{"gene":"SFTPD","run_date":"2026-06-10T07:46:31","timeline":{"discoveries":[{"year":1989,"finding":"SP-D (CP4) was purified from rat bronchoalveolar lavage and characterized as a collagenous glycoprotein (43 kDa reduced) synthesized by type II pulmonary epithelial cells, containing 4-hydroxyproline, hydroxylysine, and collagenous Gly-X-Y triplet sequences; it assembles via disulfide-bonded trimers into high-molecular-mass complexes.","method":"Reverse-phase HPLC purification, amino acid analysis, gas-phase N-terminal microsequencing, two-dimensional IEF/SDS-PAGE, gel filtration","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct biochemical characterization with multiple orthogonal methods (sequencing, chromatography, electrophoresis) establishing primary structure and quaternary assembly","pmids":["2675969"],"is_preprint":false},{"year":1991,"finding":"Human SP-D contains a C-type lectin carbohydrate recognition domain (CRD) with calcium-dependent saccharide binding; the CRD is highly homologous to mannose-binding subfamily C-type lectins and contains four conserved cysteine residues; the N-terminal domain encodes a collagenous region with 59 uninterrupted Gly-X-Y triplets and a single N-linked glycosylation consensus site.","method":"cDNA library screening with degenerate oligonucleotide probe, DNA sequencing, N-terminal microsequencing of peptides, immunoprecipitation of translation products","journal":"Archives of biochemistry and biophysics","confidence":"High","confidence_rationale":"Tier 1 / Strong — cDNA sequencing combined with peptide microsequencing and immunoprecipitation establishing domain structure and lectin family membership","pmids":["1898081"],"is_preprint":false},{"year":1992,"finding":"SP-D binds glucosylceramide (GlcCer) exclusively among a panel of glycolipids tested, in a calcium-dependent manner requiring Ca2+ (not Mg2+); this binding occurs via the lectin domain as shown by antibody inhibition and competitive displacement.","method":"TLC overlay binding with 125I-labeled SP-D, competitive inhibition assays, antibody blocking","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct binding assay with multiple controls (competition, antibody inhibition, ion substitution), single lab","pmids":["1530650"],"is_preprint":false},{"year":1992,"finding":"The major glycolipid ligand of SP-D in lung surfactant is phosphatidylinositol (PI); SP-D binds PI in a calcium-dependent, saccharide-inhibitable manner and can recognize PI presented in lipid bilayers (liposomes).","method":"2D-TLC overlay with radioiodinated SP-D, lipid purification and mass spectrometry identification, Percoll density gradient with liposomes","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — purification of the endogenous ligand to homogeneity with MS identification, reconstituted bilayer binding with multiple orthogonal controls","pmids":["1457414"],"is_preprint":false},{"year":1992,"finding":"SP-D is localized in type II cells (endoplasmic reticulum predominantly), Clara cells (secretory granules at cell periphery distinct from lamellar bodies), and alveolar macrophages (endocytotic compartments); SP-D and SP-A co-localize in the same Clara cell granules but SP-D is confined to the periphery while SP-A is distributed throughout.","method":"Immunogold labeling electron microscopy, double-labeling with anti-SP-A, lysosomal marker co-staining, endocytotic tracer (BSAG) experiments","journal":"The journal of histochemistry and cytochemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — high-resolution immunogold EM with double-labeling and multiple controls establishing subcellular compartmentalization","pmids":["1527377"],"is_preprint":false},{"year":1994,"finding":"SP-D assembles as homopolymers of four identical trimeric subunits (dodecamers) with four rod-like arms of ~46 nm, stabilized by interchain disulfide bonds in the N-terminal domain; higher-order multimers (up to 8 molecules) are also observed; trimeric collagenous fragments are released by pepsin digestion, and trimeric subunits by sulfhydryl reduction.","method":"Electron microscopy (freeze-drying), hydrodynamic analysis, pepsin digestion, bacterial collagenase digestion, sulfhydryl reduction under non-denaturing conditions, gel filtration","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple orthogonal structural and biochemical methods establishing quaternary architecture in a single rigorous study","pmids":["8006040"],"is_preprint":false},{"year":1994,"finding":"SP-D potently inhibits hemagglutination activity of influenza A viruses (IAV), causes viral aggregation, enhances neutrophil binding of IAV, and boosts neutrophil respiratory burst; these effects are mediated by the calcium-dependent carbohydrate-binding property of SP-D and SP-D was at least 10-fold more potent than SP-A or MBL in hemagglutination inhibition.","method":"Hemagglutination inhibition assay, viral aggregation assay, neutrophil binding assay, respiratory burst assay, EDTA/calcium competition experiments","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple functional assays with mechanistic dissection (calcium dependence, carbohydrate competition) replicated with human, rat, and recombinant SP-D","pmids":["8040272"],"is_preprint":false},{"year":1995,"finding":"SP-D acts as a chemoattractant for human neutrophils and monocytes via its carbohydrate recognition domain (CRD); migration is dose-dependent (peak at ~10⁻¹¹ M), inhibited by maltose but not lactose, and blocked by antibodies to the C-terminal lectin domain, demonstrating that SP-D binds specific saccharide-recognizing sites on these cells.","method":"Modified Boyden chamber assay, checkerboard analysis, maltose/lactose competition, antibody blocking, HL-60 differentiation assay","journal":"American journal of respiratory cell and molecular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple cell types tested, mechanistic dissection with sugar competition and antibody inhibition, dose-response established","pmids":["7695920"],"is_preprint":false},{"year":1996,"finding":"Recombinant human SP-D (rhSP-D) multimers of dodecamers show several-fold greater potency than purified dodecamers alone in causing IAV aggregation and protecting neutrophils from viral deactivation, demonstrating CRD valency-dependent interactions; the propensity to form multimers is determined by the primary structure of human SP-D.","method":"Recombinant protein expression in CHO-K1 cells, gel filtration chromatography, electron microscopy, hemagglutination inhibition assay, neutrophil protection assay","journal":"The American journal of physiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — purified oligomeric forms compared in multiple functional assays, primary-structure basis for multimerization established","pmids":["8944718"],"is_preprint":false},{"year":1999,"finding":"The trimeric carbohydrate recognition domain (CRD) of SP-D is sufficient for neutrophil chemotactic activity (peak at 10⁻¹⁰ M), and this activity is abolished by 20 mM maltose but not lactose, indicating that the saccharide-binding function of the CRD mediates the chemoattractant effect.","method":"Recombinant CRD expression in E. coli, gel filtration, Boyden chamber chemotaxis assay, maltose/lactose competition, preincubation cross-inhibition experiments","journal":"The American journal of physiology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — recombinant domain expressed and tested with mechanistic sugar-competition controls, single lab","pmids":["9887065"],"is_preprint":false},{"year":2000,"finding":"SP-D deficiency in mice results in multiple abnormalities in surfactant metabolism: 3–4-fold excess surfactant lipids in airspaces, faster conversion of large- to small-aggregate surfactant, faster clearance of SP-D itself (t½ 7 h vs. 13 h in wild-type), and ~3-fold higher net clearance rates of saturated phosphatidylcholine, indicating SP-D regulates surfactant lipid homeostasis.","method":"SP-D gene-targeted knockout mice, radiolabeled lipid incorporation and clearance, bronchoalveolar lavage fractionation, surface tension measurement","journal":"American journal of physiology. Lung cellular and molecular physiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — comprehensive metabolic analysis with multiple orthogonal measurements in KO mice","pmids":["10956621"],"is_preprint":false},{"year":2000,"finding":"Pulmonary-specific expression of SP-D in SP-D-deficient mice corrects the surfactant phospholipid accumulation and decreases phosphatidylcholine incorporation, confirming SP-D acts locally in the lung to regulate alveolar phospholipid homeostasis; overexpression of SP-D in wild-type mice does not alter lung morphology or macrophage abundance.","method":"Transgenic mice expressing rat SP-D under the human SP-C promoter, breeding with SP-D-/- mice, bronchoalveolar lavage lipid analysis, lung morphology","journal":"American journal of physiology. Lung cellular and molecular physiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — rescue genetics in transgenic mice with quantitative lipid measurements establishing local action","pmids":["10666121"],"is_preprint":false},{"year":2001,"finding":"Disulfide cross-linked SP-D oligomers (dodecamers) are required in vivo for regulation of surfactant phospholipid homeostasis and prevention of emphysema and foamy macrophages; monomeric/trimeric SP-D (Cys15/20→Ser mutant) fails to rescue the SP-D null phenotype and, when expressed in wild-type mice, reduces endogenous oligomers and induces emphysema and foamy macrophages without phospholipid abnormalities.","method":"Transgenic mice expressing Cys→Ser mutant SP-D, bronchoalveolar lavage lipid analysis, lung histology (emphysema quantification), Western blot","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — gain-of-function and dominant-negative transgenic experiments with multiple phenotypic readouts, mechanistic domain mapping in vivo","pmids":["11278637"],"is_preprint":false},{"year":2001,"finding":"SP-D and GM-CSF regulate surfactant phospholipid homeostasis by distinct, approximately additive mechanisms: SP-D deficiency does not impair alveolar macrophage degradation of DPPC (unlike GM-CSF deficiency), and GM-CSF expression in SP-D/GM-CSF double-null mice corrects GM-CSF-dependent abnormalities but not the SP-D-dependent emphysema and foamy macrophages.","method":"Double knockout mice (SP-D-/-, GM-CSF-/-), GM-CSF transgenic rescue, bronchoalveolar lavage lipid analysis, macrophage DPPC degradation assay, lung histology","journal":"American journal of physiology. Lung cellular and molecular physiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis with double-KO and rescue, multiple orthogonal functional readouts","pmids":["11504698"],"is_preprint":false},{"year":2001,"finding":"SP-D counteracts the inhibitory effect of SP-A on phospholipid secretion by alveolar type II cells by competing with SP-A for high-affinity binding sites on type II cells; this competition is mediated via SP-D-associated lipids (activity destroyed by butanol extraction) and involves direct SP-A–SP-D binding on nitrocellulose.","method":"Alveolar type II cell secretion assay, 125I-SP-A competitive binding, antibody blocking, heat inactivation, butanol lipid extraction, nitrocellulose binding overlay","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple binding and functional assays in single lab, SP-D-associated lipids identified as mechanistic mediator","pmids":["1930130"],"is_preprint":false},{"year":2002,"finding":"SP-D loss-of-function in SP-D-/- mice results in persistent T cell activation specifically in the lung (increased CD4+ and CD8+ T cells expressing CD69 and CD25, increased BAL CD4 lymphocytes), with upregulated IL-12 and IL-6, while splenic T cells remain unactivated, indicating SP-D plays a local immunoregulatory role suppressing T cell activation.","method":"SP-D gene-targeted mice, flow cytometry (CD4, CD8, CD69, CD25 markers), RNase protection assay, RT-PCR, ELISA for cytokines, morphometric analysis","journal":"American journal of respiratory cell and molecular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — KO mice with multiple orthogonal immunological readouts, lung-specific vs. systemic comparison","pmids":["12091242"],"is_preprint":false},{"year":2002,"finding":"Human SP-D binds Mycoplasma pneumoniae with high affinity via its carbohydrate recognition domain: binding requires Ca2+, is inhibited by mannose/glucose/maltose/inositol, and a tandem CRD mutant (E321Q/N323D) fails to bind M. pneumoniae lipids; protease treatment does not abolish Ca2+-dependent binding, indicating lipid components of the bacterial membrane are the primary SP-D targets.","method":"Solid-phase binding assays, Ca2+/EDTA competition, sugar competition, protease pretreatment, 2D-TLC overlay with purified lipids, site-directed mutagenesis of CRD","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — site-directed mutagenesis combined with multiple binding assays and ligand identification","pmids":["11916969"],"is_preprint":false},{"year":2002,"finding":"The carbohydrate recognition domains (CRDs) of both SP-D and conglutinin are required for antiviral host defense in vivo: an SP-D/conglutinin CRD chimera corrects phospholipid accumulation and IAV clearance defects in SP-D-/- mice but does not rescue emphysema or ongoing inflammation, demonstrating that CRD-mediated antimicrobial function and lipid homeostasis/emphysema prevention involve distinct molecular mechanisms.","method":"Transgenic chimeric protein expression in SP-D-/- mice, bronchoalveolar lavage lipid analysis, IAV clearance assay, inflammatory cell quantification, lung histology","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — domain-swap chimera in vivo with multiple functional readouts dissecting distinct SP-D functions","pmids":["11956209"],"is_preprint":false},{"year":2002,"finding":"SP-D conditional replacement in adult SP-D-/- mice restores alveolar SP-D within 3 days and corrects phospholipid abnormalities and macrophage dysfunction, but once emphysema is established it is not reversed, demonstrating that SP-D-dependent surfactant lipid homeostasis and emphysema are mechanistically dissociated processes.","method":"Doxycycline-inducible conditional transgenic SP-D expression in SP-D-/- mice, bronchoalveolar lavage lipid analysis, lung morphology at different ages","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional rescue genetics with temporal control, multiple phenotypic readouts","pmids":["12163500"],"is_preprint":false},{"year":2004,"finding":"NFATc3 and TTF-1 (thyroid transcription factor-1) synergistically activate the Sftpd promoter via a calcineurin/NFAT-dependent enhancer; NFATc3 and TTF-1 co-immunoprecipitate from mouse lung epithelial cells and physically interact in vitro; cyclosporin-sensitive NFAT sites in the Sftpd promoter were identified by gel supershift and DNase I footprinting.","method":"Promoter reporter assays, gel supershift, DNase I protection, co-immunoprecipitation, in vitro interaction assay, cyclosporin/VIVIT inhibitor treatments, ionomycin/PMA stimulation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstituted protein interaction (co-IP and in vitro), promoter mapping with mutagenesis-equivalent inhibitor experiments, multiple orthogonal methods","pmids":["15173172"],"is_preprint":false},{"year":2005,"finding":"A common SFTPD polymorphism at codon 11 (Met11Thr) influences SP-D oligomerization: Thr/Thr11 individuals lack the highest molecular weight multimeric form of SP-D in serum, have lower serum SP-D levels, and recombinant Thr11 SP-D also fails to form multimers; high-molecular-weight SP-D multimers preferentially bind intact IAV and bacteria, while monomeric SP-D preferentially binds isolated LPS.","method":"SFTPD genotyping, gel filtration chromatography, atomic force microscopy, ELISA, solid-phase binding assays with mannan, IAV, LPS, bacteria, recombinant protein expression","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — genotype-phenotype with recombinant protein validation, AFM structural characterization, multiple functional binding assays, replicated in two cohorts","pmids":["15661913"],"is_preprint":false},{"year":2006,"finding":"The collagenous domain of SP-D is required for regulation of pulmonary macrophage activation, airspace remodeling, and surfactant lipid homeostasis in vivo, but is not required for assembly of disulfide-stabilized oligomers nor for the innate immune response to influenza A in vivo; a collagen-deletion mutant corrects viral clearance but fails to rescue emphysema and foamy macrophages in Sftpd-/- mice.","method":"Transgenic expression of collagen-domain deletion mutant (rSftpdCDM) in wild-type and Sftpd-/- mice, carbohydrate binding assay, bacterial aggregation assay, IAV clearance assay, bronchoalveolar lavage lipid analysis, lung histology","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — domain deletion mutant in vivo with multiple functional readouts dissecting collagen domain-dependent vs. -independent functions","pmids":["16787926"],"is_preprint":false},{"year":2006,"finding":"Phenylalanine 335 (Phe-335) in the human SP-D CRD mediates ring-stacking interactions with aromatic glycosides and extended saccharides (maltotriose, p-nitrophenyl-maltoside) but not with maltose or glucose; substitution with leucine reduces affinity for these ligands while tyrosine or tryptophan substitutions restore it; alanine substitution abolishes binding to mannan or maltose supports; crystal structures show Phe-335 stacking with terminal glucose or nitrophenyl rings.","method":"Site-directed mutagenesis, surface plasmon resonance/binding affinity measurements, crystallographic analysis of CRD complexes with maltotriose and p-nitrophenyl-maltoside","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure combined with mutagenesis and quantitative binding assays","pmids":["16636058"],"is_preprint":false},{"year":2006,"finding":"SP-D-deficient mice gain significantly more body weight than wild-type mice (90 mg/week more on normal chow) and have 17% higher fat percentage, and serum SP-D levels are inversely associated with BMI and waist circumference in humans, indicating SP-D participates in regulation of systemic lipid/energy homeostasis.","method":"Spd-/- mouse feeding study with body weight time-course, fat percentage measurement, twin population cohort analysis with multiple regression for serum SP-D vs. BMI/weight/waist","journal":"Scandinavian journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO mouse phenotyping combined with population study, but mechanistic pathway not fully elucidated","pmids":["17083619"],"is_preprint":false},{"year":2006,"finding":"SP-D-deficient (Sftpd-/-) mice show NH2-terminal and collagenous domain requirement for SP-D-dependent regulation of surfactant homeostasis: neither full-length SP-A, nor an NH2-rSftpa/d chimera (SP-A NH2+collagen with SP-D neck+CRD), rescues the increased phosphatidylcholine, emphysema, or macrophage infiltration in Sftpd-/- mice; furthermore, emphysema in Sftpd-/- mice is not caused solely by MMP-9 or MMP-12 overexpression.","method":"Transgenic expression of SP-A and chimeric proteins in Sftpd-/- mice, bronchoalveolar lavage lipid analysis, lung morphology, double-KO with Mmp9-/- and Mmp12-/-","journal":"American journal of physiology. Lung cellular and molecular physiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — domain-swap chimeras and multiple KO combinations establishing structural requirements for in vivo function","pmids":["16500946"],"is_preprint":false},{"year":2009,"finding":"SP-D binds the membrane-proximal immunoglobulin-like domain (D3) of SIRPα and SIRPβ in a calcium- and carbohydrate-dependent manner via specific N-glycosylated residues on D3; this binding site is distinct from the CD47 binding site on the membrane-distal D1 domain; SP-D also binds SIRPα on human neutrophils.","method":"Domain-deleted SIRPα mutant binding assays, N-glycosylation site mutagenesis, solid-phase binding assays, binding to differentiated neutrophil-like cells","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — mutagenesis (domain deletion and N-glycosylation site mutation) combined with multiple binding assays, single lab","pmids":["22511785"],"is_preprint":false},{"year":2009,"finding":"Trimeric and multimeric forms of SP-D show distinct ligand binding: trimeric SP-D subunits bind LPS, PGN, and endogenous lipoproteins (LDL, oxLDL, HDL) with greater affinity, while multimeric SP-D preferentially binds mannan and LTA; the two forms are only partially interconvertible with distinct disulfide crosslinking patterns.","method":"ManNAc-affinity chromatography isolation of trimeric SP-D, gel filtration, solid-phase ELISA binding assays, non-reducing SDS-PAGE disulfide analysis","journal":"Molecular immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — purified distinct oligomeric forms compared in multiple binding assays, single lab","pmids":["19577304"],"is_preprint":false},{"year":2012,"finding":"MMP-9 cleaves SP-D in vitro and this cleavage abrogates its innate immune functions including bacterial aggregation and enhancement of phagocytosis by alveolar macrophages; however, MMP-9-cleaved SP-D retains LPS binding activity whereas neutrophil elastase (NE)-cleaved SP-D does not, indicating distinct cleavage sites and differential loss of function.","method":"In vitro protease cleavage assay, bacterial aggregation assay, macrophage phagocytosis assay, solid-phase LPS binding ELISA, calcium-concentration-dependent cleavage comparison","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro cleavage with multiple functional readouts, single lab","pmids":["22860023"],"is_preprint":false},{"year":2018,"finding":"SP-D assembles into a mixture of trimers, hexamers, dodecamers, and higher-order oligomers; dodecamers account for >50% by mass; dodecamer formation is stabilized by non-covalent, ionic, and hydrophobic interactions between N-terminal domains of two hexamers; acidic conditions increase dodecamer proportion with conformational compaction.","method":"Atomic force microscopy (AFM), non-reducing SDS-PAGE, gel filtration, pH manipulation experiments with full-length recombinant human SP-D and patient-derived proteinosis SP-D","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — AFM structural characterization with quantitative analysis combined with biochemical methods, replicated in recombinant and natural protein","pmids":["29626540"],"is_preprint":false},{"year":2018,"finding":"SP-D directly binds eosinophil membranes and inhibits extracellular DNA trap (EET) formation in a concentration- and carbohydrate-dependent manner; S-nitrosylation of SP-D (simulating iNOS-mediated oxidative modification in asthmatic airways) causes de-oligomerization and abolishes its ability to suppress EET formation.","method":"Recombinant SP-D binding to eosinophils (confocal imaging), EET formation assay, in vivo SP-D S-nitrosylation in allergic/ozone-challenged mice, in vitro iNOS-mediated S-nitrosylation, non-reducing SDS-PAGE oligomer analysis","journal":"Journal of leukocyte biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (in vitro binding, in vivo mouse model, in vitro post-translational modification) establishing mechanism","pmids":["29733456"],"is_preprint":false},{"year":2019,"finding":"SP-D suppresses LPS-mediated NETosis in human neutrophils by binding to LPS; SP-D deficiency in mice leads to excess NET formation in LPS-inflamed lungs; NETs inhibit surface-active properties of lung surfactant in the absence of SP-D; SP-D can reverse NET-mediated surfactant inhibition and restore its biophysical properties.","method":"SP-D binding to LPS (solid-phase assay), NETosis quantification in human neutrophils, Sftpd-/- mouse LPS model, surfactant biophysical function assay, NET-surfactant inhibition experiments","journal":"Communications biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vitro mechanism (LPS binding), in vivo validation in KO mice, and functional restoration assay, multiple orthogonal approaches","pmids":["31872075"],"is_preprint":false}],"current_model":"SP-D (SFTPD) is a dodecameric (four homotrimeric subunits) collagenous C-type lectin secreted by type II pneumocytes and Clara cells that binds phosphatidylinositol, glucosylceramide, LPS, and pathogen surface glycoconjugates via its calcium-dependent CRD (with Phe-335 mediating aromatic ring-stacking interactions), aggregates and neutralizes pathogens including influenza A virus, chemoattracts neutrophils and monocytes via the CRD, interacts with SIRPα/β D3 domains to modulate innate immune cell signaling, suppresses LPS-induced NET formation and protects surfactant function, and—through its N-terminal/collagenous domains forming disulfide-stabilized oligomers—regulates alveolar surfactant phospholipid homeostasis and prevents emphysema in vivo; its transcription is driven by a calcineurin/NFATc3–TTF-1 complex, and MMP-9 cleavage or S-nitrosylation-induced de-oligomerization abolishes its immune functions."},"narrative":{"mechanistic_narrative":"SP-D (SFTPD) is a secreted collagenous C-type lectin synthesized by type II pneumocytes and Clara cells that serves dual roles in innate pulmonary immunity and alveolar surfactant homeostasis [PMID:2675969, PMID:1527377]. Each polypeptide combines an N-terminal collagenous region of uninterrupted Gly-X-Y triplets with a C-type lectin carbohydrate recognition domain (CRD) that binds saccharides in a calcium-dependent manner [PMID:1898081]; trimers assemble through interchain disulfide bonds in the N-terminal domain into cruciform dodecamers and higher-order multimers, an architecture that determines functional valency [PMID:8006040, PMID:29626540]. Through its CRD the protein binds endogenous surfactant lipids—phosphatidylinositol and glucosylceramide—as well as microbial surfaces, neutralizing influenza A virus by aggregation and potentiating neutrophil antiviral responses, with multimerization markedly enhancing potency [PMID:1457414, PMID:1530650, PMID:8040272, PMID:8944718]. The CRD also confers direct chemoattractant activity toward neutrophils and monocytes via saccharide-recognizing cell-surface sites [PMID:7695920, PMID:9887065] and engages the membrane-proximal D3 domain of SIRPα/SIRPβ to modulate innate immune cell signaling [PMID:22511785]. SP-D restrains injurious neutrophil and eosinophil extracellular trap formation by binding LPS and cell membranes, thereby protecting surfactant biophysical function [PMID:31872075, PMID:29733456]. In vivo, disulfide-crosslinked oligomers are required for regulation of surfactant phospholipid pools and prevention of emphysema and foamy-macrophage accumulation; loss of SP-D causes lipid accumulation, persistent lung T-cell activation, and airspace remodeling [PMID:10956621, PMID:11278637, PMID:12091242], and the antimicrobial CRD function is mechanistically separable from collagen-domain-dependent lipid homeostasis and emphysema prevention [PMID:11956209, PMID:16787926]. Transcription of Sftpd is driven by a calcineurin/NFATc3–TTF-1 complex [PMID:15173172], while MMP-9 cleavage or S-nitrosylation-induced de-oligomerization abolishes its immune functions [PMID:22860023, PMID:29733456].","teleology":[{"year":1989,"claim":"Establishing that SP-D is a collagenous glycoprotein assembling from disulfide-bonded trimers defined the protein class and quaternary basis for all later function.","evidence":"HPLC purification, microsequencing, and electrophoresis of rat BAL protein","pmids":["2675969"],"confidence":"High","gaps":["Higher-order assembly geometry not yet defined","No ligand or functional activity yet assigned"]},{"year":1991,"claim":"Cloning revealed a calcium-dependent C-type lectin CRD joined to a collagenous N-terminal domain, explaining how a collagenous protein could function as a sugar-recognizing molecule.","evidence":"cDNA library screening, sequencing, and peptide microsequencing of human SP-D","pmids":["1898081"],"confidence":"High","gaps":["Physiological saccharide ligands not identified","CRD residues mediating specificity unknown"]},{"year":1992,"claim":"Identifying phosphatidylinositol and glucosylceramide as calcium-dependent CRD ligands showed SP-D recognizes endogenous surfactant lipids, not only microbial sugars, and defined its localization across pulmonary cell compartments.","evidence":"TLC overlay binding with radioiodinated SP-D, MS lipid identification, immunogold EM","pmids":["1457414","1530650","1527377"],"confidence":"High","gaps":["Functional consequence of lipid binding unresolved","Structural basis of lipid versus sugar recognition unknown"]},{"year":1994,"claim":"Defining the dodecameric four-armed architecture and demonstrating potent influenza A neutralization linked quaternary structure to CRD-mediated antiviral defense.","evidence":"Electron microscopy, pepsin/collagenase digestion, hemagglutination inhibition and neutrophil assays","pmids":["8006040","8040272"],"confidence":"High","gaps":["Receptor mediating neutrophil effects not identified","Quantitative valency-potency relationship not yet established"]},{"year":1995,"claim":"Showing SP-D is a CRD-dependent chemoattractant for neutrophils and monocytes extended its role from pathogen aggregation to active recruitment of immune cells.","evidence":"Boyden chamber chemotaxis with sugar competition and antibody blocking","pmids":["7695920"],"confidence":"High","gaps":["Cell-surface receptor for chemotaxis not identified","Signaling pathway downstream unknown"]},{"year":1996,"claim":"Demonstrating that multimers of dodecamers exceed dodecamers in antiviral potency established CRD valency as a tunable determinant of function encoded in primary structure.","evidence":"Recombinant SP-D oligomer fractionation, EM, and antiviral assays","pmids":["8944718"],"confidence":"High","gaps":["Molecular driver of higher-order multimerization not pinpointed"]},{"year":1999,"claim":"Showing the isolated trimeric CRD suffices for chemotaxis localized the chemoattractant function to the lectin domain independent of the collagenous stalk.","evidence":"Recombinant CRD expression and Boyden chamber assay with sugar competition","pmids":["9887065"],"confidence":"High","gaps":["Receptor still unidentified","Single-lab in vitro result"]},{"year":2000,"claim":"Knockout and lung-specific rescue established that SP-D regulates alveolar surfactant phospholipid homeostasis acting locally within the lung.","evidence":"Sftpd-/- mice and SP-C-promoter transgenic rescue with radiolabeled lipid clearance","pmids":["10956621","10666121"],"confidence":"High","gaps":["Mechanism linking SP-D to lipid clearance unresolved","Cellular target of lipid regulation unclear"]},{"year":2001,"claim":"Domain and epistasis genetics showed disulfide-crosslinked oligomers are required in vivo to prevent emphysema and that SP-D and GM-CSF act through distinct additive pathways.","evidence":"Cys→Ser mutant transgenics, SP-D/GM-CSF double-null mice, BAL lipid and histology","pmids":["11278637","11504698","1930130"],"confidence":"High","gaps":["Molecular pathway from de-oligomerization to emphysema unknown","How SP-D competes with SP-A at type II cell binding sites mechanistically unresolved"]},{"year":2002,"claim":"Conditional, chimeric, and domain-swap rescue experiments demonstrated that CRD-mediated antimicrobial defense and collagen/N-terminal-dependent lipid homeostasis and emphysema prevention are mechanistically dissociable functions, and that SP-D suppresses local T-cell activation.","evidence":"Doxycycline-inducible and SP-D/conglutinin CRD chimera transgenics, M. pneumoniae binding with CRD mutagenesis, Sftpd-/- immunophenotyping","pmids":["12163500","11956209","11916969","12091242"],"confidence":"High","gaps":["Why established emphysema is irreversible unknown","Mechanism of T-cell suppression undefined"]},{"year":2004,"claim":"Identifying the calcineurin/NFATc3–TTF-1 complex on the Sftpd promoter defined the transcriptional control of SP-D expression.","evidence":"Promoter reporter assays, DNase I footprinting, co-IP and in vitro interaction of NFATc3 and TTF-1","pmids":["15173172"],"confidence":"High","gaps":["Upstream signals activating calcineurin in vivo unclear","Additional regulatory inputs not mapped"]},{"year":2005,"claim":"Linking a Met11Thr polymorphism to defective multimerization tied SP-D oligomeric state to ligand selectivity, with multimers favoring intact pathogens and monomers favoring isolated LPS.","evidence":"SFTPD genotyping, AFM, recombinant protein, and solid-phase binding assays","pmids":["15661913"],"confidence":"High","gaps":["Functional clinical consequence of polymorphism not established in this corpus"]},{"year":2006,"claim":"Domain-deletion, chimeric, and structural studies dissected collagen-domain-dependent macrophage/airspace regulation from CRD function, identified Phe-335 as the aromatic ring-stacking residue for extended saccharides, and revealed a systemic role of SP-D in lipid/energy homeostasis.","evidence":"Collagen-deletion and SP-A/SP-D chimera transgenics, CRD crystallography with mutagenesis, Sftpd-/- body weight and human BMI cohort","pmids":["16787926","16636058","16500946","17083619"],"confidence":"High","gaps":["Mechanism connecting SP-D to systemic fat regulation undefined","How collagen domain controls macrophage activation unknown"]},{"year":2009,"claim":"Identifying the SIRPα/β D3 binding site and distinct oligomer-dependent ligand profiles refined how SP-D signals to immune cells and how its assembly state selects targets.","evidence":"SIRP domain-deletion and N-glycosylation mutagenesis, binding assays, trimer/multimer fractionation","pmids":["22511785","19577304"],"confidence":"High","gaps":["Downstream SIRP signaling consequences not defined","Trimer/multimer interconversion in vivo unclear"]},{"year":2012,"claim":"Showing MMP-9 cleavage abolishes bacterial aggregation and phagocytosis while sparing LPS binding established proteolytic inactivation as a regulatory and pathological mechanism with site-specific functional outcomes.","evidence":"In vitro MMP-9 and neutrophil elastase cleavage with functional readouts","pmids":["22860023"],"confidence":"Medium","gaps":["In vivo relevance of cleavage not established","Cleavage sites not mapped at residue resolution"]},{"year":2019,"claim":"Demonstrating that SP-D binds LPS and cell membranes to suppress neutrophil and eosinophil extracellular trap formation, and that S-nitrosylation-induced de-oligomerization abolishes this, connected SP-D oligomeric integrity to protection of surfactant biophysical function.","evidence":"NETosis/EET assays, Sftpd-/- LPS lung models, in vitro S-nitrosylation, surfactant function assays","pmids":["31872075","29733456"],"confidence":"High","gaps":["Receptor/membrane determinants of trap 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(Monogenea: Dactylogyridae) and a redescription of D. anus in siluriform catfish from Brazil.","date":"2017","source":"Journal of helminthology","url":"https://pubmed.ncbi.nlm.nih.gov/28382887","citation_count":36,"is_preprint":false},{"pmid":"23220073","id":"PMC_23220073","title":"Linking surfactant protein SP-D and IL-13: implications in asthma and allergy.","date":"2012","source":"Molecular immunology","url":"https://pubmed.ncbi.nlm.nih.gov/23220073","citation_count":35,"is_preprint":false},{"pmid":"12396020","id":"PMC_12396020","title":"Structural requirements for SP-D function in vitro and in vivo: therapeutic potential of recombinant SP-D.","date":"2002","source":"Immunobiology","url":"https://pubmed.ncbi.nlm.nih.gov/12396020","citation_count":35,"is_preprint":false},{"pmid":"20447075","id":"PMC_20447075","title":"SP-D and regulation of the pulmonary innate immune system in allergic airway changes.","date":"2010","source":"Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology","url":"https://pubmed.ncbi.nlm.nih.gov/20447075","citation_count":34,"is_preprint":false},{"pmid":"12396011","id":"PMC_12396011","title":"Lung surfactant protein D (SP-D) and the molecular diverted descendants: conglutinin, CL-43 and CL-46.","date":"2002","source":"Immunobiology","url":"https://pubmed.ncbi.nlm.nih.gov/12396011","citation_count":34,"is_preprint":false},{"pmid":"25631080","id":"PMC_25631080","title":"DNA vaccine molecular adjuvants SP-D-BAFF and SP-D-APRIL enhance anti-gp120 immune response and increase HIV-1 neutralizing antibody titers.","date":"2015","source":"Journal of virology","url":"https://pubmed.ncbi.nlm.nih.gov/25631080","citation_count":34,"is_preprint":false},{"pmid":"15274124","id":"PMC_15274124","title":"High resolution mass spectrometric alveolar proteomics: identification of surfactant protein SP-A and SP-D modifications in proteinosis and cystic fibrosis patients.","date":"2004","source":"Proteomics","url":"https://pubmed.ncbi.nlm.nih.gov/15274124","citation_count":34,"is_preprint":false},{"pmid":"22860023","id":"PMC_22860023","title":"MMP-9 cleaves SP-D and abrogates its innate immune functions in vitro.","date":"2012","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/22860023","citation_count":33,"is_preprint":false},{"pmid":"27782361","id":"PMC_27782361","title":"YKL-40, CCL18 and SP-D predict mortality in patients hospitalized with community-acquired pneumonia.","date":"2016","source":"Respirology (Carlton, Vic.)","url":"https://pubmed.ncbi.nlm.nih.gov/27782361","citation_count":32,"is_preprint":false},{"pmid":"9887065","id":"PMC_9887065","title":"Recombinant SP-D carbohydrate recognition domain is a chemoattractant for human neutrophils.","date":"1999","source":"The American journal of physiology","url":"https://pubmed.ncbi.nlm.nih.gov/9887065","citation_count":32,"is_preprint":false},{"pmid":"29733456","id":"PMC_29733456","title":"Oxidative damage of SP-D abolishes control of eosinophil extracellular DNA trap formation.","date":"2018","source":"Journal of leukocyte biology","url":"https://pubmed.ncbi.nlm.nih.gov/29733456","citation_count":31,"is_preprint":false},{"pmid":"19577304","id":"PMC_19577304","title":"Multimeric and trimeric subunit SP-D are interconvertible structures with distinct ligand interaction.","date":"2009","source":"Molecular immunology","url":"https://pubmed.ncbi.nlm.nih.gov/19577304","citation_count":31,"is_preprint":false},{"pmid":"16636058","id":"PMC_16636058","title":"Contributions of phenylalanine 335 to ligand recognition by human surfactant protein D: ring interactions with SP-D ligands.","date":"2006","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/16636058","citation_count":31,"is_preprint":false},{"pmid":"26849628","id":"PMC_26849628","title":"Surfactant Proteins SP-A and SP-D Ameliorate Pneumonia Severity and Intestinal Injury in a Murine Model of Staphylococcus Aureus Pneumonia.","date":"2016","source":"Shock (Augusta, Ga.)","url":"https://pubmed.ncbi.nlm.nih.gov/26849628","citation_count":30,"is_preprint":false},{"pmid":"17158597","id":"PMC_17158597","title":"SP-D-deficient mice are resistant to hyperoxia.","date":"2006","source":"American journal of physiology. Lung cellular and molecular physiology","url":"https://pubmed.ncbi.nlm.nih.gov/17158597","citation_count":30,"is_preprint":false},{"pmid":"19880212","id":"PMC_19880212","title":"The human lung surfactant proteins A (SP-A) and D (SP-D) interact with apoptotic target cells by different binding mechanisms.","date":"2009","source":"Immunobiology","url":"https://pubmed.ncbi.nlm.nih.gov/19880212","citation_count":30,"is_preprint":false},{"pmid":"1930130","id":"PMC_1930130","title":"Surfactant protein D (SP-D) counteracts the inhibitory effect of surfactant protein A (SP-A) on phospholipid secretion by alveolar type II cells. Interaction of native SP-D with SP-A.","date":"1991","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/1930130","citation_count":30,"is_preprint":false},{"pmid":"31665737","id":"PMC_31665737","title":"Evaluation of the Diagnostic Efficacies of Serological Markers KL-6, SP-A, SP-D, CCL2, and CXCL13 in Idiopathic Interstitial Pneumonia.","date":"2019","source":"Respiration; international review of thoracic diseases","url":"https://pubmed.ncbi.nlm.nih.gov/31665737","citation_count":29,"is_preprint":false},{"pmid":"18785967","id":"PMC_18785967","title":"Association of polymorphisms in the human surfactant protein-D (SFTPD) gene and postnatal pulmonary adaptation in the preterm infant.","date":"2008","source":"Acta paediatrica (Oslo, Norway : 1992)","url":"https://pubmed.ncbi.nlm.nih.gov/18785967","citation_count":27,"is_preprint":false},{"pmid":"36341812","id":"PMC_36341812","title":"Surfactant protein D (SP-D) as a biomarker of SARS-CoV-2 infection.","date":"2022","source":"Clinica chimica acta; international journal of clinical chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/36341812","citation_count":26,"is_preprint":false},{"pmid":"19439011","id":"PMC_19439011","title":"Elevated plasma surfactant protein D (SP-D) levels and a direct correlation with anti-severe acute respiratory syndrome coronavirus-specific IgG antibody in SARS patients.","date":"2009","source":"Scandinavian journal of immunology","url":"https://pubmed.ncbi.nlm.nih.gov/19439011","citation_count":26,"is_preprint":false},{"pmid":"11350793","id":"PMC_11350793","title":"GM-CSF mediates alveolar macrophage proliferation and type II cell hypertrophy in SP-D gene-targeted mice.","date":"2001","source":"American journal of physiology. Lung cellular and molecular physiology","url":"https://pubmed.ncbi.nlm.nih.gov/11350793","citation_count":26,"is_preprint":false},{"pmid":"34501312","id":"PMC_34501312","title":"Serial Measurements of Circulating KL-6, SP-D, MMP-7, CA19-9, CA-125, CCL18, and Periostin in Patients with Idiopathic Pulmonary Fibrosis Receiving Antifibrotic Therapy: An Exploratory Study.","date":"2021","source":"Journal of clinical medicine","url":"https://pubmed.ncbi.nlm.nih.gov/34501312","citation_count":26,"is_preprint":false},{"pmid":"17420052","id":"PMC_17420052","title":"Expression sites of the collectin SP-D suggest its importance in first line host defence: power of combining in situ hybridisation, RT-PCR and immunohistochemistry.","date":"2007","source":"Molecular immunology","url":"https://pubmed.ncbi.nlm.nih.gov/17420052","citation_count":26,"is_preprint":false},{"pmid":"21935489","id":"PMC_21935489","title":"Assessment of the antiviral properties of recombinant porcine SP-D against various influenza A viruses in vitro.","date":"2011","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/21935489","citation_count":26,"is_preprint":false},{"pmid":"29626540","id":"PMC_29626540","title":"Supramolecular Assembly of Human Pulmonary Surfactant Protein SP-D.","date":"2018","source":"Journal of molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/29626540","citation_count":24,"is_preprint":false},{"pmid":"21601013","id":"PMC_21601013","title":"SP-A1, SP-A2 and SP-D gene polymorphisms in severe acute respiratory syncytial infection in Chilean infants.","date":"2011","source":"Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases","url":"https://pubmed.ncbi.nlm.nih.gov/21601013","citation_count":24,"is_preprint":false},{"pmid":"22511785","id":"PMC_22511785","title":"Surfactant protein D (Sp-D) binds to membrane-proximal domain (D3) of signal regulatory protein α (SIRPα), a site distant from binding domain of CD47, while also binding to analogous region on signal regulatory protein β (SIRPβ).","date":"2012","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/22511785","citation_count":24,"is_preprint":false},{"pmid":"14742512","id":"PMC_14742512","title":"Binding and agglutination of Streptococcus pneumoniae by human surfactant protein D (SP-D) vary between strains, but SP-D fails to enhance killing by neutrophils.","date":"2004","source":"Infection and immunity","url":"https://pubmed.ncbi.nlm.nih.gov/14742512","citation_count":24,"is_preprint":false},{"pmid":"19340882","id":"PMC_19340882","title":"Genetic variants in surfactant, pulmonary-associated protein D (SFTPD) and Japanese susceptibility to ulcerative colitis.","date":"2009","source":"Inflammatory bowel diseases","url":"https://pubmed.ncbi.nlm.nih.gov/19340882","citation_count":22,"is_preprint":false},{"pmid":"20230362","id":"PMC_20230362","title":"Surfactant protein (SP)-A and SP-D as antimicrobial and immunotherapeutic agents.","date":"2010","source":"Recent patents on anti-infective drug discovery","url":"https://pubmed.ncbi.nlm.nih.gov/20230362","citation_count":21,"is_preprint":false},{"pmid":"24697551","id":"PMC_24697551","title":"Surfactant protein SP-D modulates activity of immune cells: proteomic profiling of its interaction with eosinophilic cells.","date":"2014","source":"Expert review of proteomics","url":"https://pubmed.ncbi.nlm.nih.gov/24697551","citation_count":21,"is_preprint":false},{"pmid":"16500946","id":"PMC_16500946","title":"Neither SP-A nor NH2-terminal domains of SP-A can substitute for SP-D in regulation of alveolar homeostasis.","date":"2006","source":"American journal of physiology. Lung cellular and molecular physiology","url":"https://pubmed.ncbi.nlm.nih.gov/16500946","citation_count":21,"is_preprint":false},{"pmid":"23841811","id":"PMC_23841811","title":"Donor surfactant protein D (SP-D) polymorphisms are associated with lung transplant outcome.","date":"2013","source":"American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons","url":"https://pubmed.ncbi.nlm.nih.gov/23841811","citation_count":21,"is_preprint":false},{"pmid":"15265076","id":"PMC_15265076","title":"The genes encoding bovine SP-A, SP-D, MBL-A, conglutinin, CL-43 and CL-46 form a distinct collectin locus on Bos taurus chromosome 28 (BTA28) at position q.1.8-1.9.","date":"2004","source":"Animal genetics","url":"https://pubmed.ncbi.nlm.nih.gov/15265076","citation_count":21,"is_preprint":false},{"pmid":"20457545","id":"PMC_20457545","title":"Serum-surfactant SP-D correlates inversely to lung function in cystic fibrosis.","date":"2010","source":"Journal of cystic fibrosis : official journal of the European Cystic Fibrosis Society","url":"https://pubmed.ncbi.nlm.nih.gov/20457545","citation_count":20,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":52684,"output_tokens":8193,"usd":0.140474,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":17937,"output_tokens":4912,"usd":0.106243,"stage2_stop_reason":"end_turn"},"total_usd":0.246717,"stage1_batch_id":"msgbatch_013LWZZT6MWaqB9qyTSeQBmU","stage2_batch_id":"msgbatch_0194ZzQaTGNpruoNJBbEMbt3","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1989,\n      \"finding\": \"SP-D (CP4) was purified from rat bronchoalveolar lavage and characterized as a collagenous glycoprotein (43 kDa reduced) synthesized by type II pulmonary epithelial cells, containing 4-hydroxyproline, hydroxylysine, and collagenous Gly-X-Y triplet sequences; it assembles via disulfide-bonded trimers into high-molecular-mass complexes.\",\n      \"method\": \"Reverse-phase HPLC purification, amino acid analysis, gas-phase N-terminal microsequencing, two-dimensional IEF/SDS-PAGE, gel filtration\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct biochemical characterization with multiple orthogonal methods (sequencing, chromatography, electrophoresis) establishing primary structure and quaternary assembly\",\n      \"pmids\": [\"2675969\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1991,\n      \"finding\": \"Human SP-D contains a C-type lectin carbohydrate recognition domain (CRD) with calcium-dependent saccharide binding; the CRD is highly homologous to mannose-binding subfamily C-type lectins and contains four conserved cysteine residues; the N-terminal domain encodes a collagenous region with 59 uninterrupted Gly-X-Y triplets and a single N-linked glycosylation consensus site.\",\n      \"method\": \"cDNA library screening with degenerate oligonucleotide probe, DNA sequencing, N-terminal microsequencing of peptides, immunoprecipitation of translation products\",\n      \"journal\": \"Archives of biochemistry and biophysics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cDNA sequencing combined with peptide microsequencing and immunoprecipitation establishing domain structure and lectin family membership\",\n      \"pmids\": [\"1898081\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1992,\n      \"finding\": \"SP-D binds glucosylceramide (GlcCer) exclusively among a panel of glycolipids tested, in a calcium-dependent manner requiring Ca2+ (not Mg2+); this binding occurs via the lectin domain as shown by antibody inhibition and competitive displacement.\",\n      \"method\": \"TLC overlay binding with 125I-labeled SP-D, competitive inhibition assays, antibody blocking\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct binding assay with multiple controls (competition, antibody inhibition, ion substitution), single lab\",\n      \"pmids\": [\"1530650\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1992,\n      \"finding\": \"The major glycolipid ligand of SP-D in lung surfactant is phosphatidylinositol (PI); SP-D binds PI in a calcium-dependent, saccharide-inhibitable manner and can recognize PI presented in lipid bilayers (liposomes).\",\n      \"method\": \"2D-TLC overlay with radioiodinated SP-D, lipid purification and mass spectrometry identification, Percoll density gradient with liposomes\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — purification of the endogenous ligand to homogeneity with MS identification, reconstituted bilayer binding with multiple orthogonal controls\",\n      \"pmids\": [\"1457414\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1992,\n      \"finding\": \"SP-D is localized in type II cells (endoplasmic reticulum predominantly), Clara cells (secretory granules at cell periphery distinct from lamellar bodies), and alveolar macrophages (endocytotic compartments); SP-D and SP-A co-localize in the same Clara cell granules but SP-D is confined to the periphery while SP-A is distributed throughout.\",\n      \"method\": \"Immunogold labeling electron microscopy, double-labeling with anti-SP-A, lysosomal marker co-staining, endocytotic tracer (BSAG) experiments\",\n      \"journal\": \"The journal of histochemistry and cytochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — high-resolution immunogold EM with double-labeling and multiple controls establishing subcellular compartmentalization\",\n      \"pmids\": [\"1527377\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"SP-D assembles as homopolymers of four identical trimeric subunits (dodecamers) with four rod-like arms of ~46 nm, stabilized by interchain disulfide bonds in the N-terminal domain; higher-order multimers (up to 8 molecules) are also observed; trimeric collagenous fragments are released by pepsin digestion, and trimeric subunits by sulfhydryl reduction.\",\n      \"method\": \"Electron microscopy (freeze-drying), hydrodynamic analysis, pepsin digestion, bacterial collagenase digestion, sulfhydryl reduction under non-denaturing conditions, gel filtration\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple orthogonal structural and biochemical methods establishing quaternary architecture in a single rigorous study\",\n      \"pmids\": [\"8006040\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"SP-D potently inhibits hemagglutination activity of influenza A viruses (IAV), causes viral aggregation, enhances neutrophil binding of IAV, and boosts neutrophil respiratory burst; these effects are mediated by the calcium-dependent carbohydrate-binding property of SP-D and SP-D was at least 10-fold more potent than SP-A or MBL in hemagglutination inhibition.\",\n      \"method\": \"Hemagglutination inhibition assay, viral aggregation assay, neutrophil binding assay, respiratory burst assay, EDTA/calcium competition experiments\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple functional assays with mechanistic dissection (calcium dependence, carbohydrate competition) replicated with human, rat, and recombinant SP-D\",\n      \"pmids\": [\"8040272\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"SP-D acts as a chemoattractant for human neutrophils and monocytes via its carbohydrate recognition domain (CRD); migration is dose-dependent (peak at ~10⁻¹¹ M), inhibited by maltose but not lactose, and blocked by antibodies to the C-terminal lectin domain, demonstrating that SP-D binds specific saccharide-recognizing sites on these cells.\",\n      \"method\": \"Modified Boyden chamber assay, checkerboard analysis, maltose/lactose competition, antibody blocking, HL-60 differentiation assay\",\n      \"journal\": \"American journal of respiratory cell and molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple cell types tested, mechanistic dissection with sugar competition and antibody inhibition, dose-response established\",\n      \"pmids\": [\"7695920\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"Recombinant human SP-D (rhSP-D) multimers of dodecamers show several-fold greater potency than purified dodecamers alone in causing IAV aggregation and protecting neutrophils from viral deactivation, demonstrating CRD valency-dependent interactions; the propensity to form multimers is determined by the primary structure of human SP-D.\",\n      \"method\": \"Recombinant protein expression in CHO-K1 cells, gel filtration chromatography, electron microscopy, hemagglutination inhibition assay, neutrophil protection assay\",\n      \"journal\": \"The American journal of physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — purified oligomeric forms compared in multiple functional assays, primary-structure basis for multimerization established\",\n      \"pmids\": [\"8944718\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"The trimeric carbohydrate recognition domain (CRD) of SP-D is sufficient for neutrophil chemotactic activity (peak at 10⁻¹⁰ M), and this activity is abolished by 20 mM maltose but not lactose, indicating that the saccharide-binding function of the CRD mediates the chemoattractant effect.\",\n      \"method\": \"Recombinant CRD expression in E. coli, gel filtration, Boyden chamber chemotaxis assay, maltose/lactose competition, preincubation cross-inhibition experiments\",\n      \"journal\": \"The American journal of physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — recombinant domain expressed and tested with mechanistic sugar-competition controls, single lab\",\n      \"pmids\": [\"9887065\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"SP-D deficiency in mice results in multiple abnormalities in surfactant metabolism: 3–4-fold excess surfactant lipids in airspaces, faster conversion of large- to small-aggregate surfactant, faster clearance of SP-D itself (t½ 7 h vs. 13 h in wild-type), and ~3-fold higher net clearance rates of saturated phosphatidylcholine, indicating SP-D regulates surfactant lipid homeostasis.\",\n      \"method\": \"SP-D gene-targeted knockout mice, radiolabeled lipid incorporation and clearance, bronchoalveolar lavage fractionation, surface tension measurement\",\n      \"journal\": \"American journal of physiology. Lung cellular and molecular physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — comprehensive metabolic analysis with multiple orthogonal measurements in KO mice\",\n      \"pmids\": [\"10956621\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Pulmonary-specific expression of SP-D in SP-D-deficient mice corrects the surfactant phospholipid accumulation and decreases phosphatidylcholine incorporation, confirming SP-D acts locally in the lung to regulate alveolar phospholipid homeostasis; overexpression of SP-D in wild-type mice does not alter lung morphology or macrophage abundance.\",\n      \"method\": \"Transgenic mice expressing rat SP-D under the human SP-C promoter, breeding with SP-D-/- mice, bronchoalveolar lavage lipid analysis, lung morphology\",\n      \"journal\": \"American journal of physiology. Lung cellular and molecular physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — rescue genetics in transgenic mice with quantitative lipid measurements establishing local action\",\n      \"pmids\": [\"10666121\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Disulfide cross-linked SP-D oligomers (dodecamers) are required in vivo for regulation of surfactant phospholipid homeostasis and prevention of emphysema and foamy macrophages; monomeric/trimeric SP-D (Cys15/20→Ser mutant) fails to rescue the SP-D null phenotype and, when expressed in wild-type mice, reduces endogenous oligomers and induces emphysema and foamy macrophages without phospholipid abnormalities.\",\n      \"method\": \"Transgenic mice expressing Cys→Ser mutant SP-D, bronchoalveolar lavage lipid analysis, lung histology (emphysema quantification), Western blot\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — gain-of-function and dominant-negative transgenic experiments with multiple phenotypic readouts, mechanistic domain mapping in vivo\",\n      \"pmids\": [\"11278637\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"SP-D and GM-CSF regulate surfactant phospholipid homeostasis by distinct, approximately additive mechanisms: SP-D deficiency does not impair alveolar macrophage degradation of DPPC (unlike GM-CSF deficiency), and GM-CSF expression in SP-D/GM-CSF double-null mice corrects GM-CSF-dependent abnormalities but not the SP-D-dependent emphysema and foamy macrophages.\",\n      \"method\": \"Double knockout mice (SP-D-/-, GM-CSF-/-), GM-CSF transgenic rescue, bronchoalveolar lavage lipid analysis, macrophage DPPC degradation assay, lung histology\",\n      \"journal\": \"American journal of physiology. Lung cellular and molecular physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis with double-KO and rescue, multiple orthogonal functional readouts\",\n      \"pmids\": [\"11504698\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"SP-D counteracts the inhibitory effect of SP-A on phospholipid secretion by alveolar type II cells by competing with SP-A for high-affinity binding sites on type II cells; this competition is mediated via SP-D-associated lipids (activity destroyed by butanol extraction) and involves direct SP-A–SP-D binding on nitrocellulose.\",\n      \"method\": \"Alveolar type II cell secretion assay, 125I-SP-A competitive binding, antibody blocking, heat inactivation, butanol lipid extraction, nitrocellulose binding overlay\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple binding and functional assays in single lab, SP-D-associated lipids identified as mechanistic mediator\",\n      \"pmids\": [\"1930130\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"SP-D loss-of-function in SP-D-/- mice results in persistent T cell activation specifically in the lung (increased CD4+ and CD8+ T cells expressing CD69 and CD25, increased BAL CD4 lymphocytes), with upregulated IL-12 and IL-6, while splenic T cells remain unactivated, indicating SP-D plays a local immunoregulatory role suppressing T cell activation.\",\n      \"method\": \"SP-D gene-targeted mice, flow cytometry (CD4, CD8, CD69, CD25 markers), RNase protection assay, RT-PCR, ELISA for cytokines, morphometric analysis\",\n      \"journal\": \"American journal of respiratory cell and molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — KO mice with multiple orthogonal immunological readouts, lung-specific vs. systemic comparison\",\n      \"pmids\": [\"12091242\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Human SP-D binds Mycoplasma pneumoniae with high affinity via its carbohydrate recognition domain: binding requires Ca2+, is inhibited by mannose/glucose/maltose/inositol, and a tandem CRD mutant (E321Q/N323D) fails to bind M. pneumoniae lipids; protease treatment does not abolish Ca2+-dependent binding, indicating lipid components of the bacterial membrane are the primary SP-D targets.\",\n      \"method\": \"Solid-phase binding assays, Ca2+/EDTA competition, sugar competition, protease pretreatment, 2D-TLC overlay with purified lipids, site-directed mutagenesis of CRD\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — site-directed mutagenesis combined with multiple binding assays and ligand identification\",\n      \"pmids\": [\"11916969\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"The carbohydrate recognition domains (CRDs) of both SP-D and conglutinin are required for antiviral host defense in vivo: an SP-D/conglutinin CRD chimera corrects phospholipid accumulation and IAV clearance defects in SP-D-/- mice but does not rescue emphysema or ongoing inflammation, demonstrating that CRD-mediated antimicrobial function and lipid homeostasis/emphysema prevention involve distinct molecular mechanisms.\",\n      \"method\": \"Transgenic chimeric protein expression in SP-D-/- mice, bronchoalveolar lavage lipid analysis, IAV clearance assay, inflammatory cell quantification, lung histology\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — domain-swap chimera in vivo with multiple functional readouts dissecting distinct SP-D functions\",\n      \"pmids\": [\"11956209\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"SP-D conditional replacement in adult SP-D-/- mice restores alveolar SP-D within 3 days and corrects phospholipid abnormalities and macrophage dysfunction, but once emphysema is established it is not reversed, demonstrating that SP-D-dependent surfactant lipid homeostasis and emphysema are mechanistically dissociated processes.\",\n      \"method\": \"Doxycycline-inducible conditional transgenic SP-D expression in SP-D-/- mice, bronchoalveolar lavage lipid analysis, lung morphology at different ages\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional rescue genetics with temporal control, multiple phenotypic readouts\",\n      \"pmids\": [\"12163500\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"NFATc3 and TTF-1 (thyroid transcription factor-1) synergistically activate the Sftpd promoter via a calcineurin/NFAT-dependent enhancer; NFATc3 and TTF-1 co-immunoprecipitate from mouse lung epithelial cells and physically interact in vitro; cyclosporin-sensitive NFAT sites in the Sftpd promoter were identified by gel supershift and DNase I footprinting.\",\n      \"method\": \"Promoter reporter assays, gel supershift, DNase I protection, co-immunoprecipitation, in vitro interaction assay, cyclosporin/VIVIT inhibitor treatments, ionomycin/PMA stimulation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstituted protein interaction (co-IP and in vitro), promoter mapping with mutagenesis-equivalent inhibitor experiments, multiple orthogonal methods\",\n      \"pmids\": [\"15173172\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"A common SFTPD polymorphism at codon 11 (Met11Thr) influences SP-D oligomerization: Thr/Thr11 individuals lack the highest molecular weight multimeric form of SP-D in serum, have lower serum SP-D levels, and recombinant Thr11 SP-D also fails to form multimers; high-molecular-weight SP-D multimers preferentially bind intact IAV and bacteria, while monomeric SP-D preferentially binds isolated LPS.\",\n      \"method\": \"SFTPD genotyping, gel filtration chromatography, atomic force microscopy, ELISA, solid-phase binding assays with mannan, IAV, LPS, bacteria, recombinant protein expression\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — genotype-phenotype with recombinant protein validation, AFM structural characterization, multiple functional binding assays, replicated in two cohorts\",\n      \"pmids\": [\"15661913\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"The collagenous domain of SP-D is required for regulation of pulmonary macrophage activation, airspace remodeling, and surfactant lipid homeostasis in vivo, but is not required for assembly of disulfide-stabilized oligomers nor for the innate immune response to influenza A in vivo; a collagen-deletion mutant corrects viral clearance but fails to rescue emphysema and foamy macrophages in Sftpd-/- mice.\",\n      \"method\": \"Transgenic expression of collagen-domain deletion mutant (rSftpdCDM) in wild-type and Sftpd-/- mice, carbohydrate binding assay, bacterial aggregation assay, IAV clearance assay, bronchoalveolar lavage lipid analysis, lung histology\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — domain deletion mutant in vivo with multiple functional readouts dissecting collagen domain-dependent vs. -independent functions\",\n      \"pmids\": [\"16787926\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Phenylalanine 335 (Phe-335) in the human SP-D CRD mediates ring-stacking interactions with aromatic glycosides and extended saccharides (maltotriose, p-nitrophenyl-maltoside) but not with maltose or glucose; substitution with leucine reduces affinity for these ligands while tyrosine or tryptophan substitutions restore it; alanine substitution abolishes binding to mannan or maltose supports; crystal structures show Phe-335 stacking with terminal glucose or nitrophenyl rings.\",\n      \"method\": \"Site-directed mutagenesis, surface plasmon resonance/binding affinity measurements, crystallographic analysis of CRD complexes with maltotriose and p-nitrophenyl-maltoside\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure combined with mutagenesis and quantitative binding assays\",\n      \"pmids\": [\"16636058\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"SP-D-deficient mice gain significantly more body weight than wild-type mice (90 mg/week more on normal chow) and have 17% higher fat percentage, and serum SP-D levels are inversely associated with BMI and waist circumference in humans, indicating SP-D participates in regulation of systemic lipid/energy homeostasis.\",\n      \"method\": \"Spd-/- mouse feeding study with body weight time-course, fat percentage measurement, twin population cohort analysis with multiple regression for serum SP-D vs. BMI/weight/waist\",\n      \"journal\": \"Scandinavian journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO mouse phenotyping combined with population study, but mechanistic pathway not fully elucidated\",\n      \"pmids\": [\"17083619\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"SP-D-deficient (Sftpd-/-) mice show NH2-terminal and collagenous domain requirement for SP-D-dependent regulation of surfactant homeostasis: neither full-length SP-A, nor an NH2-rSftpa/d chimera (SP-A NH2+collagen with SP-D neck+CRD), rescues the increased phosphatidylcholine, emphysema, or macrophage infiltration in Sftpd-/- mice; furthermore, emphysema in Sftpd-/- mice is not caused solely by MMP-9 or MMP-12 overexpression.\",\n      \"method\": \"Transgenic expression of SP-A and chimeric proteins in Sftpd-/- mice, bronchoalveolar lavage lipid analysis, lung morphology, double-KO with Mmp9-/- and Mmp12-/-\",\n      \"journal\": \"American journal of physiology. Lung cellular and molecular physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — domain-swap chimeras and multiple KO combinations establishing structural requirements for in vivo function\",\n      \"pmids\": [\"16500946\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"SP-D binds the membrane-proximal immunoglobulin-like domain (D3) of SIRPα and SIRPβ in a calcium- and carbohydrate-dependent manner via specific N-glycosylated residues on D3; this binding site is distinct from the CD47 binding site on the membrane-distal D1 domain; SP-D also binds SIRPα on human neutrophils.\",\n      \"method\": \"Domain-deleted SIRPα mutant binding assays, N-glycosylation site mutagenesis, solid-phase binding assays, binding to differentiated neutrophil-like cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — mutagenesis (domain deletion and N-glycosylation site mutation) combined with multiple binding assays, single lab\",\n      \"pmids\": [\"22511785\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Trimeric and multimeric forms of SP-D show distinct ligand binding: trimeric SP-D subunits bind LPS, PGN, and endogenous lipoproteins (LDL, oxLDL, HDL) with greater affinity, while multimeric SP-D preferentially binds mannan and LTA; the two forms are only partially interconvertible with distinct disulfide crosslinking patterns.\",\n      \"method\": \"ManNAc-affinity chromatography isolation of trimeric SP-D, gel filtration, solid-phase ELISA binding assays, non-reducing SDS-PAGE disulfide analysis\",\n      \"journal\": \"Molecular immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — purified distinct oligomeric forms compared in multiple binding assays, single lab\",\n      \"pmids\": [\"19577304\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"MMP-9 cleaves SP-D in vitro and this cleavage abrogates its innate immune functions including bacterial aggregation and enhancement of phagocytosis by alveolar macrophages; however, MMP-9-cleaved SP-D retains LPS binding activity whereas neutrophil elastase (NE)-cleaved SP-D does not, indicating distinct cleavage sites and differential loss of function.\",\n      \"method\": \"In vitro protease cleavage assay, bacterial aggregation assay, macrophage phagocytosis assay, solid-phase LPS binding ELISA, calcium-concentration-dependent cleavage comparison\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro cleavage with multiple functional readouts, single lab\",\n      \"pmids\": [\"22860023\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"SP-D assembles into a mixture of trimers, hexamers, dodecamers, and higher-order oligomers; dodecamers account for >50% by mass; dodecamer formation is stabilized by non-covalent, ionic, and hydrophobic interactions between N-terminal domains of two hexamers; acidic conditions increase dodecamer proportion with conformational compaction.\",\n      \"method\": \"Atomic force microscopy (AFM), non-reducing SDS-PAGE, gel filtration, pH manipulation experiments with full-length recombinant human SP-D and patient-derived proteinosis SP-D\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — AFM structural characterization with quantitative analysis combined with biochemical methods, replicated in recombinant and natural protein\",\n      \"pmids\": [\"29626540\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"SP-D directly binds eosinophil membranes and inhibits extracellular DNA trap (EET) formation in a concentration- and carbohydrate-dependent manner; S-nitrosylation of SP-D (simulating iNOS-mediated oxidative modification in asthmatic airways) causes de-oligomerization and abolishes its ability to suppress EET formation.\",\n      \"method\": \"Recombinant SP-D binding to eosinophils (confocal imaging), EET formation assay, in vivo SP-D S-nitrosylation in allergic/ozone-challenged mice, in vitro iNOS-mediated S-nitrosylation, non-reducing SDS-PAGE oligomer analysis\",\n      \"journal\": \"Journal of leukocyte biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (in vitro binding, in vivo mouse model, in vitro post-translational modification) establishing mechanism\",\n      \"pmids\": [\"29733456\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"SP-D suppresses LPS-mediated NETosis in human neutrophils by binding to LPS; SP-D deficiency in mice leads to excess NET formation in LPS-inflamed lungs; NETs inhibit surface-active properties of lung surfactant in the absence of SP-D; SP-D can reverse NET-mediated surfactant inhibition and restore its biophysical properties.\",\n      \"method\": \"SP-D binding to LPS (solid-phase assay), NETosis quantification in human neutrophils, Sftpd-/- mouse LPS model, surfactant biophysical function assay, NET-surfactant inhibition experiments\",\n      \"journal\": \"Communications biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vitro mechanism (LPS binding), in vivo validation in KO mice, and functional restoration assay, multiple orthogonal approaches\",\n      \"pmids\": [\"31872075\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"SP-D (SFTPD) is a dodecameric (four homotrimeric subunits) collagenous C-type lectin secreted by type II pneumocytes and Clara cells that binds phosphatidylinositol, glucosylceramide, LPS, and pathogen surface glycoconjugates via its calcium-dependent CRD (with Phe-335 mediating aromatic ring-stacking interactions), aggregates and neutralizes pathogens including influenza A virus, chemoattracts neutrophils and monocytes via the CRD, interacts with SIRPα/β D3 domains to modulate innate immune cell signaling, suppresses LPS-induced NET formation and protects surfactant function, and—through its N-terminal/collagenous domains forming disulfide-stabilized oligomers—regulates alveolar surfactant phospholipid homeostasis and prevents emphysema in vivo; its transcription is driven by a calcineurin/NFATc3–TTF-1 complex, and MMP-9 cleavage or S-nitrosylation-induced de-oligomerization abolishes its immune functions.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"SP-D (SFTPD) is a secreted collagenous C-type lectin synthesized by type II pneumocytes and Clara cells that serves dual roles in innate pulmonary immunity and alveolar surfactant homeostasis [#0, #4]. Each polypeptide combines an N-terminal collagenous region of uninterrupted Gly-X-Y triplets with a C-type lectin carbohydrate recognition domain (CRD) that binds saccharides in a calcium-dependent manner [#1]; trimers assemble through interchain disulfide bonds in the N-terminal domain into cruciform dodecamers and higher-order multimers, an architecture that determines functional valency [#5, #28]. Through its CRD the protein binds endogenous surfactant lipids—phosphatidylinositol and glucosylceramide—as well as microbial surfaces, neutralizing influenza A virus by aggregation and potentiating neutrophil antiviral responses, with multimerization markedly enhancing potency [#3, #2, #6, #8]. The CRD also confers direct chemoattractant activity toward neutrophils and monocytes via saccharide-recognizing cell-surface sites [#7, #9] and engages the membrane-proximal D3 domain of SIRPα/SIRPβ to modulate innate immune cell signaling [#25]. SP-D restrains injurious neutrophil and eosinophil extracellular trap formation by binding LPS and cell membranes, thereby protecting surfactant biophysical function [#30, #29]. In vivo, disulfide-crosslinked oligomers are required for regulation of surfactant phospholipid pools and prevention of emphysema and foamy-macrophage accumulation; loss of SP-D causes lipid accumulation, persistent lung T-cell activation, and airspace remodeling [#10, #12, #15], and the antimicrobial CRD function is mechanistically separable from collagen-domain-dependent lipid homeostasis and emphysema prevention [#17, #21]. Transcription of Sftpd is driven by a calcineurin/NFATc3–TTF-1 complex [#19], while MMP-9 cleavage or S-nitrosylation-induced de-oligomerization abolishes its immune functions [#27, #29].\",\n  \"teleology\": [\n    {\n      \"year\": 1989,\n      \"claim\": \"Establishing that SP-D is a collagenous glycoprotein assembling from disulfide-bonded trimers defined the protein class and quaternary basis for all later function.\",\n      \"evidence\": \"HPLC purification, microsequencing, and electrophoresis of rat BAL protein\",\n      \"pmids\": [\"2675969\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Higher-order assembly geometry not yet defined\", \"No ligand or functional activity yet assigned\"]\n    },\n    {\n      \"year\": 1991,\n      \"claim\": \"Cloning revealed a calcium-dependent C-type lectin CRD joined to a collagenous N-terminal domain, explaining how a collagenous protein could function as a sugar-recognizing molecule.\",\n      \"evidence\": \"cDNA library screening, sequencing, and peptide microsequencing of human SP-D\",\n      \"pmids\": [\"1898081\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological saccharide ligands not identified\", \"CRD residues mediating specificity unknown\"]\n    },\n    {\n      \"year\": 1992,\n      \"claim\": \"Identifying phosphatidylinositol and glucosylceramide as calcium-dependent CRD ligands showed SP-D recognizes endogenous surfactant lipids, not only microbial sugars, and defined its localization across pulmonary cell compartments.\",\n      \"evidence\": \"TLC overlay binding with radioiodinated SP-D, MS lipid identification, immunogold EM\",\n      \"pmids\": [\"1457414\", \"1530650\", \"1527377\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of lipid binding unresolved\", \"Structural basis of lipid versus sugar recognition unknown\"]\n    },\n    {\n      \"year\": 1994,\n      \"claim\": \"Defining the dodecameric four-armed architecture and demonstrating potent influenza A neutralization linked quaternary structure to CRD-mediated antiviral defense.\",\n      \"evidence\": \"Electron microscopy, pepsin/collagenase digestion, hemagglutination inhibition and neutrophil assays\",\n      \"pmids\": [\"8006040\", \"8040272\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Receptor mediating neutrophil effects not identified\", \"Quantitative valency-potency relationship not yet established\"]\n    },\n    {\n      \"year\": 1995,\n      \"claim\": \"Showing SP-D is a CRD-dependent chemoattractant for neutrophils and monocytes extended its role from pathogen aggregation to active recruitment of immune cells.\",\n      \"evidence\": \"Boyden chamber chemotaxis with sugar competition and antibody blocking\",\n      \"pmids\": [\"7695920\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cell-surface receptor for chemotaxis not identified\", \"Signaling pathway downstream unknown\"]\n    },\n    {\n      \"year\": 1996,\n      \"claim\": \"Demonstrating that multimers of dodecamers exceed dodecamers in antiviral potency established CRD valency as a tunable determinant of function encoded in primary structure.\",\n      \"evidence\": \"Recombinant SP-D oligomer fractionation, EM, and antiviral assays\",\n      \"pmids\": [\"8944718\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular driver of higher-order multimerization not pinpointed\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Showing the isolated trimeric CRD suffices for chemotaxis localized the chemoattractant function to the lectin domain independent of the collagenous stalk.\",\n      \"evidence\": \"Recombinant CRD expression and Boyden chamber assay with sugar competition\",\n      \"pmids\": [\"9887065\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Receptor still unidentified\", \"Single-lab in vitro result\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Knockout and lung-specific rescue established that SP-D regulates alveolar surfactant phospholipid homeostasis acting locally within the lung.\",\n      \"evidence\": \"Sftpd-/- mice and SP-C-promoter transgenic rescue with radiolabeled lipid clearance\",\n      \"pmids\": [\"10956621\", \"10666121\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism linking SP-D to lipid clearance unresolved\", \"Cellular target of lipid regulation unclear\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Domain and epistasis genetics showed disulfide-crosslinked oligomers are required in vivo to prevent emphysema and that SP-D and GM-CSF act through distinct additive pathways.\",\n      \"evidence\": \"Cys→Ser mutant transgenics, SP-D/GM-CSF double-null mice, BAL lipid and histology\",\n      \"pmids\": [\"11278637\", \"11504698\", \"1930130\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular pathway from de-oligomerization to emphysema unknown\", \"How SP-D competes with SP-A at type II cell binding sites mechanistically unresolved\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Conditional, chimeric, and domain-swap rescue experiments demonstrated that CRD-mediated antimicrobial defense and collagen/N-terminal-dependent lipid homeostasis and emphysema prevention are mechanistically dissociable functions, and that SP-D suppresses local T-cell activation.\",\n      \"evidence\": \"Doxycycline-inducible and SP-D/conglutinin CRD chimera transgenics, M. pneumoniae binding with CRD mutagenesis, Sftpd-/- immunophenotyping\",\n      \"pmids\": [\"12163500\", \"11956209\", \"11916969\", \"12091242\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Why established emphysema is irreversible unknown\", \"Mechanism of T-cell suppression undefined\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Identifying the calcineurin/NFATc3–TTF-1 complex on the Sftpd promoter defined the transcriptional control of SP-D expression.\",\n      \"evidence\": \"Promoter reporter assays, DNase I footprinting, co-IP and in vitro interaction of NFATc3 and TTF-1\",\n      \"pmids\": [\"15173172\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Upstream signals activating calcineurin in vivo unclear\", \"Additional regulatory inputs not mapped\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Linking a Met11Thr polymorphism to defective multimerization tied SP-D oligomeric state to ligand selectivity, with multimers favoring intact pathogens and monomers favoring isolated LPS.\",\n      \"evidence\": \"SFTPD genotyping, AFM, recombinant protein, and solid-phase binding assays\",\n      \"pmids\": [\"15661913\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional clinical consequence of polymorphism not established in this corpus\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Domain-deletion, chimeric, and structural studies dissected collagen-domain-dependent macrophage/airspace regulation from CRD function, identified Phe-335 as the aromatic ring-stacking residue for extended saccharides, and revealed a systemic role of SP-D in lipid/energy homeostasis.\",\n      \"evidence\": \"Collagen-deletion and SP-A/SP-D chimera transgenics, CRD crystallography with mutagenesis, Sftpd-/- body weight and human BMI cohort\",\n      \"pmids\": [\"16787926\", \"16636058\", \"16500946\", \"17083619\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism connecting SP-D to systemic fat regulation undefined\", \"How collagen domain controls macrophage activation unknown\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Identifying the SIRPα/β D3 binding site and distinct oligomer-dependent ligand profiles refined how SP-D signals to immune cells and how its assembly state selects targets.\",\n      \"evidence\": \"SIRP domain-deletion and N-glycosylation mutagenesis, binding assays, trimer/multimer fractionation\",\n      \"pmids\": [\"22511785\", \"19577304\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream SIRP signaling consequences not defined\", \"Trimer/multimer interconversion in vivo unclear\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Showing MMP-9 cleavage abolishes bacterial aggregation and phagocytosis while sparing LPS binding established proteolytic inactivation as a regulatory and pathological mechanism with site-specific functional outcomes.\",\n      \"evidence\": \"In vitro MMP-9 and neutrophil elastase cleavage with functional readouts\",\n      \"pmids\": [\"22860023\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vivo relevance of cleavage not established\", \"Cleavage sites not mapped at residue resolution\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Demonstrating that SP-D binds LPS and cell membranes to suppress neutrophil and eosinophil extracellular trap formation, and that S-nitrosylation-induced de-oligomerization abolishes this, connected SP-D oligomeric integrity to protection of surfactant biophysical function.\",\n      \"evidence\": \"NETosis/EET assays, Sftpd-/- LPS lung models, in vitro S-nitrosylation, surfactant function assays\",\n      \"pmids\": [\"31872075\", \"29733456\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Receptor/membrane determinants of trap suppression unresolved\", \"In vivo extent of S-nitrosylation regulation under disease unclear\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The cell-surface receptors mediating SP-D chemotaxis and immune-cell signaling, and the molecular pathway linking SP-D oligomeric state to emphysema and systemic lipid homeostasis, remain undefined.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No identified receptor for chemoattractant activity\", \"Mechanistic link from de-oligomerization to airspace remodeling unknown\", \"Pathway connecting SP-D to systemic energy homeostasis uncharacterized\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0030246\", \"supporting_discovery_ids\": [1, 2, 3, 6, 16, 22]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [3, 2, 26]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [25, 30, 29, 14]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [0, 5, 28]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005576\", \"supporting_discovery_ids\": [0, 4]},\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [4]},\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [6, 7, 25, 30, 15]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [10, 11, 13, 23]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [19]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"SIRPA\", \"SIRPB1\", \"SFTPA1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}