title: EF-hand domains of MCFD2 mediate interactions with both LMAN1 and coagulation factor V or VIII.
abstract: Combined deficiency of factor V and factor VIII (F5F8D) is a bleeding disorder caused by mutations in either LMAN1 or MCFD2. LMAN1 (ERGIC-53) and MCFD2 form a Ca(2+)-dependent cargo receptor that cycles between the endoplasmic reticulum (ER) and the ER-Golgi intermediate compartment for efficient transport of FV/FVIII from the ER to the Golgi. Here we show that the C-terminal EF-hand domains are both necessary and sufficient for MCFD2 to interact with LMAN1. MCFD2 with a deletion of the entire N-terminal non-EF hand region still retains the LMAN1-binding function. Deletions that disrupt core structure of the EF-hand domains abolish LMAN1 binding. Circular dichroism spectroscopy studies on missense mutations localized to different structural elements of the EF-hand domains suggest that Ca(2+)-induced folding is important for LMAN1 interaction. The EF-hand domains also mediate the interaction with FV and FVIII. However, mutations in MCFD2 that disrupt the tertiary structure and abolish LMAN1 binding still retain the FV/FVIII binding activities, suggesting that this interaction is independent of Ca(2+)-induced folding of the protein. Our results suggest that the EF-hand domains of MCFD2 contain separate binding sites for LMAN1 and FV/FVIII that are essential for cargo receptor formation and cargo loading in the ER.
authors.affiliation: Genomic Medicine Institute, Lerner Research Institute, Cleveland Clinic Foundation, OH 44195, USA.
authors.forename: Chunlei
authors.initials: C
authors.lastname: Zheng
authors.forename: Hui-hui
authors.initials: HH
authors.lastname: Liu
authors.forename: Jiahai
authors.initials: J
authors.lastname: Zhou
authors.forename: Bin
authors.initials: B
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000378:metabolism; D002522:Chlorocebus aethiops; D002942:Circular Dichroism; D020832:EF Hand Motifs; D005165:Factor V / Q000235:genetics / Q000378:metabolism*; D005169:Factor VIII / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D037241:Mannose-Binding Lectins / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017384:Sequence Deletion; D014162:Transfection; D033921:Vesicular Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: C084004:LMAN1 protein, human; C475146:MCFD2 protein, human; D037241:Mannose-Binding Lectins; D008565:Membrane Proteins; D033921:Vesicular Transport Proteins; D005165:Factor V; D005169:Factor VIII; D002118:Calcium