PMID- 10471296
OWN - NLM
STAT- MEDLINE
DCOM- 19990928
LR  - 20131121
IS  - 0006-2960 (Print)
IS  - 0006-2960 (Linking)
VI  - 38
IP  - 35
DP  - 1999 Aug 31
TI  - Partial glycosylation at asparagine-2181 of the second C-type domain of human
      factor V modulates assembly of the prothrombinase complex.
PG  - 11448-54
AB  - 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.
FAU - Kim, S W
AU  - Kim SW
AD  - Division of Hematology, Department of Medicine, Duke University Medical Center,
      Durham, North Carolina 27710, USA.
FAU - Ortel, T L
AU  - Ortel TL
FAU - Quinn-Allen, M A
AU  - Quinn-Allen MA
FAU - Yoo, L
AU  - Yoo L
FAU - Worfolk, L
AU  - Worfolk L
FAU - Zhai, X
AU  - Zhai X
FAU - Lentz, B R
AU  - Lentz BR
FAU - Kane, W H
AU  - Kane WH
LA  - eng
GR  - HL43106/HL/NHLBI NIH HHS/United States
GR  - HL45916/HL/NHLBI NIH HHS/United States
GR  - HL54939/HL/NHLBI NIH HHS/United States
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - United States
TA  - Biochemistry
JT  - Biochemistry
JID - 0370623
RN  - 0 (Peptide Fragments)
RN  - 0 (Recombinant Proteins)
RN  - 0RH81L854J (Glutamine)
RN  - 65522-14-7 (Factor Va)
RN  - 7006-34-0 (Asparagine)
RN  - 9001-24-5 (Factor V)
RN  - 9035-58-9 (Thromboplastin)
RN  - EC 3.2.1.- (Glycoside Hydrolases)
RN  - EC 3.2.1.- (glycanase)
SB  - IM
MH  - Animals
MH  - Asparagine/genetics/*metabolism
MH  - COS Cells
MH  - Cell Membrane/chemistry/metabolism
MH  - Factor V/biosynthesis/genetics/isolation & purification/*metabolism
MH  - Factor Va/chemistry/genetics/isolation & purification/metabolism
MH  - Glutamine/genetics
MH  - Glycoside Hydrolases/metabolism
MH  - Glycosylation
MH  - Humans
MH  - Mutagenesis, Site-Directed
MH  - Peptide Fragments/biosynthesis/genetics/*metabolism
MH  - Protein Binding/genetics
MH  - Protein Structure, Tertiary
MH  - Recombinant Proteins/chemistry/isolation & purification/metabolism
MH  - Thromboplastin/*metabolism
MH  - Transfection
EDAT- 1999/09/02 00:00
MHDA- 1999/09/02 00:01
CRDT- 1999/09/02 00:00
PHST- 1999/09/02 00:00 [pubmed]
PHST- 1999/09/02 00:01 [medline]
PHST- 1999/09/02 00:00 [entrez]
AID - 10.1021/bi991275y [doi]
AID - bi991275y [pii]
PST - ppublish
SO  - Biochemistry. 1999 Aug 31;38(35):11448-54. doi: 10.1021/bi991275y.