STRUCTURAL REQUIREMENTS FOR DIMERIZATION, GLYCOSYLATION, SECRETION, AND BIOLOGICAL FUNCTION OF VPF VEGF/

Vascular permeability factor (VPF) also known as vascular endothelial growth factor (VEGF), is a dimeric protein that affects endothelial ce ll (EC) and vascular functions including enhancement of microvascular permeability and stimulation of EC growth. To investigate the structur al features of VPF/VEGF necessary for efficient dimerization, secretio n, and biological activities, we employed site-directed mutagenesis wi th a Cos-1 cell expression system. Several cysteine residues essential for VPF dimerization were identified by mutation analysis of the Cys- 25, Cys-56, and Cys-67 residues. Mutant VPF isoforms lacking either of these cysteines were secreted as monomers and were completely inactiv e in both vascular permeability and endothelial cell mitotic assays. V PF Cys-145 mutant protein was efficiently secreted as a glycosylated, dimeric polypeptide, but had a reduction in biological activities. The site of N-linked glycosylation was directly identified as Asn-74, whi ch, when mutated produced an inefficiently secreted dimeric protein wi thout post-translational glycosylation, yet maintained full vascular p ermeability activity. Finally, we found that one VPF mutant isoform Cy s-101 was not secreted and this mutant functioned as a dominant-negati ve suppressor of wild-type VPF secretion as demonstrated by co-express ion assays in Cos-1 cells.