We read with great interest the manuscript of Zhang et al. They provide preliminary evidence for a predicted novel peptide-binding region in the ectodomain of nicastrin (Nct), which could have a potential role in substrate recruitment to the γ-secretase complex.

We would like to comment on a few specific points in the manuscript:

1. While the inhibitory effect of the anti-Nct antibody and the mutagenesis data provide evidence for the involvement of Nct ectodomain in γ-secretase activity and suggest a possible role for Nct in substrate recruitment, alternative interpretations, such as steric hindrance caused by the binding of the antibody or effects on the overall conformation of the complex, are also possible, and have not been excluded or discussed in this study. As the authors point out, it is likely that only crystallization studies will provide a definitive answer to this question, and, in fact, we completely agree with this conclusion.

2. In our previous study, we proposed that the nicastrin ectodomain was necessary for maturation of γ-secretase, but not for...  Read more

We read with great interest the manuscript of Zhang et al. They provide preliminary evidence for a predicted novel peptide-binding region in the ectodomain of nicastrin (Nct), which could have a potential role in substrate recruitment to the γ-secretase complex.

We would like to comment on a few specific points in the manuscript:

1. While the inhibitory effect of the anti-Nct antibody and the mutagenesis data provide evidence for the involvement of Nct ectodomain in γ-secretase activity and suggest a possible role for Nct in substrate recruitment, alternative interpretations, such as steric hindrance caused by the binding of the antibody or effects on the overall conformation of the complex, are also possible, and have not been excluded or discussed in this study. As the authors point out, it is likely that only crystallization studies will provide a definitive answer to this question, and, in fact, we completely agree with this conclusion.

2. In our previous study, we proposed that the nicastrin ectodomain was necessary for maturation of γ-secretase, but not for its activity (Chávez-Gutiérrez et al., 2008). In reference to nicastrin as a substrate acceptor, the authors state of our findings:

“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. However, the methods used to calculate specific activity in the latter report had several significant technical limitations....”

This statement reiterates a criticism initially raised by Dries and colleagues (Dries et al., 2009), and we would like to take the opportunity to respond to these comments here. This criticism emerges from a misunderstanding of how the specific activities (activity per unit mass of total γ-secretase) of the WT and E332A-mutant γ-secretase complexes were calculated.

In Chavez-Gutiérrez et al., 2008, we stated that “for specific activities AICD was normalized against Ps1 CTF and PEN-2 present in the reactions.” Actually, AICD product was normalized to the average of Ps1 CTF and PEN-2 levels present in the reactions and not to the sum of Ps1 CTF and PEN-2 levels, as assumed by Dries et al. In fact, Figure 5 (Chávez-Gutiérrez et al., 2008) shows in white bars the levels of WT- and E332A-Nct γ-secretase complexes and, accordingly, the legend indicates “γ-secretase levels as average of Ps1 CTF and Pen-2 levels, assessed by Western blot in the in vitro reactions.” We would like to highlight that Ps1 CTF and PEN-2 levels are decreased to a very similar extent in the Nct mutant complexes, as indicated by the error bars (white bars). Therefore, our work shows that rescue of Nct-deficient cells with the E332A-Nct mutant restores approximately 10 percent of the -secretase complex levels, relative to rescue with WT-Nct. Most importantly, our study demonstrated that the -secretase complexes that contain the E332A-Nct are as active as the WT-Nct -secretase complexes.

3. Zhang et al. also claim in the discussion that “Dries et al. established that complexes expressed in Sf9 cells that contained the E333A NCT were indeed inactive.”

According to the model proposed by Shah et al., the E332-NCT is the counterpart of a glutamate residue (anionic binding site) involved in the recognition of the free amino group of substrates and inhibitors in aminopeptidases. Importantly, mutation of this glutamate in aminopeptidases leads to complete enzymatic inactivation (Luciani et al., 1998; Vazeux et al., 1998). Thus, if the Shah model is valid for Nct, a drastic effect on the activity of the mutant E332A-NCT γ-secretase complex is expected. However, in actuality, Dries et al. reported a mild effect on the activity of the E333A-NCT γ-secretase complex. The E332A substitution in NCT lowers AICD production by a maximum of 30 percent relative to the WT-NCT, when γ-secretase assembly is overcome in Sf9 cells. This result contrasts with the critical role assigned to the E333 residue by Shah et al. and, in fact, supports our findings (Chávez-Gutiérrez et al., 2008).

In conclusion, it is clear that the substrate binding site in γ-secretase will remain a hot topic of research for the time to come, especially since it might be possible that specific modulation of such a site could provide alternative therapies. As said, we will probably need to wait for the atomic structure of the γ-secretase.

References:
Chavez-Gutierrez L, Tolia A, Maes E, Li T, Wong PC, de Strooper B (2008) Glu(332) in the Nicastrin ectodomain is essential for gamma-secretase complex maturation but not for its activity. J Biol Chem 283(29): 20096-105. Abstract

Dries DR, Shah S, Han YH, Yu C, Yu S, Shearman MS, Yu G (2009) Glu-333 of nicastrin directly participates in gamma-secretase activity. J Biol Chem 284(43): 29714-24. Abstract

Luciani N, Marie-Claire C, Ruffet E, Beaumont A, Roques BP, Fournie-Zaluski MC (1998) Characterization of Glu350 as a critical residue involved in the N-terminal amine binding site of aminopeptidase N (EC 3.4.11.2): insights into its mechanism of action. Biochemistry 37(2): 686-92. Abstract

Shah S, Lee SF, Tabuchi K, Hao YH, Yu C, LaPlant Q, Ball H, Dann CE, 3rd, Sudhof T, Yu G (2005) Nicastrin functions as a gamma-secretase-substrate receptor. Cell 122(3): 435-47. Abstract

Vazeux G, Iturrioz X, Corvol P, Llorens-Cortes C (1998) A glutamate residue contributes to the exopeptidase specificity in aminopeptidase A. Biochem J 334: 407-13. Abstract

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