Abstract
Parasitic nematodes manufacture various carbohydrate-linked phosphorylcholine (PCh)-containing molecules, including ES-62, a protein with an N-linked glycan terminally substituted with PCh. The PCh component is biologically important because it is required for immunomodulatory effects. We showed that most ES-62 was bound to a single protein, C-reactive protein (CRP), in normal human serum, displaying a calcium-dependent, high-avidity interaction and ability to form large complexes. Unexpectedly, CRP binding to ES-62 failed to efficiently activate complement as far as the C3 convertase stage in comparison with PCh-BSA and PCh-containing Streptococcus pneumoniae cell wall polysaccharide. C1q capture assays demonstrated an ES-62-CRP-C1q interaction in serum. The three ligands all activated C1 and generated C4b to similar extents. However, a C2a active site was not generated following ES-62 binding to CRP, demonstrating that C2 cleavage was far less efficient for ES-62-containing complexes. We proposed that failure of C2 cleavage was due to the flexible nature of carbohydrate-bound PCh and that reduced proximity of the C1 complex was the reason that C2 was poorly cleaved. This was confirmed using synthetic analogues that were similar to ES-62 only in respect of having a flexible PCh. Furthermore, ES-62 was shown to deplete early complement components, such as the rate-limiting C4, following CRP interaction and thereby inhibit classical pathway activation. Thus, flexible PCh-glycan represents a novel mechanism for subversion of complement activation. These data illustrate the importance of the rate-limiting C4/C2 stage of complement activation and reveal a new addition to the repertoire of ES-62 immunomodulatory mechanisms with possible therapeutic applications.
Original language | English |
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Pages (from-to) | 11939-53 |
Number of pages | 15 |
Journal | Journal of Biological Chemistry |
Volume | 291 |
Issue number | 22 |
Early online date | 4 Apr 2016 |
DOIs | |
Publication status | Published - 27 May 2016 |
Keywords
- Binding Sites
- C-Reactive Protein
- Carbohydrate Conformation
- Complement Activation
- Complement C2
- Complement C3-C5 Convertases
- Complement C4
- Complement Pathway, Classical
- Helminth Proteins
- Humans
- Phosphorylcholine
- Surface Plasmon Resonance
- Journal Article
- Research Support, Non-U.S. Gov't