Tomasini-Johansson B R, Kaufman N R, Ensenberger M G, Ozeri V, Hanski E, Mosher D F
Department of Medicine and the Molecular and Cellular Pharmacology Program, University of Wisconsin, Madison, Wisconsin 53706, USA.
J Biol Chem. 2001 Jun 29;276(26):23430-9. doi: 10.1074/jbc.M103467200. Epub 2001 Apr 25.
F1 is an adhesin of Streptococcus pyogenes which binds the N-terminal 70-kDa region of fibronectin with high affinity. The fibronectin binding region of F1 is comprised of a 43-residue upstream domain and a repeat domain comprised of five tandem 37-residue sequences. We investigated the effects of these domains on the assembly of fibronectin matrix by human dermal fibroblasts, MG63 osteosarcoma cells, or fibroblasts derived from fibronectin-null stem cells. Subequimolar or equimolar concentrations of recombinant proteins containing both the upstream and repeat domains or just the repeat domain enhanced binding of fibronectin or its N-terminal 70-kDa fragment to cell layers; higher concentrations of these recombinant proteins inhibited binding. The enhanced binding did not result in greater matrix assembly and was caused by increased ligand binding to substratum. In contrast, recombinant or synthetic protein containing the 43 residues of the upstream domain and the first 6 residues from the repeat domain exhibited monophasic inhibition with an IC(50) of approximately 10 nm. Truncation of the 49-residue sequence at its N or C terminus caused loss of inhibitory activity. The 49-residue upstream sequence blocked incorporation of both endogenous cellular fibronectin and exogenous plasma fibronectin into extracellular matrix and inhibited binding of 70-kDa fragment to fibronectin-null cells in a fibronectin-free system. Inhibition of matrix assembly by the 49-mer had no effect on cell adhesion to substratum, cell growth, formation of focal contacts, or formation of stress fibers. These results indicate that the 49-residue upstream sequence of F1 binds in an inhibitory mode to N-terminal parts of exogenous and endogenous fibronectin which are critical for fibronectin fibrillogenesis.
F1是化脓性链球菌的一种粘附素，它能与纤连蛋白的N端70 kDa区域高亲和力结合。F1的纤连蛋白结合区域由一个43个残基的上游结构域和一个由五个串联的37个残基序列组成的重复结构域构成。我们研究了这些结构域对人皮肤成纤维细胞、MG63骨肉瘤细胞或源自纤连蛋白缺失干细胞的成纤维细胞组装纤连蛋白基质的影响。含有上游和重复结构域或仅重复结构域的重组蛋白的亚等摩尔或等摩尔浓度增强了纤连蛋白或其N端70 kDa片段与细胞层的结合；这些重组蛋白的更高浓度则抑制结合。结合增强并未导致更大的基质组装，而是由配体与基质结合增加引起的。相反，含有上游结构域的43个残基和重复结构域的前6个残基的重组或合成蛋白表现出单相抑制，IC50约为10 nm。在其N端或C端截短49个残基的序列会导致抑制活性丧失。49个残基的上游序列阻止内源性细胞纤连蛋白和外源性血浆纤连蛋白掺入细胞外基质，并在无纤连蛋白的系统中抑制70 kDa片段与纤连蛋白缺失细胞的结合。49聚体对基质组装的抑制对细胞与基质的粘附、细胞生长、粘着斑的形成或应力纤维的形成没有影响。这些结果表明，F1的49个残基上游序列以抑制模式结合到外源性和内源性纤连蛋白的N端部分，这些部分对纤连蛋白纤维形成至关重要。
