Isolation of cDNAs encoding secreted and transmembrane proteins from Schistosoma mansoni by a signal sequence trap method.
Surface and secreted proteins of schistosomes orchestrate the basic physiologic requirements of a parasitic existence. These proteins are often exposed to host tissues during penetration, migration, feeding, and immune evasion, and they are obvious targets for control strategies. Signal sequence trap (SST) represents a novel approach that selects for cDNAs encoding secreted and surface proteins with N-terminal signal peptides, so we constructed a randomly primed adult Schistosoma mansoni cDNA library fused to a signalless reporter gene encoding placental alkaline phosphatase. The library was used to transfect COS-7 cells, which were then assayed for the presence of reporter at the cell surface. Eighteen S. mansoni cDNA fragments were isolated and sequenced. Expression profiles of the novel clones were determined for different developmental stages; some transcripts were restricted to single-sex adult worms, while others were ubiquitously distributed. Most clones contained signal peptides or signal anchors as determined by the SignalP algorithm. Open reading frames (ORFs) were categorized as follows: (i) previously identified S. mansoni cDNAs encoding proteins of known function; (ii) cDNAs encoding proteins of known function in other organisms but novel for Schistosoma; (iii) S. mansoni expressed sequence tags (ESTs) of unknown function; and (iv) completely novel ORFs without homologues (including ESTs) from any phylum. Clones of particular interest included tetraspanins similar to human cell surface antigens, a protein kinase, and ORFs transcribed in the antisense orientation to previously characterized S. mansoni cDNAs. This is the first report describing the use of SST as a tool for identifying secreted proteins from any pathogenic organism.
Smyth
Danielle
D
Molecular Parasitology Laboratory, Australian Centre for International and Tropical Health and Nutrition, The Queensland Institute of Medical Research and The University of Queensland, Brisbane, Queensland 4029, Australia. =
McManus
Donald P
DP
Smout
Michael J
MJ
Laha
Thewarach
T
Zhang
Wenbao
W
Loukas
Alex
A
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D018076:DNA, Complementary / Q000302:isolation & purification*; D018509:DNA, Helminth / Q000302:isolation & purification*; D020224:Expressed Sequence Tags; D005260:Female; D015801:Helminth Proteins / Q000235:genetics* / Q000502:physiology; D008297:Male; D008565:Membrane Proteins / Q000235:genetics* / Q000502:physiology; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D021382:Protein Sorting Signals; D012550:Schistosoma mansoni / Q000737:chemistry / Q000235:genetics*; D014158:Transcription, Genetic
D018076:DNA, Complementary; D018509:DNA, Helminth; D015801:Helminth Proteins; D008565:Membrane Proteins; D021382:Protein Sorting Signals