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