EtMIC4: a microneme protein from Eimeria tenella that contains tandem arrays of epidermal growth factor-like repeats and thrombospondin type-I repeats

Tomley, Fiona M.; Billington, Karen; Bumstead, Janene M.; Clark, Julie D.; Monaghan, Paul

doi:10.1016/s0020-7519(01)00255-7

Cited by 67 publications

(55 citation statements)

References 26 publications

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“…The E. coli strain NMR554, transformed by plasmid pRep4 has been described previously [9]. [7][8][9] The coding sequences for cbEGF [7][8][9] (amino acids 300-429) were PCR-amplified from plasmid pFT1 [8] using Pfu polymerase and oligonucleotide primers EGFfw 7-9 (5 -TAGTAGGGATCCATAGAAGGACGATCAGCAGTGGA-TATCGACGAGTGCTCCAAG-3 ), which incorporates a Bam HI site (underlined), factor Xa cleavage site (double underlined) and two spacer amino acids (A, V) upstream of the start of the cbEGF 7 sequence; and EGFrv 7-9 (5 -TAGTAGAAGCTTCTATTAACTGCACACCTTTCCGTC-GCC-3 ), which incorporates a Hind III site (underlined) and two stop codons downstream of the cbEGF 9 sequence. The authenticity of the amplified product was confirmed by DNA sequencing and it was ligated into plasmid expression vector pQ30 (Qiagen) in frame with the N-terminal (His) 6 tag.…”

Section: Methodsmentioning

confidence: 99%

“…The protein was then lyophilised and suspended at 2 mg ml −1 in an in vitro refolding buffer consisting of 50 mM Tris, pH 8.3, 3 mM l-cysteine, 0.3 mM l-cystine and incubated for 72 h at 4 • C. The refolded protein was re-purified by RP-HPLC, lyophilised, then suspended in 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM CaCl 2 and digested with bovine factor X (Amersham Pharmacia) overnight at 37 • C at a 1:1000 enzyme to protein ratio by weight to remove the (His) 6 tag. The cleaved cbEGF [7][8][9] was purified over a MonoQ column (Pharmacia) using a gradient of 0-0.5 M NaCl in 50 mM Tris-HCl, pH 7.3 and subjected to a final round of RP-HPLC. The concentration of cbEGF [7][8][9] was calculated from its theoretical molar extinction coefficient and absorbance at 280 nm wavelength.…”

Section: Purification Of the Cbegf 7-9 Tripletmentioning

confidence: 99%

“…The cleaved cbEGF [7][8][9] was purified over a MonoQ column (Pharmacia) using a gradient of 0-0.5 M NaCl in 50 mM Tris-HCl, pH 7.3 and subjected to a final round of RP-HPLC. The concentration of cbEGF [7][8][9] was calculated from its theoretical molar extinction coefficient and absorbance at 280 nm wavelength.…”

Section: Purification Of the Cbegf 7-9 Tripletmentioning

confidence: 99%

“…Recombinant cbEGF [7][8][9] was suspended at 0.6 mg ml −1 in 20 mM Tris-HCl, pH 7.5 containing CaCl 2 at 1 mM, 250 M, 62 M or 7 M or containing 10 mM EGTA. Circular dichroism spectra were recorded from 260 to 190 nm by use of a Jasco J-710 spectropolarimeter.…”

Section: Circular Dichroismmentioning

confidence: 99%

“…In this study, we have expressed and purified a prototypical cbEGF triplet (Fig. 1) from the E. tenella EtMIC4 protein, a 240 kDa multi-modular, transmembrane protein that contains 31 tandemly arrayed EGFs [8]. We have used the recombinant protein to investigate the calcium binding properties of this cbEGF triplet, which gives an insight into the likely functional role of cbEGFs within EtMIC4 and other apicomplexan microneme proteins.…”

Section: Introductionmentioning

confidence: 99%

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Calcium binding activity of the epidermal growth factor-like domains of the apicomplexan microneme protein EtMIC4

Periz

Gill

Knott

et al. 2005

Molecular and Biochemical Parasitology

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Microneme proteins are secreted from apicomplexan parasites during invasion of host cells and they play crucial roles in parasite–host cell adhesion. EtMIC4 is a 240 kDa transmembrane protein from Eimeria tenella that contains 31 tandemly arranged epidermal growth factor (EGF), like repeats within its extracellular domain. The majority of these repeats have calcium binding (cb) consensus sequences. Little is known about cbEGFs in apicomplexan parasites but their presence in microneme proteins suggests that they may contribute to parasite–host interactions. To investigate the potential role of cbEGFs we have expressed and correctly refolded a cbEGF triplet from EtMIC4 (cbEGF7–9) and demonstrated that this triplet binds calcium. Circular dichroism spectroscopic analysis of cbEGF7–9 demonstrates that the molecule undergoes a gradual change in conformation with increasing levels of calcium. In the presence of calcium, the triplet becomes resistant to proteolytic degradation by a variety of proteases, a characteristic feature of cbEGF repeats from higher eukaryotic proteins, such as fibrillin, suggesting that calcium binding induces the formation of a rigid conformation. Moreover, mass spectrometric mapping of the cleavage sites that are protected by calcium shows that these sites are located both close to and distant from the calcium binding sites, indicating that protection is not due to steric hindrance by calcium ions, but rather due to the overall conformation adopted by the triplet in the presence of calcium. Thus, the tandemly-arranged cbEGF repeats within EtMIC4 provide a mechanism whereby, in the calcium-rich extracellular environment, the molecule could adopt a protease-resistant, rigid structure that could favour its interaction with host cell ligands

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Section: Methodsmentioning

confidence: 99%

Section: Purification Of the Cbegf 7-9 Tripletmentioning

confidence: 99%

Section: Purification Of the Cbegf 7-9 Tripletmentioning

confidence: 99%

Section: Circular Dichroismmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Calcium binding activity of the epidermal growth factor-like domains of the apicomplexan microneme protein EtMIC4

Periz

Gill

Knott

et al. 2005

Molecular and Biochemical Parasitology

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Defining the protein repertoire of microneme secretory organelles in the apicomplexan parasite Eimeria tenella

et al. 2003

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The apicomplexan pathogen Eimeria causes coccidiosis, an intestinal disease of chickens, which has a major welfare and economic impact on the poultry industry. There is an urgent need to identify molecules that are rational targets for drug design and novel vaccines against coccidiosis. Apicomplexan secretory organelles, including micronemes and rhoptries, are essential for invasion of the host intestinal epithelium and establishment of parasitism. However, relatively little is known about the precise molecular function of these organelles, partly because few organelle proteins have been characterized. In this study, proteomics tools have been harnessed to define the protein repertoire of micronemes. Purified microneme proteins from Eimeria tenella sporozoites were excised from two-dimensional (2-D) gels and analyzed using matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) and chemically assisted fragmentation (CAF)-MALDI with de novo sequencing. Peptide mass profiles were searched against the NCBI non-redundant (nr) database and against Eimeria-specific databases using the Mascot search algorithm, resulting in the identification of 37 of 96 spots excised from the 2-D gels. In addition, we have found CAF-MALDI to be a useful adjunct for identifying proteins, without the need for tandem MS. This global approach to protein characterization will be vital to gain greater understanding of the processes involved in apicomplexan host cell invasion.

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Proteomic comparison of four Eimeria tenella life‐cycle stages: Unsporulated oocyst, sporulated oocyst, sporozoite and second‐generation merozoite

et al. 2009

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We report the proteomes of four life cycle stages of the Apicomplexan parasite Eimeria tenella. A total of 1868 proteins were identified, with 630, 699, 845 and 1532 found in early oocysts (unsporulated), late oocysts (sporulated), sporozoites and second-generation merozoites, respectively. A MudPIT shotgun approach identified 812 sporozoite, 1528 merozoite and all of the oocyst proteins, whereas 2D gel proteomics identified 230 sporozoite and 98 merozoite proteins. Comparing the invasive stages, we find moving junction components RON2 in both, whilst AMA-1 and RON4 are found only in merozoites and AMA-2 and RON5 are only found in sporozoites, suggesting stage specific moving junction proteins. During early oocyst to sporozoite development, refractile body and most ‘glideosome’ proteins are found throughout, whilst microneme and most rhoptry proteins are only found after sporulation. Quantitative analysis indicates glycolysis and gluconeogenesis are the most abundant metabolic groups detected in all stages. The mannitol cycle ‘off shoot’ of glycolysis was not detected in merozoites but was well represented in the other stages. However, in merozoites we find more protein associated with oxidative phosphorylation, suggesting a metabolic shift mobilising greater energy production. We find a greater abundance of protein linked to transcription, protein synthesis and cell cycle in merozoites than in sporozoites, which may be residual protein from the preceding massive replication during schizogony.

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EtMIC4: a microneme protein from Eimeria tenella that contains tandem arrays of epidermal growth factor-like repeats and thrombospondin type-I repeats

Cited by 67 publications

References 26 publications

Calcium binding activity of the epidermal growth factor-like domains of the apicomplexan microneme protein EtMIC4

Calcium binding activity of the epidermal growth factor-like domains of the apicomplexan microneme protein EtMIC4

Defining the protein repertoire of microneme secretory organelles in the apicomplexan parasite Eimeria tenella

Proteomic comparison of four Eimeria tenella life‐cycle stages: Unsporulated oocyst, sporulated oocyst, sporozoite and second‐generation merozoite

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