Affinage

MCFD2

Multiple coagulation factor deficiency protein 2 · UniProt Q8NI22

Length
146 aa
Mass
16.4 kDa
Annotated
2026-04-28
21 papers in source corpus 12 papers cited in narrative 11 extracted findings

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

MCFD2 is a soluble, calcium-dependent EF-hand protein that functions as the direct cargo-binding subunit of the LMAN1(ERGIC-53)–MCFD2 cargo receptor complex, mediating ER-to-Golgi transport of coagulation factors V and VIII and α1-antitrypsin. MCFD2 is intrinsically disordered in the absence of calcium and folds upon Ca²⁺ binding to its two C-terminal EF-hand motifs, which contain separable binding sites for LMAN1 and for FV/FVIII cargo (PMID:18590741, PMID:20007547). LMAN1 retains MCFD2 in the early secretory pathway and serves primarily as a membrane shuttle, while MCFD2 is the cargo-selective subunit: it is dispensable for cathepsin recruitment but essential for efficient FV/FVIII secretion, and overexpression of MCFD2 alone can rescue FV/FVIII export even in LMAN1-deficient cells (PMID:17010120, PMID:36490287). Knockout mice lacking MCFD2 exhibit reduced plasma FV, FVIII, and α1-antitrypsin levels, with hepatocyte ER accumulation of α1-antitrypsin confirming a physiological role in secretory cargo export (PMID:29735583).

Mechanistic history

Synthesis pass · year-by-year structured walk · 9 steps
  1. 2005 High

    Establishing MCFD2 as a stoichiometric partner of LMAN1 and demonstrating its direct, calcium-dependent interaction with FVIII resolved how combined FV/FVIII deficiency arises from mutations in two separate genes.

    Evidence Reciprocal co-immunoprecipitation and cross-linking in cell lysates

    PMID:15886209

    Open questions at the time
    • Whether MCFD2 contributes to cargo selectivity beyond FV/FVIII was unknown
    • Structural basis of the MCFD2–LMAN1 interaction was unresolved
    • The calcium-dependence mechanism at the molecular level was not defined
  2. 2006 High

    Demonstrating that MCFD2 is secreted in the absence of LMAN1 and is dispensable for cathepsin cargo binding established that MCFD2 confers cargo selectivity for FV/FVIII rather than acting as a general co-receptor.

    Evidence siRNA knockdown of LMAN1/MCFD2 with immunofluorescence and cargo-binding assays

    PMID:17010120

    Open questions at the time
    • Which domains of MCFD2 mediate cargo versus LMAN1 binding was unknown
    • In vivo relevance of MCFD2 loss had not been tested in an animal model
  3. 2008 High

    Solving the NMR structure of MCFD2 revealed that it is intrinsically disordered without calcium and folds upon Ca²⁺ binding to its EF-hands, explaining why the MCFD2–LMAN1 interaction is calcium-dependent and why disease mutations cause structural disorder.

    Evidence NMR structure determination and circular dichroism of wild-type and disease mutants

    PMID:18590741

    Open questions at the time
    • The atomic details of the MCFD2–LMAN1 interface were still unknown
    • Whether the disordered N-terminal region has any functional role was unresolved
  4. 2009 High

    Mapping the EF-hand domains as both necessary and sufficient for LMAN1 and FV/FVIII binding — yet showing these are separable sites — established that MCFD2 simultaneously engages its receptor and its cargo through distinct surfaces.

    Evidence Deletion mutagenesis combined with co-immunoprecipitation and CD spectroscopy

    PMID:20007547

    Open questions at the time
    • The structural basis of the cargo-binding site on MCFD2 was not resolved
    • Whether conformational flexibility of MCFD2 contributes to cargo recognition was speculative
  5. 2010 High

    Crystal structures of the LMAN1-CRD/MCFD2 complex defined the binding interface on the N-terminal β-sheet of the LMAN1 CRD, distinct from its sugar-binding site, and showed that LMAN1 oligomerization is required for ER exit and cargo receptor function.

    Evidence X-ray crystallography combined with site-directed mutagenesis and co-immunoprecipitation

    PMID:20138881 PMID:20817851

    Open questions at the time
    • No structure of MCFD2 bound to FV or FVIII cargo was available
    • Mechanism by which oligomerization enables ER exit was not determined
  6. 2018 High

    MCFD2-knockout mice confirmed the in vivo requirement for FV/FVIII secretion and extended the cargo repertoire to α1-antitrypsin; epistasis with LMAN1 knockout revealed an alternative LMAN1-independent secretion pathway.

    Evidence Gene-targeted single and double knockout mice with plasma factor assays and hepatocyte ER fractionation

    PMID:29735583

    Open questions at the time
    • Identity of the alternative LMAN1-independent secretion pathway was unknown
    • Whether additional cargo proteins depend on MCFD2 in vivo was not surveyed
  7. 2020 Medium

    High-resolution crystallographic snapshots revealed conformational plasticity of MCFD2 within the complex, suggesting this flexibility enables accommodation of diverse cargo ligands.

    Evidence Multiple crystal forms of the ERGIC-53 CRD/MCFD2 complex at 1.60 Å resolution

    PMID:32356523

    Open questions at the time
    • No direct mutagenesis of the proposed flexible cargo-binding surface was performed
    • Whether conformational plasticity is functionally required for cargo binding was not tested
  8. 2022 High

    Demonstrating that LMAN1–MCFD2 is a cargo receptor for α1-antitrypsin via the second N-glycan of AAT, while AAT binds LMAN1 independently of MCFD2, defined a glycan-dependent transport mode distinct from the MCFD2-dependent FV/FVIII pathway.

    Evidence CRISPR knockout and rescue in HepG2 and HEK293T cells, glycosylation site mutagenesis, co-immunoprecipitation

    PMID:35322856

    Open questions at the time
    • Whether MCFD2 contributes to AAT binding cooperativity with LMAN1 or is merely a co-factor for ER exit was unclear
    • The full repertoire of LMAN1-dependent versus MCFD2-dependent cargo was not defined
  9. 2023 Medium

    Showing that MCFD2 overexpression alone rescues FV/FVIII secretion in LMAN1-null cells redefined MCFD2 as the primary cargo-binding and transport subunit, with LMAN1 serving as a membrane shuttle rather than a co-receptor.

    Evidence Rescue overexpression in LMAN1/MCFD2-deficient cell lines with FV/FVIII secretion assays

    PMID:36490287

    Open questions at the time
    • How MCFD2 exits the ER without LMAN1 when overexpressed was not mechanistically resolved
    • Single-lab finding; independent confirmation is needed
    • Whether endogenous-level MCFD2 can function without LMAN1 in vivo was not tested

Open questions

Synthesis pass · forward-looking unresolved questions
  • The structural basis of MCFD2's direct interaction with FV/FVIII cargo and the mechanism by which MCFD2 can mediate ER exit independently of LMAN1 remain unresolved.
  • No structure of MCFD2 in complex with FV or FVIII has been determined
  • The alternative LMAN1-independent export mechanism is uncharacterized
  • A comprehensive cargo proteome for MCFD2 has not been defined

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0038024 cargo receptor activity 4 GO:0060090 molecular adaptor activity 3
Localization
GO:0005783 endoplasmic reticulum 2 GO:0005576 extracellular region 1
Pathway
R-HSA-9609507 Protein localization 5 R-HSA-109582 Hemostasis 3 R-HSA-5653656 Vesicle-mediated transport 3
Partners
F5F8LMAN1SERPINA1
Complex memberships
LMAN1–MCFD2 cargo receptor complex

Evidence

Reading pass · 11 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2005 MCFD2 is retained in the endoplasmic reticulum through its interaction with LMAN1 (ERGIC-53), and endogenous LMAN1 and MCFD2 are present primarily in complex with each other at 1:1 stoichiometry. MCFD2 is not required for oligomerization of LMAN1. Both LMAN1 and MCFD2 interact specifically with coagulation factor VIII via its B domain through calcium-dependent protein-protein interactions, and the MCFD2-FVIII interaction is independent of LMAN1-MCFD2 complex formation. Co-immunoprecipitation, cross-linking-immunoprecipitation assay, Western blot The Journal of biological chemistry High 15886209
2006 In the absence of ERGIC-53 (LMAN1), MCFD2 is secreted rather than retained, confirming that LMAN1 is required for ER retention of MCFD2. Knockdown of MCFD2 has no effect on LMAN1 localization. MCFD2 is dispensable for the binding of cathepsin Z and cathepsin C to ERGIC-53, indicating MCFD2 specifically recruits coagulation factors V and VIII to the ERGIC-53 complex. siRNA knockdown, yellow fluorescent protein fragment complementation, immunofluorescence Traffic (Copenhagen, Denmark) High 17010120
2007 The interaction of MCFD2 with ERGIC-53 (LMAN1) is calcium-dependent; at calcium concentrations below 0.2 mM the interaction becomes significantly weaker. MCFD2 binding to ERGIC-53 enhances the sugar-binding ability of ERGIC-53. Two disease-causing MCFD2 missense mutations show 3–4 orders of magnitude lower binding affinity for ERGIC-53. Surface plasmon resonance, flow cytometry with fluorescent ERGIC-53 probe, glycan-competition assay Blood High 18056485
2007 Deletion of only 3 C-terminal residues (ΔS-L-Q) from MCFD2 impairs binding to ERGIC-53 due to modification of the 3D structure of MCFD2, establishing that the C-terminal integrity of MCFD2 is required for ERGIC-53 interaction. Biochemical binding assay, structural analysis of mutant Blood Medium 17971482
2008 The solution structure of human MCFD2 determined by NMR shows that MCFD2 is disordered in the apo (calcium-free) state and folds into a structured protein upon binding of Ca2+ to its two C-terminal EF-hand motifs, while retaining some N-terminal disorder. Disease-causing missense mutants are predominantly disordered even in the presence of calcium, explaining the calcium dependence of the MCFD2-ERGIC-53 interaction. NMR structure determination, circular dichroism spectroscopy of mutants Journal of molecular biology High 18590741
2009 The C-terminal EF-hand domains of MCFD2 are both necessary and sufficient for interaction with LMAN1; deletion of the entire N-terminal non-EF-hand region does not affect LMAN1 binding. The EF-hand domains also mediate interaction with FV and FVIII, but mutations that abolish LMAN1 binding (and disrupt tertiary structure) still retain FV/FVIII binding, indicating the EF-hand domains contain separate binding sites for LMAN1 and for FV/FVIII. Deletion mutagenesis, co-immunoprecipitation, circular dichroism spectroscopy Blood High 20007547
2010 Crystal structure of the LMAN1-CRD/MCFD2 complex reveals that LMAN1 interacts with MCFD2 through its N-terminal beta sheet of the carbohydrate recognition domain (CRD). The CRD contains distinct, separable binding sites for MCFD2 and for cargo proteins (FV/FVIII): mutations in the first beta sheet abolish MCFD2 binding without affecting mannose binding, while mutations in the Ca2+- and sugar-binding sites disrupt FV/FVIII interaction without affecting MCFD2 binding. Monomeric LMAN1 mutants cannot exit the ER and cannot bind MCFD2, indicating oligomerization is required for cargo receptor function. Crystal structure, site-directed mutagenesis, co-immunoprecipitation Blood / FEBS letters High 20138881 20817851
2018 MCFD2-deficient mice generated by gene targeting show reduced plasma FV and FVIII, with levels lower than in LMAN1-deficient mice. Doubly deficient (LMAN1/MCFD2 null) mice match the higher FV/FVIII levels of LMAN1-deficient mice, suggesting an alternative LMAN1-independent secretion pathway exists. LMAN1 and MCFD2 single deficiency also reduces plasma α1-antitrypsin (AAT) and causes AAT accumulation in hepatocyte ER, demonstrating a shared role in AAT ER exit. Gene targeting (knockout mice), plasma coagulation factor assays, hepatocyte ER fractionation Blood advances High 29735583
2020 Crystallographic snapshots of the ERGIC-53 CRD/MCFD2 complex at 1.60 Å resolution reveal that MCFD2 exhibits significant conformational plasticity whereas ERGIC-53-CRD does not, suggesting that MCFD2's structural flexibility underlies its ability to accommodate diverse polypeptide cargo ligands. X-ray crystallography (multiple crystal forms, 1.60 Å resolution) Acta crystallographica. Section F, Structural biology communications Medium 32356523
2022 LMAN1-MCFD2 is a cargo receptor for ER-to-Golgi transport of α1-antitrypsin (AAT). LMAN1 or MCFD2 knockout HepG2 and HEK293T cells show reduced AAT secretion and elevated intracellular AAT due to delayed ER-to-Golgi transport. Secretion defects are rescued by wild-type but not mutant LMAN1 or MCFD2. The interaction of LMAN1 with the second N-glycan of AAT is critical; loss of this glycosylation site abolishes LMAN1-dependent secretion. Co-IP in MCFD2 KO cells shows AAT interacts with LMAN1 independently of MCFD2. CRISPR/Cas9 knockout cells, secretion/chase assay, co-immunoprecipitation, glycosylation site mutagenesis The Biochemical journal High 35322856
2023 Overexpression of wild-type or mutant MCFD2 alone is sufficient to rescue FV/FVIII secretion defects in LMAN1-deficient cells, indicating that MCFD2 carries out cargo binding and transport and that LMAN1 primarily serves as a shuttling carrier for MCFD2. LMAN1 carbohydrate-binding mutations only partially reduce FV/FVIII transport, indicating N-glycan binding is not essential for FV/FVIII cargo transport. Overexpression of both LMAN1 and MCFD2 does not further increase FV/FVIII secretion, indicating the complex is not rate-limiting. LMAN1/MCFD2 deficient cell lines, rescue overexpression experiments, coagulation factor secretion assays Blood advances Medium 36490287

Source papers

Stage 0 corpus · 21 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2005 LMAN1 and MCFD2 form a cargo receptor complex and interact with coagulation factor VIII in the early secretory pathway. The Journal of biological chemistry 113 15886209
2005 Combined deficiency of factor V and factor VIII is due to mutations in either LMAN1 or MCFD2. Blood 86 16304051
2006 Cargo selectivity of the ERGIC-53/MCFD2 transport receptor complex. Traffic (Copenhagen, Denmark) 65 17010120
2010 Molecular basis of LMAN1 in coordinating LMAN1-MCFD2 cargo receptor formation and ER-to-Golgi transport of FV/FVIII. Blood 50 20817851
2007 The sugar-binding ability of ERGIC-53 is enhanced by its interaction with MCFD2. Blood 46 18056485
1997 The locus for combined factor V-factor VIII deficiency (F5F8D) maps to 18q21, between D18S849 and D18S1103. American journal of human genetics 36 9245995
2008 New insights into multiple coagulation factor deficiency from the solution structure of human MCFD2. Journal of molecular biology 30 18590741
2009 EF-hand domains of MCFD2 mediate interactions with both LMAN1 and coagulation factor V or VIII. Blood 23 20007547
2010 Crystal structure of the LMAN1-CRD/MCFD2 transport receptor complex provides insight into combined deficiency of factor V and factor VIII. FEBS letters 22 20138881
2007 Deletion of 3 residues from the C-terminus of MCFD2 affects binding to ERGIC-53 and causes combined factor V and factor VIII deficiency. Blood 20 17971482
2005 Mutations in the MCFD2 gene and a novel mutation in the LMAN1 gene in Indian families with combined deficiency of factor V and VIII. American journal of hematology 20 16044454
2018 Analysis of MCFD2- and LMAN1-deficient mice demonstrates distinct functions in vivo. Blood advances 19 29735583
2022 LMAN1-MCFD2 complex is a cargo receptor for the ER-Golgi transport of α1-antitrypsin. The Biochemical journal 16 35322856
2007 Mutations in the MCFD2 gene are predominant among patients with hereditary combined FV and FVIII deficiency (F5F8D) in India. Haemophilia : the official journal of the World Federation of Hemophilia 14 17610559
2011 Analysis of newly detected mutations in the MCFD2 gene giving rise to combined deficiency of coagulation factors V and VIII. Haemophilia : the official journal of the World Federation of Hemophilia 12 21492322
2023 Separate roles of LMAN1 and MCFD2 in ER-to-Golgi trafficking of FV and FVIII. Blood advances 10 36490287
2022 Effect of co-overexpression of the cargo receptor ERGIC-53/MCFD2 on antibody production and intracellular IgG secretion in recombinant Chinese hamster ovary cells. Journal of bioscience and bioengineering 8 35963666
2020 Crystallographic snapshots of the EF-hand protein MCFD2 complexed with the intracellular lectin ERGIC-53 involved in glycoprotein transport. Acta crystallographica. Section F, Structural biology communications 8 32356523
2008 The first case of combined coagulation factor V and coagulation factor VIII deficiency in Poland due to a novel p.Tyr135Asn missense mutation in the MCFD2 gene. Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis 8 18685427
2012 Unveiling the unfolding pathway of F5F8D disorder-associated D81H/V100D mutant of MCFD2 via multiple molecular dynamics simulations. Journal of biomolecular structure & dynamics 5 22208273
2024 RNAi targeting LMAN1-MCFD2 complex promotes anticoagulation in mice. Journal of thrombosis and thrombolysis 1 39222205