Molecular cloning and primary sequence of a cysteine protease expressed by Haemonchus contortus adult worms

Cox, George N.; Pratt, Dickson; Hageman, Robert; Boisvenue, R. J.

doi:10.1016/0166-6851(90)90093-2

Cited by 103 publications

(44 citation statements)

References 12 publications

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“…According to molecular weight, optimum pH and the inhibition by E64, the proteases identified here seem to be cysteine proteases similar to those described previously in different ovine strains of the parasite [12,13]. A cysteine protease of 35 kDa was first reported by Cox et al [5] as a major component of protein extracts isolated from ovine H. contortus adult worms.…”

Section: Discussionsupporting

confidence: 84%

Immunoprotection in goats against Haemonchus contortus after immunization with cysteine protease enriched protein fractions

et al. 2004

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-Haemonchus cysteine proteases, because of their apparent critical function in worm physiology, are considered important candidates in the immunological control of haemonchosis in sheep. Only limited information is, however, available on the immunoprotective properties of these molecules in goats. In the present study cysteine proteases of Haemonchus contortus adult worms isolated from a goat strain (Gran Canaria, Spain) were enriched by affinity chromatography and evaluated as immunoprotective antigens against caprine haemonchosis. The eggs per gram of faeces averaged over the whole experiment for unvaccinated goats (550 ± 13.5) was significantly greater (P < 0.001) than that of vaccinated goats (61 ± 2.9). Accordingly, the worm burden was significantly different between the groups (P < 0.05), with mean values of 247.5 ± 43.8 and 762.5 ± 78.3 worms per animal in the immunized and nonimmunized goats, respectively. The percentage of egg (89%) and worm (68%) reduction approached those attained with other immunogens used in sheep. Haemonchus contortus / cysteine proteases / immunoprotection / goats

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Section: Discussionsupporting

confidence: 84%

Immunoprotection in goats against Haemonchus contortus after immunization with cysteine protease enriched protein fractions

et al. 2004

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“…It is likely that the cathepsin B-like enzymes evolved before the divergence of the platyhelminthes. (24), mouse (24), rat (25), Schistosoma (26), and Haemonchus (27). *, Amino acids in the active site; =, conserved in the propeptide region ofthe first four proteins; -, similar in the first four proteins where any pair of amino acids within the group have a score of 4 or greater on the scale of Feng et al (14); ., gaps introduced to maximize alignment; boldface type, residues conserved in all known cysteine proteases.…”

Section: Discussionmentioning

confidence: 99%

Two distinct gene subfamilies within the family of cysteine protease genes.

Karrer

Peiffer

DiTomas

1993

Proc. Natl. Acad. Sci. U.S.A.

347

248

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A cDNA clone for a physiologically regulated Tetrahymena cysteine protease gene was sequenced. The nucleotide sequence predicts that the clone encodes a 336-amino acid protein composed of a 19-residue N-terminal signal sequence followed by a 107-residue propeptide and a 210-residue mature protein. Comparison of the deduced amino acid sequence of the protein with those of other cysteine proteases revealed a highly conserved interspersed amino acid motif in the propeptide region of the protein, the ERFNIN motif. The motif was present in all of the cysteine proteases in the data base with the exception of the cathepsin B-like proteins, which have shorter propeptides. Differences in the propeptides and in conserved amino acids of the mature proteins suggest that the ERFNIN proteases and the cathepsin B-like proteases constitute two distinct subfamilies within the cysteine proteases.The cysteine proteases are a family of enzymes that play an important role in intracellular protein degradation. These proteases and their cDNA clones have been isolated from phylogenetically diverse organisms ranging from slime mold to mammals. The tertiary structures of two plant cysteine proteases, papain and actinidin, have been solved (1, 2). The enzymes have two protein domains that come together to form the active site. Amino acid sequence homologies suggest this double domain structure is conserved in the animal thiol proteases cathepsins B, H, and L (3).The phylogenetic range of organisms for which the sequence of cysteine protease genes are known was extended by determination of the sequence of a cDNA clone for a gene from a ciliated protozoan, Tetrahymena thermophila.t Comparison of the deduced amino acid sequence to those of known cysteine proteases revealed the presence of an amino acid motif in the propeptide region consisting of highly conserved amino acids interspersed with variable residues. The motif was present in 15 of 20 cysteine proteases in the EMBL/GenBank data base (August 1992). The five proteases that lacked the motif were all cathepsin B-like enzymes. Recognition of the differences in the propeptide region prompted comparison of the mature proteins. Alignment of the amino acid sequences of the proteases as two separate groups allowed identification of amino acids that are highly conserved among the proteases with the propeptide motif or among the cathepsin B-like proteases but strikingly different between the two groups. We suggest that the proteins with the interspersed motif and the cathepsin B-like proteases represent two distinct classes of cysteine proteases that can be distinguished by both propeptide and mature protein structure. MATERIALS AND METHODSTetrahymena clone pCyP (formerly BC11) is a cDNA clone of an RNA that is expressed in starved, but not growing, cells (4, 5). The clone was isolated from a cDNA library of RNA from starved cells cloned into the Pst I site of pUC9 (4). DNA fragments were subcloned into pBluescript for sequencing. The sequence was scanned for open reading frames by usi...

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“…At the least, the presence of a secretory signal and the reactivity of RAG1-monospecific and anti-rRAG1 mouse antibodies to components of BPES antigen suggest that RAG1 is a component of the ES antigen. Excretory-secretory antigens in parasites serve several functions, such as in feeding, penetration of the host, tissue migration, reproduction, and immune evasion (5,16,21). Functional assays are required to determine the function of this protein in B. procyonis.…”

Section: Vol 17 2010mentioning

confidence: 99%

Molecular Cloning of an Immunogenic Protein of Baylisascaris procyonis and Expression in Escherichia coli for Use in Developing Improved Serodiagnostic Assays

Dangoudoubiyam

Vemulapalli

Hancock

et al. 2010

Clin Vaccine Immunol

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Larva migrans caused by Baylisascaris procyonis is an important zoonotic disease. Current serological diagnostic assays for this disease depend on the use of the parasite's larval excretory-secretory (ES) antigens. In order to identify genes encoding ES antigens and to generate recombinant antigens for use in diagnostic assays, construction and immunoscreening of a B. procyonis third-stage larva cDNA expression library was performed and resulted in identification of a partial-length cDNA clone encoding an ES antigen, designated repeat antigen 1 (RAG1). The full-length rag1 cDNA contained a 753-bp open reading frame that encoded a protein of 250 amino acids with 12 tandem repeats of a 12-amino-acid long sequence. The rag1 genomic DNA revealed a single intron of 837 bp that separated the 753-bp coding sequence into two exons delimited by canonical splice sites. No nucleotide or amino acid sequences present in the GenBank databases had significant similarity with those of RAG1. We have cloned, expressed, and purified the recombinant RAG1 (rRAG1) and analyzed its diagnostic potential by enzyme-linked immunosorbent assay. Anti-Baylisascaris speciesspecific rabbit serum showed strong reactivity to rRAG1, while only minimal to no reactivity was observed with sera against the related ascarids Toxocara canis and Ascaris suum, strongly suggesting the specificity of rRAG1. On the basis of these results, the identified RAG1 appears to be a promising diagnostic antigen for the development of serological assays for specific detection of B. procyonis larva migrans.

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Molecular cloning and primary sequence of a cysteine protease expressed by Haemonchus contortus adult worms

Cited by 103 publications

References 12 publications

Immunoprotection in goats against Haemonchus contortus after immunization with cysteine protease enriched protein fractions

Immunoprotection in goats against Haemonchus contortus after immunization with cysteine protease enriched protein fractions

Two distinct gene subfamilies within the family of cysteine protease genes.

Molecular Cloning of an Immunogenic Protein of Baylisascaris procyonis and Expression in Escherichia coli for Use in Developing Improved Serodiagnostic Assays

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