SPPL3 cleavage of glycosyltransferases regulates their activity

The GxGD intramembrane-cleaving aspartylprotease signal peptide peptidase-like 3 (SPPL3) is a Golgi-resident, multi-pass membrane protein that is highly conserved among multicellular eukaryotes pointing to a pivotal physiological function in the Golgi network. Recently, the key branching enzyme N-acetylglucosaminyltransferase V (GnT-V) and other medial/trans Golgi glycosyltransferases were identified as first physiological SPPL3 substrates. SPPL3-mediated endoproteolysis releases these enzymes from their type II membrane anchors resulting in a subsequent secretion of the respective enzymes’ ectodomain. To systematically identify further SPPL3 substrates we used the secretome protein enrichment with click sugars (SPECS) method that makes secretomes of cultured cells accessible to mass spectrometric analyses. SPECS analyses of SPPL3-deficient and SPPL3-overexpression cell culture models revealed numerous additional SPPL3 candidate substrates and their subsequent biochemical validation underscores the role of SPPL3 in secretion of these proteins. In line with our previous observations, all novel SPPL3 substrates adopt a type II topology and the majority localizes to the Golgi network and is implicated in Golgi function. Importantly, most of the novel SPPL3 substrates catalyze the modification of N-linked glycans but also of O-glycans and glycosaminoglycans. Hence, SPPL3 emerges as a crucial player of Golgi function and the newly identified SPPL3 substrates will be instrumental to investigate the molecular mechanisms underlying the physiological function of SPPL3 in the Golgi network and in vivo.