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CDNA cloning and functional expression of the Schistosoma mansoni protective antigen triose-phosphate isomerase.

ABSTRACT: M.1 monoclonal antibody has previously been shown to passively transfer partial resistance to schistosome infection within mice and to recognize a 28-kDa antigen that has peptide sequence homology with triose-phosphate isomerase (TPI; D-glyceraldehyde-3-phosphate ketol-isomerase, EC 5.3.1.1). We have now isolated the complete coding DNA for Schistosoma mansoni TPI and confirmed that this cDNA encodes the 28-kDa antigen recognized by M.1. The predicted translation product has strong homology with other TPIs, particularly from higher eukaryotes, and the sequence homology is greatest in regions known to form the active site. The complete coding DNA has been expressed within an Escherichia coli host to produce high levels of soluble, recombinant S. mansoni TPI protein. The product is recognized and purified by the M.1 antibody and is a functional TPI with an intrinsic specific activity comparable to that of rabbit and yeast TPI.

SUBMITTER: Shoemaker C

PROVIDER: S-EPMC48549 | biostudies-other | 1992 Mar

REPOSITORIES: biostudies-other

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cDNA cloning and functional expression of the Schistosoma mansoni protective antigen triose-phosphate isomerase.

Shoemaker C C Gross A A Gebremichael A A Harn D D

Proceedings of the National Academy of Sciences of the United States of America 19920301 5

M.1 monoclonal antibody has previously been shown to passively transfer partial resistance to schistosome infection within mice and to recognize a 28-kDa antigen that has peptide sequence homology with triose-phosphate isomerase (TPI; D-glyceraldehyde-3-phosphate ketol-isomerase, EC 5.3.1.1). We have now isolated the complete coding DNA for Schistosoma mansoni TPI and confirmed that this cDNA encodes the 28-kDa antigen recognized by M.1. The predicted translation product has strong homology with ...[more]

PMID: 1542681

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Project description:Schistosomiasis is endemic in over 70 countries, in which more than 200 million people are infected with the various schistosome species. Understanding the physiological processes underlying key developmental events could be useful in developing novel chemotherapeutic reagents or infection intervention strategies. Calmodulin is a small, calcium-sensing protein found in all eukaryotes and, although the protein has been previously identified in various Schistosoma mansoni stages and implicated in egg hatching and miracidia transformation, few molecular and functional data are available for this essential protein. Herein, we report the molecular cloning, expression, and functional characterization of calmodulin in the miracidia and primary sporocyst stages of S. mansoni. Two transcripts, SmCaM1 and SmCaM2, were cloned and sequenced, and a recombinant SmCaM1 protein was expressed in Escherichia coli and used to generate anti-CaM antibodies. The 2 protein sequences were highly conserved when compared to other model organisms. The alignment of the predicted proteins of both SmCaM1 and SmCaM2 exhibited 99% identity to each other and 97-98% identity with mammalian calmodulins. Analysis of steady-state transcript abundance indicate that the 2 calmodulin transcripts differ in their stage-associated expression patterns, although the CaM protein isotype appears to be constitutively expressed during early larval development. Application of RNAi to larval parasites results in a "stunted growth" phenotype in sporocysts with 30 and 35% reduction in transcript abundance for SmCaM1 and SmCaM2, respectively, and a corresponding 35% reduction in protein level after incubation in double-stranded RNA. Differential expression of CaM transcripts during early larval development and a growth defect-inducing effect associated with partial transcript and protein inhibition as a result of RNAi suggest a potentially important role of calmodulin during early larval development.

Dataset Information

CDNA cloning and functional expression of the Schistosoma mansoni protective antigen triose-phosphate isomerase.

Publications

cDNA cloning and functional expression of the Schistosoma mansoni protective antigen triose-phosphate isomerase.

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