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C1-inhibitor potentiation by glycosaminoglycans

Bos, G.A.C.

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Citation for published version (APA):Bos, G. A. C. (2003). C1-inhibitor potentiation by glycosaminoglycans.

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Download date: 23 Jun 2020

Chapterr 7

Potentiationn mechanism of Cl-Inhibitor differss from that of other serpins

Inekee Bos, Eric Eldering, Jan Pieter Abrahams andd Erik Hack

C H A P T E RR 7

ABSTRACT T

Likee a number of other serpins, Cl-inhibitor (Cl-Inh) is potentiated by heparin and otherr glycosaminoglycans. Here, we report that residues Lys: ,̂ Arg:sz, and Argvs of Cl-Inh,, located close to the postulated secondary binding site for Cls, are important for potentiation.. Known mechanisms of serpin potentiation are less likely to be involved in Cl-Inhh potentiation: (1) titration experiments did not support a template mechanism; (ii) stabilityy of the central P-sheet was unaffected by heparin; (iii ) no loop expulsion or PI exposuree was observed upon incubation with heparin; (iv) the major part of the unique N-terminall domain could be deleted without affecting potentiation. Together these data suggestt a novel mechanism of serpin potentiation for Cl-Inh, that is likely to involve residuess close to Cl-Inh s secondary Cls binding site.

100 0

NOVELL POTENTIATION MECHANISM FOR C 1 -INH

T T XX he serpin Cl-Inhibitor (Cl-Inh) is an inhibitor of several inflammatory cascades such

ass the classical pathway of complement and the contact system of coagulation 19. Becausee of its anti-inflammatory properties, administration of Cl-Inh has been evaluated ass a therapy of inflammatory conditions like the vascular leakage syndrome, bone marroww transplantation, sepsis and acute myocardial infarction (reviewed by Caliezi et al.,, ia). These studies revealed that high doses of Cl-Inh are needed to induce an anti-inflammatoryy effect in vivo. Thee inhibitory activity of several serpins including Cl-Inh can be enhanced by heparin orr other glycosaminoglycans (GAGs) 1U:. For Cl-Inh, the mechanism of potentiation by heparinn is unknown. Administration of GAG-potentiated Cl-Inh may help to reduce the dosee of Cl-Inh in inflammatory conditions. We have evaluated the use of Cl-Inh potentiatedd by dextran sulphate in a rat model, and found that the non-covalent complex off Cl-Inh and this artificial GAG is unstable in vivo, probably due to rapid dissociation andd clearance of the GAG °. A better understanding of the potentiation mechanism of Cl-Inhh by GAGs may aid in developing improved Cl-Inh variants useful in a therapeuticall setting. Serpinss like antithrombin III (ATM), heparin cofactor II (HOI) and protein C inhibitor (PCI)) also have enhanced inhibitory activity in the presence of GAGs. In general, potentiationn results from two mechanisms. The template mechanism implies that the GAGG molecule serves to assemble the protease and the serpin next to each other in an appropriatee orientation. The second mechanism involves a conformational change in the serpinn upon binding of the GAG making the inhibitor more reactive towards the protease. . Thee mechanism of potentiation of ATII I is well characterised, in particular since the complexx of ATII I with a specific pentasaccharide has been crystallised 14,15. The heparin bindingg sites in ATII I are located in the A-, D- and P-helices, and in a cleft formed by the N-terminus.. Upon binding of a core-pentasaccharide to the ATII I molecule, a small (i-strandd is pushed out of the central (3-sheet, enhancing exposure of the reactive site. This leadss to a 300-fold increase in inhibitory activity towards factor Xa. The potentiation towardss thrombin requires at least 18 saccharides because a template mechanism is also involvedd i6"18. Since the reactive site loop (RSL) of Cl-Inh is 5 amino acids shorter than thatt of ATIII , the mechanism of potentiation of Cl-Inh by GAGs is unlikely to involve mobilisationn of the RSL, and, therefore, must be different from that of ATIII . Thee crystal structures of heparin cofactor II (HOI) and the Michaelis complex of HCII-Seri95->Alaa thrombin suggest that GAG binding also induces expulsion of the RSL in H OII 19. Heparin binds to residues in the A- and the D-helix of HOI, which are conservedd in ATII I and contribute to heparin binding. However, reactivity of HCII with thrombinn requires also an intact N-terminus, even in the presence of GAGs. The structuree of the N-terminal acidic tail has not been elucidated yet, but a mechanism involvingg allosteric activation has been proposed to explain involvement of this part of HCIII in heparin binding. This includes expulsion of the RSL, closure of the central (3-sheet,, and consequently release of the acidic tail which is now free to recruit thrombin.

101 1

CHAPTERR 7

Forr Cl-Inh it has been demonstrated that inhibition of target proteases is independent of thee unique N-terminal domain JC-:i, but whether the N-terminal domain is involved in potentiationn of Cl-Inh by GAGs is unknown. Thee heparin binding regions of protein C inhibitor (PCI) are located in the A- and the H-helix,, but only helix H-mutations result in altered rates of inhibition. The crystal structuree of cleaved PCI suggests that more residues are involved in the positive electrostaticc potential " implying that the heparin-binding region may be larger than previouslyy anticipated. Based on the surface electrostatic properties a co-occupation mechanismm is proposed; PCI and activated protein C initially bind to different sites on heparinn and then each migrates to form a complex through occupation of the same site onn heparin. Heree we describe our studies on several possible mechanisms of potentiation of Cl-Inh byy heparin. Exposure of the reactive site loop, a classic template mechanism, conformationall change of the central [3-sheet, and involvement of the first 98 amino acids off the N-terminal domain could all be excluded. However, the positively charged residuess Lys2»4, Arg:s7 and Arg37s close to the secondary Cls binding site were involved in potentiation. .

MATERIALSS AND METHODS

Materials Materials Plasmaa Cl-Inh (Cetor) was obtained from Sanquin (Amsterdam, the Netherlands). Biotinylatedd polyclonal rabbit antibodies against human Cl-Inh, and the monoclonal antibodiess (mAb) RII (against human Cl-Inh), and Kokl2 (specific for cleaved or complexedd human Cl-Inh) have been described before 23"25. Cls for ELISAs was purified andd biotinylated (Cls-BT) as described before 2\ Streptavidin coupled to polymerised horseradishh peroxidase (poly-HRP) was obtained from Sanquin (Dept. Immune Reagents).. Streptavidin coupled to monomeric horseradish peroxidase was obtained from Amershamm Pharmacia Biotech (Uppsala, Sweden). Cls for kinetic assays was purchased fromm Calbiochem (La Jolla, CA, USA). Heparinn was purchased from Leo Pharma (Breda, the Netherlands). The kit to determine antithrombinn activity (Coamatic® antithrombin) and the chromogenic substrate S2314 weree obtained from Chromogenix (Milano, Italy). The chromogenic assay for measurementt of Cl-Inh activity (Berichrom® Cl-Inhibitor) was purchased from Dade Behringg (Marburg, GmbH, Germany). Cl-Inh N-terminal deletion mutants have been describedd before 20. The mutants Lys:s4^Gly, Arg2S7^Gly, and Arg378->Gly were producedd with site-directed mutagenesis and expressed in P. pastoris, as described before :b.. Custom primers for site-directed mutagenesis were purchased from Invitrogen Life Technologiess (Carlsbad, CA, USA).

102 2

N O V E LL POTENTIATION MECHANISM FOR C1 -INH

ELISAs ELISAs Cl-Inhh antigen, active Cl-Inh, cleaved and inactive Cl-Inh were determined as described beforee 1Z27.

PeptidylPeptidyl arginine deiminase treatment Thesee experiments were performed in a similar set-up as described before for ATII I 14: Cl-Inhh or ATII I (10 uM) were incubated with varying concentrations of peptidyl argininee deiminase (PADI) in 100 mM Tris-HCl, 5 raM CaCb, pH 7.4, for 16 hours at 37°CC in the presence or absence of heparin (50 uM). The reaction was stopped by additionn of 50 mM EDTA. Residual inhibitory activity was tested with chromogenic assayss for ATII I or Cl-Inh activity.

FormationFormation of multimers measured with dynamic light scattering Multimerr formation was assessed by incubation of Cl-Inh (10 uM) for 15 minutes at 57°CC in the presence or absence of heparin (50 (iM). Protein size was determined by analysiss of the particle radius with dynamic light scattering (DynaPro 99, Protein Solutions,, NJ, USA).

PeptidePeptide insertion into the central fi-sbeet. AA custom peptide with the sequence N-acetyl-VEAAAASAISVAR, mimicking the sequencesequence of the RSL, was purchased from Invitrogen Life Technologies (Carlsbad, CA, USA).. The peptide was dissolved at 1.1 mg per ml in water with 0.11 % acetonitril, 0.039 %% NHUOH. Cl-Inh (10 uM) in the presence or absence of heparin (50 uM) was incubatedd overnight at 37°C with varying amounts of the peptide (15 - 500 nM). The residuall amount of active Cl-Inh was determined with the ELISA for active Cl-Inh.

Site-directedSite-directed mutagenesis Site-directedd mutagenesis was used to generate mutants of Cl-Inh. Mutagenesis was done byy splice-overlap PCR (modified from :s). The sequence of the primers was ACA ACA TTTT GAT CCC GGG AAA ACC AGA ATG GA for LyS2S4->Gly, GAT CCC AAG AAAA ACC GGT ATG GAA CCC TTT CAC for Arg:a7->Gly and CTC CTA ACA CTAA CCC GGG ATC AAA GTG ACG ACC for Arg37S->Gly.

PotentiationPotentiation by heparin Potentiationn of Cl-Inh by heparin was studied by incubation of 2 nM Cls with 2 or 4 nMM Cl-Inh and 3.5 mM chromogenic substrate S2314 in the presence or absence of heparin.. To analyse the possibility of a template mechanism, concentrations of 0.0125 -12500 U per ml of heparin were used. To determine the potentiation of various Cl-Inh mutants,, a limiting concentration of heparin, i.e. 0.125 U per ml, was used. The amount off Cl-Inh used in these experiments was determined by titrating Cl-Inh (2, 4, and 8 nM) againstt active Cls (2 nM). It was determined in separate experiments that under these conditions,, with a limiting heparin concentration, potentiation by heparin decreases the substratee conversion after 6 hours (data not shown). The effect of heparin on the activity

CHAPTERR 7

off the Cl-Inh variants was subsequently determined at the Cl-Inh concentration that resultedd in inhibition similar to inhibition by plasma Cl-Inh.

RESULTSS AND DISCUSSION

ReactiveReactive site exposure of Cl-Inh is not altered by heparin Bindingg of heparin to ATII I results in an altered exposure of the reactive site residue argininn at the Pl-position of the reactive site loop, as was deduced from enhanced sensitivityy to peptidyl arginine deiminase (PADI) '4. This enzyme cleaves off the imine groupp of the arginine at PI, thereby inactivating the serpin. Figure 1 shows ATII I and Cl-Inhh activity after incubation of the serpin with a varying amount of PADI. In the presencee of heparin, lower concentrations of PADI were required to decrease ATII I activity.. The Km of the reaction of PADI with ATII I decreased in the presence of heparinn (0.12 versus 0.032 uM PADI + /- heparin, respectively). The Km of the reaction off PADI with Cl-Inh was lower and irrespective of the presence of heparin (0.025 and 0.0211 uM PADI + /- heparin, respectively). These results indicated that the exposure of thee Pl-Arg in the reactive site loop of Cl-Inh was unaffected by heparin, and intrinsicallyy more accessible than Pl-Arg of ATIII .

I tt is therefore unlikely that potentiation of Cl-Inh requires a substantial rearrangement off the reactive site loop. The observation that the RSL of Cl-Inh is 5 amino acids shorter thann that of ATII I gives further support against such a mechanism. In a separate study wee have investigated the effect of RSL elongation on inhibitory activity, but this led to lesss effective inhibition (Bos et al., 2003, Chapter 5).

'5. . ID D [/> >

> > O O

re re

00 0.05 0.1 0.15 0.2 0.25

peptidylarginin ee deiminas e (u M)

Figuree 1 Hepari nn does not alter susceptibilit y of C1-lnh for PADI C1-lnhh or ATIII as a control (each at 10 uM) with and without heparin (50 uM) were incubated withh an increasing amount of PADI (x-axis). which removes the imine-group from the active site.. P1-Arg. Residual serpin activity was then measured with a chromogenic assay, and is depictedd on the y-axis. Similar results were obtained in three separate experiments.

104 4

ATI I ATIIII + heparin

Inh h nhh + heparin

N O V E LL POTENTIATION MECHANISM FOR C1 -INH

DoeDoe conformation of the central fi-sbeet is not affected by heparin

Heparinn can be postulated to induce a conformational change of Cl-Inh, which results in ann altered stability of the central P-sheet, as has been described for ATII I ' \ This mechanismm was investigated in several ways. First, the effect of heparin on the heat stabilityy of Cl-Inh was analysed. As many other serpins, Cl-Inh forms multimers at elevatedd temperatures as a result of insertion of the RSL of one molecule into the central p-sheett of another. An altered conformation of the central p-sheet probably induces a differentt stability at higher temperatures resulting in a change in the rate of multimer formation.. A temperature of 57°C was chosen since in pilot experiments the size of multimerss of normal plasma Cl-Inh incubated for 2 hours at different temperatures increasedd in a linear fashion between 50 and 60°C (data not shown). Cl-Inh multimerizationn at 57°C was unchanged in the presence of heparin (figure 2). Anotherr way to investigate the stability of the central (3-sheet is by analysing the effect of insertionn of a peptide mimicking the reactive site loop sequence into the central P-sheet. AA peptide with the same sequence as the proximal part of the RSL was incubated with Cl-Inhh as described in materials and methods. No shift in dose-response curves of the percentagee of active Cl-Inh versus the peptide concentration occurred in the presence of heparinn (figure 3). The Kd of the Cl-Inh*peptide complex (0.12 and 0.15 mM in presence orr absence of heparin), was irrespective of the presence of heparin. These data altogether suggestedd that the conformation of the central P-sheet was not affected by heparin.

CII -ti

577 C

O h h '' H>tain

57=C C

Figuree 2 Hepari nn does not alter multime r formatio n of C1-lnh C1-lnhh (10 (.iM) was incubated for 15 minutes at C in the presence or absence of heparin (500 ,uM). During this period C1-lnh multimers were formed. Particle size of the multimers wass measured with dynamic light scattering, the radius of the particles is depicted in the y-axis. .

105 5

CHAPTERR 7

16a a

oo ' ' ' 00 0.1 0.2 0.3 0.4 0.5 0.6

[peptide ]] (mM) p - -- peptide — peptide + heparin

Figuree 3 Hepari nn does not alter peptid e insertio n int o the centra l |i-shee t of C1-lnh C1-lnhh (10 (.iM) was incubated with a varying concentration of a peptide mimicking the reactivee site loop sequence (x-axis) in presence or absence of heparin (50 jiM). Remaining C1-lnhh activity was measured with an ELISA for active C1-lnh and plotted on the y-axis. Similarr results were obtained in 2 separate experiments.

PotentiationPotentiation of Cl-Inh by heparin is independent of the non-essential part of the unique N-terminalterminal domain Becausee the N-terminal domain of heparin co f act or II is probably involved in heparin bindingg 19 and because Cl-Inh also has a unique N-terminal domain, we analysed the potentiationn of two N-terminal deletion mutants of Cl-Inh. These two mutants start at aminoo acid 76 and 98 and have completely normal activity towards Cls, kallikrein and Xll aa in vitro " " ' . This shows that the first 98 amino acids are not essential for inhibition. However,, the two disulphide bridges between Cysioi and Cysics and the serpin are essentiall to maintain the metastable conformation " . For that reason we could not delete thee entire N-terminal domain, which involves the first 115 amino acids. Table I shows thee inhibition of C ls by Cl-Inh mutants in the presence or absence of heparin. The effectt of heparin on the N-terminal domain deletion mutants was similar to its effect on wild-typee Cl- Inh, hence this part of the N-terminus of Cl-Inh is not involved in potentiation. .

106 6

N O V E LL P O T E N T I A T I O N M E C H A N I S M F O R C 1 - I N H

Tablee I Thee majo r par t o f th e uniqu e N-termina l domai n of C1-ln h is no t involve d in potentiation .. C1s (2nM) with C1-lnh (2 or 4 nM) and 3.5 mM chromogenic substrate were incubatedd with or without a limiting amount of heparin (0.125 U/ml). Under these conditions C1-lnhh potentiation leads to a decrease in substrate conversion. For each sample the absorptionn at 405 nm after 6 hours was expressed as a percentage of the absorption without C1-lnh.. This percentage substrate conversion is depicted in the table.

%% substrate conversion

Plasmaa C1-Inh WTrhC1-lnh h rhC1-lnh76* * rhC1-lnh98* *

Noo heparin

76% % 76% % 81% % 82% %

Heparin n

50% % 57% % 50% % 59% %

.1255 U/ml

*rhC1-lnh,/6andd rhC1-lnh98 are deletion mutants starting at amino acid 76 andd 98 respectively, thereby lacking the majority of the unique N-terminal domain.2D D

0.00001 1

[Heparin]] (U/ml)

Figuree 4 AA templat e mechanis m is unlikel y to dominat e potentiatio n C1ss (2 nM) was incubated with 2 nM of C1-lnh, chromogenic substrate and increasing concentrationss of heparin (x-axis). The substrate conversion after 6 hours is depicted by the A^oss on the y-axis. This experiment was performed twice with similar results.

AA classic template mechanism, can not explain potentiation

Clss can bind to heparin 29"31, although the literature is contradictory on this issue J2,33. Separate,, independent binding of both serpin and protease is a hallmark of the classical templatee mechanism. A typical feature of the classic template mechanism as observed for A TT and thrombin 'A is a bell-shaped curve when the concentration of GAG is plotted againstt the inhibitory activity of the serpin 35, as at higher heparin concentrations the serpinn and the protease wil l bind to separate heparin molecules. When increasing heparin concentrationss were tested, no bell-shaped curve was observed in the case of Cl-Inh and Clss (figure 4), arguing against a template mechanism. The same results were obtained whenn another heparin preparation was tested (Celsus Laboratories, O H, USA). Involvementt of some form of template mechanism can not be completely excluded becausee both Cls and Cl-Inh seem to bind heparin. But based on these data, it is unlikelyy to play a dominant role in the enhanced inhibition.

1 0 7 7

CHAPTERR 7

Anotherr characteristic of the classic template mechanism is the minimum length requirementt of the heparin molecules. Low molecular weight dextran sulphate (5 kDa) is veryy effective in potentiation of Cl-Inh 12, and moreover, in pilot experiments we observedd potentiation by pentasaccharides (data not shown). Altogether these data indicatee that a classic template mechanism cannot explain potentiation of Cl-Inh by heparin. .

MutationMutation of residues Lys2X4, Arg287 and Argun of Cl-Inh affects heparin-induced potentiation Cl-Inhh activity can only be potentiated towards Cls and coagulation factor XIa, not towardss coagulation factor Xll a and kallikrein 1h. Hence involvement of a secondary protease-bindingg site in the potentiation mechanism can be postulated. Based on a 3D-modell of Cl-Inh we proposed 3 possible heparin-binding regions ,7. Region 1 is located closee to PI and the secondary Cls binding site '8. Region 2 is located on the opposite side off Pi behind the hinge region, and region 3 is located around helix D. Region 3 is currentlyy under investigation by other researchers w. We did not find evidence of an AT-typee potentiation, which would imply region 3, and because region 2 is too far from the Clss binding site we investigated region 1. Regionn 1 comprises Lys284, Arg>87 and Arg378. To evaluate a potential role of these residuess in heparin-dependent potentiation of Cl-Inh, they were mutated into glycine. Sequencee analysis indicated that mutant Arg:87-»Gly carried an additional mutation in thee N-terminal domain: Pro7f,^Leu. Since we had shown this part of the N-terminal domainn not to be involved in inhibitory activity 20 and heparin potentiation (table I) we analysedd this double mutant instead. We confirmed these results with another double mutantt of Arg:s7^Gly, carrying the additional mutation Leu2t,8^Phe. The location of thee possible heparin binding amino acids is depicted in figure 5a. Mutation of residues Lys:w4,, Arg:87 or Arg^s in region 1 into glycine significantly impaired potentiation by heparinn {figure 5b). Since single substitutions already showed an inhibitory effect on heparinn potentiation, multiple residues appear to be directly involved. A combination of mutationss might be required to fully block the effect of heparin. Based on the surface electrostaticc potential we propose that residues Lys:7t, Lys2w and Lys.iso might also be involved. . Thee involvement of a secondary protease binding site is not unique, but also shown for thrombin,, which interacts w7ith the N-terminal acidic tail of HCII 19. A striking feature off heparin potentiation of Cl-Inh is that it regulates the inhibitory activity towards some target-proteasess like Cls and factor XIa, but not others such as factor Xll a and kallikrein12'6,, postulating a role for the Cls binding site in potentiation. Thee involvement of region 1 in heparin potentiation of other serpins has not been demonstratedd so far, suggesting that this region is unique for Cl-Inh. We confirmed this byy analysis of the surface electrostatic potential of this region in the crystal structures of ATT (1AZX, ,4), HCII (1JMJ, ,9), and PCI (1LQ8, "). None of these serpins showed a positivelyy charged region at corresponding sites, indicating that involvement of region 1 inn heparin-mediated potentiation is unique for Cl-Inh.

1 0 8 8

N O V E LL POTENTIATION MECHANISM FOR C1 -INH

Lyss 284

Argg 378

plasmaa Cl-lnh

W T r h C H n h h

C1-lnhh K284G

C1-lnhh R287G

Cl-lnnn R287G

Figuree 5 Thee residues Lys284, Arg287 and Arg 378 o f C1-ln h are involve d in hepari n b ind ing a.. Location of possible heparin binding residues in the 3D-structure b.. C1s (2nM) with plasma C1-lnh, rhC1-lnh, C1-lnh Lys284 > Gly. C1-lnh Arg2S7^GIy or C l -lnhh Arg3?8_, Gly (4 nM) and 3.5 mM chromogenic substrate were incubated with increasing concentrationss of heparin. Under these conditions C1-lnh potentiation leads to a decrease in substratee conversion. For each sample the absorption at 405 nm after 6 hours was expressedd as a percentage of the absorption without C1-lnh. This is depicted on the y-axis. Thee experiment was repeated with a different substrate with similar results.

1 0 9 9

CHAPTERR 7

ACKNOWLEDGEMENTS S Fundedd by a Landsteiner Bloodtransfusion Research grant. We are grateful to Andre Hannemaa and Karel Eckman for their help with the activity measurements for Cl-Inh andd ATIII . We thank Maxim Kuil for his explanations of and help with the dynamic lightlight scattering and Ed Nieuwenhuys for his help with calculations.

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25.. Nuijens, J.H., Eerenberg-Belmer, A.J., Huijbregts, C.C, Schreuder, W.O., Felt-Bersma, R.J., Abbink, J.J., Thijs,, L.G., Hack, C.E. (1989). Proteolytic inactivation of plasma CI- inhibitor in sepsis. J.ClinJnvest. 84: 443-450. .

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