Total Chemical Synthesis of Glycosylated VEGF-A and evaluation of an optimal sugar for bioactivity  — ASN Events
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  2. SESSION 3: Peptide and protein modifications
  3. Total Chemical Synthesis of Glycosylated VEGF-A and evaluation of an optimal sugar for bioactivity 

Total Chemical Synthesis of Glycosylated VEGF-A and evaluation of an optimal sugar for bioactivity  (#11)

Paul WR Harris 1 2 , Sung Yang 2 , Lyn M Wise 3 , Gabriella Stuart 3 , Yuxin Wang 2
  1. School of Chemical Sciences and Maurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland, Auckland 1142, New Zealand
  2. School of Biological Sciences, The University of Auckland, Auckland 1142, New Zealand
  3. Department of Pharmacology and Toxicology, The University of Otago, Otago, New Zealand

Proteins are the fastest growing class of pharmaceuticals for the treatments of many diseases, from cancers to immune disorders. To exert their therapeutic effect, most protein drugs require glycosylation, with one or more carbohydrate molecules (glycans) attached to their surface to improve stability and solubility.1 Changes in glycan coverage can greatly affect the efficacy, half-life, and immunogenicity of proteins as therapeutics.2

In this study, we have focused on the glycoprotein, vascular endothelial growth factor (VEGF), a 192 amino acid residue protein.3  Recombinant VEGF is used clinically to stimulate blood vessel formation in ischemic, injured and bioengineered tissues. VEGF contains a single conserved N-glycan, while several variants differ in the O-glycosylation pattern. Recombinantly-produced VEGF is correctly folded and bioactive despite the heterogeneous glycosylation states,4 although glycan differences appear to impact protein solubility,5 proteolysis6 and receptor binding.7

The total chemical synthesis was undertaken by native chemical ligation8 in a convergent synthesis with polypeptide fragments equipped with thioesters and  containing multiple different glycans at Asn-75. These were folded into the 3-D homodimer active protein and assessed for receptor binding, bioactivity, and immunogenicity and compared to native, partial and unglycosylated forms.

 

 

  1. 1. Sola, R. J.; Griebenow, K., Effects of Glycosylation on the Stability of Protein Pharmaceuticals. J. Pharm. Sci. 2009, 98 (4), 1223-1245.
  2. 2. Costa, A. R.; Rodrigues, M. E.; Henriques, M.; Oliveira, R.; Azeredo, J., Glycosylation: impact, control and improvement during therapeutic protein production. Critical Reviews in Biotechnology 2014, 34 (4), 281-299.
  3. 3. Ferrara, N.; Gerber, H. P.; LeCouter, J., The biology of VEGF and its receptors. Nat Med 2003, 9 (6), 669-76.
  4. 4. (a) Cohen, T.; Gitay-Goren, H.; Neufeld, G.; Levi, B. Z., High levels of biologically active vascular endothelial growth factor (VEGF) are produced by the baculovirus expression system. Growth Factors 1992, 7 (2), 131-8; (b) Walter, D. H.; Hink, U.; Asahara, T.; VanBelle, E.; Horowitz, J.; Tsurumi, Y.; Vandlen, R.; Heinsohn, H.; Keyt, B.; Ferrara, N.; Symes, J. F.; Isner, J. M., The in vivo bioactivity of vascular endothelial growth factor vascular permeability factor is independent of N-linked glycosylation. Lab. Invest. 1996, 74 (2), 546-556.
  5. 5. Yeo, T. K.; Senger, D. R.; Dvorak, H. F.; Freter, L.; Yeo, K. T., Glycosylation Is Essential for Efficient Secretion but Not for Permeability-Enhancing Activity of Vascular-Permeability Factor (Vascular Endothelial Growth-Factor). Biochem. Biophys. Res. Commun. 1991, 179 (3), 1568-1575
  6. 6. a) Plouet, J.; Moro, F.; Bertagnolli, S.; Coldeboeuf, N.; Mazarguil, H.; Clamens, S.; Bayard, F., Extracellular cleavage of the vascular endothelial growth factor 189 amino acid form by urokinase is required for its mitogenic effect. J. Biol. Chem. 1997, 272 (20), 13390-13396; (b) Roth, D.; Piekarek, M.; Paulsson, M.; Christ, H.; Bloch, W.; Krieg, T.; Davidson, J. M.; Eming, S. A., Plasmin modulates vascular endothelial growth factor-A-mediated angiogenesis during wound repair. Am. J. Pathol. 2006, 168 (2), 670-84
  7. 7. Inder, M. K.; Wise, L. M.; Fleming, S. B.; Mercer, A. A., The C-terminus of viral vascular endothelial growth factor-E partially blocks binding to VEGF receptor-1. The FEBS journal 2008, 275 (1), 207-17; (b) Keyt, B. A.; Nguyen, H. V.; Berleau, L. T.; Duarte, C. M.; Park, J.; Chen, H.; Ferrara, N., Identification of vascular endothelial growth factor determinants for binding KDR and FLT-1 receptors - Generation of receptor-selective VEGF variants by site-directed mutagenesis. J. Biol. Chem. 1996, 271 (10), 5638-5646.
  8. 8. Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. H., Synthesis of Proteins by Native Chemical Ligation. Science 1994, 266 (5186), 776-779;
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