10 research outputs found
Comparison of the rates of deamidation, diketopiperazine formation, and oxidation in recombinant human vascular endothelial growth factor and model peptides
In this work, we examine the way in which stability information obtained from studies on small model peptides correlates with similar information acquired from a protein. The rates of deamidation, oxidation, and diketopiperazine reactions in model peptide systems were compared to those of recombinant human vascular endothelial growth factor (rhVEGF). The N-terminal residues of rhVEGF, a potent mitogen in angiogenesis, are susceptible to the aforementioned reactions. The degradation of the peptides L-Ala-L-Pro-L-Met (APM) and Gly-L-Gsn-L-His-L-His (GQNHH), residues 1–3 and 8–12 of rh VEGF, respectively, and rhVEGF were examined at pH 5 and 8 at 37°C. Capillary electrophoresis and high-performance liquid chromatography (HPLC) stability-indicating assays were developed to monitor the degradation of the penta- and tripeptides, respectively. The degradation of rhVEGF was determined by tryptic mapping and quantified by RP-HPLC. The rates of degradation of both peptides and the protein followed apparent first-order kinetics and increased with increasing pH. The tripeptide APM underwent diketopiperazine formation (Ala-Prodiketopiperazine) and oxidation of the Met residue, whereas the pentapeptide GQNHH degraded via the deamidation pathway. The results indicate that the rates of deamidation and oxidation of the protein are comparable to those observed in the model peptides at both pH values. However, the rate of the diketo-piperazine reaction was slower in the protein than in the model peptide, which may be the result of differences in the cis-trans equilibrium of the X-Pro peptide bonds in the 2 molecules
Production of Soluble Human Vascular Endothelial Growth Factor VEGF-A165-Heparin Binding Domain in Escherichia coli
Mechanism of Selective VEGF-A Binding by Neuropilin-1 Reveals a Basis for Specific Ligand Inhibition
Inhibition of FLT1 ameliorates muscular dystrophy phenotype by increased vasculature in a mouse model of Duchenne muscular dystrophy
Enhanced sensitivity of VEGF detection using catalase-mediated chemiluminescence immunoassay based on CdTe QD/H2O2 system
Biophysical Studies of the Induced Dimerization of Human VEGF Receptor 1 Binding Domain by Divalent Metals Competing with VEGF-A
Finding New Partnerships: The Function of Individual Extracellular Receptor Domains in Angiogenic Signalling by VEGF Receptors
Vascular endothelial growth factors (VEGFs) constitute a family of polypeptides regulating blood and lymphatic vessel development. VEGFs bind to type V receptor tyrosine kinases (RTKs), VEGFR-1, VEGFR-2, and VEGFR-3, but also bind directly to neuropilins and heparan sulphate glycosaminoglycans (HSPG), or indirectly to co-receptors such integrins and semaphorins. VEGFR activation results from ligand-induced dimerisation, which is mediated by the extracellular receptor domain (ECD). Recent studies established that dimerisation is necessary, but not sufficient, for receptor activation, since it was shown that only distinct orientations of receptor monomers give rise to active receptor dimers that are capable to instigate transmembrane signalling. Additional complexity in VEGFR signalling arises from association with specific co-receptors, which is determined by ligand- and ECD-specific interaction domains.
In the following, the role of the different extracellular subdomains in VEGFR activation and signalling is discussed. We give an overview of the mechanistic concepts arising from recent structural studies that led to the development of novel allosteric receptor inhibitors and discuss their possible application in therapies aimed at pathological angiogenesis