Single chain variable fragment against nicastrin inhibits the gamma-secretase activity

J Biol Chem. 2009 Oct 9;284(41):27838-27847. doi: 10.1074/jbc.M109.055061. Epub 2009 Aug 14.

Abstract

Gamma-secretase is a membrane protein complex that catalyzes intramembrane proteolysis of a variety of substrates including the amyloid beta precursor protein of Alzheimer disease. Nicastrin (NCT), a single-pass membrane glycoprotein that harbors a large extracellular domain, is an essential component of the gamma-secretase complex. Here we report that overexpression of a single chain variable fragment (scFv) against NCT as an intrabody suppressed the gamma-secretase activity. Biochemical analyses revealed that the scFv disrupted the proper folding and the appropriate glycosyl maturation of the endogenous NCT, which are required for the stability of the gamma-secretase complex and the intrinsic proteolytic activity, respectively, implicating the dual role of NCT in the gamma-secretase complex. Our results also highlight the importance of the calnexin cycle in the functional maturation of the gamma-secretase complex. The engineered intrabodies may serve as rationally designed, molecular targeting tools for the discovery of novel actions of the membrane proteins.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Amyloid Precursor Protein Secretases / antagonists & inhibitors*
  • Amyloid Precursor Protein Secretases / chemistry
  • Amyloid Precursor Protein Secretases / genetics
  • Amyloid Precursor Protein Secretases / immunology
  • Amyloid Precursor Protein Secretases / metabolism*
  • Animals
  • Antibodies / genetics
  • Antibodies / immunology*
  • Cell Line
  • Enzyme Inhibitors / metabolism
  • Humans
  • Immunoglobulin Variable Region / genetics
  • Immunoglobulin Variable Region / metabolism*
  • Indolizines / metabolism
  • Membrane Glycoproteins / chemistry*
  • Membrane Glycoproteins / genetics
  • Membrane Glycoproteins / immunology*
  • Molecular Sequence Data
  • Protein Binding
  • Protein Conformation
  • Protein Folding
  • Protein Structure, Tertiary
  • Sequence Alignment

Substances

  • Antibodies
  • Enzyme Inhibitors
  • Immunoglobulin Variable Region
  • Indolizines
  • Membrane Glycoproteins
  • nicastrin protein
  • Amyloid Precursor Protein Secretases
  • castanospermine