TY - JOUR

T1 - Single-chain vascular endothelial growth factor variant with antagonist activity

AU - Boesen, Thomas P

AU - Soni, Bobby

AU - Schwartz, Thue W

AU - Halkier, Torben

N1 - Keywords: Amino Acid Sequence; Cells, Cultured; Cloning, Molecular; Dimerization; Electrophoresis, Polyacrylamide Gel; Endothelial Growth Factors; Endothelium, Vascular; Humans; Intercellular Signaling Peptides and Proteins; Lymphokines; Mitosis; Molecular Sequence Data; Mutagenesis, Site-Directed; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

PY - 2002

Y1 - 2002

N2 - Vascular endothelial growth factor is a specific endothelial cell mitogen that is essential for the formation of the vascular system but in the adult individual is involved in several pathological conditions, including cancer. It is a homodimeric protein that activates its receptor by binding two receptor molecules and inducing dimerization. By mixing two vascular endothelial growth factor monomers, each with different substitutions, heterodimers with only one active receptor binding site have previously been prepared. These heterodimers bind the receptor molecule but are unable to induce dimerization and activation. However, preparation of heterodimers is cumbersome, involving separate expression of different monomers, refolding the mixture, and separating heterodimers from homodimers. Here we show that a fully functional ligand can efficiently be expressed as a single protein chain containing two monomers. Single-chain vascular endothelial growth factor is functionally equivalent to the wild-type protein. By monomer-specific mutagenesis, one receptor binding site was altered. This variant competitively and specifically antagonizes the mitogenic effect of the wild-type protein on endothelial cells. The results obtained with the single-chain antagonist show the feasibility of the single-chain approach in directing alterations to single specific regions in natural homodimeric proteins that would be impossible to target in other ways.

AB - Vascular endothelial growth factor is a specific endothelial cell mitogen that is essential for the formation of the vascular system but in the adult individual is involved in several pathological conditions, including cancer. It is a homodimeric protein that activates its receptor by binding two receptor molecules and inducing dimerization. By mixing two vascular endothelial growth factor monomers, each with different substitutions, heterodimers with only one active receptor binding site have previously been prepared. These heterodimers bind the receptor molecule but are unable to induce dimerization and activation. However, preparation of heterodimers is cumbersome, involving separate expression of different monomers, refolding the mixture, and separating heterodimers from homodimers. Here we show that a fully functional ligand can efficiently be expressed as a single protein chain containing two monomers. Single-chain vascular endothelial growth factor is functionally equivalent to the wild-type protein. By monomer-specific mutagenesis, one receptor binding site was altered. This variant competitively and specifically antagonizes the mitogenic effect of the wild-type protein on endothelial cells. The results obtained with the single-chain antagonist show the feasibility of the single-chain approach in directing alterations to single specific regions in natural homodimeric proteins that would be impossible to target in other ways.

U2 - 10.1074/jbc.M204107200

DO - 10.1074/jbc.M204107200

M3 - Journal article

C2 - 12151391

VL - 277

SP - 40335

EP - 40341

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 43

ER -