The single-copy gene of human basic fibroblast growth factor (bFGF) encodes four co-expressed isoforms, with an apparent molecular weight (M(r)) of 24 kD, 22.5 kD, 22 kD, and 18 kD, co-translated from a single mRNA. As a tool for the study of the role exerted by the different bFGF isoforms in the biology of endothelial cells, human recombinant 24-kD bFGF was produced and purified from transformed Escherichia coli cells. To this purpose, the novel CUG start codon present in human bFGF cDNA and responsible for the synthesis of 24-kD bFGF was mutagenized to the classic AUG start codon. Transient expression of the mutagenized cDNA in simian COS-1 cells, followed by immunolocalization and subcellular fractionation, resulted in the synthesis of high levels of 24-kD bFGF, which localizes in the cell nucleus as an intact protein. When the same 24-kD bFGF cDNA was expressed in E. coli, the recombinant protein was purified to homogeneity by heparin-Sepharose and ion-exchange chromatography. Recombinant 24-kD bFGF was similar to recombinant 18-kD bFGF in receptor-binding activity and in inducing cell proliferation, plasminogen activator production, and chemotactic movement in cultured endothelial cells. In agreement with the in vitro observations, 24-kD bFGF and 18-kD bFGF exerted a similar angiogenic response when assayed in vivo in the rabbit cornea. Experiments performed with the radiolabeled molecule demonstrated that 24-kD bFGF has an intrinsic ability to bind to high-affinity receptors when added to endothelial GM 7373 cell cultures. Receptor-bound 24-kD bFGF is internalized within the cell and associates with the nucleus with kinetics similar to 18-kD bFGF. Internalized 24-kD bFGF is first processed to the 18-kD form via a chloroquine-insensitive pathway and then to smaller fragments into the lysosomal compartment. At variance with the data obtained in transfected COS-1 cells, only limited amounts of exogenous internalized 24-kD bFGF associates with the nucleus in the intact form, mostly of the nuclear-bound molecule being represented by the processed 18-kD protein and by smaller degradation products. In conclusion, human recombinant 24-kD bFGF exerts a biological response in endothelial cells similar to 18-kD bFGF both in vitro and in vivo. Our data point to a different intracellular behavior of the high-molecular-weight bFGF isoform when added exogenously to cultured cells or when produced endogenously in transfected cells.

A., G., C., U., M., R., Ziche, M., M., P. (1994). Interaction of high-molecular-weight basic fibroblast growth factor with endothelium: biological activity and intracellular fate of human recombinant M(r) 24,000 bFGF. JOURNAL OF CELLULAR PHYSIOLOGY, 161(1), 149-159 [10.1002/jcp.1041610118].

Interaction of high-molecular-weight basic fibroblast growth factor with endothelium: biological activity and intracellular fate of human recombinant M(r) 24,000 bFGF.

ZICHE, MARINA;
1994-01-01

Abstract

The single-copy gene of human basic fibroblast growth factor (bFGF) encodes four co-expressed isoforms, with an apparent molecular weight (M(r)) of 24 kD, 22.5 kD, 22 kD, and 18 kD, co-translated from a single mRNA. As a tool for the study of the role exerted by the different bFGF isoforms in the biology of endothelial cells, human recombinant 24-kD bFGF was produced and purified from transformed Escherichia coli cells. To this purpose, the novel CUG start codon present in human bFGF cDNA and responsible for the synthesis of 24-kD bFGF was mutagenized to the classic AUG start codon. Transient expression of the mutagenized cDNA in simian COS-1 cells, followed by immunolocalization and subcellular fractionation, resulted in the synthesis of high levels of 24-kD bFGF, which localizes in the cell nucleus as an intact protein. When the same 24-kD bFGF cDNA was expressed in E. coli, the recombinant protein was purified to homogeneity by heparin-Sepharose and ion-exchange chromatography. Recombinant 24-kD bFGF was similar to recombinant 18-kD bFGF in receptor-binding activity and in inducing cell proliferation, plasminogen activator production, and chemotactic movement in cultured endothelial cells. In agreement with the in vitro observations, 24-kD bFGF and 18-kD bFGF exerted a similar angiogenic response when assayed in vivo in the rabbit cornea. Experiments performed with the radiolabeled molecule demonstrated that 24-kD bFGF has an intrinsic ability to bind to high-affinity receptors when added to endothelial GM 7373 cell cultures. Receptor-bound 24-kD bFGF is internalized within the cell and associates with the nucleus with kinetics similar to 18-kD bFGF. Internalized 24-kD bFGF is first processed to the 18-kD form via a chloroquine-insensitive pathway and then to smaller fragments into the lysosomal compartment. At variance with the data obtained in transfected COS-1 cells, only limited amounts of exogenous internalized 24-kD bFGF associates with the nucleus in the intact form, mostly of the nuclear-bound molecule being represented by the processed 18-kD protein and by smaller degradation products. In conclusion, human recombinant 24-kD bFGF exerts a biological response in endothelial cells similar to 18-kD bFGF both in vitro and in vivo. Our data point to a different intracellular behavior of the high-molecular-weight bFGF isoform when added exogenously to cultured cells or when produced endogenously in transfected cells.
1994
A., G., C., U., M., R., Ziche, M., M., P. (1994). Interaction of high-molecular-weight basic fibroblast growth factor with endothelium: biological activity and intracellular fate of human recombinant M(r) 24,000 bFGF. JOURNAL OF CELLULAR PHYSIOLOGY, 161(1), 149-159 [10.1002/jcp.1041610118].
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11365/30459
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