title: Structural characterization of carbohydrate binding by LMAN1 protein provides new insight into the endoplasmic reticulum export of factors V (FV) and VIII (FVIII).
abstract: LMAN1 (ERGIC-53) is a key mammalian cargo receptor responsible for the export of a subset of glycoproteins from the endoplasmic reticulum. Together with its soluble coreceptor MCFD2, LMAN1 transports coagulation factors V (FV) and VIII (FVIII). Mutations in LMAN1 or MCFD2 cause the genetic bleeding disorder combined deficiency of FV and FVIII (F5F8D). The LMAN1 carbohydrate recognition domain (CRD) binds to both glycoprotein cargo and MCFD2 in a Ca(2+)-dependent manner. To understand the biochemical basis and regulation of LMAN1 binding to glycoprotein cargo, we solved crystal structures of the LMAN1-CRD bound to Man-α-1,2-Man, the terminal carbohydrate moiety of high mannose glycans. Our structural data, combined with mutagenesis and in vitro binding assays, define the central mannose-binding site on LMAN1 and pinpoint histidine 178 and glycines 251/252 as critical residues for FV/FVIII binding. We also show that mannobiose binding is relatively independent of pH in the range relevant for endoplasmic reticulum-to-Golgi traffic, but is sensitive to lowered Ca(2+) concentrations. The distinct LMAN1/MCFD2 interaction is maintained at these lowered Ca(2+) concentrations. Our results suggest that compartmental changes in Ca(2+) concentration regulate glycoprotein cargo binding and release from the LMAN1·MCFD2 complex in the early secretory pathway.
authors.affiliation: Genomic Medicine Institute, Cleveland Clinic, Cleveland, Ohio 44195, USA.
authors.forename: Chunlei
authors.initials: C
authors.lastname: Zheng
authors.forename: Richard C
authors.initials: RC
authors.lastname: Page
authors.forename: Vaijayanti
authors.initials: V
authors.lastname: Das
authors.forename: Jay C
authors.initials: JC
authors.lastname: Nix
authors.forename: Edvard
authors.initials: E
authors.lastname: Wigren
authors.forename: Saurav
authors.initials: S
authors.lastname: Misra
authors.forename: Bin
authors.initials: B
authors.lastname: Zhang
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D019556:COS Cells; D002118:Calcium / Q000378:metabolism; D002240:Carbohydrate Sequence; D002522:Chlorocebus aethiops; D018360:Crystallography, X-Ray; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005165:Factor V / Q000235:genetics / Q000378:metabolism*; D005169:Factor VIII / Q000235:genetics / Q000378:metabolism*; D005998:Glycine / Q000235:genetics / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008358:Mannose / Q000737:chemistry / Q000378:metabolism*; D037241:Mannose-Binding Lectins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D033921:Vesicular Transport Proteins / 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; D006639:Histidine; D005165:Factor V; D005169:Factor VIII; D008358:Mannose; D002118:Calcium; D005998:Glycine
keywords: Calcium-binding Proteins; Carbohydrate-binding Protein; Coagulation Factors; ERGIC; Endoplasmic Reticulum (ER); Golgi; Hemorrhagic Disorders; Hemostasis; Membrane Trafficking; Protein Secretion