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 N-linked 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.

Fig. 1.

Secretome analysis of HEK293 cells ectopically expressing active SPPL3. A, Pie chart of all glycoproteins which have been identified in at least 4 out of 5 biological replicates of the HEK293 secretome subdivided according to their topology and the presence of a transmembrane domain. B, Volcano plot: Plotted is the negative lg of the p value (y axis) of log2 LFQ values versus log2 LFQ ratio of SPPL3 overexpressing and control cells (x axis) of a given identified protein. The interspaced light gray line defines the significance level of p = 0.05 (Student's t test, multiple hypothesis testing was not applied). Almost exclusively type II membrane proteins clustered in the upper right quadrant upon SPPL3 overexpression. The previously identified substrate MAN1B1 (9) is given in bold. C, Pie chart of all proteins that were significantly increased in secretomes of HEK293 cells overexpressing SPPL3.

Fig. 2.

Immunoblot validation of novel SPPL3 candidate substrates identified in HEK293 cells overexpressing catalytically active SPPL3. Levels of secreted (s) and cellular XylT2 (A), HS6ST2 (B), HS6ST1 (C), β3GalT6 (D), Sgk196 (E), and EXTL3 (F) were analyzed by immunoblotting in TCA-precipitated conditioned supernatants (sup) and whole-cell lysates, respectively, obtained from HEK293 cells. Where indicated, cells were transiently transfected with nontargeting control siRNA pools (CTRL) or with siRNA pools specific for SPPL3 (20 nm). siRNA pools targeting the individual substrates were transfected to control for antibody specificity. SPPL3 was ectopically expressed in a HEK293 cell line stably transfected with SPPL3 under control of a doxycycline-sensitive repressor. To induce SPPL3 overexpression, media were supplemented with doxycycline (+ Dox) throughout the experiment, whereas control cells were left untreated (− Dox). In all panels calnexin was used as a loading control. **, Sgk196 specific band of unknown nature.

Fig. 3.

Secretome analysis of Sppl3-deficient MEF. A, Pie chart of all glycoproteins which have been identified in at least four out of five biological replicates of the MEF secretome subdivided according to their topology and the presence of a transmembrane domain. B, Volcano plot: Plotted is the values of the negative lg of the p value of log2 intensity ratios (y axis) versus the log2 ratio of intensity values of Sppl3 knockout MEF and wild type MEF (x axis) of a given identified protein. The interspaced light gray line defines the significance level of p = 0.05. Type II membrane proteins were almost exclusively reduced and thus clustered in the left half of the volcano. The previously identified substrates are given in bold, those also identified in the SPPL3 overexpression secretome analysis in italics.

Fig. 4.

Immunoblot validation of novel candidate Sppl3 substrates identified in Sppl3-deficient MEF. Selected type II membrane protein hits (A, B) and the type I membrane protein Integrin α5 (C, D) identified in the MEF secretome analysis (Fig. 3 and Table II) were validated by immunoblotting using specific antibodies in Sppl3-deficient MEF (A, C) and in HEK293 cells (B, D). Secreted (s) and cellular amounts of the respective protein hits were analyzed in TCA-precipitated conditioned supernatants (sup) and whole-cell lysates, respectively. Sppl3 knock-down and overexpression in HEK293 cells (B, D) were achieved as detailed in Fig. 2. The previously identified Sppl3 substrates GnT-V (gene name: MGAT5) and β4GalT1 (gene name: B4GALT1) were used as controls. In all panels calnexin was used as a loading control. *, nonspecific band.

Fig. 5.

Cleavage site analysis of SPPL3 substrates. Selected semitryptic (red) and selected tryptic peptides (green) identified in the secretome analyses are highlighted within the context of the respective full-length precursor and its TMD. An arrowhead indicates nontryptic cleavage sites. TMDs are shown by dashed lines with Uniprot annotations given in green and TMHMM v2.0 predictions (http://www.cbs.dtu.dk/services/TMHMM/) in blue. Nontryptic cleavage events are highlighted by a red arrowhead. MGAT5 and B4GALT1 were identified as SPPL3 substrates previously and were confirmed in this study (9) and ATP1B1, MGAT1, and MGAT2 were identified as novel candidate substrates. B4GALT3 and A4GAT peptides were detected in proteomic datasets but no statistically significant differences in secretion following SPPL3 overexpression or Sppl3 knockout were observed. hs, human; mm, murine.

Fig. 6.

Schematic of the heparan sulfate biosynthesis. Heparan sulfate biosynthesis on serine residues of proteoglycans is illustrated according to (41). Enzymes involved in the individual steps are indicated. The tri-saccharide precursor generated by GlucAT1 activity can also give rise to chondroitin and dermatan sulfate glycosaminoglycans (not depicted). Heparan sulfate polymerization is catalyzed by successive addition of β1,4GlcA and α1,4GlcNAc by the EXT1/EXT2 heterodimer. SPPL3 substrates identified in the secretome analysis in HEK293 cells overexpressing SPPL3 (red boxes), in Sppl3-deficient MEF (green boxes), and in both (box filled with red-to-green gradient) are highlighted.