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Expression and characterization of α-methylacyl CoA racemase from Anisakis simplex larvae.

Kim BJ, Kim SM, Cho MK, Yu HS, Lee YS, Cha HJ, Ock M - Korean J. Parasitol. (2012)

Bottom Line: A. simplex Amacr showed a high degree of homology compared to Amacr orthologs from other species.Amacr mRNA was highly and constitutively expressed regardless of temperature (10-40℃) and time (24-48 hr).The Amacr protein produced in large quantities from the ventriculus is probably responsible for many functions in the development and growth of A. simplex larvae.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, Kosin University College of Medicine, Busan 602-703, Korea.

ABSTRACT
Larval excretory-secretory products of Anisakis simplex are known to cause allergic reactions in humans. A cDNA library of A. simplex 3rd-stage larvae (L3) was immunoscreened with polyclonal rabbit serum raised against A. simplex L3 excretory-secretory products to identify an antigen that elicits the immune response. One cDNA clone, designated as α-methylacyl CoA racemase (Amacr) contained a 1,412 bp cDNA transcript with a single open reading frame that encoded 418 amino acids. A. simplex Amacr showed a high degree of homology compared to Amacr orthologs from other species. Amacr mRNA was highly and constitutively expressed regardless of temperature (10-40℃) and time (24-48 hr). Immunohistochemical analysis revealed that Amacr was expressed mainly in the ventriculus of A. simplex larvae. The Amacr protein produced in large quantities from the ventriculus is probably responsible for many functions in the development and growth of A. simplex larvae.

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Complete nucleotide and amino acid sequences (A) and phylogenetic analysis (B) of α-methylacyl CoA racemase (Amacr) from Anisakis simplex L3 (GenBank; HQ662605). The Amacr gene consists of 1,414 bp and 418 amino acids. The start codon (ATG), stop codon (TAA), and poly-adenylation signal (aataaa) are marked with an asterisk. The phylogenetic analyses were conducted in MEGA4 [23] using the maximum Parsimony method. The A. simplex Amacr was grouped with the nematode Caenorhabditis elegans.
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Figure 2: Complete nucleotide and amino acid sequences (A) and phylogenetic analysis (B) of α-methylacyl CoA racemase (Amacr) from Anisakis simplex L3 (GenBank; HQ662605). The Amacr gene consists of 1,414 bp and 418 amino acids. The start codon (ATG), stop codon (TAA), and poly-adenylation signal (aataaa) are marked with an asterisk. The phylogenetic analyses were conducted in MEGA4 [23] using the maximum Parsimony method. The A. simplex Amacr was grouped with the nematode Caenorhabditis elegans.

Mentions: We selected 1 clone from the final round of immunoscreening, designated as Asim37, which was the most consistently reactive with the polyclonal anti-Anisakis ES mouse sera during the screening process. The Amacr clone had an insert size of 1,412 bp and yielded 1 open reading frame (ORF) that encoded for a 418 amino acid protein with a predicted molecular weight of 46.7 kDa (GenBank; HQ662605). The complete sequence of the Amacr clone was identified as a α-methylacyl CoA racemase ortholog, a member of the CoA-transferase family III (Fig. 2A). Sequence analysis showed that the cDNA included 5' (32 bp) and 3' (188 bp) untranslated sequences. The cDNA construct contained an ATG start codon, a TAA stop codon, and a poly-A tail that comprised of 31 adenine residues. A typical polyadenylation signal was also present in 13 nucleotides upstream of the poly-A sequence. Interestingly, the A. simplex Amacr amino acid sequence was longer than that of other species at the N-terminus. Sequence homology analysis of the Anisakis Amacr gene displayed a homology of 36-50% and highly conserved amino acid sequences compared with 19 Amacr orthologs of different species (data not shown). The sequence analysis and phylogenetic analysis classified A. simplex Amacr as the most closely related to the C. elegans group (Fig. 2B).


Expression and characterization of α-methylacyl CoA racemase from Anisakis simplex larvae.

Kim BJ, Kim SM, Cho MK, Yu HS, Lee YS, Cha HJ, Ock M - Korean J. Parasitol. (2012)

Complete nucleotide and amino acid sequences (A) and phylogenetic analysis (B) of α-methylacyl CoA racemase (Amacr) from Anisakis simplex L3 (GenBank; HQ662605). The Amacr gene consists of 1,414 bp and 418 amino acids. The start codon (ATG), stop codon (TAA), and poly-adenylation signal (aataaa) are marked with an asterisk. The phylogenetic analyses were conducted in MEGA4 [23] using the maximum Parsimony method. The A. simplex Amacr was grouped with the nematode Caenorhabditis elegans.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3375458&req=5

Figure 2: Complete nucleotide and amino acid sequences (A) and phylogenetic analysis (B) of α-methylacyl CoA racemase (Amacr) from Anisakis simplex L3 (GenBank; HQ662605). The Amacr gene consists of 1,414 bp and 418 amino acids. The start codon (ATG), stop codon (TAA), and poly-adenylation signal (aataaa) are marked with an asterisk. The phylogenetic analyses were conducted in MEGA4 [23] using the maximum Parsimony method. The A. simplex Amacr was grouped with the nematode Caenorhabditis elegans.
Mentions: We selected 1 clone from the final round of immunoscreening, designated as Asim37, which was the most consistently reactive with the polyclonal anti-Anisakis ES mouse sera during the screening process. The Amacr clone had an insert size of 1,412 bp and yielded 1 open reading frame (ORF) that encoded for a 418 amino acid protein with a predicted molecular weight of 46.7 kDa (GenBank; HQ662605). The complete sequence of the Amacr clone was identified as a α-methylacyl CoA racemase ortholog, a member of the CoA-transferase family III (Fig. 2A). Sequence analysis showed that the cDNA included 5' (32 bp) and 3' (188 bp) untranslated sequences. The cDNA construct contained an ATG start codon, a TAA stop codon, and a poly-A tail that comprised of 31 adenine residues. A typical polyadenylation signal was also present in 13 nucleotides upstream of the poly-A sequence. Interestingly, the A. simplex Amacr amino acid sequence was longer than that of other species at the N-terminus. Sequence homology analysis of the Anisakis Amacr gene displayed a homology of 36-50% and highly conserved amino acid sequences compared with 19 Amacr orthologs of different species (data not shown). The sequence analysis and phylogenetic analysis classified A. simplex Amacr as the most closely related to the C. elegans group (Fig. 2B).

Bottom Line: A. simplex Amacr showed a high degree of homology compared to Amacr orthologs from other species.Amacr mRNA was highly and constitutively expressed regardless of temperature (10-40℃) and time (24-48 hr).The Amacr protein produced in large quantities from the ventriculus is probably responsible for many functions in the development and growth of A. simplex larvae.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, Kosin University College of Medicine, Busan 602-703, Korea.

ABSTRACT
Larval excretory-secretory products of Anisakis simplex are known to cause allergic reactions in humans. A cDNA library of A. simplex 3rd-stage larvae (L3) was immunoscreened with polyclonal rabbit serum raised against A. simplex L3 excretory-secretory products to identify an antigen that elicits the immune response. One cDNA clone, designated as α-methylacyl CoA racemase (Amacr) contained a 1,412 bp cDNA transcript with a single open reading frame that encoded 418 amino acids. A. simplex Amacr showed a high degree of homology compared to Amacr orthologs from other species. Amacr mRNA was highly and constitutively expressed regardless of temperature (10-40℃) and time (24-48 hr). Immunohistochemical analysis revealed that Amacr was expressed mainly in the ventriculus of A. simplex larvae. The Amacr protein produced in large quantities from the ventriculus is probably responsible for many functions in the development and growth of A. simplex larvae.

Show MeSH
Related in: MedlinePlus