{"gene":"C1R","run_date":"2026-04-28T17:12:38","timeline":{"discoveries":[{"year":1976,"finding":"C1r functions as a serine protease zymogen that undergoes proteolytic cleavage to become activated; activation converts each 95,000 Da chain into disulfide-linked subunits of 60,000 and 35,000 Da and enables C1r to cleave and activate C1s. C1r also serves as the physical link between C1q and C1s in macromolecular C1, requiring C1q and calcium for binding to sensitized erythrocytes.","method":"Biochemical fractionation, SDS-PAGE, radiolabeled binding assays, functional reconstitution of C1","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 1–2 — multiple orthogonal biochemical methods, replicated across two papers (PMID 814163 and PMID 1249422)","pmids":["814163","1249422"],"is_preprint":false},{"year":1976,"finding":"Activation of C1r by proteolytic cleavage is inhibited by calcium ions (which stabilize the proenzyme form), C1 inactivator (which forms a complex with the light chain of activated C1r), polyanethol sulfonate, and DFP. Activation is triggered when C1r is incorporated into the C1 complex with C1q and aggregated IgG.","method":"Biochemical activation assays, SDS-PAGE analysis of cleavage products, inhibitor titration experiments","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 1–2 — detailed biochemical dissection with multiple inhibitors and conditions, replicated","pmids":["1249422"],"is_preprint":false},{"year":1977,"finding":"Activated C1r (C1-r) is a serine protease with a catalytic B chain (~27,000 Da) homologous to the trypsin family of serine proteases; it hydrolyses lysine and tyrosine ester bonds but cleaves only C1s as a protein substrate, demonstrating highly restricted proteolytic specificity.","method":"Amino acid composition analysis, N-terminal sequencing, synthetic ester substrate assays","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 — direct enzymatic characterization with multiple substrate assays, foundational paper","pmids":["869924"],"is_preprint":false},{"year":1983,"finding":"The catalytic B chain of activated C1r contains 242 amino acids with a serine protease active site triad (His-39, Asp-94, Ser-191 by chymotrypsinogen numbering); it lacks the histidine-loop disulfide bridge found in other serine proteases and has five half-cystine residues.","method":"Direct amino acid sequence determination by chemical fragmentation and sequencing","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 — complete amino acid sequence with active-site residue identification","pmids":["6303394"],"is_preprint":false},{"year":1985,"finding":"C1r undergoes autocatalytic (autolytic) activation involving two intermolecular reactions: a DFP-insensitive zymogen-catalyzed step and a DFP-sensitive enzyme-catalyzed step, consistent with activation proceeding through a conformational isomer intermediate before full proteolytic cleavage.","method":"Kinetic analysis of activation time-course, DFP inhibition experiments, surface radiolabeling","journal":"Journal of biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 — kinetic and biochemical dissection in a single lab; supported by later structural work","pmids":["2987200"],"is_preprint":false},{"year":1986,"finding":"The C1r polypeptide precursor of 705 amino acids contains an N-terminal non-catalytic A chain with an EGF-like domain, two pairs of internal repeats (one pair related to C1s N-terminal sequence; the other related to complement factor B and beta2-glycoprotein I), and a C-terminal B chain encoding the serine protease domain homologous to the trypsin family.","method":"cDNA sequencing of full-length C1r transcript from human liver and HepG2 libraries","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 — complete cDNA sequence defining domain architecture, confirmed by two independent groups (PMID 3021205 and PMID 3030286)","pmids":["3021205","3030286"],"is_preprint":false},{"year":1987,"finding":"The A chain of activated C1r (446 residues) contains a beta-hydroxyaspartic acid residue at position 150 within an EGF consensus sequence, two carbohydrate chains attached at Asn-108 and Asn-204, and is subdivided by autolytic cleavage into three fragments (alpha, beta, gamma); fragment gamma is disulfide-linked to the catalytic B chain.","method":"Complete amino acid sequence determination from autolytic fragments and chemical cleavage peptides","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 — complete protein sequence determination with PTM identification","pmids":["3036070"],"is_preprint":false},{"year":1988,"finding":"The human C1R and C1S genes are located on chromosome 12p13 in a closely linked tail-to-tail arrangement separated by approximately 9.5 kb, consistent with gene duplication from a common ancestor; both genes are primarily expressed in liver.","method":"cDNA sequencing, genomic DNA blotting, in situ hybridization, RNA blot analysis","journal":"Proceedings of the National Academy of Sciences","confidence":"High","confidence_rationale":"Tier 2 — multiple genomic and expression methods confirming chromosomal organization","pmids":["2459702"],"is_preprint":false},{"year":1994,"finding":"Ca2+ ions suppress C1r autoactivation by an intramolecular mechanism involving the Ca2+-binding alpha-region; this inhibitory effect is released in the C1 complex by a signal originating in C1q and transmitted through the C1q/C1r interface. Incorporation of C1r into the C1s-C1r-C1r-C1s tetramer fully stabilizes C1r in proenzyme form, and interaction with intact C1q (but not collagen-like C1q fragments) is required to trigger activation above 25°C.","method":"Spontaneous activation assays, sedimentation analysis, Ca2+ titration, reconstitution of C1 with truncated C1q fragments","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1–2 — reconstitution experiments with defined fragments and quantitative activation kinetics","pmids":["8042996"],"is_preprint":false},{"year":1997,"finding":"The catalytic domain of C1r (CCP1-CCP2-SP) forms a non-covalent homodimer in a head-to-tail arrangement; chemical cross-linking and homology modeling identified an intramonomer cross-link between Lys426 (CCP module V) and the C-terminal Asp688 (SP domain) and an intermonomer cross-link between Gly280 and Glu493, supporting a model where CCP module V interacts with the SP domain on the side opposite to the active site.","method":"Chemical cross-linking with EDC, CNBr cleavage, N-terminal sequencing, mass spectrometry, homology modeling","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 — cross-linking combined with MS and structural modeling","pmids":["9174342"],"is_preprint":false},{"year":1998,"finding":"The EGF-like module of C1r (residues 123–175) has a well-ordered C-terminal part with canonical EGF fold and a disordered large N-terminal loop between the first two cysteines; Ca2+ at 80 mM did not significantly alter the EGF module structure, indicating Ca2+ binding is mediated by a different mechanism in the full-length protein.","method":"NMR structure determination, restrained molecular dynamics calculations","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 — NMR structure with functional implication from Ca2+ experiments","pmids":["9477945"],"is_preprint":false},{"year":2000,"finding":"In human serum, C1r and C1s are found exclusively in complex with C1q and not with MBL, while MASP-1, MASP-2, and MAp19 are found exclusively with MBL; C1r-deficient serum shows no complement activation on IgG-coated surfaces, confirming that C1r is essential for classical pathway activation via antibody complexes.","method":"Gel-permeation chromatography of serum fractions, ELISA, complement activation assays with deficient sera","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 — reciprocal biochemical separation and functional deficiency assay confirming pathway specificity","pmids":["10878362"],"is_preprint":false},{"year":2001,"finding":"Autoactivation and C1s cleavage are intrinsic properties of the C1r SP domain alone; the CCP2 module significantly increases proteolytic activity toward C1s and stabilizes the SP domain structure; CCP1 is required for dimer formation but is not required for autoactivation or C1s cleavage. Dimerization of C1r is not a prerequisite for autoactivation.","method":"Recombinant expression of domain-deletion fragments in E. coli, renaturation, esterolytic and C1s-cleavage assays, differential scanning calorimetry, sedimentation analysis","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 1–2 — domain deletion mutagenesis combined with functional and biophysical assays","pmids":["11673533"],"is_preprint":false},{"year":2001,"finding":"Both CCP1/2-SP and CCP2-SP fragments of C1r activate autolytically (confirmed by recovery as two-chain proteases while R446Q and S637A mutants remain single-chain zymogens), proving C1r activation is an autolytic process. CCP1 drives dimerization (5.5 S) but is not required for self-activation; relative C1s-cleavage efficiency is C1r < CCP1/2-SP < CCP2-SP, showing CCP1 is not involved in C1s recognition.","method":"Baculovirus expression, site-directed mutagenesis (R446Q, S637A), sedimentation velocity, C1s cleavage assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — reconstitution with active-site and cleavage-site mutagenesis; first direct proof of autolytic mechanism","pmids":["11445589"],"is_preprint":false},{"year":2002,"finding":"Crystal structure of the zymogen and active catalytic domain (CCP1-CCP2-SP) of C1r shows a homodimer with head-to-tail arrangement where the active site of one monomer and the cleavage site (Arg446) of its partner are at opposite ends, demonstrating that a mechanical stress disrupting the CCP1-SP interface is required to trigger C1r activation in the C1 complex.","method":"X-ray crystallography to 2.9 Å (zymogen, S647A mutant) and crystal structures of active CCP2-SP domain","journal":"The EMBO journal / Structure","confidence":"High","confidence_rationale":"Tier 1 — crystal structures with functional validation of activation mechanism","pmids":["11823416","12429092"],"is_preprint":false},{"year":2004,"finding":"C1r-like protein (C1r-LP), a complement-related serine protease, mediates proteolytic cleavage of prohaptoglobin in the endoplasmic reticulum; mutation of the active-site Ser abolished cleavage; substituting Gly for Arg-161 in proHp blocked cleavage; RNAi knockdown of C1r-LP reduced proHp cleavage by up to 45% in HepG2 cells. (Note: This is C1r-LP, a paralog/related protein, not C1r itself, but the abstract identifies it as a distinct gene.)","method":"Coexpression in COS-1 cells, active-site mutagenesis, substrate mutagenesis, RNAi knockdown, in vitro cleavage assay with purified proteins","journal":"Proceedings of the National Academy of Sciences","confidence":"Medium","confidence_rationale":"Tier 1 — but concerns C1r-LP (a distinct paralog), not C1r itself; included as mechanistic finding about a C1r-related enzyme","pmids":["15385675"],"is_preprint":false},{"year":2007,"finding":"Crystal structure of the entire active catalytic region (CCP1-CCP2-SP) of human C1r shows head-to-tail dimer similar to the zymogen; an enzyme-product complex in the crystal lattice reveals a critical S1-P1 salt bridge between Asp631 and Arg446, and intermolecular interaction between CCP2 and SP domain, supporting a split-and-reassembly model for C1r autoactivation without large-scale C1q arm movements.","method":"X-ray crystallography of full active catalytic region","journal":"Molecular immunology","confidence":"High","confidence_rationale":"Tier 1 — crystal structure of activated form providing atomic-level mechanism","pmids":["17996945"],"is_preprint":false},{"year":2009,"finding":"The C1q-binding sites of the C1s-C1r-C1r-C1s tetramer reside primarily in the CUB1 and CUB2 modules of C1r (high affinity) and CUB1 of C1s (lower affinity); these sites involve acidic residues that also serve as Ca2+ ligands; the EGF module of C1r (Glu137-Glu-Asp139) does not participate in C1q binding, contrary to earlier models. Altogether six binding sites interact with reactive lysines of C1q stems.","method":"Site-directed mutagenesis of C1r and C1s, surface plasmon resonance binding assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 — systematic mutagenesis combined with SPR quantification","pmids":["19473974"],"is_preprint":false},{"year":2010,"finding":"Ca2+ binding to the CUB2 domain of C1r induces folding from a disordered to a compact structure (Kd ~430 μM); partial Ca2+ saturation of CUB2 in blood provides conformational flexibility required for C1 activation; Ca2+-free CUB2 shows intrinsically disordered character, and Ca2+ significantly suppresses autoactivation of native C1r zymogen.","method":"Recombinant domain production, isothermal titration calorimetry, NMR, activation assays with native C1r","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — ITC, NMR, and functional assays in combination","pmids":["20178990"],"is_preprint":false},{"year":2010,"finding":"Mass spectrometry-based surface accessibility mapping of the C1r/C1s tetramer in isolation versus in assembled C1 showed that the C1s CUB1-EGF-CUB2 interaction domains, distant in the free tetramer, associate with each other in the C1 complex; the C1s serine protease domain is partly positioned inside the C1q cone in the proenzyme form.","method":"Chemical modification of Lys residues, quantitative MS-based accessibility comparison","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 — label-free semi-quantitative MS with chemical cross-linking","pmids":["20592021"],"is_preprint":false},{"year":2013,"finding":"C1r demonstrates clear active-site specificity for Gln at P2 and Ile at P1' positions; these residues are present in the physiological C1s activation site, and their removal reduces C1r-mediated C1s activation; introduction of Gln at P2 of the non-substrate MASP-3 enables its efficient cleavage by C1r. The CCP (complement control protein) domains of C1r provide exosite interactions that contribute to efficient C1s cleavage.","method":"Phage display library substrate specificity assay, mutagenesis of C1s activation site, MD simulations, structural modeling","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 — phage library combined with mutagenesis and structural modeling","pmids":["23589288"],"is_preprint":false},{"year":2013,"finding":"Recombinant C1q variants with mutations at LysB61 and LysC58 in the collagen-like stems show markedly reduced interaction with the C1s-C1r-C1r-C1s tetramer by surface plasmon resonance, demonstrating that LysB61 and LysC58 form key salt bridges with acidic Ca2+ ligands of C1r and C1s CUB domains during C1 assembly.","method":"Recombinant C1q expression in HEK293-F cells, site-directed mutagenesis, SPR binding assays, C1r/C1s activation assay","journal":"Proceedings of the National Academy of Sciences","confidence":"High","confidence_rationale":"Tier 1–2 — mutagenesis plus quantitative SPR in reconstituted system","pmids":["23650384"],"is_preprint":false},{"year":2016,"finding":"Heterozygous missense or in-frame insertion/deletion mutations in C1R (affecting subunit interfaces or inter-domain hinges) cause periodontal Ehlers-Danlos syndrome; pathogenic C1r variants are associated with intracellular retention and mild ER enlargement, indicating that proper C1r folding and secretion are required for connective tissue homeostasis.","method":"Human genetics (whole-exome sequencing of 19 families), immunofluorescence for ER morphology","journal":"American journal of human genetics","confidence":"High","confidence_rationale":"Tier 2 — genetic discovery in large cohort with cellular phenotyping","pmids":["27745832"],"is_preprint":false},{"year":2018,"finding":"Crystal structure of the CUB1-EGF-CUB2 heterodimer of C1r and C1s reveals an antiparallel L-shaped heterodimer with Ca2+ at the interface from each subunit; contacts involving all three domains make heterodimer formation more favorable than homodimer formation, explaining preferential heterotetramer assembly. Structural data support a model where activation is driven by separation of C1r-C1s dimer pairs when C1q binds to a surface.","method":"X-ray crystallography of C1r-C1s CUB1-EGF-CUB2 complex","journal":"Proceedings of the National Academy of Sciences","confidence":"High","confidence_rationale":"Tier 1 — crystal structure with comprehensive structural and biophysical analysis","pmids":["29311313"],"is_preprint":false},{"year":2019,"finding":"Knockdown of C1r in cutaneous squamous cell carcinoma (cSCC) cells inhibited ERK1/2 and Akt signaling, promoted apoptosis, and suppressed xenograft tumor growth and vascularization in vivo, demonstrating a tumor-cell-autonomous role for C1r in promoting cSCC growth independent of the complement pathway.","method":"siRNA knockdown in cSCC cell lines, Western blot for signaling pathways, xenograft tumor model","journal":"The British journal of dermatology","confidence":"Medium","confidence_rationale":"Tier 2–3 — KD with defined signaling readout in cell and in vivo models; mechanism downstream of C1r not fully resolved","pmids":["31049937"],"is_preprint":false},{"year":2019,"finding":"C1R mutations causing periodontal EDS result in constitutive intracellular activation of C1r and/or C1s; patient-derived fibroblasts secrete activated C1s and activate externally added C4 in the absence of microbial triggers, demonstrating that pEDS variants cause autonomous complement cascade activation rather than loss of C1 complex assembly.","method":"Overexpression in HEK293T cells, Western blot, size exclusion chromatography, SPR, ELISA of patient fibroblast supernatants, C4 activation assay","journal":"Frontiers in immunology","confidence":"High","confidence_rationale":"Tier 1–2 — multiple orthogonal methods including functional complement activation assay in patient cells","pmids":["31749804"],"is_preprint":false},{"year":2019,"finding":"C1r deletion in renal tubular epithelial cells (C1r-null mice) reduced C1s expression, C3 fragment formation, and kidney fibrosis after folic acid injury; in vitro experiments showed C1s expression is dependent on C1r expression; interferon-γ induces co-expression of both proteases.","method":"Global C1r knockout mouse, folic acid nephropathy model, immunohistochemistry, in situ hybridization, in vitro renal epithelial cell assays","journal":"American journal of physiology. Renal physiology","confidence":"Medium","confidence_rationale":"Tier 2 — genetic KO with defined in vivo fibrosis phenotype; mechanism largely through complement C3 activation","pmids":["31509012"],"is_preprint":false},{"year":2021,"finding":"CRISPR/Cas9 knockout of C1r in cSCC cells decreased proliferation, migration, and invasion through collagen type I, suppressed MMP-1, MMP-10, MMP-12, and MMP-13 production, and reduced collagen degradation and xenograft tumor invasion in vivo, placing C1r upstream of MMP-13-driven matrix remodeling in cSCC.","method":"CRISPR/Cas9 knockout, mRNA-seq, Western blot for MMPs, invasion assay through collagen I, xenograft model","journal":"The Journal of investigative dermatology","confidence":"High","confidence_rationale":"Tier 1–2 — CRISPR KO with transcriptomic and functional validation in vitro and in vivo","pmids":["34756877"],"is_preprint":false},{"year":2021,"finding":"Crystal structure of the Borrelia burgdorferi BBK32 C-terminal domain in complex with activated human C1r reveals that BBK32 inhibits C1r by occluding the S1 and S1' substrate-binding subsites; B loop-interacting residues of BBK32 also contribute to full inhibitory activity, as validated by site-directed mutagenesis combined with SPR and complement function assays.","method":"X-ray crystallography, SAXS, site-directed mutagenesis, SPR, complement activation assays","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 1 — structure plus mutagenesis validation of binding interface","pmids":["34759015"],"is_preprint":false},{"year":2021,"finding":"The C1r2s2 protease heterotetramer stabilizes C1q binding to surface-bound IgG; the contribution of C1r2s2 to C1q-IgG stability differs between IgG subclasses, and hexamer-enhancing IgG mutations improve C1q complex stability both with and without C1r2s2, mechanistically linking the proteases to avid C1 complex assembly on IgG-opsonized surfaces.","method":"SPR binding assays, antibody engineering, complement-dependent phagocytosis assays with human neutrophils","journal":"Proceedings of the National Academy of Sciences","confidence":"High","confidence_rationale":"Tier 2 — SPR with engineered antibody variants and functional neutrophil assay","pmids":["34155115"],"is_preprint":false},{"year":2024,"finding":"In hepatocellular carcinoma, C1r acts as a tumor suppressor; C-reactive protein (CRP) binds to C1r, and when C1r is downregulated (via DNMT1/DNMT3a-mediated promoter methylation), free CRP activates NF-κB signaling, upregulating HIF-1α and increasing glycolysis to promote aggressive HCC behavior.","method":"CRISPR/siRNA knockdown, RNA-seq, methylation-specific PCR, co-immunoprecipitation (CRP-C1r interaction), dual-luciferase reporter assay, xenograft model","journal":"Molecular carcinogenesis","confidence":"Medium","confidence_rationale":"Tier 2–3 — co-IP identifying CRP as C1r binding partner, combined with reporter and in vivo data; single lab","pmids":["39150096"],"is_preprint":false},{"year":1999,"finding":"One active C1r subunit within the C1r homodimer is sufficient for full hemolytic activity of the C1 complex; stabilization of one C1r monomer as a zymogen (R446Q mutant) does not abolish activity when paired with wild-type C1r in a mixed dimer, and C1r monomers exchange between dimers even at 4°C.","method":"Baculovirus expression of zymogen-stabilizing C1r mutants, reconstitution hemolytic assay, mixed-dimer experiments","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 1–2 — active-site and cleavage-site mutagenesis with functional reconstitution","pmids":["9916740"],"is_preprint":false}],"current_model":"C1r is a modular serine protease zymogen that assembles with C1s into a Ca2+-dependent C1s-C1r-C1r-C1s heterotetramer via CUB-EGF-CUB domain interactions, associates with the recognition protein C1q through high-affinity CUB1/CUB2-mediated contacts with reactive lysines on C1q collagen stems, undergoes autolytic self-activation (driven by mechanical stress from C1q target binding that disrupts a Ca2+-regulated CCP1-SP interface) via cleavage of the Arg446-Ile447 bond to generate the active two-chain enzyme, which then cleaves and activates C1s at a Gln(P2)-Arg-Ile(P1') site (also using CCP domain exosites), thereby initiating the classical complement cascade; beyond complement, tumour-cell-derived C1r promotes cSCC invasion through upregulation of matrix metalloproteinases (MMP-1, -10, -12, -13) and activates ERK1/2/Akt signalling, while in HCC it acts as a tumour suppressor by sequestering CRP and preventing NF-κB/HIF-1α-driven glycolysis."},"narrative":{"teleology":[{"year":1976,"claim":"Establishing that C1r is a serine protease zymogen activated by proteolytic cleavage into disulfide-linked subunits answered the fundamental question of how the classical pathway is enzymatically initiated, and revealed that C1r bridges C1q to C1s in a Ca2+-dependent manner.","evidence":"Biochemical fractionation, SDS-PAGE, radiolabeled binding assays, functional C1 reconstitution","pmids":["814163","1249422"],"confidence":"High","gaps":["Cleavage site identity unknown","Whether activation is autolytic or requires an exogenous protease was unresolved","Domain architecture undefined"]},{"year":1977,"claim":"Demonstrating that activated C1r cleaves only C1s among tested protein substrates established C1r as a highly specific protease rather than a general trypsin-like enzyme, defining its singular role as the activator of C1s.","evidence":"Amino acid composition, N-terminal sequencing, synthetic ester substrate assays","pmids":["869924"],"confidence":"High","gaps":["Molecular basis for substrate restriction unknown","Active-site sequence not yet determined"]},{"year":1986,"claim":"Determination of the complete cDNA sequence and domain architecture (CUB1-EGF-CUB2-CCP1-CCP2-SP) resolved how C1r combines recognition/interaction domains with a catalytic module and explained homology with C1s, suggesting gene duplication origin.","evidence":"Full-length cDNA sequencing from liver/HepG2 libraries, confirmed by two independent groups","pmids":["3021205","3030286","2459702"],"confidence":"High","gaps":["Functions of individual domains not experimentally tested","C1q binding site not mapped"]},{"year":1994,"claim":"Reconstitution experiments with truncated C1q fragments showed that Ca2+ suppresses C1r autoactivation intramolecularly, that the C1s–C1r–C1r–C1s tetramer stabilizes the proenzyme, and that intact C1q globular heads plus collagen stems are required to transmit the activation signal — resolving the question of how C1r activation is held in check until target recognition.","evidence":"Spontaneous activation assays, sedimentation, Ca2+ titration, reconstitution with C1q fragments","pmids":["8042996"],"confidence":"High","gaps":["Nature of the C1q-transmitted mechanical signal unknown","Ca2+-binding domain not identified"]},{"year":2001,"claim":"Domain-deletion and active-site mutagenesis (R446Q, S637A) proved that C1r activation is autolytic rather than requiring an external protease, that autoactivation and C1s cleavage are intrinsic to the SP domain, and that CCP2 enhances C1s cleavage while CCP1 drives dimerization but is dispensable for catalysis.","evidence":"Recombinant domain fragments, site-directed mutagenesis, sedimentation, cleavage assays in E. coli and baculovirus systems","pmids":["11445589","11673533"],"confidence":"High","gaps":["Structural basis for the CCP1–SP interface restraining activation unknown","How dimerization relates to activation in the C1 complex unresolved"]},{"year":2002,"claim":"Crystal structures of zymogen and active C1r catalytic region revealed a head-to-tail homodimer where the active site and cleavage site of partner monomers are maximally separated, establishing that mechanical stress disrupting the CCP1–SP interface is the trigger for autoactivation within the C1 complex.","evidence":"X-ray crystallography at 2.9 Å (zymogen S647A mutant) and active CCP2-SP structures","pmids":["11823416","12429092"],"confidence":"High","gaps":["Source and magnitude of mechanical stress in physiological C1 not measured","Full C1 complex structure unavailable"]},{"year":2010,"claim":"Mapping C1q-binding sites to acidic Ca2+-ligand residues in C1r CUB1 and CUB2 (high affinity) and C1s CUB1 (lower affinity), and identifying reactive lysines LysB61/LysC58 on C1q stems as their partners, resolved the molecular interface holding the C1 complex together.","evidence":"Site-directed mutagenesis, SPR, ITC, NMR of CUB2, recombinant C1q variants","pmids":["19473974","20178990","23650384"],"confidence":"High","gaps":["Stoichiometry of engagement with all six C1q stems not fully resolved","Role of partial Ca2+ saturation of CUB2 in vivo unclear"]},{"year":2013,"claim":"Phage-display substrate profiling and mutagenesis defined the stringent P2-Gln/P1'-Ile specificity of C1r's active site and demonstrated that CCP domains contribute exosite interactions for efficient C1s cleavage, explaining why C1r is restricted to C1s as its sole natural substrate.","evidence":"Phage display library, C1s activation-site mutagenesis, MD simulation","pmids":["23589288"],"confidence":"High","gaps":["Atomic structure of the C1r–C1s enzyme–substrate complex not determined","Whether CCP exosite contacts are essential or merely rate-enhancing unclear"]},{"year":2016,"claim":"Discovery that heterozygous C1R mutations cause periodontal Ehlers–Danlos syndrome linked C1r to connective-tissue homeostasis, and subsequent work showed that pEDS variants cause constitutive intracellular C1r/C1s activation and autonomous complement activation rather than loss of C1 complex assembly.","evidence":"Whole-exome sequencing of 19 families, immunofluorescence, patient fibroblast C4-activation assays, overexpression in HEK293T","pmids":["27745832","31749804"],"confidence":"High","gaps":["How complement activation in connective tissue leads to periodontitis and skin fragility unknown","Whether intracellular activation damages the ER or acts extracellularly unresolved"]},{"year":2018,"claim":"The crystal structure of the C1r–C1s CUB1-EGF-CUB2 heterodimer revealed an antiparallel L-shaped architecture with Ca2+ at each interface, explaining preferential heterotetramer over homodimer assembly and supporting a model where surface binding separates C1r–C1s pairs to trigger activation.","evidence":"X-ray crystallography of the C1r–C1s interaction domain complex","pmids":["29311313"],"confidence":"High","gaps":["Full-length C1 complex structure still unavailable","Dynamics of pair separation upon C1q bending not captured"]},{"year":2021,"claim":"Beyond complement, CRISPR knockout of C1r in cutaneous SCC cells established a complement-independent tumor-promoting function: C1r drives MMP-1/10/12/13 expression, collagen degradation, ERK1/2–Akt signaling, and xenograft invasion; conversely, in HCC, C1r sequesters CRP to suppress NF-κB/HIF-1α–driven glycolysis, acting as a tumor suppressor.","evidence":"CRISPR KO and siRNA in cSCC lines, mRNA-seq, Western blot, xenograft models; co-IP of CRP–C1r, reporter assays, methylation-specific PCR in HCC","pmids":["34756877","31049937","39150096"],"confidence":"Medium","gaps":["Whether the oncogenic function requires C1r catalytic activity or is scaffold-mediated unknown","CRP–C1r interaction awaits structural characterization","Single-lab findings for both cancer contexts"]},{"year":2021,"claim":"The crystal structure of Borrelia BBK32 bound to activated C1r revealed that pathogen-derived inhibitors block C1r by occluding the S1/S1' substrate-binding subsites, providing atomic-level insight into immune evasion and a template for therapeutic C1r inhibitor design.","evidence":"X-ray crystallography, SAXS, site-directed mutagenesis, SPR, complement assays","pmids":["34759015"],"confidence":"High","gaps":["Whether other microbial C1r inhibitors use the same mechanism unknown","No small-molecule inhibitor of C1r yet designed from this template"]},{"year":null,"claim":"A complete atomic structure of the full C1 complex (C1q bound to the C1s–C1r–C1r–C1s tetramer) is still lacking, and the precise mechanical forces and conformational changes that relay C1q target recognition to C1r autoactivation remain unresolved; additionally, the complement-independent roles of C1r in connective tissue homeostasis and cancer require mechanistic dissection of whether they depend on its catalytic activity or scaffold functions.","evidence":"","pmids":[],"confidence":"Low","gaps":["Full C1 complex structure not determined","Force transduction mechanism from C1q to C1r activation site not measured","Catalytic vs. non-catalytic roles in pEDS pathogenesis and cancer not distinguished"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,2,12,13,20]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[2,3,4]}],"localization":[{"term_id":"GO:0005576","term_label":"extracellular region","supporting_discovery_ids":[0,11,29]},{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[22,25]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[0,1,8,11,20,28]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[22,25,27,30]}],"complexes":["C1 complex (C1q:C1r2:C1s2)","C1s-C1r-C1r-C1s tetramer"],"partners":["C1S","C1QA","C1QB","C1QC","CRP","SERPING1"],"other_free_text":[]},"mechanistic_narrative":"C1r is a modular serine protease zymogen that initiates the classical complement pathway by undergoing Ca2+-regulated autoactivation and subsequently cleaving and activating its sole physiological substrate, C1s, within the C1 macromolecular complex. The proenzyme assembles with C1s into a Ca2+-dependent C1s–C1r–C1r–C1s heterotetramer via CUB1-EGF-CUB2 domain interactions, which binds C1q collagen stems through high-affinity salt bridges between acidic Ca2+-coordinating residues in the C1r CUB domains and reactive lysines (LysB61, LysC58) on C1q [PMID:19473974, PMID:23650384, PMID:29311313]. Upon C1q recognition of immune complexes, mechanical disruption of the CCP1–SP interface in the head-to-tail C1r homodimer triggers autolytic cleavage at Arg446–Ile447, generating the active two-chain enzyme whose SP domain exhibits stringent specificity for Gln(P2)–Arg–Ile(P1') in C1s, augmented by CCP2-mediated exosite contacts [PMID:11823416, PMID:11445589, PMID:23589288]. Heterozygous missense mutations in C1R cause periodontal Ehlers–Danlos syndrome through constitutive intracellular activation of C1r/C1s and autonomous complement activation rather than loss of complex assembly [PMID:27745832, PMID:31749804]."},"prefetch_data":{"uniprot":{"accession":"P00736","full_name":"Complement C1r subcomponent","aliases":["Complement component 1 subcomponent r"],"length_aa":705,"mass_kda":80.1,"function":"Serine protease component of the complement C1 complex, a multiprotein complex that initiates the classical pathway of the complement system, a cascade of proteins that leads to phagocytosis and breakdown of pathogens and signaling that strengthens the adaptive immune system (PubMed:17996945, PubMed:19473974, PubMed:29449492). C1R catalyzes the first enzymatic step in the classical complement pathway: it is activated by the C1Q subcomplex of the C1 complex, which associates with IgG or IgM immunoglobulins complexed with antigens to form antigen-antibody complexes on the surface of pathogens (PubMed:29449492, PubMed:34155115). Immunoglobulin-binding promotes the autocatalytic cleavage and activation of C1R (PubMed:11445589, PubMed:11673533, PubMed:17996945, PubMed:20178990, PubMed:6254570, PubMed:6271784). Activated C1R then cleaves and activates C1S, the second protease of the classical complement pathway (PubMed:11445589, PubMed:11673533, PubMed:6271784). It is unclear if C1R activates C1S within single, strained C1 complexes or between neighboring C1 complexes on surfaces (PubMed:28104818, PubMed:29311313, PubMed:29449492)","subcellular_location":"Secreted; Cell surface","url":"https://www.uniprot.org/uniprotkb/P00736/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/C1R","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":70,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/C1R","total_profiled":1310},"omim":[{"mim_id":"617174","title":"EHLERS-DANLOS SYNDROME, PERIODONTAL TYPE, 2; EDSPD2","url":"https://www.omim.org/entry/617174"},{"mim_id":"613785","title":"COMPLEMENT COMPONENT 1, r SUBCOMPONENT; C1R","url":"https://www.omim.org/entry/613785"},{"mim_id":"613783","title":"COMPLEMENT COMPONENT C1s DEFICIENCY; C1SD","url":"https://www.omim.org/entry/613783"},{"mim_id":"609862","title":"TRANSMEMBRANE PROTEASE, SERINE 6; TMPRSS6","url":"https://www.omim.org/entry/609862"},{"mim_id":"609342","title":"COBALAMIN-BINDING INTRINSIC FACTOR; CBLIF","url":"https://www.omim.org/entry/609342"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Uncertain","locations":[{"location":"Nucleoplasm","reliability":"Uncertain"},{"location":"Cytosol","reliability":"Uncertain"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"liver","ntpm":1733.3}],"url":"https://www.proteinatlas.org/search/C1R"},"hgnc":{"alias_symbol":[],"prev_symbol":[]},"alphafold":{"accession":"P00736","domains":[{"cath_id":"2.60.120.290","chopping":"27-143","consensus_level":"high","plddt":91.9099,"start":27,"end":143},{"cath_id":"2.60.120.290","chopping":"194-307","consensus_level":"high","plddt":92.3955,"start":194,"end":307},{"cath_id":"2.10.70.10","chopping":"309-374","consensus_level":"medium","plddt":93.0565,"start":309,"end":374},{"cath_id":"2.40.10.10","chopping":"469-703","consensus_level":"medium","plddt":88.5196,"start":469,"end":703}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P00736","model_url":"https://alphafold.ebi.ac.uk/files/AF-P00736-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P00736-F1-predicted_aligned_error_v6.png","plddt_mean":87.0},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=C1R","jax_strain_url":"https://www.jax.org/strain/search?query=C1R"},"sequence":{"accession":"P00736","fasta_url":"https://rest.uniprot.org/uniprotkb/P00736.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P00736/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P00736"}},"corpus_meta":[{"pmid":"1541831","id":"PMC_1541831","title":"The 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pro-C1s.","date":"2005","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/15527420","citation_count":13,"is_preprint":false},{"pmid":"12396000","id":"PMC_12396000","title":"Structure, function and molecular genetics of human and murine C1r.","date":"2002","source":"Immunobiology","url":"https://pubmed.ncbi.nlm.nih.gov/12396000","citation_count":12,"is_preprint":false},{"pmid":"8676886","id":"PMC_8676886","title":"Functional effects of domain deletions in a multidomain serine protease, C1r.","date":"1996","source":"Molecular immunology","url":"https://pubmed.ncbi.nlm.nih.gov/8676886","citation_count":12,"is_preprint":false},{"pmid":"9777414","id":"PMC_9777414","title":"Structural and functional studies on C1r and C1s: new insights into the mechanisms involved in C1 activity and assembly.","date":"1998","source":"Immunobiology","url":"https://pubmed.ncbi.nlm.nih.gov/9777414","citation_count":12,"is_preprint":false},{"pmid":"2552984","id":"PMC_2552984","title":"Biosynthesis of C1r and C1s subcomponents.","date":"1989","source":"Behring Institute Mitteilungen","url":"https://pubmed.ncbi.nlm.nih.gov/2552984","citation_count":11,"is_preprint":false},{"pmid":"25445868","id":"PMC_25445868","title":"The diagnostic role of signal peptide-C1r/C1s, Uegf, and Bmp1-epidermal growth factor domain-containing protein 1 in non-ST-elevation acute coronary syndrome.","date":"2014","source":"The American journal of emergency medicine","url":"https://pubmed.ncbi.nlm.nih.gov/25445868","citation_count":11,"is_preprint":false},{"pmid":"9916740","id":"PMC_9916740","title":"One active C1r subunit is sufficient for the activity of the complement C1 complex: stabilization of C1r in the zymogen form by point mutations.","date":"1999","source":"Journal of immunology (Baltimore, Md. : 1950)","url":"https://pubmed.ncbi.nlm.nih.gov/9916740","citation_count":11,"is_preprint":false},{"pmid":"1270407","id":"PMC_1270407","title":"The role of C1 esterase inhibitor in the activation of C1r, a subcomponent of the first component of complement from human plasma.","date":"1976","source":"Journal of biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/1270407","citation_count":11,"is_preprint":false},{"pmid":"2550362","id":"PMC_2550362","title":"FN-C1q and C1 INH C1r-C1s complexes as indicators of complement activation in patients with chronic lymphocytic leukaemia.","date":"1989","source":"Immunology letters","url":"https://pubmed.ncbi.nlm.nih.gov/2550362","citation_count":10,"is_preprint":false},{"pmid":"36012546","id":"PMC_36012546","title":"Autoantibodies against Complement Classical Pathway Components C1q, C1r, C1s and C1-Inh in Patients with Lupus Nephritis.","date":"2022","source":"International journal of molecular 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glycolysis.","date":"2024","source":"Molecular carcinogenesis","url":"https://pubmed.ncbi.nlm.nih.gov/39150096","citation_count":8,"is_preprint":false},{"pmid":"12686506","id":"PMC_12686506","title":"A novel murine complement-related gene encoding a C1r-like serum protein.","date":"2003","source":"Molecular immunology","url":"https://pubmed.ncbi.nlm.nih.gov/12686506","citation_count":8,"is_preprint":false},{"pmid":"33193398","id":"PMC_33193398","title":"Complement C1q Interacts With LRP1 Clusters II and IV Through a Site Close but Different From the Binding Site of Its C1r and C1s-Associated Proteases.","date":"2020","source":"Frontiers in immunology","url":"https://pubmed.ncbi.nlm.nih.gov/33193398","citation_count":8,"is_preprint":false},{"pmid":"12187480","id":"PMC_12187480","title":"C1r-C1s-C1inhibitor (C1rs-C1inh) complex measurements in tears of patients before and after penetrating keratoplasty.","date":"2002","source":"Current eye research","url":"https://pubmed.ncbi.nlm.nih.gov/12187480","citation_count":8,"is_preprint":false},{"pmid":"7939409","id":"PMC_7939409","title":"Non-coordinated biosynthesis of early complement components in a deficiency of complement proteins C1r and C1s.","date":"1994","source":"Scandinavian journal of immunology","url":"https://pubmed.ncbi.nlm.nih.gov/7939409","citation_count":8,"is_preprint":false},{"pmid":"15358180","id":"PMC_15358180","title":"A novel human dendritic cell-derived C1r-like serine protease analog inhibits complement-mediated cytotoxicity.","date":"2004","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/15358180","citation_count":7,"is_preprint":false},{"pmid":"12914573","id":"PMC_12914573","title":"The human complement component C1R gene: the exon-intron structure and the molecular basis of allelic diversity.","date":"2003","source":"Annals of human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/12914573","citation_count":7,"is_preprint":false},{"pmid":"30025171","id":"PMC_30025171","title":"A Chinese family with periodontal Ehlers-Danlos syndrome associated with missense mutation in the C1R gene.","date":"2018","source":"Journal of clinical periodontology","url":"https://pubmed.ncbi.nlm.nih.gov/30025171","citation_count":7,"is_preprint":false},{"pmid":"11231310","id":"PMC_11231310","title":"Identification of cDNA encoding a serine protease homologous to human complement C1r precursor from grafted mouse skin.","date":"2001","source":"The Journal of investigative dermatology","url":"https://pubmed.ncbi.nlm.nih.gov/11231310","citation_count":7,"is_preprint":false},{"pmid":"9008307","id":"PMC_9008307","title":"Inefficient assembly limits transport and cell surface expression of HLA-Cw4 molecules in C1R.","date":"1996","source":"Tissue antigens","url":"https://pubmed.ncbi.nlm.nih.gov/9008307","citation_count":7,"is_preprint":false},{"pmid":"2338498","id":"PMC_2338498","title":"Trimer and tetramer complexes containing C1 esterase inhibitor, C1r and C1s, in serum and synovial fluid of patients with rheumatic disease.","date":"1990","source":"Journal of immunological methods","url":"https://pubmed.ncbi.nlm.nih.gov/2338498","citation_count":7,"is_preprint":false},{"pmid":"3006820","id":"PMC_3006820","title":"C1r serine proteinase of human complement: a case of intramolecular autolytic activation.","date":"1985","source":"Bioscience reports","url":"https://pubmed.ncbi.nlm.nih.gov/3006820","citation_count":7,"is_preprint":false},{"pmid":"2572213","id":"PMC_2572213","title":"Human complement C1r and C1s proteins and genes: studies with molecular probes.","date":"1989","source":"Behring Institute 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human C1r complement molecule.","date":"1982","source":"Molecular immunology","url":"https://pubmed.ncbi.nlm.nih.gov/6285181","citation_count":4,"is_preprint":false},{"pmid":"2850274","id":"PMC_2850274","title":"Genetic polymorphism of human C1R subcomponent of the first complement component in the Japanese population.","date":"1988","source":"Forensic science international","url":"https://pubmed.ncbi.nlm.nih.gov/2850274","citation_count":4,"is_preprint":false},{"pmid":"32082741","id":"PMC_32082741","title":"Diagnostic value of signal peptide-Complement C1r/C1s, Uegf, and Bmp1-epidermal growth factor domain-containing protein 1 on serum and tissue samples in non-small cell lung cancer.","date":"2018","source":"Turk gogus kalp damar cerrahisi dergisi","url":"https://pubmed.ncbi.nlm.nih.gov/32082741","citation_count":4,"is_preprint":false},{"pmid":"2987200","id":"PMC_2987200","title":"Autocatalytic activation of C1r subcomponent of the first component of human complement.","date":"1985","source":"Journal of biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/2987200","citation_count":3,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":48696,"output_tokens":7679,"usd":0.130636},"stage2":{"model":"claude-opus-4-6","input_tokens":11450,"output_tokens":3314,"usd":0.21015},"total_usd":0.340786,"stage1_batch_id":"msgbatch_01TqNM6p6FsoMBT2jzQTwKBr","stage2_batch_id":"msgbatch_01Ya473eco8sTHbvRymQaU8o","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1976,\n      \"finding\": \"C1r functions as a serine protease zymogen that undergoes proteolytic cleavage to become activated; activation converts each 95,000 Da chain into disulfide-linked subunits of 60,000 and 35,000 Da and enables C1r to cleave and activate C1s. C1r also serves as the physical link between C1q and C1s in macromolecular C1, requiring C1q and calcium for binding to sensitized erythrocytes.\",\n      \"method\": \"Biochemical fractionation, SDS-PAGE, radiolabeled binding assays, functional reconstitution of C1\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — multiple orthogonal biochemical methods, replicated across two papers (PMID 814163 and PMID 1249422)\",\n      \"pmids\": [\"814163\", \"1249422\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1976,\n      \"finding\": \"Activation of C1r by proteolytic cleavage is inhibited by calcium ions (which stabilize the proenzyme form), C1 inactivator (which forms a complex with the light chain of activated C1r), polyanethol sulfonate, and DFP. Activation is triggered when C1r is incorporated into the C1 complex with C1q and aggregated IgG.\",\n      \"method\": \"Biochemical activation assays, SDS-PAGE analysis of cleavage products, inhibitor titration experiments\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — detailed biochemical dissection with multiple inhibitors and conditions, replicated\",\n      \"pmids\": [\"1249422\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1977,\n      \"finding\": \"Activated C1r (C1-r) is a serine protease with a catalytic B chain (~27,000 Da) homologous to the trypsin family of serine proteases; it hydrolyses lysine and tyrosine ester bonds but cleaves only C1s as a protein substrate, demonstrating highly restricted proteolytic specificity.\",\n      \"method\": \"Amino acid composition analysis, N-terminal sequencing, synthetic ester substrate assays\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — direct enzymatic characterization with multiple substrate assays, foundational paper\",\n      \"pmids\": [\"869924\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1983,\n      \"finding\": \"The catalytic B chain of activated C1r contains 242 amino acids with a serine protease active site triad (His-39, Asp-94, Ser-191 by chymotrypsinogen numbering); it lacks the histidine-loop disulfide bridge found in other serine proteases and has five half-cystine residues.\",\n      \"method\": \"Direct amino acid sequence determination by chemical fragmentation and sequencing\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — complete amino acid sequence with active-site residue identification\",\n      \"pmids\": [\"6303394\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1985,\n      \"finding\": \"C1r undergoes autocatalytic (autolytic) activation involving two intermolecular reactions: a DFP-insensitive zymogen-catalyzed step and a DFP-sensitive enzyme-catalyzed step, consistent with activation proceeding through a conformational isomer intermediate before full proteolytic cleavage.\",\n      \"method\": \"Kinetic analysis of activation time-course, DFP inhibition experiments, surface radiolabeling\",\n      \"journal\": \"Journal of biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — kinetic and biochemical dissection in a single lab; supported by later structural work\",\n      \"pmids\": [\"2987200\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1986,\n      \"finding\": \"The C1r polypeptide precursor of 705 amino acids contains an N-terminal non-catalytic A chain with an EGF-like domain, two pairs of internal repeats (one pair related to C1s N-terminal sequence; the other related to complement factor B and beta2-glycoprotein I), and a C-terminal B chain encoding the serine protease domain homologous to the trypsin family.\",\n      \"method\": \"cDNA sequencing of full-length C1r transcript from human liver and HepG2 libraries\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — complete cDNA sequence defining domain architecture, confirmed by two independent groups (PMID 3021205 and PMID 3030286)\",\n      \"pmids\": [\"3021205\", \"3030286\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1987,\n      \"finding\": \"The A chain of activated C1r (446 residues) contains a beta-hydroxyaspartic acid residue at position 150 within an EGF consensus sequence, two carbohydrate chains attached at Asn-108 and Asn-204, and is subdivided by autolytic cleavage into three fragments (alpha, beta, gamma); fragment gamma is disulfide-linked to the catalytic B chain.\",\n      \"method\": \"Complete amino acid sequence determination from autolytic fragments and chemical cleavage peptides\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — complete protein sequence determination with PTM identification\",\n      \"pmids\": [\"3036070\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1988,\n      \"finding\": \"The human C1R and C1S genes are located on chromosome 12p13 in a closely linked tail-to-tail arrangement separated by approximately 9.5 kb, consistent with gene duplication from a common ancestor; both genes are primarily expressed in liver.\",\n      \"method\": \"cDNA sequencing, genomic DNA blotting, in situ hybridization, RNA blot analysis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple genomic and expression methods confirming chromosomal organization\",\n      \"pmids\": [\"2459702\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"Ca2+ ions suppress C1r autoactivation by an intramolecular mechanism involving the Ca2+-binding alpha-region; this inhibitory effect is released in the C1 complex by a signal originating in C1q and transmitted through the C1q/C1r interface. Incorporation of C1r into the C1s-C1r-C1r-C1s tetramer fully stabilizes C1r in proenzyme form, and interaction with intact C1q (but not collagen-like C1q fragments) is required to trigger activation above 25°C.\",\n      \"method\": \"Spontaneous activation assays, sedimentation analysis, Ca2+ titration, reconstitution of C1 with truncated C1q fragments\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — reconstitution experiments with defined fragments and quantitative activation kinetics\",\n      \"pmids\": [\"8042996\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"The catalytic domain of C1r (CCP1-CCP2-SP) forms a non-covalent homodimer in a head-to-tail arrangement; chemical cross-linking and homology modeling identified an intramonomer cross-link between Lys426 (CCP module V) and the C-terminal Asp688 (SP domain) and an intermonomer cross-link between Gly280 and Glu493, supporting a model where CCP module V interacts with the SP domain on the side opposite to the active site.\",\n      \"method\": \"Chemical cross-linking with EDC, CNBr cleavage, N-terminal sequencing, mass spectrometry, homology modeling\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — cross-linking combined with MS and structural modeling\",\n      \"pmids\": [\"9174342\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"The EGF-like module of C1r (residues 123–175) has a well-ordered C-terminal part with canonical EGF fold and a disordered large N-terminal loop between the first two cysteines; Ca2+ at 80 mM did not significantly alter the EGF module structure, indicating Ca2+ binding is mediated by a different mechanism in the full-length protein.\",\n      \"method\": \"NMR structure determination, restrained molecular dynamics calculations\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — NMR structure with functional implication from Ca2+ experiments\",\n      \"pmids\": [\"9477945\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"In human serum, C1r and C1s are found exclusively in complex with C1q and not with MBL, while MASP-1, MASP-2, and MAp19 are found exclusively with MBL; C1r-deficient serum shows no complement activation on IgG-coated surfaces, confirming that C1r is essential for classical pathway activation via antibody complexes.\",\n      \"method\": \"Gel-permeation chromatography of serum fractions, ELISA, complement activation assays with deficient sera\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal biochemical separation and functional deficiency assay confirming pathway specificity\",\n      \"pmids\": [\"10878362\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Autoactivation and C1s cleavage are intrinsic properties of the C1r SP domain alone; the CCP2 module significantly increases proteolytic activity toward C1s and stabilizes the SP domain structure; CCP1 is required for dimer formation but is not required for autoactivation or C1s cleavage. Dimerization of C1r is not a prerequisite for autoactivation.\",\n      \"method\": \"Recombinant expression of domain-deletion fragments in E. coli, renaturation, esterolytic and C1s-cleavage assays, differential scanning calorimetry, sedimentation analysis\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — domain deletion mutagenesis combined with functional and biophysical assays\",\n      \"pmids\": [\"11673533\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Both CCP1/2-SP and CCP2-SP fragments of C1r activate autolytically (confirmed by recovery as two-chain proteases while R446Q and S637A mutants remain single-chain zymogens), proving C1r activation is an autolytic process. CCP1 drives dimerization (5.5 S) but is not required for self-activation; relative C1s-cleavage efficiency is C1r < CCP1/2-SP < CCP2-SP, showing CCP1 is not involved in C1s recognition.\",\n      \"method\": \"Baculovirus expression, site-directed mutagenesis (R446Q, S637A), sedimentation velocity, C1s cleavage assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstitution with active-site and cleavage-site mutagenesis; first direct proof of autolytic mechanism\",\n      \"pmids\": [\"11445589\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Crystal structure of the zymogen and active catalytic domain (CCP1-CCP2-SP) of C1r shows a homodimer with head-to-tail arrangement where the active site of one monomer and the cleavage site (Arg446) of its partner are at opposite ends, demonstrating that a mechanical stress disrupting the CCP1-SP interface is required to trigger C1r activation in the C1 complex.\",\n      \"method\": \"X-ray crystallography to 2.9 Å (zymogen, S647A mutant) and crystal structures of active CCP2-SP domain\",\n      \"journal\": \"The EMBO journal / Structure\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structures with functional validation of activation mechanism\",\n      \"pmids\": [\"11823416\", \"12429092\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"C1r-like protein (C1r-LP), a complement-related serine protease, mediates proteolytic cleavage of prohaptoglobin in the endoplasmic reticulum; mutation of the active-site Ser abolished cleavage; substituting Gly for Arg-161 in proHp blocked cleavage; RNAi knockdown of C1r-LP reduced proHp cleavage by up to 45% in HepG2 cells. (Note: This is C1r-LP, a paralog/related protein, not C1r itself, but the abstract identifies it as a distinct gene.)\",\n      \"method\": \"Coexpression in COS-1 cells, active-site mutagenesis, substrate mutagenesis, RNAi knockdown, in vitro cleavage assay with purified proteins\",\n      \"journal\": \"Proceedings of the National Academy of Sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 — but concerns C1r-LP (a distinct paralog), not C1r itself; included as mechanistic finding about a C1r-related enzyme\",\n      \"pmids\": [\"15385675\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Crystal structure of the entire active catalytic region (CCP1-CCP2-SP) of human C1r shows head-to-tail dimer similar to the zymogen; an enzyme-product complex in the crystal lattice reveals a critical S1-P1 salt bridge between Asp631 and Arg446, and intermolecular interaction between CCP2 and SP domain, supporting a split-and-reassembly model for C1r autoactivation without large-scale C1q arm movements.\",\n      \"method\": \"X-ray crystallography of full active catalytic region\",\n      \"journal\": \"Molecular immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure of activated form providing atomic-level mechanism\",\n      \"pmids\": [\"17996945\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"The C1q-binding sites of the C1s-C1r-C1r-C1s tetramer reside primarily in the CUB1 and CUB2 modules of C1r (high affinity) and CUB1 of C1s (lower affinity); these sites involve acidic residues that also serve as Ca2+ ligands; the EGF module of C1r (Glu137-Glu-Asp139) does not participate in C1q binding, contrary to earlier models. Altogether six binding sites interact with reactive lysines of C1q stems.\",\n      \"method\": \"Site-directed mutagenesis of C1r and C1s, surface plasmon resonance binding assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — systematic mutagenesis combined with SPR quantification\",\n      \"pmids\": [\"19473974\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Ca2+ binding to the CUB2 domain of C1r induces folding from a disordered to a compact structure (Kd ~430 μM); partial Ca2+ saturation of CUB2 in blood provides conformational flexibility required for C1 activation; Ca2+-free CUB2 shows intrinsically disordered character, and Ca2+ significantly suppresses autoactivation of native C1r zymogen.\",\n      \"method\": \"Recombinant domain production, isothermal titration calorimetry, NMR, activation assays with native C1r\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — ITC, NMR, and functional assays in combination\",\n      \"pmids\": [\"20178990\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Mass spectrometry-based surface accessibility mapping of the C1r/C1s tetramer in isolation versus in assembled C1 showed that the C1s CUB1-EGF-CUB2 interaction domains, distant in the free tetramer, associate with each other in the C1 complex; the C1s serine protease domain is partly positioned inside the C1q cone in the proenzyme form.\",\n      \"method\": \"Chemical modification of Lys residues, quantitative MS-based accessibility comparison\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — label-free semi-quantitative MS with chemical cross-linking\",\n      \"pmids\": [\"20592021\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"C1r demonstrates clear active-site specificity for Gln at P2 and Ile at P1' positions; these residues are present in the physiological C1s activation site, and their removal reduces C1r-mediated C1s activation; introduction of Gln at P2 of the non-substrate MASP-3 enables its efficient cleavage by C1r. The CCP (complement control protein) domains of C1r provide exosite interactions that contribute to efficient C1s cleavage.\",\n      \"method\": \"Phage display library substrate specificity assay, mutagenesis of C1s activation site, MD simulations, structural modeling\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — phage library combined with mutagenesis and structural modeling\",\n      \"pmids\": [\"23589288\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Recombinant C1q variants with mutations at LysB61 and LysC58 in the collagen-like stems show markedly reduced interaction with the C1s-C1r-C1r-C1s tetramer by surface plasmon resonance, demonstrating that LysB61 and LysC58 form key salt bridges with acidic Ca2+ ligands of C1r and C1s CUB domains during C1 assembly.\",\n      \"method\": \"Recombinant C1q expression in HEK293-F cells, site-directed mutagenesis, SPR binding assays, C1r/C1s activation assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — mutagenesis plus quantitative SPR in reconstituted system\",\n      \"pmids\": [\"23650384\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Heterozygous missense or in-frame insertion/deletion mutations in C1R (affecting subunit interfaces or inter-domain hinges) cause periodontal Ehlers-Danlos syndrome; pathogenic C1r variants are associated with intracellular retention and mild ER enlargement, indicating that proper C1r folding and secretion are required for connective tissue homeostasis.\",\n      \"method\": \"Human genetics (whole-exome sequencing of 19 families), immunofluorescence for ER morphology\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic discovery in large cohort with cellular phenotyping\",\n      \"pmids\": [\"27745832\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Crystal structure of the CUB1-EGF-CUB2 heterodimer of C1r and C1s reveals an antiparallel L-shaped heterodimer with Ca2+ at the interface from each subunit; contacts involving all three domains make heterodimer formation more favorable than homodimer formation, explaining preferential heterotetramer assembly. Structural data support a model where activation is driven by separation of C1r-C1s dimer pairs when C1q binds to a surface.\",\n      \"method\": \"X-ray crystallography of C1r-C1s CUB1-EGF-CUB2 complex\",\n      \"journal\": \"Proceedings of the National Academy of Sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure with comprehensive structural and biophysical analysis\",\n      \"pmids\": [\"29311313\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Knockdown of C1r in cutaneous squamous cell carcinoma (cSCC) cells inhibited ERK1/2 and Akt signaling, promoted apoptosis, and suppressed xenograft tumor growth and vascularization in vivo, demonstrating a tumor-cell-autonomous role for C1r in promoting cSCC growth independent of the complement pathway.\",\n      \"method\": \"siRNA knockdown in cSCC cell lines, Western blot for signaling pathways, xenograft tumor model\",\n      \"journal\": \"The British journal of dermatology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — KD with defined signaling readout in cell and in vivo models; mechanism downstream of C1r not fully resolved\",\n      \"pmids\": [\"31049937\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"C1R mutations causing periodontal EDS result in constitutive intracellular activation of C1r and/or C1s; patient-derived fibroblasts secrete activated C1s and activate externally added C4 in the absence of microbial triggers, demonstrating that pEDS variants cause autonomous complement cascade activation rather than loss of C1 complex assembly.\",\n      \"method\": \"Overexpression in HEK293T cells, Western blot, size exclusion chromatography, SPR, ELISA of patient fibroblast supernatants, C4 activation assay\",\n      \"journal\": \"Frontiers in immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — multiple orthogonal methods including functional complement activation assay in patient cells\",\n      \"pmids\": [\"31749804\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"C1r deletion in renal tubular epithelial cells (C1r-null mice) reduced C1s expression, C3 fragment formation, and kidney fibrosis after folic acid injury; in vitro experiments showed C1s expression is dependent on C1r expression; interferon-γ induces co-expression of both proteases.\",\n      \"method\": \"Global C1r knockout mouse, folic acid nephropathy model, immunohistochemistry, in situ hybridization, in vitro renal epithelial cell assays\",\n      \"journal\": \"American journal of physiology. Renal physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO with defined in vivo fibrosis phenotype; mechanism largely through complement C3 activation\",\n      \"pmids\": [\"31509012\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"CRISPR/Cas9 knockout of C1r in cSCC cells decreased proliferation, migration, and invasion through collagen type I, suppressed MMP-1, MMP-10, MMP-12, and MMP-13 production, and reduced collagen degradation and xenograft tumor invasion in vivo, placing C1r upstream of MMP-13-driven matrix remodeling in cSCC.\",\n      \"method\": \"CRISPR/Cas9 knockout, mRNA-seq, Western blot for MMPs, invasion assay through collagen I, xenograft model\",\n      \"journal\": \"The Journal of investigative dermatology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — CRISPR KO with transcriptomic and functional validation in vitro and in vivo\",\n      \"pmids\": [\"34756877\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Crystal structure of the Borrelia burgdorferi BBK32 C-terminal domain in complex with activated human C1r reveals that BBK32 inhibits C1r by occluding the S1 and S1' substrate-binding subsites; B loop-interacting residues of BBK32 also contribute to full inhibitory activity, as validated by site-directed mutagenesis combined with SPR and complement function assays.\",\n      \"method\": \"X-ray crystallography, SAXS, site-directed mutagenesis, SPR, complement activation assays\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — structure plus mutagenesis validation of binding interface\",\n      \"pmids\": [\"34759015\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"The C1r2s2 protease heterotetramer stabilizes C1q binding to surface-bound IgG; the contribution of C1r2s2 to C1q-IgG stability differs between IgG subclasses, and hexamer-enhancing IgG mutations improve C1q complex stability both with and without C1r2s2, mechanistically linking the proteases to avid C1 complex assembly on IgG-opsonized surfaces.\",\n      \"method\": \"SPR binding assays, antibody engineering, complement-dependent phagocytosis assays with human neutrophils\",\n      \"journal\": \"Proceedings of the National Academy of Sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — SPR with engineered antibody variants and functional neutrophil assay\",\n      \"pmids\": [\"34155115\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"In hepatocellular carcinoma, C1r acts as a tumor suppressor; C-reactive protein (CRP) binds to C1r, and when C1r is downregulated (via DNMT1/DNMT3a-mediated promoter methylation), free CRP activates NF-κB signaling, upregulating HIF-1α and increasing glycolysis to promote aggressive HCC behavior.\",\n      \"method\": \"CRISPR/siRNA knockdown, RNA-seq, methylation-specific PCR, co-immunoprecipitation (CRP-C1r interaction), dual-luciferase reporter assay, xenograft model\",\n      \"journal\": \"Molecular carcinogenesis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — co-IP identifying CRP as C1r binding partner, combined with reporter and in vivo data; single lab\",\n      \"pmids\": [\"39150096\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"One active C1r subunit within the C1r homodimer is sufficient for full hemolytic activity of the C1 complex; stabilization of one C1r monomer as a zymogen (R446Q mutant) does not abolish activity when paired with wild-type C1r in a mixed dimer, and C1r monomers exchange between dimers even at 4°C.\",\n      \"method\": \"Baculovirus expression of zymogen-stabilizing C1r mutants, reconstitution hemolytic assay, mixed-dimer experiments\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — active-site and cleavage-site mutagenesis with functional reconstitution\",\n      \"pmids\": [\"9916740\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"C1r is a modular serine protease zymogen that assembles with C1s into a Ca2+-dependent C1s-C1r-C1r-C1s heterotetramer via CUB-EGF-CUB domain interactions, associates with the recognition protein C1q through high-affinity CUB1/CUB2-mediated contacts with reactive lysines on C1q collagen stems, undergoes autolytic self-activation (driven by mechanical stress from C1q target binding that disrupts a Ca2+-regulated CCP1-SP interface) via cleavage of the Arg446-Ile447 bond to generate the active two-chain enzyme, which then cleaves and activates C1s at a Gln(P2)-Arg-Ile(P1') site (also using CCP domain exosites), thereby initiating the classical complement cascade; beyond complement, tumour-cell-derived C1r promotes cSCC invasion through upregulation of matrix metalloproteinases (MMP-1, -10, -12, -13) and activates ERK1/2/Akt signalling, while in HCC it acts as a tumour suppressor by sequestering CRP and preventing NF-κB/HIF-1α-driven glycolysis.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"C1r is a modular serine protease zymogen that initiates the classical complement pathway by undergoing Ca2+-regulated autoactivation and subsequently cleaving and activating its sole physiological substrate, C1s, within the C1 macromolecular complex. The proenzyme assembles with C1s into a Ca2+-dependent C1s–C1r–C1r–C1s heterotetramer via CUB1-EGF-CUB2 domain interactions, which binds C1q collagen stems through high-affinity salt bridges between acidic Ca2+-coordinating residues in the C1r CUB domains and reactive lysines (LysB61, LysC58) on C1q [PMID:19473974, PMID:23650384, PMID:29311313]. Upon C1q recognition of immune complexes, mechanical disruption of the CCP1–SP interface in the head-to-tail C1r homodimer triggers autolytic cleavage at Arg446–Ile447, generating the active two-chain enzyme whose SP domain exhibits stringent specificity for Gln(P2)–Arg–Ile(P1') in C1s, augmented by CCP2-mediated exosite contacts [PMID:11823416, PMID:11445589, PMID:23589288]. Heterozygous missense mutations in C1R cause periodontal Ehlers–Danlos syndrome through constitutive intracellular activation of C1r/C1s and autonomous complement activation rather than loss of complex assembly [PMID:27745832, PMID:31749804].\",\n  \"teleology\": [\n    {\n      \"year\": 1976,\n      \"claim\": \"Establishing that C1r is a serine protease zymogen activated by proteolytic cleavage into disulfide-linked subunits answered the fundamental question of how the classical pathway is enzymatically initiated, and revealed that C1r bridges C1q to C1s in a Ca2+-dependent manner.\",\n      \"evidence\": \"Biochemical fractionation, SDS-PAGE, radiolabeled binding assays, functional C1 reconstitution\",\n      \"pmids\": [\"814163\", \"1249422\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cleavage site identity unknown\", \"Whether activation is autolytic or requires an exogenous protease was unresolved\", \"Domain architecture undefined\"]\n    },\n    {\n      \"year\": 1977,\n      \"claim\": \"Demonstrating that activated C1r cleaves only C1s among tested protein substrates established C1r as a highly specific protease rather than a general trypsin-like enzyme, defining its singular role as the activator of C1s.\",\n      \"evidence\": \"Amino acid composition, N-terminal sequencing, synthetic ester substrate assays\",\n      \"pmids\": [\"869924\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis for substrate restriction unknown\", \"Active-site sequence not yet determined\"]\n    },\n    {\n      \"year\": 1986,\n      \"claim\": \"Determination of the complete cDNA sequence and domain architecture (CUB1-EGF-CUB2-CCP1-CCP2-SP) resolved how C1r combines recognition/interaction domains with a catalytic module and explained homology with C1s, suggesting gene duplication origin.\",\n      \"evidence\": \"Full-length cDNA sequencing from liver/HepG2 libraries, confirmed by two independent groups\",\n      \"pmids\": [\"3021205\", \"3030286\", \"2459702\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functions of individual domains not experimentally tested\", \"C1q binding site not mapped\"]\n    },\n    {\n      \"year\": 1994,\n      \"claim\": \"Reconstitution experiments with truncated C1q fragments showed that Ca2+ suppresses C1r autoactivation intramolecularly, that the C1s–C1r–C1r–C1s tetramer stabilizes the proenzyme, and that intact C1q globular heads plus collagen stems are required to transmit the activation signal — resolving the question of how C1r activation is held in check until target recognition.\",\n      \"evidence\": \"Spontaneous activation assays, sedimentation, Ca2+ titration, reconstitution with C1q fragments\",\n      \"pmids\": [\"8042996\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Nature of the C1q-transmitted mechanical signal unknown\", \"Ca2+-binding domain not identified\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Domain-deletion and active-site mutagenesis (R446Q, S637A) proved that C1r activation is autolytic rather than requiring an external protease, that autoactivation and C1s cleavage are intrinsic to the SP domain, and that CCP2 enhances C1s cleavage while CCP1 drives dimerization but is dispensable for catalysis.\",\n      \"evidence\": \"Recombinant domain fragments, site-directed mutagenesis, sedimentation, cleavage assays in E. coli and baculovirus systems\",\n      \"pmids\": [\"11445589\", \"11673533\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis for the CCP1–SP interface restraining activation unknown\", \"How dimerization relates to activation in the C1 complex unresolved\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Crystal structures of zymogen and active C1r catalytic region revealed a head-to-tail homodimer where the active site and cleavage site of partner monomers are maximally separated, establishing that mechanical stress disrupting the CCP1–SP interface is the trigger for autoactivation within the C1 complex.\",\n      \"evidence\": \"X-ray crystallography at 2.9 Å (zymogen S647A mutant) and active CCP2-SP structures\",\n      \"pmids\": [\"11823416\", \"12429092\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Source and magnitude of mechanical stress in physiological C1 not measured\", \"Full C1 complex structure unavailable\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Mapping C1q-binding sites to acidic Ca2+-ligand residues in C1r CUB1 and CUB2 (high affinity) and C1s CUB1 (lower affinity), and identifying reactive lysines LysB61/LysC58 on C1q stems as their partners, resolved the molecular interface holding the C1 complex together.\",\n      \"evidence\": \"Site-directed mutagenesis, SPR, ITC, NMR of CUB2, recombinant C1q variants\",\n      \"pmids\": [\"19473974\", \"20178990\", \"23650384\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry of engagement with all six C1q stems not fully resolved\", \"Role of partial Ca2+ saturation of CUB2 in vivo unclear\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Phage-display substrate profiling and mutagenesis defined the stringent P2-Gln/P1'-Ile specificity of C1r's active site and demonstrated that CCP domains contribute exosite interactions for efficient C1s cleavage, explaining why C1r is restricted to C1s as its sole natural substrate.\",\n      \"evidence\": \"Phage display library, C1s activation-site mutagenesis, MD simulation\",\n      \"pmids\": [\"23589288\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic structure of the C1r–C1s enzyme–substrate complex not determined\", \"Whether CCP exosite contacts are essential or merely rate-enhancing unclear\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Discovery that heterozygous C1R mutations cause periodontal Ehlers–Danlos syndrome linked C1r to connective-tissue homeostasis, and subsequent work showed that pEDS variants cause constitutive intracellular C1r/C1s activation and autonomous complement activation rather than loss of C1 complex assembly.\",\n      \"evidence\": \"Whole-exome sequencing of 19 families, immunofluorescence, patient fibroblast C4-activation assays, overexpression in HEK293T\",\n      \"pmids\": [\"27745832\", \"31749804\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How complement activation in connective tissue leads to periodontitis and skin fragility unknown\", \"Whether intracellular activation damages the ER or acts extracellularly unresolved\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"The crystal structure of the C1r–C1s CUB1-EGF-CUB2 heterodimer revealed an antiparallel L-shaped architecture with Ca2+ at each interface, explaining preferential heterotetramer over homodimer assembly and supporting a model where surface binding separates C1r–C1s pairs to trigger activation.\",\n      \"evidence\": \"X-ray crystallography of the C1r–C1s interaction domain complex\",\n      \"pmids\": [\"29311313\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full-length C1 complex structure still unavailable\", \"Dynamics of pair separation upon C1q bending not captured\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Beyond complement, CRISPR knockout of C1r in cutaneous SCC cells established a complement-independent tumor-promoting function: C1r drives MMP-1/10/12/13 expression, collagen degradation, ERK1/2–Akt signaling, and xenograft invasion; conversely, in HCC, C1r sequesters CRP to suppress NF-κB/HIF-1α–driven glycolysis, acting as a tumor suppressor.\",\n      \"evidence\": \"CRISPR KO and siRNA in cSCC lines, mRNA-seq, Western blot, xenograft models; co-IP of CRP–C1r, reporter assays, methylation-specific PCR in HCC\",\n      \"pmids\": [\"34756877\", \"31049937\", \"39150096\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether the oncogenic function requires C1r catalytic activity or is scaffold-mediated unknown\", \"CRP–C1r interaction awaits structural characterization\", \"Single-lab findings for both cancer contexts\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"The crystal structure of Borrelia BBK32 bound to activated C1r revealed that pathogen-derived inhibitors block C1r by occluding the S1/S1' substrate-binding subsites, providing atomic-level insight into immune evasion and a template for therapeutic C1r inhibitor design.\",\n      \"evidence\": \"X-ray crystallography, SAXS, site-directed mutagenesis, SPR, complement assays\",\n      \"pmids\": [\"34759015\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether other microbial C1r inhibitors use the same mechanism unknown\", \"No small-molecule inhibitor of C1r yet designed from this template\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"A complete atomic structure of the full C1 complex (C1q bound to the C1s–C1r–C1r–C1s tetramer) is still lacking, and the precise mechanical forces and conformational changes that relay C1q target recognition to C1r autoactivation remain unresolved; additionally, the complement-independent roles of C1r in connective tissue homeostasis and cancer require mechanistic dissection of whether they depend on its catalytic activity or scaffold functions.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Full C1 complex structure not determined\", \"Force transduction mechanism from C1q to C1r activation site not measured\", \"Catalytic vs. non-catalytic roles in pEDS pathogenesis and cancer not distinguished\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 2, 12, 13, 20]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [2, 3, 4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005576\", \"supporting_discovery_ids\": [0, 11, 29]},\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [22, 25]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [0, 1, 8, 11, 20, 28]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [22, 25, 27, 30]}\n    ],\n    \"complexes\": [\n      \"C1 complex (C1q:C1r2:C1s2)\",\n      \"C1s-C1r-C1r-C1s tetramer\"\n    ],\n    \"partners\": [\n      \"C1S\",\n      \"C1QA\",\n      \"C1QB\",\n      \"C1QC\",\n      \"CRP\",\n      \"SERPING1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}