Proc. Natl. Acad. Sci. USAVol. 89, pp. 1548-1552, March 1992Immunology

Transmission-blocking antibodies recognize microfilarial chitinasein brugian lymphatic filariasis

(Brugia malyi/parasitic nematodes/PCR)

JULIET A. FUHRMAN*t, WILLIAM S. LANEt, RANDALL F. SMITH§, WILLY F. PIESSENS*,AND FRANCINE B. PERLERt*Department of Tropical Public Health, Harvard School of Public Health, 665 Huntington Avenue, Boston, MA 02115; tNew England Biolabs, Inc., 32 TozerRoad, Beverly, MA 01915; *Harvard University Microchemistry Facility, 16 Divinity Avenue, Cambridge, MA 02138; and §Molecular Biology ComputerResearch Resource, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115

Communicated by Phillips W. Robbins, November 12, 1991

ABSTRACT Brugia malayi is a parasitic nematode thatcauses lymphatic filariasis in humans. The monoclonal anti-body MF1, which mediates clearance of peripheral microfila-remia in a gerbil infection model, recognizes two stage-specificproteins, p70 and p75, in B. malayi microrflariae. cDNA codingfor the MF1 antigen was sequenced, and the predicted proteinsequence shows significant similarities to chitinases from bac-teria and yeast. When microfilarial extracts and purifiedpreparations of the MF1 antigen were tested for chitinaseactivity, strong bands of chitin-degrading activity comigratedin SDS/PAGE with p70 and p75 and showed a reduction-dependent mobility shift characteristic of the MFl antigen.Thus, the MF1 antigen is microfrarial chitinase, which mayfunction to degrade chitin-containing structures in the micro-filaria or in its mosquito vector during parasite developmentand transmission.

Lymphatic filariasis afflicts nearly 100 million people world-wide (1). Of the three species of lymphatic filariids that infecthumans, Brugia malayi can most readily be maintained in thelaboratory by passage between gerbils (another natural host)and a mosquito vector. Thus, B. malayi has been useful forstudying pathogenesis and transmission of filariasis at themolecular level.

Passive transfer of the monoclonal antibody MF1 waspreviously shown to mediate the transient clearance of firststage larvae, or microfilariae (mf), from the peripheral bloodof gerbils infected with B. malayi (2). The MF1 antibodyrecognizes two stage-specific proteins (p70 and p75) in ex-tracts of mf that appear only after the mf have matured forseveral days in the vertebrate host (3). The appearance of theMF1 antigen corresponds with the onset of the parasite'sability to infect the mosquito (3).

It was thus of great interest to define more precisely therole of the MF1 antigen in filarial development and trans-mission. With the expectation that the proteins' sequencesmight clarify their function, we obtained partial amino acidsequences for p70 and p75 and then used this information toisolate and to sequence the corresponding cDNA.1 We reporthere that the predicted protein sequence of the MF1 antigenhas several regions of significant similarity to bacterial andyeast chitinases. Furthermore, the native MF1 antigen isdemonstrated to degrade chitin in vitro.

MATERIALS AND METHODSParasite Material. Gerbils (Meriones unguiculatus) were

infected intraperitoneally with B. malayi either in our ownfacilities or at the Filariasis Repository Research Service,

Athens, GA. mfwere purified and proteins were extracted asdescribed (3). Poly(A) mRNA was isolated from purified mfaccording to Cox and Smulian (4). Genomic DNA was madefrom adult B. malayi as described (5).

Protein Purification and Amino Acid Sequencing. The MF1antigen was partially purified from extracts of mf by DE-52chromatography as described (6). N-terminal sequence anal-ysis was performed on the combined p70 and p75 followingelution with 0.5 M NaCI from DE-52, using an ABI model477A protein sequencer. The sample was subjected to auto-mated Edman degradation using the program NORMAL-i,which was modified using the manufacturer's recommenda-tions for faster cycle time (37 min) by decreasing dry-downtimes and increasing reaction cartridge temperature to 530Cduring coupling. The resultant phenylthiohydantoin aminoacid fractions were manually identified using an on-line ABImodel 120A HPLC apparatus and Shimadzu CR4A integra-tor.

Trypsin, Asp-N, and Glu-C (sequencing grade, BoehringerMannheim) were used to digest the separated p70 and p75.Proteolytic digests were performed directly on nitrocelluloseaccording to Aebersold et al. (7), using DE-52-purified ma-terial that was separated by SDS/PAGE (8, 9) and transblot-ted (10). Following proteolytic digestion, peptides were sep-arated by narrow-bore reverse-phase HPLC on a Vydac 2.1mm x 150 mm C18 column. The gradient employed was amodification of that described by Stone et al. (11). Briefly,where buffer A was 0.06% trifluoroacetic acid/H20 andbuffer B was 0.055% trifluoroacetic acid/acetonitrile, a gra-dient of 5% buffer B at 0 min, 33% buffer B at 63 min, 60%obuffer B at 95 min, and 80% buffer B at 105 min with a flowrate of 0.15 ml/min was used. CNBr peptides were generatedby cleavage of the separated p70 and p75 in gel slicesfollowing SDS/PAGE, using 7 mg of CNBr per ml in 70%6formic acid. Cleavage products were then electrophoresedand transblotted to poly(vinylidene difluoride) (Immobilon,Millipore) according to Matsudaira (12), stained briefly withCoomassie blue, excised, and sequenced directly.

Glycosidase treatment was performed on total mf extracts,using N-glycanase and 0-glycanase from Genzyme, accord-ing to the supplier's protocols.

Library Screening, DNA Sequencing, and PCR Amplifica-tion. MF1-specific DNA probes were synthesized by PCRamplification of B. malayi genomic DNA using degenerateprimers derived from the protein sequence of the N terminus(sense strand, primers P1 and P4) and a tryptic peptide(antisense strand, primers P5 and P6). PCR amplificationswere performed using Taq polymerase (Cetus), and amplifi-cation products were purified from low-melting-point agarose

Abbreviation: mf, microfilaria(e).$The sequence reported in this paper has been deposited in theGenBank data base (accession no. M73689).

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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(SeaKem, FMC). The genomic probes were 32P-labeled bynick-translation (13) and used to screen a B. malayi mfcDNAlibrary in Agtll (gift of Timothy Nilsen, Case WesternReserve University, Cleveland).

MF1-specific single-stranded cDNA was synthesized frommf mRNA with the cDNA Synthesis Plus kit (Amersham),using an antisense primer (P21) derived from the DNAsequence of phage 4A2 (see Results). First strand cDNA wasthen PCR amplified using two primer combinations. ThecDNA fragment 52.4 was generated using P48 and P21, andfragment 4'V was generated using PSL and P21.2 (see Fig. 3).

Oligonucleotides for PCR amplifications and for sequenc-ing primers were synthesized on a Biosearch model 8750DNA synthesizer or were purchased from New EnglandBiolabs. The cDNA clones and PCR-amplified cDNA prod-ucts were sequenced in both directions using a nested set ofoligonucleotide primers and single-stranded DNA templates(14). All nucleotide sequencing was performed by the dide-oxy method using Sequenase Version 2.0 (United StatesBiochemical) according to the manufacturer's protocol.Computer Sequence Analyses. Protein sequence data base

searches were conducted with the GENINFLO BLAST NetworkService provided by the National Center of BiotechnologyInformation, National Library of Medicine, National Insti-tutes of Health (15). Multisequence alignments were per-formed using the PATTERN-INDUCED MULTI-ALIGNMENT pro-gram (R. F. Smith and T. F. Smith, Molecular BiologyComputer Research Resource, 1990) based on the patternconstruction algorithm previously described (16).

Chitinase Activity Assays. Assays for chitinolytic activitywere performed in SDS gels cast with glycol chitin accordingto Trudel and Asselin (17). Extracts of native mf proteinswere prepared as described (6), but in the absence ofproteaseinhibitors. Under these conditions, p70 and p75 consistentlymigrate with a lower molecular mass than previously de-scribed for extracts prepared in the presence of proteaseinhibitors. Following electrophoresis, proteins were rena-tured in the gel by overnight incubation at 37°C in 1% TritonX-100 (Boehringer Mannheim, 789-704). Areas of digestedchitin were visualized as dark bands on a fluorescent back-ground after staining with Calcofluor M2R (17).

RESULTSProtein Sequence of the Native Antigen. The MF1 mono-

clonal antibody detects two bands in Western blots withmobilities corresponding to approximately 70 and 75 kDaunder reducing conditions (6). The HPLC profiles of trypticdigests of p70 and p75 are highly homologous, with mostmajor peaks shared between the two (Fig. 1). Three trypticpeptides, as well as two CNBr peptides, from p75 hadsequences identical to peptides of the same mobility from p70(Fig. 2). N-terminal sequence analysis of a mixture ofp70 andp75 generated a single amino acid residue at each cycle (Fig.2). CNBr peptides derived from the individual proteinsgenerated the same N-terminal sequence (YVRG-YY), sug-gesting that p70 and p75 are identical at the N terminus. Thesecomparisons indicate a high degree of primary structuresimilarity between p70 and p75.To examine differences between the primary structures of

p70 and p75, we sequenced the double peak (retention time= 67 min) indicated by the arrow in the p75 profile (Fig. 1A)and the corresponding single peak (retention time = 67 min)from the p70 profile. The p70 peak gave a single sequence(GYGGAFIWALDFDDFTGK). The p75 double peak gavethis sequence and a secondary sequence in addition (GPY-PLLNAISSELEGESEN-). This suggested that the second-ary sequence could be part of a peptide unique to the p75molecule.

E 100A

-10,10 20 30 40 50 60 70

60 B50-

40

:D30E 20

10

0--10

10 20 30 40 50 60 70Time (min)

FIG. 1. Vydac C18 HPLC profiles of tryptic digests of isolated p75(A) and p70 (B). The arrow indicates the mobility of the peptideunique to the p75 map that was partially sequenced (amino acids377-395, see Fig. 3). mAU, milliabsorbance units.

Isolation of a cDNA Clone Coding for the C Terminus of theMF1 Antigen. Degenerate oligonucleotide primers were syn-thesized corresponding to the N-terminal region (sense) anda tryptic peptide shared by p70 and p75 (antisense). Theseprimers (P1, P4, P5, and P6, Fig. 3) were used to amplifygenomic DNA from B. malayi by PCR. Combinations of P1and P5 or P6 generated a 2.3-kilobase (kb) amplificationproduct, whereas combinations of P4 and P5 or P6 generateda 2.0-kb product. This suggests the presence of an intronbetween P1 and P4. The 2.3-kb product was partially se-quenced, and the predicted amino acid sequence of oneregion matched the sequence of a tryptic peptide from p75(amino acids 207-221) distinct from that used to construct theoligonucleotide primers. This strongly implicated the 2.3-kb

N-terminus p75/p70YVRG-YYTNW AQYRDGEGKF

Tryptic peptides, p75GYGGAFIWAL DFDDFTGK*NNFDGFDLDW EYPVGVAEQH A**KNNFDGFDLIIIGIPMYAQ GWTLDNP'ETVHQEGVGA YMVK

GPYPLLNAIS SELEGESEN

GTIDGSGDQWYG-DELGDSKPF E-NDEDTEWS KGGEVSGIGIFN TEFAADYWAS

LVEAMK

NFDLLFLMSY DLHGS-ETNPAGGTASY -EIS-GK

CNBr peptides, p75YVRG-YYTNW AQYRDGEGKF LPGNI'YSAVT5YAQG

LPGNIPNGL- T-ILYAFAKV D

Tryptic peptides, p7OGYGGAFIWAL DFDDFTGK*NNFDGFDLD- EY**

YLK

-IIGIPMYAQ G-TLD'ETVHQEGVGA YMVK

Asp-N peptides, p70DGSYNVESLG KNFDQWYGYDNPSETAI-A AASRPS-A

Glu-C peptides, p70AMKTAFVE

GVGAYMVKGD Q-YGYDN

CNBr peptides, p70YVRG-YYDN- -QY'YSAVTKLRET DPGLKVLLSY G-YNF'YAQGWTLDNP SETAIGKTAFVEEAKT SGKQRLL

FIG. 2. Peptide sequences from p75 and p70. Peptides markedwith identical superscripts have equivalent retention times (trypticpeptides) or molecular masses on SDS/PAGE (CNBr peptides).

Immunology: Fuhrman et al.

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1550 Immunology: Fuhrman et al.

amplified genomic DNA fragment as part ofthe coding regionfor the MF1 antigen.The 2.3- and 2.0-kb DNA fragments were used to probe a

B. malayi microfilarial cDNA library in Agtll. Clone 4A2contained the largest insert of all hybridizing clones. Theinsert was sequenced and found to encode seven peptidessequenced from native p75, including the peptide unique tothe p75 tryptic profile (Fig. 3). No discrepancies could befound between the amino acid sequences of the nativepeptides and the deduced sequence of clone 4A2. The openreading frame of 718 nucleotides was followed by a stopcodon, a 50-nucleotide untranslated region, and a poly(A)tail.The 4A2 insert encoded sequences from p70 and p75 (Figs.

2 and 3). It was therefore used as a probe for Southern-blotted, EcoRI-restricted genomic DNA (from B. malayi) totest the possibility that two separate genes code for p70 andp75. Two bands (1.4 and 3.7 kb) hybridized strongly, and twobands (0.8 and 3.1 kb) hybridized very weakly with the4A2-derived probe (data not shown). PCR amplification ofgenomic DNA spanning the region coding for 4A2 generateda single 2.5-kb product, which yielded a 1.4-kb productfollowing digestion with EcoRI (data not shown). Althoughthis suggests that the two strongly hybridizing bands (1.4 and3.7 kb) are part of a single gene coding for these proteins, wecannot rule out the possibility that the weakly hybridizingbands represent a second gene coding for an MF1-reactivemolecule.

Sequencing the 5' End of the mRNA by PCR Amplification.None of the isolated cDNA clones contained sequence en-coding the N terminus of the MF1 protein. Therefore, the

remainder of the sequence was obtained from unclonedcDNA amplified in two steps by PCR. First strand cDNA wassynthesized using mf mRNA and an antisense primer fromthe 4A2 phage insert (P21, Fig. 3). The cDNA was thenamplified by PCR using P48 (corresponding to sequenceobtained from the 2.3-kb genomic amplification product) andP21. The sequence of this amplified cDNA (fragment 52.4,Fig. 3) yielded an open reading frame of 814 nucleotides. Thefinal 132 nucleotides overlapped with the 5' end of the 4A2insert, indicating that the EcoRI site at the beginning of the4A2 insert was derived from the MF1 cDNA and not fromlinker addition. The deduced amino acid sequence of 52.4corresponded to six peptides sequenced from native p75,including the last three residues from the N-terminal se-quence. One discrepancy was noted at amino acid residue156, which was predicted to be a glutamate from the nucle-otide sequence but appeared as glutamine in the trypticpeptide.To obtain the 5' terminus of the message, we amplified the

first strand cDNA using a forward primer (PSL, Fig. 3)corresponding to the sequence of the trans-spliced leaderRNA from B. malayi (18) and a reverse primer from theantisense strand of 52.4 (P21.2). This yielded fragment 4'V(Fig. 3), which contained an open reading frame of 516nucleotides beginning 28 nucleotides from the 3' end of thespliced leader, with a potential 22-residue signal peptidepreceding the N terminus of the mature protein.Sequence Similarity to Chitinase and Enzymatic Activity of

Native MF1. A search of the Protein Identification Resource(release 27.0), Swiss-Prot (European Molecular Biology Lab-oratory, release 17.0), and Translated GenBank (release 64.3)

nt

cDNA

1 200 400 600 800 1000 1200 1400I I I I I I

4'V EcoRI

52-4

i

i4A2

B

tAAE A:':-A'-AG A "

A G C Y T N W A 5 r P G E G K F S. P G N r F N C' T * *,

-- - - -;A-'.--AA"' ,A-A, --;ATAA:,--:A - - - - -A-AAAAA---AAAALGIA -_LAAA4;,e,AFF i' F. '1 N , A ; 4 N A GA A YA A A V T KT K E T N p

::-'NFI.hA; A : X ANF'rGF

A''-A--AAAA -AA- 'A-i'7A'-':AAAAAA A:ACA.AAA'A AA-,A A.'-A''"k-G- -.''WFT:.AA-.AFG AA ;X h..LA.....AA..A.AAA 'A

D w E YA P V V A A SE H A m 1. Y E AAM-NL F MAYA L A A A A.

*f' 'CA'PI.AI40GWAP.L...F....A...A.A-: AT AS AT A 4A- -AA. 's-: AA

A"ATA"-'AAALAZ~~~~~j A:-AA ','A,CAGA'I AAAAAT AA

AAYT IIAG S Y NrAAG A M 4H

A ':TAhGA :.-'A-- AA';:AGA.-'AXA:,A'A"A'..GAALePPAArATA'AATrAAITGAATTT'f AX;ATC-AATTGGAAA';A-A;:;AAA A;,,T A'(A A::AAAAA''::GE V S G F AtFL F A A D Y N A S A P A: A:

*AATA '.AL -GAA ' G ~ GA _ATA ALA *A-ACAAA -a AA.''A.'-A.AA.;-"A.'-AT:' Af:'Y A AS r AF s A' AR

AAAG:A' A A ~ 'A A' A A.' -A AA:' GAG' 'CA'' -GA' A A: - -A -GA' -- -A -',A':.:T:'*r,':A 'ArAIAAA;AA.....................M, t C x A ; A 'T;:;1T...........A'Csv sTfmA TAS 'GG T( JG TuTTCATAT..................... . ... . ....T T AC ;('. AA: AAh 7: A . 1: .A AA-A.

o- '' M Y ~~~~~~~~~~~wrF "::0 F'

V &YG '3 A F I W A L D F DD r G K C S G pG P ? L N A

*'A A' ni7' -AA.'g .';A .'.' A ,A;;.| A.: '--:,AAA- AA..A.:A-`-'a *LA./AA ': A .' A .A'A:.sA-.:.I;AA'.A:. :AAhA- .,AA.........................\A AM'A A...-A 7':A!% - AAA A

hA' 1.A-AAhAA-Ah .A A 'A.-. A r A sA:r.FAA A'.. -AA s A ?ALA ;.;- e hA lAAf e';A - -A r ,A A-

.., . ,|, , .e, A; -AA~~a:AhA-.A''hAh'.. .'-:AA-''AAA'-:-- T ::'AA.'.?s*.- ".A -A

FIG. 3. (A) Alignment ofcDNA fragments used to se-

quence the MF1 message. 4A2 is aAgtll clone. 52-4 and 4'V arecDNA fragments amplified frommRNA using primers P48 and P21or PSL and P21.2, respectively.Nucleotide numbering begins withthe first nucleotide of the initiationcodon. (B) Nucleotide sequenceand deduced amino acid sequencefor the MF1 antigen. Nucleotidenumbering corresponds to that inA. Amino acid numbering beginsat the initiating methionine, withthe mature N terminus beginningat amino acid 23. Lowercase let-ters on the nucleotide line repre-sent untranslated regions. Primersused for PCR amplifications are

marked as arrows above the nu-cleotide line, with open arrows

denoting degenerate primers de-rived from protein sequence andclosed arrows denoting nondegen-erate primers corresponding toknown nucleotide sequence. TheEcoRI site is overscored on thenucleotide line. The consensus

poly(A) signal is underlined. Low-ercase letters on the amino acidline represent the predicted signalpeptide. The N-terminal sequencederived from the combined p70and p75 is underlined with dashes----). Peptides sequenced fromnative p75 are shaded, and pep-tides sequenced from native p70are boxed. A triangle marks thestart of the peptide unique to p75tryptic profile.

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1 15 16 30 31 45 46 60 61 75 76 901 MF1 --------------- ---YVRGCYYTNWAQ YRDGEGKFLPGNIPN GLCTHILYAFAKVDE LGDSKPFEWNDEDTE WSKGMYSAVTKLRET

*L*2 CHI1$BACCI 62/VADIDPTKVTHINYA FADICWNGIHGNPDP SGPNPVTWTCQNEKS QTINVPNGTIVLGDP WIDTGKTFAGDTWDQ PIAGNINQLNKLKQT3 CHIB$SERMA ------MSTRKAVIG YYFIPTNQINNYTET DTSVVPFPVSNITPA KAKQLTHINFSFLDI NSNLECAWDPATNDA KARDVVNRLTALKAH4 CHIA$SERMA 173/NFTVDKIPAQNLTHL LYGFIPICGGNGIND SLKEIEGSFQALQRS CQGREDFKISIHDPF AALQKAQKGVTAWDD PYKGNFGQLMALKQA5 KTXA$KLULA 349/DYCDKKSSTTGAPGT DGCFSNCGYGSTSNV KSSTFKKIAYWLDAK DKLAMDPKNIPNGPY DILHYAFVNINSDFS IDDSAFSKSAFLKVT

1 MF1

2 CHI1$BACCI3 CHIBSSERMA4 CHWIASERMA5 KTXA$KLULA

IMFI

2 CHI1$BACCI3 CHIMBSERMA4 CHIASSERMA5 KTXASKLULA

1 MF1

2 CHII$BACCI3 CHIBSSERMA4 CHIASSERMA5 KTXA$KLULA

1MF1

2 CHI1$BACCI3 CHIBSSERMA4 CHIASSERMA5 KTXA$KLULA

IMF12 CHI1$BACCI3 CHIB$SERMA4 CHIASSERMAS KTXASKLULA

1 MF1

2 CHI1SBACCI3 CHIB$SERMA4 CHIASSERMA5 KTXA$KLULA

90 105 106 120 121 135 136 150 151 165 166 180NPGLXVLLSYGGYNF G IFTGIA------ KSAQK TERFIKSAl AFLRKN-NFDGFDLDW EYPV4AEEHAKLVE AMKT--AFV------** * * S*GG* *l** F * **** *DG*D*DW E*P * *NPNLKTIISVGGWTW SN FSDVAA------ -TAAT EVFANSAV DFLRKY-NFDGVDLDW EYPVYSGLDGNSKRP EDKQNYTLLLSKIRENPSLRIMFSIGGWYY SNI LGVSHANYVNAV KTPAA TKFAQSCV RIMKDY-GFDGVDIDW EYPQA .EVDGFIAAL QEIR--TLL-----NHPDLKILPSIGGWTL SDPFFFMG.------- -DK DRFVGSVK EFLQTWKFFDGVDIDW EFPGC KGANPNLGSP QDGETYVLLMKELRASS--XKIPSFGGWDF S PSTYT-IFRNAV KTDQN NTFANNLI NFMNKY-NLDGIDLDW EYPGPDIPDIPADD SSSG-SNYL-----T

181 195 196 210 211 225 226 240 241 255 256 270--EEAKTSGKQRL]L TAAVSA GTI--DG SYNVESLGK- DL LFLMSYDLHGS -KN VDLHGKLHPTXGE-- ---------------

*L * A *** * D * *M*YD* G ** *KLDAAGAVDGKKY 14L TIASGA TYAAN-- TELAKIAA-- W INIMTYDFNGA -KI SAHNAPLNYDPAAS- ---------------

QQTIADGRQALPYqL TIAGAGC 4FFLSRYY SKLAQIVA-- DY INLMTYDLAGP -KI TNHQAALFGDAAGPT FYNALREANLGWSWEMLDQLSAETGRKY EL TSAISAC KDKI---- DKVAYNVAQN MDH IFLMSYDFYGAF LKN LGHQTALNARPGS-- ---------------

FLKLLKGKMPSGK' LSIAIPS WYL---- KNFPISDIQNI YMWMTYDIHGI-EY GKANSYINCHTP--- ---------------

271 285 286 300 301 315 316 330 331 345 346 360-------VSGIGIFN TEFAADYWAS MPK EKIIIGIPMYAQGW LDNPSETAIGAAASR PSSASKTNPAGG--- -------------TA

** ~ * K** G * Y***** * * *

----AAGVPDANTFN VAAGAQGHLDA GVPA AKLVLGVPFYGRGW GCAQAGNGQYQTCTG GSSVGTWEAGSF--- -------F------DFELTRAFPSPFSLTVD -AAVQQHLMMGVPS AKIVMGVPFYGRAF GVSGGNGGQYSSHST PGEDPYPNADYWLVG CDECVRDKDPRIASY----------RHRLH HGERRECAAG VKP GKIVVGTAMYGRGWI GVNGYQNNIPFTGTH RAVKGTWENGIV--- --------------D-----------RKEIE DA--IKMLDK GVKF NKVFGGVANYGRSY MVNTNCYNYGCGFQR EGGNSRDMTNTP--- --GVLSDSEIIDIDS

361 375 376 390 391 405 406 420 421 435 436 450SYWEICKYLKEGGKE TVHQEGV YMVKGD Q--WYGYDNEETI I KMKWLKEKGYGGAFI WALDFDDF KSCGK GPYPLLNAISSELEG**** ** * D **IW D** * *

YDLEANYINKNGYTR YWNDTAK YLYNAS NKRFISYDDAES Y KTAYIKSKGLGGAMF WELSGDRNK rL-QNK LKADLPTGGTVPPVDRQLEQMLQGNYGYQR LWNDKTK YLYHAQ NGLFVTYDDAESF Y KAKYIKQQOLGGVMF WHLGODNRN ----D LLAALDRYFNAADYDYRQIASQFMSGEWQY TYDATAE YVFKPS TGDLITFDDARS A KGKYVLDKQLGGLFS WEIDADNGD --LNS MNASLGNSAGVQ---SDKKNDRWVDTNTDC IFMKYDGNSSWPK SR-YDLEDMKN F AGTSLWAANYFKHDE WKNDEDD ----D TEDPFDEENVYFDVY

451 465 466 480 481 495 496 510 511 525 526 540ESENPEITTEEPSIT ETEAYETDETEETSE TEAYDTDETEETSET EATTYDTDETEGQEC PERDGLFPHPTDCHL FIQCANNIAYVMQCP

TTAPSVPGNARSTGV TANSVTLAWNASTDN VGVTGYNVYNGANLA TSVTGTTATISGLTA GTSYTFTIKAKDAAG NLSAASNAVTVSTTADSQLDMGTGLRYTGV G--PGNLPIMTAPAY VPGTTYAQGALVSYQ GYVWQTKWGYITSAP GSDSAWLKVGRLA-- ---------------

DCKNKAGYDLDNPVY GCRLETAINI I IWNG TESVNTVLNILNDYD NYIKYYEALTRAHYD SVMEKYEKWLFEEDG YYTYYTDVDGDDI II

541 555 556 580ATTFFNDAIKVCDHM TNAPDTCI-------

QPGGDTQAPTAPTNL ASTAQTTSS ITLSWT\

TPPDKKKRDYIQEKY SFEKEFMMSQNMTEL\

FIG. 4. Amino acid sequence alignment ofthe MF1 protein with three bacterial chitinases and one yeast chitinase: chitinase Al ofB. circulans(Swiss-Prot locus CHI1$BACCI), chitinases B and A of S. marcescens (CHIB$SERMA and CHIA$SERMA), and killer toxin RF2 a subunitof K. lactis (KTXA$KLULA). Major regions of primary sequence similarity shared among this set are shown boxed. Identical residuesconserved in the set are shown as uppercase letters beneath sequence 1; conservative amino acid groups are indicated by asterisks.

data bases revealed significant sequence similarities betweenthe MF1 protein sequence and several chitinases (E.C.3.2.1.14). An alignment of the MF1 protein sequence withthree bacterial chitinases (chitinase Al of Bacillus circulans,chitinases A and B of Serratia marcescens) and one yeastchitinase [killer toxin RF2 a subunit of Kluyveromyces lactis(19)] shows seven major regions of primary sequence con-servation (shown as boxed regions in Fig. 4). Following thelast conserved region, the MF1 sequence diverges and con-tains an acid-rich triplication of 14 amino acids (starting atreference position 466, Fig. 4).To test the MF1 antigen for chitinase activity, extracts of

mf were electrophoresed in SDS gels containing glycol chitin(17). Chitinase activity was localized to two prominent bandsthat copurified with the MF1 antigen in DE-52 chromatog-raphy and comigrated with the MF1 antigen in one-dimensional SDS/PAGE, demonstrating the reduction-dependent shift in mobility characteristic of the MF1 antigen(Fig. 5; ref. 6).

Since the predicted molecular mass (=55 kDa) for thetranslation product of the cDNA sequence is lower than thatobserved for the enzymatically active bands in SDS/PAGE,we tested the possibility that glycosylation could account forthe discrepancy. Following treatment with 0-glycanase, butnot N-glycanase, a sizeable shift in molecular mass wasobserved for p70 and p75 by Western blot (Fig. 5).

DISCUSSIONTransmission of lymphatic filariasis in nature depends on thepersistence of mf in the peripheral blood of infected verte-

brates and on the capacity of those mf to infect and developin the insect vector. Female adults of Brugia spp. shed livemf, which retain a remnant of their eggshells as the extracu-ticular sheath (20). This sheath is maintained while the mfcirculate in the vertebrate bloodstream but is lost during theinitial phases of development in the arthropod vector.

2-ME ---+ + + +-

-..v|- 95

5.R'E

- +

-95 5 -95.5

5-55-43

2 3 4 5 6 7 8 9 10 1112 13

FIG. 5. Copurification of microfilarial chitinase with the MF1antigen. Molecular masses are indicated in kDa. Lanes 1-6, chitinaseactivity is demonstrated following electrophoresis in glycol chitin-containing SDS/polyacrylamide gels for total microfilarial extract(lanes 1 and 6) and DE-52-purified fractions of extracts eluted with0.3 M (lanes 2 and 5) and 0.5 M NaCl (lanes 3 and 4), electrophoresedin the absence (lanes 1-3) or presence (lanes 4-6) of 2-mercaptoeth-anol (2-ME, - or +). Lanes 7-10, silver-stained SDS/PAGE (lanes7 and 8) and Western blot (lanes 9 and 10) of 0.5 M NaCl fractionelectrophoresed in the absence (lanes 8 and 9) and presence (lanes 7and 10) of2-mercaptoethanol. Lanes 11-13, total microfilarial extractfollowing sham (lane 11), N-glycanase (lane 12), or 0-glycanase (lane13) treatment was Western blotted and developed with the MF1monoclonal antibody.

Immunology: Fuhrman et al.

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1552 Immunology: Fuhrman et al.

Exsheathment is necessary for subsequent molting and de-velopment of the filarial larvae in the vector.We previously demonstrated that the microfilarial sheath

contains oligomers of f,1-4-linked N-acetylglucosamineand that sheath morphogenesis can be altered by inhibitors ofchitin synthesis (21). It is thus of great interest to find a larvalchitinase whose temporal appearance coincides with theonset of microfilarial infectivity for the mosquito. The chiti-nase could be involved in either the degradation of microfi-larial structures, such as the sheath, or the interaction withchitinous mosquito structures that the mf must disrupt orpenetrate during infection. The peritrophic membrane is achitinous structure elaborated in the mosquito midgut fol-lowing a bloodmeal, but mf appear to escape from the midgutlong before this membrane is well formed (22). The possibilitythat mosquitoes secrete a midgut lectin that can inhibitinfection was suggested by Phiri and Ham (23), who demon-strated enhanced microfilarial penetration of the midgut inthe presence of N-acetylglucosamine. A microfilarial chiti-nase could generate inhibitory oligosaccharides and promoteinfection by a similar mechanism.

Chitinase activity was previously described in Onchocercagibsoni (24). In this filarial species, the activity is present inthe gravid female adult and presumably acts to remodel chitinfibrils in the developing eggshell or to free the developing mffrom their eggshells prior to birth. The mf of Onchocerca spp.are born without sheaths and therefore do not need a mech-anism for exsheathment in the vector. Brugian mf do nothatch from their eggshells in utero but seem to remodel theshell and elongate it ultimately to form the sheath (20). Thisremodeling may also involve chitinase, but this enzymeshould be antigenically distinct from the chitinase sequencedhere, as the MF1 antibody does not react with extracts ofadult female B. malayi (6).The sequence reported here was compiled from three

overlapping sequences: the cDNA clone 4A2 (nucleotides794-1606), the cDNA PCR product 52.4 (nucleotides 112-925), and the cDNA PCR product 4'V (nucleotides -48-516).The 5' and 3' untranslated regions are short, and a poly(A)addition signal (ATAAAA) occurs at nucleotides 1547-1552.In sequencing the 5' terminus of the message, we utilized aprimer specific for the spliced leader present on some, but notall, mRNAs in B. malayi. Since this technique selectivelyamplified SL-containing messages, any versions of the MF1message not containing this leader would not have beendetected. Nevertheless, at least a subpopulation of the mes-sages coding for this antigen does contain this spliced leaderand a signal peptide preceding the mature N terminus.Although the complete cDNA sequence was compiled fromthree separate fragments, the extensive overlap at the nucle-otide and peptide levels supports the contiguity of the se-quence. Furthermore, a unique 1.5-kb fragment can be am-plified from microfilarial cDNA using primers correspondingto the sequence coding for p70/p75 N terminus (forward) andthe 3' end of the 4A2 clone upstream of the poly(A) tail(reverse) (M. Southworth, personal communication).

Chitinase activity is demonstrated here for both proteins,p70 and p75, recognized by the MF1 antibody. However, therelationship between p70 and p75 is still unclear. Two sep-arate messages could exist for p75 and p70, or posttransla-tional modification (such as C-terminal truncation) couldaccount for the difference in their apparent molecularmasses. Since O-glycanase treatment caused equivalent mo-bility shifts for both proteins, we cannot at this point ascribethe difference between the two to this particular class ofposttranslational modification.The region ofthe sequence that spans amino acids 377-453,

which includes the acid-rich triplication, is characterized bya high PEST score [where PEST indicates proline (P),

glutamic acid (E), serine (S), and threonine (T); ref. 25],suggesting a calcium-binding site. p70 and p75 were previ-ously shown to bind calcium (6), and this region ofthe proteincould be responsible for that property. The translation of thefull cDNA predicts a molecular mass of -55 kDa for theencoded protein, which is smaller than the molecular massfor either protein recognized by the MF1 antibody. Asdemonstrated in Fig. 5, 0-glycosylation accounts for part ofthis discrepancy. In addition, the very acidic composition ofthis PEST region could result in anomolous migration in SDSgels. Expression of the recombinant protein from full-lengthcDNA should resolve this question.As chitin is absent from the vertebrate host, chitin syn-

thesis and processing offer potential targets for chemother-apy, and the enzymes involved in chitin metabolism could besuitable candidates for vaccine development. The demon-stration of chitinase activity for the purified MF1 antigen andthe availability of the nucleotide sequence coding for theenzyme favor its analysis as a therapeutic target in this andother parasitic helminths.

We thank Dr. Sen Dissanayake for expert advice, Sherry Roemerfor sequencing the genomic PCR product, and Dr. Maurice W.Southworth for helpful discussions. Peptide preparation and se-quencing were performed by Renee Robinson and Ruth Davenportat the Harvard Microchemistry Facility. Materials used in this studywere provided by a National Institute of Allergy and InfectiousDiseases supply contract (Al 02642), U.S.-Japan Cooperative Med-ical Science Program. This work was supported in part by a NationalInstitute of Allergy and Infectious Diseases grant (Al 24858) toJ.A.F.

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