Two Schistosoma mansoni cDNAs encoding ATP-binding cassette (ABC) family proteins

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Molecular and Biochemical Parasitology 65 (1994) 351-356

BIOCHEMICAL PARASITOLOGY

Short communication Two Schistosoma mansoni cDNAs encoding ATP-binding cassette (ABC) family proteins Irene B. Bosch a, Zhen-Xi Wang a, Liang-Feng Tao b, Charles B. Shoemaker a'* ~Department of Tropical Public Health, Harvard School of Public Health, 665 Huntington Avenue, Boston, MA 02115, USA, bCenter for Tropical Diseases, University of Massachusetts at Lowell, Lowell, MA, USA

Received 29 December 1993; accepted 8 February 1994

Key words: Schistosoma mansoni; cDNA; ATP-cassette; P-glycoprotein

Adult schistosomes interact closely with their m a m m a l i a n hosts as they perform survival functions such as the uptake of nutrients and the disposal of toxic metabolites. We are interested in studying the host-interactive proteins that perform these essential functions to understand how the parasite avoids a damaging immune attack directed at these proteins, and to test their potential as vaccine targets. Metabolite disposal, along with the export of certain biomolecules, is commonly performed by members of the ATP-binding cassette (ABC) family of proteins which are found within organisms spanning the broad evolutionary range from microbes to vertebrates. These proteins are invariably integral m e m b r a n e proteins and contain one or two 'cassettes', each cassette generally having 6 membrane-spanning do~ponding 4914.

author. Tel.: 617-432-1339; Fax: 617-738-

Note: Nucleotide sequence data reported in this paper have

been submitted to GenbankT M data base with the accession nos. L26286 and L26287. Abbreviations: MDR, multi-drug resistance; ABC, ATP-binding cassette; PCR, polymerase chain reaction.

0166-6851/94/$7.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 1 6 6 - 6 8 5 1 ( 9 4 ) 0 0 0 3 9 - P

mains and an intracellular A T P binding domain (reviewed in [1,2]). This ATP-binding domain is well-conserved through evolution and contains several short amino acid sequence motifs that are nearly invariant in all members of the family. A subset of ABC proteins, the P-glycoproteins, was originally discovered as being responsible for the multi-drug resistance ( M D R ) phenotype found in m a n y m a m m a l i a n tumors. This group of proteins has two ATP-binding cassettes, each with six transmembrane domains, fused in tandem. M a n y other m a m m a l i a n members of this class of protein have since been identified, some of which are not associated with drug resistance but whose normal function is thought to be the removal or export of lipophilic molecules from within organs and cells (reviewed in [3,4]). P-glycoprotein homologues have also been identified within lower eukaryotes, sometimes associated with drug resistance [1]. Here we report the isolation of fulllength c D N A s encoding two different ATP-cassette family proteins from Schistosoma mansoni including one that is structurally homologous with m a m m a l i a n P-glycoproteins. Two opposing oligonucleotide primers were prepared [5] which were designed to hybridize to

352

LB. Bosch et al./Molecular and Biochemical Parasitology 65 (1994) 351-356

different conserved motifs within the A T P binding domain of ABC proteins (see legend to Fig. 1). A polymerase chain reaction (PCR) using these oligonucleotides and an S. m a n s o n i c D N A template produced a single amplified D N A fragment of the expected size (about 420 bp). The amplified D N A was radiolabeled and used as a probe to screen a 2-5-kb size-selected adult w o r m c D N A library [6]. Two positive hybridizing c D N A clones were obtained from about 100,000 recombinant phage screened. Restriction mapping indicated that the clones were different and D N A sequencing confirmed that they encode two different ABC protein homologues. The clones were used as specific probes to rescreen the c D N A library and multiple additional clones of each class were isolated and characterized. As a result, the complete coding D N A for both S. m a n s o n i ABC protein homologues, named SMDR1 and SMDR2, was obtained and sequenced. The predicted amino acid sequences of both proteins are shown in Fig. 1 in alignment with the sequence of the murine P-glycoprotein, M M D R 1 [7]. S M D R I consists of six predicted transmembrane spans with a single A T P binding domain and has a size of about 76 kDa. As such, S M D R 1 is similar to ABC proteins such as the bacterial export proteins [8], the T-cell transporters responsible for the import of peptides for M H C presentation [9,10] and the peroxisomal m e m b r a n e protein [11]. This predicted structure of SMDR1 and its strong homology to the ATPbinding domain of other ABC proteins (Fig. 1) strongly suggests that SMDR1 performs a transport function within schistosomes. But an extensive database search revealed no outstanding homologies to other known members of the ABC protein family which could permit predictions as

to the type of molecule SMDR1 transports in schistosomes. The second S. m a n s o n i ATP-binding cassette protein homologue, SMDR2, is predicted to be a 140-kDa protein with 12 transmembrane spans and two ATP-binding domains. In terms of its primary sequence and predicted structure S M D R 2 is distinctly homologous with P-glycoproteins. The homology is particularly strong within the A T P binding domain but remains significant within the hydrophobic regions of the molecule (Fig. 1). In addition, alignment of S M D R 2 with P-glycoprotein, M M D R 1 , requires very few gaps to maximize homology and the predicted transmembrane domains align almost exactly. Therefore this protein clearly belongs to the P-glycoprotein subfamily of A T P cassette proteins and, most likely, performs export functions similar to those suggested for m a m m a l i a n P-glycoproteins. Interestingly, the only significant gap found by aligning S M D R 2 with M M D R 1 is within the major extracellular domain in which S M D R 2 is truncated relative to M M D R 1 . If, in fact, S M D R 2 is a host-exposed protein, perhaps serving an export function, the reduction in size of the major extrafacial domain of this protein m a y be an evolutionary adaptation of schistosomes to reduce the protein exposure to the host and thereby minimize a possibly damaging immune response. Steady state m R N A levels for SMDR1 and S M D R 2 were compared in whole cercariae, cercarial tails, schistosomula and adult worms of different genders by northern analysis. The results, shown in Fig. 2A, demonstrate that SMDR1 is expressed within both larval and adult stages but is only weakly detected within cercarial tails. This observation is consistent with our recent findings that cercarial tails contain disproportionately high

Fig. 1. Homology between S. mansoni P-glycoprotein homologues and murine P-glycoprotein 1. Synthetic oligonucleotides (same as used in Wilson et al. [5]) were used in a PCR with S. mansoni cDNA as described by Skelly et al. [12]. The resulting amplified DNA product was radiolabeled [19] and used to screen an adult S. mansoni cDNA library. Positive cDNA clones from the first screen were used as probes to rescreen the cDNA library and eventually lead to the isolation of the complete coding sequence for two different ABC protein homologues (see text). DNA sequencing was done by the method of Sanger et al. [20] and all coding sequence was obtained from both DNA strands and at least two independent isolates. The open reading frame translation from both S. mansoni ATP protein homologues, SMDR1 and SMDR2, are displayed and aligned with the murine P-glycoproteinhomologue, MMDR1 [7]. Shading indicates sequence identity. Gaps have been introduced into the sequences (dashes) to maximize homology. Predicted transmembrane domains are underlined and the conserved potential extracellular, N-linked glycosylationsites are double underlined.

I.B. Bosch et al./Molecular and Biochemical Parasitology 65 (1994) 351-356

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Fig. 2. Expression of SMDR1 and S M D R 2 m R N A within larval and adult schistosomes. Poly(A) + R N A was obtained from cercariae (C), purified cercarial tails (T), transformed schistosomula (S), adult male worms (M) and adult female worms (F) and used for northern blot analysis as previously described [12]. The northern blots were successively hybridized with SMDR1 and S M D R 2 specific probes. Each probe fragment was prepared by P C R and then radiolabeled as previously described [12]. The probe fragments were from the coding sequences of SMDR1 (aa 24t~581) and S M D R 2 (aa 876-1204). The same northern blots were also hybridized with various control probes so as to demonstrate that each of the lanes contained similar a m o u n t s of m R N A (control blots shown in [12]). Molecular weight estimates based on mobility relative to molecular weight standards are noted in kb.

levels of transcripts for glycogen metabolic enzymes [12] and often low or undetectable levels of other transcripts (e.g., hexokinase [12] and two glucose transporters [13]). Northern analysis of SMDR2 expression within the different schistosome life cycle stages demonstrates that this m R N A is found primarily within adult females (Fig. 2B). Transcription, though, is not unique to females as the northern blot identi-

ties some expression of SMDR2 m R N A in adult males and PCR studies, using SMDR2-specific oligonucleotide primers and various schistosome cDNA templates (not shown), indicate at least some detectable expression of SMDR2 within cercariae and schistosomula. Overexpression of P-glycoprotein homologues has been associated with drug resistance in several parasites including Plasmodium, Leishmania

LB. Bosch et al./Molecular and Biochemical Parasitology 65 (1994) 351-356

a n d E n t a m o e b a [5,14-16]. W e tested for evidence o f gene a m p l i f i c a t i o n a n d e n h a n c e d expression o f S M D R 2 within two different s c h i s t o s o m e isolates, M A P a n d H36, r e p o r t e d l y r e s i s t a n t to the r e l a t e d drugs, h y c a n t h o n e a n d o x a m n i q u i n e [17,18]. F i r s t we c o n f i r m e d the d r u g resistance o f the strains b y t r e a t i n g infected a n i m a l s ( 8 / g r o u p ) with either 100 m g / k g o x a m n i q u i n e o r 80 m g / k g h y c a n t h o n e a n d c o m p a r i n g w o r m survival with t h a t o f the d r u g sensitive BH strain. W o r m r e c o v e r y o f the two d r u g resistant strains was effectively u n c h a n g e d b e t w e e n d r u g - t r e a t e d o r u n t r e a t e d mice a l t h o u g h w o r m r e c o v e r y was r e d u c e d a p p r o x i m a t e l y 80% in the B H strain following d r u g t r e a t m e n t . N e x t we p e r f o r m e d g e n o m i c S o u t h e r n blots a n d n o r t h ern blots on D N A a n d R N A i s o l a t e d f r o m the t w o d r u g resistant strains a n d the B H strain. U s i n g a n S M D R 2 - s p e c i f i c p r o b e , p r e p a r e d as d e s c r i b e d in Fig. 2, we find no evidence o f i n c r e a s e d S M D R 2 gene c o p y n u m b e r o r increased S M D R 2 t r a n s c r i p tion ( d a t a n o t shown). Thus, we find no relationship b e t w e e n h y c a n t h o n e a n d o x a m n i q u i n e resistance in s c h i s t o s o m e s a n d S M D R 2 expression.

Acknowledgements T h e a u t h o r s wish to t h a n k Drs. D. W i r t h a n d C. W i l s o n for the use o f the M D R - s p e c i f i c P C R oligos. These studies were f u n d e d , in p a r t , b y N a t i o n a l Institutes o f H e a l t h G r a n t A I 2 8 4 9 9 (C.S.), the U n i t e d N a t i o n s D e v e l o p m e n t P r o g r a m m e / W o r l d B a n k / W H O Special P r o g r a m for R e s e a r c h a n d T r a i n i n g in T r o p i c a l Diseases (C.S.) a n d U S A I D g r a n t #02-0315 ( L - F . T). I.B. is a fellow o f the C o n s e j o N a c i o n a l de I n v e s t i g a c i o n e s Cientificas de Venezuela.

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