A novel Syk-family tyrosine kinase from Schistosoma mansoni which is preferentially transcribed in reproductive organs☆
Introduction
Schistosomes are digenean parasites which have a complex life cycle including two free living larval stages (miracidium, cercaria). Adult worms survive for decades in the blood stream of their mammalian hosts causing schistosomiasis (bilharzia) in humans (Basch, 1991, Chitsulo et al., 2000). Within the trematodes, schistosomes represent the only family being dioecious. A remarkable feature of this parasite is that the sexual maturation of the female depends on a close pairing contact with the male (Kunz, 2001). Upon pairing the reproductive organs of the female differentiate, a prerequisite for egg production. To increase the knowledge of the molecular biology of schistosomes, international efforts are made to identify genes with key cellular functions that could represent new targets for drug design or vaccination (Johnston et al., 1999).
Growth and development of parasites require permanent communication processes with the environment to control differentiation. In the animal kingdom, differentiation processes are mediated by specific classes of signalling cascades involving conserved molecules. Among these, the cellular tyrosine kinases (TKs) play key roles as transmitter molecules forwarding incoming signals to a variety of downstream targets. Cellular TKs are involved in pathways of eukaryotes controlling diverse cellular processes such as adhesion, migration, proliferation, differentiation, cytoskeletal alteration and survival (Thomas and Brugge, 1997, Smithgall et al., 1998, Mano, 1999).
Among the cellular TKs, the mammalian members of the Syk family, Syk and ZAP-70, are involved in the differentiation of haematopoietic cells transducing signals from a variety of immune receptors (Turner et al., 2000). Due to their absence from the Caenorhabditis elegans genome (Ruvkun and Hobert, 1998) and their specialized role in mammals, it was assumed that this class of kinases evolved recently. However, the isolation of a syk gene from Hydra indicates an early metazoan evolution (Steele et al., 1999).
To understand molecular processes during the development of schistosomes, research has concentrated on the identification of signalling molecules and the elucidation of putative signal transduction mechanisms. During the last years, a number of different molecules have been identified which belong to conserved signal-transduction cascades (Shoemaker et al., 1992, Escriva et al., 1997, Davies et al., 1998, Freebern et al., 1999, Inal, 1999, Kampkotter et al., 1999).
Recently, we started an attempt to identify TKs in schistosomes by a polymerase chain reaction (PCR) approach with degenerate primers directed against conserved regions of the catalytic domain (Kapp et al., 2001). Several novel TK-molecules were isolated which belong to different families. Among them, a Fyn-like TK has been found which is transcribed in the larval stages and in adults (Kapp et al., 2001). Here we present the characterization of another molecule, TK4, a cellular TK of the Syk family which has not been described in schistosomes yet.
Section snippets
Parasite stock
A Liberian strain of Schistosoma mansoni was maintained in Biomphalaria glabrata as intermediate host and in Syrian golden hamsters (Mesocricetus auratus) as final host (Grevelding, 1995). Adult worms were obtained by perfusion at day 42 post infection. Females and males were separated with a fine brush and stored under liquid nitrogen. Unisexual worm populations were generated by monomiracidial infections as described elsewhere (Grevelding, 1999).
Isolation of nucleic acids
Deoxyribonucleic acid (DNA) from female and
Cloning of the TK4 cDNA from S. mansoni
RT-PCR experiments were performed with degenerate primers directed against highly conserved regions within the TK domain (VHRD and WE). For primer design, the preferred codon usage of S. mansoni was taken into consideration. Total RNA from adult male worms was taken as template. An amplification product of approximately 200 bp was detected by electrophoresis that matched the predicted distance between the conserved VHRD and WE boxes. The PCR amplicon was gel-purified and cloned. About 150
Discussion
Within the large group of cytoplasmic TKs, the class of Syk-TKs is characterized by the existence of two SH2 domains and a catalytic TK domain. Sequence and data base analyses demonstrated that TK4 from S. mansoni belongs to this class of molecules, although significant differences have been found. It is striking that TK4 represents the longest Syk molecule found so far, being more than twice as long as other molecules of this class. This peculiarity is due to an elongated hinge region between
Acknowledgements
The receptor tyrosine kinase clone p17-2 from X. maculatus was kindly provided by Professor Schartl (Würzburg, Germany). The authors thank Dr Karl Köhrer and Dr Sibylle Scheuring from the Centre for Biological and Medical Research Düsseldorf for support with the automated sequencing, Simone Bork for technical help, Stefan Sroka as well as Didina David for technical assistance, and the Deutsche Forschungsgemeinschaft (Grants Ku 282/15-1 and GR 1549/1-1) for financial support. J. Knobloch is
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Note: Sequences reported here are available from DDBJ/EMBL/GenBank under the accession number AJ421472.
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Both these authors contributed equally to this work.