Two Clusters of Acidic Amino Acids at the NH2- Terminus of Complement Component C4 a'-Chain Are Important for C2 Binding

A thesis submitted in confonnity with the requirements for the degree of Master of Science

University of Toronto The author has grantecl a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant B la National Li'braiy of Caaada to Biblioth&quenationale du Canada de reprduce, loan, distniute or sell reproduire, pdter, disûiiuer ou copies of this thesis in microform, vendre des copies de cette thèse sous pper or electronic formats. la forme de microfiche/fb, de reproduction sur papier ou sur format électronique.

The author retains ownership of the L'auteur CoIlServe la propriéte du copyright in tbis thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantiai exttacts hmit Ni la Wse ni des extreits substantiels may be printed or otherwise de celle-ci ne doivent être imprimds nptoduced without the author's ou autrem«it reproduits sans son pe~mission. autorisation. Thesis Title: Two Clustas of Acidic Amino Acids at the m-Tnminus of Complement Component C4 a'-Chaui Are Impatant fm C2 Binding.

Submitted by: Qunb Department of Biochemistry, University of Toronto Master of Science, 2000

Cleavage of complement component C3 into C3a and C3b is a critical step in the activation of the complement system and conseqwntly the role of this system in host defense. The readon is catalyzed by two homologous enzymes, the ciassicai and alternative pathway C3 coavertases, which bave the compositions C4b2a and CJbBb, nspectively. The goal of this thesis was to identify sequeaces in humaa complement component C4 that contriiute to the binding of C2. Revious mapping studies using monoclonal antibodies have suggested that the 738-826 segment at the NH2-terminus of C4 m'-Chain is involveci in the interaction with WBP, a iigand of C4b which is antagonietic to the binding of C2. Mapping studies on C3, puticularly using the sitedirected mutagenesis approach, have also identifid an acidic amho acid segment at the NHpterminu of C3 a'-chah as being essentiai fa the interaction with frctor B. the altemative pathway C2 andogue. Due to the fûnfiioctionrl similrrity between C4 and C3 and the sigpificant sequence similarity at the NBptermini of th& nspective a'-chains,this segment of C4 was therefore hypothesized to conmie to the binding of C2. In otder to test this hypothesis, sevd independent mapping approaches were employed including sitedirected mutagenesis, synthetic peptide mimetics and anti-peptide antibodies. As a resuit of replacing subsets of the charged residues witbin eitha of the two acidic amino acid clusters at the NHp terminus of C4b a'- (specifically 7~EED-749DEDD) with theù neutnl isatezic amides, teduced C2 binding llcfivity wu obsaved Using a synthetic peptide comsponding to the 740- 756 segment of C4 as a competitor with C4b for C2 binding, it was found that the peptide inhiited C2 binding to ceii-associated C4b in a dosedependent manner. Finaüy, it was show that rntiiy nised a~ainstUs peptide segment was able to recognize its epitope(s) in the con- of the intact C4b moleoule and to block the C4b-C2 interaction in a specific and concentration-dependent mamer. Taken together, the collective results from the three complementary mapping approaches sttongly suggest thaî the NHptemiinal acidic residue-rich segment of C4 a'-chaincontn'butes iniportsnîly to the interaction with C2.

Table of Contents

4mUTER 1 INTRODUCTION ,bb...... ,....b..b..,...... 1 1.1 GENERAL~NTRODUCJII:ON TO TEE COMPLEUENTSYSTEM ...... 2 1 Il Discowy of the @stem ...... 2 1.1.2 Functronul Signijicance of the System ...... e...... 2 13 CHEMISTRYAND BIOLOGYOF C4 ...... 5 2.1 Expression of C4 ...... 5 1.2.2 Smccnae and Biosynhesis of Cl ...... 5 1.3 STRUC~JREAND CHEMICALCHARACTERlZATION OF THE ?HIOESTER BONDOF C4 ...... 8 1.3. Stnrcftrre und Functibn of the ~foestsrBond ...... 8 1.3.2 Redon Mechuntm of the îRioester Bond ...... 10 1.3.3 Ch4micol Repctfons of the Tliiwstw Bond ...... 11 1.4 TKECOMPLEMENTA~~~ATIONPATHWAYS...... 14 1.4.1 The CIiusical Pathway of Cornplentent Acttvation ...... 14 42 ~ektinPu~oyofCo~IemmtActivation...... 16 1.4.3 Ilre Almative Patbay of Coinpement Activation ...... 17 1.4.4 TheFonnationoftkeMenibmneAtfrickCo~1plex~C)...... 18 1.5 CONTROLOF THE COMPLEMENTSYSIZM ...... e...... 20 5.1 F~1~t0rI-diured~odationondReguI~1tion...... 22 1.5.2 C4-Bindiing nOwin ...... 24 I.5.3 FCIC~O~H ...... 24 I .5.4 Cornplentent Rereptor 1 (C'/CD35)...... 25 1.6 SAdUW'MEs BerwePr THE CLASSICAL AND TIlE ALTERNATNE PATHWAYc3 CONVERTASES ...... 26 1.6.1 Aaenibly and Functfon of the C3 Co11yiert41sm...... 0...26 1.6.2 Homology of C2 and FitB ,...... 28 1.7 PROJECTRATIONALE ..~.....~...... 30 2.2.1 Rad10Iabelingof C4 ...... 36 2.3 CELL CULTUREMEDIA...... ,...... 37 2.4 S-C ...... 38

2.5 ..s~....*...~.rn.....***..-..~~~sem~e.~~~~~~~~~~~..~~~..e.....~.m..... *.0.39 2.5.1 Generation and MpCatbn of Anti-PqW &n'bodies ...... Ce....e...... 39 2.5.2 Meur~ursnerit ofAntisenun Ti- und @x@ities ...... 39 2.6 DNA -0DS ...... m...... C...... b...... 4 2.6. f Generatlon of Recombinant CI and Si&WctedMutagenesis ...... 40 2.6.2 Co~.0nofGSTGSTC4F~on&efn...... -...... 43 2.7 ~RESSIONAND~ANIITI'ATIONOFRE~~C~...... , ...... 44 2.7.1 TrangfectjOn of COSI1 mils ...... 44 2.7.2 MdlicI;abeIing Btosyntheric Chur(~~terizatdon.and Immunoprec@itation ...... 44 2.7.3 Quantitative Measurement of Skmted Recombinant C4 ...... 45 2.8 ~RESSIONOFG~T~~~~SIONPIW~EIN...... 45 29 FUNCTIONALASSAYS ...... 2.9.1 Clarscal Pathway-Dependent Hemolytic Assay ...... *...**.....46

2.9.2 Fhn'd-Phme C2-&pendentHemoiytic Inhibition hsay ...... O 47 2.9.3 Solid-Phase C2-Dspen&nt Henioiytic Inhibithn Assay ...... 47 2.9.4 SyntheticPepndeCollf~etitionhsoy...... 48 2.9.5 Antibày Blocking Assays ...... 48

3.1 T)IES~~E-DIRECTEDMUTAGENESISAPPROACH...... 51 3.1.1 Eigression andhduaton ...... 51 3.1.2 Hemo&tic Activicy ...... m...... 51 3.1.3 Biosyntditic Roctssr'ng and Smctptibiliv to Cleavaige by C% ...... 54 3.1.4 C2 Binding A&@ ...... 56 3 -2 THEGST&PEPTIDE FUSIONPROTEIN APPROACH ...... 61 3.3 THEPEPTIDE MlMenc APPROACH...... 63 3.4 TEBLOCKINGANT~BODYAPPROACH...... 65 3.4.1 Detsclion ofAntfsert~n lîms and 2@c@cifes ...... 65 3.42 Efect of Anti-Pqtii#e Anriaody on the C2-Dependdent Ass~~...... 67 3.4.3 Efiaof Ang-Pqtide Antfbd) on the C4bC2 Bidlig hw*uctron ...... 71

4.1 DrSCZfSSION ...... 76 42 CON~USIONSAND FUTURE ~CTIONS...... 82 List of Flgpns and Tables

Figure 1.1 The biologicalcoasequences of the activation of the complement systemc ...... 4 Figue 1 3 Biosynthetk processing and activation of human C4...... 7 Fi- 1.3 ChemidreactiopsofthethioestabondofC4...... 12 Figure 1.4 Themajapath~aysofcomplementactivati~n...... 15 Figue 1.5 Initiation, ampiifidon and deactivation of the altemative pathway ...... 19 Figure 1.6 Contml of the coqlement system by complement mguiatory proteins ...... 21 Figure 1.7 Activationandpmteolytic&gndPtionofC4andC3 ...... 23 Figure 1.8 Assembly of the classicai and eltemative pathway C3 and C5 convertases...... 27 Figure 1.9 Roposed binding sites within the 727-767 segment of C3 a'-chah identifiai by site- âirected mutagenesis ...... 32 Figun 1.10 Sequence alignment of the NH2-teminal @on of human C3 and C4 ...... 33

Chapter 2 Materiah and hlethoâs Figure 2.1 Site-directad mutagenesis by ovalap extension PCR ...... 41

Table 1 Nomenclraae and amino acid sequences of the 744-752 segment mutants examined in the present study ...... 52 Figure 3.1 Comparison of the hemolytic activities of recombinant wild-type and mutant C4 moldes..*...... 53 Figure 3.2 Biosynthetic processing and suscepti'bility to ~fscleavage of recombinant wild-type and mutant C4 molecules...... 55 Figure 3.3 Quantitative anaîysis of biosynthetic processing of recombinant wild-type and mutant C4 molecules ...... 57 Figure 3.4 Binâing of C2 to recombinant wild-type and mutant CryCH3NH2) molecules ...... 59 Figure 3.5 Relative C2 binding ability of recomôinrnt wild-type and mutant clyCH3NH2) mIecllles .*...... *...... Figure 3.6 Solubility of GST and GSTUMon peptide and binding of GST and GST-C4 Mon peptide to giutathione kads...... 62 Figure 3.7 Bindiag of C2 to C4 segment M*-Vapg expressed as a GST Monprotein bcmd to glutathione ôeads ...... 64 Fi- 3.8 Inhi'bitim of C2 binding to EAC4b by synthetic peptide...... *...... 66 Figure 3.9 Wonof the unîisemm titre ...... 68 Figure 3.10 Detectionof the untisenuntitre and bindiug specificity to ClyCHm?)...... 69 Figure 3.1 1 Iiitu'bition of C2dependent hemoiytic activity by mti-peptide anti...... 70 Figure 3.12 A Control Ibpednmt to Dctect Cmy Ovaof Anti-C47*7% fiom the Fht Set of EACQb Ceb to the Second Set ...... C.œ...... C..~...... 73 Figure 3.13 rphWonofC2bindingto EACQbby rnti-peptidernt'body-...... 74 vii

a2-r~croglobulin bovine senun albiimin methylaminetreated C4 a 9 kDa peptide cleaved by CTS ftom th m-teuninus of C4 a-chain C4 im A, isotype B mature 3chUn C4 after the removd of a 9 kDa fragment at the NH2- terminus of a-chah C4BP Wbinding protein CCP complement cormol protein CRI, cR2,CR3 complemmt receptor type 1, type 2, type 3 DAF âecay accelerating fador dNTP deoxynucleotide triphosphate DMEM Dulbecco's modifieci Eagle's medium dithiothreitol hemolysin-sensitued sheep erythrocytes hemo1ysin-sensitized sheep erythrocytes bearing coqlement camponent Cl hemolysin-sensitizeâ sheep aythracytes beriring complement wmponent C4b hemolysin-sensitized sheep erythrocytes bearllig complement component C4b and C2 HRF homologous restriction facta m isaptopyl ~IEthiogalactopyranoside KLH keyhole limpet hemocyanin LHR long homologow repeat MAC membrane attack complex MASP MBGassociated protease MBL mannose binding pteh MCP membrane cohctor protein MHC major histocompaûiility cumplex MIRL membrane inhiiitor of reactive lysis OW2 iodine-oxidized C2 PBS phosphate bufféred saline PCR polymense chah naction PGA pyroglutamic acid PMSF phenylmethylsulfonyl fluaride rC4(a3NH2) methylaminetreated recombinant C4 RCA RIA RT SCR SDS-PAGE sodium dodscyl sulfate polyac~ylamidegel elecarophoresis SGVB SVB containing gelatin SLE systemic lupus erythematoms SRBC sheep red blood ceil SVB low imi smngth VBS containing sucroee TCA trichlOrOBCdic acid VBS vetoaalbufferedsaline vWA von Wilii'btand factorA Chapter 1

Introduction 1.1 Cenerai Inttoduction to the Complement System

1.lm 1 Discovery of the System

In the late 19~cenhay, complement was discovered as a heat-labile component present in fresh noanal serum that wuable to lyse bactena in viw meiffer, 1894) or in vitro (Bdet, 1895) in the presence of heat-inactivited immune senim. This substance was initiaily tenned alexin. Borda (1895) merdemonstrated that the lytic or bactericidal action of immune senun required two factors, a heat-stable factor present oniy in immune se- wbich was tenned sensitizer, now known as antibody, and alexin, a heat-labile factor present also in nonimmune senun, now known as complernent. Subsequent studies by Bordet (Bouldan. 1910) indicated that immune lysis also included the phenomenon of hemolysis of erythrocytes by immune senim.

In the 1910~~it was recognized that complement, far ftom king a singie substance. is a multiple component system (Ferrata, 1907). The system is made up of both insoluble euglobulins and soluble globulins present in normal sera of many animal species. Since then, more than 30 complement proteins have been identified due to the advances in protein chemise and immunochemistry techniques. The system is now hown to consist of a group of serum proteins and celi membrane proteins that intnaa in a complex cdemechanism leading to a wide variety of important biologicai mpon8es.

1.12 Functiond Sip*ficaaceof the System

Complement, as its name implies, was once considered to be augmenthg or complementing-the hemolytic and rntiôactenal activities of aatibody. When fiirther undersîanding of the complement system wu avaiiable, it was mognized thrt the system wur not mefeLy an accessory Wrbut a very important system in its own nght that played a leadhg role in immune &f- against infection.

To protect the host hminfdous pathogens, the complement system has to be activami because many key components in the system circulate in the plasma as fllnctionally inactive precmor' molecules. Activation of the complernent system te& to a wide variety of important biologicai responses (Figure 1.1). These nspunses inch& cdlysis upon the forniaton of membrane attack complex (MAC), immune complex clearance, complement-mediated viral neutralization, opsonization which may result in subsequent phagocytosis, release of various vasactive chernicals which induœ ianammatiioii, and modulation of humoral immunity.

The system caa be activated by three pathways, tedthe classical pathway, the le& pathway and the alternative pathway. Wbereas the classical pathway is primarily activated by antigen-anti'body complexes, both the alternative pathway and the lectin pathway can be initiated in an antiWy-independent manner, and are thus signifiant for eariy defense of the host againsi bactena and viruses. In addition to its role in innate immupity, the complement system also plays a role in the humorel fesponses of the adaptive immune system by replathg the activation of B lymphocytes (Fischer et al., 1996). Because of the importance of complement, an inhedted or acquired deficiency in rny one component of the system is frequendy associated with either an increased suscephiiiity to recurrent infections or antoimmune diseases that result Born diminished clearance of circulating immune complexes.

The fourbi component of humui complement (C4) plays a critical role in activating the clrssicai pathway as it is a subunit of the ciassicai pathway C3 coweztaiw. C4 ais0 has a mie in the BCfiVation of the altetnatiive pathway because C3b Wtedvia the clrssicai pathwry can act as a nidus for the initiation of the alternative pathway. Fucther evidence of its dtifbnctiopality cLnssxcnt(ci,cr,cz), LtCTIN(=/MMP, C4 C2) 0RALTtRNATIVE(B,DvC3) PATHWAX OF ACTIVATION

Activation of the classicai, latin or alternative pathway results in the cleavage of the third compnent of complement (C3)into C3a and C3b. C3a is an anaphylatoxin thrit is bound by a spccif~ccellular receptot and mediates an ian-tory response. Nmtlyactivatecl Qb hm the transient ab'ity to transacylate onto nuciwphilic acœptor surfhcm wkre it has mrny biological col~~quences.Fint, C3b can bind and modulate C.5 for cleavage, Idhg to the formaîion of membrane attack cornplex (MAC), which can effit CytbIysis. Second, dsposition of ab ai& in immune cornplex solubilizatxon and viril neuüaüzation. ThVd, Qb w be degraded by saine pmtmse f~tot1 in the pnsence of appiopfiite cofadot proteins into iC3b and =dg. Ubanâ iC3b are Ligaads for corn lement 1ioooptots type 1 (CRI) and type 3 (CR3), mFvely* C3b and iC3b together with tEeu ltctptors play a mijar dein opsonhation whch leads to nibeqmt pbgocytosis. C3dg b recopkd by cornplemtnt receptor type 2 (CW) and plays a ide in immmoregulation. can be seen in clinicai cases of C4 deficiencies, which msult in lupus-iike syndromes, kidney dysfhction, recumnt iafiztions and Unmnne coqladiaerses (ceviewed by Colten, 1992).

1.2.1 Expression of (34

C4 cuculates in human senim at a concentration of approximately 0.64mg/ml and is primarily produced in the üver by hepatocytes (reviewed by Alper and Rom, 1976; Morris et al., 1982). To a lesser extent, C4 is also synthesized by a vpriety of other cells including fibroblasts (Stecha and Thorbecke, 1967), macrophages and mononuclear phagocytes (Colten et al., 1979), monocytes (Whaiey, 1980; Kuiics et ai., 1990; Tsukamoto et al., 19920; 1992b) and epitheiial ceils (Feucht et ai., 1989; Zhou et al., 1993). The extrabepatic tissues that generate C4 include kidney, lung, spleen, brain and rnamrnary glands (Cox and Robins, 1988). It has ken reporteci that acute inflammatoty response, tissue injury, and treatmnit with intafetony, a potent mediatm of infiammation, nsult in a 2-3-fold inaecse in C4 plasma concentration (Johansson et ai.. 1972).

1.22 Smictun and Biosynthesis of C4

The genes encodiDg coinplaneot components C4, C2 and fr*or B are located within the major histocompab'bility complex (MHC) class Di region on the short ann of human chrmome6 (Carn,U et ai., 1984). In humans, C4 exists in two hinctional isofarms, C4A and C4B, and the genes codiag for the two C4 isotypes are separated fmm each other by about 10 kb on chromosome 6 (Campbell and Bentiey, 1985). Sequence dysisdemonstmted that C4A and C4B are highiy consend, there being more than 990h identity between the DNA nucleotide sequaces of the two genes and between the rmino acid sequences of the two proteins @elt a ai., 1985). C4 is by frr the most polymaphic of the complement pmteins. At least 35 distinct aiieles have been reveaîed by biochemical or inmnuiologicaî tests (Carroli and Alper, 1987).

The primaiy etructun of C4, deduced from the cDNA sequence, consists of 1,722 Pmino acids (8elt et al., 1984) and has been characterized as a giycoprotein of-205 kDa cansisting of three ditmifide-linlced polypeptide chains: a (95 kDa), P (75 kDa) and y (33 LDo) (Schrn'ber and Müller-Eberhard, 1974; Gigii et al., 1977). C4 is initially ttanslated as the single chain prrciasor, preprOlC4. The order of the chains in the precunror is NH2-~-a.y-CûûH(Goldberger et al., 1980; Goldberger and Colten, 1980; Parker et al., 1980; Carroll and Porter, 1983; ûgata et al., 1983) (Figure 1.2). Foliowing the removai of the signal peptide upon translocation to the endoplasmic reticulum, pro44 undergoes Merpost-translational processing involving glyoosylation (Ibhtthews et d., 1982; Chan and Atkinson, 1985), formation of in- and inter- chain diwilfide bonds (Schm'kr and Müiier-Eberhard, 1974; Gigli et al., 1977) and genexation of an intemal thioester bond (Karp, 19830). Mer king transporteci to the Golgi apparatus, pro- C4 is sulfated (Karp, 1983b), two tetrs-basic peptides linking and ay are cleaved by an endoprotease which is likely a member of the furin famly (Goldberger and Colten, 1980; Oda, 1992) and is fiiialîy secreted iato the plasma as mature multichain C4. Following secretion, the protein is Mer processed by the rmuwal of a dCOOH-terminal hgrnent of 5 kDa of the a-chah by a plasma prote85e (Kerp et al., 1982; Chan et al., 1983). It was demonstrated that the secreted and the plasma foms of C4 possess similtu hemolytic activity (Chan and Atkinson,

1984). C4 caculotes in the plusma as an inactive -en. I)uring activation of the classical pathwry, proteolytic cleavage in the a-chiin of C4 by ds yields a large C4b fiagrnent (191 kDa) and a smaii C4a fhgmnt (9 Da), tbe latter king a weak anaphylatoxin (Schreiber and MWer-Eberhad, 1974). The C4b fragment possesses stable interaction sites for other complement protein ligands as weii as the traasient ability to home cavalently attacheci to the surfie of the target ceii or immune cornplex. The covalent bindiag is mediated via an intramolecular thioester bond prisent within the C4d region of a-chain. Details of the stniapn . . a-chah (93 kDa) - COOH W.tur8 C4 I

C4 is synthesized as a sin& chria pre-pmC4 preculsot molccule. Witb the endoplasmic nticulum, the signal peptide is nmoved, four high-niannose oligosacchacides are added, inter- and intra- chah disuifide bonds as well as an htmmolecular thiomter bond are formel. The and bction of the thioester bond are descnied in Section 1.3 in pater detail. Currently the thteedimensional stwtm of C4 is iinhiown.

1.3 Structure rnd Chernicd Chuwctetlon of the Thbester Bon& of C4

1.3.1 Structure and Function of the Thioester Bond

Complement components C3, C4 and CS play a central role at key amplification steps in the complement activation pathways (reviewed by Law and Reid, 1995). It has been demonstrateci that these three complement components and the semm protease inhibitor a?- mricroglobulip (a2-M)constitute a family of proteins that are closely related stnicturally and are encoded by genes that evolved fkom a commoa ancestor (Sottnip-Jensen et al., 1985; Cempbeii et al., 1988). More impomtly, C3, C4 and al-M shan an identical intrachain thioester smicnire, wwhh enables them to bind covalently to target molecules (Tack et ai., 1980; Pangbum and Mûller-Eberhard, 1980; Campbel et al., 1981; Sottnip-Jensen et al., 1981).

The thioester bond pnsent in C3, C4 and a2-M is formed between the suifhydryl pup of cysteine and the carbonyl pupof glutemine. These two amino acids are separeted by only two residues, glycine and glutamate, in the primary stnicture (revieweâ by Tack, 1983). In native C4, the intemal thioester bond is fodbetween Cys991 and 01@ in the C4d region of a-chaint (Janatova and Tack, 1981). The mechanism through which the thioester is formed during biosynthesis remains uncertain as the Iiteratwe contains evidence both in favour of spontaneous thioester formation (Pangbum, 1992) and in favour of protein facilitrted thioester formation (Auetbach et ai., 1990). The covalent attachment of complement proteins C3 and C4 to their mget acceptm is a critical step in the çleamce of pathogens ('ewedby Law and Reid, 1995). In the case ofC4, the integrity of the thioester bond is essential for both mediating the covalent binding and maintaining the native confi*onaistate of C4 (rm,1983; Israc a al., 1998).

It has ban shown that upon proteolytic cleavage of C3 to C3b and C4 to C4b, a conform~~tio~change occurs in these moledes @senman and Cooper, 1981; Isenman and Kelis, 1982) such that the intramolecular tbioester bond becomes exposed on the surface. The solvent-exposed thioester bond cm be attacked by nucleophiles on target molecules or hydrolysecl by water (Law and Levine, 1977; Law et al., 1979; 1984; Hostetter et al, 1982; Isenman and Young, 1984). Consequently, a covalent bond is fodbetween the acyl group of the thioester and the adno or hydroxyl groups of the target. Following proteolytic activation, C3 and C4 undergo pronounced conformational changes, which ultimately result in the acquisition by C3 and C4 of numerous protein interaction sites that are not present in the native molecules. Covalent attachment also occurs in a sllnilar manner between a2-M and the activating protease (Howd et al., 1983).

The thioester proteins show sipificant differences in their reactivity and binding preferences. Studies using isolatecl complement proteins have shown that the C4B isotype of human C4 and C3 predomhantly form ester bonds with hydroxyl groups on carbohydntes, whems the C4A isotype and a?-M primarily form amide bonds with proteins (Dodds and Law, 1988; Ren et al., 1995). The chemicai mechanislfls of the reaction on descri'bed in Section 1.3.2. Mirent nactivity may leaâ to diffaent mles of the C4A and C4B isaypes in the elimination of

Merént pathogenic targets. For instance, using the carbohydrate-rich sheep red ceîî as a target, the C4B isotype bas ban show to be 34fold more hemolyticaiiy efficient than is C4A @&muanand Young, 1984; Law a al., 1984). In contra& at least in assays using punfied protein, the C4A isotype shows greater covalent bindiag to imniunog10buii.n in immune complexes (Law et al, 1984; Kishae et ai., 1988). However, more ment studies in senun fül to show as large a difference between the human C4 isotypes in binding b immune complexes, mggesting that other sem proteins may as weil affect the bindhg propensities of the C4 isofamur (Reilly, 1999). Neverthelesa, thae remiins the obsewation that men partial âeficieiicy states of UACO* susceph'bility to the development of the immune complex disease systemic lupus erythematowui (SLE) (Kemp et al., 1987; Briggs et al., 1991), although there is no definitive proof that it is the ciifference in the covalent binding properties of the isotypes of human C4 that is the causative factor. l Redon Mecbaaism of the Thioester Bond

Demonstration of the binding specificity of the human C4 isotypes provided important dues to ceveal the reaction mechanism of the thioester bond. Sequence anaiysis showed that C4A and C4B differ by only four amino aciâs between residues 1,lO1 and 1,106, which lie -100 residues COOH-terminal to the thioester site, suggesting that these residues are responsible for the binding specificity (Belt et al., 1984; Yu et al., 1986). By ushg sitadireaed mutagenesis, it has been established that the key residue is at position 1,106 (Cadi et al., 1990; Sepp et al., 1993). Molecules with His at position 1,106 (C4B) have C4B-lüre covdent binding properties, whereas those with an Asp (C4A), Asn or Ala are C4A-me. Sequeaces of the isotype region of C4A and C4B, as well as the corresponding region of C3 and a2-M (Belt et ai., 1984; de Bnlljn and Fey, 1985; Kin et al., 1985) are show below. The covalent binding between C3 or C4 and acteptor molecules involves a transacylation reactioa whereby the thioester cuboayl ûansacylates ont0 an acceptor molecule nucleophile. However, it is now known that the donrnechanism of C4B and C3 is différent fkom dut of C4A and pfobably a2-M. h the cese of C4A, which contains a non-nucleaphilic midue (Asp) at position 1,106, the thioester mcts directly with target nucleophiles. Thenfore, the naction with the mon nucleophiiic amino gmups is âornin~tand hydrolysis is nlatively slow @odds et al., 1996). If the residue at position 1,106 is nucleophilic (such as His in C4B and C3), it has been determined that the binding reaction consists of a two-step catalytic mechanhm that facilitates both hydroxyl group trmsacylation and rapid hydrolysis @odds a al., 1996; Law and Dodds, 1997; Gadjeva a al., 1998). Specifically, upon activation, histidine 1,106 first attacks the thioester carbonyl and fom an acyl-imiâazole intermediate. The released thiolate anion of the cysteine nsidue then acts as a general base to catalyze the second step of the reaction between the acyl-imidazole intermediate and the acceptor hydroxyl-bearing molecule. The proposed reaction mechanisms are consistent with the observations that the wlife (T112) of activated C4A is about 10 s and whereas that of C4B is very short (cl s) (Sepps et al., 1993). These mechanisms ensure a relatively rapid hydrolysis of the thioester bond and thus the deposition of C4b is ümited to the site of activation.

1.3.3 Chernical Reactions of the Thioester Bond

Besides the covalent binding reaction, a the compebing hydrolysis mction descri'bed above, conformational changes pmduced by 6scleawge resuit in the rcquisition by C4b of stable interaction sites far protein Ligands such as C2 (Mililex-Eberhard et al., 1%7) aad C4 binding protein (CIIBP) (Gïgli et ai., 1979). The majority of nascent C4b is hydrolysed and nmiins in the fluid-phase and whiie thîoester-hydrolysed C4b can no longer attach to a met, the fluid-phase molecule ntiins the interaction sites for coqlement ligands such as C2 and

Figure 1.3 Chemid mctiona of the Ubcricr bond otC4. U n proteolytic cleavage of native C4, the C4a peptide is released hmn the NH2-tenninu OF" the a-chain and nascent C4b undergoes a conformationai change which rendete the inüamolecular thioester bond highly susceptible to nucleopbilic attack. The transiently activatecl thioester bond cm mediate depmFtionof C4b onto the hydroxyl or amino groups of target duesby üansacyhtion, whereas the mjority of ment C4b is hydrolysed and temains in the fluid-phase. The thioester in native C4 cm be attacked by small nucleophiles such as methylamine, or chaotropes such as KBr may mediate its direct hydrolysis, leading to the fimnation of thioester-cleaved but a-chain-mtact C4 decules. These molecules are termed C4b-like molecules because the conformation and binding interactions of these molecules resemble those of C4b. Iii the wesence of denaturanc heating of native C4 leads to an autolytic cleavage in the acboin, which nsults in the eneration of two autolytic fkagments of 54 kDa and 40 kDa, respectively and the !ornuton of a pyroglutamic neid (PGA) n the N-terminus of the 54 kDa hgment. It has ban iliustrated that treatment of native C4 with nucLeophiles (methylamine or hydraplle), chaotropes a fepeated &ePpg and thawing leads to a conf'tionai change similar to that see~afk proteolytic activation by 6s(Janatova et al., 1980; Janatova and Teck, 198 1; von Zabern et al., 1981; 1982; Xsenman and Kells, 1982). Ptoducts of such treatment are thioester-bond-cleaved but a-chah-intact C4 (Figure 1.3). These molecules are tamed C4blike molecules in view of their coiifonnational and hctional similanty with C4b. Isenman and Kelis (1982) firrther showed that methylamine-treoted C4 (CX(CH3NH2)) L aimost equipotent with C4b in its ability to bind C2 in the fluid-phase, although the rate of the nucleophile-induced codo11118tional change was much slower than tbat resulting fkom proteolytic cleavage.

As an indication of the bigh reactivity of the thioester structure, heating of native C4 in the presence of denaturapt wili cause a portion of the molecule to undergo an autolytic cleavage reaction (Davies and Sim, 198 1; Sim and Sim, 1983) (Figure 1.3). In this reaction the carbonyl group of ~in994is attacked by the peptide bond nitrogen of the same residue, leading to the fodonof a fivemembaed imide ring (pyrrolidhe carboxylic acid) and the release of a thiol. Hydrolysis of the imide resuits in the cleavage of a-chain and two autolytic fkagments of 54 kDa and 40 kDa respectively are generated with the f-tion of a new pyroglutamic acid (PGA) at the NH2-terminus of the 54 kDa fhgment.

The sections above have discussed the biochemistry of C4, since this is the mejor focus of my thesis project. However, to be appdated in the context of the complement system as a whole, and especially to highlight the paraiieiisms between the roles of C4 and C3 as subunits of the ciassicai and aitemative pathway C3 conveftases nspeaively, the foilowing Secfions proYide a more generai ovenriew of the complement pathways and th& ngulaton. 1.4 The Completnent ACavatbn Pathways

Complanent is a oomplex mixrRrn of plaama proteins which are activateâ sequentiaiiy, many by conversion of a proteolytic ymogen into an active proteinase. There are three pathways by which the eeector fiinctions of complement cm be activated. nie interacting pathways are tamed the classicai, the lectin md the altemative pathways, respectively. The end product of each activation pathway is an enzyme capable of cleaving C5 (CS convertase). Advatad CS then initiates the terminal route of the complement system, which le* to the fmtion of the membrane a- colaplex (MAC)(Figure 1.4).

1.4.1 The Classical Pathway of Coqlement Activation

The classical pathway is primarily initiated by the interaction of Clq with anti'body- antigen aggregates and is composed of four pl- proteins, C 1, C4, C2 and C3. Many other substances, however, in the absence of antibody, such as DNA, mitochondrial membranes, C- reactive protein and some extraceliular matrix proteins are als~able to react with Clq leading to the complement activation (reviewed by Volanakis et al., 1990).

The f?irstcomplement component (Cl) (740 ma) contains one molecule of Clq and two rnoIecuIes each of Clr and Ch,association of the components rrquiring calcium ions (reviewed by Arlaud and Theilens, 1993). Faanti'body-dependent activation, the initiai step involves the binding of two or more globuh herds of Clq to the Fc portion of the imniiinoglobulin, resuiting in distortion of the Clq colagen-iike stem region @mon, 1993). The distortiond change dCCULnLIg in Clq i tmsmitted to the serine protmse praenymcs Clr and Cls which are bound to the Clq stem portion. This results in prirwise ymogen aidoactivation of CIr wWn the Clq(Cla(Cls)2 complex, and then C!in bim cle8ves both Cls subunits (reviewed by Co1cunb The classical pathway of complement activation can bc initiated by both antibody-âepcndmt and antibody-independent mechanians, and is a ascsde of reactiom involving complement components Cl, C4 and C2. The ldnactivation pathway is initiated in an antibody-independent msnner and involves MBUMASP, C4 and C2. The altemative pathway of completnent rtivation is initiated by an antibody-inâcpenht mechanian and involves complement components frdor D,factor B and C3, which fulfffl ml- that am ~aiogousto tbeir classical pathway counterparts Cls, C2 and C4, nrpectively. AU thpathwa s muit in the formation of thtir respective C3 convertase complexes, C4b2a and QbBb, whic1 cleave Q into CJa and C3b. Activated ab binds to C4ô2a and QbBb, ldhgto the eneration of CS convertase complexes. C4b2a3b and QbBb3b, rrspectively. These complexes f iad and modulate CS to dow its clervage into C5. by proteases C2a or of CS the the and C5b serine Bb. Activation initiates rursembly7 of terminal complement compontnts. Cm,into the membrane attack complex (MM). et al., 1984; Schumaker et ai., 1986; 1987). Activated Cls (as) thus acpuins protease BCtivity towmds complement component C4.

CTS cleaves C4 in the achain, releasing a 77 amho acid NHpterminal fragment, C4a (Schrei and MüIIer-Eberhard, 1974). The major cleavage fiagrnent C4b is extnmly mctive with nucleophiles and cm bind fidy to suitable surfaces (Law and Levine, 1977). Upon activation, C4b acquires the ability to bind C2 in the pnsence of magnesium ions. C4b is also capable of interacting with nasceat C3b, with CS, and with the complement regdatory proteuis C4binding protein (C4BP), complement receptor 1 (CRI), membrane cofactor protein (MCP), decay accelerating factor PAF) and factor 1(reviewed by Lambris et al., 199%).

C2 (102 kDa) is a saine protease proenzyme and consists of a single polypeptide chah Binding of C2 to C4b leads to the cleavage of C2 by C& into C2a (70 a),which expresses protease activity, and C2b (30 kDa), a non-catalytic fragment (Polley and MùUer-Eberhard, 1968; Kerr, 1980). Foilowiag cleavage, C2a remains non-covalently associated with C4b to fom a C4b2a complex, the classical pathwry C3 convertase (hm, 1980; Mliller-Eberhard a ai., 1967). This complex then cleaves C3 to C3a and C3b. C3b subsequently binds covalently to C4b to fom a CS convertase C4b2a3b, as the C4b3b heterodimer provides a high ffiity binding site for CS (T'beeet al., 1987).

1.4 The Lectin Pathway of CompIement Activation

Recently, it has been recognized that molecules der than Clq also participate in activating the comp1ement system, one of these being mannose-binding le& (MBL). MBL nsembles Clq in stnicture, hoving giobulor h& and coiîagenous regims, md is able to wibstitute for Clq in complement activation. MBL recogeizes terminai mannose and N- acetyIgiucoaunine stn~amson yerst, brctai. and vimses via its globular hetde which coosisi of &+depen&nt C-type lectin carbohydrate recognition domains. Such taminal sugars are rare in the hota and thus MBL does not bind to host tissue. The coliagen-lile segments of MBL are aseociated with a serine protease cailed MEIGassociated serine pcote88e (MASP), which is amicturally sllnilar to Clr and Cls. Upon activation, MASP acts like ds to cleave Cs and C2, resuiting in the fomution of C4b2a complex having C3 convertase activity (Matsushita and Fujita, 1992; Matsushita et al., 1993). It should be noted that MBLMSP triggers the compiement system in an anticbody-independent manner, which enables the ho& to geoerate inmediate defense against invading pathogens.

4 The Alternative Pathway of Complement Activation

The alternative pathway of complement activation provides a natural defense system against rnicroorganiisms and pathogens which operates independently of specific antibody. The pathway can be activatecl by bacteria, Wuses, parasites, virus-infected cells and both eumyotic and procaryotic ce11 producfs (reviewed by Taylor, 1993). The alternative pathway activates the complement syetem in a marner simiiar to the ciasticai pathway and invoives proteins that ue analogous to their classical pathway counterparts, namely fmor B (93 LDa) (the C2 analogue), facta D (24 kDa) (the ~fsanalogue) and C3 (185 ma) (the C4 analogue).

It has ken generally accepted that the alternative pathway is continuously uudergoing spontaneous activation at a slow rate (reviewed by Pangburn and Müller-Eberhard, 1984). This regults ftom C3 king able to undergo spontaneous thiaster hydrolysis, which yields an active but uncleaved C3b-like structure, tamd Q(H2O) (Isenman et al., 1981). In the pnsence of mageeshm ions, this molede binde to haor B in the fluid phase, rendering it susceptible to cleavage to Ba and Bb by serine protease facta D. This reaction leads to the formation of a fluid-ph C3 convertase C3(w)Bb, which is able to cleave C3 to C3a and C3b. Nmnt

C3b can then k deposîted on nucleaphilic acceptor surfoces by ûansacylibion and thefbmi surface-bound C3 convatase, C3bBb. Hence an ampüfication lcmp is famed becawe once C3b is deposited on a 8Ul'f&ce, it has the potentiai to fonn a new altanative pathway C3 convertase and in this way generate large quantities of C3b (Figure 1.5). The CS convertase of the aitemative pathway (C3bBb3b) is subsequently famed by the bhding of a C3b molede to the surfiicebound C3bBb.

It has been established that activation and amplification of the aitemative pathway is subject to a control mechanism: whether amplification or inactivation occurs depends on the nahue of the daceto which C3b is attached. It is important to mention that upon cleavage, C3b undergoes conformational changes and thereby aquires the capacity to bind multiple ligands, including CS and factor B as described above and some regulatory proteins such as factor Ef, f8ctor 1, C4BP, CRI, MCP, DAF and properdin (rwiewed by Lambris et al., 1998). Whereas facta H, fmar I, C4BP and properdin are soluble saum proteins, CRI, MCP and DAF an membrane-associated molecules found on host tissue and blood celis. With respect to the propagation of the complement pathways on a surface, the mechanian of discrimination between invading targets and seEtissues is accomplished by the action of these regdatory proteins. The biiiding specificities and biologicaî activities of the various complunent control proteins are discussed in Section 1.S.

1.4.4 The Formation of the Membrane Anack Cornplex (MAC)

The ossembly of the terminal complement components is initiated by the CS convertase- mediated formation of CSb, and then proceeds via the sequential assembly of CSb with components Cd, C7,Cû and C9 (reviewed by Müller-Eberhard, 1988). These Wonslead to the generatim of a heteropol~*ccompiex, referred to as the MAC, which has the capacity to C30)*C C3(H2O) - B Ruid-Wase Altemative Pathway C3 Convertase C3

factor 1 + COfiactor ,<--*D iC3b

Surfkce-Bound Alternative Pauiway C3 Converîase

Alternative Pahtway CS Convertase

Initiation of the aiternative activation pathway occun in the fiuid-phase whsa a thiosster- hydidyzed C3 decule, C3(H20), Vm& and mddates a moiecule of factot B to lowits cleavagt by factor D. Tbi, msuits in the formation of a fluid- hase alternative pathway Q convertase, C3(w)Bb. Q(H2O)Bb clcaves C3 to C3b, wbicE is able to deposit mdomly onto nucleophiüc acceptai nidim by transacylation. Tha C3b can then p on to interad with factor B and the proteo1ytic cleavage by fwtor D leads to the formation of sWface-bou11d aitemative pathway C3 convertase, QbBb. Erh Qb deposited onto the activatin suifaa bas the poteritid to fom a new C3 convertawe, wbich subsequentiy gentrates new Qf moIecuies. thus forming an amplification loop. Priapagation and deactivation of the altedvt pathway is controiied by the complement regdatory pmteins. 15 Contn,l of the Compïemtllt Syrbm

Activation of the complement system initiates a seqwnce of biachemid nrctions, each component activating the next in a cascade manner. This cascade mechanian allows considerable ampiüication to ocmin the system, Le., a single activation step CM lead to the production of numemus active molecules. In ordet to prevent excessive activation and damage to host cells, a stringent control mechanism is essential. Multiple inhibitory proteins have evolved to influence and modulate the activity of the enzymes.

It bas been demon~eatedthat the C3 and CS convertases are controiied in three ways. First, the subunits with protease activity, C2a and Bb, spontaneously dissociate hmC4b and C3b and do not re-bind. In th& free fonns, C2a and Bb no longer have significant proteolytic activity for their nomial protein substrates. The second control mechanism involves proteins with decay-accelerating activity, namely C4BP, futor H, CRI and DAF. These proteins cm bind to the C4b2a or C3bBb complexes and melerate the dissociation of their respective serine protease moieties. Thini, C4b and C3b can be irreversiôly inactivated by the serine protease factor 1. The action of factor 1requires the presence of protein cofactors, including soluble proteins C4BP and factor H, and membrane-associated proteins CR1 and MCP (Figure 1.6). This group of proteins are membas of the regulators of complement activation (RCA) fdy.

The RCA femily consists of a homologous group of proteins that are encoded at a single chromasoonrl location (chtomosorne l), the RCA cluster (Reid et al., 1986; Campbell et al., 1988). These proteins are composed mainly of a tandemly cepe8ted motif of approrcimately 60 amino acids, which is characterized by the conservation of approximntely 10-15 residues and is tamed short consensus mpeat (SCR)a amplement control protein (CCP). --mm>C3 C3 c5 C6-9 CONVERTASES CûWERTASES C5 -b CSb

The complsmeot conûol pmteins shown in bold &ers regdate the activation and degradation of the complemcot system by either ptevcnting the formation, mlmting the dccay, or in combination with fstor I. ineversibly de@ng C3 and C5 conv~mof the beccomplement activation pathways. The fiiaction of factar 1, and cofBctors C4BP, fhctor H and CR1 that are of importance to the project rationale of this thesis are dimssed individuaiiy in Section 1.S. 1- 1J -4. i .S. 1 Factor 1-rriediated Degradation and Regulstion

Factor 1(88 kDa) is an active saine proteinase in plasmi consisting of a heavy ch& (50 kDa) and a catalytic iight chah (38 Da) (reviewed by Sim et al., 1993). With the aid of cofactors, factor 1 can inactivate C3KS convertases by cleaving C4b or C3b into smaller fkagments. Nagasawa a al. (1980) demonsüated that factor 1was capable of cleaving C4b to an intermediate iC4b, followed by merdegradation to C4c and C4d (45 kDa) (Figure 1.7). Similarly, fmor 1 cleaves C3b to iC3b and C3f (Davis and Hurison, 1982). Under certain conditions, iC3b is fûrther degraded into subhpents C3c and C3dg (Ruddy and Austen, 1971; Gitlin et al., 1975; Davis et al., 1984) (Figure 1.7). CRI is the only cofactor that can mediate this so-called thûd factor I cleawge under physiological conditionsybut at low ionic strength, factor H is also an acceptable 1-co&ctor for this cleavage. Factor 1-mediated cleavage thezeby reguiates complement activation and prevents the over-expression of C3 and CS convertase enzymes. It should be noted that two of the degradation products of C3b, narnely iC3b and C3dg, are ligands for the Ieukocyte complement receptors CR3 (iC3b) and CR2 (both iC3b and

C3dg). CR3 on phagocytic celle wich as neutrophils and macrophages mediates aîtachment and clearance of iC3b opsonized antigen by the phagocyte. CR2 is present on B aUs and fouiculat. denciritic cells and the interaction of iC3WC3dg cdmtigen with CR2 plays an impatant role in the generation of an optimil antii'body nsponse a-st the target dgen(reviewed by Carroll, 1998). if if -+ ww,- CL,8 4' IIP 4' Fipm 1.7 Activatton and proteoiytic degradation of CI and C3. Activation of native C4 by the serine rotease asreleases the C4a peptide from the MI2- terminus of the achain, and rduces CS b, which is composed of the a'chain disul hide-liked to the and y-chins. Factor P in the presence of a rqnate cof8Ctors iacluding Cf BP9 CR1 and MCP, clcaves the dehain of C4b to produce iC4r Factor 1 ean fùrther degrade iC4b into Cb and W. Similady, activation of peptide from the NH2- terminus of the achain, a'-chah disulphiâe-linked to the B-chain. Factor 1, in including factor H, CR1 and MCP, cleaves the roduce iC3b. Factor 1 can merdegrade of mediating this futor I cleavage under phy C4BP is a large piasrna protein (570 kDa) consisting of men identicai a-chahs and a unique Bohain linked together by disuifide bridges (Scharftaein et ai., 1978; Hilîup and Dahlbiick, 1988). The a- and B-chahs contain eight and three CCP modules, respeaively . (Dahlbiiclt, 1991). Electron microscopy and X-ray mering studies have show11 that C4BP bas a spida-like ofghtion with a central con and the seven a-chains formiag extended tentacles (DahlbW and Mûlier-Eberhard, 1984; Wb&& et al., 1983; Perkins et al., 1986).

C4BP bas the ability to intemt with C4b. It has been shown that C4BP cm trap nascent C4b and prevent the ab42interaction, thenby dm-regulating the formation of the classical pathway C3 convertase (Viiiiers et ai., 1982). C4BP also accelerates the decay rate of the C4b2a complex and mediates the cleavage and inactivation of C4b by factor 1 (Gigli et al., 1979; Fujita and Tamura, 1983). Additionally, C4BP has been shown to meas a cokctor to factor 1 in C3b degradation (Nagasawa and Stroud, 1977). Recently, it has been suggested that a patch of positively cbuged nsidues, at the interface bmeen the a-chah CCP1 and CCP2 domains, plays an importaat role in the interaction between C4BP and C4b (Villoutreix et al., 1998).

Factor H (155 kDa) is a plasma glycaprotein comprishg 20 CCP domains (Ripoche et ai., 1988; Bo& a ai., 1995). X-ray scattering data indicate that fscta H, in solution, is a long extended molecule of 77-87 nm in length (Pkins et al., 199 1).

Factor H is a veqr important reguiata of aitemative pathway activation. Binding of factor H to C3b inhi'bits the interaction of C3b with hctor B and CS, and promotes the dissociation of the alternative potbway C3 and CS mvettases (Pingburn md MW-- 1978; Whaley and Ruddy, 1976; Isenmzul et al., 1980; Pangbm, et al., 1977). The ability of the alternative pathway components to disctiminnte mets as either activators or non-activators is mediated by diffemt binding properties of fhctor H to dace-bound C3b molecules. It has been qorted that fgota H has an apparent affinity for C3b bound to non-activators 8-10 times gniter thrn that fa C3b ôound to activators (HOtstmZLIlll et al., 1985). In aâdition, fMor H acts as r cofactot for the factor 1-rnediated degradation of C3b. hie to the genetic homology and firnctionai simiisnty, factor H can be considered as the altanative pathway analogue of C4BP. Mousstudies have suggested that faorH reacts with at leut two sites in C3, one in the C3d region and one near the NR2-temiinus of a'-chah (Lambris et al., 1988; Koistinen et ai., 1989; Oran and Isenman, 1999). On the f8Cf0r H side of the binding interface, it has been shown that segments coasisting of SCRs 14,640 and 16-20 aîl bind to C3b (Shomu and Pangbum, 1996) although only SCRP 1-4 are reqyired for cof-r activity. Additionally, it is lcnown that SCRs 7 and 20 contain binding sites for pdyanions (Blackmore et al., 1996; 1998). It is believed that the latter sites promote bindiog of factor H to C3b on sialic acidiich sufaces, thereby accounting for the long standing observation that sialic acid-rich surfaces are poor activators of the aiternative pathway .

1.SA Complement Receptor 1 (CRIKD3 5)

CRI (160-250 kDa) is a polymoqhic glycoprotein that binds C3b and C4b strongly and C3c and iC3b weakiy (reyiewed by Lambris, 1988). CR1 is membme-assocjated and is present on a variety of cells includllig erythtocytes, polymorphonuclear Leukocytes, monocytes, manophages and denciritic celis (Fearon and Won& 1983). It hm been shown bat CR1 contains 4 long homologous qmting (LHR-A,B, C, D) units which are each comprised of 7 SCRs (Klickstein et al., 1987). The C4b and C3b bindiiig sites in CR1 are distinct. Wh- C4b hm a single bhdipe site mim'maHy consisting of the first 3 SCRs of LHR-A, there are two eqirivalent binding sites for C3b located reapectively within the first 3 SCR U13its of ïXR-B and LEIR-C (rwiewed by Ahem and Rosengwd, 1998).

The biologid eff'of CR1 are diverse and depend on the naaire of the ligands as weii as on the ce11 type expnssing the receptor. CRI is able to increuie the decay of the C3 convertase or act as a cofactor fot f8Ctor 1 in both pathways (Iida and Nussenzweig, 1981; Weismpn et ai., 1990). However, it is believed that the major role of CRI, particularly on erythrocytes, is to prome the clearance of C3bspsonized immune complexes by transporting them to macrophages that reside in the tiver.

1.6 Stmgiritïes Betweea the Ciadcal and the Alternative Pathway C3 Convertaws

C3 haa been shown to hold a key position in the complement system because its protedytic cleavage products result in a wide mge of biological responses (reviewed by Law and Reid, 1995). The cleavage of C3 is therefore a criticai step in the coqlement pathways.

1.6.1 Assembly and Funaion of the C3 Convertases

The activation of C3 is caîalyzed by two stnicturauy homologous enzymes, the classical and the alternative pathwry C3 convertases, having the compositions C4b2a and C3bBb, respectively (Figure 1.8). The activities of both C3 c~nvertasesare subseqyently modfied by the binding of rdditionai C3b to baome CS convertases. The subcomponent C2a acts as the catalytic eubunit fa the ciassicai pathway C3 and CS convertsses. Simünrly, the fkagment Bb provides the cataiytic subunit fot the ewalent C3 and CS convertases of the alternative pathway. In view of the fact that C2a and Bb are only active when bound to C4b and C3b, reqectively (MüUer-Ebeirbatd et ai., 1967; Vogt et 1,1975), one might consider that C4b and Claasicd Pathway Alternative Pathway C3 and CS Convertases C3 and CS Convertases

Covalent Bond

Figure 1J AssemMy of the durid and alternative pathway C3 and CS convertam. The classicai pathway C3 convertase is a complex composed of C4b and C2a. The binding of additional C3b to îhe C3 convertase resuits in the formation of the classical athwa CS convertase, a trimolecuiar CO lex CO osed of C4b, C2a and C3b. diie CL is noncovalently associated with C4"g , the CSm% inding site is composed of the covalentiy bonded heterodimer C4b-C3b. In the aitemative pathway of complement acîivation, C3 convertase is a complur composd of C3b and Bb. The bindiag of another C3b molede to the C3 convertase results in the formation of the alternative pathway CS convertase, which is also a trimoleculot complex composed of two molecules of C3b aad Bb. Again, Bb is noncovalentiy associated with C3b, while the alternative pathway CS binding sites are located on the covalently boaded homodimer C3b-C3b. C3b hction as wfBctors for the nspective enymatic activities of the classicai and alternative pathway CUCS convertases.

Regulation of the ciassical and aitemative pathway C3 and CS convertases is aIao vay simiiar. In the ciassicai and altemative pathways, factor 1irreversibly inactivates Wb and ab, respectively. C4BP and hctor H serve as cofactors for factor I-mediated degradation and accelerate the decay rate of the classicai and aitemative pathway C3 convatase, respectively. On menibnmes, DAF causes dissociation of Cilb2a and C3bBb and MCP seives as a cof8ctor for fa*ot 1in the regdation of both pathways (Nicholson-Weller et al., 1982; Seya et al., 1986). In addition, CR1 not only serves in both pathways to dissociate the convertases but also acts as a cof- for factor 1-mediated degradation. Therefore, in temu of the structure, fuaction and Ligand binding reactions of the proteins involved in each pathway, C4 of the classical pathway and C3 of the altemaiive pathway are homologues of one another.

1.62 Homology of C2 and Factor B

C2 (102 Ha) and factor B (90 kDa) are two glycoproteins in the human complement system which show both structural and functional simikity. They act as fiinctional analogues in the activation of C3 of the classical and alternative pathway, respectively. C2 and fwor B an each comprised of a singie polypeptide chah and share rrom common features in biosyntheds and p08t-translationai modification, suggesting that they folha similu biosynthetk pathway (Matthews a al., 1982). Tmsmidon electron mkoscopy studies suggest that C2 and faor B each consist of three discrste globuiar domains of approximately similar size (-40 A in diameter) (Smith et al., 1984). Fragments C2a and Bb each contain two domains connected by a short üaLa segment. Secpence compm*sonanalysis abows that the sequenœ simila+ity ôetween C2 and faaB extends aver the entire Length ofthe two proains and is 39?? in terms of identities and 5036 if one incIudes consenative residue replucements (Bentley, 1986). The Seqllence Mershows that C2 and fator B have a similor moddar structure consisting fiom Na-to COOH-terminus of three short consensus repe~ts(Sm) of the type found in the complernent reguiatary proteins encoded in the RCA locus a von Wihidfactor A (vWF A) doaipin and a serine protease domiin. The theSCR domains cover most of the C2b and Ba fkagments, respectively, whereas C2a and Bb are each composed of a von WiilibraDd A domain and a saine protease domain. The genes ooding for C2 and factor B have been mapped to the HLA class HI ngion on the short ami of hllrnan chromosome 6 and are less thaa 500 base pairs apart (Carroll et al., 1984; Campbell and Bentley, 1985). The close linkage of the genes suggests that they arose by gene duplication Rom a single gene encoding an ancestral molecule. Fiatber evidence is avaiiable fiom evolutiopary studies in phylogeneticaliy distant vextebrates, including iamprey (Nonaka et ai., 1994), zebrafish (Seeger et al., 1996), Japanese madeka fish (Kwoda et al., 1996), rainbow trout (Sunyer et al., 1998) and Xenop (Kato a al., 1995), where the molecules are equally nlated to the m.mmnlian factor B and C2 proteins and in the case of one rainbow trout BE2 isoform, the molwule has been shown to have fbctional activity in both the clessicai and dtemative paîhways.

The extensive simiiarities in structure9bction and sequence of the two proteins indicate their common role in ligand binding reactions. It bas been shown that the Netemiinal SCR domrins of C2 (ab)and hictor B (Ba) contain binding sites fot C4b (ûglesby et al., 1988) and C3b (Veda a al., 1987; Pryzdial and Isenman, 1987), respectively. In fragments C2a and Bb, the NH2-terminal vWF A domains have been suggested to be able to associate with moldes C4b adC3b, reqectively (Horiuchi et al., 199 1; Sanchez-Cod et al., 1990). This suggested that the initial binding of C2 to C4b or hictar B to C3b involves at least two distinct binding sites, one in C2a or Bb and one in C2b or Ba. A thirâ C3b binding site in -or B ha9 ken meppsd to the COOH-taminai serine protease dmuin of Bb portion (-ris and Millier-Eberhard, 1984).

(hi the otbn band, there hLs been no clear evidence suggestive of the Ubbinding activity of the saineptotersedomahofC2i. It is important to mention that pnor matment of C2 with iodine leads to enhanced stability and activity of the enzyme C3 convertase (PoUey and Müiler-Eberhard, 1967). This inrreased activity is the nsult of a tighter binding of C2a to C4b, which pfevents dissociation of the C2a and inmases the haElSe of the convertase (Kerr, 1980). The effet of iodine has been attniiuted to oxidation of the ftee thiol group of Cys241 (Padces et ai., 1983). This reaction nsuits in the nmoval of thiol Born the dace of C2 and subsequent accpisition by C2 of a conformational state most favorable for the interaction with C4b (Honuchi et al., 1991). (hi the 0thhand, factor B does not have an equivalent free thiol residue and iodine treatment of frctor B does not mect its hemolytic activity.

Given the special fiinctional significance of the classical pathway C3 convertase, we have been interested in Merunderstanding the biochemistry of this bimolecular entity and specifically in locaiizing a C2 binding site within C4b. In the absence of information on the th-dimensional structure of C4, our proposai of a candidate C2 binding site in C4 was @ded by avrilable inâirect evidence.

The first due to the locrlitatioii of a C2 binding site in C4b came hma moaoclonai anhisnidy. Ichibora and coworkers (1986) have shown that a monoclonal rntiiody raised against human C4b inhibits the binding of C2 as well as C4BP to C4b. Another monoclonal antiistudy suggested that residues Ala738-Arg826 at the NH2-tenninus of C4 aiChain are inqnut~tfor the inîeraction with C4BP (Hessing a al, 1990~1990b). These Snrdies therefore indirectly suggest that the NH2-teminus of C4 aB-cûainis involved in C2 biading, as well as CllBP biading. More indirect evidence came fnmn @es aimed at localipng ligand binding sites in C3, since C4 shares sequence and hctional similari*tywith C3 (mtewed in Becherer et al., 1989). Studies using synthaic peptides (GMU and Millier-Eberhard, 1985; Fishelwn, 1991; Becherer et al., 1992). anti-peptide antibodies (Bechaer a al., 1992), monoclonal ~tiies(Becherer et ai., 1992), engineeced chimeric moledes (hmbris et al., 1996) and recombinant mutant proteins (Tauiguchi-Sidle and Isenman, 1994; Oran and Isenman, 1999) have reached a cornmon conclusion drat a 42 amho acid segment at the NQ-teminus of C3b a'-cliaincontains binding sites for factor B, ndoi H, CR1 and CR.3. In particuhr, sitedkected mutigenesis studies canied out in this lob merdemonstratecl that a segment near the NHptermiLlus of C3 akhain, which contaias the negatively charged residues ODE and 736~~(human pro-C3 numbering where nsidue number 1 is the fint amino acid of p-chain and the order of the chah in the pro- moiecule is b),is involved in the interaction of C3b with factor B, CR1 and CR3 (Taniguchi- Sidle and Isenman, 1994) (Figure 1.9). This hding is in agreement with previous observations that the interactions beiween coqlement proteins and their ligands are enhanced under reduced ionic strength conditions, implicating a role for ionic forces in these protein-protein interactions.

Sequence cornparisons of humen C4 and C3 show that the C4 al-chain NH2-tenninal residues Glu744-Glu768 share a high degree of sequence similarity with the correspondhg nsidues in C3 a'ahain (Asp730-01~754). Whezws the ovedi sequence identity of the two proteins is 29%, the identity over the NH2-terminal segment encompassing 28 residues is 54% (64% if consewative amino acid replacements am incluâed) (Figure 1.10). Of particular interest was the conservation of the negatively charged dueten, thrt in the case of C3 had ken shown to mediate intetactions with otha complement prateins. Specifically, 73- of C3 comsponds to 744~~~ofC4 and 73%~ of C3 corresponds to the middie pair of $Cidic residues in the C4 cluster 749DEDD. Secluence cornparisons ammg différent species of C4 rnd C3 have shown that the two clusten, of negatively charged residues are genemliy consaved. In üght of the fiinctid similntiîy between the and C3b-B interactions, we pmpodthat the sequence Sm is the ha-terminal midue ofC3 a'-chin. Raidues that have becn identifid by mvious sito-dinctod mutapesis studies in tbir iab to afkt the interactions with frtor B. Pactor H. CR1 and CR3 am ~ndicatedb undctlincd &tkts. The shrrdcd bars tepment îhe boudaries of the fidor B and fmt H bii& g sites determinad by an oveda thetîcp 'der ratch shclson. 1991). With mpcct m its Icdicmr idivity. CR~b~kfterrntn#katm%at di F-r I-dpcd ~la~ag~w- a- by tbe mutation n ~huped.mino ridSI it rhc indicatcd rtsidue(s), whenu CR1 in outlinc k~sindiates an &ect d on the thi t6 factor b mediateci elcavage as illusirstcd in Figure 1.7 lcading to the formation of Acand C3dg. C3a' 764 ISTKLMNIFL KDSITTWEIL AVSMSDKKGf CVADPFEVTV 803

Ch' 778 LWLPDSLTTW EIHGLSLSKT KGICVATPVQ LRVFREFHLH 8 18

Rqirc 1.10 Sequecc dgnment of the N-terminai won of bumm C3 and Cd

ie the fust midue of C3 akhain, ~~38is the fitnsidue of CI) al-chain. Sequence identitim am indicatcd by @la.The ullcletlined &dues denote the most auciai residues for factor B. CR1 and CR3 binding to C3 as detemineci from pnvious mutagenesis studies by Taai chiaidle (1994). The two charge clusters of intemt in this thesis are Ciepictcdm bo flèttcts* simiianty between the respective NH2-termini of C4 and C3 a'-chains was suggestive of wmilru ligand binding sites in these two molecules. Thenfore, it is our hypothesis that the negatively charged nsidues 744EED and 749DE~Dat the NH2-te~minusof C4 a'chain are involved in the interaction with C2 and the aim of this thesis pmject is to test this hypathesis.

To directly assess the contn'bution of this C4 segment to C2 binding, a sitedirected mutagenesis approach was exnployed, in which the candidate acidic amino aciâs (Glu and Asp) were nplaced with th& isosteric amides (Gln and Asn) within the context of the intact C4B imtype molecule. The mutagenesis projeci wu initiated by Roger Ebanlcs, a pnvious graduate snident in the lab, who had obcained some preüminary results suggesting that the two acidic clusters were indeed involveci in mediating C2 binding to C4b (Ebanlrs, 1995). However, there was at le- one important mutant missing from the analysis and the C2 binding assay results were not as definitive as they needed to be. Accordingly, for my thesis pmject 1have extended the site-directed mutagenesis studies in which a more wmplete series of mutants were assessed for their respective C2 binding abiîities as well as hemolytic activities and coiifonnational propdes. Unlike earlier C3 mapping studies perfomd in this lab where the mutagenesis approach was able to corroborate or nfute sites proposed on the basis of synthetic peptide mimetics or anti-peptide mti'body blocking studies, no such saidies had been done to msp C2 binding sites in C4. Accordingly, 1 dso canied out glutathione S-tmsferase (GST) fusion protein binding assays, synthetic peptide mimetic inhi'bition assays and rnti-peptide anti'body blocking eqmiments as complementary approaches &signeci to fiuther test our hypothesis that the NHptemU acidic segment of C4 ag-cbainphys a dein the binding of C2. Chapter 2

Materials and Methods The foiiowing diethyl barbiturate (veronai)-NaCl buffers were used (Rapp and Bateos, 1963): VBS, 4 mM veronal, 0.15 M NaCl, 0.15 mM CaC12, and 0.5 mM MgC12, pH 7.2 (p = 0.15); GVB, VBS containing 0.1% gelak, GVB-M~~+~GVB containing SmM MgC12; GVBE, GVB containhg 10 mM EDTA; SVB, low ionic streagth VBS made isotonic with sucrose, 4mM vaonai, 0.06 M NaCl, 0.15 mM CaC12,0.5 mM MgCL2, and 0.17 M sucrose, pH 72(p = 0.0; SVB-M~'+,SVB containing 5 mM MgC12; SGVB, SVB contriining 0.1% gelatin; SGVB-Mg2+, SGVB containing 5mM MgC12.

(Gigli et al., 1976), C2 (Nagasawa and Stroud, 1977b) and C4 (Tack a al., 1981) were purified fkom fimh fkozea human plasma as described previously. A bctionally pure human Cl reagent, used in the prepmtion of complement component cellular intemediates, was prepared fiom a euglobulin precipitation of whole human senun (Cooper and Mûller-Eberhard, 1968). C4(CH3N&), a nucleophile-rnodifed C4 molecule, was obtained as descriied previously (Isenmm and Kelis, 1982) by treating putüied human C4 with 0.1 M methylamine, pH 8.0 at 37OC for 6 h. Guws pig camplement (Sigma Chernical Co., St. Louis, MO) was diluted 50-fold in GVBE to obtain a C3-9 reagent.

Pmtein ndioiodination was performeâ by the lactoperoxidase procedure descn'bed prevtCous1yby Marchalonis (1%9). Typidy, 50 pl of up to 250 pg of C4 was mixed with 10 pl 1M NaH2P04, pK 73 and 1/80 wliK (enzyme/protein) of lrctoperoxidase (1 mglml) (Si- CherniocalCo.). 13pi of [lsI]-~a(100 mCi/ml) (Amersbrm Canada, Oalnrille, Ontub) and 3 pL of (1/4,000 dilution in Hz0 of a 30% w/w stock) were added to initiate protein radioiodination. nie donmateriais were incubated at RT fot 10 min, at which bune 500 pi of phosphate buffered saline (PBS) containing 0.02 % NaN3 and 5 pi 0.1 M KI were aWto stop the reaction. Separation of unincorporated [12SI]-~awas accomplished by incubating the solution at RT for 30 min with 500 pl BSA-saturatecl PBS-washed Dowex-1 (Sigma Chernical Co.), wbich only bound free [12SI]-Na but not 125I-pmteh. In orda to detennine the fhction of protein-associated lzsl, 1 pl of labeled C4 was mixed with 100 pi BSA (1 mglml), foilowed by precipitation with 500 fl of 8% trichloroacetic acid (TCA). Genenlly, this procedure yields greater than 95% TCA precipitable mdioactivity (pellet/pellet + supernatant) and typical specific activities of 106 cpdpg protein.

The high glucose formulation of Dulbecco's modified Eagle's medium @MEM) supplemented with 2mM L-glutamine, 100 unitdm1 of peniciliidstreptomycin was the basal tirunie cuiture medium used in this study. The pH of the medium was rnaintained by 5% Ce in a humidified incubator. COS4 cells were maintained in DMEM supplemented with 10% heat- imctivatd fetal calf serum (Fa,Life Technologies hc., Grand Island, NY) (complete DMEM). DMEM/DEAE-Dexttsn transfection medium consisted of DMEM containing 50 mM Tris, pH 8.O and 0.5 mghl DEAE-Deman. The chloroquine concentration in complete DMEM/chloroquine was 100 PM. The growth medium for transfected COS4 cells was coqlete DMEM. Two days befon cuiture superaatants were to be hawested, the celis were washed in phosphate bflisaline PBS) and then incubated in FCS-fne DMEM, this medium being complete DMEM in which the 10% FCS was replaced with 1% Nutridomi-HU (Boehringer Mannheim, Montreai, Quebec, Canada). In the metaiK,tic iabeling expairnent to k demi below, both the basal DMEM and the Ma- end Cys-&e DMEM were supplemented with 4% K76-COOH-treated FCS and 1% Nutn'dorna (DhEWK76). K76-COOH is a monocarboxylic acid derivative of K-76, which is isolated and purifieci hmthe culaire su-& su-& of a fimgus, Smhyboobys mmplementi nov. sp. K-76 (Myalliln a al., 1980). K76- CûûH was found to be able to imversiily inactivate bovine factor 1(Hong et al., 1981). The produre foiK76-CûûH tnitmmt of FCS has been demiprwiou~ly (T'aniguchi-Sidle and Iseoman, 1994).

2.4 Synthetic Peptides

The synthetic peptide EILQEEDLIDEDDIPVR, comsponding to residues 740-756 of mature C4 (C4740-7561, was obtained ftom two independent sources, respectively Procyon Biopharma Inc. (London, ûntario, Canada) and the Alberta Peptide Institute (API, Edmonton, Alberta, Canada). The Procyon peptide had a COOH-terminal cysteine-amide residue in place of the naturally occumng senne and the API peptide had at its COOH-terminus the photoactivatabk amino acid derivative oniithine-amidebewoylbellzoate. The API peptide was also suppüed as phot~osslinkeâconjugates of keyhole limpet hemocyanin (KLH) and bovine senm albumin (BSA), each substituted with on average 17 molecules of peptide per moleaile of derprotein. The KLH- and BSA-peptide conjugates were used respectively for rabbit immunizations and for anti-peptide anti'body detection and purification (see below). The peptides SIERPDSAPPRVGDT and EDPOKQLYNVEATSY, nspectively corresponding to residues 455-469 of mature C4 p-chah and residues 1198-1212 of human C3, were obtained hmChiron Mimotopes (Raleigh, NC) fa previous stuàies and were used as control peptides. Rior to use, alî peptides were lyophilized ftom H20sennl times to nmwe traces of voîatile compoulids and then dissolved in H20to yield a concentration of abait 700 W. The exact concentration wu, detenaiaed spectropb~caîlyat 220 mn using an E km,imof 100. 2.5 Antibodiea

Rabbit anti-aheep red blood celi stroma, rabbit polyclonal IgG against human C4, rabbit polyclonai IgG awst human C3c and goat aikabe-phosphatase-conjugated anti'body against rabbit IgG wmpwchased nOm Sigma Chernical Company.

2.5.1 Generation and Purification of Anti-Peptide Anh'bodies

Antiies against the synthetic peptide C4740-756 were raised in rabbits by subcutaneous injections of 500 pg of the KLH-peptide conjugate, initially in complete FreunCs adjuvant, and at two subsequent 2 week intervals in incoqlete Freund's adjuvant. The immunizations and the collection of the rabbit antisera were done by technical staff in the Department of Comparative Medicine, Faculty of Medicine, University of Toronto, in accordance with the guideiines of the Canadian Council on Animal Cam. The rabbit IgG was fractionated hm0th- serum proteins by ioncxchange chromatography on QAE Sephadex A-50 as descni previously (Joustni and Lundgren, 1969) and diaiyzed against PBS, pH 73. An immunoadsorbent colwmi was prepated by coupling the BSA-Wpeptide conjugate to MOel15(Bio-Rad Laboratories, Herdes, CA) according to the manufhmmds instructions. The affinity mririx was equilibrated with PBS, pH 7.3 and the IgG fiaction of the rabbit rntisenim was applied to the colmm by gravity flow. Foiiowing washing in PBS, the bound antibodies were eluted with 100 mM glycine, pH 2.5 and neutrabd to pH 8.0 by the addition of 1.O M phosphate buffer, pH 8.0. The pooled anti-peptide anti'body fiactions were dialyzed extensively against sVB-M~~+containing 0.02% sodium azide.

Titration and bindhg specificities of the imnnine semwere detemiined by direct enzyme ünked immunoadsorbent assay @LISA). Briefly, polystyrene microtiter piates (Stanrtedt Inc., St. Laurent, Quebec) were coated overnight at 4OC with BSA-C4-peptide conjugate (10 pg/d in 0.01 M NaHCO3,pH 9.8,200 pheli). The pkswere washed with MaA (PBS, pH 7.4,0.05% whBSA) and blocked with Blotto (5% skim milk in PBS-Ween 20,0.1%, 220 Wwell) at 37OC for 2 h. After wasbiiig, the wells wen incuôated with vrrying concentrations of antiserum in barnA at 3f°C foi 2 h (100 pllweli). A starting concentration of

1 : 100 dilution in buffer A was used and a 5-fold dilution series was set up. Foliowing wsiphing, Boat allcalinephosphatase-conjugated anb'body agaW rabbit IgG (1 : 5,000 in buffkr A, 100 plhnrell) was applied and incubateci for 2 h. Following washing, bound conjugated antibody was detected by the addition of p-nitrophenyl phosphate (1 mg/ml in 0.1 M diethanolamine, pH 9.8; Sigma Chernicals Co.) and the absorbance at 405 nm was measured using a Mode1 450 Microplate Reader (BieRad Labratories, Richmond, CA). To detefmjIle the binding activity of the antisenun to nucleophile-modified C4, a direct ELISA on plates coated with methylamine- treated C4 was @ormed simiMy as bas been descriaed above.

2.6 DNA Methods

2.6.1 Generation of Recombinant C4 and Site-Directed Mutagenesis

The cDNA expression plasmid pSV-C4B, coâing for the wild-type human C4B isotype unda the control of an SV40 promuter, has been âescn'bed previously (Carroll et al., 1990). The plrsmid pUC-C4-5' was useâ as an intermeâiate vector, consistiag of a 2.8 kb 5' &ï 1-&oRI hgment of the C4B cDNA cloaed into pUC19. Site-directed mutants were produced by the overlap entention polymerase chah reaction (FCR.) mutagenesis method (Ho et al., 1989) uhg the pmfknriing enzyme Vent DNA polymecase @lm Eoglaod Biolabs, Beverly, MA) and pSV- C4B as the wiîd-type tanplate. The methd is ülusûated in Figure 2.1. "folnl81:d" 5'-flanking mutagenic trrimet I oligonucleotide 1

to be mtated is indicated sites are iadicateâ by 'Y ". and 3'-flaaking primas aAer with the appropriate restriction enzymes (Eco4mI and &RI)* 4. Ihe digestion produa is subcloneû into pUC4%5', which contains unique restrichCwsites fa EcdllII and bRi. The vecta pUWis firrtber di- ~ith &lI and &!URI. 5 The SaWEcoRI digestion product is subcIoneâ into similpiy testfltcted expression vectorpSV-CXB, The overlap extension technique involves the primiry PCR donsand a second primer extension reaction. Briefly, in primary PUb two fragments of the target sequence are amplifieci. Each resction uses one extemal flaaling primer that hybridws et one end of the target secpence and one opposing-oriented internai mutagenic primer that hybridizes at the site of the mutation and contains the mismatched bases. The 5'4lanking primer was designed to be upsûeam of a unique dctionenzyme site (Eco4îIU)and the 3'-flanking primer was domstream of rnother unique restriction site (&RI). Thefore, one of the resulting PCR products consistai of the "forwarâ'' mutagenic oligoaucleotide and the restriction site for EcoRI and the other fralpnent containeci the "backward" mutagenic oligonucleotide and the enzyme site for Eco4RII. By using the two flanLing primers, the pnimary PCR firigments CMbe fused by denaturing and annealhg in a secondary PCR naction. The overlap PCR allows one sûand ftom each fiagrnent to act as a primer on the other, and extension of this overlap results in the mutant product which is then amplified by the 5'- and 3'-flanLing primar.

Specifically, amplification of DNA fragments hmthe plasmid template pSV-C4B was acbieved by adding 20 ng of template DNA, 100 pmol of each primer, 6 pidNTPs (2.5 mM), 10 pi 10 x Vent polymeme bWa (10 mM KCl, 10 mM (NH&S04,2 mM Mg?SO4, O. 1 % Triton Rlûû, 20 mM Tris-HCl,pH 8.8) and 1 unit of Vent DNA polymerase in a final volume of 100 pl. The samples were subject to 30 cycles of denaturation (1 min, 94OC), annealhg (1 min, 52OC) and extension (1 min, 72OC) using the GeneAmp PCR system 2400 (Perkin Elmer Corporation, Nomaik, Connecticut). The products of the reaction were andyzed by size- fhctionating the DNA by electraphoresis tbrough an agarose gel and the band of the appropriate size was cut from the gel and purified ushg the Geneclean II kit (BIOIOI, Inc., Vista, CA) aecording to the manuf'tureis instructions. The pudieci PCR fhgments were subsequcntiy used as substrates in the ovahp extension PCR The reaction conditions were identical ta those uedintheprimuy PCR. The resulting secondary PCR product is a 932-bp hgment encompassing unique restriction sites for Eco4lIII and EcoRI (at bases 2,132 and 2,788 of the C4B cDNA, respectively), and it was digestecl with these enzymes to genaste a 657-bp maitition-containing fiagrnent which couid be mbc1oned into similariy digested pUCC4-Y, whm the sites for Eco4710 and hRIare also unique (the internrpdiate vector step is requVcd because W7UI is na a unique digestion site in pSV-C4B). Foiiowing confimution of the desûed mutation(s), and the absence of any undesired mufations within the 657-bp target sequence by stranddenaturation dideoxy-sequencing (T7 polymerese sequencing kit, Amersham Pharmacia Biotech, Inc.), pUC- C4-5' was restricted with SalI and EcoRI to produce a 2780-bp fiagment encompassing the mutation($). The SaNEcoRI mutation-contttining fragment was then exchanged for the comsponding wild-type region in pSV-C4B.

2.62 Construction of GST-C4 Fusion Rotein

Plasmid @EX-2T (Invitmgen Corporation, Carlsbad, CA) was used as the glutathione S- msferase (GST) fusion expression vector and the cDNA endhg the NH2-terminal5 1-amino acid hgment of C4B a'4ah(Aia738-Va1788) was inserted into the unique Bumm and &OH cloning sites of pGEX-2T by PCR subcloning. Plasmid pSV-C4B was used as the PCR template, with appropriate primers desigaed as foliows. The sense primer contains m enzyme site for BmHI and nucleotides 2399-23 17 and the anti-sense primer is composed of an enzyme site fa EcoRI, a stop codon and nucleotides 2.45 l-2,430. The PCR reaction and the product purification were paformed as descn'bed above. The resulting fiagment was subject to digestion with BonHI and EcoRI to create a 162-bp fragment, which was subsequently subcloned into similrsly digesteci pOEX-ZT. Sequencing of the recombinant DNA was performed as descn'bed above and cOllfiLmed the in &me insetfion of DNA coding for a CûûKtaminil extension of the GST Mon portion. 2.7 Eqression rad Quntktion of Recombinant C4

2.7.1 TmsMonof COS4 cells

The Mcan green monkey kiâney COS4 ceiï üne wur used to express the recombinant C4 proteins. The ceils were transientiy transfeoted by the DEAE-Dextran method essentially as desm'bed in our eariia studits (Ebankset al., 1992). Briefly, 100- plates were seeded with 1 x 106 cells priar to transfection. Mer ovmight incubation, cells were 40-60% confluent. Foliowing washing with PBS, 4.5 ml DMEMDEAE-Dextraa transfecton &um and 15 pg of pSV-C4B plssmid were added. After 404nof incubation at 37OC, the transfection medium was replaced with an equal volume of complete DMEM/chloroquine and the cells were incubateci for 2-3 h. Foiiowing rernoval of this medium and washing of the cells in PBS, the celis were allowed to grow in 9 ml coqlete DMEM for 72 h, aftet which time the medium was changed to FCS-fiee DMEM. Recombinant Ckontaining supernatants were harvested atter a Mer48-72 h of incubation.

2.7.2 MetaboLic Labebg, Biosynthetic Cbracterization, and Iuununopredpitation

Afk being aliowed to grow for 72 h in complete DMEM,transfected ceils were washed in PBS and then incubateci for 1 h in Met- and Cys-&e DMEM (ïCN Biomedicalq Costa Mesa, CA) containing 4% K76treated FCS and 1% Nutridoma-HU (3 ml per 1Wmmplate) in order to deplete Uiteraal staes of methionine and cysteine. The mxhm was then supplemented with 250 pCi of 3~~-mefhionin&sS=cysteine(ICN Biochemicals TransLabel, -1300 Cilmmd). Afkr 5 h of incubation, an eqdvolume of Met- and Cys-8UfXicient DMEM/K76 was added, and the incubatim was continued overnight. To assess the biosynthetic processing and C% cleavability of the recombinant C4, metabolidy bledsupematants wen immunoprecipitatedwith rabbit Co*),both with and withaut prior treatment of the supemritrillts with 6s(2pg/ml, 1 h, 37OC). The buffets and waeh procediaes for the immiinop&pitations have ban descri'bed pmviously (Cole et al., 1985). AU samples wae analyzed on 8% SDS-PAGEunder reducing conditions, foliowed by autoflumgraphy rndhphosphorimage analysis. Quantitative messurement of band intensities on phosphobaged gels was accomplished using a Storm 860 scanna (Molecuiar Dynrmics Inc.) rad pixel peak profile integration using the program IPLabGe12.0 f.

2.7.3 Quantitative Masurement of Sented Recombinant C4

The concentration of recombinant C4 in dialyzeâ culture supematants of transfeded cells was determined by a competitive sold-phase radioimmunmsay (RIA) (Harlow and Laue, 1988) using i*%labeled purified human C4 as the probe and rabbit polyclonal IgG anti-humen C4 as the capture antibody adsorbed to opaque polystyrene microtiter plates (Parkard Instruments, Meridan, CT). Mfied human C4 was used to obtain a standard curve. Radioactiviry wos measured by liquid scintillation counting directly in the plates using a TopCount instrument

(ParkardInstniments). ,

The DHSa sûain of Eschenèkia coli was used for GST-C4 fuaion protein expression. Due to the Wubility of the GST fusion protein, a niixed detergent solubiiization protocol was employed to facilitate bindhg of the GST protein product to glutathione beads (Frangioni and Neel, 1993). Briefly, production of the Mon protein was done by diluthg the oveniight culture of the tnnsfibacteria in îiesh LB medium (1 :50) and aiîowing the bacteria to grow for 1.5 h at 37OC befme adding isoptopyl B-ITthiogalactopymoside (IPTG, BioShop Cuirdr Inc., Bialiagton. Ontario) to 0.1 mM ARer induction with ïPTG and growth farnother 4 h, the ceils

were washed and tesuspenâed in 135 j,û STE (100 mM Tris, pH 8.0, 150 mM NaCl, 1 mM EDTA) containhg 100 p@nllysozyme. Foiiowhg 15 min incubation at O°C, dithiothreitol @TT, BioShop Canada Iuc.) (5 mM) and pmtease inhibitor phenylmetbyl-SUlfonyI fluoride (PMSF, ICN Biochemicals, Ino., Cleveland, Ohio) (2 mM) were added and the bacteria were lysed by the addition of N-lourylsarcosine (sarkoqd) to 1.5%. Celis were then dissupted by sonication on ice (3 x 10 s with I min between bursts). Mer centrifiigation, the supematants were collected and Triton X-100 (Sigma Chernical Co.) was added to 4%. Subsequently, the supernatrnts were incubated with 100 pl of glutathione sepharose 48 (Phamacia LKB Biotechnology) at 4OC for 15 min. Foiiowing washing with cold PBS and centrifugation at 4OC, the supemstants were discardeci and proteins attached to the beads were visualized on 10% SDS- PAGE under reducing conditions. Glutathione beads bearing equal amount of OST as control beads wen produceâ similarly as desrnid above.

2.9.1 Classicai Pathway-Dependent Hemolytic Assay

The hemolytic activity of recombinant C4 in ~~~-~~''-dial~zednansfection supemtants was determineci by using EACI, iodineoxidized C2 (OVC2) and C3-9 reagent as descrii previously (Cooper and MWer-Eberhard, 1%8; Ebanks et al., 1992). Afta corrrcting for ôackground, the de- of specific lysis was converied to "2"units where Z = -Ln(l-fhctional lysis) and is physicaliy equal to the nuniber of hemoLyticaiiy effdve molecules per e@myte. Comparisoaa of activity were made on the baeis of Z units per amount of immiinochemiicrly deteunined rC4. 2.92 Fluid-Phase C2-Dependent Eemolytic Inhtibition Assay

Secreted recombinant C4 was converted to wa3NH2),a C4b-Lüte molecule, by tnating the culture supematants tnrmtransW cebwith 0.1 M methylamine, pH 8.0 for 6 h at 37OC (Isenman and Keiis, 1982). The methylamine-treated supernatana were then dialyzed extensively against SVB-M~~+and concentrated to approximptely 118 of original volume by using either Centricon- 100 concentraters (Amicon hc., Beverly, MA) or Ulaaftee- 15 Centrifugai Filter Devices (Miiiipore Corp., Bedford, MA). Following this procedure, the concentration of the rC4(CH3NH2) was daennined by a cornpetitive radioimmunoassay as descnbed above. Iodinesxidized C2 in SGVB-Mg2+ in an amount sunicient to generate approximately 80% hemolysis in the absence of inhibitor protein in a C2 hemolytic assay to be descnied below (typicaiiy 16 ng of C2) was incubated with variable amounts of WCH3NH2) in a total volume of 200 pl of SGVB-M~~+at O°C for 5 min. EAC4b alls (1.5 x 109, prepared as descriid pmiously (Eba and Isenman, 1994), were added and the tubes were further incubated at O°C for 10 min, ot which thethe ceb wmwashed with cold GVB-~g2? The resulting EAC4b2 celis were resuspended in 200 pl of GVB-M~~+contsining excess Cl nagent and incubateci for 10 min at 30°C. Lysis was developed by the addition of C3-9 reagent (1 ml) at 37°C for 45 min. Mer spinning down ualysed ceils, the degree of lysis was detennined by measuring the absotbance of the supernatant at 412 llll~ Hemolytic dota were used to caiculate the relative C2 bindhg ability of each dX(CH3NH2) species by comparing the IHsOof a given molecule to that of the wild-type molecule, whm IHsois defined as the amount of rC4(CH3NH2) giving 50% inhi'bition of hemolytic activity.

2.93 Soiid-Pbase C2-Dependent H-1yb:c Inhi'bition Assay

Diffetent amunts (10 pi and 50 pl) of gIutathime beaâs and beads bearing equal amnmt of either GST or GST-Wpeptide Mon protein as judged fmm SDS-PAGE anaiysis of the beads were incubated with 16 ng of qC2in SGVB-Mg2+ (100 pl) for 10 min at O°C. Afta centrifûgation, constant amounts of the supematants (50 pl) were transferred to rnother tube. The hemolytic activity of C2 remahhg in the supematants wae then determîaed by the C2 hemolytic aswy descn'kd above*

2.9.4 Synthetic Peptiâe Cornpetition Assay

Varying amounts of synthetic peptide in 160 IJ of H20 were mixexi with 40 pl of 5 x SGVB-M~~+and these solutions were then incubated with 16 ng of WC2 for 10 min at O°C. EAC4b cells (15 x 10') were then added and the remaining steps of the assay were done exactly as descrii above fa the fluid-phase Cz-dependent hemolytic inhibition by rC4(CH3NH2).

Erkt ofAnti-Pqtide Antibody on the C2-Lkpendent Hemoiytic Assay In this assay, various amomts of mti-peptide anti'body were incubated with EAC4b cells (1 J x 107) in 65 pl SOVB-Mg*+at O OCfor 90 min, at which time 16 ng of WC2 were added and the assay volume wes increased to 200 pI with SGVB-M~*+.After 10 min of incubation at O°C, all samples were centrifigeci and the supematants were discarde& The resuiting EAC4b2 WSwere treateâ with Cl and C3-9 nagents and the degree of lysis was determineci as descrii above.

Efiof Anti-Pqtide Antibtx& ON the C4bC2 Bincing In~t~ion

A second blocking assay using the mti-C47*-756 nagent was designed to restrict the effixt of the antihly wlely to the ab42 interaction step. A preihimy assay was perfotmed to cOannn that the anti-0&740-756 antitibody did na affixtthe degree of hemolysis beyond the C4b62 iatrraction step. In me proup of tuôes (tubes A)# EAC4b cells (1 r 108) were hcuba!ed with various amounts of anti-Ot74e7# for 90 min at O°C, foliowed by washing with cold GVB- M$+. The cells were then resuspended in 100 pi SGVB-MgZ+ and afk 10 min incubation at O°C, the cells were pelieted. In the second group of tubes (tubes B), EA cens (1 x 108) were inCUb8fed fa 10 min at O°C with WC2 in an amount SuffiCient to generate approrrimately 8W hemolysis in a nsiduai C2 hemolytic assay to be dernibelow (typically 23 ng of CZ), foiioweâ by centrifbgation. Fixed ~lll~unts(95 pl) of the respective supematants from tubes A were traosferred to another tube, ond mixed with the supemtants from tubes B (95 pl). The supematants were then supplemented with SGVB-M~~+to give a total volume of 200 pl and the hemolytic activity of C2 was detemhed as descrihi above.

To directiy assess the potential blocking effect of the anti-peptide antiiy on the C4b- C2 interaction, EAC4b cells (1 x IO*), in SuffiCient amount to bind virtuaiiy al1 of a test quantity of C2, were incubated with various amounts of anti-C4740-7% for 90 min at O°C, followed by washing with cold GvB-M~~?The ceb were then resuspended in 100 pl of SGVB-M~~+and a fixed amount of oW2was added. The amount of oJW2had been pre-determined to yield appmximately 80% hemolysis in the same assay to be descn'bod below by pre-incubating with EA cells (1 x 104 in the absence of antiiy (typicaiiy 23 ng of C2). Mer incubation fa 10 min et O°C, the cells were centrifugecl and fixeci amounts (95 pi) of the respective supematants were tnnsferred to another tube. The wipanatuits wem then supplemented with SGVB-Mg2+ to give a total volume of 200 pl and incubated with hesh EAC4b cells (1.5 x 107) for 10 min at O°C. The hemolytic activity of C2 capturecl on the mndset of celis was then determined by the addition of Cl and C3-C9 magents as descn'bed above* Chapter 3

Results

AU results shown in this chaptex represent my work. Mutants 'WED, 744QQN,749~Q~~ and 749NQNN were available as complete expression plasmids from Roger Ebanks, a pnvious graduate -&nt in this lab. Mutants 744QQD, T49DQD~and 749DE~Dwere wnmcted to an intermediate stage by Roger Eboaks and were completed by me. Mutant 7*EEN was entirely oonstnicted by me. 3.1 The Site-Directaï Mutagencd, Approreh

Si&dmtedmutagenesis was the fint amchanployed in this study to assess the role in C2 binding of the two negatively chprped Mino acid clusters at the NK~te&u of C4 a'- chah. The candidate midues ('44-749DEDD) were systematicaiiy replacecl with their isoatenc amides rnd both the wiid-type and mutant cDNAs were expressed and examiaed for their biosynthetk and functional activities including level of secretion, post-translational processing, activation by Cïs, hemolytic activity, and C2 binding ability. The mutant C4 molecules constructecl and the nomenclature of these mutsnts are shown in Table 1,

3.1.1 Expression and Production

The various rC4B isofype derivatives were expressecl transiently in COS4 cells and Wested 5 to 6 days aAer transfection in saum fne medium. The expression levels in the culture supemtants measured by competitive RIA showed that the protein production of ail the ncombinaat C4 molecules were at levels comparable to wild type (-1.5-2 pg/ml at the thne of hawesting). This suggested that there was no selective intracelîular retention or &gradation of any of the engineereû mutant C4 molecules due to misfolding.

3.2 Hemolytic Activity

The C4 hemolytic actïvities of the wild-type aad mutant C4 molecules were detetmind in a purely classical pathway-dependent hhemlytic asry. The Lesuts are shown in Figure 3.1. It can be seen that ali of the C4 mutants disphyed differentially reduced hemoLytic activity relative to the wüd-type. 744EEN showed less thm a 2-fold de- in hemolytic activity, 7- and

749DQDD displayed approximateLy 3-fold luwer acîbiîy îhan did wild-type, '44~~~showed an Table I

Nomendatpre and Amho Acld Sequences of Native C4B and the 744- 752 Segment Mutants Examlned in tbb Study.

Abbreviated Designation of Seqwnce of molecules between Moldes residues 744 and 752

r-p'49 D END I

The residues changed are indicated in bold letters. The designations shown in the left column are used hughout the thesis in the text In the figures, only the thredfout-letter designations are used due to space limitation. Mutant Nam

Hernolytic activîty of rC4B was assessed in a purely ciassiciil pathwaydopendent hernolytic assay. Dilution series of culnite supmatants containing the various rC4 molecules weia incubateci with MC1 ceîîs (1J x 101) at 308C for 15 min. After washing, the ctUs wen resuspended in Zûû pl SBVG-Mg2+ and incubated with OW2(250 ng) at 30°C for 7 min. Hemolysis was developed by the addition d C-EDTA (1 ml) and the degne of lyds wudetermined by measiing absorbanœ at 412 am8 CompuWns of hem01 'c acaivity were IM& on the basis of Z unit3 pcr amount (ng) of iC4, where Z = -ln(l-r" ractional lysis). This was detemincd fiwi a plot of Z vs. ngs rC4B addeci, generaîly 5 to 6 &ta points, and repssion analysis using the MacCw6t pprolgim. The pmpm estimatr.c the cmassdated with esch dope and thme generally were lest th10% of the vaiue of the dope. in the cases of highly compromised mutants, the emdd k as much as 1596. 8-fold defa and 7MQQN, 749DEND, 749DQ~and 74%1~~~were sevenly compromised with a pater than 15-fold hemnlytk defa.

The hemolytic defects of the C4 mutants obsaved in the hemolytic assry might be the result of a defect in the ability of these mutants to bind C2. However, otha factors including defm in biosynthetic processing and the mdecule's ability to be EiCfivateà by C~Scould also affect the uitimate hemolytic fuiictioiiality of C1).

3-13 Biosynthetic Rocessing and Susceptibility to Cleavage by CTS

To investigate if the mutation(s) introduced into the protein has produced pronounced chmges in the ovdfolding of the protein, the biosynthetic processing and susceptiibility to cleavage by c'Cs of the recombinant C4 moIecules were assessed. COS-1 cells that were tramfected transientiy with wild-type or mutant C4 cDNAs were labeled metabolicslly with 3%- mahionine/3%-cysteine. C4 in the culture supematants was then immunoprecipitated with anti- human C4, either with or without prior treatment of the supematants with excess CTS, followed by SDS-PAGEand phosphorimage anaiysis. Figure 3.2 shows the results of this qmiment. As has been observed previously (Ebanka et al., 1992; Ebanks and Isenman, 1995), the processing of C4 to the mature tbreechain fami in COS-1 cells is incomplete. Apart from the a-,p-, and y- chah, the processing pcoâucts include a large amount of pur intenneâiate, as well as a lesser mount of pmC4. Additiody, processed a-chain exists in the serum as two fonns, as (the secreted fonn) and ap (the plasma form). as is cleaved into ap afkthe remval of a -5 kDa COOH-terminal b-nt by a plasma protease (Chan a ai., 1983), wbich in our case cornes fmm the K76-treated facaif senun in the medium employed for this expairnent As shown in

Figure 32, aertment of wüd-type C4 with CE@ves rise to bands cocfesponding to als-and amp- chah It can k seen tbrt whereas somemutmt C4 proteins showai wüd-typbüke bduvia with A WT(EED) EEN QE D QQD QQN - 4- 4 - + - +- + cïs

No DNA C WT(DEDD) DQDD DEN0 DQND NQNN

Ii + 9 + - +- +- + cïs

a's Q'P

Phosphorimsge of 8% nducing SDS-PAGE of metabolidly labled C4, immuaopnci itatsd with rabbit 1gG @tLhuman CI from transfcctcd COS4 cc11 supcmatmts. Mi(-) ad&r (+) mentwîth Cis (2 pglml). Punels A Md B show rrspectively the immunoprcipitaîion of the 74EED cluster mutants and of the 7qr)DEDD clustcr mutants. "No DNAw sefers to the immuwpdpiîation of labeled supertll~atltshm mock-tmsfii COS-1 œUs. "Curcftrs to the coatrol immpmpncipitation of wild-typc rC4B aipmtaats with rabbit IgG anti-human ac* respect to bth prooessing and CTS cleavabiiityPsome of the mutants were processed and claved less eficiently than wa9 wild-type C4 (iiee below).

To analyze qwtitatively the extent of biosynthetic processing, the gels shown Ui Figure 3.2 were phospharimsgeâ, and the ratio of the a-chain (abp)band pixel intensity over the totaî pixel intensity of al1 of the chahs in each of the C% (-) lanes was calculated. The results are shown in Figure 3.3. It con be seen that 744EEN, 7uQEDP7uQQD and 749DQDD were processed at levels comparable to the wild-type control. In contrast, 7uQQN, 749DEND, 749DQND and 749NQNN showed a 2-3-fold defect in biosynthetic processing. It can also be seen in Figure 32 that whereas 744EEN,7e)QEQ 744- and ~~WDDshowed wild-type-like behavior upon C% treatment, 7uQQN, 749DEND, 749- and 74?NQNN were relatively resistant to the cleavage by C~S.These results suggested that in mutants 744EEN, ~~)QED, 744QQD and 749DQDDP the gross protein structure was not significantly perturbed by the changes in the acidic amino acids. On the other hand, by these two criteria, the various mutation(s) in molecules 7uQQN, 749DEND, 749DQND and 749NQNN had a measurable effect on the conformation of these proteins. Since the incompletely processed fonns of C4 are hemolytically inactive (Chan and Atkinson, 1984), the defects of these mutants in post- translational proteolytic processing in this latter group of mutants may in part account for their impaired hemolytic activities.

In order to more ditsctly assess the C2 binding ability of recombinant C4 moleles, a fluid-phase C2 hemolytic inhi'bition rrssry was pafanried. To avoid the problem of C'Scleavage resistance displayed by some mutants, the cuitacre supematants were reacted with small nucleophile mefhyiamine because this treatment of native C4 leads to the acquisition by C4 of a C4blike c4iifozmartion @exmm aad Keb, 1982). In the fluid ph,this C4biike molde is The gels shown in Figure 3.2 were phosphorimaged, and the ratio of the a-cJain band pixel intensity over the total pixel intensiv of dl the chahs in the respective C 1s (-) lanes was calculatcd and plotted appmximately equivalent to C4b in its ability to bind C2. Thus, culture supematants containhg various EUI~OU~Sof rnethylamine-moâüied ncombînant wild-type or mucllit C4 in SGVBI)Mg2+ were ailowed to react in the fluid ph= with a constant amount of C2, the amount of C2 having been pre-detennined to yield approximately 80% hemolysis in a C2 hemolytic assay. Uncomplexeci C2 was thm captured by EAC4b cells made with excess pdedC4. Following washing, hemolysis of these EAC4b2 cells was developed by the addition of Cl and subsequetltly CEDTA as a source of C3-9. Therefore, by meamhg hemolytic activity of mbound C2 in the fluid phase, one can assess the C2 binding ability of the rC4 without the requirement for tC4 deposition onto the sheep red blood cell (SRBC) sufkce which in turn would require cleavage ày as. If the C2 binding ability of the C4 mutants has been impaire& the mutant C4(CH3NH2) molecules wiil not be able to bind C2 in the pre-incubation atep and thus no decnase of C2-dependent hemolysis will be observed.

The resuits of one repnsentative fluid-phase expeiunent for the 7"EED cluster mutants and the 749DEDD cluster mutants an shown in Figures 3.4A and 3.4B, respectively. It can be seen that there is increased inhibition of C2 hemolytic activity when increasing arnounts of wild- type C4 were added. Ali the mutants made in both clusters displayed lower C2 binding activity than did wild-type C4. Figure 3.5 Summtvizes the resuits of thne hemolytic assays perfonned on culture media fkom three separate tranefection expaMents and displays the dota relative to wild- type as the C2 binding capacities of the various mutant C4 molecules. These results suggest that both negatively charged amino acid clusters are involved in mediating C2 binding and generally indicate that multiple mutations nsult in a cumulative defect. When compared to the overaü C4 hemolytic activity data described above (Figure 3.1), it was noted that there was a good correlation between the relative C2 binding capacity and the relative hanolytic activity for each mutant C4 molecule. Since 744EEN. 744QED, T44QQ1> and 749DQDD have wild-type-like behanot in processing and cleavability by as, the hemolytic def- of these mutllnts are pndominantiy a result of theu diminished ability to bind C2. This strongly suggests that the rCI(CH3NH2) (ng) Figure 3.4 Bindfng of C2 to recombinant wild-type and mutant C4(CH3NE3 moIecules. The ability of nucleophile-modifiecl C4 to bind C2 was assessed in a fluid-phase C24ependent hemolvtic inhibition- asw. Dilution series of culture suaernatants coniaininn the iranous ~c~(C&NH~) moleoules were incubated at O°C for 5 rninkith WC2 in SGm-~g2+at a concentration sufncient to generate -80% hemolysis in the absence of inhiibitor protein (16 n . EAC4b cells (1 x 107) were added and the incubation was continued for 10 min at O°C. The ceP s were then washed and treated with Cl and subsequently C-EDTA. Panels A and B depict a representative experiment for the 744E~cluster mutants and the "~DEDD cluster mutants, respectively. The dahd Iim represent the degree of CPdependent hemolytic activity in sham pmcubation of C2 with noaC1) containing dialysed culture medium. Mutant Name

FfOnra 3.5 Reiative C2 bhbgrbUity of recombinant wüà-type and mutant U(CH3NH3 rnoiecdts.

The ability of nucleophilc-modifieci C4 to biad ÇL was assascd in a fluid-phase C2dependent hemolytic inhibition assay. The relative C2 binding ability of each C4(CH3MI?) species was obtained by comptaring the Mso of a given C4 mutant to that of the wild-type molcculc, where Mm is defincd as the amount of rC4(CH3NH2) giving 5û% inhibition of hemolytic activity. The nsuits show the means and SD of tbne independent experiments. 744EED and ~~~DEDDclustets at the NH2-te1minus of C4 a'-chain do indeed contribute to the binding interaction with C2. In the case of mutants 744QQN. 749DEND, 74mND and 74%JQNN, which showed a 2-3-fold defa in processing and a greater than 15-fold hemolytic defect, the reduced hemolytic activities of these mutants uose hmthe combineci resuîts of impaved pst-translational processing and nduoed C2 bhding ability. Thus, these mutagenesis results are consistent with our hypothesis of a defor the mCdicchisicrs in binding C2, however, these data on their own are not defiinitive and require conoboration fnw other experimental duections.

3.2 The GST-CCPeptide Fusion Robin Approrch

The second approach employed in an attempt to locate a C2 binding site in C4 was to aasess the interaction between CZ and a GST fusion protein bearing the target segment of C4. The NH2-terminal5 1 amino acid fragment (Aia'38-Va1789 of C4 a'-chah was fused to GST as a COOH-terminal extensioa and the chimera was allowed to bind to glutathione-bearing agarose beads. Despite the fact that GST is a highly soluble protein whea expressed in bacteria, it was found that the GST-C4 fusion product formeâ inclusion bodia and was essentially absent fnmi the supematants of standard bacterial lysates produced by either sonication or French Ress treatmeut (Figure 3.6, Panels A and B). In an attempt to overcome this problem, bacteria expressing the GST-C4 fusion constnict, as weil as those expressing cootrol GST were lysed by sonidon in the prerience of the detergent N-iaurylsarcosine (1.5%). Triton X-100 (4%) wrs then adâed to make a non-dePILtUtibg mixed micelle and the remaining soluble material was adsorbeci ont0 glutathione-conjugated beads (Frangîoni a al., 1993). Foilowing washing, the arnouut of protein dsorbed was assessedby boiling an aliquot of the beads in SDS sample bufk folidby SDS-PAGEanalysb. After visual inspection of the relative amomts of GSTaand GST adsorbed per fixed volume of beads, the amount of GST bound per voIume of bead suspension was equrlized with tbat of the GST-04 sample by apptopriately dilidiillg the GST- A B TSP TSP 205 - 205 - 116- 116- 97.4- 97.4- 66 - 66 - 45 - 45 - 29 - +GSTa Fusion Peptide

+GST-C4 Fusion Peptide +Gsr

Figure 3.6 Saiubiiity d GST and GST-CI hision peptide rad Madi- of GST uid GSTahi011 pepadc b gîutathione beaàs.

The NH2-tmninal 51 amiw acid fragment (Ala738-Vd788)of C4 a'chain was fuscd to GST as a COOH-terminal extension. Poneis A md B show the solubility of GST end GST-C4 fusion peptide, respcctively. "T","Sm end "PRindicate the total ce11 lysate, the supernatant, aud the pellet, respectively. The cells wen disruptcâ by sonication in thia expriment. GST and GST-C4 fusion peptide were subsequtntly bound to gldoae beads by empIoying a mixd detergent solubilizaîion protocol. Pancl C shows the dative amounts of GST and GST44 fusion peptide bouad to çompiuable volumes of XI% kad suspensions. bepds with non-protein containing giutritbione beads. Panel C of Figure 3.6 shows the relative gmdlltlts of GST64 and GST bouad to comparable volumes of 50% bead suspensions. It can be seen that comparable levels of adsorbed GST-C4 and GST per volume of beads had ban achieved. A solid-phm CL-dependent hemolytic inhibition assay was then performed on glutathione beads bearhg GST-Wpeptide fusion protein (GS~-C473*-788) and pppropriate control bas, Le., beads bearing GST ody and glutathione beods bea~gno adsorbai protein. The experiment was done in a manner siimlar to the fluid-phase hemolytic inhibition assay. Variable amounts of beads bearing GST-C4 Mon protein and control beads were aliowed to nad with an amount of human C2 sufncient to generate approximately 80% hemolysis in a C2 hemolytic assay. Afta centrifiigation, residual &e C2 in the supernatant was measund in a hdyticassay.

nie results of such an experiment are shown in Figure 3.7. It can be seen that then was no specific binding of C2 to the GSTC4 fusion protein. Any obsewed decnase in C2 activity relative to the control correlates with the volume of the beads employed and is the same for aU three types of beada, suggesting that the glutathione beads may interact with C2 non-specifically. Although the results of this experiment did not corroborate the mutagenesis results, it mry have foüed for technical reasons relating to the insolubility of the GST-C4 fusion molecule. For exemple9the tendency of GST-CI), but not GST, to aggregate into inclusion bodies may reveal that interaction of the fbsion protein with itself or with GST would effectively limit its accessabiüty for interaction with C2. Alternatively, the concentration of GST-C4 fusion protein that couid be achieved on the bads mry bave been too low to bind C2 effectively.

In otdet to fiirther investigate the importance of the rn-tenninai seqyence in the interaction with a,a peptide cocfe8~011dingto the NH2-temhî 17 amino aciûs ofC4 a%hain C2 kagant lOul SOyl 1Ojd 50ul tOuI SOy!

Amount of 8eads Addeâ (pl)

The C2-binding ability of GSW4 hLPion piotein was din a solid- hase CZ hemolytic inhibition -y. Glutathionc kado, kads karing GST and beads bearing Gd TOC4 hision protein wem incutmtcd with OWC2 (16 ng) at VC for 10 min. After centtihgation, unbourd C2 in the supernatant was tmsîicdto another tube and incubated with EAC4b alls (1.5 x 107) for 10 min at WC. The œlls wen then washod d pmvidcd with Cl followed by C-EXYïA. Tbe mdts show the meam of trvo experiments. "C2" nfers to the hemolytic activity of C2 in the absence of any W. "Rcagent!" reftrs to the backgrwnd hemolysis wben no C2 wac prrsent. (C47w756) was synthesized and aseessed for its ability to inhibit C2 binding to EAC4b celis in a competition asmy. This segment, while encompassing the acidic nsidue clusters, avoids the hydrophobie stretches that would bave been present in the 51 residue GST fusion protein and may have contnito the hsolubilïty problem.

In the competition assay, various amounts of synthetic peptides were allowed to incubate with a limiting amount of C2 at O°C, EAC4b ceils made with excess purified human C4 were then added to capture the uncomplexed C2 and hemolysis of the resultant EAC4b2 cells was developed by the addition of Cl and C3-9 ragent. If the synthetic peptide competes with celL- associated C4b for the binding of C2, reduceâ C2-dependent hemolysis will be obsewed. The TeSults âepicted in Figure 3.8 show that the synthetic peptide ~4740-756inhibited C2 binding to EAC4b ceUs in a concentrationdependent mannet, with the 5W inhibition point requiring approximately 0.2 mM of peptide, while the conml peptide derived fiom a segment of C3d had no effect on this interaction. Similar C2 binding was obsewed using an independently synthesizeâ peptide haGg the same C4 74-756 sequence and a second control peptide derived hma C4 bhain segment was still negative in the assay (data not shown).

3.4.1 Detedon of Antisenim Titers and Spscificities

To merexamine the C2 binding capacity of the NH2-terminal a'chain segment, a polyclonai rabbit antibody wuraid against the syllthetic peptide Cq710-756 and tested for its ability to *'bit the interaction between C4b and C2. Before any fimctional assays wae ded out, the specifîcity and titre of the Ulimnine saum haâ been assessed. Since the KLH-peptide configate was useci as the immunogen, the BSA-peptide conjugate was then used to eneen for the anti-peptide uia'bodies. Thus, a direct ELISA protoc01 on miCFOQiterplates coried with BSA- Concentration of Inhibitor (M)

Various amounts of synthetic 'de (C47*7s, Aiôerta Pe 'de htitute sample were incubated with WC2 (16 ng) at !?'C for 10 min, at which thne &C4b ceb (15 x 10b were adàd and the tubwem fdcrincukted at OOC for 10 min. Foiiowing wasbin ,the cels were üeated with excers Cl and hemolysis was developed by the addition of C dA (1 ml). The n~n-~cpeptide employd cornsponds to the l,l!Sl3l2 region of humau C3. conjugated peptide was performed Detection of the binding of antibody to BSA-conjugated peptide was achieved by the addition of goat aikaline-phosphatrsaconjugatedanti'body against rabbit IgG. The results of tbis experiment am shown in Figure 3.9. It shows that the immune saum recognized the peptide and haâ an end point titre of 1/300,000, indicating that the rabbit had mounted a strong immune nsponse against the peptide antigen. By contrast, the pre- immune saum showed no such reactivity.

To determine whether the anti-peptide antii'body was capable of recoghhg its epitope(s) within the context of the rest of the C4 molecule, ancsther direct ELISA protocol on plates coated with methylamine-treated C4 was perfotmed Figure 3.10 shows that the antisaum alw reacted with CX(CH3NH2) specincally, although the titer was lower with an end point of 1112,500. The pre-immune serum as a control showed no binding $Ctivity towards cs(CH3m).

The rabbit immune serum was then IgG fhctionated and the purification of the specific anti-peptide anti'bodies was achieved by immunoaffinity chromatography on a rein presenting the BSA-C4 peptide conjugate. The ability of the irnmunoofnnity-purified anti-peptide mtibodies to block the interaction between C4b and C2 was then assessed,

3.4.2 Effecî of Anti-Peptide Anti'body on the C2-Dependent EIemolytic Assay

EACQb celle, made with urcess purifiai C4, were pre-incubated with various uiiounts of specific anti-peptide antibody or with control non-specific nbbit IgG. The celis were then incubateâ with a ümitiiig qpantity of CL, sufEcient to give 80% hemîysis in control assrys, and hemolysis was developed ôy the addition of excess Cl and GEDTA If the rntiçr) mibody bl& the C2 binding capacity of C4b, reâuced C2 hé11~1yticactivity wiii be observed. Figure 3.1 1 depicts the results of diia qmiment It an be seen that the mti-peptide antiibody inhibited C2dependent hemolysis in a dossdependent tnanner, while the non-spdic IgG mtrol haû no The dire of the nbbit semm immunizcd agai~tthe W-C4 peptide conjugatc was assesscd by ushg a direct EUSA pmtocoL on plates matecl with BSA-C4 peptide conju ate. The background level of biding of the immune wrum io plates coated ody with BSA is dso indicated, as W the biidiiig of pm-immune senim io the BSA-C4 peptide-coatcd plates. The binding sptcificity anâ titre of the rabbit immune scnrm was aswssed by using a dirrct ELiSA pmtoc01 on plates costed with CX(CHNH2)(10 &mi in 0.01M NaHC03, pH 9.8, aW)pîhueU). Mtrr a blocking with Bloüo, the wclis wmincukted with the immune se= for 2 h at 3TC. FoUowin wmhing, gort ~bphosp~njugakdmtibody against rabbit 16(1 : 5,000 in b Aer A, 100 pYwe11) wrr aâdd and detection of bound conjugated antibody wurhicved by the ddition of the rubstntt pniûopbcnyl phosphate (1 mg/d in 0.1 M diethanalamine, pH 9.8,100 pVweU). The absohnce was rneasurod at 405 m. Varying arnounts of anti-peptide rntibodics (anti4%7&7%) wcre pfc-incubatcd with EAC4b ceîis (1.5 x 107) at O°C for 90 min, at which the WC2 (16 ng) was added and the incubation was continued for 10 min at WC. After centrifu 'on, the cells wcre tnatcd 6th excess Cl and hemolysis was developsd by the addition of r-EDTA (1 ml). "C 1@ &ers to the fraction of IgG that ran through the immmdity columa adsorbcd with BSA-~#)-'~ conjugete. effect in this assay. Although the reSuIts of this antiiy blocking eqxknmt suggested that the pnti-C474oo7*6 antibody mi@ inhiiit the hemolysis by blocking the C2 binâing site in C4b, these results on the* own were not definitive because we couid not exclude the possi'bility that this antibody mi@ interfere with the CS convertase subunit fûnctionality of C4b. To overcome this problem, a second type of antiibody inhr'bition expedment was designed to restrict the efffect of the antiC4740-756 antiiy solely to the UbC2interaction step.

3.4.3 Effect of Anti-Peptide Antiibody on the C4bC2 Binding Interaction

In the ontibody hemolytic inhibition assay describeci above, the C2dependent hemolysis was developed in the presence of both celî-associatecl and residual unbound anti-C4740-756. In the second assay, a wash was included afbr the antiibody pre-incubation step to remove mbound antiibody. Following incubation of the washed celis with a fixed quantity of C2, the hemolytic activity of uncomplexed C2 in the supernatant was measured with fresh EAC4b to avoid the potentiai effects of C4b-asm~iatedantibody at the CS convertase stage. The gorl of these changes made in tbis assay was to measurr the C2-dependent hemolysis in an antiiy-fne environment. Nevatheless, thece was a smail possibility that antibody bound to the first set of EAC4b ceils could partly dissociate during the C2 incubation step. It might then be carried across in the supernatant together with the unbound C2 and affect the binding of bis C2 to the second set of EAC4b celis, or possibly the CS convertase subunit activity of the C4b on the second set of EAC4b alls. To detannne whether such a "carry-over" phenornenon would be a pmblem in our assay, the follawiag prelhhuy experiment was done.

Various amounts of uiti-C4740-756 were allowed to pre-incubate with EAC4b ceiis, followed by washing. The ceiis were then resuspended in 100 pi SGVB-M~~+.After with a limiting unount of C2. The amount of C2 had been pn-determineci to generate approxhately 80% hemolysis in a residual C2 hemolytic assay when proincubwd with EA ceiis in the absence ofthe antii. The CZdependeat hemolysis was then meawrnd by ushg a set of fresh EAC4b ceb. Cl and GEDTA. The reaults of this arpaimnt are ahown in Figure

3.12. It can be seen that at aii amdunts of ~~~~~756 anployed with the ntst set of EAC4ô ceils, there was indcient "canyiwer"of antiibody to have any inhi'bitory effed on the C2- dependent lysis of the second set of ceils. Baseâ on these msults, we were confident that in this muitistep assay the effects of the anti-peptide anh'body would be limited to the C2 bindiag step on the first set of EAC4b cells.

In the expriment shown in Figure 3.13, EAC4b cells were pn-incubated with the anti- C4740-756 or with the control non-specific IgG. Folîowing washing, the cebwere incubated with a ümiting amount of C2. The ceUs were then pelleted and the hemolytic activity of the midual C2 in the supernatant was assessed using an aliquot of fresh EAC4b cells. 'Tbus, if the mti-C4740-756 anh'body blocks the interaction between C2 and cellassociated C4b in the pre- incubation step, the C2 remains in the supernatant and cm be measured in a C2 hemolytic May using kshEAC4b celis. It is shown in Fi- 3.13 bat the anti-C47w56 inhi'bited the C4b-C2 intedon in a dosedependent mamer, while the control IgG at an amount equivalent to the highest amount of the anti-C4740"56 employed had no ability to block this interaction. Cumuiatively, these antibody blocking arperiments show that the rntibody raised against the 740-756 segrnent of C4 a'cbain can inhiiit the binding of C2 to C4b. Amount of ~nti-C4740-~56IgG Added to First Set of EAC4b (pg)

Figure 3.12 Effcet of miduai ad-c47~756onthe formation of CS convertase.

Various amounts of anti-c47@7% wen incubated with EAC4b cells (1 x 108) for 90 min at O°C. After washing with cold GVB-~g2+,the cells were resuspended in 100 pl SGVB-Mg2+. Following centrifugation, the supernatants containing residual an~i-C4'@~%were collected and incubated for 10 min at O°C with oxYC2. The arnount of C2 had been pn-detemiincd to enerate -80% hemolysis in the absence of the antibody when pn-incubated with EA cells (1 x 10 0) for 90 min at O°C (23 ng). The mixture was then incubated with a set of bsh EAC4b cells (1 x 107) for 10 min at O°C. Following washing, the cells were tnated with excess Cl followed by C-EIYïA. O 0.7 1.4 2.7 5.5 10.9 21.9 43.8 87.5 87.5

Amount of IgG Aded to FMSet of EAC4b (pg)

Various arnounts of anti-peptide antibodies (a11ti-C4'~~56)wem pn-incubated with MC& cens (1 x 108) at WC for 90 min. The cells were washed with cold GVB-Mg2+. followeâ by the addition of OXYC2 (23 ng). After incubation for 10 min at O°C, the cells wcn ccntrifugtd and the supematants were coiiected. The hemolytic activity of miduai C2 was chmcterized using fmh EAC4b ceiis (15 x IO'), Cl and C-EDTA as a source of C3-9. "C IgW =fers to the fraction of IgG that ran through the immunomnity colurnn adsotbed with BSA-C47-7% conjugate. Chapter 4

Discussion The aim of this shidy was to test the hypothesis dut an acidic residue-rich segment near the I?@-temiinus of C4ô al-chain contains a binding site for C2. It was suggested pmriously by monoclonal anti'body studies (Hessing a ai., 1990q 1990b) that a stretch of 89 amino aci& spenning the 738-826 region at the NH2-terminw of C4 a'-chain was involved in C4BP biading, although no specifio amino acid residues within ihis segment were identifie& Since C4BP and C2 were hown to be antagonistic Ligands of C4b, the 738-826 segment of C4b a'-&& was by extension a candidate for containhg a C2 binding site. Recentiy, a sitedrected mutagenesis mdy on the NHpterminal segment of C3 a'-chah has shown that the negatively charged residuea 730~~and 736EE contn'bue to the interactions of C3b with factor B and CRI, and that of iC3b with CR3 (Taniguchi-Sidle and Isenmao, 1994). In light of the sequence similarity between the NH2-termini of C4 and C3 a'-chains, the present study focused on the two clusters of acidic amino aciâs spanning the 744-752 region of C4 al-chain and examined the role of these highiy consaved charged nsidues in C2 binding.

Severai independent experimeatal approaches were used in testiag our hypothesis including sitedirected mutagenesis, the use of a synthetic peptide mimetic, the use of an ami- peptide anb'body, and the use of a GST-C4 fusion protein. With the exception of the GST-C4 fusion appmach, which may have failed for technicai nasons (see below), the combined resuits hmthe 0ththne approaches strongly suggest that the N&-tenninal segment of C4 a1-chain, and in parti& the two clusters of negatively ch- amino acids 744EED and '~~DEDD, contn'bute impatrntly to the interaction with C2.

The sitedicected mtageneds upptouch was the first mefhod chosen for this study becciuse mutagenesis aîiows one to examine the effect of one or sevd spacinc midues on a pirticuiar activity wîthin the con= of the physioIogidy relevant fam of C4. In this sbdy, isostenc amide substitutions were inttoduced instead of the wuai alaninesam because Gin and Am are sûuddly smiüot to the c-ed residues Glu and Asp, nepectively, and are therefore not expectod to cause major changes in the folding of recombinant C4 molecules. A loss of îbnction as a nwilt of the isosteric axnide substitution cm be intqretted a~ the negative charge p se king requlled for the C2 bindiag interaction, on the condition that the mutation did not ais0 cause any significant long range confdonaiperturbations. The copfo~~~tionalintegrity of the recombinant molecules was therefore also assessed by examiaing the level of secretion, the extent of pst-translational proteolytic processing and the ascleavability of the variow rC4 moleCuies.

interpretation of the site-directed mutagenesis data con be organized into two groups.

Halfof the mutants examineci (744EEN,7*QED, 744- and ~~~DQDD)displayed wild-type- like secretion, processing and cleavability by 6sand yet also showed 2-8-fold defa in C2 binding. Thus, the defects in the respective C2 binding abilities of these mutants can be considered as direct evidence of the detrimental effects caused by the loss of charge mutations. This consequently suggests that residues E'*, ~746and E~Jdirectly conmbute to the binding interaction with C2. On the other hand, halfof the mutants C-N, 7%END, ~~%QNDand 74?NQNN) while showing normal secretion kel, indicating that they were not grossly misfoldod, did show a 23-fold defect in processing, as weii as resistence to C~Scleavage (Figure 3.3). This suggests that the mutations hdintroduced some degree of perturbation of the ovd stnicture of these recombinant molecules, which of neassity clouds the interpretation of th& C2 binding behaviow. Nevertheless, a much greater defect in C2 binding of these mutants was obsewed (Figure 3.5) thmuid be accounted for by the decmse in the concentration of the 3- chah form of C4(CH3NH2) that would have been present dirring the C2 binding assiy.

SpeoificaUy, 749DEND and 744QQN showed -15-fold and -30-fold defects in C2 binding nspectiveIy, and ~~~DQNDand ~~WQNN alma& lost theu C2 binding abilities completely (CM% residuaî activity). This suggests that although the conformational pembation introduced by the isosteric amide substitution in these mutants mybe a contributory fsctor, it is unlikely that this effect on its own can account for the observed very substaotiai loss of C2 binding Wty.

The mutagenesis study has therefore tentatively identified the two acidic cluters of residues located near the NH2-terminus of C4 a*-chahas king essentid for the binding of C2. In consideration of the pmrious &mondon that the two acidic amino acid clusters at the NHptenainus of C3 dahain (~QEand 736EE) are involved in the binding of fmtor B to C3b (Toniguchi-Sidle and Isenman, 1994) and the functional homology between the C3b-factor B interaction and the C4b-C2 interaction, the pnsent finding is consistent with the evolutionary relationship between the classical and alternative pathway C3 convertases. C4 nsidues ~750and D'si, both of which appear to be necesspy for the interaction with C2, comspond to the C3 residues ~736and ~737,whose mutation has been shown to diminish the C3b-B binding activity by 3-fold. However, whereas the C3 residues 730~~only play a minor role in the binding of factor B, it appears that the two acidic clusters ~MEEDand 749~~~~axe equalIy important in mediating the C4W2 interaction.

The observations from the mutagenesis study that were suggestive of a functional sipnincance of the NH2-terminal acidic clustsrs in C2 binding were comborated by the synthetic peptide cornpetition experiments and anti'body blocking assays. A synthetic peptide encompassing nsidues 740-756 wu shown to be able to inhibit the C4b-C2 interaction in a specifïc and dosedependent maMer and an antibody raised agaiast this peptide segment was abcapable of blocking the binding of C2 to C4b.

The peptide appmh has been used extensively in bindiag site mapping studies because of its simplicity, however it involves the ri& of both Wse n-tive and fuse positive dts. A negative ndt with a peptide is inconc1usive because there mry k a stnd oonfodond dependence of the binding interaction that is not attainable by the &oit peptide. However, even in the case of a positive result, such as is the prisent case, there is some ri& of this being a false positive remît if the peptide displays an array of si& chahs which would nounaüy not be accessible in the intact protein. In fact, this is the major nuion why anti-peptide ant'badies oh do not noogPize the intact protein Rom which the peptide was deriveci (Stanfield a al., 1990; Spangln, 1991). However, in ios study the anti-C4740-756anti'body not only ôound the peptide but dso ncognized nucleophile-modüied C4. Furthemores this anh'body was able to bind tell- associated C4b and in so doing block the C4b-C2 interaction in a concentrationdepeadent rnanner. Therefore, in this case, the mimetic peptide is likely to reflect the ûue role of the comsponding seqwnce of authentic C4b in C2 bindhg.

The mti-c4740-756 antibody hm been shown to be epitope-specific in the context of C4b and capable of blocking the binding of C2 to all-bound C4b. but antiiodies may exert their inhibitory effkct not only by directly blocking the specific antigenic determinant, but also by indirect Mchindrance. As independent but complementary mapping appmches, both the site- directed mutagenesis and the synthetic peptide mimetic approaches nstnct the effects on the C4b-C2 binding interaction to the N&-terminal acidic residue-rich segment. Positive resuits fnw these studies in tum suggest that the inhibitory ability of the ami-peptide anhibody is a direct, rather than an indirect effect on this intaaction.

Taken together, the results fkom various xnapping studies involving the use of recombinant moledes, synthetic peptides and anti-peptide antiiodies argue for the direct puticipation of the NH2-terminal acidic clustem in the biading interaction with C2. Although the observations hmthe GSTX4738-78%Mon pratein binding eqeriments corried out in this study are discordant with au argument, the negative results may be false because the hion peptide wueither not present at a sufticient concentration to mediate C2 binding a the relevant peptide segment wrs inaccessible to the C2 in the context of the fimion molede. The insolubiiity of the GST-CQ fusion protein provides strong evidence for this viewpoiat, since GST on its own is a very sduble protein. Whereas the 740-756 peptide segment is quite hydrophilic, the COOH-teminai halfof the 738-788 fusion segment contains sevdhydrophobie segments which may &t in either selfdationor association with the GST fuson putna.

Human complement composent C2 and its alternative pathway analogue factor B each contains 3 SCR domains, a vWF A ddnand a serine protease domain. Previous studies aimed at locaiizing C4b or C3b binding sites in C2 or faor B have presented &dence that C4b or C3b binds to at least two sites in C2 or fator B, respectively, one in the SCR domains of C2b or Ba and the other in the vWF A domain of C2a or Bb (Oglesby et al., 1988; Horiuchi et al., 1991; Ueda et al., 1987; Ryzâiai and benman, 1987; Sanchez-Cod et al., 1990). In addition, the serine protease &main of Bb has been shown to be able to bind C3b (Lambris and Müiler- Eberhard, 1984), howeva, to date there has been no fodevidence suggestive of a C4b- binding 8Ctivity within the saine protease domain of C2a

It has been hypodiesized that the SCR-containing C2b portion of C2 is important in the assembly of the classical pathway C3 convertase (Oglesby et al., 1988). Like many complement protein-protein interactions, the binding of C4b to C2b is enhanced under low ionic mngth conditions (Kerr, 1980), implicating a role for ionic forces in the C4b-CZb binding reaction. This is consistent with the involvement of the acidic residue-rich segment near the MI2-te1mi.n~ of C4 aa-chauiin C2 binding. Revious investigations on the binding interactions between C4b a C3b and their ligand proteins are rlso suggestive of an ionic intenction between SCR-motif- contliining protsins and the negatively chargecl residues at the NH2-termini of both C4 and C3 a'- chain8. On the C3b side of the interaction, a mutagenesis study on the acidic residues within the 727-767 segment of C3 ag-chinbLP shmthat the negatively charged side chains are impartrnt fa the interaction of C3b with facta H and CRI, both of which com'st of SCR &a entirely (Onii and ïsenman, 1999). Substitution of rwidues 736E.Eôy th& iwsteric amides not ooly abolished the binding of C3b by CRI, but aiso markediy inprirsd the interaction between C3b and fUor B (T8Piguch.i-Sidieand Xsenman, 1994). Considerhg that the stnicaaal darmin which

CR1 and f-0~ B possess in cornmon is the SCR motif, it is tempting to speculate that it is the SCR-containing Ba region off- B that binds to the mDdicsegment at the N'Hz-t&w of C3b aW-chain.ûn the derside of the interfi, it bas been shown that the binding activity of CR1 to C3b or C4b con be enhanced by nduciag negative ciurge rnd/or increasing positive charge in the SCR domoins of CRI, suggesting that CR1 binds C3 and C4 tbrough ionic interactions (Krych et al., 1998). Another obsewation in îine with the above evidence is that a cluster of positively charged nsidues, at the interf'ae between the first and second SCR modules of C4BP, has been predicted to play an important role in C4b binding, indicating an electropositive interface on C4BP and an electronegative intahce on C4b (Villoutreix et al., 1998; Blom et al., 1999). A mutagenesis saidy on this patch of basic amino acids fiutber showd that replacement of the basic nsidues by neutral ones led to a signifiant decrease in C4b- binding ability. Cmulatively, these data at least indirectly suggest that the interaction bnween C2 and the acidic residue-rich segment at the NHptexminus of C4 a'-chah involves the SCR domeins of the C2b hgment.

As descn'bed by pmrious stuâies, the assembly of the classical and alternative pathway C3 convertases requins the presence of rnagnesium ions. ~g2+may act eithet as a bndging molede or an allosteric effector in initiating the C4M2 or C3b-B interactions. In the C3b-B interaction, the binding site of M$+ has been locaiized on Bb (Fishelson et ai., 1983) and the C3b-Ba interaction shows no metai ion dependence (Ryzdial and Isemaa, 1987). Unîike the C3b-Ba interaction, the eqyivaient classicai pathway interaction between C4b and C2b is Mgz+- âependent (Kerr, 1980). Thus it is possible that one a more of the carboxylate pups within the 744-752 segment of C4b a'chain Save as a cooodinate ligand for the metai ion, which in tum fatatesbridge fonnation between C4b and C2b. 4.2 ConclUdbu rnd Future Dinctiona

In summary, the collective data fkom the sitedirected mutagenesis studies and the synthetic peptide and anti-peptide anhibody experiments provide atrong evidence that the widic residues 744EED and ~~~D near the NH2-temunus of C4 a'-chain are cnicial fathe binding interaction with C2. We have fiirther predicted that the binding reaction between C2 and the acidic amino acid segment of C4 involves the SCR domains of C2b. Future work will exmine the role of these acidic residues, as well as residues immediately cuboxyl-terminal of this segment, in the interaction of C4b with the SCR motifdominated proteins CR1 and C4BP. Accdng to the mutagenesis study on the compondiag region of C3 a'-chah, although the fmmB and CR1 sites of interaction partialiy ovalap, the charged residue contacts reqyired by CR1 extend over a wider portion of the 727-767 segment than is the case for factor B (Taniguchi-Sidle and Isenrmn, 1994; ûcan and Isenmen, 1999). Specifically, these acidic amino rcids include the 730D~pair, the 7%~pair, E747 and the ~SEDpair. In terms of the putative binding sites in C4b for C4BP, both sitodirected mutagenesis and peptide mimetic studies of C3b have shown that tbe contact residues for factor H are located -7 amino acids C8TbOxyC teminal of those for factor B (Chan and Isenman, 1999; Fisbelson, 1991) and one migbt therefore predict the same spatial disaibution of C2 and C4BP binding sites within the NHp terminal segment of C4b a'-chah In this respect, a syathetic peptide cumponding to the 740- 757 segment of C4 a'-chain wuemployai in an attempt to locaiize a C4BP binding site, but this peptide faiied to inbibit the binding of C4b to C4BP (BLom et ai., 1999). However, it may have ban iucking the COOH-tenninsi segment nece8882y for mediating binding to C4BP. In con- using the viroually identicai peptide, we were able to show the inhi'bitmy e&et of this peptide on the interaction between C4b and C2. nius die apparent divergence of the binding sites for C2 and C4BP camsponds to what we see in the altanative paihway rnelogw, actar B and hcrar II, whoe binâing sites in C3b are separable, despite the antagoniam ktwan the= Ch the C2 side of the interaction, -dies using recombinant fkagmeats of C2, is., recombinant C2b and recombinant C2 vWF A domain, wiîl be necessary to definitively identify which structural domain of C2 is involved in the binding interaction mediated by the NHpterminai acidic segment of C4b ce%hain.

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