Engineering of an FGF–proteoglycan fusion protein with heparin-independent, mitogenic activity

Abstract

In the absence of heparan sulfate (HS) on the surface of target cells, or free heparin (HP) in the vicinity of their receptors, fibroblast growth factor (FGF) family members cannot exert their biological activity and are easily damaged by proteolysis. This limits the utility of FGFs in a variety of applications including treatment of surgical, burn, and periodontal tissue wounds, gastric ulcers, segmental bony defects, ligament and spinal cord injury. Here we describe an FGF analog engineered to overcome this limitation by fusing FGF-1 with HS proteoglycan (PG) core protein. The fusion protein (PG–FGF-1), which was expressed in Chinese hamster ovary cells and collected from the conditioned medium, possessed both HS and chondroitin sulfate sugar chains. After fractionation, intact PG–FGF-1 proteins with little affinity to immobilized HP and high-level HS modification, but not their heparitinase or heparinase digests, exerted mitogenic activity independent of exogenous HP toward HS-free Ba/F3 transfectants expressing FGF receptor. Although PG–FGF-1 was resistant to tryptic digestion, its physiological degradation with a combination of heparitinase and trypsin augmented its mitogenic activity toward human endothelial cells. The same treatment abolished the activity of simple FGF-1 protein. By constructing a biologically active proteoglycan–FGF-1 fusion protein, we have demonstrated an approach that may prove effective for engineering not only FGF family members, but other HP-binding molecules as well.

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Figure 1: Structure and characterization of PG–FGF-1.
Figure 2: Separation of PG–FGF-1 subpopulations differing in their affinity for HP.
Figure 3: PG–FGF-1 Fraction U is mitogenic toward FR-Ba/F3 cells without addition of HP.
Figure 4: PG–FGF-1 is resistant to proteolytic digestion.
Figure 5: Mitogenicity of PG–FGF-1 toward HUVEC is potentiated by physiological degradation.

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Acknowledgements

We thank Drs. Chie Matsuda and Syuichi Oka, and Ms. Kazuko Miyakawa at NIBH for technical assistances and discussions. The text was edited by Dr. William Goldman at MST Editing Company. This study was supported by a AIST project of Complexed Carbohydrate to T.I., M.A., and M.S. and by a STA-COE grant and a AIST-HFSP grant to T.I. A.Y. was supported by a NEDO postdoctoral fellowship.

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Correspondence to Toru Imamura.

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Yoneda, A., Asada, M., Oda, Y. et al. Engineering of an FGF–proteoglycan fusion protein with heparin-independent, mitogenic activity. Nat Biotechnol 18, 641–644 (2000). https://doi.org/10.1038/76487

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