α1-Antichymotrypsin Present in Therapeutic C1-Inhibitor Products Competes with Selectin–Sialyl LewisX InteractionFunding S.H., A.L.H.E. and M.W. were supported by the European Union (Seventh Framework Program High Glycan project, grant number: 278535). R.E., L.D.N., D.R., G.v.M., D.W. and S.Z. were supported (in part) by research funding from ViroPharma Inc. (part of Shire group of companies).
16 January 2018
20 September 2018
19 November 2018 (eFirst)
Background C1-inhibitor (C1-inh) therapeutics can reduce neutrophil activity in various inflammatory conditions. This ‘novel’ anti-inflammatory effect of C1-inh is attributed to the tetrasaccharide sialyl LewisX (SLeX) present on its N-glycans. Via SLeX, C1-inh is suggested to interact with selectins on inflamed endothelium and prevent neutrophil rolling. However, C1-inh products contain plasma glycoprotein α1-antichymotrypsin (ACT) as a co-purified protein impurity.
Objective This article investigates the contribution of ACT to the effects observed with C1-inh.
Materials and Methods We have separated C1-inh and ACT from a therapeutic C1-inh preparation and investigated the influence of these proteins on SLeX–selectin interactions in a specific in vitro model, which makes use of rolling of SLeX-coated beads on immobilized E-selectin.
Results We find that ACT and not C1-inh, shows a clear sialic acid-dependent interference in SLeX–selectin interactions, at concentrations present in C1-inh therapeutics. Furthermore, we do not find any evidence of SLeX on C1-inh using either Western blotting with anti-SLeX antibodies (CSLEX1 and KM93) or by mass spectrometric analysis of N-glycans. C1-inh reacts weakly to antibody HECA-452, which detects a broad range of selectin ligands, but ACT gives a much stronger signal, suggesting the presence of a selectin ligand on ACT.
Conclusion The ‘novel’ anti-inflammatory effects of C1-inh are unlikely due to SLeX on C1-inh and can in fact be due to SLeX-like glycans on ACT, present in C1-inh products. In view of our results, it is important to assess the role of ACT in vivo and revisit past studies performed with commercial C1-inh.
R. Engel acquired, analysed and interpreted the data, designed the study and wrote the manuscript. L. Delvasto-Nuñez acquired, analysed and interpreted the data and wrote the manuscript. D. Wouters and S. Zeerleder conceived and designed the study, interpreted the data and reviewed the manuscript. D. Roem and G. van Mierlo performed experiments. S. Holst, A. L. Hipgrave Ederveen and M. Wuhrer acquired, analysed and interpreted mass spectrometric data of N-glycans. J.D. van Buul advised on flow model and reviewed the manuscript.
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