Alternative splicing creates sex-specific transcripts and truncated forms of the furin protease in the parasite Dirofilaria immitis.
ABSTRACT Many extracellular proteins are activated by specific cleavage with an endoprotease. In nematodes, several proteins are cleaved after RX(K/R)R, the recognition site for the subtilisin-like proprotein convertases, furin and blisterase. To characterize furin in the parasitic nematode Dirofilaria immitis, we determined the sequence of the difur gene and its multiple transcripts. The gene spans 11 kb; encodes 16 exons and has a complex pattern of alternative splicing which generates at least 16 distinct mRNAs. The major transcript is a 4.4 kb mRNA which codes for a protein of 834 aa with an unusually long prodomain of 254 aa. Sex-specific splice variants of difur were observed by RT-PCR. The three female-specific and five male-specific transcripts are the first reported examples of sex-specific splicing in parasitic nematodes. This suggests that nematodes have sex-specific factors which regulate RNA splicing. Other splice variants are predicted to alter the phosphorylation and localization of the protease. Alternative splicing after the prodomain encodes a truncated protein that may be an inhibitor and/or substrate of Difurin.
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ABSTRACT: Chorismate mutase (CM) secreted from the stylet of plant-parasitic nematodes plays an important role in plant parasitism. We isolated and characterized a new nematode CM gene (Gr-cm-1) from the potato cyst nematode, Globodera rostochiensis. The Gr-cm-1 gene was found to exist in the nematode genome as a single-copy gene that has two different alleles, Gr-cm-1A and Gr-cm-1B, both of which could give rise to two different mRNA transcripts of Gr-cm-1 and Gr-cm-1-IRII. In situ mRNA hybridization showed that the Gr-cm-1 gene was exclusively expressed within the subventral oesophageal gland cells of the nematode. Gr-cm-1 was demonstrated to encode a functional CM (GR-CM-1) potentially having a dimeric structure as the secreted bacterial *AroQ CMs. Gr-cm-1-IRII, generated by retention of intron 2 of the Gr-cm-1 pre-mRNA through alternative splicing (AS), would encode a truncated protein (GR-CM-1t) lacking the CM domain with no CM activity. The quantitative real-time reverse transcription-PCR assay revealed that splicing of the Gr-cm-1 gene was developmentally regulated; Gr-cm-1 was up-regulated whereas Gr-cm-1-IRII was down-regulated in early nematode parasitic stages compared to the preparasitic juvenile stage. Low-temperature SDS-PAGE analysis revealed that GR-CM-1 could form homodimers when expressed in Escherichia coli and the dimerization domain was retained in the truncated GR-CM-1t protein. The specific interaction between the two proteins was demonstrated in yeast. Our data suggested that the novel splice variant might function as a dominant negative isoform through heterodimerization with the full-length GR-CM-1 protein and that AS may represent an important mechanism for regulating CM activity during nematode parasitism.Molecular and Biochemical Parasitology 09/2008; 162(1):1-15. DOI:10.1016/j.molbiopara.2008.06.002 · 2.24 Impact Factor
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ABSTRACT: Many of the filarial proteases involved in critical physiological functions are expressed in stage-specific manner and belong to various mechanistic classes. Setaria cervi, a bovine filarial parasite express different classes of proteases. This parasite shows strong antigenic cross-reactivity with human filarial parasites Wuchereria bancrofti and Brugia malayi. Somatic extracts of S. cervi microfilariae (mf) and adult stages as well as their excretory–secretory (ES) products were screened for the presence of different classes of proteases using general (casein, bovine hemoglobin) and class specific substrates. Detergent-soluble extracts of male and female worms were also screened. Significant enzyme activity was detected in ES products both at pH5.0 and 7.0 with casein. Cathepsin B-like activity was found to be much higher in membrane-bound extract than in the crude-soluble extract. However, it was also found to be actively secreted by both mf and adult worms. Cathepsin D-like activity assayed at pH3.0 was very low both in somatic extract as well as in ES products. Collagenase activity at neutral pH showed higher levels, both in somatic extract and ES products. Cathepsin L-like activity was detected only in crude-soluble extract but was below detectable limit in ES products. Leucine aminopeptidase activity was significant both in crude-soluble extract and ES products. This study, thus, might be helpful for a better understanding of host–parasite interaction and identification of appropriate virulence factors that may be targeted as vaccine and/or drug targets against lymphatic filariasis.Parasitology Research 06/2009; 104(6):1399-1405. DOI:10.1007/s00436-009-1336-x · 2.33 Impact Factor