Molecular and Biochemical Parasitology, 58 (1993) 161 - 164 161 ~z-~ 1993 Elsevier Science Publishers B.V. All rights reserved. / 0166-6851,/93/$06.00

MOLBIO01920

Shor t C o m m u n i c a t i o n

Cloning and sequencing of a complete myosin heavy chain cDNA from Schistosoma mansoni

a* • b* David Weston , Jut ta Schmltz , W. Michael K e m p a and Werner Kunz b aTexas A & M University, Department of Bioh)gy, Laboratory of Parasitology, College Station, TX, USA; and

blnstitut ffir Genetik, Heinrich-Heine-Universit?t't Dffsseldo(f, Dft~seldorJ~ Germany

(Received 6 October 1992; accepted 28 October 1992)

Key words: Schistosoma mansoni; Myosin; Recombinant DNA; D N A sequence

Many Schistosoma mansoni-derived contrac- tile proteins such as myosin heavy chain, paramyosin, and tropomyosin, play impor- tant roles in the immune responses of infected hosts [1-5]. Schistosome tropomyosin elicits immunodominant responses in chronically infected humans and mice vaccinated with irradiated ceracariae [6]. Schistosome myosin heavy chain and paramyosin elicit immunodo- minant antibody responses in infected mice and rats, yet neither molecule exists on the surface of the schistosome tegument [1,4]. When purified recombinant or native para- myosin is intradermally injected into mice, partial protective immunity against schisto- some challenge infection is induced [3]. Injec- tion of myosin, however, does not induce similar protection [7], although both mole- cules share similarities in amino acid sequence and similar or-helical secondary structure [3,8]. Rapidly developing recombinant DNA techni-

Correspondence address: W. Kunz, institut ffir Genetik, Heinrich-Heine-Universit/it Diisseldorf, Universitiitsstrasse 1, W-4000 Diisseldorf, Germany. Tel. and Fax: (211) 311 2333.

*Note: These two authors contributed equally to this work.

Note: Nucleotide sequence data reported in this paper have been submitted to the GenBank T M with the accession number L01634.

ques allow for the cloning of these antigens for potential immunodiagnostic [9] and vaccine [3- 4] reagent testing. In the present study we report the cloning and sequence characteriza- tion of a complete myosin heavy chain cDNA from S. mansoni.

Recently, a partial cDNA (SM20) from a Brazilian strain adult S. mansoni ;tZAP cDNA expression library (library BR) was isolated and characterized as encoding an antigen shared with its intermediate host, Biomphalar- ia glabrata (D. Weston and W.M. Kemp, manuscript submitted). The 2.l-kb insert of SM20, identified as a partial myosin heavy chain cDNA, was used to rescreen library BR yielding 29 positives out of 2.4 x 105 recombinants screened. Two clones, SMmhc3 and SMmhcl8, containing 1321-bp and 3062- bp inserts respectively, were sequenced in their entirety. Additional clones from a Liberian female S. mansoni 2ZAPII cDNA expression library (library L) were obtained in the following manner. Female S. mansoni crude protein extract was fractionated by SDS- PAGE, transferred to nitrocellulose, and probed with rabbit antiserum directed against the same protein extract. Monospecific poly- clonal antiserum, affinity purified from the immunodominant 205-kDa band by a pre- viously described procedure [10-1 I], was used

162

1 Kb I I

I I SMmhcl8

I I I I SMmhc3 Sm200-9

I I I I SM20

l I SmM7

SmM8

Fig. 1. Schematic diagram of SMmhc restriction map and alignments of completely sequenced overlapping cDNA clones. The eDNA coding region is shown in grey and the non-coding regions in white block. The selected restriction enzymes arc B, BamHl; D, Dral; H, H/ndlll; X, Xbal, Xh, Xhol', E, EcoRl; A, Accl; S, Sail. The single 1940 amino acid open reading frame begins with the start methionine at position 121 and ends at position 5940. The conserved polyadenylation signal, AATAAA, lies 35 bp downstream of the poly(A) ~ tail. All library screening, cloning, and DNA ,sequencing was performed using well established techniques [16]. The Genetics Computer Group (GCG) sequence analysis software package [17] was used to

analyze the nucleotide and deduced amino acid sequences.

to screen library L. Of 4.5 × 105 recombinant plaques screened 5 immunoreactive clones were obtained. The largest clone, SM200-9, with a 4032-bp cDNA insert, and two smaller clones, SmM7 and SmM8, with 447-bp and 1013-bp inserts, respectively, were sequenced in their entirety. Together the above mentioned clones were overlapped to give a complete myosin heavy chain cDNA (Fig. 1). Computer assisted analysis of the 6986-bp myosin cDNA (SMmhc) revealed a single open reading frame encoding a 1940-amino acid protein with a predicted molecular weight of approx. 220000. Comparison of the SMmhc peptide sequence with myosin sequences of other species yielded alignment scores ranging from 65.5% to 49.3% identity, the highest score being the myosin from the adductor muscle of the bay scallop Aequipecten irradians [12] (Fig. 2).

It should also be noted that all fully sequenced clones (Fig. 1), as well as all partially sequenced clones ( not shown) in this study, were in full agreement at the nucleotide level. Although the partial myosin sequence reported by Grossman et al.[2] is in agreement with our reported sequence, the partial amino acid sequence derived from a

Puerto Rican strain and reported by Newport et al.[1] displays some differences from the SMmhc sequence in this report. It is likely that these differences reflect strain and/or popula- tion genetic polymorphisms since previously reported data [1,2], in addition to unpublished data from one of our laboratories, strongly argues the existence of a single gene copy for schistosome myosin. Another possibility is that multiple myosin isoforms may exist by way of alternative splicing of this single gene, al- though more data are needed to appropriately address this question.

Myosin is a multimeric protein that is localized in muscle tissue and occurs in the cytoplasm of virtually all eukaryotic cells [13]. Myosins are categorized on whether their heavy chains form monomers or dimers: myosins I and myosins I1, respectively. Myosin II is a large hexameric protein complex of about 500 kDa composed of two myosin heavy chains, two alkali light chains and two regulatory light chains [14]. The myosin II heavy chain consists of two structural domains, the globular head and the s-helical rod. The globular head contains ATP and actin binding sites, whereas the rod

SM --MDP•DPDFKYLGVDRKALLKE-LANFDSKN••WVEDEKEGYVLAD•KDTTGDT•TV-ALKDGSEKKVKKDDAQQVNPPKFFL•EDMANLTHLNDA•VLENLR I 0 0 Aeq MNI.F ..... Q..A .... K.M..QT.A..G.KNC..P ..... FAS.E.QSSK..E...KIVA.S.TRT ..... I.SM ..... EKL ..... M.Y..E .... Y... 104

SM ARYYRQL•YTYSGLFCVAVNPYKRFP•YTEQVALKYKGKRRGEMPPHIF•I•DNAYHNML•DREN•SILITGE•GAGKTENTKKVI•YFAwA-AASKKED 200

Aeq S..TSG .......... I ..... R.L .... DS.IA..R...KT.I...L..VA .... Q..VT ...... C .................. M.L.K..C.VK..DE 205

+-J&~-4-

SM DD~KK-GTLED~I~QANPVLEAYGNAKTTRNNNS~RFGKFIRIHFGTTGKIAGADIEHYLLEKSRVVSQMKGERNYHIFYQLL~TYGSKYHDKLLVQTD~ 300

Aeq EA.D..E.S ..... I ................................ p .......... T ........ TY.QSA ......... IC.NAIPELN.VM..TP.S 306

SM ALY~INQGELTID~VDD~EEMKLCDEAFEVLGFNDDEKL~LFKCTTSI~NMGEMKFKQRP~EEQAF.ADGTAEAEKVAFLLGVNAKDLLTSFLKPKVKVG 400

Aeq G ........ C..V.NI..V..F ....... DI...TKE..Q.M .... A..LH ................ $ ............ C.I..G...KAL ........ 406

SM TEFVTKGQNLNQVTYAV~`~LLAK~LYNRMFGWLVARVNKTLDTKVKRQFFIGVLDIAGFEIFTENGFEQICINYTNERLQQFFNHHMFVLEQEEYKREKIQ 500

Aeq ..M ...... M...VNS.G ....... D...N...R ......... A..NYY ............ DF.S...L .................. I ....... K.G.A 506

SM WTF~DFGMDLQACIDL~EKPMGIL~ILEEEC~VPKASDQTFL~KLYDNHLGK~PNFTKP-KPPKPGHVKAHFELHHYAG~VPYTITGWLEKNKDPLND~w 600

Aeq .E ......... M ................... MF...D.KS.QD...Q..M..NRM .... G..TR.NQGP .......... N...S ........... I.EN.. 607

SM ALLGDSKDPLVSNLF .... TPwGEPGKKTKGG~FLTvT~MHRE~LNKLMKNLQ~T~P~FIRCIVPNEFKQPGVIDAHLVLHQLHCNGVLEG~R~cRKGFPN~M 700

Aeq .... A..E...AE..KAPEE.AG.GKK..G.SSA.Q.ISAV ............ Y..H.H.V...I...L .... LV..E ...... Q ................ S.L 711 t *

SM ~Y~EFKQRY~ILAPNVIPDGFVDGRQVTEKILEATQLDKNLYQCGNTKVFFKAGTL~LEDLRDDKLNGII~LFQAEIRGYLMRKQYKKLQDQRVALTLM 800

Aeq ............... A..Q ..... KT.S .... AGL.M.PAE.RL.T ........ V.GN..EM..ER.SK...M...H ..... I..A ........ IG.SVI 811

SM QRN~RKYLVLRNWPWWRLYTKVKPMLNIARQEEEMKKAAEELAKLKEEYEKLEKLKKELEEQNVTVLQQKNDLFLQLQTEQD~LADAEEK~KLVLQRGD 900

Aeq ...... W ...... Q..K..S .... L.S ......... EQLKQMD.M..DLA.T.RI .......... L.E ........... LE..MG.Q..RVE..IM.KA. 911

SM MEQR~K~LEERLADEEDQAANLTEV~(KKM~AEIE~LKKDVEDLE~SLQKAEQEKQTKDNQIRTLQSEMAQQDEMIGKLNKDKKNLEEQNKRTQEALQAEE I000

Aeq F.SQ ........ L .... A..D.EGI .... E.DNAN .... IG...NT ...... D.AH ..... S...G.I$ .... H ...... E..A...A..K.SDS ..... 1011

SM DKVNHLNKLKAKLESTLDEMEENLAREQK I RGDVEKSKRKLEGD LKATQETVDDLERVKRDLEEQLRRKEAE IGGLSGKFEDEQGLVAQLQRK i KELQTR II00

Aeq . .C ........... Q~%...L.D..E..K.V ...... A...V.Q...5...N.E ....... E...NV ....... SS.NS. L .... N..S .......... A. iiii

SM IQELEEDLEAERAAR•KAEKSRQQLESELEEwDRLEEQDGATAAQ•DLTKKREAELMKLKRDLEDTRLQNEQAIATMRKK•SDA•NELAD•LDQANKAK 1200

Aeq .E .... E ..... N..A.V..Q.AE.NR .... LGE.. D.AG... S.. IE. N ....... L. IR....EAS.. H.AQ.SAL...HQ..A.. M...V..LQ.V. 1211

SM AKAEKERSQFKAELDDAHNQVDS I ~(A/(LNSEKTVKALESQLQEVSVKLDEATRNLNEOASTKARS SQEVSELQRQLEEAESQLSQLNK i KQQLSAQLEE 1300

Aeq S.L. . DKKDL.R.M.. LES. MTHN.. N.GC... VM. QF... MSDLN~/~. EDSQ. SI . . LQ.Q.S. LQA.N.D.T .... D.. HRV.V.S.E.S...S...D 1311

SM ARHS LE DE SRMKAKLNGEVRNLTSDLDSLRETLEEEQSAKGD LQRQLQKLQGELQQLRSRGGGGGDVRSEEVEE LKRKMNAK i PALESEAESAKSKCGQL 1400

Aeq . .R... E. T.ARS..QN .... MHA.M.AI..Q ..... ES. S.V .... S.ANN. I..W..KFESE.AN. T.. L. DQ... LLG. LSEA.QTT.A.NA..SA. 1411

SM EKTKAR LQGELEDLMVDVERANGLASQLERKQNNFNRTLAEWQKKYADSQAELENAQRDARGQSTE i FRLKAQLEEVHEQMEGLRRENKN LSDE i HDLTE 1500

Aeq . .A.S...Q .... MSIE. D...ASVN.M.K..RA.DK.T .... A.VNSL. S .... S.KES..Y.A. LY. I..SI..YQDSIGA ........ A ....... D 1511

SM QLGE GG RSVHE I DKNRRR LEMEKE EL Q~JLLEEAE SA LE QEEAKVQRAQ LEMS Q I RQE i DRRLAE KEEEFEAT RK N H QR2~4E S QQAS LEAEGKGKAEAMRV 1600

Aeq ..S ..... T..L..A ................... G ......... M ..... IATV.N...K.IQ ...... DN..R ..... L..M ....... A .... D...I 1611

SM KKKLEQD I NE LEVS LDGANRA/:~/tEQEENVKKFQQQVRE LQSQLEDDQRQRDDLREQFQAAERRATVLAGE LDELRI ALDQAERSRK IAEAERAEA.SDRAT 1700

Aeq ............. A..AS..GK..M.KT..RY...I..M.TSI.EE ..... EA..SYNM .... C. LMS..VE...A..E .... A..ASDN. L.D.N..VN 1711

SM EM STQTAS LAAQKRKLEAD LA/LMQAD LEEAANEAKQAD ERAK]LKMADSA/~VFEE i RQEQE H TQHVEKA!~tKQLE i QVKE LMARLED SES GAMK NGRKAMGK 1800

Aeq . LTS.VS.VQG ...... G.IN...T. . D.MHG.L.G .... C ...... A.. LAD. L.A..D.SNQ...V..N..5 .... FQI..DEA.ASSL. G. K. MIQ. 1811

SM LEQRV~LETEL~QRRHGETQKNLRKVDRgJ4KE I S LQAEEDKKSHDRMQELVEK LQGK I KTYKRQVQEAEE i AA i NLAKYRK I QHE i EDAEERADQAE 1900

Aeq . .S..H...A..DN ..... A ..... M..A...L..L~.. D..R.NQ~. L...ID..NA .... F .... E ............... A...L.E ...... T.D 1911

SM QALQKLRAKNRSSVSTARGVSPAPLGGLDMLIVLVNQOPV 1940

Aeq ST... F... $ ..... VQ. SSVSVSASN 1938

Fig. 2. Amino acid alignment of myosin from A. irradians with SMmhc. The FastA output using single capital letter amino acid designations with periods representing positions of identity. Bold asterisks denote the conserved cysteines, the bold caret

designates Pro s24, and the conserved ATP binding site is shown.

164

domain assembles with a second heavy chain to form a myosin dimer [14]. The schistosome myosin heavy chain conforms to the myosin lI group as predicted by sequence similarity (Fig. 2) and by a Chou-Fasman plot of secondary structure (not shown). In clone SMmhc Pro s24 marks the end of the globular head domain and the beginning of the ~-helical rod that ends with a non-helical 'tailpiece" of 19 amino acids beginning with Pro 1922. Within the globular head, the domain spanning amino acids 172 179 contains a highly conserved ATP binding site, in addition to conserved Cys ~'s2 and Cys 6'~2, likely to be the reactive cysteines required for ATPase activity [14] (Fig. 2). The rod portion of the schistosome myosin contains the heptad repeat pattern in which the first and fourth residues are usually hydro- phobic (ref. 15; not shown), as demonstrated in previous reports of partial schistosome MHC clones [1,2].

In support of the above sequence analysis, affinity-purified antibodies raised against the protein product of the 2gtl I MHC expression clone SmMS, containing part of the MHC rod sequence, specifically bind to the muscle layer of adult schistosomes (unpublished data). Taken together the data presented in this report suggest that the cDNA sequence SMmhc represents a muscle myosin heavy chain from S. mansoni.

Acknowledgements

The helpful advice of Thomas Didion and Markus Maniak is gratefully aknowledged. Supported by the Department of Naval Research, NIH AI-26505: Texas Advanced Research Program #010366-100, the Deutsche Forschungsgemeinschaft (Ku 282/ 13-1) and by the UNDP/World Bank/WHO Special Program for Research and Training in Tropical Diseases.

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