title: Preparation of human vascular endothelial growth factor-D for structural and preclinical therapeutic studies.
abstract: Vascular endothelial growth factor-D (VEGF-D), a secreted angiogenic and lymphangiogenic glycoprotein, enhances tumor growth and metastasis in animal models, and its expression correlates with metastasis and poor patient outcome in some cancers - it is therefore considered a target for novel anti-cancer therapeutics. The definition of the structure of the complex of VEGF-D bound to its receptors would be beneficial for design of inhibitors of VEGF-D signaling aimed at restricting the growth and spread of cancer. In addition, there is interest in using VEGF-D protein for therapeutic angiogenesis and lymphangiogenesis in the settings of cardiovascular diseases and lymphedema, respectively. However, VEGF-D has proven difficult to express and purify in a highly bioactive form due to a tendency to exist as monomers rather than bioactive dimers. Here we describe a protocol for expression and purification of mature human VEGF-D, and a mutant thereof with reduced glycosylation, potentially suitable for preclinical therapeutic and structural studies, respectively. The degree of glycosylation in mature VEGF-D was reduced by eliminating one of the two N-glycosylation sites, and expressing the protein in Lec3.2.8.1 cells which had reduced glycosylation capacity. Mature VEGF-D and the glycosylation mutant were each enriched for the biologically active dimeric form by optimizing the separation of dimer from monomer via gel filtration, followed by conversion of remaining monomers to dimers via treatment with cysteine. The glycosylation mutant of VEGF-D intended for structural studies preserved all the cysteine residues of mature VEGF-D, in contrast to previous structural studies, exhibited comparable receptor binding to mature VEGF-D and might facilitate structural studies of the VEGF-D/VEGFR-3 complex.
authors.affiliation: Ludwig Institute for Cancer Research, P.O. Box 2008, Royal Melbourne Hospital, Victoria 3050, Australia.
authors.forename: Natalia
authors.initials: N
authors.lastname: Davydova
authors.forename: Victor A
authors.initials: VA
authors.lastname: Streltsov
authors.forename: Sally
authors.initials: S
authors.lastname: Roufail
authors.forename: George O
authors.initials: GO
authors.lastname: Lovrecz
authors.forename: Steven A
authors.initials: SA
authors.lastname: Stacker
authors.forename: Timothy E
authors.initials: TE
authors.lastname: Adams
authors.forename: Marc G
authors.initials: MG
authors.lastname: Achen
mesh_terms: D002460:Cell Line; D003460:Crystallization; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006031:Glycosylation; D006801:Humans; D009154:Mutation; D011485:Protein Binding; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D042643:Vascular Endothelial Growth Factor D / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*
chemical_list: D042643:Vascular Endothelial Growth Factor D