Elucidating the roles of Alzheimer disease-associated proteases and the signal-peptide peptidase-like 3 (SPPL3) in the shedding of glycosyltransferases

Abstract

The Golgi resident glycosyltransferases (GTs) are membrane-bound glycoproteins but are frequently found as soluble proteins in biological fluids where their function remains largely unknown. Previous studies have established that the release of these proteins involved Alzheimer disease-associated proteases such as {beta}-secretases (BACE1 and BACE2) and the intramembrane-cleaving aspartyl proteases Presenilins 1 and 2. Recent studies have involved another intramembrane-cleaving enzyme, the signal peptide peptidese-like-3 (SPPL3). Except for the latter, the two former studies mostly addressed particular cases of GTs, namely ST6Gal-I (BACEs) or GnT-V (Presenilins). Therefore the question still remains as which of these secretases is truly responsible for the cleavage and secretion of GTs. We herein combined the 3 proteases in a single study with respect to their abilities to release 3 families of GTs encompassing three N-acetylglucosaminyltransferases, two fucosyltransferases and two sialyltransferases. Green fluorescent protein (gfp)-fused versions of these GTs were virally transduced in mouse embryonic fibroblasts devoid of BACEs, Presenilins or SPPL3. We found that neither BACE nor Presenilins are involved in the shedding of these glycosyltransferases, while SPPL3 was involved in the cleavage and release of some but not all GTs. Notably, the {gamma}- secretase inhibitor DFK-167 was the only molecule capable of significantly decreasing glycosyltransferase secretion, suggesting the involvement of {gamma}-secretase(s), yet different from Presenilins but comprising SPPL3 among other proteases still to be identified. Using confocal microscopy, we show that SPPL3 selectivity towards GTs relays not only on sequence specificity but also depends on how GTs distribute in the cell with respect SPPL3 during their cycling within and outside the Golgi.