate that TG2-mediated crosslinking of FN significantly increases Young's modulus (of elasticity), an observation of increased rigidity having important implications with respect to the biological role of ECM protein-crosslinking in cell signaling and guiding cell differentiation.
Nelea
Valentin
V
Faculty of Dentistry, McGill University, Montreal, QC, Canada, H3A 2B2.
Nakano
Yukiko
Y
Kaartinen
Mari T
MT
D000818:Animals; D002417:Cattle; D003432:Cross-Linking Reagents / Q000378:metabolism*; D005353:Fibronectins / Q000097:blood* / Q000737:chemistry*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D008027:Light; D018625:Microscopy, Atomic Force; D008970:Molecular Weight; D011487:Protein Conformation / Q000187:drug effects; D017510:Protein Folding; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D012542:Scattering, Radiation; D011503:Transglutaminases / Q000378:metabolism*
D003432:Cross-Linking Reagents; D005353:Fibronectins; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D019204:GTP-Binding Proteins