Partial glycosylation at asparagine-2181 of the second C-type domain of human factor V modulates assembly of the prothrombinase complex.
Thrombin-activated factor Va exists as two isoforms, factor Va(1) and factor Va(2), which differ in the size of their light chains and their affinity for biological membranes. The heterogeneity of the light chain remained following incubation of factor Va with N-glycanase. However, we found that the factor V C2 domain, which contains a single potential glycosylation site at Asn-2181, was partially glycosylated when expressed in COS cells. To confirm the structural basis for factor Va(1) and factor Va(2), we mutated Asn-2181 to glutamine (N2181Q) and expressed this mutant using a B domain deletion construct (rHFV des B) in COS cells. Thrombin activation of N2181Q released a light chain with mobility identical to that of factor Va(2) on SDS-PAGE. The functional properties of purified N2181Q were similar to those of factor Va(2) in prothrombinase assays carried out in the presence of limiting concentrations of phosphatidylserine. The binding of human factor Va(1) and factor Va(2) to 75:25 POPC/POPS vesicles was also investigated in equilibrium binding assays using proteins containing a fluorescein-labeled heavy chain. The affinity of human factor Va(2) binding to POPC/POPS vesicles was approximately 3-fold higher than that of factor Va(1). These results indicate that partial glycosylation of factor V at asparagine-2181 is the structural basis of the light chain doublet and that the presence of this oligosaccharide reduces the affinity of factor Va for biological membranes.
Kim
S W
SW
Division of Hematology, Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA.
Ortel
T L
TL
Quinn-Allen
M A
MA
Yoo
L
L
Worfolk
L
L
Zhai
X
X
Lentz
B R
BR
Kane
W H
WH
D000818:Animals; D001216:Asparagine / Q000235:genetics / Q000378:metabolism*; D019556:COS Cells; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D005165:Factor V / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015943:Factor Va / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D005973:Glutamine / Q000235:genetics; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013925:Thromboplastin / Q000378:metabolism*; D014162:Transfection
D010446:Peptide Fragments; D011994:Recombinant Proteins; D005973:Glutamine; D015943:Factor Va; D001216:Asparagine; D005165:Factor V; D013925:Thromboplastin; D006026:Glycoside Hydrolases; C020668:glycanase