Molecular Immunology 71 (2016) 161–165 Contents lists available at ScienceDirect Molecular Immunology j ourna l ho me pa ge: www.elsevier.com/locate/molimm Short communication C1 Inhibitor as a glycoprotein: The influence of polysaccharides on its function and autoantibody target Arije Ghannam a,∗ , Pauline Sellier a , Olivier Fain b,c , Ludovic Martin c,d , Denise Ponard c,e , Christian Drouet c,f a KininX SAS, Grenoble, France b Internal medicine department, Saint Antoine Hospital, AP-HP, DHU i2B, Paris 6 University, Paris, France c Centre de Référence des Angioedèmes CREAK, CHU Grenoble, CHU Angers and AP-HP, Paris, France d L’UNAM Université, Dermatology department, University Hospital, Angers, France e CHU Grenoble, Laboratoire d’Immunologie, Grenoble, France f Université Joseph Fourier, GREPI EA-UJF 7408, Grenoble, France a r t i c l e i n f o Article history: Received 29 December 2015 Received in revised form 8 February 2016 Accepted 10 February 2016 Keywords: Acquired angioedema C1 inhibitor Contact phase Glycosylation a b s t r a c t C1 Inhibitor (C1Inh), a member of the Serine proteinase inhibitor family, is the most heavily glycosy- lated plasma protein. This work investigated the impact of C1Inh glycosylation on its function regarding protease targets and autoantibody binding. C1Inh deglycosylation was found to affect its function with O-linked polysaccharides, but not with N-linked polysaccharides, in controlling the contact phase but not C1s target, thus indicating the N-terminal domain’s involvement in C1Inh function. Instructive sam- ples demonstrated that O-deglycosylation strongly suppressed autoantibody binding, suggesting the polysaccharide motif is an antibody target. The autoantibodies did not directly affect C1Inh function. © 2016 Elsevier Ltd. All rights reserved. 1. Introduction C1 Inhibitor (C1Inh) is a multi-functional Serine protease inhibitor (serpin), which operates by inactivating various Serine proteases in different plasmatic cascades, including the comple- ment (classical pathway C1r and C1s; lectin pathway MASP1 and MASP2), contact (Factor XII and kallikrein), coagulation (Factor XI and thrombin), and fibrinolytic (tPA and plasmin) systems. C1Inh is the primary control of contact phase, i.e., the kinin forming sys- tem and its deficiency is well known as the cause of a rare genetic disorder, namely hereditary angioedema (HAE). Its primary disease manifestation consists of swelling caused by fluid leaking from the blood vessels into connective tissue. C1Inh is a glycoprotein of 478 amino acid residues which undergoes extensive post-translational modification, with 45% polysaccharides by weight bearing six N- linked carbohydrates and 14 potential O-glycosylation sites, seven of which have been verified by carbohydrate analysis. C1Inh is thus one of the most heavily glycosylated plasma proteins. Most of its Abbreviations: AAE, acquired angioedema; C1Inh, C1 Inhibitor; C1Inh-HAE, hereditary angioedema with C1 Inhibitor deficiency; FXII, Factor XII; FXIIa, activated Factor XII; HAE, hereditary angioedema; HK, high-molecular-weight kininogen. ∗ Corresponding author at: KininX SAS, 8 rue Duployé, F-38000 Grenoble, France. E-mail address: arije.ghannam@kininx.com (A. Ghannam). sugars are located in the N-terminal non-serpin domain (residues 1–120) (Bock et al., 1986) and only three N-linked oligosaccha- rides are attached to the serpin domain, through Asn 216 , Asn 231 , and Asn 330 (Koles et al., 2004; Beinrohr et al., 2007). The precise function of these carbohydrate groups on C1Inh interaction with target proteases, except C1s, has not yet been investigated. Previous findings indicated that the LPS-binding site of C1Inh was located within its N-terminal 97 amino acid residues (Liu et al., 2003) and that this binding process was dependent on the presence of N-linked carbohydrate at Asn 3 , Asn 47 , and Asn 59 , not the residues in the serpin domain (Liu et al., 2004). Our study sought to investigate whether O- or N-deglycosylation affects (1) C1Inh function towards contact-phase or C1s targets and (2) anti-C1Inh autoantibody binding. 2. Methods 2.1. Patients Serum samples from adult patients who had confirmed angioedema diagnosis (acquired angioedema, AAE; n = 9). All pro- cedures were performed according to the principles of the Helsinki declaration and French ethical policies governing the use of the biological sample collection (Ministry of Health authorization: http://dx.doi.org/10.1016/j.molimm.2016.02.007 0161-5890/© 2016 Elsevier Ltd. All rights reserved.