title: 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.
authors.affiliation: From the DZNE - German Center for Neurodegenerative Diseases, Munich, Germany; Institute for Advanced Study, Technische Universität München, Garching, Germany;
authors.forename: Peer-Hendrik
authors.initials: PH
authors.lastname: Kuhn
authors.affiliation: Institute for Metabolic Biochemistry, Ludwig-Maximilians University Munich, Munich, Germany;
authors.forename: Matthias
authors.initials: M
authors.lastname: Voss
authors.affiliation: Institute for Metabolic Biochemistry, Ludwig-Maximilians University Munich, Munich, Germany;
authors.forename: Martina
authors.initials: M
authors.lastname: Haug-Kröper
authors.affiliation: Biochemical Institute, Christian-Albrechts University Kiel, Olshausenstrasse 40, D-24118 Kiel, Germany;
authors.forename: Bernd
authors.initials: B
authors.lastname: Schröder
authors.affiliation: Institute of Toxicology and Genetics, KIT, Campus North, Hermann-von-Helmholtz-Platz 1, D-76344 Eggenstein-Leopoldshafen, Germany Munich Cluster for Systems Neurology (SyNergy), Munich, Germany;
authors.forename: Ute
authors.initials: U
authors.lastname: Schepers
authors.affiliation: Institute of Toxicology and Genetics, KIT, Campus North, Hermann-von-Helmholtz-Platz 1, D-76344 Eggenstein-Leopoldshafen, Germany Munich Cluster for Systems Neurology (SyNergy), Munich, Germany;
authors.forename: Stefan
authors.initials: S
authors.lastname: Bräse
authors.affiliation: From the DZNE - German Center for Neurodegenerative Diseases, Munich, Germany; Institute for Metabolic Biochemistry, Ludwig-Maximilians University Munich, Munich, Germany; Munich Cluster for Systems Neurology (SyNergy), Munich, Germany;
authors.forename: Christian
authors.initials: C
authors.lastname: Haass
authors.affiliation: From the DZNE - German Center for Neurodegenerative Diseases, Munich, Germany; Institute for Advanced Study, Technische Universität München, Garching, Germany; Munich Cluster for Systems Neurology (SyNergy), Munich, Germany; Neuroproteomics, Klinikum rechts der Isar, Technische Universität München, Munich, Germany.
authors.forename: Stefan F
authors.initials: SF
authors.lastname: Lichtenthaler
authors.affiliation: From the DZNE - German Center for Neurodegenerative Diseases, Munich, Germany; Institute for Metabolic Biochemistry, Ludwig-Maximilians University Munich, Munich, Germany; regina.fluhrer@med.uni-muenchen.de stefan.lichtenthaler@dzne.de.
authors.forename: Regina
authors.initials: R
authors.lastname: Fluhrer
mesh_terms: D016282:Aspartic Acid Endopeptidases / Q000378:metabolism*; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans
chemical_list: D016282:Aspartic Acid Endopeptidases; C513914:signal peptide peptidase like 3, human
references.citation: EMBO J. 2012 Jul 18;31(14):3157-68
references.pmid: 22728825
references.citation: BMC Bioinformatics. 2014;15:207
references.pmid: 24946880
references.citation: Elife. 2014;3. doi: 10.7554/eLife.03943
references.pmid: 25279697
references.citation: Science. 2002 Jun 21;296(5576):2215-8
references.pmid: 12077416
references.citation: Nat Protoc. 2006;1(6):2856-60
references.pmid: 17406544
references.citation: J Biol Chem. 2008 Jan 18;283(3):1644-52
references.pmid: 17965014
references.citation: BMC Bioinformatics. 2009;10:48
references.pmid: 19192299
references.citation: Cell Mol Life Sci. 2008 May;65(9):1311-34
references.pmid: 18239854
references.citation: Nucleic Acids Res. 2013 Jul;41(Web Server issue):W459-64
references.pmid: 23729472
references.citation: J Cell Biol. 2014 Jun 23;205(6):847-62
references.pmid: 24958774
references.citation: Biochemistry (Mosc). 2002 Jul;67(7):826-35
references.pmid: 12139484
references.citation: J Biol Chem. 2003 Apr 25;278(17):14865-71
references.pmid: 12473667
references.citation: Clin Chim Acta. 2002 Mar;317(1-2):199-202
references.pmid: 11814476
references.citation: EMBO J. 2014 Dec 17;33(24):2890-905
references.pmid: 25354954
references.citation: Mol Cell. 2000 Sep;6(3):625-36
references.pmid: 11030342
references.citation: Biochim Biophys Acta. 2013 Dec;1828(12):2828-39
references.pmid: 24099004
references.citation: J Biol Chem. 2012 Feb 10;287(7):5156-63
references.pmid: 22194595
references.citation: J Invest Dermatol. 1999 Jun;112(6):919-24
references.pmid: 10383739
references.citation: Beilstein J Org Chem. 2013;9:56-63
references.pmid: 23399592
references.citation: J Cell Biol. 2005 Mar 14;168(6):855-62
references.pmid: 15767459
references.citation: Hum Mutat. 2012 Aug;33(8):1261-6
references.pmid: 22539336
references.citation: J Biol Chem. 2009 Feb 27;284(9):5662-70
references.pmid: 19114711
references.citation: J Proteome Res. 2011 Apr 1;10(4):1794-805
references.pmid: 21254760
references.citation: Nat Biotechnol. 2008 Dec;26(12):1367-72
references.pmid: 19029910
references.citation: Annu Rev Genet. 2008;42:191-210
references.pmid: 18605900
references.citation: J Biol Chem. 2008 Oct 31;283(44):29627-31
references.pmid: 18650432
references.citation: J Biol Chem. 2012 Dec 21;287(52):43401-9
references.pmid: 23132852
references.citation: Mol Biol Cell. 2011 Aug 15;22(16):2810-22
references.pmid: 21697506
references.citation: Mol Cell Biol. 2015 Jan;35(2):451-67
references.pmid: 25384971
references.citation: Biochem J. 1997 Jul 15;325 ( Pt 2):351-7
references.pmid: 9230113
references.citation: PLoS Biol. 2008 Oct 21;6(10):e257
references.pmid: 18942891
references.citation: Elife. 2014;3. doi: 10.7554/eLife.03941
references.pmid: 25279699
references.citation: Nat Rev Mol Cell Biol. 2002 Jul;3(7):509-19
references.pmid: 12094217
references.citation: Hum Mol Genet. 2002 May 1;11(9):1037-44
references.pmid: 11978763
references.citation: J Biol Chem. 2001 Dec 21;276(51):48189-95
references.pmid: 11551958
references.citation: Mol Cell. 2002 Oct;10(4):735-44
references.pmid: 12419218
references.citation: Science. 2013 Aug 23;341(6148):896-9
references.pmid: 23929950
references.citation: Mol Cell. 2009 Dec 25;36(6):1048-59
references.pmid: 20064469