Functional Characterization of the Presenilin Homologue SPE-4

The C. elegans transmembrane sperm protein SPE-4 is the most distant homologue of presenilin (PS), which represents the catalytic subunit of γ-secretase, an intramembranecleaving aspartyl protease complex. Besides PS, γ-secretase consists of three other components, nicastrin (NCT), APH-1 and PEN-2. γ-Secretase catalyzes the intramembrane cleavage of Alzheimer’s disease-associated APP and many other type I transmembrane proteins including Notch, a major physiological γ-secretase substrate, which is required for cell differentiation during development and in adulthood. PS contains two functional aspartates within its transmembrane domains (TMDs) 6 and 7. The active site motif residing in TMD 7 of PS is an unusual GxGD motif, which presents a novel signature motif of a number of intramembrane cleaving aspartyl proteases. Despite its low homology to PS, the two functional aspartates including the GxGD active site motif are conserved in SPE-4 indicating a proteolytic function of this C. elegans PS homologue. In this study, the putative proteolytic activity of SPE-4 was examined. Because it was difficult to analyze the endogenous SPE-4 in C. elegans due to its temporally and spatially limited expression pattern, mammalian cells were chosen as experimental system. Surprisingly, when SPE-4 was expressed in mammalian cells, it did not support γ-secretase complex formation suggesting that SPE-4 cannot interact with human γ-secretase complex components. Consistent with this observation, SPE-4 did not process a recombinant APP-based γ- secretase substrate in vitro. These results indicated that SPE-4 cannot function as a protease alone and/or might have its own partner protein(s) and/or own substrate(s) in C. elegans. SPE-4 was incorporated into a γ-secretase complex only when its C-terminal region had been exchanged with the corresponding region of PS1, which is known to be required for γ-secretase complex assembly. Although the chimeric protein SPE-4/PS1c showed an interaction with NCT, it failed to support APP processing. This was due to the failure of an efficient interaction with PEN-2 thus resulting in incomplete γ-secretase complex formation. This result suggested a requirement of other PS domains for assembly of an active γ-secretase complex in addition to its C-terminus. Because SPE-4/PS1c failed to be incorporated into a functional γ-secretase complex, the proteolytic function of the putative SPE-4 active site domain was subsequently directly assessed by an active site domain exchange approach with PS. To this end, an active site chimeric protein PS1/SPE- 46/7, in which TMDs 6 and 7 of PS1 had been replaced by those of SPE-4, was constructed. This chimeric protein PS1/SPE-46/7 underwent normal γ-secretase complex formation and was able to process APP, demonstrating that the putative active site of SPE-4 has a proteolytic function.

Surprisingly, despite its substantial activity in APP processing, PS1/SPE-46/7 did not support Notch processing. Mapping the residue responsible for the Notch processing defect revealed that a single amino acid at position x of the GxGD active site motif in TMD 7 of PS affects Notch processing.

During the progress of this study, the presence of two substrate-binding sites in the γ- secretase complex was reported. One site is provided by NCT, which mediates the initial substrate recognition by recognizing the length of the ectodomain-shedded substrates. The other binding site, termed docking site, was mapped very close to the active site within a distance of three amino acids. The results of this study identifying a single amino acid critical for APP/Notch substrate selectivity, which is only two amino acids away from the active site aspartate supports the concept of a substrate-docking site close to the active site, and suggests that the GxGD active site motif region may be a part of it.

Taken together, the findings in this thesis suggest a proteolytic function of SPE-4 in C. elegans and indicate an important role of the GxGD active site motif of PS in substrate identification in addition to its role in the catalytic function of γ-secretase.