Glu(332) in the Nicastrin ectodomain is essential for gamma-secretase complex maturation but not for its activity.
The gamma-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 alpha-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 alpha-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 alpha-amino-terminal binding function, remained fully active. When we introduced the previously characterized E332A mutation, we found strongly decreased gamma-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 gamma-secretase activity. Consequently, these studies indicate that the main role of the Glu(332) is in the maturation and assembly of gamma-secretase rather than in the recognition of the substrates.
Chávez-Gutiérrez
Lucía
L
Center for Human Genetics, KULeuven, and Department for Molecular and Developmental Genetics, VIB4, Herestraat 49, Leuven, Belgium.
Tolia
Alexandra
A
Maes
Elke
E
Li
Tong
T
Wong
Philip C
PC
de Strooper
Bart
B
D000595:Amino Acid Sequence; D053829:Amyloid Precursor Protein Secretases / Q000737:chemistry / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D017124:Conserved Sequence; D015870:Gene Expression; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D013379:Substrate Specificity
D008562:Membrane Glycoproteins; C413604:nicastrin protein; D018698:Glutamic Acid; D053829:Amyloid Precursor Protein Secretases