The sugar-binding ability of ERGIC-53 is enhanced by its interaction with MCFD2.
Combined deficiency of factors V and VIII (F5F8D) is a bleeding disorder caused by mutations in LMAN1 or MCFD2. LMAN1 encodes ERGIC-53, a cargo receptor with an L-type lectin domain, and MCFD2 is a EF-hand-containing protein. We prepared a biotinylated, soluble form of ERGIC-53, which we labeled with R-phycoerythrin conjugated streptavidin. By flow cytometry, sERGIC-53-SA bound to HeLaS3 cells in the presence of calcium but only after preincubation with MCFD2. Treating the cells with endo H or incubating them with high mannose-type oligosaccharides, especially M(8B), abrogated sERGIC-53-SA binding. Surface plasmon resonance experiments demonstrated that MCFD2 specifically bound to sERGIC-53 and 2 MCFD2 mutants found in F5F8D patients had a K(a) that was 3 or 4 orders of magnitude lower for sERGIC-53 than for wild-type MCFD2. The K(a) of sERGIC-53 and MCFD2 was measured at several pH values and calcium concentrations, and we found that at a calcium concentration less than 0.2 mM, this interaction became significantly weaker. These results demonstrate that the binding of ERGIC-53 to sugar is enhanced by its interaction with MCFD2, and defects in this interaction in F5F8D patients may be the cause for reduced secretion of factors V and VIII.
Kawasaki
Norihito
N
Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Chiba, Japan.
Ichikawa
Yoko
Y
Matsuo
Ichiro
I
Totani
Kiichiro
K
Matsumoto
Naoki
N
Ito
Yukishige
Y
Yamamoto
Kazuo
K
D001483:Base Sequence; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D017931:DNA Primers; D005166:Factor V Deficiency / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006467:Hemophilia A / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D037241:Mannose-Binding Lectins / Q000235:genetics* / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D037121:Plant Lectins / Q000378:metabolism*; D011134:Polysaccharides / Q000378:metabolism; D011485:Protein Binding; D033921:Vesicular Transport Proteins / Q000378:metabolism*
D017931:DNA Primers; C041384:Datura stramonium lectin; C084004:LMAN1 protein, human; C475146:MCFD2 protein, human; D037241:Mannose-Binding Lectins; D008565:Membrane Proteins; D037121:Plant Lectins; D011134:Polysaccharides; D033921:Vesicular Transport Proteins; C063117:snowdrop lectin