Toivanen Pyry I, Nieminen Tiina, Viitanen Lenita, Alitalo Annamari, Roschier Miia, Jauhiainen Suvi, Markkanen Johanna E, Laitinen Olli H, Airenne Tomi T, Salminen Tiina A, Johnson Mark S, Airenne Kari J, Ylä-Herttuala Seppo
Department of Biotechnology and Molecular Medicine, A. I. Virtanen Institute for Molecular Sciences, FI-70211 Kuopio, Finland.
J Biol Chem. 2009 Jun 5;284(23):16037-48. doi: 10.1074/jbc.M109.001123. Epub 2009 Apr 14.
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.
血管内皮生长因子（VEGF）家族成员在血管生成和淋巴管生成中起关键作用。当正常血流受损时，它们是诱导心肌和骨骼肌血管形成的潜在治疗药物。VEGF/血小板衍生生长因子蛋白超家族的大多数成员以共价结合的反平行二聚体形式存在。然而，VEGF-D的成熟形式（VEGF-D(DeltaNDeltaC)）主要是非共价二聚体，尽管在VEGF家族其他成员中形成亚基间二硫键的半胱氨酸残基（Cys-44和Cys-53）在VEGF-D中也保守。此外，VEGF-D在亚基界面处还有一个额外的半胱氨酸残基（Cys-25）。在我们的VEGF-D(DeltaNDeltaC)模型指导下，对亚基界面处的半胱氨酸进行突变，以研究这些残基对VEGF-D(DeltaNDeltaC)结构和功能特性的影响。发现保守的半胱氨酸Cys-44和Cys-53对VEGF-D(DeltaNDeltaC)的功能至关重要。更重要的是，用各种氨基酸取代二聚体界面处的Cys-25提高了重组VEGF-D(DeltaNDeltaC)的活性，并增加了二聚体与单体的比例。具体而言，取代为疏水性氨基酸Ile、Leu和Val，与其他VEGF中发现的氨基酸等效，对重组VEGF-D(DeltaNDeltaC)的活性影响最为有利。这些突变体活性的增加主要是由于蛋白质的稳定。这项研究使我们能够更好地理解控制VEGF-D生物活性的结构决定因素。这里描述的VEGF-D(DeltaNDeltaC)的新型变体是需要诱导血管形成的治疗应用的潜在药物。
