Identification and cloning of a novel tetraspanin (TSP) homologue from Brugia malayi

Abstract

This is the first report of a tetraspanin (TSP)-like molecule in the lymphatic filarial parasites. Expressed sequence tag (EST) database search for TSP like molecules in the filarial genome resulted in three significant EST hits (two partial ESTs from Brugia malayi and one full length EST from Wuchereria bancrofti). The full length gene cloned from B. malayi showed significant similarity to Caenorhabditis elegans TSP and human TSP and hence the gene was named B. malayi TSP (BmTSP). Subsequent Genbank analysis with the predicted ORF of BmTSP showed additional homologous genes reported from Schistosoma mansoni and Taenia solium parasites. Structural analyses showed that BmTSP has four transmembrane domains and other conserved domains such as CCG and two other critical cysteine residues present within the large extracellular loop similar to other reported TSPs. In addition, putative post-translational modifications such as N-glycosylation, protein kinase c phosphorylation, casein kinase II phosphorylation and N-myristoylation sites have been found in BmTSP sequence. Further, PCR analyses showed that BmTSP is differentially transcribed, with highest level of expression being present in the adult stages followed by L3 and mf stages. This study thus describes a novel TSP cloned from B. malayi, its putative functions in cuticle biogenesis and role in protective immunity.

Figure 1

Multiple alignments (CLUSTAL) of the amino acid sequences of TSP family of proteins from B. malayi (BmTSP, accession no. EF397425), W. bancrofti (WbTSP, accession no. CD455836), H. sapiens (HsCD81, accession no. NP_004347), H. sapiens (HsCD9, accession no. NP_001760), S. mansoni (SmTSP-2, accession no. AF521091), T. solium (TsTSP, accession no. AY211879) and C. elegans (CeTSP-15, accession no. Q9XVM9). (★) Indicates identical amino acids, (:) indicates strongly similar amino acids and (.) indicates weakly similar amino acids.

Figure 2

Phylogenetic tree analysis of TSP family of proteins. The tree distances were generated according to the ClustalW algorithm, and the tree was constructed using TreeTop (Phylip program). Phylogenetic tree analysis shows that filarial TSPs are closely related with distantly separated from trematode (S. mansoni) and cestode (T. solium) parasites.

Figure 3

Expression of BmTSP mRNA in various life cycle stages of B. malayi. A. BmTSP and BmGAPDH transcripts were amplified by PCR from the cDNA libraries of various life cycle stages (L3, adult and Mf) of B. malayi using primers specific for BmTSP and BmGAPDH. Expression of BmGAPDH was used as an internal house keeping control gene. PCR products were resolved on a 1% agarose gel and stained with ethidium bromide. B. Band intensity was normalized to BmGAPDH PCR products and values were calculated using NIH image software. Data represented in B is average of values from three similar experiments.