mapping of the minimal domain encoding a conformational epitope by λ phage surface display: factor...

Ž .Journal of Immunological Methods 224 1999 89–99

Mapping of the minimal domain encoding a conformationalepitope by l phage surface display: factor VIII inhibitor

antibodies from haemophilia A patients

Ichiro Kuwabara a, Hiroko Maruyama a, Seiki Kamisue b, Midori Shima b,Akira Yoshioka b, Ichi N. Maruyama a,)

a Department of Cell Biology, MB-30, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USAb Department of Pediatrics, Nara Medical UniÕersity, 840 Shijo-cho, Kashihara, Nara 634, Japan

Received 16 December 1998; accepted 15 January 1999

Abstract

Haemophilia A patients who receive repeated transfusion of fVIII concentrates often develop inhibitor alloantibodies,resulting in reduced efficacy of the therapy. Determination of fVIII epitopes for the alloantibodies is essential for anunderstanding of their inhibitory effect on blood coagulation. Random fragments of fVIII displayed on l phage particleswere selected using two patient plasmas immobilized onto the surface of a microtiter plate. A set of clones defined theminimal domain that consisted of 157 amino acid residues including cysteine at both boundaries. The minimal domainabsorbed most of the binding activities of the plasmas to fVIII, suggesting that the domain contains a major determinant forthe plasmas. Site-directed mutagenesis and chemical denaturation of the domain confirmed that a tertiary structure formed bythe disulfide bridge was recognized by the antibodies. The epitope domain defined overlaps with fVIII binding sites to vWfand phospholipid, and may play an important role in blood coagulation. Thus, the bacteriophage l surface display may beuseful for mapping the minimal folding domain of various protein antigens that contain a conformational epitope. q 1999Elsevier Science B.V. All rights reserved.

Keywords: Bacteriophage l; Surface expression; Fusion protein; Affinity selection

AbbreÕiations: bp, nucleotide base pairs; BSA, bovine serumalbumin; DTT, dithiothreitol; ELISA, enzyme-linked immuno-sorbent assay; fVIII, blood coagulation factor VIII; Gal-3, humanlectin galectin-3; IgG, immunoglobulin G; PAGE, polyacrylamidegel electrophoresis; PBS, phosphate-buffered saline; PCR, poly-merase chain reaction; PEG, polyethylene glycol; pfu, plaque-for-ming unit; PMSF, phenylmethanesulfonyl fluoride; SDS, sodiumdodecylsulfate; vWf, von Willebrand factor; X-Gal, 5-bromo-4-chloro-3-indolyl-b-D-galactoside

) Corresponding author. Tel.: q1-619-784-2012; Fax: q1-619-784-9740; E-mail: [email protected]

1. Introduction

Antibodies often recognize three-dimensional,discontinuous structures of protein antigens. For themapping of such conformational epitopes, sophisti-cated technology, such as X-ray crystallographyŽ .Amit et al., 1986; Davies and Cohen, 1996 ordeuterium exchange analysis by nuclear magnetic

Ž .resonance Paterson et al., 1990; Zvi et al., 1995 ,has been used for analysis of the limited number ofantibody–antigen complexes. Alternatively, libraries

0022-1759r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved.Ž .PII: S0022-1759 99 00012-5

( )I. Kuwabara et al.rJournal of Immunological Methods 224 1999 89–9990

of random peptides displayed on the surface of fila-Žmentous bacteriophage Scott and Smith, 1990;

. ŽCortese et al., 1995 or chemically synthesized Fodor.et al., 1991; Pinilla et al., 1996 have been utilized

for the analysis of epitopes, but have been provenless successful in case of conformational epitopesŽ .Luzzago et al., 1993 . Instead, after mutagenesis

Žsuch as deletion Mehra et al., 1986; Ware et al.,. Ž1992 or alanine scanning Jin et al., 1992; Lubin et

.al., 1997 of cDNA encoding antigens of interest,modified antigens have been widely analysed. Over-lapping synthetic peptides, which cover the entiresequence of an antigen, have also been used to

Ž .define its conformational epitopes peptide scanningŽ .Geysen et al., 1987; Loomans et al., 1998 . How-ever, these approaches require substantial efforts andare often inefficient for the determination of theminimal folding domain encoding conformationalepitopes.

Display of the random fragments of protein anti-gens on the surface of filamentous bacteriophageshas more recently been introduced for epitope map-

Žping Petersen et al., 1995; van Zonneveld et al.,.1995; Wang et al., 1995 . The random fragment

libraries have been reported to be more efficient forepitope mapping compared to the random peptide

Ž .libraries Fack et al., 1997; Kuwabara et al., 1997 .However, the random fragment libraries constructedwith the filamentous phage vectors have not beenused for the determination of epitopes that span alarge portion of antigen molecules and that havediscontinuous or conformational structures. The fila-mentous phage vectors rely on the ability of fusionproteins to translocate across the plasma membrane.Some proteins and their fragments fused to the phagecoat proteins may interfere with the passage of thefusion product from the bacterial cytoplasm to

Ž .periplasm Malik et al., 1996; Santini et al., 1998 .Ž .We Maruyama et al., 1994; Mikawa et al., 1996

Ž .and others Dunn, 1995; Sternberg and Hoess, 1995have developed surface expression vectors based onbacteriophage l, whose coat proteins fold up andassemble in the cytoplasm. The vectors are designedto express fusion proteins on the surface of the phagetail or head, and have been successfully used toexpress both cytoplasmic and secreted proteins.

The fVIII is an essential glycoprotein for bloodcoagulation, acting as a cofactor in the factor X

Ž .activation complex van Dieijen et al., 1981 . Defectin this protein results in haemophilia A, a commonhereditary bleeding disorder. Patients who receiverepeated transfusion of fVIII concentrates often, inapproximately 25% of the patients, develop inhibitoralloantibodies against fVIII, resulting in reduced effi-

Ž .cacy of the therapy Ehrenforth et al., 1992 . Theinhibitor antibodies often interfere with the binding

Žof fVIII to vWf, phospholipid and factor IXa Arai etal., 1989; Shima et al., 1993, 1995; Saenko andScandella, 1995; Fijnvandraat et al., 1998; Zhong et

.al., 1998 .Epitopes recognized by fVIII inhibitor antibodies

have extensively been studied. The antibodies pre-Ždominantly bind the A2 andror C2 domain Healey

et al., 1995; Scandella et al., 1995; Shima et al.,.1995; Prescott et al., 1997 . A3–C1 domains and

small regions rich in acidic amino acid residuesoutside the domains are also targets for the inhibitorsŽFulcher et al., 1987; Scandella et al., 1989; Foster etal., 1990; Tiarks et al., 1992; Fijnvandraat et al.,

.1998; Zhong et al., 1998 . In total, at least sixdifferent epitope sites for the inhibitors have beenidentified so far. These epitopes have mainly beendetermined by the expression of fVIII fragments inbacteria or cultured cells followed by analysis usingSDS-PAGE or immunoprecipitation. Discrepancy be-tween epitope sites determined by these approaches

Žhas been reported for inhibitor antibodies Scandella.et al., 1992 . In this report, we describe an efficient

approach to mapping of the minimal folding domainwithin the fVIII molecule that is recognized byinhibitor antibodies, using the l surface expression

Ž .vector lfoo f usion on the outside . The resultsobtained from the present work are also comparedwith the previous ones.

2. Materials and methods

2.1. Library construction

The plasmid construct pSP-VIII that containsŽ . Žfull-length fVIII cDNA 7272 bp in length Toole et

.al., 1984 was obtained from the American TypeŽ .Culture Collection Rockville, MD . The plasmid

DNA, ;10 mg, was digested with 10 ng of DNase IŽ .Boehringer Mannheim, Indianapolis, IN in 100 ml

( )I. Kuwabara et al.rJournal of Immunological Methods 224 1999 89–99 91

Žof buffer 50 mM Tris–HCl, pH 7.4, 10 mM MnCl ,2.50 mgrml BSA for 15 min at 158C. The resulting

random fragments were separated by 2% agarose gelelectrophoresis. A portion of agarose containing DNAfragments, 50–200 bp or 100–2000 bp in sizes, wasexcised and DNA fragments were eluted from agarose

Ž .by a GENECLEAN II kit BIO 101, Vista, CAaccording to the manufacturer’s protocol. The differ-ent DNA size ranges were used for the constructionof libraries having short and long inserts, respec-tively. The ends of the purified DNA, ;2 mg, were

Žblunted by 20 units of T DNA polymerase New4.England Biolabs, Beverly, MA in 50 ml of buffer

Ž50 mM Tris–HCl, pH 8.0, 5 mM MgCl , 5 mM2.DTT, 100 mM each of dNTPs, 50 mgrml BSA for

1 h at 158C. An aliquot, 0.5 mg, of the resultantDNA was ligated in 20 ml of buffer containing20-fold molar excess of adaptor oligonucleotidesŽ .Kuwabara et al., 1997 and 400 units of T DNA4

Ž .ligase New England Biolabs . Unligated adaptorswere removed by 2% agarose gel electrophoresis asdescribed above, and ;30 ng of purified DNAfragments were ligated with 1 mg of lfooDc DNAŽ .Mikawa et al., 1996 which had been digested withBamHI and EcoRI. The ligation mixture was pack-

Žaged using MaxPlax Epicentre Technologies, Madi-. 7son, WI . The resultant libraries consisted of ;10

independent recombinants when titrated with Es-Žcherichia coli strain JM105 Yanisch-Perron et al.,

.1985 . Library amplification was performed by infec-wtion of E. coli strain Q447 mcrA mcrB hsdR

Ž . xD lac-proAB argE rif mal . General manipulationof DNA and phage was carried out as describedŽ .Ausubel et al., 1987; Sambrook et al., 1989 .

2.2. Library screening

ŽPrior to panning, wells of a microtiter plate Im-.mulon 4, Dynatech Laboratories, Chantilly, VA were

coated overnight at 48C with a patient plasma, case 1or case 2, diluted to 1r3000 or 1r500 with PBS,respectively, and preblocked with blocking bufferw Ž . Ž .5% wrv non-fat dry milk, 0.1% vrv Tween-20,

Ž . x0.05% wrv sodium azide in PBS for 1 h at roomtemperature. Libraries were grown with an E. colihost, TG1, to produce fusion proteins on the surfaceof the phage particle as previously describedŽ .Maruyama et al., 1994 . After complete lysis of 2

ml of the library culture, PMSF was added at a finalconcentration of 2 mM. Phages were precipitated by

wthe addition of 1r4 volumes of a solution 26%Ž . Ž .wrv PEG Mw;8000; Fisher, Fair Lawn, NJ ,

x2.6 M NaCl , and suspended in 100 ml of bindingwbuffer 5% non-fat dry milk, 0.1% Tween-20, 0.1%

xsodium azide in PBS supplemented with 2 mMPMSF. The phage suspension, 50 ml, was applied tothe microtiter well and incubated overnight at 48C.Unbound phages were removed by washing at room

Žtemperature three times with washing buffer 5%.non-fat dry milk, 0.5% Tween-20 in PBS and then

Žtwice with second washing buffer 10 mM Tris–HCl,.pH 7.5, 5 mM MgSO , 0.2 M NaCl, 10 mM CaCl .4 2

Phages were eluted from the well with 50 ml of aŽcollagenase solution 20 units in second washing

.buffer for 1 h at 378C. An aliquot, 40 ml, of theeluate was used to infect 2 ml TG1 culture for thesecond round of affinity selection and likewise there-after. The eluate was also assayed for the ratio of

Ž . Žblue vector phage to white plaques recombinant.phage by plating with JM105 in the presence ofŽ .X-Gal 5 Prime–3 Prime, Boulder, CO . General

procedures for phage culture and titration have beenŽdescribed previously Sambrook et al., 1989; Mikawa

.et al., 1996 .

2.3. Immunostaining of phage plaques

Phage plaques formed on the lawn of JM105 wereŽlifted onto nitrocellulose membrane filters Schleicher

.& Schuell, Keene, NH , and the filters were pre-blocked with blocking buffer for 30 min at roomtemperature. The filters were then stained with pa-tient plasmas diluted to 1r2000 with blocking buffer,and mouse anti-human IgG conjugated with alkaline

Ž .phosphatase GG-5, Sigma, St. Louis, MO as sec-ondary antibody. Colour development was performed

Žin buffer 100 mM Tris–HCl, pH 9.5, 100 mM.NaCl, 5 mM MgCl supplemented with 330 mgrml2

nitroblue tetrazolium and 165 mgrml 5-bromo-4-Žchloro-3-indolyl phosphate BIO-RAD, Hercules,

.CA . Intensity of bands was analysed by a computerŽsoftware Gel-Pro Analyzer, Media Cybernetics, Sil-

.ver Spring, MD after capturing the band imageŽusing a photodocumentation station Gel Print 2000i,

.BioPhotonics, Ann Arbor, MI . General proceduresfor plaque lifting and immunostaining have been

Ž .described previously Ausubel et al., 1987 .


2.4. DNA sequencing

DNA sequences of phage clones were determineddirectly from their plaques, according to the manu-

Ž .facturer’s protocols Stratagene, La Jolla, CA usingCyclist Exo-Pfu DNA sequencing kit.

2.5. Site-directed mutagenesis

Site-directed mutagenesis of cysteine residues wasŽ .performed by PCR Saiki et al., 1988 , using primer

pairs, fVIIIf and fVIIIr, fVIIIfm and fVIIIr, fVIIIfand fVIIIrm, and fVIIIfm and fVIIIrm, and pSP-VIII

Ž .as template. Each reaction 20 ml contained 1.0 ngof the pSP-VIII DNA, 0.5 mM primers, 200 mMeach of dNTPs, 2.5 units of Taq DNA polymeraseŽ . wFisher , and PCR buffer 50 mM KCl, 10 mM

Ž .Tris–HCl, pH 8.3, 1.5 mM MgCl , 0.001% wrv2xgelatin . Temperature cycling conditions used were

25 cycles of denaturation for 30 s at 948C, annealingfor 30 s at 558C, and extension for 30 s at 728C. Thesequence of the primers used were as follows: fVIIIfŽ X .5 -CGCCAAGCTTTAGTTGCAGCATGCC , fVII-

Ž X .Ifm 5 -CGCCAAGCTTTAGTGCTAGCATGCC ,Ž XfV IIIr 5 G G CCA G A A TTCTTA TCA G TC -

.CTGTGCCTCGCAGCCCAGAACC , fVIIIrmŽ X5 GGCCAGAATTCTTATCAGTCCTGTGCCTCA-

.CGCCCAGAACC . The PCR products were di-gested with HindIII and EcoRI and cloned intolfooDc as described above. The sequence of theclones was also confirmed by DNA sequencing asdescribed above.

2.6. Absorption of antibodies with epitope phage

Epitope and vector phages were grown in 50 mlŽ .CY medium Maruyama et al., 1994 supplemented

with 3 mM MgCl and 0.5 ml of overnight culture2

of TG1. After complete lysis, 0.25 mg of DNase IŽ .and 0.5 mg of RNase A Boehringer were added to

the culture and incubated for 10 min at 378C. PhagesŽ .were precipitated by the addition of 7% wrv PEG

and 0.65 M NaCl at final concentrations, and sus-pended in 0.5 ml of PBS. Each haemophilia-A pa-tient plasma was diluted to 1r10,000 with PBS andthe phage suspension, and incubated overnight at48C. After removing phages by PEG precipitation,

Žsupernatant was subjected to ELISA Engvall and

.Perlmann, 1971 to measure the amount of remainingŽantibodies. Wells of a microtiter plate Immulon 4,

.Dynatech were coated with 100 mgrml recombi-Ž .nant fVIII KOGENATE, Miles, Elkhart, IN in 100

ml of PBS overnight at 48C and then preblocked withblocking buffer. Patient plasmas, 50 ml each, ab-sorbed with the phage as described above wereapplied to the wells and incubated for 1.5 h at roomtemperature. After washing the wells three timeswith blocking buffer, 50 ml of mouse anti-humanIgG monoclonal antibody conjugated with alkaline

Ž .phosphatase Sigma , which was diluted to 1r10,000with blocking buffer, was added to the plate. Afterincubation for 1 h at room temperature, the wellswere washed three times with blocking buffer andthen twice with PBS. Colour development was per-

Žformed at room temperature in 0.1 ml buffer 100.mM Tris–HCl, pH 9.5, 100 mM NaCl, 5 mM MgCl2

Ž .supplemented with 2 mgrml Sigma-104 Sigma .Under the conditions described above, colour devel-opment at 405 nm was not affected by PEG, and waslinearly proportional to incubation time up to severalhours and patient plasma concentrations ranging from1r1000 to 1r100,000 dilutions. Data were themeans"S.E.M. of three separate measurements.

3. Results

3.1. Construction and selection of epitope libraries

We constructed random fVIII fragment librariesfrom plasmid DNA containing the full length offVIII cDNA. After digestion of the plasmid withDNase I, cleaved DNA fragments consisting of 50–200 bp and 100–2000 bp in sizes were isolated byagarose gel electrophoresis, and were separatelycloned into the lfoo vector to generate libraries withsmall- and large-inserts, respectively. Libraries werescreened with plasmas from two haemophilia pa-tients, case 1 and case 2. The patients were multi-transfused with human plasma-derived fVIII concen-trates, and developed high titre inhibitor antibodies,3200 and 3100 Bethesda units per ml, respectivelyŽ .Shima et al., 1995 . As the selection cycle pro-ceeded in the screening of the large-insert library,reactive phages with either case 1 or 2 plasma wereincreased in number, and dominated in the phage


Ž .population after a few cycles Fig. 1 . In contrast,reactive clones were not at all enriched by thescreening of the small-insert library with either ofthe plasmas. These results suggested that the size ofepitope fragments recognized by the plasmas might

Žbe larger than estimated sizes ;16–66 amino acid.residues encoded by the small-inserts library and

that the epitopes might not be linear or sequential.

3.2. Sequence analysis of epitope clones

The phage clones, 20 each, isolated from thelarge-insert library by the case 1 and case 2 plasmas

Ž .were analysed by DNA sequencing Fig. 2 and alsoŽconfirmed to be positive by immunostaining Fig.

.3A . Among the positives, a few clones had markedlyweak, -5% of total, reactivity with the plasmascompared with the majority having strong reactivityas shown in Fig. 3A, and were, therefore, separatelyanalysed. Alignment of sequences encoded by theseclones along with that of fVIII defined a common

Fig. 1. Affinity selection of fVIII fragment libraries. The large-in-sert library was selected four cycles by haemophilia-A case 1 andcase 2 plasmas immobilized to microtiter wells. Approximately1010 pfu of the phage library were applied to the wells and boundphages were eluted by collagenase. After amplification of theeluted phages by infection to E. coli, phages were subjected to thesecond affinity selection, and likewise thereafter. After each cycleof selection, approximately 1000 pfu of phages were plated, liftedonto a membrane filter, and stained with the plasmas for monitor-ing the enrichment of positive phages in the populations asdescribed in Section 2.

sequence consisting of 157 amino acid residueswithin the C2 domain of fVIII. This large size as anepitope site is consistent with the result in which nopositive clone was recovered from the small-insertlibrary.

The sequence located between Cys-2174 and Asp-2330 in the fVIII sequence was shared by all theclones that had strong reactivity with both of thecase 1 and 2 plasmas, indicating that the two in-hibitor antibodies recognized the same domain withcysteine residues, Cys-2174 and Cys-2326, at amino-and carboxyl-terminal boundaries; there is no othercysteine in the C2 domain of fVIII. Interestingly, theamino-terminal Cys-2174 was deleted in clones thathad markedly weak reactivities with the antibodies.This suggested that the two cysteine residues mightform a disulfide bridge essential for the domain toform a structure recognized by the antibodies. Thecarboxyl-terminus of many clones, 6 out of 14,terminated at the position of Asp-2330, indicatingthat the four amino acid residues after Cys-2326might also be important for binding to the antibodies.In fact, since Gln-2329 are conserved in all the fVIIImolecules defined in animals such as human, pig,

Ž .dog and mice Cameron et al., 1998 , this residuemay be critical for the fVIII structure or function.

3.3. Absorption of antibodies in plasmas by epitopeclone

In this epitope screening, we have identified onlya single epitope domain and failed to isolate clonesspecifying other regions of the fVIII molecule. Thescreening was conducted under the conditions wherethe clones with a greatly reduced, less than 5% oftotal, reactivity, such as a35 and a37 in Fig. 3A,could be recovered. Therefore, the epitope domaindefined might be a sole determinant recognized bythe case 1 and 2 plasmas. Indeed, the minimaldomain clones, a2 in Fig. 2, absorbed ;90% ofantibodies specific to fVIII in the patient plasmasŽ .Fig. 4 . The results are consistent with the previousobservations; when the fVIII molecule was digestedwith peptidases, the case 1 and 2 plasmas onlyreacted with fVIII fragments containing the C2 do-

Ž .main Shima et al., 1995 and there are inhibitorscompletely neutralized by fVIII fragments containing

Ž .C2 Scandella et al., 1989 . Furthermore, three out of


Ž .Fig. 2. Nucleotide sequence of inserts from affinity-purified phage. A Domain structure of the fVIII molecule based on the publishedŽ .sequence Wood et al., 1984 . The mature fVIII protein consisting of 2332 amino acid residues is cleaved intracellularly to form a heavy

chain composed of A1, A2, and B domains and a light chain of A3, C1, and C2 domains. The secreted heterodimer is proteolyticallyactivated by thrombin or factor Xa to a heterotrimer consisting of the A1 and A2 domains and the light chain; the B domain is not required

Ž . Ž .for function and is lost after activation Vehar et al., 1984; Fay et al., 1991 . B Nucleotide sequence of affinity purified clones by case 1and 2 patient plasmas. Portions of the nucleotide and amino acid sequences of fVIII are shown on top. Twenty clones each from the twogroups were sequenced, and clones with a unique sequence were shown on the left with ‘a.’ A common nucleotide sequence among the

5clones is omitted and the boundaries are shown by ‘ .’ Clones with significantly weak reactivity with the plasma in each group are shownunder a dotted line. The 5X and 3X end extensions of nucleotide sequences encoded by some clones are indicated by ‘q numbers.’ Theminimal domain sequence shared by all the clones having strong reactivity is indicated by bold letters.

four other plasmas tested also bound to the minimalŽ .domain data not shown . These are consistent with

the recent observation in which 83% of the patientŽplasmas recognized the C2 domain Prescott et al.,

.1997 .

3.4. Cysteine disulfide bridge for epitope formation

To examine a role for the two cysteine residues inantibody recognition, each of the two residues orboth were replaced with alanine and the reactivity ofthe modified domain was examined by immuno-staining of phage plaques lifted onto a nitrocellulosefilter. As shown in Fig. 3A, all the mutants hadmarkedly reduced activities. The production and sta-bility of the mutant epitopes were indistinguishable

from that with the native sequence, when the fusionproteins prepared from purified phage particles were

Ž .analysed by SDS-PAGE data not shown . Theseresults again suggest that the two cysteine residuesform a disulfide bridge and are essential for recogni-tion by the antibodies.

3.5. Chemical denaturation of epitope

The minimal domain was also analysed by prob-ing with reagents that denature protein conformation.Phage plaques expressing the native or modifieddomains were also lifted to membrane filters that hadbeen treated with SDS or DTT. SDS or DTT treat-ment of the filters markedly reduced the reactivity of

Ž .the native domain with the antibodies Fig. 3B . The


Fig. 3. Immunostaining of phage plaques expressing fVIII epi-Ž .topes. A Library clones purified by affinity selection with case 2

patient plasma were streaked on the E. coli JM105 lawn, trans-ferred to a nitrocellulose filter, and stained with 4000-fold dilutedcase 2 plasma. Note that markedly weak reactivity of clones a37and a35 with the plasma is observed. Phage plaques expressingthe minimal domain, from Cys-2174 to Asp-2330, of fVIII withnative or modified sequences were also stained using the sameprocedures, and are shown on the right. A clone with a substitu-tion of Cys-2174 with alanine is designated as C2174A, likewise,Cys-2326 as C2326A. The lfoo clone with the native minimal

Ž . Ž .domain of fVIII ‘M.D.’ or without insert ‘Vector’ is alsoŽ .shown as a reference. B Phage encoding the minimal domain or

its alanine-replaced mutant was also lifted onto membrane filtersŽ .that had been soaked in 4% SDS ‘qSDS’ or 200 mM DTT

Ž .‘qDTT’ and then dried. Phage plaques were lifted onto theŽ . Ž .treated filter and were stained as in A . A reference phage Gal-3

was also lifted onto the same filters, and stained with a mono-clonal antibody, 1H11, which recognizes a linear, non-conforma-tional epitope of the human lectin galectin-3. The same resultswere also obtained, using other four plasmas including case 1.

weak reactivity of the modified domain was notchanged by the filter treatment and was comparableto that of the native epitope blotted onto the treatedfilters. In this experiment, we also used a phageclone expressing Gal-3 as a reference. The Gal-3sequence contains a linear or sequential epitope rec-ognized by a specific monoclonal antibody, 1H11

Ž .Kuwabara et al., 1997 . The Gal-3 epitope liftedonto the SDS- or DTT-treated filter retained a full

Ž .binding activity to the antibody Fig. 3B , indicatingthat the filter treatment did not affect the antibody–antigen interaction. This also confirmed that thephage proteins were lifted to and retained on theSDS- or DTT-treated filter. The markedly weak reac-tivity of the native domain transferred to the filtertreated with the reducing reagent is consistent withthe results from the alanine-replaced mutants, andindicates that the disulfide bridge plays a crucial rolein the formation of a conformational structure. Inaddition, the conformation is also SDS-sensitive.

The results described above confirm that the anti-bodies recognize the tertiary structure of the epitopedomain, for which a disulfide bridge between thetwo cysteine residues is essential. Furthermore, thefusion proteins expressing the epitope domain had adifferent mobility in the presence or absence ofb-mercaptoethanol, when analysed by SDS-PAGEŽ .data not shown . This also suggests the existence ofthe disulfide bond between Cys-2174 and Cys-2326in the absence of the reducing agent. The disulfidebridge was predicted from an analysis on cyanogenbromide cleaved-fragments of the recombinant fVIII

Fig. 4. Absorption of antibodies in patient plasmas with epitopephage. Plasmas, cases 1 and 2, were mixed with the minimal

Ž . Žepitope phage epitope phage or lfoo vector phage control.phage , and their remaining binding activities to fVIII immobi-

lized to microtiter wells were measured by ELISA as described inSection 2.


protein, although the paucity of the material recov-ered from the high performance liquid chromatogra-

Žphy prevented direct confirmation McMullen et al.,.1995 . Therefore, fVIII in blood is also likely to

have the disulfide bridge between residues Cys-2174and Cys-2326.

4. Discussion

In the present study, we have successfully usedthe l phage surface expression vector for the deter-mination of the minimal domain of fVIII recognizedby the inhibitor antibodies. This is the first instancein which a phage display library of antigen frag-ments has been used for the mapping of a largefolding domain that forms a three-dimensional struc-ture recognized by antibodies. The minimal domaindetermined consists of 157 amino acid residues span-ning most of the fVIII C2 domain including cysteineresidues at both of its boundaries. We have alsofound that the disulfide bridge formed by these twocysteine residues is essential for recognition by thefive plasmas tested in this work, and this is the firstdirect observation showing the importance of thedisulfide bridge in the C2 domain for recognition byfVIII inhibitor antibodies. The reactivity of the do-main with the antibodies was also abolished by theSDS treatment of the filter under the condition inwhich the cysteine disulfide bridge is intact. There-fore, the epitope recognized by the antibodies used inthis work is likely conformational or discontinuous.Amino acid residues scattered around in the minimaldomain may be brought together by the disulfidebridge and other forces such as ionic and hydropho-bic interactions. It is equally possible that linear orsequential residues constrained by the disulfide bridgemay also be recognized by the plasmas. It is notnecessary that the five plasmas used here recognizeexactly the same epitope. Different epitope sites onthe three-dimensional structure of the minimal do-main may be recognized.

Epitopes recognized by eleven inhibitors includ-ing the plasmas studied here have previously beenmapped to a region spanning residues 2248–2312 of

Ž .fVIII Scandella et al., 1995; Shima et al., 1995 .This epitope site overlaps with the one determined inthis study. However, it is considerably shorter and

does not include the cysteine residues essential forthe antibody binding. The previous epitope mappingemployed SDS-PAGE, including a reducing agent,for the separation of epitope fragments. Under theseconditions, the native three-dimensional structure ofthe fVIII epitope should be disrupted. Furthermore,in this epitope mapping, we have failed to isolate aclone shorter than the clones that have markedlyweak activities. The amino-terminus of all the weak-est clones started from the 2176th methionine residueof the C2 domain, suggesting that the methionine ora residue nearby is essential for the weakest reactiv-ity. Therefore, if any, a clone that encodes the shortepitope previously determined should have weakeractivity than the weakest clones isolated in this work.These results suggest that the epitope site previouslydetermined may also lack other residues in additionto the cysteines. Alternatively, the epitope site maybind separate antibodies in the plasmas that recog-nize completely denatured structure of fVIII, sincefVIII concentrates used for infusion therapy are likelyto contain inactive, denatured factors.

The fVIII C2 domain has been shown to interactŽwith vWF and phospholipid Shima et al., 1993;

.Saenko and Scandella, 1997 . Based on inhibitionassay using synthetic peptides, the binding site offVIII to phospholipid has been localized to its car-boxyl-terminal region, residues 2303–2332, within

Ž .the C2 domain Foster et al., 1990 . This syntheticŽpeptide also inhibits fVIII binding to vWf Saenko

.and Scandella, 1995 . The fVIII binding site to phos-pholipid and vWf overlaps with the minimal domaindetermined in this study. However, amino acidresidues responsible for fVIII binding to inhibitorantibodies, vWf or phospholipid remain to be deter-mined. Starting from the minimal domain of fVIIIthat has been determined in this work, amino acidresidues that directly interact with the plasmas, vWfor phospholipid may also be defined by random

Žmutagenesis as previously described Jespers et al.,.1997 . The minimal domain expressed on the l

phage may be randomly mutated by the error-proneŽ .PCR Cadwell and Joyce, 1994 or using mutagenic

Ž .synthetic oligonucleotides Maruyama et al., 1995 .After selection of phage clones that express thedomains correctly folded, phage clones with no orweak activity may be negatively selected by affinitychromatography using the plasmas, vWf or phospho-


lipid, respectively. DNA sequence analysis of theclones should identify residues critical for the bind-ing. Residues involved in antibody binding may notcompletely overlap with those responsible for fVIIIbinding to vWf and phospholipid. By mutagenesis ofthe non-overlapping residues, it may be possible toconstruct modified fVIII molecules that does notbind C2-specific inhibitors but have normal pro-coagulant activity. Such fVIII molecules may also beless immunogenic for infusion therapy of haemophiliaA patients.

The l phage coat proteins fold up and assembleinto the phage particle in the cytoplasm of E. coli.The reducing condition in the bacterial cytoplasmprevents the formation of cysteine disulfide bonds,although it is known that the bonds are formed in

Ž .particular mutants Derman et al., 1993 . After as-sembly, the phage breaks bacterial cell walls and isreleased into culture media, where disulfide bondsmay be formed as we have shown in this report.Hence, l phage surface display can be efficientlyused for mapping of the minimal folding domain thatcontains a conformational epitope of cytoplasmic orsecreted proteins with disulfide bridges. However,epitope sites that are post-translationally modified bysuch a mechanism as glycosylation or sulfation maynot be efficiently determined by the approach usinglfoo.

Acknowledgements

We are grateful to Margaret Huflejt, Mona Jazay-eri, Jerry Ware, Nalin Kumar, Takanori Moriki andMasataka Nakamura for critical readings of themanuscript. This work was supported in part byTaitec.

References

Amit, A.G., Mariuzza, R.A., Phillips, S.E.V., Poljak, R.J., 1986.Three-dimensional structure of an antigen–antibody complex

˚at 2.8 A resolution. Science 233, 747.Arai, M., Scandella, D., Hoyer, L.W., 1989. Molecular basis of

factor VIII inhibition by human antibodies. Antibodies thatbind to the factor VIII light chain prevent the interaction offactor VIII with phospholipid. J. Clin. Invest. 83, 1978.

Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman,

J.G., Smith, J.A., Struhl, K., 1987. Current Protocols inMolecular Biology, Wiley-Interscience, New York.

Cadwell, R.C., Joyce, G.F., 1994. Mutagenic PCR. PCR MethodsApplic. 3, S136.

Cameron, C., Notley, C., Hoyle, S., McGlynn, L., Hough, C.,Kamisue, S., Giles, A., Lillicrap, D., 1998. The canine factorVIII cDNA and 5X flanking sequence. Thromb. Haemost. 79,317.

Cortese, R., Monaci, P., Nicosia, A., Luzzago, A., Felici, F.,Galfre, G., Pessi, A., Tramontano, A., Sollazzo, M., 1995.´Identification of biologically active peptides using randomlibraries displayed on phage. Curr. Opin. Biotechnol. 6, 73.

Davies, D.R., Cohen, G.H., 1996. Interactions of protein antigenswith antibodies. Proc. Natl. Acad. Sci. U.S.A. 93, 7.

Derman, A.I., Prinz, W.A., Belin, D., Beckwith, J., 1993. Muta-tions that allow disulfide bond formation in the cytoplasm ofEscherichia coli. Science 262, 1744.

Dunn, I.S., 1995. Assembly of functional bacteriophage lambdavirions incorporating C-terminal peptide or protein fusionswith the major tail protein. J. Mol. Biol. 248, 497.

Ehrenforth, S., Kreuz, W., Scharrer, I., Linde, R., Funk, M.,Gungor, T., Krackhardt, B., Kornhuber, B., 1992. Incidence of¨ ¨development of factor VIII and factor IX inhibitors inhaemophiliacs. Lancet 339, 594.

Engvall, E., Perlmann, P., 1971. Enzyme-linked immunosorbentŽ .assay ELISA . Quantitative assay of immunoglobulin G. .

Immunochemistry 8, 871.Fack, F., Hugle-Dorr, B., Song, D., Queitsch, I., Petersen, G.,¨ ¨

Bautz, E.K.F., 1997. Epitope mapping by phage display:random versus gene-fragment libraries. J. Immunol. Methods206, 43.

Fay, P.J., Haidaris, P.J., Smudzin, T.M., 1991. Human factorVIIIa subunit structure. Reconstitution of factor VIIIa from theisolated A1rA3–C1–C2 dimer and A2 subunit. J. Biol. Chem.266, 8957.

Fijnvandraat, K., Celie, P.H.N., Turenhout, E.A.M., ten Cate,J.W., van Mourik, J.A., Mertens, K., Peters, M., Voorberg, J.,1998. A human alloantibody interferes with binding of factorIXa to the factor VIII light chain. Blood 91, 2347.

Fodor, S.P.A., Read, J.L., Pirrung, M.C., Stryer, L., Lu, A.T.,Solas, D., 1991. Light-directed, spatially addressable parallelchemical synthesis. Science 251, 767.

Foster, P.A., Fulcher, C.A., Houghten, R.A., Zimmerman, T.S.,1990. Synthetic factor VIII peptides with amino acid se-quences contained within the C2 domain of factor VIII inhibitfactor VIII binding to phosphatidylserine. Blood 75, 1999.

Fulcher, C.A., de Graaf Mahoney, S., Zimmerman, T.S., 1987.FVIII inhibitor IgG subclass and FVIII polypeptide specificitydetermined by immunoblotting. Blood 69, 1475.

Geysen, H.M., Rodda, S.J., Mason, T.J., Tribbick, G., Schoofs,P.G., 1987. Strategies for epitope analysis using peptide syn-thesis. J. Immunol. Methods 102, 259.

Healey, J.F., Lubin, I.M., Nakai, H., Saenko, E.L., Hoyer, L.W.,Scandella, D., Lollar, P., 1995. Residues 484–508 contain amajor determinant of the inhibitory epitope in the A2 domainof human factor VIII. J. Biol. Chem. 270, 14505.

Jespers, L., Jenne, S., Lasters, I., Collen, D., 1997. Epitope´


mapping by negative selection of randomized antigen librariesdisplayed on filamentous phage. J. Mol. Biol. 269, 704.

Jin, L., Fendly, B.M., Wells, J.A., 1992. High resolution func-tional analysis of antibody–antigen interactions. J. Mol. Biol.226, 851.

Kuwabara, I., Maruyama, H., Mikawa, Y.G., Zuberi, R.I., Liu,F.-T., Maruyama, I.N., 1997. Efficient epitope mapping bybacteriophage lambda surface display. Nature Biotechnol. 15,74.

Loomans, E.E., Nijholt, L.J., Cremers, A.J., Paulij, W.P., Schie-len, W.J., 1998. Epitope mapping and serodiagnosis using

Ž .Ata- and Lys 7 peptides. Biochim. Biophys. Acta 1379, 273.Lubin, I.M., Healey, J.F., Barrow, R.T., Scandella, D., Lollar, P.,

1997. Analysis of the human factor VIII A2 inhibitor epitopeby alanine scanning mutagenesis. J. Biol. Chem. 272, 30191.

Luzzago, A., Felici, F., Tramontano, A., Pessi, A., Cortese, R.,1993. Mimicking of discontinuous epitopes by phage-dis-played peptides: I. Epitope mapping of human H ferritin usinga phage library of constrained peptides. Gene 128, 51.

Malik, P., Terry, T.D., Gowda, L.R., Langara, A., Petukhov, S.A.,Symmons, M.F., Welsh, L.C., Marvin, D.A., Perham, R.N.,1996. Role of capsid structure and membrane protein process-ing in determining the size and copy number of peptidesdisplayed on the major coat protein of filamentous bacterio-phage. J. Mol. Biol. 260, 9.

Maruyama, I.N., Maruyama, H.I., Brenner, S., 1994. Lambda foo:a lambda phage vector for the expression of foreign proteins.Proc. Natl. Acad. Sci. U.S.A. 91, 8273.

Maruyama, I.N., Mikawa, Y.G., Maruyama, H.I., 1995. A modelfor transmembrane signalling by the aspartate receptor basedon random-cassette mutagenesis and site-directed disulfidecross-linking. J. Mol. Biol. 253, 530.

McMullen, B.A., Fujikawa, K., Davie, E.W., Hedner, U., Ezban,M., 1995. Locations of disulfide bonds and free cysteines inthe heavy and light chains of recombinant human factor VIIIŽ .antihemophilic factor A . Protein Sci. 4, 740.

Mehra, V., Sweetser, D., Young, R.A., 1986. Efficient mapping ofprotein antigenic determinants. Proc. Natl. Acad. Sci. U.S.A.83, 7013.

Mikawa, Y.G., Maruyama, I.N., Brenner, S., 1996. Surface dis-play of proteins on bacteriophage lambda heads. J. Mol. Biol.262, 21.

Paterson, Y., Englander, S.W., Roder, H., 1990. An antibodybinding site on cytochrome c defined by hydrogen exchangeand two-dimensional NMR. Science 249, 755.

Petersen, G., Song, D., Hugle-Dorr, B., Oldenburg, I., Bautz,¨ ¨E.K.F., 1995. Mapping of linear epitopes recognized by mono-clonal antibodies with gene-fragment phage display libraries.Mol. Gen. Genet. 249, 425.

Pinilla, C., Appel, J.R., Houghten, R.A., 1996. Tea bag synthesisof positional scanning synthetic combinatorial libraries andtheir use for mapping antigenic determinants. Methods Mol.Biol. 66, 171.

Prescott, R., Nakai, H., Saenko, E.L., Scharrer, I., Nilsson, I.M.,Humphries, J.E., Hurst, D., Bray, G., Scandella, D., Recombi-nate and kogenate study groups, 1997. The inhibitor antibodyresponse is more complex in hemophilia A patients than in

most nonhemophiliacs with factor VIII autoantibodies. Blood89, 3663.

Saenko, E.L., Scandella, D., 1995. A mechanism for inhibition offactor VIII binding to phospholipid by von Willebrand factor.J. Biol. Chem. 270, 13826.

Saenko, E.L., Scandella, D., 1997. The acidic region of the factorVIII light chain and the C2 domain together form the highaffinity binding site for von Willebrand factor. J. Biol. Chem.272, 18007.

Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R.,Horn, G.T., Mullis, K.B., Erlich, H.A., 1988. Primer-directedenzymatic amplification of DNA with a thermostable DNApolymerase. Science 239, 487.

Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular cloning:a laboratory manual, 2nd edn. Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, NY.

Santini, C., Brennan, D., Mennuni, C., Hoess, R.H., Nicosia, A.,Cortese, R., Luzzago, A., 1998. Efficient display of an HCVcDNA expression library as C-terminal fusion to the capsidprotein D of bacteriophage lambda. J. Mol. Biol. 282, 125.

Scandella, D., Mattingly, M., de Graaf, S., Fulcher, C.A., 1989.Localization of epitopes for human factor VIII inhibitor anti-bodies by immunoblotting and antibody neutralization. Blood74, 1618.

Scandella, D., Timmons, L., Mattingly, M., Trabold, N., Hoyer,L.W., 1992. A soluble recombinant factor VIII fragment con-taining the A2 domain binds to some human anti-factor VIIIantibodies that are not detected by immunoblotting. Thromb.Haemost. 67, 665.

Scandella, D., Gilbert, G.E., Shima, M., Nakai, H., Eagleson, C.,Felch, M., Prescott, R., Rajalakshmi, K.J., Hoyer, L.W.,Saenko, E., 1995. Some factor VIII inhibitor antibodies recog-nize a common epitope corresponding to C2 domain aminoacids 2248 through 2312, which overlap a phospholipid-bind-ing site. Blood 86, 1811.

Scott, J.K., Smith, G.P., 1990. Searching for peptide ligands withan epitope library. Science 249, 386.

Shima, M., Scandella, D., Yoshioka, A., Nakai, H., Tanaka, I.,Kamisue, S., Terada, S., Fukui, H., 1993. A factor VIIIneutralizing monoclonal antibody and a human inhibitor al-loantibody recognizing epitopes in the C2 domain inhibitfactor VIII binding to von Willebrand factor and to phos-phatidylserine. Thromb. Haemost. 69, 240.

Shima, M., Nakai, H., Scandella, D., Tanaka, I., Sawamoto, Y.,Kamisue, S., Morichika, S., Murakami, T., Yoshioka, A.,1995. Common inhibitory effects of human anti-C2 domaininhibitor alloantibodies on factor VIII binding to von Wille-brand factor. Br. J. Haematol. 91, 714.

Sternberg, N., Hoess, R.H., 1995. Display of peptides and proteinson the surface of bacteriophage l. Proc. Natl. Acad. Sci.U.S.A. 92, 1609.

Tiarks, C., Pechet, L., Anderson, J., Mole, J.E., Humphreys, R.E.,1992. Characterization of a factor VIII immunogenic site usingfactor VIII synthetic peptide 1687–1695 and rabbit anti-peptideantibodies. Thromb. Res. 65, 301.

Toole, J.J., Knopf, J.L., Wozney, J.M., Sultzman, L.A., Buecker,J.L., Pittman, D.D., Kaufman, R.J., Brown, E., Shoemaker, C.,


Orr, E.C. et al., 1984. Molecular cloning of a cDNA encodinghuman antihaemophilic factor. Nature 312, 342.

van Dieijen, G., Tans, G., Rosing, J., Hemker, H.C., 1981. Therole of phospholipid and factor VIIIa in the activation ofbovine factor X. J. Biol. Chem. 256, 3433.

van Zonneveld, A.-J., van den Berg, B.M.M., van Meijer, M.,Pannekoek, H., 1995. Identification of functional interactionsites on proteins using bacteriophage-displayed random epi-tope libraries. Gene 167, 49.

Vehar, G.A., Keyt, B., Eaton, D., Rodriguez, H., O’Brien, D.P.,Rotblat, F., Oppermann, H., Keck, R., Wood, W.I., Harkins,R.N. et al., 1984. Structure of human factor VIII. Nature 312,337.

Wang, L.-F., Plessis, D.H.D., White, J.R., Hyatt, A.D., Eaton,B.T., 1995. Use of a gene-targeted phage display randomepitope library to map an antigenic determinant on the blue-tongue virus outer capsid protein VP5. J. Immunol. Methods178, 1.

Ware, J., MacDonald, M.J., Lo, M., de Graaf, S., Fulcher, C.A.,

1992. Epitope mapping of human factor VIII inhibitor antibod-ies by site-directed mutagenesis of a factor VIII polypeptide.Blood Coagul. Fibrinolysis 3, 703.

Wood, W.I., Capon, D.J., Simonsen, C.C., Eaton, D.L., Gitschier,J., Keyt, B., Seeburg, P.H., Smith, D.H., Hollingshead, P.,Wion, K.L. et al., 1984. Expression of active human factorVIII from recombinant DNA clones. Nature 312, 330.

Yanisch-Perron, C., Vieira, J., Messing, J., 1985. Improved M13phage cloning vectors and host strains: nucleotide sequencesof the M13mp18 and pUC19 vectors. Gene 33, 103.

Zhong, D., Saenko, E.L., Shima, M., Felch, M., Scandella, D.,1998. Some human inhibitor antibodies interfere with factorVIII binding to factor IX. Blood 92, 136.

Zvi, A., Kustanovich, I., Feigelson, D., Levy, R., Eisenstein, M.,Matsushita, S., Richalet-Secordel, P., Regenmortel, M.H., An-´glister, J., 1995. NMR mapping of the antigenic determinantrecognized by an anti-gp120, human immunodeficiency virusneutralizing antibody. Eur. J. Biochem. 229, 178.

mapping of the minimal domain encoding a conformational epitope by λ phage surface display: factor...

Documents