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`Volume 71
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`;
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`March 2016
`
`”“l”“ili“‘“ill”ll
`MOLECULAR
`
`
`
`
`
`
`
`
`IMMUNOLOGY UW.
`
`l
`
`
`
`Contents
`
`Review
`
`R.Yazdani, M. Fatholahi,
`M. Ganjalikhani-Hakemi, H. Abolhassani,
`G. Azizi, K.M. Hamid, N. Rezaei and
`A. Aghamohammadi
`
`Regular Articles
`
`N. Szarvas, A. Szilégyi, D. Csuka, B. Takécs,
`K. Rusai,T. Muller, K. Arbeiter, M. Ftéti,
`A. Haris, L. Wagner, S. Térék, K. Kelen,
`A.J. Szabé, G.S. Reusz, B.P. Morgan and
`Z. Prohaszka
`
`1
`
`10
`
`Role of apoptosis in common variable immunodeficiency
`and selective immunoglobulin A deficiency
`
`Genetic analysis and functional characterization of novel
`mutations in a series of patients with atypical hemolytlc
`uremic syndrome
`
`Y. Ma, F. Han, J. Liang, J.Yang, J. Shi, J. Xue, 23
`L.Yang,Y. Li, M. Luo, Y. Wang, J. Wei and
`X. Liu
`
`A species-specific activation of Toll-like receptor signaling
`in bovine and sheep bronchial epithelial cells triggered
`by Mycobacterial infections
`
`G.Tadepalli, A.K. Singh, K. Baiakrishna,
`H.S. Murali and H.V. Batra
`
`S. Ghosh, M. Sarkar, T. Ghosh, I. Guha,
`A. Bhuniya, A. Saha, S. Dasgupta, S. Barik,
`A. Bose and R. Baral
`
`X. Wang,Y. Wei, H. Xiao, X. Liu, Y. Zhang,
`G. Han, G. Chen, c. Hou, L. Zhang, N. Ma,
`B. Shen, Y. Li, C.E. nguagu and R. Wang
`
`34
`
`42
`
`54
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`Immunogenicity and protective efficacy of Bruce/la
`abortus recombinant protein cocktail (rOmp19 + rP39)
`against B. aborfus 544 and B. meiitensis 16M infection
`in murine model
`
`Neem leaf glycoprotein promotes dual generation of
`central and effector memory CD8’r T cells against
`sarcoma antigen vaccine to induce protective anti-tumor
`immunity
`
`Pre-existing CD19-independent GL7“ Breg cells are
`expanded during inflammation and in mice with lupus-like
`disease
`
`(Contents continued on inside back cover)
`
`Available onlim:4Sx§m r cripnrpr‘lirpfl'.cnm
`Sci NFlTIONFlL LIE 0F MEDICINEEB/SOO
`ORDER it:
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`ABSTRACTED/ INDEXED/ IN. Excerp Med, index Me oc—50356-—nc
`15543152
`ACSA, CAB Inter Cambridge Scientific Absti 1L5 5843883
`ISI/BIOMED Dataiziase, Scientific Citation Ir MOLECULAR IllMUNOLOGYE/NONMEMBERS/
`
`ation database Scoopt 2016‘V0LUMEz71
`(I)
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`(Contents continued from outside back cover)
`
`3. Gorgoglione, E. Zahran, N.G.H.Taylor,
`S-W. Feist, J. Zou and C.J. Secombes
`
`3. Chang, C. He, H. Zhou, X. Kong, H. Xie,
`L. XIa and J. Van
`
`3- Kay and T. Madan
`
`X. Ben, L. Wang and X. Wu
`
`RM. Elbehidy, D.M.Youssef, A.s. El—Shal,
`S-M. Shalaby, H.S. Sherbiny, L.M. Sherief
`and NE. Akeel
`
`C--H.Weng, S. Gupta, P. Geraghty, R. Foroniy
`and AB. Pernis
`
`S-A. Jaradat, S. Caccia, R. Rawashdeh,
`M- Melhem, A. AI-Hawamdeh, T. Carzaniga
`and H. Haddad
`P. latropoulos, M. Noris, c. Mele, R. Piras,
`5- Valoti, E. Bresin, M. Curreri, E. Mondo, A. Zito,
`‘5,- Gamba, S. Bettoni, L. Murer,
`- Fremeaux—Bacchi, M. Vivarelli, F. Emma,
`E- Daina and G. Remuzzi
`E-B- Handing, LG. Shabalin, K. Szlachta,
`.A. Majorek and W. Minor
`;- De Silva, P. Dhanapala, T. Doran,
`-L.K. Tang and C. Suphioglu
`C, Liongue, T. Taznin and A.C. Ward
`
`111 LUZar, P. Molek, M. sitar, P. Koroéec,
`- KOsnik, B. Strukelj and M. Lunder
`P- Gao, M.Yuan, x. Ma, w. Jiang, L. Zhu,
`-Wen, J. Xu, 0. Liu and H. An
`
`M. Villalba, F. Fredericksen, c. Otth and
`- Olavarria
`Short Communication
`A- Ghannam, P. Sellier, o. Fain, L. Martin,
`- Ponard and C. Drouet
`
`64
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`78
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`87
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`98
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`107
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`115
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`123
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`131
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`143
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`152
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`166
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`176
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`184
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`192
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`161
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`Comparative study of CXC chemokines modulation
`in brown trout (Salmo trutta) following infection with a
`bacterial or viral pathogen
`
`The effect of Toll-like receptor 4 on [32-glycoprotein
`l-induced B cell activation in mouse model
`
`Fertility defects in Surfactant associated protein D
`knockout female mice: altered ovarian hormone profile
`
`A potential link between TSLP/TSLPR/STATS and TLR2/
`MyD88/NFKB-p65 in human corneal epithelial cells for
`Aspergil/us fumigatus tolerance
`MicroFlNA-21 as a novel biomarker in diagnosis and
`response to therapy in asthmatic children
`
`Cigarette smoke inhibits ROCK2 activation in T cells and
`modulates IL-22 production
`
`Hereditary angioedema in a Jordanian family with a novel
`missense mutation in the C1-inhibitor N-terminal domain
`
`Complement gene variants determine the risk of
`immunoglobulin—associated MPGN and CB glomerulopathy
`and predict long-term renal outcome
`
`Crystal structure of equine serum albumin in complex
`with cetirizine reveals a novel drug binding site
`
`Molecular and immunological analysis of hen’s egg yolk
`allergens with a focus on YGP42 (Gal d 6)
`
`Signaling via the CytoR/JAK/STAT/SOCS pathway:
`Emergence during evolution
`Identification and characterization of major cat allergen
`Fel d 1 mimotopes on filamentous phage carriers
`
`regulates
`positively
`Fli-1
`factor
`Transcription
`lipopolysaccharide-induced interleukin-27 production in
`macrophages
`
`Transcriptomic analysis of responses to cytopathic bovine
`viral diarrhea virus-1 (BVDV-1) infection in MDBK cells
`
`Inhibitor as a glycoprotein: The influence of
`C1
`polysaccharides on its function and autoantibody targe
`
`Page 6 of 11
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`

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`Molecular immunology 71 (2016) 161-165
`
`,x
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`
`
`Contents lists available at ScienceDirect
`
`Molecular Immunology
`
`MOLECULAR
`IMMUNOLOGY
`r m v... .flflmw
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`
`
`
`
`
`journal homepage: www.e|sevier.com/locate/molimm
`
`
`Short communication
`
`C1 inhibitor as a glycoprotein: The influence of polysaccharides on its @CmsMa,k
`function and autoantibody target
`
`Arije Ghannamar", Pauline Selliera, Olivier Fain W, Ludovic Martin“, Denise Ponard Cie,
`Christian Drouetcrf
`a KininX 5A5. Grenoble, France
`‘7 internal medicine department, SaintAntolne Hospital, AP—HP, DHU iZB, Paris 6 University, Paris. France
`° Centre de Reference des Angioedemes CREQK. CHU Grenoble. CHU Angers and AP—HP, Paris, France
`‘1 L'UNAM Université, Dermatology department, University Hospital, Angers, France
`” CHU Grenoble, Laboratoire d‘lmmunologle. Grenoble. France
`’ Universitéjoseph Fourier, GREPI EA-UJF 7408, Grenoble, France
`
`ARTICLE INFO
`
`ABSTRACT
`
`
`Article history:
`Received 29 December 2015
`Received in revised form 8 February 2016
`Accepted 10 February 2016
`Available online 18 February 2016
`
`C1 inhibitor (Cllnh), a member of the Serine proteinase inhibitor family, is the most heavily giycosy-
`lated plasma protein This work investigated the impact of C1 lnh glycosyiation on its function regarding
`protease targets and autoantibody binding. Clinh degiycosylation was found to affect its function with
`O-linked polysaccharides, but not with N—iinked polysaccharides. in controlling the contact phase but
`not C15 target, thus indicating the N-terminal domain‘s involvement in C1lnh 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 C1lnh function.
`© 201 G Elsevler Ltd. All rights reserved.
`
`Keywords:
`Acquired angloedema
`C1 inhibitor
`Contact phase
`Glycosylation
`M
`
`1. Introduction
`
`inhibitor (Clinh) is a muitl-functionai Serine protease
`C1
`inhibitor (serpin), which operates by inactivating various Serine
`proteases in different plasmatic cascades. including the comple-
`ment (classical pathway Clr and C15: lectin pathway MASPl and
`MASPZ), contact (Factor Xil and kallikrein), coagulation (Factor Xi
`and thrombin), and fibrinolytic (tPA and plasmin) systems. Clinh
`is the primary control of contact phase, i.e., the kinin forming sys-
`tem and its deficiency is well known as the cause ofa 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. C1lnh 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
`ofwhich have been verified by carbohydrate analysis. C1 lnh is thus
`
`Abbreviations: AAE. acquired angioedema: C1 lnh. C1 Inhibitor: C1 inh—HAE.
`hereditary angioedema with C1 inhibitor deficiency; FXii, Factoeri; FXiia. activated
`Factor X11; HAE, hereditary angioedema; HK. high-molecular-weight kininogen.
`* Corresponding author at: KinlnX SAS, 8 rue Duployé, F—38000 Grenoble, France.
`E-maii address: arijeghannamdikininxcom (A. Ghannam).
`
`http://dx.doi.org/iO.1016/j.moiimm.2016.02.007
`016l—5890/© 2016 Elsevler Ltd. All rights reserved.
`
`Page 7 of 11
`
`one of the most heavily glycosylated plasma proteins. Most of its
`sugars are located in the N-terminal non—serpin domain (residues
`1—120) (Bock et ai.. 1986) and only three N—linked oligosaccha-
`rides are attached to the serpin domain, through Asn215, Am”,
`and Asn33° (Koles et a1., 2004; Beinrohr et al., 2007).
`The precise function of these carbohydrate groups on Cllnh
`interaction with target proteases. except Cls, has not yet been
`investigated. Previous findings indicated that the LPS—binding site
`ofC1inh was located within its N—termina197 amino acid residues
`(Liu et ai., 2003) and that this binding process was dependent on
`the presence of N—linked carbohydrate at A5113, Am“, and Mn”,
`not the residues in the serpin domain (Liu et al., 2004).
`Our study sought to investigate whether 0— or N—deglycosylation
`affects (1 ) C1 lnh function towards contact—phase or C1s targets and
`(2) anti—C1 lnh 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
`
`Page 7 of 11
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`

`

`162
`
`A. Ghannam amt/Molecular Immunology 71 (2016) 161—165
`
`declaration and French ethical policies governing the use of the
`biological sample collection (Ministry of Health authorization:
`DC—2008—634), with all patients having provided informed consent
`to participate in the investigation using biological assays. All blood
`donors (healthy controls) answered a standard questionnaire. All
`data was anonymously processed.
`
`PBS/0.1% Tween buffer and blocking with PBS/BSA 2%. plates were
`incubated for 1h at 370C with patient‘s serum. After washing,
`plates were incubated for 1h at 37¢C with HRP-conjugated anti-
`human immunoglobulins (Abliance. Compiegne. France). The assay
`was developed with Tetramethylbenzidine (Sigma—Aldrich, Saint
`Quentin Fallavier, France) as a chromogenic substrate.
`
`2.2. Cilnh deglycosylation
`
`3. Results and discussion
`
`in order to investigate the impact of the polysaccharides on
`C11nhfunction. 1mg/ml of purified C1lnh (Berinert®; CSL Behring
`GmbH, Paris, France) was treated with a combination of 12.5 U/ml
`N-giycanase (PNGase F, New England Biolabs, Evry, France) which
`removes Asparagine-N—glycans, 1 Wm] O-glycanase (Roche Diag-
`nostics GmbH. Mannheim, Germany) which hydrolyses Serine- or
`Threonine—O—glycans. and 0.1 U/ml neuraminidase (Roche Diagnos-
`tics GmbH, Mannheim, Germany) which hydrolyses neuraminic
`acid residues overnight at 37°C in a buffer containing 10mM
`sodium phosphate. pH 6. Deglycosylated Cilnh was subjected to
`Western blot analysis, as described below.
`
`2.3. Ci lnh function assays
`
`a) To control contact phase proteases: C1 inh function was assessed
`via titration using the residual amidase activity (Pro—Phe-Arg—
`pNA) ofthe contact phase target (Ghannam et al., 2015).
`b) To control C15 target: Clinh function was assessed via titra-
`tion using the residual esterase activity (benzoyl-Arg ethyl ester)
`(Drouet et al.. 1988).
`
`2.4. Immunological assays
`
`2.4.1. Complex-formation assay
`A C11nh—KK complex—formation assay was used to determine
`whether C1lnh deglycosylation interfered with its control to con—
`tact phase proteases: C1lnh species (10 ug) were incubated with
`reconstituted contact phase proteases (equimolar amounts) at
`370C for 30min in 150mM NaC], 25 mM Tris—HCl pH 7.8, result—
`ing in a complex with KK. then the samples were subjected to
`sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-
`PAGE) analysis. Contact-phase proteins FXIia, pKK and HK were
`obtained from (Enzyme Research. Swansea. UK).
`The same procedure was followed for Clinh-Cis complex-
`formation assay to determine whether Cllnh deglycosyiation
`interfered with the proteinase—inhibitory function of C1lnh. C1lnh
`species (10 Mg) Were incubated with C15 protease (10 pg) at 373 for
`30min in 20 mM Tris-HCl, 41 mM glycine pH 8.5 buffer. Samples
`were then subjected to SDS-PAGE.
`
`2.4.2. C1lnh immunoblot
`C1lnh species and C1lnh complexes were submitted to 8%
`polyacrylamide SDS—PAGE followed by transfer onto nitrocellulose
`membrane (Hybond, GE HealthCare, Chalfont St. Giles, UK). sat—
`uration with 5% non-fat—dry milk in TBS-T buffer (150 mM NaCl.
`10mM Tris. pH 7.5. 0.01% Tween20), The blot was probed with
`anti-C1lnh (Binding Site, Birmingham. UK) in TBS-T buffer, using
`enhanced chemiluminesence (Clarity Western ECL substrate, Bio-
`rad. Hercules, USA).
`
`2.4.3. Binding ofanti-Cilnh autoantibody to Cilnh species
`C11nh species were incubated with patient serum samples in
`order to detect autoantibody binding. using a direct Enzyme-
`linked immunosorbent assay (ELISA) slightly modified from the
`technique used in Alsenz et a1. (1987). Briefly, 96-well plates
`(Dominique Dutscher, Brumath France) were coated overnight
`with purified C1lnh species (0.25 rig/well). After washing with
`
`Page 8 of 11
`
`3.1. O—glycosylated structures play a role in KK control by C1lnh
`
`We added a combination ofglycosidase enzymes to C1lnh. then
`conducted SDS-PAGE to validate the decreased relative molecu-
`lar mass (Mr) (~70 to ~90kDa) of the deglycosylated proteins
`(Fig. 1A). C1lnh function was then evaluated in terms of its con-
`trol of contact phase. C1lnh digested by combined neuraminidase
`and N—glycanase, exhibited partially diminished control compared
`to that ofnative C11nh.O-glycanase was then added. combined with
`either neuraminidase or neuraminidase and N-glycanase, causing
`complete loss ofC1lnh control of contact phase proteases (Fig. 1B).
`O-glycanase was used to release the Gal B(1—3) GaiNAc unit from
`O-glycans. Bonds to Serine as well as to Threonine are hydrolyzed.
`Substituents on the disaccharide. e.g. sialic acid in the great major-
`ity of soluble glycoproteins, prevent the hydrolysis and therefore
`must be removed enzymatically using neuraminidase.
`To strengthen these results. purified contact phase components
`were incubated with the different deglycosylated C1lnh species.
`SDS—PAGE analysis revealed that the native C1lnh, but not the fully-
`deglycosylated species. formed a complex with kallikrein (KK).
`Neuraminidase- and N—glycanase—treated C1lnh also formed a com—
`plex with KK. However. when O—glycanase was used, C1lnh was not
`able to form a complex with l<l( (Fig. 1C. lanes 5 and 9). These results
`suggest O-glycosylated structures play a role in C1lnh control of KK.
`Our results are in line with a previous study by the Patston group in
`2004 (Ravindran et al.. 2004), where C1lnh lost control of KK when
`truncated at its N—terminus, in the setting of endothelial cells.
`We nextinvestigated the impactofCiInh deglycosylation on C15
`protease control. The different C1lnh species were incubated with
`C15 protease and analyzed by means of SDS-PAGE. in all analyses.
`C1 lnh formed a complexwith C15(Fig.1D).AftertestingClinhfunc-
`tion using chromogenic substrate (Drouet et al.. 1988). digested
`C1 lnh kept control ofC1s. though its capacity was diminished when
`O—glycanase was used (Fig. 1E). These results suggest that deglyco-
`sylated C1lnh retains both its ability to form complexes with C15
`and its inhibitory function, as reported in earlier reports (Liu et a1..
`2004: Reboul et al., 1987).
`Previous studies have shown that post-translational modifica-
`tions of serpins, i.e. glycosylation, affect their protease specificity.
`A good example for this is in the case of the Protein C Inhibitor
`(PCI) Serpin. for which glycan trees have been proposed to reg—
`ulate the association with the protease. as demonstrated by
`the accelerated reaction of the deglycosylated PC! with throm-
`bin (Sun et al.. 2008). Our observations support these authors‘
`findings that the significant glycosylation impact has in modify-
`ing serpin—protease interaction. However, deglycosylated Ciinh
`species exhibited decreased serpin function towards contact phase
`proteases. This is congruent with the strongly decreased inhibi—
`tion of thrombin and plasma KK due to PC] following complete
`removal of N—acetylneuraminic acid and galactose. as well as the
`trimannosyl core from biantennary N—glycans (lzutani et al., 2001).
`As Clinh interaction with the C15 protease was not affected by
`polysaccharides, unlike its interaction with KK, this effect could be
`protease-specific. The O-linked amino acid residues are all found
`within the non-serpin N-terminal domain. Given that O-glycanase
`digestion altered C1lnh-KK interaction. it could be hypothesized
`
`Page 8 of 11
`
`

`

`A. Glrmmarn etal./Moleculnr Immunology 71 (2016) 161-165
`
`163
`
`
` A Cllnh
`N-Glycosldase
`_
`+
`+
`_
`O—Glycosidase
`-
`+
`.
`+
`Neuraminidase
`-
`+
`+
`+
`
`‘
`
`Mr(kDa)
`
`C‘llnh ~—> ‘
`dCllnh i}
`
`v-—-—
`
`“ “ 100
`70
`55
`
`c
`Cllnh/KK
`.....
`“WWW
`»
`+
`-
`+
`.
`+
`_.
`
`—
`-
`-
`r
`
`—
`.
`.,
`.
`
`i
`
`r
`.
`t
`
`+
`+
`+
`;,
`
`+
`.
`4
`
`,
`
`.
`+
`+
`.
`
`.
`+
`+
`+
`
`B
`
`N-Glycosidase
`O-Glycosidase
`Neuraminidase
`FPH
`
`Cllnh-KK —+
`
`Cllnh —§
`
`“fi' —~-~
`
`u
`
`dCllnh —+
`
`N—Glycosidase
`O-lecosidase
`Neuraminidase
`C15
`
`W ,,
`'
`~
`-
`4—
`
`'
`.
`
`Cllnh/C1s protease
`WWWW WW
`+
`+
`l»
`+
`+
`~
`+
`+
`r
`—
`vi-
`
`~'
`..
`.1
`
`.
`
`.
`Cllnh-Cls ~+ ‘
`
`Cllnh “P
`dCllnh ~~>
`
`_,
`
`v
`
`.
`
`Mr
`170
`130
`100
`
`70
`55
`
`~
`+
`+
`,
`
`4.»
`~
`v
`‘.
`
`with
`
`W
`
`—
`+
`+
`4,
`
`Mr
`170
`130
`
`100
`7°
`55
`
`g;
`f:
`g
`E
`E
`E
`E
`4;
`"3
`m
`i
`
`
`
`i
`lor,,
`0
`
`i'
`1
`
`-i
`2
`C1lnh(pmol)
`
`.777 ~
`3
`
`4
`
`.Cllnh
`ACllnhtOrGlycanasctN-Glycanase+Neuramlnidase
`C1]nh+N-Glycanase+Neuraminidase
`C1lnh+0~Glycanese+Neuraminldase
`
`
`‘
`
`V
`
`l
`l
`
`1.00
`
`E
`E
`:3 0.75
`g
`E
`E 0.50 i
`E
`i
`f,
`
`u
`g 025
`
`0.00 r
`
`i
`
`0
`
`
`
`1
`
`Cllnhipmol)
`
`3
`
`Cllnh
`O
`......‘..... Clinh+0~Glycanasei‘N-GlycanasefNeLlramlnlnase
`C1inh+N-lecanase+Neuramininase
`CLInn+0—lecanaseweuramininase
`
`Fig. 1. Deglycosylation ofCllnh: effect on reactivity with contact phase and Cls proteases.
`[Al SDS-PAGE analysrs of deglycosylated Cllnh (dCllnlr): Cilnlr was treated with a combination of neuraminidase with N—glycanase and/or Oeglycanase overnight at 37 7C.
`Cllnh species were analyzed by SDS-PAGE. The different relative molecular mass (M.-) is consistent wrth previously published data (Liu er al. 2004; Reboul et al, 1987). (B)
`Effect ofdeglycosylated Cllnh on the formation ofCllnh—KK complexes: Cllnh species were incubated with equirnolar amounts of reconstituted contact phase proteases
`(Prekallrkreln: pKK: activated Factor X“: FXlla: high molecular weight kininogen: HK; this mixture is identified FPH in the figure] at 37 ‘C for 30min, resulting in Cllnh-KK
`complex, the samples were then subjected to SDS—PAGE analysis. (C) Effect ofdeglycosylated Ci lnh on its function to control contact phase pr'ateases. Tire different species
`of Cilnh, prepared as outlined above. were incubated with contact phase purified proteins. Le, the mixture FPH, and Cllnli function was then evaluated as described by
`Ghannam et al. [Channarn et al. 2015). (D) Effect ofdeglycosylated Cilnh on the formation ofCllnh—Cls complex: Cllnh species (10 pg) were incubated with (is protease
`(10 pg) at 37 ‘C for 30 min, following which the samples were submitted for EDS-PAGE analysis. E) Effect Ordeglycosylated Cllnh on its function using Cls protease as target
`and chromogenic substrate, data are expressed as ratio as described in Drouet et al. (1988).
`
`Page 9 of 11
`
`Page 9 of 11
`
`

`

`164
`
`)HOO
`
`A. Gilannam emf/Molecularlmmunology 71 (2016‘) 161465
`
`B
`100
`
`I1 iiI11.
`
`.1
`
`i l
`
` I
`
`I.1:
`ii
`(‘Ii1
`l
`
`l
`l
`l
`
`1
`.
`
`
`
`1
`2E
`'
`i
`25
`1
`‘3;
`1H
`1“
`lgM IglVl
`igA
`lgA
`IgA
`lgG
`(P7)
`(P8)
`(P6)
`(P5)
`(P4)
`(P3)
`Auto-antibody isotype
`
`
`
`
`
` \lU1 KKactivityremaining(Vmax%) NU1U1O
`
`O
`
`IgIVI
`(P9)
`
`O
`
`1
`
`2
`
`3
`
`4
`
`I Clinh
`
`C1Inh(pmoi)
`
`\lCOlD000
`
`CHO
`Ln 0
`
`
`
` HO Bindingratio(Abs%ofnativeClinh
`
`
`
`U.) D
`NO
`
`o8
`
`E
`73F'1
`IgG
`lgG
`(P2)
`(P1)
`lCllnh
`
`i1 Clinh'tNeuraminidase+N—Glycanase
`
`C1|nh+Neuraminidase+N<Glycanase+O~Glycanase
`
`I3C1!nh+Neuraminidase+O—Glycanase
`
`0 Clinh+plasma ctrl without autoAbs
`Clinh-tplasma with autoAbs lgG (P1)
`C1|nh+plasma with autoAbs lgA (P4)
`Cllnh+plasma with autoAbs lgM (P7)
`Fig. 2. Anti-C1 Inhibitor (Cilnh) autoantibodies: Cilnh polysaccharide moiety as target and impact on Cllnh function,
`(A) Binding of anti-C 1 Inh autoantibody on native and deglycosylated Clinh species. C 1 Inh species were incubated with acquired angioedema patient serum samples in order
`to detect autoantibody binding using a direct ELISA. Deglycosylation was examined using native C1 lnh as a reference. Nine patient samples (Pl—P9) were tested as illustrative
`examples, and antibody binding was expressed as binding ratio. Serum samples from healthy donors exhibit values of binding ratio <5 for the three isotypes. (B) Effect of
`anti—Cllnh autoantibodies on purified Cilnh Function. Three patient plasma samples (one illustrative patient per isotype‘) were incubated with increased concentrations of
`purified C1 Inh; C1 Inh function was tested using the conditions described above (Ghannam et at, 2015)
`
`the non—serpin domain of Cllnh could contribute to its
`that
`function, in addition to its already established role in the clearance
`ofserpin from the blood (Minta, 1981).
`
`3.2. Polysaccharides are targets for anti-Cilnh autoantibodies in
`acquired angioedema (AAE) patients
`
`i.e., not hereditary.
`Rare cases of acquired angioedema (AAE),
`are primarily associated with lymphoproliferative or autoimmune
`disorders. Anti—Clinh antibodies have been detected in approxi—
`mately 40% of these patients. In the rare cases where samples have
`been tested, C1 Inh control ofcontact phase (Ghannam et a1., 2015)
`was found decreased in patient plasma samples with anti-Clinh
`antibody—associated AAE The question arised whether polysac—
`charides could be a target for the autoantibodies by analyzing
`AAE patient plasma samples with different autoantibody isotypes
`(n = 9). Autoantibody binding to Cl lnh species was tested by means
`of ELISA. Antibody binding was strongly decreased following CI Inh
`digestion by neuraminidase and 04 or/and N—glycanases, particu-
`larly with O—glycanase combined enzyme digestions. This decrease
`was independent of the autoantibody isotype (Fig. 2A). We next
`tested the impact of the autoantibodies on Clinh control of the
`contact phase target. Purified Cllnh was incubated in the presence
`of normal or AAE patient samples. Regardless ofthe immunoglob—
`ulin isotype. Clinh function was not affected by the autoantibody
`(Fig. 28). These results indicate that the polysaccharides represent
`a target for the autoantibodies which have been tested, but that
`the antibody did not directly affect the function of supplemented
`Clinh.
`
`and Hull. 1991; Gowda and Davidson, 1994). Such altered
`conformation following O—linked deglycosylation could also
`account for the