Structural requirements of anticoagulant protein S for its binding to the complement regulator c4b-binding protein
(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)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/339952
- author
- Giri, TK ; Linse, Sara LU ; Garcia de Frutos, Pablo LU ; Yamazaki, Tomio LU ; Villoutreix, BO and Dahlbäck, Björn LU
- organization
- publishing date
- 2002
- type
- Contribution to journal
- publication status
- published
- subject
- in
- Journal of Biological Chemistry
- volume
- 277
- issue
- 17
- pages
- 15099 - 15106
- publisher
- American Society for Biochemistry and Molecular Biology
- 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
- 2016-04-01 11:42:49
- date last changed
- 2022-01-26 17:06:32
@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 = {{American Society for Biochemistry and Molecular Biology}},
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}},
doi = {{10.1074/jbc.M103036200}},
volume = {{277}},
year = {{2002}},
}