Glu332 in the Nicastrin Ectodomain Is Essential for γ-Secretase Complex Maturation but Not for Its Activity

Abstract

The ␥-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 (Glu 333 in human) serving as the anionic binding site for the ␣-amino-terminal group of substrates. The putative binding site is located within the aminopeptidase-like domain of Nicastrin. The Glu 332 is proposed to function as the counterpart of the exopeptidase Glu located in the active site of these peptidases. Although Glu 332 could bind the ␣-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 ␣-amino-terminal binding function, remained fully active. When we introduced the previously characterized E332A mutation, we found strongly decreased ␥-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, Glu 332 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 ␥-secretase activity. Consequently, these studies indicate that the main role of the Glu 332 is in the maturation and assembly of ␥-secretase rather than in the recognition of the substrates.

Identification of a tetratricopeptide repeat-like domain in the nicastrin subunit of γ-secretase using synthetic antibodies

“…A region of the NCT ECD, termed the DAP domain, and an essential glutamate (E333) within this domain was shown to be critical for the substrate binding, presumably via a salt-bridge interaction to the exposed amino terminus of the substrate (7). This model was challenged by the demonstration that expression of NCT harboring an E333A mutation in NCT −/− cells leads to inefficient oligosaccharide maturation of the NCT variant and reduced levels of mature γ-secretase complexes, but that the specific activity of the remaining complexes was no different from that of complexes containing WT NCT (11). However, the methods used to calculate specific activity in the latter report had several significant technical limitations, and a subsequent report by Dries et al (10) established that complexes expressed in Sf9 cells that contained the E333A NCT were indeed inactive.…”

“…In this case, glutamate 333 (E333) within the "DAP" domain (DYIGS and peptidase; residues 261-502) was shown to be critical for substrate binding and delivery to the catalytic site (7,10). However, it has been argued that E333 may play an alternative role in enhancing NCT maturation through the secretory pathway (11). A recent study demonstrated that a Notch substrate was processed, albeit weakly, in NCT-deficient (NCT −/− ) fibroblasts treated with a proteasome inhibitor, suggesting that NCT was not absolutely required for γ-secretase activity (12).…”

Activation and intrinsic γ-secretase activity of presenilin 1

“…For example, a mutation of Glu333 in NCT was shown to reduce γ-secretase activity, evidence that was used to support a role of NCT as substrate receptor within the γ-secretase complex (8). In contrast, other studies have reported that the same mutation reduced the formation of the γ-secretase complex (9). Furthermore, a recent report showed the PS1-PEN2-Aph1 complex, in the absence of NCT, is catalytically active under conditions wherein proteosomal degradation is inhibited (10).…”

“…Endoproteolysis of the newly synthesized approximately 52 kDa PS protein to generate amino-terminal (NTF) and carboxyl-terminal (CTF) fragments is a critical step for formation of active γ-secretase complexes (9). Previous studies indicated that PS is responsible for γ-secretase activity and endoproteolytic cleavage (termed presenilinase or PSase) because mutagenesis of two putative catalytic residues of PS1 at Asp257 and Asp385 abolished both γ-secretase and PSase activity (11).…”

Trafficking and Proteolytic Processing of APP

“…Little is known about the biological function of NCT, APH-1, and PEN-2. NCT is probably required as a sizeselecting substrate receptor (Shah et al 2005;Dries et al 2009), although recent findings may challenge such a function (Chavez-Gutierrez et al 2008;Martin et al 2009). PEN-2 apparently facilitates PS endoproteolysis into its active heterodimeric state and stabilizes PS within the g-secretase complex (Hasegawa et al 2004;Prokop et al 2004).…”