Assembly of fibronectin molecules with mutations or deletions of the carboxyl-terminal type I modules.
Fibronectin is a large modular protein that is assembled into fibrils in a stepwise process that involves the binding of soluble fibronectin to the cell surface and formation of fibronectin multimers that are stabilized by disulfides. Fibronectin contains two types of disulfide-containing repeat modules, types I and II. The type I modules form units that mediate binding to assembly sites (I-1 through I-5), mediate binding to gelatin (I-6 through I-9 plus the type II modules), or have no known function other than fibrin binding (I-10 through I-12). All type I modules contain four cysteines that are disulfide-linked in a 1-3, 2-4 arrangement, except for I-12 that contains six cysteines disulfide-bonded in an unknown arrangement. I-12 contains the consensus sequence Cys-Xaa-Yaa-Cys found in a number of proteins involved in disulfide exchange reactions [Holmgren, A. (1985) Annu. Rev. Biochem. 54, 237; Boniface, J. J., & Reichert, L. E., Jr. (1990) Science 247, 61]. We explored the role of I-12 and adjacent type I modules of fibronectin in matrix assembly. We generated mutant fibronectins in which the second and sixth or fifth and sixth cysteine residues in I-12 were changed to serines (CS mutants) or that contained deletions of the 12th (delta 12) or 10th through 12th (delta 10-12) type I modules. Expression of I-12 as a fusion protein with the gelatin binding part of fibronectin indicated that this module folds independently and that the most likely disulfide pairing is 1-4, 2-6, 3-5.(ABSTRACT TRUNCATED AT 250 WORDS)
Sottile
J
J
Department of Medicine, University of Wisconsin, Madison 53706.
Mosher
D F
DF
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D005109:Extracellular Matrix / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D005353:Fibronectins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017353:Gene Deletion; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D021382:Protein Sorting Signals / Q000235:genetics; D017433:Protein Structure, Secondary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D013462:Sulfur Radioisotopes; D014162:Transfection
D005353:Fibronectins; D009838:Oligodeoxyribonucleotides; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D013462:Sulfur Radioisotopes