May 2005
Volume 46, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2005
Mutational Analysis of the Heparin– and Neuropilin–1–Binding Domains of VEGF164
Author Affiliations & Notes
  • D. Krilleke
    Drug Discovery Group, Eyetech Pharmaceuticals, Inc., Woburn, MA
  • Y.S. Ng
    Drug Discovery Group, Eyetech Pharmaceuticals, Inc., Woburn, MA
  • D.T. Shima
    Drug Discovery Group, Eyetech Pharmaceuticals, Inc., Woburn, MA
  • Footnotes
    Commercial Relationships  D. Krilleke, Eyetech Pharmaceuticals E; Y.S. Ng, Eyetech Pharmaceuticals E; D.T. Shima, Eyetech Pharmaceuticals E.
  • Footnotes
    Support  Eyetech Pharmaceuticals
Investigative Ophthalmology & Visual Science May 2005, Vol.46, 1362. doi:
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      D. Krilleke, Y.S. Ng, D.T. Shima; Mutational Analysis of the Heparin– and Neuropilin–1–Binding Domains of VEGF164 . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1362.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Abstract: : Purpose: The heparin–binding domain in the C–terminal of VEGF164 and VEGF188 confers isoform–specific biological properties in vascular development and disease. The aim of this study is to define the specific binding sites for heparin and the isoform–specific receptor Np–1 in VEGF164. The biological activity and binding to heparin and Np–1 by different VEGF164 heparin–binding domain mutants (1, 2, 3 basic amino acids were replaced with alanines) was evaluated. Methods: Wild type and mutated VEGF164 proteins were produced using the inducible yeast Pichia Pastoris protein production system. The biological activity of the different VEGF164 mutants was compared with wild type by qRT–PCR analysis of VEGF–induced tissue factor gene expression. The heparin filter–binding assay was used to determine the apparent binding affinity (KD) of VEGF–heparin interaction. Np–1 binding affinity was determined using a VEGF/VEGF–NP–1 competition assay. Data from the binding assays were analyzed by non–linear regression to determine KD and IC50 values. Results: All mutant VEGF proteins showed biological activity comparable to that of wild–type as determined by VEGF–induced tissue factor expression. Heparin–binding assays revealed critical amino acids on one side of the C–terminal domain (K30, R35, R39, R46 and R49) and an adjacent loop region (R13, R14) with residues R13/R14/R49 serving as a putative minimal heparin binding site. The mutational analysis showed that Np–1 binding was less strong in mutants that are deficient in heparin–binding indicating a partial overlap of the binding sites for heparin and Np–1. Conclusions: These results suggest electrostatic and protein sequence–specific components in the VEGF–heparin interaction and confer unique biological functions for VEGF. Deletion of the critical residues identified in this study generates VEGF164 mutants that lack heparin–binding and therefore cell surface– and matrix–binding activity. These heparin–binding deficient VEGF mutants can help to characterize the mechanism of action of the heparin–binding VEGF isoforms, and help explore the therapeutic potential of interfering with the heparin–binding domain of VEGF.

Keywords: protein structure/function • growth factors/growth factor receptors 
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