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 cell (EC) and vascular functions including enhancement of microvascular permeability and stimulation of EC growth. To investigate the structural features of VPF/VEGF necessary for efficient dimerization, secretion, and biological activities, we employed site-directed mutagenesis with 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 inactive in both vascular permeability and endothelial cell mitotic assays. VPF 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, which, when mutated produced an inefficiently secreted dimeric protein without post-translational glycosylation, yet maintained full vascular permeability activity. Finally, we found that one VPF mutant isoform Cys-101 was not secreted and this mutant functioned as a dominant-negative suppressor of wild-type VPF secretion as demonstrated by co-expression assays in Cos-1 cells.
Claffey
K P
KP
Department of Pathology, Beth Israel Hospital, Boston, MA 02215.
Senger
D R
DR
Spiegelman
B M
BM
D000818:Animals; D001011:Aorta; D001483:Base Sequence; D002199:Capillary Permeability; D002417:Cattle; D002455:Cell Division; D002461:Cell Line, Transformed; D002522:Chlorocebus aethiops; D017077:Culture Media, Conditioned; D016228:Endothelial Growth Factors / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology*; D004730:Endothelium, Vascular / Q000166:cytology; D015870:Gene Expression; D006031:Glycosylation; D006168:Guinea Pigs; D007668:Kidney; D008222:Lymphokines / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000494:pharmacology; D013329:Structure-Activity Relationship; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D017077:Culture Media, Conditioned; D016228:Endothelial Growth Factors; D008222:Lymphokines; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors