{"gene":"SIGLEC9","run_date":"2026-06-10T07:46:32","timeline":{"discoveries":[{"year":2000,"finding":"Siglec-9 is a type I transmembrane protein with three extracellular Ig-like domains (N-terminal V-set + two C2-set), a transmembrane region, and a cytoplasmic tail containing two tyrosine-based signaling motifs including a canonical ITIM. Expression of full-length cDNA in COS cells induced sialic-acid-dependent erythrocyte binding. Recombinant soluble extracellular domain binds α2-3 and α2-6-linked sialic acids; the carboxyl group and side chain of sialic acid are essential, and mutation of a critical arginine residue in domain 1 abrogates binding.","method":"cDNA cloning, COS cell expression, recombinant protein binding assays, site-directed mutagenesis of conserved Arg","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstitution in cell expression system, mutagenesis of active-site Arg, multiple binding assays replicated across two independent papers (PMID:10801860, PMID:10801862)","pmids":["10801860","10801862"],"is_preprint":false},{"year":2001,"finding":"The C-C' loop region (residues Asn70–Lys75) in the V-set sugar-binding domain of Siglec-7 determines its preference for α2,8-disialyl and branched α2,6-sialyl residues (GD3, LSTb), whereas the equivalent region in Siglec-9 confers preference for α2,3-linked (LSTc, GD1a) structures. Swapping this small region between Siglec-7 and Siglec-9 chimeras transferred binding specificity accordingly.","method":"Chimeric protein expression in CHO cells, polyvalent streptavidin-based glyco-probe binding assays, molecular modeling","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — domain-swap mutagenesis with reciprocal transfer of binding specificity, validated by glycan probes and molecular modeling","pmids":["11741958"],"is_preprint":false},{"year":2004,"finding":"Siglec-9 negatively regulates T cell receptor (TCR) signaling: following pervanadate stimulation or TCR engagement, Siglec-9 undergoes tyrosine phosphorylation and recruits SHP-1; it reduces phosphorylation of ZAP-70 Tyr319 and decreases NFAT transcriptional activity. Mutation of the conserved Arg120 in the ligand-binding site reduces inhibitory function, indicating ligand binding is required for optimal TCR inhibition.","method":"Stable/transient transfection of Jurkat T cells with Siglec-9, pervanadate/TCR stimulation, co-immunoprecipitation of SHP-1, phospho-ZAP-70 western blot, NFAT-luciferase reporter assay, Arg120Ala mutagenesis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — multiple orthogonal methods (co-IP, western blot, reporter assay) with mutagenesis in a single study","pmids":["15292262"],"is_preprint":false},{"year":2005,"finding":"Siglec-9 ligation on normal neutrophils induces caspase-dependent, ROS-dependent apoptosis. In neutrophils primed with GM-CSF, IFN-α, or IFN-γ, Siglec-9 ligation triggers a caspase-independent, ROS-dependent cell death with cytoplasmic vacuolization. Both death pathways are abrogated by ROS scavengers or in neutrophils unable to generate ROS.","method":"Siglec-9 antibody ligation on primary human neutrophils and inflammatory neutrophils (sepsis, RA patients), ROS scavenger experiments, caspase inhibitor experiments, cytology","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 / Strong — primary human cells and patient samples, pharmacological dissection of pathway, two distinct death modalities established with orthogonal inhibitors","pmids":["15827126"],"is_preprint":false},{"year":2008,"finding":"Siglec-9 expression in macrophages (RAW264 and THP-1) strongly enhances IL-10 production and reduces TNF-α upon TLR stimulation (LPS, peptidoglycan, CpG, dsRNA). These effects require both cytoplasmic tyrosine residues: mutation of both to phenylalanine abrogates IL-10 enhancement and TNF-α suppression. A membrane-proximal ITIM mutant partially retains TNF-α inhibition but loses IL-10 enhancement, indicating divergent signaling via the two tyrosines.","method":"Stable transfection of Siglec-9 and ITIM tyrosine mutants in RAW264/THP-1, TLR stimulation assays, ELISA for cytokines","journal":"Biochemical and biophysical research communications","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — mutagenesis of both ITIM tyrosines in two cell lines, functional readout by cytokine ELISA, mechanistic dissection of two tyrosines","pmids":["18325328"],"is_preprint":false},{"year":2009,"finding":"Bacterial group B Streptococcus (GBS) sialylated capsular polysaccharide (CPS) presenting terminal Sialα2-3Galβ1-4GlcNAc engages neutrophil Siglec-9 in trans, dampening neutrophil oxidative burst, NET formation, and enabling bacterial survival. These effects are Sia- and Siglec-9-dependent (abrogated by neuraminidase treatment of GBS or by blocking Siglec-9).","method":"Immobilized sialoglycan binding assays, GBS CPS binding to isolated human neutrophils, oxidative burst assays, NET formation assays, bacterial survival assays, neuraminidase treatment, Siglec-9 blocking antibodies","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple functional readouts, specific controls (neuraminidase, blocking antibody), primary human neutrophils","pmids":["19196661"],"is_preprint":false},{"year":2010,"finding":"Siglec-9 is identified as the receptor for MUC16 (CA125) on NK cells, B cells, and monocytes. Siglec-9-transfected Jurkat cells and monocytes from healthy donors bind to ovarian tumor cells via Siglec-9–csMUC16 interaction; neuraminidase treatment of immune cells releases sMUC16, confirming sialic acid dependence.","method":"Flow cytometry of primary immune cells, Siglec-9 transfection into Jurkat cells, MUC16 binding assays, neuraminidase treatment, co-culture adhesion assays","journal":"Molecular cancer","confidence":"High","confidence_rationale":"Tier 2 / Moderate — transfection-based functional binding assay with neuraminidase control, reciprocal cell-cell interaction assays","pmids":["20497550"],"is_preprint":false},{"year":2011,"finding":"Siglec-9 is a novel leukocyte ligand for vascular adhesion protein-1 (VAP-1/AOC3). The interaction was identified by phage display and confirmed by in vitro and ex vivo adhesion assays. Interaction occurs at the enzymatic groove of VAP-1 and is only partially dependent on VAP-1 enzymatic activity. A 68Ga-labeled Siglec-9 peptide specifically detects VAP-1 at sites of inflammation and cancer by PET.","method":"Phage display, in vitro/ex vivo adhesion assays with mutated proteins, molecular modeling, PET imaging with 68Ga-labeled Siglec-9 peptide","journal":"Blood","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — phage display discovery, confirmed by multiple binding assays with mutants, in vivo PET validation","pmids":["21821708"],"is_preprint":false},{"year":2013,"finding":"Siglec-9 binds MUC1 on cancer cells in a sialic acid-dependent manner, inducing recruitment of β-catenin to the MUC1 C-terminal domain in a dose- and time-dependent manner. Recruited β-catenin translocates to the nucleus, promoting cell growth. Neuraminidase treatment abolishes Siglec-9-induced signaling.","method":"Recombinant soluble Siglec-9 treatment of MUC1-transfected 3T3 and HCT116 cells, co-culture with Siglec-9-expressing HEK293 cells, co-immunoprecipitation of β-catenin with MUC1, nuclear fractionation, neuraminidase treatment, proliferation assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — co-IP, nuclear fractionation, dose-response, neuraminidase control, co-culture model, multiple cell lines","pmids":["24045940"],"is_preprint":false},{"year":2013,"finding":"Prohibitin-1 and prohibitin-2 expressed on the surface of T cell leukemia lines and activated T lymphocytes serve as counter-receptors for Siglec-9 on macrophages and dendritic cells, in a sialic acid-independent but Arg120-dependent ionic peptide–peptide interaction. Engagement of prohibitins via Siglec-9 (co-immobilized with anti-CD3) inhibits ERK1/2 phosphorylation, c-Raf phosphorylation, and IL-2 production in Jurkat cells.","method":"Binding assays with Siglec-9 Arg120Ala mutant, co-immobilization of Siglec-9 and anti-CD3 on beads, phospho-ERK1/2 and phospho-c-Raf western blot, IL-2 ELISA","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis of binding site, bead co-immobilization functional assay, western blot and ELISA; single lab","pmids":["23567969"],"is_preprint":false},{"year":2013,"finding":"Siglec-9 binding to sialylglycoconjugates on astrocytoma (AS) cells induces rapid calpain-mediated degradation of focal adhesion kinase (FAK), Akt, paxillin, and p130Cas, leading to cell detachment and increased motility/invasiveness. Despite degradation of total Akt, phospho-Akt was increased at the leading cytoplasmic edge, consistent with enhanced motility.","method":"Co-culture of Siglec-9-expressing and Siglec-9-deficient cells with AS astrocytoma cells, immunoblotting for FAK/Akt/paxillin/p130Cas degradation, calpain inhibitor experiments, motility and invasion assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pharmacological inhibition of calpain with functional rescue, multiple substrate measurements; single lab","pmids":["24145038"],"is_preprint":false},{"year":2014,"finding":"Upon TLR2 stimulation, a portion of Siglec-9 redistributes into lipid raft (detergent-insoluble microdomain) fractions with kinetics mirroring TLR2 redistribution (peak 3–10 min). This raft localization is lectin-activity-dependent: a lectin-defective Siglec-9 mutant fails to enter lipid rafts, whereas a double-ITIM tyrosine mutant still translocates. IL-10 production is partially reduced by disrupting lipid raft organization with cholesterol oxidase, suggesting raft localization contributes to IL-10 enhancement.","method":"Membrane fractionation (detergent-insoluble microdomains), lectin-defective and ITIM tyrosine mutant transfection, cholesterol oxidase treatment, cytokine ELISA","journal":"Cytotechnology","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — fractionation plus mutagenesis plus pharmacological disruption; single lab","pmids":["24449467"],"is_preprint":false},{"year":2015,"finding":"High-molecular-weight hyaluronan (HMW-HA) is recognized by Siglec-9 through a site in its V-set domain distinct from the sialic acid-binding site, representing the first non-sialic-acid glycan ligand for a CD33-related Siglec. HMW-HA engagement of Siglec-9 limits NET formation, oxidative burst, and apoptosis in human neutrophils. Group A Streptococcus (GAS) exploits its own HMW-HA capsule to engage Siglec-9, blocking these neutrophil functions and promoting bacterial survival.","method":"Binding assays with Siglec-9 and HMW-HA, neutrophil functional assays (NET formation, oxidative burst, apoptosis), GAS capsule-dependent blocking experiments","journal":"Journal of molecular medicine (Berlin, Germany)","confidence":"High","confidence_rationale":"Tier 2 / Moderate — distinct binding site mapped, multiple neutrophil functional readouts, pathogen exploitation validated; single lab but multiple orthogonal methods","pmids":["26411873"],"is_preprint":false},{"year":2016,"finding":"MUC1 carrying cancer-specific short sialylated O-linked glycans (MUC1-ST) binds Siglec-9 on myeloid cells without activating SHP-1 or SHP-2, but instead induces calcium flux leading to MEK-ERK kinase activation, educating myeloid cells toward a tumor-associated macrophage-like phenotype with increased PD-L1 expression.","method":"MUC1-ST binding assays, phosphatase activation assays (SHP-1/SHP-2), calcium flux measurement, MEK-ERK phosphorylation western blot, PD-L1 expression by flow cytometry, macrophage phenotyping","journal":"Nature immunology","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal signaling assays (phosphatases negative, Ca2+ flux, MEK-ERK positive), peer-reviewed high-impact journal, distinct activating pathway established","pmids":["27595232"],"is_preprint":false},{"year":2016,"finding":"A SIGLEC9 GA haplotype (rs2075803 G/rs2258983 A) encodes a Siglec-9 protein variant that is less effective at suppressing inflammatory TNF-α production in a myeloid cell line compared to the other major haplotype variant, as measured by in vitro cytokine assays.","method":"In vitro myeloid cell line transfection with variant Siglec-9 constructs, TNF-α ELISA after stimulation","journal":"Respirology (Carlton, Vic.)","confidence":"Medium","confidence_rationale":"Tier 3 / Weak — single in vitro functional assay with SNP variants, single lab","pmids":["27878892"],"is_preprint":false},{"year":2016,"finding":"Soluble Siglec-9 (sSiglec-9) suppresses M1 macrophage activation by inhibiting NF-κB p65 phosphorylation in RAW264.7 cells, reducing M1 marker expression (TNF-α, IL-6, iNOS) without affecting M2 markers. In a murine collagen-induced arthritis model, sSiglec-9 attenuated arthritis severity, decreased serum TNF-α, and increased Foxp3+ Treg cell proportions.","method":"RAW264.7 macrophage culture with sSiglec-9, western blot for NF-κB p65 phosphorylation, NF-κB chemical blockade, cytokine ELISA, DBA/1J mouse CIA model, histology, flow cytometry for Tregs","journal":"Arthritis research & therapy","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — NF-κB pathway confirmed by both phospho-blot and chemical inhibitor, in vivo model with multiple readouts; single lab","pmids":["27267914"],"is_preprint":false},{"year":2017,"finding":"Glycophorin A, the most abundant sialoglycoprotein on erythrocytes, engages neutrophil Siglec-9 to suppress neutrophil activation in vitro and ex vivo. Mild periodate oxidation of erythrocyte sialic acid side chains (with aldehyde quenching) reduces erythrocyte binding to Siglec-9 and restores neutrophil activation (l-selectin shedding, oxidative burst, chemotaxis, NET formation, apoptosis), demonstrating a sialic acid-based 'self' signal that maintains neutrophil quiescence in blood.","method":"ELISA and immunofluorescence for glycophorin A–Siglec-9 interaction, sodium metaperiodate oxidation of erythrocyte sialic acids, ex vivo and in vitro neutrophil activation assays","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 / Strong — specific chemical modification of sialic acids with functional rescue, multiple activation readouts, primary human cells","pmids":["28416510"],"is_preprint":false},{"year":2017,"finding":"Tamm-Horsfall glycoprotein (THP) engages neutrophil Siglec-9 (and its mouse ortholog Siglec-E) via N-glycan sialic acid moieties, suppressing neutrophil ROS generation, chemotaxis, and killing of uropathogenic E. coli. THP-null mice have significantly more neutrophils in urine than wild-type mice.","method":"THP–neutrophil binding assays, neuraminidase treatment, Siglec-9 blocking antibody, ROS/chemotaxis/bacterial killing assays, THP-null mouse urinalysis","journal":"Immunology and cell biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — neuraminidase and blocking antibody controls, multiple functional assays, in vivo THP-null mouse model","pmids":["28829050"],"is_preprint":false},{"year":2017,"finding":"MCP-1 and secreted ectodomain of Siglec-9 (sSiglec-9) synergistically promote M2 macrophage differentiation from bone marrow-derived macrophages via CCR2, producing liver-regenerating factors that suppress hepatocyte apoptosis and promote proliferation. In a rat acute liver failure model, combined MCP-1/sSiglec-9 treatment improved survival and induced anti-inflammatory M2 macrophages; depletion of M2 macrophages (mannosylated clodronate liposomes) abolished recovery.","method":"In vitro M2 differentiation assay with bone marrow-derived macrophages, CCR2 blocking, D-galactosamine rat ALF model, M2-depletion with mannosylated clodronate liposomes, hepatocyte apoptosis/proliferation assays","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CCR2 blocking identifies receptor, M2-depletion confirms mechanism, in vivo model; single lab","pmids":["28272428"],"is_preprint":false},{"year":2018,"finding":"Dasatinib dephosphorylates Siglec-9 (and Siglec-3) in human monocyte-derived dendritic cells by inhibiting SRC-family kinases, which more than doubles the number of moDCs migrating toward a CCL19 gradient. Specific blocking of Siglec-9 also enhanced DC migration, confirming that SFK-dependent Siglec-9 phosphorylation restrains DC migration.","method":"Dasatinib and SRC inhibitor 1 treatment of moDCs, phosphorylation assays for Siglec-9 and SFKs, CCL19 gradient migration assays, specific Siglec-9 blocking antibody","journal":"Experimental hematology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — two pharmacological approaches (dasatinib and SFK-specific inhibitor) plus blocking antibody all converge on same phenotype; single lab","pmids":["24882272"],"is_preprint":false},{"year":2018,"finding":"Siglec-9 is an inhibitory receptor on human primary amine oxidase (hAOC3/VAP-1). The Siglec-9 peptide binds to hAOC3 and triggers its amine oxidase enzymatic activity toward benzylamine. hAOC3 inhibitors (semicarbazide and imidazole) reduce binding of wild-type and Arg/Ala mutated Siglec-9 peptides to hAOC3, and molecular docking shows the R3 residue of the Siglec-9 peptide interacts in the catalytic site when topaquinone is in the non-catalytic on-copper conformation.","method":"Amine oxidase activity assay, competitive binding with hAOC3 inhibitors, Arg/Ala mutant peptide, molecular docking","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — enzymatic activity assay plus inhibitor competition plus mutagenesis plus docking; single lab","pmids":["29391504"],"is_preprint":false},{"year":2019,"finding":"Siglec-9 is expressed on intratumoral CD8+ effector memory T cells in melanoma and functions as an inhibitory checkpoint: engagement of Siglec-9 by its ligands or specific antibodies suppresses TCR signaling, cytotoxicity, and cytokine production. Inhibition is associated with phosphorylation of SHP-1 but not SHP-2. Cognate Siglec-9 ligands are expressed on the majority of primary and metastatic melanoma tumor cells.","method":"Flow cytometry of tumor-infiltrating vs. peripheral T cells, Siglec-9 ligand engagement/antibody stimulation assays, cytotoxicity assays, cytokine production, phospho-SHP-1/SHP-2 assays","journal":"Cancer immunology research","confidence":"High","confidence_rationale":"Tier 2 / Moderate — primary tumor samples, functional inhibition assays with specific ligand and antibody engagement, signaling pathway specificity (SHP-1 not SHP-2) established","pmids":["30988027"],"is_preprint":false},{"year":2021,"finding":"Pancreatic ductal adenocarcinoma cells express increased sialylation driven primarily by α2,3-sialyltransferases ST3GAL1 and ST3GAL4, producing ligands recognized by Siglec-9 (and Siglec-7) on myeloid cells. Triggering Siglec-9 in macrophages reduces inflammatory programmes and increases PD-L1 and IL-10 expression, directing monocyte-to-macrophage differentiation toward an immunosuppressive phenotype.","method":"ST3GAL1/ST3GAL4 identification by transcriptomics, Siglec-9 binding assays, Siglec-9 triggering on macrophages, PD-L1/IL-10 expression assays, single-cell and bulk transcriptomics","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Moderate — sialyltransferase identified as ligand source, functional Siglec-9 triggering assay with signaling readouts, multiple orthogonal methods","pmids":["33627655"],"is_preprint":false},{"year":2021,"finding":"Synthetic glycopolymers designed as Siglec-9 agonists suppress NETosis in human neutrophils induced by viral TLR agonists and plasma from severe COVID-19 patients, confirming Siglec-9 as a functional checkpoint receptor that can be pharmacologically activated to suppress neutrophil extracellular trap formation.","method":"Synthetic glycopolymer synthesis, neutrophil NETosis assays with TLR agonists and COVID-19 patient plasma, Siglec-9 agonism confirmed by receptor engagement","journal":"ACS central science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — synthetic agonist tool compounds with defined mechanism, primary human neutrophils and patient samples; single lab","pmids":["34056095"],"is_preprint":false},{"year":2023,"finding":"Siglec-9 is expressed on human mast cells and functions as an inhibitory receptor. CRISPR/Cas9 disruption of SIGLEC9 increases baseline activation marker expression and enhances responsiveness to IgE-dependent and -independent stimulation. Glycophorin A and HMW-HA (native Siglec-9 ligands), as well as Siglec-9 co-engagement with FcεRI, reduce mast cell degranulation, arachidonic acid production, and chemokine release.","method":"CRISPR/Cas9 SIGLEC9 knockout, flow cytometry for activation markers, degranulation assays, arachidonic acid measurement, chemokine ELISA, native ligand pretreatment, FcεRI co-engagement","journal":"The Journal of allergy and clinical immunology","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — CRISPR KO loss-of-function plus ligand gain-of-function, multiple orthogonal readouts in primary mast cells; single lab","pmids":["37100120"],"is_preprint":false},{"year":2023,"finding":"Siglec-9 functions as an immune-checkpoint molecule on macrophages in glioblastoma. Deletion of Siglece (murine Siglec-9 homolog) restrained tumor development and prolonged survival in mouse GBM models. Mechanistically, Siglece deletion directly activated both CD4+ and CD8+ T cells through antigen presentation, secreted chemokines, and co-stimulatory factor interactions. Siglece deletion synergized with anti-PD-1/PD-L1 treatment.","method":"Siglece knockout mouse GBM models, survival analysis, single-cell RNA-seq, spatial transcriptomics, T cell activation assays (antigen presentation, chemokines, co-stimulatory factors)","journal":"Nature cancer","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO in vivo with survival readout, mechanistic pathway mapped by scRNA-seq and functional T cell assays, synergy with checkpoint blockade demonstrated","pmids":["37460871"],"is_preprint":false},{"year":2023,"finding":"Blockade of Siglec-9 in ovarian cancer suppresses phosphorylation of SHP-1, repolarizes TAMs toward an antitumorigenic phenotype, and restores cytotoxic CD8+ T cell activity in vitro and ex vivo. Siglec-9 blockade synergizes with anti-PD-1 antibody to enhance CD8+ T cell cytotoxicity.","method":"Siglec-9 blocking antibody treatment of primary ovarian cancer TAMs, phospho-SHP-1 assay, macrophage phenotyping by flow cytometry, CD8+ T cell cytotoxicity assays (in vitro and ex vivo), anti-PD-1 combination","journal":"Journal for immunotherapy of cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — SHP-1 phosphorylation mechanistically linked to blocking, multiple functional readouts; single lab","pmids":["37709296"],"is_preprint":false},{"year":2024,"finding":"SARS-CoV-2 Omicron spike sequence FAPFFAF (positions 371–377) confers enhanced binding to Siglec-9 on macrophages, impairing phagocytosis and antigen presentation. A phenylalanine-to-serine mutation at position 375 (F375S) reverts this to the ancestral-strain sequence, abolishes enhanced Siglec-9 binding, and restores macrophage uptake and immunogenicity of Omicron RBD nanoparticles.","method":"Reverse mutagenesis (F375S) in Omicron spike, Siglec-9 binding assays, macrophage phagocytosis assays, antigen presentation assays, RBD nanoparticle immunization in mice/rabbits/macaques","journal":"Nature immunology","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — site-specific mutagenesis with direct mechanistic rescue, multiple species immunization, functional phagocytosis and antigen presentation assays","pmids":["38454157"],"is_preprint":false},{"year":2024,"finding":"ST3GAL4 is identified as the main driver of Siglec-9 ligand synthesis in AML cells by integrated CRISPR genomic screening and bioinformatics. CRISPR-Cas9 KO of ST3GAL4 dramatically reduces Siglec-9 ligand expression. Mass spectrometry shows Siglec-9 primarily binds N-linked sialoglycans on AML cells. ST3GAL4 KO enhances AML cell sensitivity to phagocytosis by Siglec-9-expressing macrophages.","method":"CRISPR genomic screen, ST3GAL4 CRISPR-Cas9 KO, Siglec-9 ligand expression assays, mass spectrometry of cell-surface glycosylation, macrophage phagocytosis assay","journal":"Leukemia","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — CRISPR screen + KO validation + mass spectrometry glycan analysis + functional phagocytosis assay; single lab, multiple orthogonal methods","pmids":["39551873"],"is_preprint":false},{"year":2024,"finding":"NMR spectroscopy and molecular dynamics revealed that Neu5Ac is accommodated between the F and G β-strands at the canonical sialic acid binding site of the Siglec-9 V-set domain. Synthetic sialoglycan modifications at C9 (MTTS scaffold) generate new interactions with hydrophobic residues at the G-G' loop and N-terminal region; C5 modifications (BTC scaffold) stabilize the B'-C loop, explaining enhanced affinity of these modified ligands.","method":"Triple-resonance 3D NMR backbone assignment of Siglec-9 V-set domain, NMR chemical shift perturbation mapping, molecular dynamics simulation","journal":"ACS chemical biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — NMR structural assignment plus MD simulation, mechanistic basis for ligand binding at atomic resolution established; single lab but rigorous method","pmids":["38321945"],"is_preprint":false},{"year":2024,"finding":"CD59 is identified as a candidate Siglec-9 ligand on prostate cancer cells by CRISPRi screen and mass spectrometry. Blocking Siglec-7/9–sialic acid interactions inhibited prostate cancer xenograft growth and increased immune cell infiltration in humanized mice.","method":"CRISPRi screen, mass spectrometry, Siglec-9 blocking in humanized mouse xenograft model","journal":"The Journal of clinical investigation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — unbiased CRISPRi screen plus MS identification plus in vivo blocking; CD59 as Siglec-9 ligand not yet independently confirmed","pmids":["39436703"],"is_preprint":false},{"year":2025,"finding":"DSG2 (Desmoglein 2) is identified as a dominant counter receptor of Siglec-9 in melanoma cells, with the interaction mediated primarily by sialic acid-bearing N-glycans on DSG2. Blocking DSG2–Siglec-9 trans-interaction significantly enhances macrophage phagocytosis of melanoma cells.","method":"Proximity labeling combined with CRISPR knockout screening, sialic acid dependency assays, macrophage phagocytosis assays","journal":"Advanced science (Weinheim, Baden-Wurttemberg, Germany)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — proximity labeling plus CRISPR screen plus functional validation; single lab, novel finding","pmids":["39813162"],"is_preprint":false},{"year":2025,"finding":"GPIbα mucin-like region carries O-linked glycans with α2,3-linked sialic acid that bind Siglec-9 in cis on platelets, acting as a 'parking brake' to suppress platelet activation. Siglec-E conditional knockout (platelet factor 4-Cre) significantly increases platelet coagulation activity in vivo and in vitro. The GPIbα ligand does not engage Siglec-9 in trans on other cells, indicating a self-modulation mechanism.","method":"Platelet factor 4-Cre:Siglec-E conditional KO mice, in vitro human platelet culture, recombinant GPIbα glycoprotein, cis vs. trans binding assays, neuraminidase treatment, platelet activation assays","journal":"Journal of thrombosis and haemostasis : JTH","confidence":"High","confidence_rationale":"Tier 2 / Moderate — conditional KO in vivo plus in vitro functional assays, ligand identity by recombinant protein, cis/trans distinction established; single lab","pmids":["40204021"],"is_preprint":false},{"year":2009,"finding":"Siglec-9 and SHP-1 physically interact in human neutrophils as shown by co-immunoprecipitation. Neonatal PMN express diminished Siglec-9 with basal phosphorylation, and GM-CSF differentially regulates Siglec-9 phosphorylation in neonatal vs. adult PMN (decreasing it in neonates, increasing it in adults), with distinct downstream survival signaling consequences.","method":"Co-immunoprecipitation of Siglec-9 and SHP-1, flow cytometry, western blot for phospho-Siglec-9, GM-CSF stimulation assays in adult and neonatal PMN","journal":"Pediatric research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP of Siglec-9/SHP-1 complex in primary neutrophils, differential phosphorylation confirmed; single lab","pmids":["19542910"],"is_preprint":false},{"year":2006,"finding":"Siglec-9 mediates rapid endocytosis of anti-Siglec-9 monoclonal antibody in AML cells and transfected rat basophilic leukemia cells, identifying it as an endocytic receptor on myeloid leukemia cells absent from normal bone marrow myeloid progenitors.","method":"Anti-Siglec-9 mAb internalization assay in primary AML cells and transfected RBL cells, flow cytometry","journal":"Leukemia research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — internalization directly demonstrated in primary AML cells and transfected cell line; single lab","pmids":["16828866"],"is_preprint":false},{"year":2015,"finding":"Siglec-9 modulates IL-4-stimulated macrophage signaling: Siglec-9 expression enhances induction of arginase-1 (Arg1) by IL-4 through the MEK-ERK pathway (not the PI-3K pathway), as ITIM tyrosine mutations abolish the Arg1 enhancement, and MEK inhibitors but not PI-3K inhibitors block the effect. Siglec-9 also enhances IL-4-induced Akt phosphorylation and ERK phosphorylation without IL-4.","method":"Stable transfection with Siglec-9 and ITIM mutants in RAW264, IL-4 stimulation, Arg1 expression assay, MEK and PI-3K inhibitors, phospho-Akt and phospho-ERK western blots","journal":"Bioscience, biotechnology, and biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ITIM mutagenesis plus pathway-selective inhibitors, defined signaling pathway; single lab","pmids":["26540411"],"is_preprint":false},{"year":2016,"finding":"Siglec-9 knockdown in human macrophages enhances LPS- and LPS/IFN-γ-induced CCR7 expression and decreases IL-4-induced CD200R expression, demonstrating that constitutively expressed Siglec-9 modulates macrophage polarization responses.","method":"Siglec-9 siRNA knockdown in primary human monocyte-derived macrophages, LPS/IFN-γ and IL-4 stimulation, CCR7 and CD200R expression by qRT-PCR and flow cytometry","journal":"Bioscience, biotechnology, and biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function knockdown with specific stimuli and phenotypic readouts; single lab","pmids":["26923638"],"is_preprint":false}],"current_model":"Siglec-9 is an inhibitory transmembrane lectin on myeloid cells, neutrophils, NK cells, and T cell subsets that recognizes α2,3- and α2,6-linked sialic acids (and non-sialylated HMW-HA) via its V-set Ig domain, recruits SHP-1 via cytoplasmic ITIM tyrosines to suppress innate and adaptive immune cell activation (dampening oxidative burst, NET formation, TCR/ZAP-70/NFAT signaling, NK cytotoxicity, and mast cell degranulation), and can also transduce activating signals (MEK-ERK, calcium flux) depending on the ligand and cell context; ligands include host sialoglycoproteins (glycophorin A, MUC1-ST, MUC16, GPIbα, DSG2, CD59), microbial sialoglycan mimics (GBS and GAS capsules), and VAP-1/AOC3 on endothelium, with ligand synthesis in cancer cells driven principally by ST3GAL4."},"narrative":{"mechanistic_narrative":"SIGLEC9 encodes an inhibitory sialic-acid-binding immunoglobulin-like lectin expressed across myeloid cells, neutrophils, T cells, mast cells, and platelets that translates recognition of host and microbial sialoglycans into immunoregulatory signals [PMID:10801860, PMID:10801862, PMID:15827126, PMID:30988027, PMID:37100120]. Its N-terminal V-set Ig domain binds both α2,3- and α2,6-linked sialic acids through a canonical site between the F and G β-strands, where a conserved domain-1 arginine (Arg120) is essential for ligand engagement, while a distinct C-C' loop region tunes glycan specificity [PMID:10801860, PMID:10801862, PMID:11741958, PMID:38321945]; the same V-set domain independently recognizes high-molecular-weight hyaluronan at a separate, non-sialic-acid site [PMID:26411873]. Upon ligand engagement and tyrosine phosphorylation of its cytoplasmic ITIM motifs, Siglec-9 recruits the phosphatase SHP-1 to dampen activating signaling, suppressing TCR/ZAP-70/NFAT signaling and T cell cytotoxicity, neutrophil oxidative burst and NET formation, and mast cell degranulation [PMID:15292262, PMID:30988027, PMID:37100120, PMID:19542910]. The two cytoplasmic tyrosines transmit divergent outputs in macrophages, enhancing IL-10 while suppressing TNF-α upon TLR stimulation [PMID:18325328, PMID:24449467]. Engagement is not uniformly inhibitory: certain cancer-associated ligands such as MUC1 bearing short sialylated O-glycans (MUC1-ST) bypass SHP recruitment to drive calcium flux and MEK-ERK activation, educating myeloid cells toward an immunosuppressive PD-L1-high tumor-associated macrophage phenotype [PMID:27595232, PMID:33627655, PMID:26540411]. A broad spectrum of counter-receptors has been mapped, including host sialoglycoproteins that enforce neutrophil and platelet quiescence (glycophorin A, Tamm-Horsfall glycoprotein, GPIbα) and tumor-cell ligands (MUC16, DSG2, CD59) whose synthesis is driven principally by the α2,3-sialyltransferase ST3GAL4 [PMID:28416510, PMID:28829050, PMID:40204021, PMID:20497550, PMID:39813162, PMID:39436703, PMID:39551873]. Sialylated bacterial capsules (group B and group A Streptococcus) and SARS-CoV-2 Omicron spike exploit Siglec-9 to evade phagocytic and neutrophil defenses, and Siglec-9 functions as an immune checkpoint whose genetic deletion or antibody blockade restores antitumor T cell and macrophage activity and synergizes with PD-1/PD-L1 blockade [PMID:19196661, PMID:26411873, PMID:38454157, PMID:37460871, PMID:37709296]. Siglec-9 also serves as a non-immune leukocyte ligand for endothelial VAP-1/AOC3 at its enzymatic groove [PMID:21821708, PMID:29391504].","teleology":[{"year":2000,"claim":"Establishing the molecular identity of Siglec-9 as a sialic-acid-binding inhibitory receptor was the foundational question, defining its domain architecture and ligand-binding determinants.","evidence":"cDNA cloning, COS-cell expression, recombinant binding assays, and mutagenesis of a conserved domain-1 arginine","pmids":["10801860","10801862"],"confidence":"High","gaps":["Cytoplasmic signaling output not yet defined","Physiological ligands in vivo not identified"]},{"year":2001,"claim":"How Siglec family members achieve distinct glycan specificities was resolved by mapping the sequence region that discriminates α2,3- versus α2,8-linked sialosides.","evidence":"Reciprocal C-C' loop domain-swap chimeras of Siglec-7 and Siglec-9 with glyco-probe binding and modeling","pmids":["11741958"],"confidence":"High","gaps":["Atomic-resolution structural basis not yet defined","Affinity for natural ligands not quantified"]},{"year":2004,"claim":"Whether Siglec-9 transmits inhibitory signals was answered by showing it suppresses TCR signaling via SHP-1 recruitment in an ITIM- and ligand-binding-dependent manner.","evidence":"Jurkat transfection, SHP-1 co-IP, phospho-ZAP-70 blots, NFAT reporter, Arg120Ala mutagenesis","pmids":["15292262"],"confidence":"High","gaps":["Endogenous T cell context not addressed","Physiological ligand triggering inhibition unknown"]},{"year":2005,"claim":"The functional consequence of Siglec-9 ligation on neutrophils was defined as induction of two distinct ROS-dependent death pathways, establishing a role in neutrophil fate.","evidence":"Antibody ligation of primary and inflammatory human neutrophils with ROS scavenger and caspase inhibitor dissection","pmids":["15827126"],"confidence":"High","gaps":["Natural ligands driving death in vivo not identified","Molecular link between ITIM signaling and ROS not mapped"]},{"year":2008,"claim":"How the two cytoplasmic tyrosines divide signaling labor was resolved by showing they differentially control IL-10 enhancement versus TNF-α suppression during TLR stimulation.","evidence":"Tyrosine-to-phenylalanine mutants in RAW264/THP-1 with TLR stimulation and cytokine ELISA","pmids":["18325328"],"confidence":"High","gaps":["Downstream effectors of each tyrosine not identified","Endogenous ligand context not tested"]},{"year":2009,"claim":"Microbial subversion of Siglec-9 was established by showing GBS sialylated capsule engages neutrophil Siglec-9 in trans to blunt antibacterial functions.","evidence":"Sialoglycan binding, neutrophil oxidative burst/NET assays, neuraminidase and blocking-antibody controls","pmids":["19196661"],"confidence":"High","gaps":["In vivo contribution to GBS pathogenesis not demonstrated","Host-pathogen specificity across Siglecs not delineated"]},{"year":2009,"claim":"The physical Siglec-9–SHP-1 complex was confirmed in primary neutrophils and shown to be developmentally regulated.","evidence":"Co-immunoprecipitation and phospho-Siglec-9 analysis in adult versus neonatal PMN with GM-CSF stimulation","pmids":["19542910"],"confidence":"Medium","gaps":["Reciprocal validation of complex stoichiometry not shown","Functional consequence of neonatal differences not fully resolved"]},{"year":2010,"claim":"Identification of MUC16 (CA125) as a Siglec-9 ligand connected the receptor to tumor immune evasion on NK cells, B cells, and monocytes.","evidence":"Flow cytometry of primary immune cells, Jurkat-Siglec-9 transfection, MUC16 binding with neuraminidase control","pmids":["20497550"],"confidence":"High","gaps":["Downstream signaling from MUC16 engagement not defined","Quantitative ligand affinity not measured"]},{"year":2011,"claim":"A non-immune adhesion function was uncovered by identifying Siglec-9 as a leukocyte ligand for endothelial VAP-1/AOC3, enabling inflammation imaging.","evidence":"Phage display, adhesion assays with mutants, modeling, and 68Ga-Siglec-9 peptide PET","pmids":["21821708"],"confidence":"High","gaps":["Signaling consequences of VAP-1 engagement on leukocytes unknown","Sialic-acid dependence of this interaction not fully resolved"]},{"year":2013,"claim":"Siglec-9 engagement was shown to actively promote tumor cell growth by inducing MUC1–β-catenin signaling, expanding its role beyond immune inhibition.","evidence":"Soluble Siglec-9 and co-culture treatment of MUC1-transfected cells, β-catenin co-IP, nuclear fractionation, neuraminidase control","pmids":["24045940"],"confidence":"High","gaps":["In vivo relevance to tumor progression not established","Receptor on the cancer-cell side beyond MUC1 not defined"]},{"year":2013,"claim":"A sialic-acid-independent counter-receptor mode was reported with prohibitins engaging Siglec-9 via an Arg120-dependent peptide interaction to inhibit ERK signaling.","evidence":"Arg120Ala mutant binding, bead co-immobilization with anti-CD3, phospho-ERK/c-Raf blots and IL-2 ELISA","pmids":["23567969"],"confidence":"Medium","gaps":["Single-lab finding without reciprocal validation","Physiological relevance of sialic-acid-independent binding unclear"]},{"year":2013,"claim":"Siglec-9 engagement on astrocytoma cells was shown to drive calpain-mediated degradation of focal-adhesion proteins, linking it to tumor cell motility.","evidence":"Co-culture immunoblotting of FAK/Akt/paxillin/p130Cas with calpain inhibitor rescue and invasion assays","pmids":["24145038"],"confidence":"Medium","gaps":["Single-lab finding requiring independent confirmation","Signaling link from Siglec-9 to calpain activation unmapped"]},{"year":2014,"claim":"Subcellular regulation of signaling was addressed by showing lectin-activity-dependent redistribution of Siglec-9 into lipid rafts contributes to IL-10 enhancement.","evidence":"Detergent-insoluble membrane fractionation, lectin-defective and ITIM mutants, cholesterol oxidase, cytokine ELISA","pmids":["24449467"],"confidence":"Medium","gaps":["Direct partners within rafts not identified","Single-lab observation"]},{"year":2015,"claim":"The first non-sialic-acid glycan ligand was identified, showing HMW-HA engages a distinct V-set site to suppress neutrophil functions, a route exploited by GAS.","evidence":"HMW-HA binding to a separate site, neutrophil functional assays, GAS capsule-dependent blocking","pmids":["26411873"],"confidence":"High","gaps":["Structural definition of the HA-binding site not resolved","Relative contribution of HA versus sialic-acid ligands in vivo unknown"]},{"year":2015,"claim":"Siglec-9 was shown to modulate alternative macrophage activation by enhancing IL-4-induced Arg1 through MEK-ERK rather than PI-3K.","evidence":"ITIM mutants in RAW264, IL-4 stimulation, pathway-selective inhibitors, phospho-ERK/Akt blots","pmids":["26540411"],"confidence":"Medium","gaps":["Single-lab finding","Ligand triggering this polarization in vivo not defined"]},{"year":2016,"claim":"A distinct activating signaling mode was established when tumor MUC1-ST was shown to drive Siglec-9 calcium flux and MEK-ERK activation, educating immunosuppressive TAMs.","evidence":"MUC1-ST binding, phosphatase assays (SHP-1/2 negative), calcium flux, MEK-ERK blots, PD-L1 flow cytometry","pmids":["27595232"],"confidence":"High","gaps":["Mechanistic switch between inhibitory and activating outputs not fully defined","Receptor proximal adaptor for calcium flux unknown"]},{"year":2016,"claim":"Loss-of-function and knockdown studies established that constitutively expressed Siglec-9 sets macrophage polarization responses and inflammatory cytokine balance.","evidence":"Siglec-9 siRNA knockdown with LPS/IFN-γ and IL-4 stimulation, CCR7/CD200R readouts; soluble Siglec-9 NF-κB inhibition with CIA model; SNP-variant cytokine assay","pmids":["26923638","27267914","27878892"],"confidence":"Medium","gaps":["Soluble versus membrane Siglec-9 mechanisms not unified","Genetic-variant functional impact tested only in vitro"]},{"year":2017,"claim":"Host 'self' sialoglycoproteins were identified as tonic suppressors of neutrophil activation, defining a sialic-acid-based self-recognition system.","evidence":"Glycophorin A and Tamm-Horsfall protein binding, periodate/neuraminidase modification, neutrophil activation rescue, THP-null mouse urinalysis","pmids":["28416510","28829050"],"confidence":"High","gaps":["Quantitative contribution of each self-ligand in vivo not weighted","Mechanism integrating multiple self-ligands unresolved"]},{"year":2018,"claim":"Src-family-kinase-dependent phosphorylation of Siglec-9 was shown to restrain dendritic-cell migration, and soluble Siglec-9/MCP-1 to drive reparative M2 macrophages.","evidence":"Dasatinib/SFK inhibitor and blocking-antibody migration assays; CCR2-blocking M2 differentiation and ALF rat model with M2 depletion","pmids":["24882272","28272428"],"confidence":"Medium","gaps":["Single-lab findings","Endogenous ligand driving DC restraint not identified"]},{"year":2018,"claim":"The Siglec-9–VAP-1 interaction was further characterized as occurring at the amine-oxidase catalytic site and capable of triggering enzymatic activity.","evidence":"Amine oxidase activity assay, inhibitor competition, Arg/Ala mutant peptide, molecular docking","pmids":["29391504"],"confidence":"Medium","gaps":["Physiological consequence of triggering VAP-1 activity unknown","Single-lab finding"]},{"year":2019,"claim":"Siglec-9 was defined as a T-cell inhibitory checkpoint in melanoma, expanding its checkpoint role to the adaptive immune compartment.","evidence":"Flow cytometry of tumor-infiltrating T cells, ligand/antibody engagement, cytotoxicity and cytokine assays, SHP-1-specific phosphorylation","pmids":["30988027"],"confidence":"High","gaps":["Identity of melanoma ligands not defined here","In vivo therapeutic blockade not tested in this study"]},{"year":2021,"claim":"The cancer-cell glycan machinery generating Siglec-9 ligands was traced to α2,3-sialyltransferases, and synthetic agonists confirmed pharmacological control of neutrophil NETosis.","evidence":"ST3GAL1/ST3GAL4 transcriptomics with Siglec-9 triggering on macrophages; synthetic glycopolymer agonists in COVID-19 neutrophil NETosis assays","pmids":["33627655","34056095"],"confidence":"High","gaps":["Single dominant sialyltransferase across cancers not generalized","In vivo efficacy of agonists/antagonists not established here"]},{"year":2023,"claim":"Genetic and antibody studies established Siglec-9 as a targetable macrophage checkpoint in glioblastoma and ovarian cancer that synergizes with PD-1/PD-L1 blockade, and as an inhibitory mast-cell receptor.","evidence":"Siglece-KO mouse GBM models with scRNA-seq; ovarian TAM blockade with phospho-SHP-1 and CD8 cytotoxicity assays; CRISPR SIGLEC9 knockout in primary mast cells with ligand gain-of-function","pmids":["37460871","37709296","37100120"],"confidence":"High","gaps":["Murine Siglece versus human Siglec-9 correspondence not fully resolved","Combination dosing and ligand-specific responses not optimized"]},{"year":2024,"claim":"New ligands and structural/glycan determinants were defined, and viral exploitation of Siglec-9 by SARS-CoV-2 Omicron spike was demonstrated.","evidence":"NMR/MD of the V-set domain; ST3GAL4 CRISPR screen and mass spectrometry in AML; CD59 CRISPRi screen; Omicron F375S spike mutagenesis with phagocytosis/antigen-presentation rescue","pmids":["38321945","39551873","39436703","38454157"],"confidence":"High","gaps":["CD59 as a ligand not independently confirmed","In vivo relevance of viral exploitation to disease severity not established"]},{"year":2025,"claim":"Additional counter-receptors were mapped, including DSG2 in melanoma and a cis self-modulatory GPIbα–Siglec-9 interaction that brakes platelet activation.","evidence":"Proximity-labeling/CRISPR screen identifying DSG2 with phagocytosis assays; platelet-specific Siglec-E conditional KO with cis/trans GPIbα binding assays","pmids":["39813162","40204021"],"confidence":"Medium","gaps":["DSG2 finding from single lab","Mechanistic distinction between cis and trans ligand engagement not fully generalized"]},{"year":null,"claim":"How a single V-set domain integrates competing host self-ligands, microbial mimics, and HMW-HA to switch between SHP-1-dependent inhibition and calcium/MEK-ERK activation in a given cell type remains unresolved.","evidence":"No single study reconciles ligand identity, cell context, and signaling output across compartments","pmids":[],"confidence":"Medium","gaps":["No unified rule predicting inhibitory versus activating output from ligand structure","Quantitative ligand competition in vivo not modeled","Proximal adaptor mediating activating calcium flux unidentified"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[0,1,12,29]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[2,4,21,24]},{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[2,13,21,24]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[12]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[2,33]},{"term_id":"GO:0038024","term_label":"cargo receptor activity","supporting_discovery_ids":[34]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,11,32]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[8]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[2,4,21,24,25]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[2,13,33,35]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[5,12,22,27]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[3]},{"term_id":"R-HSA-109582","term_label":"Hemostasis","supporting_discovery_ids":[32]}],"complexes":[],"partners":["PTPN6","MUC1","MUC16","AOC3","GYPA","DSG2","GP1BA","CD59"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9Y336","full_name":"Sialic acid-binding Ig-like lectin 9","aliases":["CDw329","Protein FOAP-9"],"length_aa":463,"mass_kda":50.1,"function":"Putative adhesion molecule that mediates sialic-acid dependent binding to cells. Preferentially binds to alpha-2,3- or alpha-2,6-linked sialic acid. The sialic acid recognition site may be masked by cis interactions with sialic acids on the same cell surface","subcellular_location":"Membrane","url":"https://www.uniprot.org/uniprotkb/Q9Y336/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/SIGLEC9","classification":"Not Classified","n_dependent_lines":1,"n_total_lines":1208,"dependency_fraction":0.0008278145695364238},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/SIGLEC9","total_profiled":1310},"omim":[{"mim_id":"606135","title":"KALLIKREIN-RELATED PEPTIDASE 14; KLK14","url":"https://www.omim.org/entry/606135"},{"mim_id":"606094","title":"SIALIC ACID-BINDING IMMUNOGLOBULIN-LIKE LECTIN 12; SIGLEC12","url":"https://www.omim.org/entry/606094"},{"mim_id":"606091","title":"SIALIC ACID-BINDING IMMUNOGLOBULIN-LIKE LECTIN 10; SIGLEC10","url":"https://www.omim.org/entry/606091"},{"mim_id":"605640","title":"SIALIC ACID-BINDING IMMUNOGLOBULIN-LIKE LECTIN 9; SIGLEC9","url":"https://www.omim.org/entry/605640"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"lymphoid tissue","ntpm":19.9}],"url":"https://www.proteinatlas.org/search/SIGLEC9"},"hgnc":{"alias_symbol":["CD329"],"prev_symbol":[]},"alphafold":{"accession":"Q9Y336","domains":[{"cath_id":"2.60.40.10","chopping":"23-141","consensus_level":"high","plddt":91.3161,"start":23,"end":141},{"cath_id":"2.60.40.10","chopping":"148-234","consensus_level":"high","plddt":86.6776,"start":148,"end":234},{"cath_id":"2.60.40.10","chopping":"241-339","consensus_level":"high","plddt":75.2008,"start":241,"end":339}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y336","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y336-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y336-F1-predicted_aligned_error_v6.png","plddt_mean":73.44},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=SIGLEC9","jax_strain_url":"https://www.jax.org/strain/search?query=SIGLEC9"},"sequence":{"accession":"Q9Y336","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9Y336.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9Y336/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y336"}},"corpus_meta":[{"pmid":"19196661","id":"PMC_19196661","title":"Molecular mimicry of host sialylated glycans allows a bacterial pathogen to engage neutrophil Siglec-9 and dampen the innate immune response.","date":"2009","source":"Blood","url":"https://pubmed.ncbi.nlm.nih.gov/19196661","citation_count":316,"is_preprint":false},{"pmid":"27595232","id":"PMC_27595232","title":"The mucin MUC1 modulates the tumor immunological microenvironment through engagement of the lectin Siglec-9.","date":"2016","source":"Nature immunology","url":"https://pubmed.ncbi.nlm.nih.gov/27595232","citation_count":312,"is_preprint":false},{"pmid":"33627655","id":"PMC_33627655","title":"Sialic acids in pancreatic cancer cells drive tumour-associated macrophage differentiation via the Siglec receptors Siglec-7 and Siglec-9.","date":"2021","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/33627655","citation_count":256,"is_preprint":false},{"pmid":"37460871","id":"PMC_37460871","title":"Siglec-9 acts as an immune-checkpoint molecule on macrophages in glioblastoma, restricting T-cell priming and immunotherapy response.","date":"2023","source":"Nature cancer","url":"https://pubmed.ncbi.nlm.nih.gov/37460871","citation_count":190,"is_preprint":false},{"pmid":"10801862","id":"PMC_10801862","title":"Siglec-9, a novel sialic acid binding member of the immunoglobulin superfamily expressed broadly on human blood leukocytes.","date":"2000","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/10801862","citation_count":190,"is_preprint":false},{"pmid":"15827126","id":"PMC_15827126","title":"Siglec-9 transduces apoptotic and nonapoptotic death signals into neutrophils depending on the proinflammatory cytokine environment.","date":"2005","source":"Blood","url":"https://pubmed.ncbi.nlm.nih.gov/15827126","citation_count":181,"is_preprint":false},{"pmid":"20497550","id":"PMC_20497550","title":"Identification of Siglec-9 as the receptor for MUC16 on human NK cells, B cells, and monocytes.","date":"2010","source":"Molecular cancer","url":"https://pubmed.ncbi.nlm.nih.gov/20497550","citation_count":172,"is_preprint":false},{"pmid":"11741958","id":"PMC_11741958","title":"A small region of the natural killer cell receptor, Siglec-7, is responsible for its preferred binding to alpha 2,8-disialyl and branched alpha 2,6-sialyl residues. A comparison with Siglec-9.","date":"2001","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/11741958","citation_count":168,"is_preprint":false},{"pmid":"15292262","id":"PMC_15292262","title":"Negative regulation of T cell receptor signaling by Siglec-7 (p70/AIRM) and Siglec-9.","date":"2004","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/15292262","citation_count":163,"is_preprint":false},{"pmid":"10801860","id":"PMC_10801860","title":"Cloning, characterization, and phylogenetic analysis of siglec-9, a new member of the CD33-related group of siglecs. Evidence for co-evolution with sialic acid synthesis pathways.","date":"2000","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/10801860","citation_count":132,"is_preprint":false},{"pmid":"30988027","id":"PMC_30988027","title":"Siglec-9 Regulates an Effector Memory CD8+ T-cell Subset That Congregates in the Melanoma Tumor Microenvironment.","date":"2019","source":"Cancer immunology research","url":"https://pubmed.ncbi.nlm.nih.gov/30988027","citation_count":130,"is_preprint":false},{"pmid":"21821708","id":"PMC_21821708","title":"Siglec-9 is a novel leukocyte ligand for vascular adhesion protein-1 and can be used in PET imaging of inflammation and cancer.","date":"2011","source":"Blood","url":"https://pubmed.ncbi.nlm.nih.gov/21821708","citation_count":104,"is_preprint":false},{"pmid":"28416510","id":"PMC_28416510","title":"Erythrocyte sialoglycoproteins engage Siglec-9 on neutrophils to suppress 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Germany)","url":"https://pubmed.ncbi.nlm.nih.gov/26411873","citation_count":73,"is_preprint":false},{"pmid":"20971061","id":"PMC_20971061","title":"Immunomodulation of monocyte-derived dendritic cells through ligation of tumor-produced mucins to Siglec-9.","date":"2010","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/20971061","citation_count":66,"is_preprint":false},{"pmid":"24045940","id":"PMC_24045940","title":"Binding of the sialic acid-binding lectin, Siglec-9, to the membrane mucin, MUC1, induces recruitment of β-catenin and subsequent cell growth.","date":"2013","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/24045940","citation_count":66,"is_preprint":false},{"pmid":"34056095","id":"PMC_34056095","title":"Synthetic Siglec-9 Agonists Inhibit Neutrophil Activation Associated with COVID-19.","date":"2021","source":"ACS central 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transmembrane protein with three extracellular Ig-like domains (N-terminal V-set + two C2-set), a transmembrane region, and a cytoplasmic tail containing two tyrosine-based signaling motifs including a canonical ITIM. Expression of full-length cDNA in COS cells induced sialic-acid-dependent erythrocyte binding. Recombinant soluble extracellular domain binds α2-3 and α2-6-linked sialic acids; the carboxyl group and side chain of sialic acid are essential, and mutation of a critical arginine residue in domain 1 abrogates binding.\",\n      \"method\": \"cDNA cloning, COS cell expression, recombinant protein binding assays, site-directed mutagenesis of conserved Arg\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstitution in cell expression system, mutagenesis of active-site Arg, multiple binding assays replicated across two independent papers (PMID:10801860, PMID:10801862)\",\n      \"pmids\": [\"10801860\", \"10801862\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"The C-C' loop region (residues Asn70–Lys75) in the V-set sugar-binding domain of Siglec-7 determines its preference for α2,8-disialyl and branched α2,6-sialyl residues (GD3, LSTb), whereas the equivalent region in Siglec-9 confers preference for α2,3-linked (LSTc, GD1a) structures. Swapping this small region between Siglec-7 and Siglec-9 chimeras transferred binding specificity accordingly.\",\n      \"method\": \"Chimeric protein expression in CHO cells, polyvalent streptavidin-based glyco-probe binding assays, molecular modeling\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — domain-swap mutagenesis with reciprocal transfer of binding specificity, validated by glycan probes and molecular modeling\",\n      \"pmids\": [\"11741958\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Siglec-9 negatively regulates T cell receptor (TCR) signaling: following pervanadate stimulation or TCR engagement, Siglec-9 undergoes tyrosine phosphorylation and recruits SHP-1; it reduces phosphorylation of ZAP-70 Tyr319 and decreases NFAT transcriptional activity. Mutation of the conserved Arg120 in the ligand-binding site reduces inhibitory function, indicating ligand binding is required for optimal TCR inhibition.\",\n      \"method\": \"Stable/transient transfection of Jurkat T cells with Siglec-9, pervanadate/TCR stimulation, co-immunoprecipitation of SHP-1, phospho-ZAP-70 western blot, NFAT-luciferase reporter assay, Arg120Ala mutagenesis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — multiple orthogonal methods (co-IP, western blot, reporter assay) with mutagenesis in a single study\",\n      \"pmids\": [\"15292262\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Siglec-9 ligation on normal neutrophils induces caspase-dependent, ROS-dependent apoptosis. In neutrophils primed with GM-CSF, IFN-α, or IFN-γ, Siglec-9 ligation triggers a caspase-independent, ROS-dependent cell death with cytoplasmic vacuolization. Both death pathways are abrogated by ROS scavengers or in neutrophils unable to generate ROS.\",\n      \"method\": \"Siglec-9 antibody ligation on primary human neutrophils and inflammatory neutrophils (sepsis, RA patients), ROS scavenger experiments, caspase inhibitor experiments, cytology\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — primary human cells and patient samples, pharmacological dissection of pathway, two distinct death modalities established with orthogonal inhibitors\",\n      \"pmids\": [\"15827126\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Siglec-9 expression in macrophages (RAW264 and THP-1) strongly enhances IL-10 production and reduces TNF-α upon TLR stimulation (LPS, peptidoglycan, CpG, dsRNA). These effects require both cytoplasmic tyrosine residues: mutation of both to phenylalanine abrogates IL-10 enhancement and TNF-α suppression. A membrane-proximal ITIM mutant partially retains TNF-α inhibition but loses IL-10 enhancement, indicating divergent signaling via the two tyrosines.\",\n      \"method\": \"Stable transfection of Siglec-9 and ITIM tyrosine mutants in RAW264/THP-1, TLR stimulation assays, ELISA for cytokines\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — mutagenesis of both ITIM tyrosines in two cell lines, functional readout by cytokine ELISA, mechanistic dissection of two tyrosines\",\n      \"pmids\": [\"18325328\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Bacterial group B Streptococcus (GBS) sialylated capsular polysaccharide (CPS) presenting terminal Sialα2-3Galβ1-4GlcNAc engages neutrophil Siglec-9 in trans, dampening neutrophil oxidative burst, NET formation, and enabling bacterial survival. These effects are Sia- and Siglec-9-dependent (abrogated by neuraminidase treatment of GBS or by blocking Siglec-9).\",\n      \"method\": \"Immobilized sialoglycan binding assays, GBS CPS binding to isolated human neutrophils, oxidative burst assays, NET formation assays, bacterial survival assays, neuraminidase treatment, Siglec-9 blocking antibodies\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple functional readouts, specific controls (neuraminidase, blocking antibody), primary human neutrophils\",\n      \"pmids\": [\"19196661\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Siglec-9 is identified as the receptor for MUC16 (CA125) on NK cells, B cells, and monocytes. Siglec-9-transfected Jurkat cells and monocytes from healthy donors bind to ovarian tumor cells via Siglec-9–csMUC16 interaction; neuraminidase treatment of immune cells releases sMUC16, confirming sialic acid dependence.\",\n      \"method\": \"Flow cytometry of primary immune cells, Siglec-9 transfection into Jurkat cells, MUC16 binding assays, neuraminidase treatment, co-culture adhesion assays\",\n      \"journal\": \"Molecular cancer\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — transfection-based functional binding assay with neuraminidase control, reciprocal cell-cell interaction assays\",\n      \"pmids\": [\"20497550\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Siglec-9 is a novel leukocyte ligand for vascular adhesion protein-1 (VAP-1/AOC3). The interaction was identified by phage display and confirmed by in vitro and ex vivo adhesion assays. Interaction occurs at the enzymatic groove of VAP-1 and is only partially dependent on VAP-1 enzymatic activity. A 68Ga-labeled Siglec-9 peptide specifically detects VAP-1 at sites of inflammation and cancer by PET.\",\n      \"method\": \"Phage display, in vitro/ex vivo adhesion assays with mutated proteins, molecular modeling, PET imaging with 68Ga-labeled Siglec-9 peptide\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — phage display discovery, confirmed by multiple binding assays with mutants, in vivo PET validation\",\n      \"pmids\": [\"21821708\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Siglec-9 binds MUC1 on cancer cells in a sialic acid-dependent manner, inducing recruitment of β-catenin to the MUC1 C-terminal domain in a dose- and time-dependent manner. Recruited β-catenin translocates to the nucleus, promoting cell growth. Neuraminidase treatment abolishes Siglec-9-induced signaling.\",\n      \"method\": \"Recombinant soluble Siglec-9 treatment of MUC1-transfected 3T3 and HCT116 cells, co-culture with Siglec-9-expressing HEK293 cells, co-immunoprecipitation of β-catenin with MUC1, nuclear fractionation, neuraminidase treatment, proliferation assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — co-IP, nuclear fractionation, dose-response, neuraminidase control, co-culture model, multiple cell lines\",\n      \"pmids\": [\"24045940\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Prohibitin-1 and prohibitin-2 expressed on the surface of T cell leukemia lines and activated T lymphocytes serve as counter-receptors for Siglec-9 on macrophages and dendritic cells, in a sialic acid-independent but Arg120-dependent ionic peptide–peptide interaction. Engagement of prohibitins via Siglec-9 (co-immobilized with anti-CD3) inhibits ERK1/2 phosphorylation, c-Raf phosphorylation, and IL-2 production in Jurkat cells.\",\n      \"method\": \"Binding assays with Siglec-9 Arg120Ala mutant, co-immobilization of Siglec-9 and anti-CD3 on beads, phospho-ERK1/2 and phospho-c-Raf western blot, IL-2 ELISA\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis of binding site, bead co-immobilization functional assay, western blot and ELISA; single lab\",\n      \"pmids\": [\"23567969\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Siglec-9 binding to sialylglycoconjugates on astrocytoma (AS) cells induces rapid calpain-mediated degradation of focal adhesion kinase (FAK), Akt, paxillin, and p130Cas, leading to cell detachment and increased motility/invasiveness. Despite degradation of total Akt, phospho-Akt was increased at the leading cytoplasmic edge, consistent with enhanced motility.\",\n      \"method\": \"Co-culture of Siglec-9-expressing and Siglec-9-deficient cells with AS astrocytoma cells, immunoblotting for FAK/Akt/paxillin/p130Cas degradation, calpain inhibitor experiments, motility and invasion assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pharmacological inhibition of calpain with functional rescue, multiple substrate measurements; single lab\",\n      \"pmids\": [\"24145038\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Upon TLR2 stimulation, a portion of Siglec-9 redistributes into lipid raft (detergent-insoluble microdomain) fractions with kinetics mirroring TLR2 redistribution (peak 3–10 min). This raft localization is lectin-activity-dependent: a lectin-defective Siglec-9 mutant fails to enter lipid rafts, whereas a double-ITIM tyrosine mutant still translocates. IL-10 production is partially reduced by disrupting lipid raft organization with cholesterol oxidase, suggesting raft localization contributes to IL-10 enhancement.\",\n      \"method\": \"Membrane fractionation (detergent-insoluble microdomains), lectin-defective and ITIM tyrosine mutant transfection, cholesterol oxidase treatment, cytokine ELISA\",\n      \"journal\": \"Cytotechnology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — fractionation plus mutagenesis plus pharmacological disruption; single lab\",\n      \"pmids\": [\"24449467\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"High-molecular-weight hyaluronan (HMW-HA) is recognized by Siglec-9 through a site in its V-set domain distinct from the sialic acid-binding site, representing the first non-sialic-acid glycan ligand for a CD33-related Siglec. HMW-HA engagement of Siglec-9 limits NET formation, oxidative burst, and apoptosis in human neutrophils. Group A Streptococcus (GAS) exploits its own HMW-HA capsule to engage Siglec-9, blocking these neutrophil functions and promoting bacterial survival.\",\n      \"method\": \"Binding assays with Siglec-9 and HMW-HA, neutrophil functional assays (NET formation, oxidative burst, apoptosis), GAS capsule-dependent blocking experiments\",\n      \"journal\": \"Journal of molecular medicine (Berlin, Germany)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — distinct binding site mapped, multiple neutrophil functional readouts, pathogen exploitation validated; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"26411873\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"MUC1 carrying cancer-specific short sialylated O-linked glycans (MUC1-ST) binds Siglec-9 on myeloid cells without activating SHP-1 or SHP-2, but instead induces calcium flux leading to MEK-ERK kinase activation, educating myeloid cells toward a tumor-associated macrophage-like phenotype with increased PD-L1 expression.\",\n      \"method\": \"MUC1-ST binding assays, phosphatase activation assays (SHP-1/SHP-2), calcium flux measurement, MEK-ERK phosphorylation western blot, PD-L1 expression by flow cytometry, macrophage phenotyping\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal signaling assays (phosphatases negative, Ca2+ flux, MEK-ERK positive), peer-reviewed high-impact journal, distinct activating pathway established\",\n      \"pmids\": [\"27595232\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"A SIGLEC9 GA haplotype (rs2075803 G/rs2258983 A) encodes a Siglec-9 protein variant that is less effective at suppressing inflammatory TNF-α production in a myeloid cell line compared to the other major haplotype variant, as measured by in vitro cytokine assays.\",\n      \"method\": \"In vitro myeloid cell line transfection with variant Siglec-9 constructs, TNF-α ELISA after stimulation\",\n      \"journal\": \"Respirology (Carlton, Vic.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single in vitro functional assay with SNP variants, single lab\",\n      \"pmids\": [\"27878892\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Soluble Siglec-9 (sSiglec-9) suppresses M1 macrophage activation by inhibiting NF-κB p65 phosphorylation in RAW264.7 cells, reducing M1 marker expression (TNF-α, IL-6, iNOS) without affecting M2 markers. In a murine collagen-induced arthritis model, sSiglec-9 attenuated arthritis severity, decreased serum TNF-α, and increased Foxp3+ Treg cell proportions.\",\n      \"method\": \"RAW264.7 macrophage culture with sSiglec-9, western blot for NF-κB p65 phosphorylation, NF-κB chemical blockade, cytokine ELISA, DBA/1J mouse CIA model, histology, flow cytometry for Tregs\",\n      \"journal\": \"Arthritis research & therapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — NF-κB pathway confirmed by both phospho-blot and chemical inhibitor, in vivo model with multiple readouts; single lab\",\n      \"pmids\": [\"27267914\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Glycophorin A, the most abundant sialoglycoprotein on erythrocytes, engages neutrophil Siglec-9 to suppress neutrophil activation in vitro and ex vivo. Mild periodate oxidation of erythrocyte sialic acid side chains (with aldehyde quenching) reduces erythrocyte binding to Siglec-9 and restores neutrophil activation (l-selectin shedding, oxidative burst, chemotaxis, NET formation, apoptosis), demonstrating a sialic acid-based 'self' signal that maintains neutrophil quiescence in blood.\",\n      \"method\": \"ELISA and immunofluorescence for glycophorin A–Siglec-9 interaction, sodium metaperiodate oxidation of erythrocyte sialic acids, ex vivo and in vitro neutrophil activation assays\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — specific chemical modification of sialic acids with functional rescue, multiple activation readouts, primary human cells\",\n      \"pmids\": [\"28416510\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Tamm-Horsfall glycoprotein (THP) engages neutrophil Siglec-9 (and its mouse ortholog Siglec-E) via N-glycan sialic acid moieties, suppressing neutrophil ROS generation, chemotaxis, and killing of uropathogenic E. coli. THP-null mice have significantly more neutrophils in urine than wild-type mice.\",\n      \"method\": \"THP–neutrophil binding assays, neuraminidase treatment, Siglec-9 blocking antibody, ROS/chemotaxis/bacterial killing assays, THP-null mouse urinalysis\",\n      \"journal\": \"Immunology and cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — neuraminidase and blocking antibody controls, multiple functional assays, in vivo THP-null mouse model\",\n      \"pmids\": [\"28829050\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"MCP-1 and secreted ectodomain of Siglec-9 (sSiglec-9) synergistically promote M2 macrophage differentiation from bone marrow-derived macrophages via CCR2, producing liver-regenerating factors that suppress hepatocyte apoptosis and promote proliferation. In a rat acute liver failure model, combined MCP-1/sSiglec-9 treatment improved survival and induced anti-inflammatory M2 macrophages; depletion of M2 macrophages (mannosylated clodronate liposomes) abolished recovery.\",\n      \"method\": \"In vitro M2 differentiation assay with bone marrow-derived macrophages, CCR2 blocking, D-galactosamine rat ALF model, M2-depletion with mannosylated clodronate liposomes, hepatocyte apoptosis/proliferation assays\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CCR2 blocking identifies receptor, M2-depletion confirms mechanism, in vivo model; single lab\",\n      \"pmids\": [\"28272428\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Dasatinib dephosphorylates Siglec-9 (and Siglec-3) in human monocyte-derived dendritic cells by inhibiting SRC-family kinases, which more than doubles the number of moDCs migrating toward a CCL19 gradient. Specific blocking of Siglec-9 also enhanced DC migration, confirming that SFK-dependent Siglec-9 phosphorylation restrains DC migration.\",\n      \"method\": \"Dasatinib and SRC inhibitor 1 treatment of moDCs, phosphorylation assays for Siglec-9 and SFKs, CCL19 gradient migration assays, specific Siglec-9 blocking antibody\",\n      \"journal\": \"Experimental hematology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — two pharmacological approaches (dasatinib and SFK-specific inhibitor) plus blocking antibody all converge on same phenotype; single lab\",\n      \"pmids\": [\"24882272\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Siglec-9 is an inhibitory receptor on human primary amine oxidase (hAOC3/VAP-1). The Siglec-9 peptide binds to hAOC3 and triggers its amine oxidase enzymatic activity toward benzylamine. hAOC3 inhibitors (semicarbazide and imidazole) reduce binding of wild-type and Arg/Ala mutated Siglec-9 peptides to hAOC3, and molecular docking shows the R3 residue of the Siglec-9 peptide interacts in the catalytic site when topaquinone is in the non-catalytic on-copper conformation.\",\n      \"method\": \"Amine oxidase activity assay, competitive binding with hAOC3 inhibitors, Arg/Ala mutant peptide, molecular docking\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — enzymatic activity assay plus inhibitor competition plus mutagenesis plus docking; single lab\",\n      \"pmids\": [\"29391504\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Siglec-9 is expressed on intratumoral CD8+ effector memory T cells in melanoma and functions as an inhibitory checkpoint: engagement of Siglec-9 by its ligands or specific antibodies suppresses TCR signaling, cytotoxicity, and cytokine production. Inhibition is associated with phosphorylation of SHP-1 but not SHP-2. Cognate Siglec-9 ligands are expressed on the majority of primary and metastatic melanoma tumor cells.\",\n      \"method\": \"Flow cytometry of tumor-infiltrating vs. peripheral T cells, Siglec-9 ligand engagement/antibody stimulation assays, cytotoxicity assays, cytokine production, phospho-SHP-1/SHP-2 assays\",\n      \"journal\": \"Cancer immunology research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — primary tumor samples, functional inhibition assays with specific ligand and antibody engagement, signaling pathway specificity (SHP-1 not SHP-2) established\",\n      \"pmids\": [\"30988027\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Pancreatic ductal adenocarcinoma cells express increased sialylation driven primarily by α2,3-sialyltransferases ST3GAL1 and ST3GAL4, producing ligands recognized by Siglec-9 (and Siglec-7) on myeloid cells. Triggering Siglec-9 in macrophages reduces inflammatory programmes and increases PD-L1 and IL-10 expression, directing monocyte-to-macrophage differentiation toward an immunosuppressive phenotype.\",\n      \"method\": \"ST3GAL1/ST3GAL4 identification by transcriptomics, Siglec-9 binding assays, Siglec-9 triggering on macrophages, PD-L1/IL-10 expression assays, single-cell and bulk transcriptomics\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — sialyltransferase identified as ligand source, functional Siglec-9 triggering assay with signaling readouts, multiple orthogonal methods\",\n      \"pmids\": [\"33627655\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Synthetic glycopolymers designed as Siglec-9 agonists suppress NETosis in human neutrophils induced by viral TLR agonists and plasma from severe COVID-19 patients, confirming Siglec-9 as a functional checkpoint receptor that can be pharmacologically activated to suppress neutrophil extracellular trap formation.\",\n      \"method\": \"Synthetic glycopolymer synthesis, neutrophil NETosis assays with TLR agonists and COVID-19 patient plasma, Siglec-9 agonism confirmed by receptor engagement\",\n      \"journal\": \"ACS central science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — synthetic agonist tool compounds with defined mechanism, primary human neutrophils and patient samples; single lab\",\n      \"pmids\": [\"34056095\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Siglec-9 is expressed on human mast cells and functions as an inhibitory receptor. CRISPR/Cas9 disruption of SIGLEC9 increases baseline activation marker expression and enhances responsiveness to IgE-dependent and -independent stimulation. Glycophorin A and HMW-HA (native Siglec-9 ligands), as well as Siglec-9 co-engagement with FcεRI, reduce mast cell degranulation, arachidonic acid production, and chemokine release.\",\n      \"method\": \"CRISPR/Cas9 SIGLEC9 knockout, flow cytometry for activation markers, degranulation assays, arachidonic acid measurement, chemokine ELISA, native ligand pretreatment, FcεRI co-engagement\",\n      \"journal\": \"The Journal of allergy and clinical immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — CRISPR KO loss-of-function plus ligand gain-of-function, multiple orthogonal readouts in primary mast cells; single lab\",\n      \"pmids\": [\"37100120\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Siglec-9 functions as an immune-checkpoint molecule on macrophages in glioblastoma. Deletion of Siglece (murine Siglec-9 homolog) restrained tumor development and prolonged survival in mouse GBM models. Mechanistically, Siglece deletion directly activated both CD4+ and CD8+ T cells through antigen presentation, secreted chemokines, and co-stimulatory factor interactions. Siglece deletion synergized with anti-PD-1/PD-L1 treatment.\",\n      \"method\": \"Siglece knockout mouse GBM models, survival analysis, single-cell RNA-seq, spatial transcriptomics, T cell activation assays (antigen presentation, chemokines, co-stimulatory factors)\",\n      \"journal\": \"Nature cancer\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO in vivo with survival readout, mechanistic pathway mapped by scRNA-seq and functional T cell assays, synergy with checkpoint blockade demonstrated\",\n      \"pmids\": [\"37460871\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Blockade of Siglec-9 in ovarian cancer suppresses phosphorylation of SHP-1, repolarizes TAMs toward an antitumorigenic phenotype, and restores cytotoxic CD8+ T cell activity in vitro and ex vivo. Siglec-9 blockade synergizes with anti-PD-1 antibody to enhance CD8+ T cell cytotoxicity.\",\n      \"method\": \"Siglec-9 blocking antibody treatment of primary ovarian cancer TAMs, phospho-SHP-1 assay, macrophage phenotyping by flow cytometry, CD8+ T cell cytotoxicity assays (in vitro and ex vivo), anti-PD-1 combination\",\n      \"journal\": \"Journal for immunotherapy of cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — SHP-1 phosphorylation mechanistically linked to blocking, multiple functional readouts; single lab\",\n      \"pmids\": [\"37709296\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"SARS-CoV-2 Omicron spike sequence FAPFFAF (positions 371–377) confers enhanced binding to Siglec-9 on macrophages, impairing phagocytosis and antigen presentation. A phenylalanine-to-serine mutation at position 375 (F375S) reverts this to the ancestral-strain sequence, abolishes enhanced Siglec-9 binding, and restores macrophage uptake and immunogenicity of Omicron RBD nanoparticles.\",\n      \"method\": \"Reverse mutagenesis (F375S) in Omicron spike, Siglec-9 binding assays, macrophage phagocytosis assays, antigen presentation assays, RBD nanoparticle immunization in mice/rabbits/macaques\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — site-specific mutagenesis with direct mechanistic rescue, multiple species immunization, functional phagocytosis and antigen presentation assays\",\n      \"pmids\": [\"38454157\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ST3GAL4 is identified as the main driver of Siglec-9 ligand synthesis in AML cells by integrated CRISPR genomic screening and bioinformatics. CRISPR-Cas9 KO of ST3GAL4 dramatically reduces Siglec-9 ligand expression. Mass spectrometry shows Siglec-9 primarily binds N-linked sialoglycans on AML cells. ST3GAL4 KO enhances AML cell sensitivity to phagocytosis by Siglec-9-expressing macrophages.\",\n      \"method\": \"CRISPR genomic screen, ST3GAL4 CRISPR-Cas9 KO, Siglec-9 ligand expression assays, mass spectrometry of cell-surface glycosylation, macrophage phagocytosis assay\",\n      \"journal\": \"Leukemia\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — CRISPR screen + KO validation + mass spectrometry glycan analysis + functional phagocytosis assay; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"39551873\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"NMR spectroscopy and molecular dynamics revealed that Neu5Ac is accommodated between the F and G β-strands at the canonical sialic acid binding site of the Siglec-9 V-set domain. Synthetic sialoglycan modifications at C9 (MTTS scaffold) generate new interactions with hydrophobic residues at the G-G' loop and N-terminal region; C5 modifications (BTC scaffold) stabilize the B'-C loop, explaining enhanced affinity of these modified ligands.\",\n      \"method\": \"Triple-resonance 3D NMR backbone assignment of Siglec-9 V-set domain, NMR chemical shift perturbation mapping, molecular dynamics simulation\",\n      \"journal\": \"ACS chemical biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR structural assignment plus MD simulation, mechanistic basis for ligand binding at atomic resolution established; single lab but rigorous method\",\n      \"pmids\": [\"38321945\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CD59 is identified as a candidate Siglec-9 ligand on prostate cancer cells by CRISPRi screen and mass spectrometry. Blocking Siglec-7/9–sialic acid interactions inhibited prostate cancer xenograft growth and increased immune cell infiltration in humanized mice.\",\n      \"method\": \"CRISPRi screen, mass spectrometry, Siglec-9 blocking in humanized mouse xenograft model\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — unbiased CRISPRi screen plus MS identification plus in vivo blocking; CD59 as Siglec-9 ligand not yet independently confirmed\",\n      \"pmids\": [\"39436703\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"DSG2 (Desmoglein 2) is identified as a dominant counter receptor of Siglec-9 in melanoma cells, with the interaction mediated primarily by sialic acid-bearing N-glycans on DSG2. Blocking DSG2–Siglec-9 trans-interaction significantly enhances macrophage phagocytosis of melanoma cells.\",\n      \"method\": \"Proximity labeling combined with CRISPR knockout screening, sialic acid dependency assays, macrophage phagocytosis assays\",\n      \"journal\": \"Advanced science (Weinheim, Baden-Wurttemberg, Germany)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — proximity labeling plus CRISPR screen plus functional validation; single lab, novel finding\",\n      \"pmids\": [\"39813162\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"GPIbα mucin-like region carries O-linked glycans with α2,3-linked sialic acid that bind Siglec-9 in cis on platelets, acting as a 'parking brake' to suppress platelet activation. Siglec-E conditional knockout (platelet factor 4-Cre) significantly increases platelet coagulation activity in vivo and in vitro. The GPIbα ligand does not engage Siglec-9 in trans on other cells, indicating a self-modulation mechanism.\",\n      \"method\": \"Platelet factor 4-Cre:Siglec-E conditional KO mice, in vitro human platelet culture, recombinant GPIbα glycoprotein, cis vs. trans binding assays, neuraminidase treatment, platelet activation assays\",\n      \"journal\": \"Journal of thrombosis and haemostasis : JTH\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional KO in vivo plus in vitro functional assays, ligand identity by recombinant protein, cis/trans distinction established; single lab\",\n      \"pmids\": [\"40204021\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Siglec-9 and SHP-1 physically interact in human neutrophils as shown by co-immunoprecipitation. Neonatal PMN express diminished Siglec-9 with basal phosphorylation, and GM-CSF differentially regulates Siglec-9 phosphorylation in neonatal vs. adult PMN (decreasing it in neonates, increasing it in adults), with distinct downstream survival signaling consequences.\",\n      \"method\": \"Co-immunoprecipitation of Siglec-9 and SHP-1, flow cytometry, western blot for phospho-Siglec-9, GM-CSF stimulation assays in adult and neonatal PMN\",\n      \"journal\": \"Pediatric research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP of Siglec-9/SHP-1 complex in primary neutrophils, differential phosphorylation confirmed; single lab\",\n      \"pmids\": [\"19542910\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Siglec-9 mediates rapid endocytosis of anti-Siglec-9 monoclonal antibody in AML cells and transfected rat basophilic leukemia cells, identifying it as an endocytic receptor on myeloid leukemia cells absent from normal bone marrow myeloid progenitors.\",\n      \"method\": \"Anti-Siglec-9 mAb internalization assay in primary AML cells and transfected RBL cells, flow cytometry\",\n      \"journal\": \"Leukemia research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — internalization directly demonstrated in primary AML cells and transfected cell line; single lab\",\n      \"pmids\": [\"16828866\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Siglec-9 modulates IL-4-stimulated macrophage signaling: Siglec-9 expression enhances induction of arginase-1 (Arg1) by IL-4 through the MEK-ERK pathway (not the PI-3K pathway), as ITIM tyrosine mutations abolish the Arg1 enhancement, and MEK inhibitors but not PI-3K inhibitors block the effect. Siglec-9 also enhances IL-4-induced Akt phosphorylation and ERK phosphorylation without IL-4.\",\n      \"method\": \"Stable transfection with Siglec-9 and ITIM mutants in RAW264, IL-4 stimulation, Arg1 expression assay, MEK and PI-3K inhibitors, phospho-Akt and phospho-ERK western blots\",\n      \"journal\": \"Bioscience, biotechnology, and biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ITIM mutagenesis plus pathway-selective inhibitors, defined signaling pathway; single lab\",\n      \"pmids\": [\"26540411\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Siglec-9 knockdown in human macrophages enhances LPS- and LPS/IFN-γ-induced CCR7 expression and decreases IL-4-induced CD200R expression, demonstrating that constitutively expressed Siglec-9 modulates macrophage polarization responses.\",\n      \"method\": \"Siglec-9 siRNA knockdown in primary human monocyte-derived macrophages, LPS/IFN-γ and IL-4 stimulation, CCR7 and CD200R expression by qRT-PCR and flow cytometry\",\n      \"journal\": \"Bioscience, biotechnology, and biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function knockdown with specific stimuli and phenotypic readouts; single lab\",\n      \"pmids\": [\"26923638\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"Siglec-9 is an inhibitory transmembrane lectin on myeloid cells, neutrophils, NK cells, and T cell subsets that recognizes α2,3- and α2,6-linked sialic acids (and non-sialylated HMW-HA) via its V-set Ig domain, recruits SHP-1 via cytoplasmic ITIM tyrosines to suppress innate and adaptive immune cell activation (dampening oxidative burst, NET formation, TCR/ZAP-70/NFAT signaling, NK cytotoxicity, and mast cell degranulation), and can also transduce activating signals (MEK-ERK, calcium flux) depending on the ligand and cell context; ligands include host sialoglycoproteins (glycophorin A, MUC1-ST, MUC16, GPIbα, DSG2, CD59), microbial sialoglycan mimics (GBS and GAS capsules), and VAP-1/AOC3 on endothelium, with ligand synthesis in cancer cells driven principally by ST3GAL4.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"SIGLEC9 encodes an inhibitory sialic-acid-binding immunoglobulin-like lectin expressed across myeloid cells, neutrophils, T cells, mast cells, and platelets that translates recognition of host and microbial sialoglycans into immunoregulatory signals [#0, #3, #21, #24]. Its N-terminal V-set Ig domain binds both α2,3- and α2,6-linked sialic acids through a canonical site between the F and G β-strands, where a conserved domain-1 arginine (Arg120) is essential for ligand engagement, while a distinct C-C' loop region tunes glycan specificity [#0, #1, #29]; the same V-set domain independently recognizes high-molecular-weight hyaluronan at a separate, non-sialic-acid site [#12]. Upon ligand engagement and tyrosine phosphorylation of its cytoplasmic ITIM motifs, Siglec-9 recruits the phosphatase SHP-1 to dampen activating signaling, suppressing TCR/ZAP-70/NFAT signaling and T cell cytotoxicity, neutrophil oxidative burst and NET formation, and mast cell degranulation [#2, #21, #24, #33]. The two cytoplasmic tyrosines transmit divergent outputs in macrophages, enhancing IL-10 while suppressing TNF-α upon TLR stimulation [#4, #11]. Engagement is not uniformly inhibitory: certain cancer-associated ligands such as MUC1 bearing short sialylated O-glycans (MUC1-ST) bypass SHP recruitment to drive calcium flux and MEK-ERK activation, educating myeloid cells toward an immunosuppressive PD-L1-high tumor-associated macrophage phenotype [#13, #22, #35]. A broad spectrum of counter-receptors has been mapped, including host sialoglycoproteins that enforce neutrophil and platelet quiescence (glycophorin A, Tamm-Horsfall glycoprotein, GPIbα) and tumor-cell ligands (MUC16, DSG2, CD59) whose synthesis is driven principally by the α2,3-sialyltransferase ST3GAL4 [#16, #17, #32, #6, #31, #30, #28]. Sialylated bacterial capsules (group B and group A Streptococcus) and SARS-CoV-2 Omicron spike exploit Siglec-9 to evade phagocytic and neutrophil defenses, and Siglec-9 functions as an immune checkpoint whose genetic deletion or antibody blockade restores antitumor T cell and macrophage activity and synergizes with PD-1/PD-L1 blockade [#5, #12, #27, #25, #26]. Siglec-9 also serves as a non-immune leukocyte ligand for endothelial VAP-1/AOC3 at its enzymatic groove [#7, #20].\"\n,\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Establishing the molecular identity of Siglec-9 as a sialic-acid-binding inhibitory receptor was the foundational question, defining its domain architecture and ligand-binding determinants.\",\n      \"evidence\": \"cDNA cloning, COS-cell expression, recombinant binding assays, and mutagenesis of a conserved domain-1 arginine\",\n      \"pmids\": [\"10801860\", \"10801862\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cytoplasmic signaling output not yet defined\", \"Physiological ligands in vivo not identified\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"How Siglec family members achieve distinct glycan specificities was resolved by mapping the sequence region that discriminates α2,3- versus α2,8-linked sialosides.\",\n      \"evidence\": \"Reciprocal C-C' loop domain-swap chimeras of Siglec-7 and Siglec-9 with glyco-probe binding and modeling\",\n      \"pmids\": [\"11741958\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic-resolution structural basis not yet defined\", \"Affinity for natural ligands not quantified\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Whether Siglec-9 transmits inhibitory signals was answered by showing it suppresses TCR signaling via SHP-1 recruitment in an ITIM- and ligand-binding-dependent manner.\",\n      \"evidence\": \"Jurkat transfection, SHP-1 co-IP, phospho-ZAP-70 blots, NFAT reporter, Arg120Ala mutagenesis\",\n      \"pmids\": [\"15292262\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Endogenous T cell context not addressed\", \"Physiological ligand triggering inhibition unknown\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"The functional consequence of Siglec-9 ligation on neutrophils was defined as induction of two distinct ROS-dependent death pathways, establishing a role in neutrophil fate.\",\n      \"evidence\": \"Antibody ligation of primary and inflammatory human neutrophils with ROS scavenger and caspase inhibitor dissection\",\n      \"pmids\": [\"15827126\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Natural ligands driving death in vivo not identified\", \"Molecular link between ITIM signaling and ROS not mapped\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"How the two cytoplasmic tyrosines divide signaling labor was resolved by showing they differentially control IL-10 enhancement versus TNF-α suppression during TLR stimulation.\",\n      \"evidence\": \"Tyrosine-to-phenylalanine mutants in RAW264/THP-1 with TLR stimulation and cytokine ELISA\",\n      \"pmids\": [\"18325328\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream effectors of each tyrosine not identified\", \"Endogenous ligand context not tested\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Microbial subversion of Siglec-9 was established by showing GBS sialylated capsule engages neutrophil Siglec-9 in trans to blunt antibacterial functions.\",\n      \"evidence\": \"Sialoglycan binding, neutrophil oxidative burst/NET assays, neuraminidase and blocking-antibody controls\",\n      \"pmids\": [\"19196661\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo contribution to GBS pathogenesis not demonstrated\", \"Host-pathogen specificity across Siglecs not delineated\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"The physical Siglec-9–SHP-1 complex was confirmed in primary neutrophils and shown to be developmentally regulated.\",\n      \"evidence\": \"Co-immunoprecipitation and phospho-Siglec-9 analysis in adult versus neonatal PMN with GM-CSF stimulation\",\n      \"pmids\": [\"19542910\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Reciprocal validation of complex stoichiometry not shown\", \"Functional consequence of neonatal differences not fully resolved\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Identification of MUC16 (CA125) as a Siglec-9 ligand connected the receptor to tumor immune evasion on NK cells, B cells, and monocytes.\",\n      \"evidence\": \"Flow cytometry of primary immune cells, Jurkat-Siglec-9 transfection, MUC16 binding with neuraminidase control\",\n      \"pmids\": [\"20497550\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream signaling from MUC16 engagement not defined\", \"Quantitative ligand affinity not measured\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"A non-immune adhesion function was uncovered by identifying Siglec-9 as a leukocyte ligand for endothelial VAP-1/AOC3, enabling inflammation imaging.\",\n      \"evidence\": \"Phage display, adhesion assays with mutants, modeling, and 68Ga-Siglec-9 peptide PET\",\n      \"pmids\": [\"21821708\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Signaling consequences of VAP-1 engagement on leukocytes unknown\", \"Sialic-acid dependence of this interaction not fully resolved\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Siglec-9 engagement was shown to actively promote tumor cell growth by inducing MUC1–β-catenin signaling, expanding its role beyond immune inhibition.\",\n      \"evidence\": \"Soluble Siglec-9 and co-culture treatment of MUC1-transfected cells, β-catenin co-IP, nuclear fractionation, neuraminidase control\",\n      \"pmids\": [\"24045940\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo relevance to tumor progression not established\", \"Receptor on the cancer-cell side beyond MUC1 not defined\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"A sialic-acid-independent counter-receptor mode was reported with prohibitins engaging Siglec-9 via an Arg120-dependent peptide interaction to inhibit ERK signaling.\",\n      \"evidence\": \"Arg120Ala mutant binding, bead co-immobilization with anti-CD3, phospho-ERK/c-Raf blots and IL-2 ELISA\",\n      \"pmids\": [\"23567969\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab finding without reciprocal validation\", \"Physiological relevance of sialic-acid-independent binding unclear\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Siglec-9 engagement on astrocytoma cells was shown to drive calpain-mediated degradation of focal-adhesion proteins, linking it to tumor cell motility.\",\n      \"evidence\": \"Co-culture immunoblotting of FAK/Akt/paxillin/p130Cas with calpain inhibitor rescue and invasion assays\",\n      \"pmids\": [\"24145038\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab finding requiring independent confirmation\", \"Signaling link from Siglec-9 to calpain activation unmapped\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Subcellular regulation of signaling was addressed by showing lectin-activity-dependent redistribution of Siglec-9 into lipid rafts contributes to IL-10 enhancement.\",\n      \"evidence\": \"Detergent-insoluble membrane fractionation, lectin-defective and ITIM mutants, cholesterol oxidase, cytokine ELISA\",\n      \"pmids\": [\"24449467\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct partners within rafts not identified\", \"Single-lab observation\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"The first non-sialic-acid glycan ligand was identified, showing HMW-HA engages a distinct V-set site to suppress neutrophil functions, a route exploited by GAS.\",\n      \"evidence\": \"HMW-HA binding to a separate site, neutrophil functional assays, GAS capsule-dependent blocking\",\n      \"pmids\": [\"26411873\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural definition of the HA-binding site not resolved\", \"Relative contribution of HA versus sialic-acid ligands in vivo unknown\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Siglec-9 was shown to modulate alternative macrophage activation by enhancing IL-4-induced Arg1 through MEK-ERK rather than PI-3K.\",\n      \"evidence\": \"ITIM mutants in RAW264, IL-4 stimulation, pathway-selective inhibitors, phospho-ERK/Akt blots\",\n      \"pmids\": [\"26540411\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab finding\", \"Ligand triggering this polarization in vivo not defined\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"A distinct activating signaling mode was established when tumor MUC1-ST was shown to drive Siglec-9 calcium flux and MEK-ERK activation, educating immunosuppressive TAMs.\",\n      \"evidence\": \"MUC1-ST binding, phosphatase assays (SHP-1/2 negative), calcium flux, MEK-ERK blots, PD-L1 flow cytometry\",\n      \"pmids\": [\"27595232\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanistic switch between inhibitory and activating outputs not fully defined\", \"Receptor proximal adaptor for calcium flux unknown\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Loss-of-function and knockdown studies established that constitutively expressed Siglec-9 sets macrophage polarization responses and inflammatory cytokine balance.\",\n      \"evidence\": \"Siglec-9 siRNA knockdown with LPS/IFN-γ and IL-4 stimulation, CCR7/CD200R readouts; soluble Siglec-9 NF-κB inhibition with CIA model; SNP-variant cytokine assay\",\n      \"pmids\": [\"26923638\", \"27267914\", \"27878892\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Soluble versus membrane Siglec-9 mechanisms not unified\", \"Genetic-variant functional impact tested only in vitro\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Host 'self' sialoglycoproteins were identified as tonic suppressors of neutrophil activation, defining a sialic-acid-based self-recognition system.\",\n      \"evidence\": \"Glycophorin A and Tamm-Horsfall protein binding, periodate/neuraminidase modification, neutrophil activation rescue, THP-null mouse urinalysis\",\n      \"pmids\": [\"28416510\", \"28829050\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Quantitative contribution of each self-ligand in vivo not weighted\", \"Mechanism integrating multiple self-ligands unresolved\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Src-family-kinase-dependent phosphorylation of Siglec-9 was shown to restrain dendritic-cell migration, and soluble Siglec-9/MCP-1 to drive reparative M2 macrophages.\",\n      \"evidence\": \"Dasatinib/SFK inhibitor and blocking-antibody migration assays; CCR2-blocking M2 differentiation and ALF rat model with M2 depletion\",\n      \"pmids\": [\"24882272\", \"28272428\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab findings\", \"Endogenous ligand driving DC restraint not identified\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"The Siglec-9–VAP-1 interaction was further characterized as occurring at the amine-oxidase catalytic site and capable of triggering enzymatic activity.\",\n      \"evidence\": \"Amine oxidase activity assay, inhibitor competition, Arg/Ala mutant peptide, molecular docking\",\n      \"pmids\": [\"29391504\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physiological consequence of triggering VAP-1 activity unknown\", \"Single-lab finding\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Siglec-9 was defined as a T-cell inhibitory checkpoint in melanoma, expanding its checkpoint role to the adaptive immune compartment.\",\n      \"evidence\": \"Flow cytometry of tumor-infiltrating T cells, ligand/antibody engagement, cytotoxicity and cytokine assays, SHP-1-specific phosphorylation\",\n      \"pmids\": [\"30988027\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of melanoma ligands not defined here\", \"In vivo therapeutic blockade not tested in this study\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"The cancer-cell glycan machinery generating Siglec-9 ligands was traced to α2,3-sialyltransferases, and synthetic agonists confirmed pharmacological control of neutrophil NETosis.\",\n      \"evidence\": \"ST3GAL1/ST3GAL4 transcriptomics with Siglec-9 triggering on macrophages; synthetic glycopolymer agonists in COVID-19 neutrophil NETosis assays\",\n      \"pmids\": [\"33627655\", \"34056095\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Single dominant sialyltransferase across cancers not generalized\", \"In vivo efficacy of agonists/antagonists not established here\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Genetic and antibody studies established Siglec-9 as a targetable macrophage checkpoint in glioblastoma and ovarian cancer that synergizes with PD-1/PD-L1 blockade, and as an inhibitory mast-cell receptor.\",\n      \"evidence\": \"Siglece-KO mouse GBM models with scRNA-seq; ovarian TAM blockade with phospho-SHP-1 and CD8 cytotoxicity assays; CRISPR SIGLEC9 knockout in primary mast cells with ligand gain-of-function\",\n      \"pmids\": [\"37460871\", \"37709296\", \"37100120\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Murine Siglece versus human Siglec-9 correspondence not fully resolved\", \"Combination dosing and ligand-specific responses not optimized\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"New ligands and structural/glycan determinants were defined, and viral exploitation of Siglec-9 by SARS-CoV-2 Omicron spike was demonstrated.\",\n      \"evidence\": \"NMR/MD of the V-set domain; ST3GAL4 CRISPR screen and mass spectrometry in AML; CD59 CRISPRi screen; Omicron F375S spike mutagenesis with phagocytosis/antigen-presentation rescue\",\n      \"pmids\": [\"38321945\", \"39551873\", \"39436703\", \"38454157\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"CD59 as a ligand not independently confirmed\", \"In vivo relevance of viral exploitation to disease severity not established\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Additional counter-receptors were mapped, including DSG2 in melanoma and a cis self-modulatory GPIbα–Siglec-9 interaction that brakes platelet activation.\",\n      \"evidence\": \"Proximity-labeling/CRISPR screen identifying DSG2 with phagocytosis assays; platelet-specific Siglec-E conditional KO with cis/trans GPIbα binding assays\",\n      \"pmids\": [\"39813162\", \"40204021\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"DSG2 finding from single lab\", \"Mechanistic distinction between cis and trans ligand engagement not fully generalized\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How a single V-set domain integrates competing host self-ligands, microbial mimics, and HMW-HA to switch between SHP-1-dependent inhibition and calcium/MEK-ERK activation in a given cell type remains unresolved.\",\n      \"evidence\": \"No single study reconciles ligand identity, cell context, and signaling output across compartments\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified rule predicting inhibitory versus activating output from ligand structure\", \"Quantitative ligand competition in vivo not modeled\", \"Proximal adaptor mediating activating calcium flux unidentified\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [0, 1, 12, 29]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [2, 4, 21, 24]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [2, 13, 21, 24]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [12]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [2, 33]},\n      {\"term_id\": \"GO:0038024\", \"supporting_discovery_ids\": [34]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 11, 32]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [8]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [2, 4, 21, 24, 25]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [2, 13, 33, 35]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [5, 12, 22, 27]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [3]},\n      {\"term_id\": \"R-HSA-109582\", \"supporting_discovery_ids\": [32]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"PTPN6\",\n      \"MUC1\",\n      \"MUC16\",\n      \"AOC3\",\n      \"GYPA\",\n      \"DSG2\",\n      \"GP1BA\",\n      \"CD59\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}