Glu332 in the nicastrin ectodomain is essential for γ-secretase complex maturation but not for its activity

Lucía Chávez-Gutiérrez, Alexandra Tolia, Elke Maes, Tong Li, Philip C. Wong, Bart De Strooper

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The γ-secretase complex is responsible for the proteolysis of integral membrane proteins. Nicastrin has been proposed to operate as the substrate receptor of the complex with the glutamate 332 (Glu333 in human) serving as the anionic binding site for the α-amino-terminal group of substrates. The putative binding site is located within the aminopeptidase-like domain of Nicastrin. The Glu332 is proposed to function as the counterpart of the exopeptidase Glu located in the active site of these peptidases. Although Glu332 could bind the α-amino-terminal group of substrates, we hypothesized, in analogy with M28-aminopeptidases, that other residues in the putative binding site of Nicastrin should participate in the interaction as well. Surprisingly, mutagenesis of these residues affected the in vivo processing of APP and Notch substrates only weakly. In addition, the E332Q mutation, which completely abolishes the anionic α-amino-terminal binding function, remained fully active. When we introduced the previously characterized E332A mutation, we found strongly decreased γ-secretase complex levels, but the remaining complex appeared as active as the wild-type complex. We confirmed in two independent in vitro assays that the specific enzymatic activity of the E332A mutant was comparable with that of the wild-type complex. Thus, Glu332 crucially affects complex maturation rather than substrate recognition. Moreover other Nicastrin mutants, designed to either impede or alter substantially the putative binding pocket, affected only marginally γ-secretase activity. Consequently, these studies indicate that the main role of the Glu332 is in the maturation and assembly of γ-secretase rather than in the recognition of the substrates.

Original languageEnglish (US)
Pages (from-to)20096-20105
Number of pages10
JournalJournal of Biological Chemistry
Issue number29
StatePublished - Jul 18 2008

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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