Functional Characterization of the Recombinant Human C1 Inhibitor Serpin Domain: Insights into Heparin Binding

Abstract

Variants of the human C1 inhibitor serpin domain containing three N-linked carbohydrates at positions 216, 231, and 330 (C1inhΔ97), a single carbohydrate at position 330 (C1inhΔ97DM), or no carbohydrate were produced in a baculovirus/insect cells system. An N-terminally His-tagged C1inhΔ97 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 KD values of 16.7 × 10−8 M (C1 inhibitor), 2.3 × 10−8 M (C1inhΔ97), and 3.6 × 10−8 M (C1inhΔ97DM). C1s also bound to heparin, with lower affinity (KD = 108 × 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.

Regulation of Complement and Contact System Activation via C1 Inhibitor Potentiation and Factor XIIa Activity Modulation by Sulfated Glycans – Structure-Activity Relationships

“…Moreover, the markedly lower activity of MMWH compared to UFH (M r 15 000) is plausible considering that UFH consists of chains with M r up to 35 000, whereas MMWH contains only 2.5% of chains with M r 12 000-15 000. In contrast to the activity of the heparin oligosaccharides VLMWH (18.2 ± 2.7% C1-INH potentiation at 6.25 μg/ml), which was in line with the results by Rossi et al [32], the inactivity of the PENTA oligosaccharides (-0.7 ± 2.8% C1-INH potentiation at 6.25 μg/ml) suggests the requirement of a minimum chain length or a minimum number of sulfate groups, respectively. The latter is concluded from the slight C1-INH potentiating effect (3.3 ± 5.3% at 6.25 μg/ml) of fondaparinux (FPX), the synthetic antithrombin-binding pentasaccharide with 8 (DS 1.60) instead of about 5 sulfate groups, which indicates additional relevance of the DS for the C1-INH potentiation.…”

“…bridging), Beinrohr et al [49] suggested a so-called sandwich-mechanism for the potentiation of C1-INH by polyanions with a heparin binding site in the contact area of the serpin-protease encounter complex. The experiments by Murray-Rust et al [50] confirmed that polyanion interactions with both C1-INH and C1s play a vital role in the mechanism of accelerating the reaction between C1s and C1-INH and both Rossi et al [32] and Rajabi et al [51] proved the binding of heparin to C1-INH as well as C1s by surface plasmon resonance.…”

“…Furthermore, a fucoidan extracted from the brown alga Ascophyllum nodosum turned out to influence the C1s inhibition by C1-INH only to a small extent [31]. Rossi et al demonstrated that the C1-INH potentiation by heparin oligomers is due to binding to both C1-INH and C1s [32]. Just recently, the effects of UFH and low-molecular weight heparins (LMWHs) on CP, LP and AP activation in absence and presence of C1-INH were compared [33].…”

Identification of a Catalytic Exosite for Complement Component C4 on the Serine Protease Domain of C1s

“…The studies provide a firm basis to guide a full elucidation of the molecular details underpinning the kinetic mechanism by which C1s interacts with C4. It is possible that the identified exosite also plays a role in the binding of highly sulfated heparin by C1s, an interaction that is vital for full regulation of the protease by the serpin, C1 inhibitor (37,38). Because unregulated complement activation underlies many inflammatory diseases, the data revealed in this study provide a basis for the design of therapeutic molecules for the targeted prevention of activation of complement via the classical pathway.…”

Pharmacology of Heparin and Related Drugs

“…Mutants with altered glycosylation in the serpin domain have been studied (Rossi et al, 2010) and are active. C1inh with an N-terminal truncation has been crystallized (2OAY.pdb), and a potential heparin binding site was identified, not at the helix D location (which is obscured by the N-terminal domain) as for AT, but at the enzyme-inhibitor interface (Beinrohr et al, 2007).…”