Novel vascular endothelial growth factor D variants with increased biological activity.
Members of the vascular endothelial growth factor (VEGF) family play a pivotal role in angiogenesis and lymphangiogenesis. They are potential therapeutics to induce blood vessel formation in myocardium and skeletal muscle, when normal blood flow is compromised. Most members of the VEGF/platelet derived growth factor protein superfamily exist as covalently bound antiparallel dimers. However, the mature form of VEGF-D (VEGF-D(DeltaNDeltaC)) is predominantly a non-covalent dimer even though the cysteine residues (Cys-44 and Cys-53) forming the intersubunit disulfide bridges in the other members of the VEGF family are also conserved in VEGF-D. Moreover, VEGF-D bears an additional cysteine residue (Cys-25) at the subunit interface. Guided by our model of VEGF-D(DeltaNDeltaC), the cysteines at the subunit interface were mutated to study the effect of these residues on the structural and functional properties of VEGF-D(DeltaNDeltaC). The conserved cysteines Cys-44 and Cys-53 were found to be essential for the function of VEGF-D(DeltaNDeltaC). More importantly, the substitution of the Cys-25 at the dimer interface by various amino acids improved the activity of the recombinant VEGF-D(DeltaNDeltaC) and increased the dimer to monomer ratio. Specifically, substitutions to hydrophobic amino acids Ile, Leu, and Val, equivalent to those found in other VEGFs, most favorably affected the activity of the recombinant VEGF-D(DeltaNDeltaC). The increased activity of these mutants was mainly due to stabilization of the protein. This study enables us to better understand the structural determinants controlling the biological activity of VEGF-D. The novel variants of VEGF-D(DeltaNDeltaC) described here are potential agents for therapeutic applications, where induction of vascular formation is required.
Department of Biotechnology and Molecular Medicine, A. I. Virtanen Institute for Molecular Sciences, FI-70211 Kuopio, Finland.
Pyry I
PI
Toivanen
Tiina
T
Nieminen
Lenita
L
Viitanen
Annamari
A
Alitalo
Miia
M
Roschier
Suvi
S
Jauhiainen
Johanna E
JE
Markkanen
Olli H
OH
Laitinen
Tomi T
TT
Airenne
Tiina A
TA
Salminen
Mark S
MS
Johnson
Kari J
KJ
Airenne
Seppo
S
Ylä-Herttuala
D000818:Animals; D001665:Binding Sites; D001783:Blood Flow Velocity; D003326:Coronary Circulation; D014644:Genetic Variation; D006321:Heart / Q000502:physiology; D006706:Homeostasis; D006801:Humans; D008958:Models, Molecular; D018482:Muscle, Skeletal / Q000098:blood supply; D009206:Myocardium / Q000378:metabolism; D018919:Neovascularization, Physiologic; D011487:Protein Conformation; D040262:Receptors, Vascular Endothelial Growth Factor / Q000502:physiology; D013329:Structure-Activity Relationship; D013552:Swine; D042643:Vascular Endothelial Growth Factor D / Q000737:chemistry / Q000235:genetics* / Q000502:physiology / Q000627:therapeutic use; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000502:physiology
D042643:Vascular Endothelial Growth Factor D; D040262:Receptors, Vascular Endothelial Growth Factor; D040301:Vascular Endothelial Growth Factor Receptor-2