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Defining the factor Xa-binding site on factor Va by site-directed glycosylation.

Steen, Mårten LU ; Villoutreix, Bruno O.; Norström, Eva LU ; Yamazaki, Tomio LU and Dahlbäck, Björn LU (2002) In Journal of Biological Chemistry 277(51). p.50022-50029
Abstract
Activated Factor V (FVa) functions as a membrane-bound cofactor to the enzyme factor Xa (FXa) in the conversion of prothrombin to thrombin, increasing the catalytic efficiency of FXa by several orders of magnitude. To map regions on FVa that are important for binding of FXa, site-directed mutagenesis resulting in novel potential glycosylation sites on FV was used as strategy. The consensus sequence for N-linked glycosylation was introduced at sites, which according to a computer model of the A-domains of FVa, were located at the surface of FV. In total, thirteen different regions on the FVa surface were probed, including sites that are homologous to FIXa-binding sites on FVIII. The interaction between the FVa variants and FXa and... (More)
Activated Factor V (FVa) functions as a membrane-bound cofactor to the enzyme factor Xa (FXa) in the conversion of prothrombin to thrombin, increasing the catalytic efficiency of FXa by several orders of magnitude. To map regions on FVa that are important for binding of FXa, site-directed mutagenesis resulting in novel potential glycosylation sites on FV was used as strategy. The consensus sequence for N-linked glycosylation was introduced at sites, which according to a computer model of the A-domains of FVa, were located at the surface of FV. In total, thirteen different regions on the FVa surface were probed, including sites that are homologous to FIXa-binding sites on FVIII. The interaction between the FVa variants and FXa and prothrombin were studied in a functional prothrombin activation assay, as well as in a direct binding assay between FVa and FXa. In both assays, the four mutants carrying a carbohydrate side chain at position 467, 511, 652, or 1683, displayed attenuated FXa binding whereas the prothrombin affinity was unaffected. The affinity towards FXa could be restored when the mutants were expressed in the presence of tunicamycin to inhibit glycosylation, indicating the lost FXa affinity to be caused by the added carbohydrates. The results suggested regions surrounding residues 467, 511, 652, and 1683 in FVa to be important for FXa binding. This indicate the enzyme:cofactor assembly of the prothrombinase and the tenase complexes to be homologous and provides a useful platform for further investigation of specific structural elements involved in the FVa-FXa complex assembly. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Journal of Biological Chemistry
volume
277
issue
51
pages
50022 - 50029
publisher
ASBMB
external identifiers
  • wos:000180028900120
  • scopus:0347997285
ISSN
1083-351X
DOI
10.1074/jbc.M205609200
language
English
LU publication?
yes
id
04b8831a-870b-4baa-b2ba-da9ecd197697 (old id 110599)
alternative location
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12384508&dopt=Abstract
date added to LUP
2007-07-27 09:24:58
date last changed
2017-01-01 05:17:31
@article{04b8831a-870b-4baa-b2ba-da9ecd197697,
  abstract     = {Activated Factor V (FVa) functions as a membrane-bound cofactor to the enzyme factor Xa (FXa) in the conversion of prothrombin to thrombin, increasing the catalytic efficiency of FXa by several orders of magnitude. To map regions on FVa that are important for binding of FXa, site-directed mutagenesis resulting in novel potential glycosylation sites on FV was used as strategy. The consensus sequence for N-linked glycosylation was introduced at sites, which according to a computer model of the A-domains of FVa, were located at the surface of FV. In total, thirteen different regions on the FVa surface were probed, including sites that are homologous to FIXa-binding sites on FVIII. The interaction between the FVa variants and FXa and prothrombin were studied in a functional prothrombin activation assay, as well as in a direct binding assay between FVa and FXa. In both assays, the four mutants carrying a carbohydrate side chain at position 467, 511, 652, or 1683, displayed attenuated FXa binding whereas the prothrombin affinity was unaffected. The affinity towards FXa could be restored when the mutants were expressed in the presence of tunicamycin to inhibit glycosylation, indicating the lost FXa affinity to be caused by the added carbohydrates. The results suggested regions surrounding residues 467, 511, 652, and 1683 in FVa to be important for FXa binding. This indicate the enzyme:cofactor assembly of the prothrombinase and the tenase complexes to be homologous and provides a useful platform for further investigation of specific structural elements involved in the FVa-FXa complex assembly.},
  author       = {Steen, Mårten and Villoutreix, Bruno O. and Norström, Eva and Yamazaki, Tomio and Dahlbäck, Björn},
  issn         = {1083-351X},
  language     = {eng},
  number       = {51},
  pages        = {50022--50029},
  publisher    = {ASBMB},
  series       = {Journal of Biological Chemistry},
  title        = {Defining the factor Xa-binding site on factor Va by site-directed glycosylation.},
  url          = {http://dx.doi.org/10.1074/jbc.M205609200},
  volume       = {277},
  year         = {2002},
}