Binding epitopes and interaction structure of the neuroprotective protease inhibitor cystatin C with beta-amyloid revealed by proteolytic excision mass spectrometry and molecular docking simulation

J Med Chem. 2009 Apr 23;52(8):2420-8. doi: 10.1021/jm801115e.

Abstract

Human cystatin C (HCC) is a protease inhibitor with a propensity to form beta-amyloid (Abeta)-like fibrils and to coassociate with amyloidogenic proteins. Recently, a specific interaction between HCC and Abeta has been found. Here, we report the identification of the Abeta and HCC binding epitopes in the Abeta-HCC complex, using a combination of selective proteolytic excision and high resolution mass spectrometry. Proteolytic excision of Abeta(1-40) on sepharose-immobilized HCC and MALDI-MS identified the epitope Abeta(17-28). On immobilized Abeta(1-40), affinity MS of HCC fragments identified a specific C-terminal epitope, HCC(101-117). Binding specificities of both epitopes were ascertained by ELISA and surface plasmon resonance and by direct electrospray MS of the HCC-Abeta epitope peptide complexes. A structure model of the HCC-Abeta complex by molecular docking simulation showed full agreement with the identified Abeta and HCC epitopes. Inhibition studies in vitro revealed Abeta-fibril inhibiting activity of the HCC(101-117)-epitope. The Abeta-HCC interacting epitopes provide lead structures of neuroprotective inhibitors for AD and HCC amyloidosis therapy.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Amyloid beta-Peptides / chemistry*
  • Binding Sites
  • Cystatin C / chemistry*
  • Enzyme-Linked Immunosorbent Assay
  • Epitopes
  • Humans
  • Models, Molecular*
  • Molecular Sequence Data
  • Peptide Fragments / chemistry
  • Spectrometry, Mass, Electrospray Ionization
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Surface Plasmon Resonance

Substances

  • Amyloid beta-Peptides
  • Cystatin C
  • Epitopes
  • Peptide Fragments