Secretome Analysis Identifies Novel Signal Peptide Peptidase-Like 3 (SPPL3) Substrates and Reveals a Role of SPPL3 in Multiple Golgi Glycosylation Pathways*

Abstract

Signal peptide peptidase-like 3 (SPPL3) is a Golgi-resident intramembrane-cleaving protease that is highly conserved among multicellular eukaryotes pointing to pivotal physiological functions in the Golgi network which are only beginning to emerge. Recently, SPPL3 was shown to control protein N-glycosylation, when 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 the catalytic ectodomains of these enzymes from their type II membrane anchors. Protein glycosylation is a multistep process involving numerous type II membrane-bound enzymes, but it remains unclear whether only few of them are SPPL3 substrates or whether SPPL3 cleaves many of them and thereby controls protein glycosylation at multiple levels. Therefore, to systematically identify SPPL3 substrates we used Sppl3-deficient and SPPL3-overexpression cell culture models and analyzed them for changes in secreted membrane protein ectodomains using the proteomics "secretome protein enrichment with click sugars (SPECS)" method. SPECS analysis identified numerous additional new SPPL3 candidate glycoprotein substrates, several of which were biochemically validated as SPPL3 substrates. All novel SPPL3 substrates adopt a type II topology. The majority localizes to the Golgi network and is implicated in Golgi functions. Importantly, most of the novel SPPL3 substrates catalyze the modification of Nlinked glycans. Others contribute to O-glycan and in particular glycosaminoglycan biosynthesis, suggesting that SPPL3 function is not restricted to N-glycosylation, but also functions in other forms of protein glycosylation. 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. Data are available via ProteomeXchange with identifier PXD001672. Molecular

NK Cell Maturation and Cytotoxicity Are Controlled by the Intramembrane Aspartyl Protease SPPL3

“…SPPL3 has recently been reported to affect glycosylation in the Golgi through inhibitory cleavage and shedding of glycosyl transferases, such as MGAT5 (21, 22). In SPPL3-deficient MEFs, MGAT5 is retained within the cell and there is an increase in higher-order glycosylation (21).…”

“…Both protease-independent and protease-dependent functions for SPPL3 have recently been described (20–22). The phenotypes observed after expression of the protease-dead SPPL3 D271A allele in NK cells reveal that NK cell maturation relies on the proteolytic activity of SPPL3, and suggest that the protease-independent function of SPPL3 in facilitating store-operated calcium entry is not required in this context.…”

“…In a protease-independent manner, SPPL3 functions in the endoplasmic reticulum (ER) in T cells to promote store-operated calcium entry in response to T cell receptor engagement by enhancing the interaction between STIM1 and Orai1 (20). SPPL3 has also been shown to function as a protease in cell lines and murine embryonic fibroblasts (MEFs) to regulate the extent of constitutive complex glycosylation by targeting several glycosylation enzymes for cleavage and shedding from the ER or Golgi (21, 22). …”

Proteolytic ectodomain shedding of membrane proteins in mammals—hardware, concepts, and recent developments

“…While all known SPPL2b substrates require processing by a canonical sheddase before SPPL2b processing can occur within the TM segment (Fluhrer et al , ; Martin et al , , ; Zahn et al , ), SPPL3 was recently found to act as a non‐canonical sheddase independently of the substrates’ ectodomain length (Fig C; Voss et al , ). Proteomic approaches have identified many substrate candidates, in particular with functions in regulation of cellular N‐glycosylation (Voss et al , ; Kuhn et al , ). By shedding of various glycosyltransferases and glycosidases, SPPL3 removes the catalytic domain of these enzymes in the Golgi.…”

Mouse brain proteomics establishes MDGA1 and CACHD1 as in vivo substrates of the Alzheimer protease BACE1

“…The contributing proteases, referred to as sheddases, are mostly themselves membrane‐bound enzymes and often do not only cleave a single substrate, but numerous different ones. Among the sheddases with multiple substrates are rhomboids, a disintegrin and metalloproteases (ADAM), signal peptide peptidase‐like 3 (SPPL3) and the beta‐site APP cleaving enzymes (BACE) BACE1 and BACE2 …”