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Article Dans Une Revue Journal of Immunology Année : 2010

Functional characterization of the recombinant human C1 inhibitor serpin domain: insights into heparin binding.

Isabelle Bally
Sarah Ancelet
  • Fonction : Auteur
Yuanyuan Xu
  • Fonction : Auteur
Véronique Frémeaux-Bacchi
Romain R Vivès
Nicole M. Thielens

Résumé

Variants of the human C1 inhibitor serpin domain containing three N-linked carbohydrates at positions 216, 231, and 330 (C1inhDelta97), a single carbohydrate at position 330 (C1inhDelta97DM), or no carbohydrate were produced in a baculovirus/insect cells system. An N-terminally His-tagged C1inhDelta97 variant was also produced. Removal of the oligosaccharide at position 330 dramatically decreased expression, precluding further analysis. All other variants were characterized chemically and shown to inhibit C1s activity and C1 activation in the same way as native C1 inhibitor. Likewise, they formed covalent complexes with C1s as shown by SDS-PAGE analysis. C1 inhibitor and its variants inhibited the ability of C1r-like protease to activate C1s, but did not form covalent complexes with this protease. The interaction of C1 inhibitor and its variants with heparin was investigated by surface plasmon resonance, yielding K(D) values of 16.7 x 10(-8) M (C1 inhibitor), 2.3 x 10(-8) M (C1inhDelta97), and 3.6 x 10(-8) M (C1inhDelta97DM). C1s also bound to heparin, with lower affinity (K(D) = 108 x 10(-8) M). Using the same technique, 50% inhibition of the binding of C1 inhibitor and C1s to heparin was achieved using heparin oligomers containing eight and six saccharide units, respectively. These values roughly correlate with the size of 10 saccharide units yielding half-maximal potentiation of the inhibition of C1s activity by C1 inhibitor, consistent with a "sandwich" mechanism. Using a thermal shift assay, heparin was shown to interact with the C1s serine protease domain and the C1 inhibitor serpin domain, increasing and decreasing their thermal stability, respectively.

Dates et versions

hal-01073615 , version 1 (10-10-2014)

Identifiants

Citer

Véronique Rossi, Isabelle Bally, Sarah Ancelet, Yuanyuan Xu, Véronique Frémeaux-Bacchi, et al.. Functional characterization of the recombinant human C1 inhibitor serpin domain: insights into heparin binding.. Journal of Immunology, 2010, 184 (9), pp.4982-9. ⟨10.4049/jimmunol.0902016⟩. ⟨hal-01073615⟩
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