title: Identification and characterization of a serpin from Eimeria acervulina.
abstract: Serpins are serine protease inhibitors that are widely distributed in metazoans but have not been previously characterized in Eimeria spp. A serpin from Eimeria acervulina was cloned, expressed and characterized. Random screening of an E.acervulina sporozoite cDNA library identified a single clone (D14) whose coding region shared high similarity to consensus structure of serpins. Clone D14 contained an entire open reading frame (ORF) consisting of 1,245 nts that encode a peptide 413 amino acids in length with a predicted molecular weight of 45.5 kDa and containing a signal peptide 28 residues in length. By Western blot analysis, polyclonal antiserum to the recombinant serpin (rbSp) recognized a major 55 kDa protein band in unsporulated oocysts and in oocysts sporulated up to 24 hr (fully sporulated). The anti-rbSp detected bands of 55 kDa and 48 kDa in sporozoites (SZ) and merozoites (MZ) respectively. Analysis of MZ secretion products revealed a single protein of 48 kDa which may correspond to secreted serpin. By immuno-staining the serpin was located in granules distributed throughout both the SZ and MZ but granules appeared to be concentrated in the parasite's anterior. Analysis of the structure predicts that the E. acervulina serpin should be an active inhibitor. However, rbSp was without inhibitory activity against common serine proteases. By Western blot analysis the endogenous serpin in MZ extracts did not form the expected high molecular weight complex when coincubated with either trypsin or subtilisin. The results demonstrate that E. acervulina contains a serpin gene and expresses a protein with structural properties similar to an active serine protease inhibitor. Although the function of the E. acervulina serpin remains unknown the results further suggest that serpin is secreted by the parasite where it may be involved in cell invasion and other basic developmental processes.
authors.affiliation: Animal Parasitic Diseases Laboratory, Animal and Natural Resources Institute, U.S. Department of Agriculture, Henry A. Wallace Beltsville Agricultural Research Center, Beltsville, Maryland, USA. raymond.fetterer@ars.usda.gov
authors.forename: R H
authors.initials: RH
authors.lastname: Fetterer
authors.forename: K B
authors.initials: KB
authors.lastname: Miska
authors.forename: M C
authors.initials: MC
authors.lastname: Jenkins
authors.forename: R C
authors.initials: RC
authors.lastname: Barfield
authors.forename: H
authors.initials: H
authors.lastname: Lillehoj
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002645:Chickens; D003001:Cloning, Molecular; D017124:Conserved Sequence; D018076:DNA, Complementary / Q000737:chemistry; D004539:Eimeria / Q000737:chemistry* / Q000235:genetics; D005260:Female; D018507:Gene Expression Regulation, Developmental; D015723:Gene Library; D008969:Molecular Sequence Data; D016053:RNA, Protozoan / Q000235:genetics; D011817:Rabbits; D016415:Sequence Alignment; D015843:Serpins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D018076:DNA, Complementary; D016053:RNA, Protozoan; D015843:Serpins