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Structural requirements of anticoagulant protein S for its binding to the complement regulator c4b-binding protein

Giri, TK; Linse, Sara LU ; Garcia de Frutos, Pablo LU ; Yamazaki, Tomio LU ; Villoutreix, BO and Dahlbäck, Björn LU (2002) In Journal of Biological Chemistry 277(17). p.15099-15106
Abstract
The vitamin K-dependent anticoagulant protein S binds with high affinity to C4b-binding protein (C4BP), a regulator of complement. Despite the physiological importance of the complex, we have only a patchy view of the C4BP-binding site in protein S. Based on phage display experiments, protein S residues 447-460 were suggested to form part of the binding site. Several experimental approaches were now used to further elucidate the structural requirements for protein S binding to C4BP. Peptides comprising residues 447-460, 451460, or 453-460 of protein S were found to inhibit the protein S-C4BP interaction, whereas deletion of residues 459-460 from the peptide caused complete loss of inhibition. In recombinant protein S, each of residues... (More)
The vitamin K-dependent anticoagulant protein S binds with high affinity to C4b-binding protein (C4BP), a regulator of complement. Despite the physiological importance of the complex, we have only a patchy view of the C4BP-binding site in protein S. Based on phage display experiments, protein S residues 447-460 were suggested to form part of the binding site. Several experimental approaches were now used to further elucidate the structural requirements for protein S binding to C4BP. Peptides comprising residues 447-460, 451460, or 453-460 of protein S were found to inhibit the protein S-C4BP interaction, whereas deletion of residues 459-460 from the peptide caused complete loss of inhibition. In recombinant protein S, each of residues 447-460 was mutated to Ala, and the protein S variants were tested for binding to C4BP. The Y456A mutation reduced binding to C4BP similar to10-fold, and a peptide corresponding to residues 447-460 of this mutant was less inhibitory than the parent peptide. A further decrease in binding was observed using a recombinant variant in which a site for N-linked glycosylation was moved from position 458 to 456 (Y456N/N458T). A monoclonal antibody (HPSf) selective for free protein S reacted poorly with the Y456A variant but reacted efficiently with the other variants. A second antibody, HPS 34, which partially inhibited the protein S-C4BP interaction, reacted poorly with several of the Ala mutants, suggesting that its epitope was located in the 451-460 region. Phage display analysis of the HPS 34 antibody further identified this region as its epitope. Taken together, our results suggest that residues 453-460 of protein S form part of a more complex binding site for C4BP. A recently developed three-dimensional model of the sex hormone-binding globulin-like region of protein S was used to analyze available experimental data. (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
17
pages
15099 - 15106
publisher
ASBMB
external identifiers
  • wos:000175203000095
  • scopus:0037177873
ISSN
1083-351X
DOI
10.1074/jbc.M103036200
language
English
LU publication?
yes
id
6e8dcbd7-192e-40d4-8d5e-53acfcab0c7e (old id 339952)
date added to LUP
2007-08-20 16:29:00
date last changed
2017-01-01 04:28:11
@article{6e8dcbd7-192e-40d4-8d5e-53acfcab0c7e,
  abstract     = {The vitamin K-dependent anticoagulant protein S binds with high affinity to C4b-binding protein (C4BP), a regulator of complement. Despite the physiological importance of the complex, we have only a patchy view of the C4BP-binding site in protein S. Based on phage display experiments, protein S residues 447-460 were suggested to form part of the binding site. Several experimental approaches were now used to further elucidate the structural requirements for protein S binding to C4BP. Peptides comprising residues 447-460, 451460, or 453-460 of protein S were found to inhibit the protein S-C4BP interaction, whereas deletion of residues 459-460 from the peptide caused complete loss of inhibition. In recombinant protein S, each of residues 447-460 was mutated to Ala, and the protein S variants were tested for binding to C4BP. The Y456A mutation reduced binding to C4BP similar to10-fold, and a peptide corresponding to residues 447-460 of this mutant was less inhibitory than the parent peptide. A further decrease in binding was observed using a recombinant variant in which a site for N-linked glycosylation was moved from position 458 to 456 (Y456N/N458T). A monoclonal antibody (HPSf) selective for free protein S reacted poorly with the Y456A variant but reacted efficiently with the other variants. A second antibody, HPS 34, which partially inhibited the protein S-C4BP interaction, reacted poorly with several of the Ala mutants, suggesting that its epitope was located in the 451-460 region. Phage display analysis of the HPS 34 antibody further identified this region as its epitope. Taken together, our results suggest that residues 453-460 of protein S form part of a more complex binding site for C4BP. A recently developed three-dimensional model of the sex hormone-binding globulin-like region of protein S was used to analyze available experimental data.},
  author       = {Giri, TK and Linse, Sara and Garcia de Frutos, Pablo and Yamazaki, Tomio and Villoutreix, BO and Dahlbäck, Björn},
  issn         = {1083-351X},
  language     = {eng},
  number       = {17},
  pages        = {15099--15106},
  publisher    = {ASBMB},
  series       = {Journal of Biological Chemistry},
  title        = {Structural requirements of anticoagulant protein S for its binding to the complement regulator c4b-binding protein},
  url          = {http://dx.doi.org/10.1074/jbc.M103036200},
  volume       = {277},
  year         = {2002},
}