{"gene":"SPPL3","run_date":"2026-04-28T20:42:08","timeline":{"discoveries":[{"year":2014,"finding":"SPPL3 is a Golgi-resident intramembrane-cleaving aspartyl protease of the GxGD type that proteolytically releases active site-containing ectodomains of glycosidases and glycosyltransferases (including N-acetylglucosaminyltransferase V, β-1,3 N-acetylglucosaminyltransferase 1, and β-1,4 galactosyltransferase 1), thereby reducing their cellular activity and altering global N-glycosylation patterns. Reduced SPPL3 expression causes hyperglycosylation; elevated SPPL3 causes hypoglycosylation.","method":"Overexpression and knockdown cell culture models, biochemical substrate cleavage assays, glycosylation profiling","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1–2 — multiple orthogonal methods (biochemical cleavage, gain/loss-of-function, glycan profiling) in single rigorous study; independently replicated","pmids":["25354954"],"is_preprint":false},{"year":2005,"finding":"SPPL3 is localized to the endoplasmic reticulum (ER), in contrast to SPPL2b which localizes to endosomes/lysosomes. Knockdown of sppl3 in zebrafish causes cell death in the CNS, and expression of a D/A mutation in the putative C-terminal active site phenocopies the sppl3 knockdown, demonstrating SPPL3 is a catalytically active GXGD-type aspartyl protease.","method":"Subcellular localization studies in cultured cells, antisense gripNA-mediated knockdown in zebrafish, active-site mutagenesis (D/A mutation)","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 — active-site mutagenesis combined with in vivo knockdown and localization; moderate evidence base","pmids":["15998642"],"is_preprint":false},{"year":2012,"finding":"SPPL3 cleaves the 18-kDa leader peptide (LP18) of the foamy virus envelope protein (FVenv), acting as a sheddase capable of cleaving substrates with large ectodomains without requiring prior shedding—unlike SPPL2a/b and γ-secretase. The SPPL3-generated cleavage product of FVenv then serves as a substrate for consecutive intramembrane cleavage by SPPL2a/b.","method":"In vitro cleavage assay with human SPPL3 and FVenv mutants, biochemical identification of cleavage products","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — direct in vitro cleavage assay with mutant substrates demonstrating unique sheddase-like activity","pmids":["23132852"],"is_preprint":false},{"year":2015,"finding":"Secretome proteomics (SPECS method) of SPPL3-deficient and SPPL3-overexpressing cells identified numerous Golgi-localized type II membrane proteins as SPPL3 substrates, extending beyond N-glycosylation to include O-glycan and glycosaminoglycan biosynthesis enzymes. SPPL3-mediated endoproteolysis releases catalytic ectodomains of these type II membrane enzymes from their membrane anchors.","method":"SPECS proteomics on SPPL3-deficient and overexpression cell culture models, biochemical validation of candidate substrates","journal":"Molecular & cellular proteomics : MCP","confidence":"High","confidence_rationale":"Tier 1–2 — systematic secretome proteomics with biochemical validation across multiple substrates","pmids":["25827571"],"is_preprint":false},{"year":2014,"finding":"SPPL3 is required downstream of T cell receptor engagement for maximal Ca2+ influx and NFAT activation in a protease-independent manner. SPPL3 enhances the signal-induced association of STIM1 and Orai1, associates with STIM1 through its transmembrane region and the CRAC activation domain (CAD), and promotes STIM1 CAD association with Orai1.","method":"Screen for NFAT activators, Ca2+ influx measurements, Co-immunoprecipitation of SPPL3 with STIM1/Orai1, catalytic mutant analysis","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP, catalytic mutant, functional Ca2+ and NFAT assays in single study","pmids":["25384971"],"is_preprint":false},{"year":2016,"finding":"SPPL3 protease activity is required cell-autonomously for efficient NK cell maturation and cytotoxicity. CRISPR/Cas9-generated knockin mice expressing catalytically compromised SPPL3 D271A phenocopy SPPL3 deletion, showing reduced CD27+CD11b+ and CD27−CD11b+ NK cell numbers and impaired tumor killing.","method":"Hematopoietic- and NK cell-specific SPPL3 deletion, CRISPR/Cas9 knockin of D271A catalytic mutant, in vivo tumor clearance and in vitro cytotoxicity assays","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 1–2 — catalytic mutant knockin combined with conditional KO and in vivo/in vitro functional assays","pmids":["26851218"],"is_preprint":false},{"year":2020,"finding":"Loss of SPPL3 augments B3GNT5 enzyme activity, resulting in upregulation of surface neolacto-series glycosphingolipids (nsGSLs) that sterically impede antibody and receptor interactions with HLA class I, diminishing CD8+ T cell activation. SPPL3 thus controls the GSL repertoire to regulate adaptive immune recognition.","method":"Iterative genome-wide CRISPR screens, GSL profiling, functional HLA-I antigen presentation assays, CD8+ T cell activation assays","journal":"Immunity","confidence":"High","confidence_rationale":"Tier 1–2 — genome-wide screens with orthogonal biochemical and functional validation; multiple methods","pmids":["33271119"],"is_preprint":false},{"year":2017,"finding":"In melanocyte-to-melanoma transformation, SPPL3-mediated activation of ADAM10 is a critical transformation event. This involves translocation of SPPL3 and ADAM10 into Rab4- or Rab27-positive endosomal compartments triggered by mutant BRAFV600E, and this endosomal translocation is inhibited by tumor suppressor PTEN.","method":"Multiepitope ligand cartography (MELC) tissue analysis, co-localization of SPPL3 and ADAM10 in endosomes, functional transformation assays in patient tissues and cell co-cultures","journal":"Science signaling","confidence":"Medium","confidence_rationale":"Tier 2–3 — co-localization and tissue analysis with functional consequence; single lab, single study","pmids":["28292959"],"is_preprint":false},{"year":2022,"finding":"N-terminomics analysis of SPPL3-deficient and active-site knock-in (D271A) HEK293 and HeLa cells identified over 20 SPPL3 substrates including novel ones (e.g., GALNT2), provided comprehensive SPPL3 cleavage sites demonstrating intramembrane proteolysis, and showed that transmembrane domain composition determines susceptibility to SPPL3 cleavage.","method":"N-terminomics (TAILS) on isogenic SPPL3 KO and D271A knockin cell lines, chimeric glycosyltransferase constructs, immunoblot validation","journal":"Cellular and molecular life sciences : CMLS","confidence":"High","confidence_rationale":"Tier 1–2 — N-terminomics with isogenic controls plus active-site mutant knockin and biochemical validation; comprehensive substrate mapping","pmids":["35279766"],"is_preprint":false},{"year":2022,"finding":"Endogenous SPPL3 tagged at the endogenous locus localizes predominantly to the mid-Golgi under steady-state conditions, co-localizing with its substrates. Co-localization alone with type II proteins in the Golgi is not sufficient for cleavage; substrate-intrinsic properties (e.g., transmembrane domain flexibility) additionally govern SPPL3-mediated intramembrane proteolysis.","method":"Genome editing to generate isogenic N- and C-terminally tagged endogenous SPPL3; co-localization with Golgi markers; drug-disruption of Golgi; co-localization with substrates vs non-substrates","journal":"Biochimica et biophysica acta. Molecular cell research","confidence":"High","confidence_rationale":"Tier 1–2 — endogenous tagging via genome editing, isogenic controls, multiple markers; rigorous localization with mechanistic inference","pmids":["36007678"],"is_preprint":false},{"year":2022,"finding":"The GxxxG motif in the transmembrane domain of GnTV (an SPPL3 substrate) regulates SPPL3-dependent cleavage efficiency. Mutations disrupting the GxxxG motif increase transmembrane helix rigidity (assessed by HDX and NMR), reducing SPPL3-dependent ectodomain shedding, while increased helix flexibility facilitates SPPL3-dependent shedding.","method":"Site-directed mutagenesis of GxxxG motif, deuterium/hydrogen exchange MS, NMR spectroscopy of transmembrane peptides, SPPL3 cleavage assays","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 1 — mutagenesis combined with biophysical structural analysis (HDX, NMR) and cleavage assays in single study","pmids":["36470941"],"is_preprint":false},{"year":2004,"finding":"C. elegans ce-imp-2 (ortholog of human SPP/IMP1 rather than IGF2BP2/IMP2) does not promote Notch (lin-12, glp-1) proteolysis or signaling, unlike presenilins, and its knockdown leads to embryonic death and abnormal molting. The molting defect is mimicked by cholesterol depletion or lrp-1 disruption and suppressed by lrp-1 derivative expression, implicating IMP/SPP proteases in lipid-lipoprotein receptor-mediated pathways.","method":"RNAi knockdown in C. elegans, genetic epistasis with Notch pathway components, cholesterol depletion, lrp-1 rescue","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 — epistasis and rescue in vivo; single study; note this concerns Ce-imp-2/SPP ortholog, functionally distinct from IGF2BP2","pmids":["15469912"],"is_preprint":false},{"year":2021,"finding":"SPPL3 expression controls the cell surface staining of CD59 through its intramembrane protease activity by suppressing neolacto-series glycosphingolipid (nsGSL) synthesis. The effect is nsGSL-dependent and not mediated by N-glycan changes, suggesting nsGSLs sterically impair CD59 accessibility.","method":"SPPL3 KO and rescue with protease-dead mutant, nsGSL inhibition, flow cytometry of CD59 and GPI-anchored proteins","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2–3 — active-site mutant and pathway inhibition with functional readout; single lab","pmids":["34303967"],"is_preprint":false},{"year":2024,"finding":"SPPL3-deficient tumor cells are less susceptible to trogocytosis by neutrophils and killing by NK cells and γδ T cells. The nsGSL-dependent SPPL3 sensitivity for trogocytosis and γδ T cell killing depends on proximity of surface receptor domains to the membrane and receptor-ligand interaction affinity; NK cell killing reduction by SPPL3 loss is nsGSL-independent.","method":"SPPL3 KO tumor cell lines, neutrophil trogocytosis assay, NK and γδ T cell killing assays, nsGSL inhibition rescue","journal":"European journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 — multiple immune effector assays with KO and chemical rescue; single study","pmids":["39655358"],"is_preprint":false}],"current_model":"SPPL3 is a mid-Golgi-resident intramembrane-cleaving aspartyl protease of the GxGD type that acts as the primary sheddase for type II membrane-anchored Golgi glycan-modifying enzymes (glycosyltransferases and glycosidases), releasing their catalytic ectodomains through intramembrane proteolysis to reduce their cellular activity and globally regulate protein N-glycosylation, O-glycosylation, and glycosphingolipid biosynthesis; by controlling the glycosphingolipid repertoire (particularly neolacto-series GSLs via B3GNT5), SPPL3 modulates immune recognition including HLA-I antigen presentation and innate immune killing, and additionally exerts a protease-independent function in T cell NFAT signaling by facilitating STIM1–Orai1 association."},"narrative":{"teleology":[{"year":2004,"claim":"Early genetic work in C. elegans showed that the SPP/IMP protease family (including the SPPL3 ortholog) is essential for viability and molting and operates independently of Notch/presenilin signaling, establishing that these GxGD proteases have distinct in vivo functions from γ-secretase.","evidence":"RNAi knockdown and genetic epistasis with Notch components in C. elegans","pmids":["15469912"],"confidence":"Medium","gaps":["C. elegans ortholog studied (ce-imp-2) is closer to SPP than to SPPL3; direct relevance to mammalian SPPL3 uncertain","substrates in worm not identified"]},{"year":2005,"claim":"Demonstration that mammalian SPPL3 is a catalytically active GxGD-type aspartyl protease whose active-site aspartate is essential for function in vivo, with zebrafish knockdown revealing a requirement for CNS cell survival.","evidence":"Active-site D/A mutagenesis, subcellular localization in cultured cells, antisense knockdown in zebrafish","pmids":["15998642"],"confidence":"High","gaps":["endogenous substrates not yet identified","initial localization placed SPPL3 in the ER rather than Golgi"]},{"year":2012,"claim":"SPPL3 was shown to act as a sheddase capable of cleaving substrates with large ectodomains without prior ectodomain trimming—unlike SPPL2a/b and γ-secretase—establishing a unique proteolytic mode within the GxGD family.","evidence":"In vitro cleavage assay using foamy virus envelope protein and mutant substrates","pmids":["23132852"],"confidence":"High","gaps":["only viral substrate tested; endogenous cellular substrates not yet addressed","cleavage site not mapped at residue resolution"]},{"year":2014,"claim":"Identification of Golgi glycosyltransferases and glycosidases as endogenous SPPL3 substrates resolved the key question of SPPL3's physiological function: it is a master regulator of cellular glycosylation by releasing the catalytic ectodomains of glycan-modifying enzymes.","evidence":"Overexpression and knockdown in cultured cells, biochemical cleavage assays, lectin-based glycosylation profiling","pmids":["25354954"],"confidence":"High","gaps":["substrate repertoire not fully defined","mechanism of substrate selectivity unknown"]},{"year":2014,"claim":"A protease-independent role for SPPL3 was discovered in T cell signaling, where it facilitates STIM1–Orai1 coupling to promote store-operated Ca²⁺ entry and NFAT activation, revealing a non-catalytic scaffolding function.","evidence":"Co-immunoprecipitation of SPPL3 with STIM1/Orai1, Ca²⁺ influx measurements, catalytic-dead mutant retains function","pmids":["25384971"],"confidence":"High","gaps":["structural basis of SPPL3–STIM1 interaction unresolved","relevance beyond Jurkat T cells not tested"]},{"year":2015,"claim":"Systematic secretome proteomics greatly expanded the SPPL3 substrate repertoire to include enzymes of O-glycan and glycosaminoglycan biosynthesis, showing SPPL3's regulatory scope extends well beyond N-glycosylation.","evidence":"SPECS proteomics on SPPL3-deficient and overexpressing cells with biochemical validation","pmids":["25827571"],"confidence":"High","gaps":["not all candidate substrates biochemically validated","in vivo confirmation of extended substrate list lacking"]},{"year":2016,"claim":"In vivo evidence established that SPPL3 protease activity is cell-autonomously required for NK cell maturation and cytotoxicity, linking its catalytic function to immune cell development.","evidence":"Hematopoietic-specific SPPL3 KO and CRISPR knockin of D271A catalytic mutant in mice; in vivo tumor clearance assays","pmids":["26851218"],"confidence":"High","gaps":["whether NK cell defect is glycosylation-dependent or involves additional substrates not determined","impact on other immune lineages not fully explored"]},{"year":2020,"claim":"SPPL3 was identified as a critical regulator of immune recognition: loss of SPPL3 elevates B3GNT5-dependent neolacto-series glycosphingolipids that sterically shield HLA-I from antibodies and TCRs, diminishing CD8⁺ T cell activation.","evidence":"Genome-wide CRISPR screens, GSL profiling, HLA-I antigen presentation and T cell activation assays","pmids":["33271119"],"confidence":"High","gaps":["relevance to in vivo tumor immune evasion not directly shown","whether SPPL3 loss also shields other surface receptors via nsGSLs not systematically tested"]},{"year":2022,"claim":"Endogenous tagging and comprehensive N-terminomics resolved SPPL3's steady-state localization to the mid-Golgi and identified >20 substrates with mapped cleavage sites, establishing that transmembrane domain composition—not mere co-localization—determines cleavage susceptibility.","evidence":"Genome editing for endogenous tagging, TAILS N-terminomics on isogenic KO and D271A knockin lines, chimeric substrate constructs","pmids":["36007678","35279766"],"confidence":"High","gaps":["no high-resolution structure of SPPL3 or SPPL3–substrate complex available","contributions of luminal/cytoplasmic domains to substrate recognition not fully dissected"]},{"year":2022,"claim":"Biophysical analysis revealed that the GxxxG motif in substrate transmembrane helices promotes helix flexibility that is required for efficient SPPL3-mediated cleavage, providing the first structural rationale for substrate selectivity.","evidence":"Site-directed mutagenesis, hydrogen-deuterium exchange MS, NMR of transmembrane peptides, cleavage assays","pmids":["36470941"],"confidence":"High","gaps":["tested on single substrate (GnTV); generalizability to other substrates not confirmed","no SPPL3 active-site structure to visualize helix unwinding during catalysis"]},{"year":2024,"claim":"The immunomodulatory scope of SPPL3 was extended to innate immunity: SPPL3-deficient tumors resist neutrophil trogocytosis and killing by NK cells and γδ T cells, with nsGSL-dependent and -independent mechanisms operating for different effector cell types.","evidence":"SPPL3 KO tumor lines, trogocytosis and cytotoxicity assays with multiple effector cell types, nsGSL inhibition rescue","pmids":["39655358"],"confidence":"Medium","gaps":["NK cell killing reduction was nsGSL-independent but alternative mechanism not identified","in vivo validation in tumor models not reported"]},{"year":null,"claim":"Key open questions include the high-resolution structure of SPPL3 (alone and with substrate), the full in vivo substrate hierarchy in different tissues, whether the protease-independent STIM1–Orai1 scaffolding function is physiologically relevant in primary T cells, and whether SPPL3-controlled glycosphingolipid shielding drives immune evasion in human tumors.","evidence":"","pmids":[],"confidence":"Low","gaps":["no cryo-EM or crystal structure of SPPL3","in vivo substrate hierarchy across tissues unresolved","protease-independent function not tested in primary cells or in vivo"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,2,3,5,8,10]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[4]}],"localization":[{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[0,3,9]},{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[1]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,3,8,10]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[5,6,13]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[4]}],"complexes":[],"partners":["STIM1","ORAI1","MGAT5","B3GNT1","B4GALT1","GALNT2","B3GNT5"],"other_free_text":[]},"mechanistic_narrative":"SPPL3 is a GxGD-type intramembrane-cleaving aspartyl protease that functions as the principal sheddase for type II membrane-anchored glycosyltransferases and glycosidases in the Golgi, thereby globally regulating protein N-glycosylation, O-glycosylation, and glycosphingolipid biosynthesis [PMID:25354954, PMID:25827571, PMID:35279766]. SPPL3 resides predominantly in the mid-Golgi, where it cleaves substrates within their transmembrane domains; cleavage efficiency is governed by substrate-intrinsic features such as transmembrane helix flexibility, with GxxxG motif-containing helices being preferentially processed [PMID:36007678, PMID:36470941]. By controlling the glycosphingolipid repertoire—particularly neolacto-series species synthesized by B3GNT5—SPPL3 modulates HLA class I antigen presentation, CD8+ T cell activation, neutrophil trogocytosis, and innate immune killing by NK cells and γδ T cells [PMID:33271119, PMID:39655358]. Independent of its protease activity, SPPL3 facilitates STIM1–Orai1 association to promote store-operated Ca²⁺ entry and NFAT activation downstream of T cell receptor engagement [PMID:25384971]."},"prefetch_data":{"uniprot":{"accession":"Q8TCT6","full_name":"Signal peptide peptidase-like 3","aliases":["Intramembrane protease 2","IMP-2","Presenilin homologous protein 1","PSH1","Presenilin-like protein 4"],"length_aa":384,"mass_kda":42.3,"function":"Intramembrane-cleaving aspartic protease (I-CLiP) that cleaves type II membrane protein substrates in or close to their luminal transmembrane domain boundaries (PubMed:16873890, PubMed:25354954, PubMed:25827571). Acts like a sheddase by mediating the proteolytic release and secretion of active site-containing ectodomains of glycan-modifiying glycosidase and glycosyltransferase enzymes such as MGAT5, B4GAT1 and B4GALT1 (PubMed:25354954, PubMed:25827571). Catalyzes the intramembrane cleavage of the envelope glycoprotein gp130 and/or the leader peptide gp18LP of the simian foamy virus independent of prior ectodomain shedding by furin or furin-like proprotein convertase (PC)-mediated cleavage proteolysis (PubMed:23132852). May also have the ability to serve as a shedding protease for subsequent intramembrane proteolysis by SPPL2A and SPPL2B of the envelope glycoprotein gp130 (PubMed:23132852). Plays a role in the regulation of cellular glycosylation processes (PubMed:25354954). Required to link T-cell antigen receptor (TCR) and calcineurin-NFAT signaling cascades in lymphocytes by promoting the association of STIM1 and ORAI1 during store-operated calcium entry (SOCE) in a protease-independent manner (PubMed:25384971)","subcellular_location":"Endoplasmic reticulum membrane; Golgi apparatus; Membrane","url":"https://www.uniprot.org/uniprotkb/Q8TCT6/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/SPPL3","classification":"Not Classified","n_dependent_lines":19,"n_total_lines":1208,"dependency_fraction":0.015728476821192054},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"HSP90B1","stoichiometry":0.2},{"gene":"TMED10","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/SPPL3","total_profiled":1310},"omim":[{"mim_id":"608240","title":"SIGNAL PEPTIDE PEPTIDASE-LIKE 3; SPPL3","url":"https://www.omim.org/entry/608240"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Vesicles","reliability":"Supported"},{"location":"Plasma membrane","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/SPPL3"},"hgnc":{"alias_symbol":["IMP2","PSL4","MGC90402","MGC126674","MGC126676","DKFZP586C1324"],"prev_symbol":[]},"alphafold":{"accession":"Q8TCT6","domains":[],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8TCT6","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q8TCT6-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q8TCT6-F1-predicted_aligned_error_v6.png","plddt_mean":77.88},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=SPPL3","jax_strain_url":"https://www.jax.org/strain/search?query=SPPL3"},"sequence":{"accession":"Q8TCT6","fasta_url":"https://rest.uniprot.org/uniprotkb/Q8TCT6.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q8TCT6/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8TCT6"}},"corpus_meta":[{"pmid":"22899010","id":"PMC_22899010","title":"Imp2 controls oxidative phosphorylation and is crucial for preserving glioblastoma 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patterns. Reduced SPPL3 expression causes hyperglycosylation; elevated SPPL3 causes hypoglycosylation.\",\n      \"method\": \"Overexpression and knockdown cell culture models, biochemical substrate cleavage assays, glycosylation profiling\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — multiple orthogonal methods (biochemical cleavage, gain/loss-of-function, glycan profiling) in single rigorous study; independently replicated\",\n      \"pmids\": [\"25354954\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"SPPL3 is localized to the endoplasmic reticulum (ER), in contrast to SPPL2b which localizes to endosomes/lysosomes. Knockdown of sppl3 in zebrafish causes cell death in the CNS, and expression of a D/A mutation in the putative C-terminal active site phenocopies the sppl3 knockdown, demonstrating SPPL3 is a catalytically active GXGD-type aspartyl protease.\",\n      \"method\": \"Subcellular localization studies in cultured cells, antisense gripNA-mediated knockdown in zebrafish, active-site mutagenesis (D/A mutation)\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — active-site mutagenesis combined with in vivo knockdown and localization; moderate evidence base\",\n      \"pmids\": [\"15998642\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"SPPL3 cleaves the 18-kDa leader peptide (LP18) of the foamy virus envelope protein (FVenv), acting as a sheddase capable of cleaving substrates with large ectodomains without requiring prior shedding—unlike SPPL2a/b and γ-secretase. The SPPL3-generated cleavage product of FVenv then serves as a substrate for consecutive intramembrane cleavage by SPPL2a/b.\",\n      \"method\": \"In vitro cleavage assay with human SPPL3 and FVenv mutants, biochemical identification of cleavage products\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — direct in vitro cleavage assay with mutant substrates demonstrating unique sheddase-like activity\",\n      \"pmids\": [\"23132852\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Secretome proteomics (SPECS method) of SPPL3-deficient and SPPL3-overexpressing cells identified numerous Golgi-localized type II membrane proteins as SPPL3 substrates, extending beyond N-glycosylation to include O-glycan and glycosaminoglycan biosynthesis enzymes. SPPL3-mediated endoproteolysis releases catalytic ectodomains of these type II membrane enzymes from their membrane anchors.\",\n      \"method\": \"SPECS proteomics on SPPL3-deficient and overexpression cell culture models, biochemical validation of candidate substrates\",\n      \"journal\": \"Molecular & cellular proteomics : MCP\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — systematic secretome proteomics with biochemical validation across multiple substrates\",\n      \"pmids\": [\"25827571\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"SPPL3 is required downstream of T cell receptor engagement for maximal Ca2+ influx and NFAT activation in a protease-independent manner. SPPL3 enhances the signal-induced association of STIM1 and Orai1, associates with STIM1 through its transmembrane region and the CRAC activation domain (CAD), and promotes STIM1 CAD association with Orai1.\",\n      \"method\": \"Screen for NFAT activators, Ca2+ influx measurements, Co-immunoprecipitation of SPPL3 with STIM1/Orai1, catalytic mutant analysis\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP, catalytic mutant, functional Ca2+ and NFAT assays in single study\",\n      \"pmids\": [\"25384971\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"SPPL3 protease activity is required cell-autonomously for efficient NK cell maturation and cytotoxicity. CRISPR/Cas9-generated knockin mice expressing catalytically compromised SPPL3 D271A phenocopy SPPL3 deletion, showing reduced CD27+CD11b+ and CD27−CD11b+ NK cell numbers and impaired tumor killing.\",\n      \"method\": \"Hematopoietic- and NK cell-specific SPPL3 deletion, CRISPR/Cas9 knockin of D271A catalytic mutant, in vivo tumor clearance and in vitro cytotoxicity assays\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — catalytic mutant knockin combined with conditional KO and in vivo/in vitro functional assays\",\n      \"pmids\": [\"26851218\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Loss of SPPL3 augments B3GNT5 enzyme activity, resulting in upregulation of surface neolacto-series glycosphingolipids (nsGSLs) that sterically impede antibody and receptor interactions with HLA class I, diminishing CD8+ T cell activation. SPPL3 thus controls the GSL repertoire to regulate adaptive immune recognition.\",\n      \"method\": \"Iterative genome-wide CRISPR screens, GSL profiling, functional HLA-I antigen presentation assays, CD8+ T cell activation assays\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — genome-wide screens with orthogonal biochemical and functional validation; multiple methods\",\n      \"pmids\": [\"33271119\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"In melanocyte-to-melanoma transformation, SPPL3-mediated activation of ADAM10 is a critical transformation event. This involves translocation of SPPL3 and ADAM10 into Rab4- or Rab27-positive endosomal compartments triggered by mutant BRAFV600E, and this endosomal translocation is inhibited by tumor suppressor PTEN.\",\n      \"method\": \"Multiepitope ligand cartography (MELC) tissue analysis, co-localization of SPPL3 and ADAM10 in endosomes, functional transformation assays in patient tissues and cell co-cultures\",\n      \"journal\": \"Science signaling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — co-localization and tissue analysis with functional consequence; single lab, single study\",\n      \"pmids\": [\"28292959\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"N-terminomics analysis of SPPL3-deficient and active-site knock-in (D271A) HEK293 and HeLa cells identified over 20 SPPL3 substrates including novel ones (e.g., GALNT2), provided comprehensive SPPL3 cleavage sites demonstrating intramembrane proteolysis, and showed that transmembrane domain composition determines susceptibility to SPPL3 cleavage.\",\n      \"method\": \"N-terminomics (TAILS) on isogenic SPPL3 KO and D271A knockin cell lines, chimeric glycosyltransferase constructs, immunoblot validation\",\n      \"journal\": \"Cellular and molecular life sciences : CMLS\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — N-terminomics with isogenic controls plus active-site mutant knockin and biochemical validation; comprehensive substrate mapping\",\n      \"pmids\": [\"35279766\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Endogenous SPPL3 tagged at the endogenous locus localizes predominantly to the mid-Golgi under steady-state conditions, co-localizing with its substrates. Co-localization alone with type II proteins in the Golgi is not sufficient for cleavage; substrate-intrinsic properties (e.g., transmembrane domain flexibility) additionally govern SPPL3-mediated intramembrane proteolysis.\",\n      \"method\": \"Genome editing to generate isogenic N- and C-terminally tagged endogenous SPPL3; co-localization with Golgi markers; drug-disruption of Golgi; co-localization with substrates vs non-substrates\",\n      \"journal\": \"Biochimica et biophysica acta. Molecular cell research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — endogenous tagging via genome editing, isogenic controls, multiple markers; rigorous localization with mechanistic inference\",\n      \"pmids\": [\"36007678\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"The GxxxG motif in the transmembrane domain of GnTV (an SPPL3 substrate) regulates SPPL3-dependent cleavage efficiency. Mutations disrupting the GxxxG motif increase transmembrane helix rigidity (assessed by HDX and NMR), reducing SPPL3-dependent ectodomain shedding, while increased helix flexibility facilitates SPPL3-dependent shedding.\",\n      \"method\": \"Site-directed mutagenesis of GxxxG motif, deuterium/hydrogen exchange MS, NMR spectroscopy of transmembrane peptides, SPPL3 cleavage assays\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — mutagenesis combined with biophysical structural analysis (HDX, NMR) and cleavage assays in single study\",\n      \"pmids\": [\"36470941\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"C. elegans ce-imp-2 (ortholog of human SPP/IMP1 rather than IGF2BP2/IMP2) does not promote Notch (lin-12, glp-1) proteolysis or signaling, unlike presenilins, and its knockdown leads to embryonic death and abnormal molting. The molting defect is mimicked by cholesterol depletion or lrp-1 disruption and suppressed by lrp-1 derivative expression, implicating IMP/SPP proteases in lipid-lipoprotein receptor-mediated pathways.\",\n      \"method\": \"RNAi knockdown in C. elegans, genetic epistasis with Notch pathway components, cholesterol depletion, lrp-1 rescue\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — epistasis and rescue in vivo; single study; note this concerns Ce-imp-2/SPP ortholog, functionally distinct from IGF2BP2\",\n      \"pmids\": [\"15469912\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"SPPL3 expression controls the cell surface staining of CD59 through its intramembrane protease activity by suppressing neolacto-series glycosphingolipid (nsGSL) synthesis. The effect is nsGSL-dependent and not mediated by N-glycan changes, suggesting nsGSLs sterically impair CD59 accessibility.\",\n      \"method\": \"SPPL3 KO and rescue with protease-dead mutant, nsGSL inhibition, flow cytometry of CD59 and GPI-anchored proteins\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — active-site mutant and pathway inhibition with functional readout; single lab\",\n      \"pmids\": [\"34303967\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"SPPL3-deficient tumor cells are less susceptible to trogocytosis by neutrophils and killing by NK cells and γδ T cells. The nsGSL-dependent SPPL3 sensitivity for trogocytosis and γδ T cell killing depends on proximity of surface receptor domains to the membrane and receptor-ligand interaction affinity; NK cell killing reduction by SPPL3 loss is nsGSL-independent.\",\n      \"method\": \"SPPL3 KO tumor cell lines, neutrophil trogocytosis assay, NK and γδ T cell killing assays, nsGSL inhibition rescue\",\n      \"journal\": \"European journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple immune effector assays with KO and chemical rescue; single study\",\n      \"pmids\": [\"39655358\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"SPPL3 is a mid-Golgi-resident intramembrane-cleaving aspartyl protease of the GxGD type that acts as the primary sheddase for type II membrane-anchored Golgi glycan-modifying enzymes (glycosyltransferases and glycosidases), releasing their catalytic ectodomains through intramembrane proteolysis to reduce their cellular activity and globally regulate protein N-glycosylation, O-glycosylation, and glycosphingolipid biosynthesis; by controlling the glycosphingolipid repertoire (particularly neolacto-series GSLs via B3GNT5), SPPL3 modulates immune recognition including HLA-I antigen presentation and innate immune killing, and additionally exerts a protease-independent function in T cell NFAT signaling by facilitating STIM1–Orai1 association.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"SPPL3 is a GxGD-type intramembrane-cleaving aspartyl protease that functions as the principal sheddase for type II membrane-anchored glycosyltransferases and glycosidases in the Golgi, thereby globally regulating protein N-glycosylation, O-glycosylation, and glycosphingolipid biosynthesis [PMID:25354954, PMID:25827571, PMID:35279766]. SPPL3 resides predominantly in the mid-Golgi, where it cleaves substrates within their transmembrane domains; cleavage efficiency is governed by substrate-intrinsic features such as transmembrane helix flexibility, with GxxxG motif-containing helices being preferentially processed [PMID:36007678, PMID:36470941]. By controlling the glycosphingolipid repertoire—particularly neolacto-series species synthesized by B3GNT5—SPPL3 modulates HLA class I antigen presentation, CD8+ T cell activation, neutrophil trogocytosis, and innate immune killing by NK cells and γδ T cells [PMID:33271119, PMID:39655358]. Independent of its protease activity, SPPL3 facilitates STIM1–Orai1 association to promote store-operated Ca²⁺ entry and NFAT activation downstream of T cell receptor engagement [PMID:25384971].\",\n  \"teleology\": [\n    {\n      \"year\": 2004,\n      \"claim\": \"Early genetic work in C. elegans showed that the SPP/IMP protease family (including the SPPL3 ortholog) is essential for viability and molting and operates independently of Notch/presenilin signaling, establishing that these GxGD proteases have distinct in vivo functions from γ-secretase.\",\n      \"evidence\": \"RNAi knockdown and genetic epistasis with Notch components in C. elegans\",\n      \"pmids\": [\"15469912\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"C. elegans ortholog studied (ce-imp-2) is closer to SPP than to SPPL3; direct relevance to mammalian SPPL3 uncertain\", \"substrates in worm not identified\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Demonstration that mammalian SPPL3 is a catalytically active GxGD-type aspartyl protease whose active-site aspartate is essential for function in vivo, with zebrafish knockdown revealing a requirement for CNS cell survival.\",\n      \"evidence\": \"Active-site D/A mutagenesis, subcellular localization in cultured cells, antisense knockdown in zebrafish\",\n      \"pmids\": [\"15998642\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"endogenous substrates not yet identified\", \"initial localization placed SPPL3 in the ER rather than Golgi\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"SPPL3 was shown to act as a sheddase capable of cleaving substrates with large ectodomains without prior ectodomain trimming—unlike SPPL2a/b and γ-secretase—establishing a unique proteolytic mode within the GxGD family.\",\n      \"evidence\": \"In vitro cleavage assay using foamy virus envelope protein and mutant substrates\",\n      \"pmids\": [\"23132852\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"only viral substrate tested; endogenous cellular substrates not yet addressed\", \"cleavage site not mapped at residue resolution\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Identification of Golgi glycosyltransferases and glycosidases as endogenous SPPL3 substrates resolved the key question of SPPL3's physiological function: it is a master regulator of cellular glycosylation by releasing the catalytic ectodomains of glycan-modifying enzymes.\",\n      \"evidence\": \"Overexpression and knockdown in cultured cells, biochemical cleavage assays, lectin-based glycosylation profiling\",\n      \"pmids\": [\"25354954\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"substrate repertoire not fully defined\", \"mechanism of substrate selectivity unknown\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"A protease-independent role for SPPL3 was discovered in T cell signaling, where it facilitates STIM1–Orai1 coupling to promote store-operated Ca²⁺ entry and NFAT activation, revealing a non-catalytic scaffolding function.\",\n      \"evidence\": \"Co-immunoprecipitation of SPPL3 with STIM1/Orai1, Ca²⁺ influx measurements, catalytic-dead mutant retains function\",\n      \"pmids\": [\"25384971\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"structural basis of SPPL3–STIM1 interaction unresolved\", \"relevance beyond Jurkat T cells not tested\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Systematic secretome proteomics greatly expanded the SPPL3 substrate repertoire to include enzymes of O-glycan and glycosaminoglycan biosynthesis, showing SPPL3's regulatory scope extends well beyond N-glycosylation.\",\n      \"evidence\": \"SPECS proteomics on SPPL3-deficient and overexpressing cells with biochemical validation\",\n      \"pmids\": [\"25827571\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"not all candidate substrates biochemically validated\", \"in vivo confirmation of extended substrate list lacking\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"In vivo evidence established that SPPL3 protease activity is cell-autonomously required for NK cell maturation and cytotoxicity, linking its catalytic function to immune cell development.\",\n      \"evidence\": \"Hematopoietic-specific SPPL3 KO and CRISPR knockin of D271A catalytic mutant in mice; in vivo tumor clearance assays\",\n      \"pmids\": [\"26851218\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"whether NK cell defect is glycosylation-dependent or involves additional substrates not determined\", \"impact on other immune lineages not fully explored\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"SPPL3 was identified as a critical regulator of immune recognition: loss of SPPL3 elevates B3GNT5-dependent neolacto-series glycosphingolipids that sterically shield HLA-I from antibodies and TCRs, diminishing CD8⁺ T cell activation.\",\n      \"evidence\": \"Genome-wide CRISPR screens, GSL profiling, HLA-I antigen presentation and T cell activation assays\",\n      \"pmids\": [\"33271119\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"relevance to in vivo tumor immune evasion not directly shown\", \"whether SPPL3 loss also shields other surface receptors via nsGSLs not systematically tested\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Endogenous tagging and comprehensive N-terminomics resolved SPPL3's steady-state localization to the mid-Golgi and identified >20 substrates with mapped cleavage sites, establishing that transmembrane domain composition—not mere co-localization—determines cleavage susceptibility.\",\n      \"evidence\": \"Genome editing for endogenous tagging, TAILS N-terminomics on isogenic KO and D271A knockin lines, chimeric substrate constructs\",\n      \"pmids\": [\"36007678\", \"35279766\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"no high-resolution structure of SPPL3 or SPPL3–substrate complex available\", \"contributions of luminal/cytoplasmic domains to substrate recognition not fully dissected\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Biophysical analysis revealed that the GxxxG motif in substrate transmembrane helices promotes helix flexibility that is required for efficient SPPL3-mediated cleavage, providing the first structural rationale for substrate selectivity.\",\n      \"evidence\": \"Site-directed mutagenesis, hydrogen-deuterium exchange MS, NMR of transmembrane peptides, cleavage assays\",\n      \"pmids\": [\"36470941\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"tested on single substrate (GnTV); generalizability to other substrates not confirmed\", \"no SPPL3 active-site structure to visualize helix unwinding during catalysis\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"The immunomodulatory scope of SPPL3 was extended to innate immunity: SPPL3-deficient tumors resist neutrophil trogocytosis and killing by NK cells and γδ T cells, with nsGSL-dependent and -independent mechanisms operating for different effector cell types.\",\n      \"evidence\": \"SPPL3 KO tumor lines, trogocytosis and cytotoxicity assays with multiple effector cell types, nsGSL inhibition rescue\",\n      \"pmids\": [\"39655358\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"NK cell killing reduction was nsGSL-independent but alternative mechanism not identified\", \"in vivo validation in tumor models not reported\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key open questions include the high-resolution structure of SPPL3 (alone and with substrate), the full in vivo substrate hierarchy in different tissues, whether the protease-independent STIM1–Orai1 scaffolding function is physiologically relevant in primary T cells, and whether SPPL3-controlled glycosphingolipid shielding drives immune evasion in human tumors.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"no cryo-EM or crystal structure of SPPL3\", \"in vivo substrate hierarchy across tissues unresolved\", \"protease-independent function not tested in primary cells or in vivo\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 2, 3, 5, 8, 10]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [0, 3, 9]},\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [1]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 3, 8, 10]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [5, 6, 13]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"STIM1\",\n      \"ORAI1\",\n      \"MGAT5\",\n      \"B3GNT1\",\n      \"B4GALT1\",\n      \"GALNT2\",\n      \"B3GNT5\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}