Different transmembrane domains determine the specificity and efficiency of the cleavage activity of the -secretase subunit presenilin

The gamma-secretase complex catalyzes the intramembrane cleavage of C99, a carboxy-terminal fragment of the amyloid precursor protein. Two paralogs of its catalytic subunit presenilin (PS1 and PS2) are expressed which are autocatalytically cleaved into an N-terminal and a C-terminal fragment during maturation of gamma-secretase. In this study, we compared the efficiency and specificity of C99 cleavage by PS1- and PS2-containing gamma-secretases. Mass spectrometric analysis of cleavage products obtained in cell-free and cell-based assays revealed that the previously described lower amyloid-beta (A beta)38 generation by PS2 is accompanied by a reciprocal increase in A beta 37 production. We further found PS1 and PS2 to show different preferences in the choice of the initial cleavage site of C99. However, the differences in A beta 38 and A beta 37 generation appear to mainly result from altered subsequent stepwise cleavage of A beta peptides. Apart from these differences in cleavage specificity, we confirmed a lower efficiency of initial C99 cleavage by PS2 using a detergent-solubilized gamma-secretase system. By investigating chimeric PS1/2 molecules, we show that the membrane-embedded, nonconserved residues of the N-terminal fragment mainly account for the differential cleavage efficiency and specificity of both presenilins. At the level of individual transmembrane domains (TMDs), TMD3 was identified as a major modulator of initial cleavage site specificity. The efficiency of endoproteolysis strongly depends on nonconserved TMD6 residues at the interface to TMD2, i.e., at a putative gate of substrate entry. Taken together, our results of C99 and the generation of A beta peptides.