Identification and function of the
EtMIC4-EtMIC5 protein complex. A novel microneme complex of Eimeria tenella
Javier Periz, Andrew Gill, Lawrence Hunt and
Fiona Tomley
Institute for Animal Health, Compton
Eimeria tenella, like other members of the
Apicomplexa, shares a conserved mechanism of motility and invasion which relies
on the sequential exocytosis of adhesive proteins from secretory organelles such
as micronemes. We have investigated the function of EtMIC4, a 240 kDa
transmembrane microneme protein of Eimeria tenella that displays a striking
multimodular organisation. EtMIC4 contains 16 TSP-1 like domains and 31 EGF-like
domains, both of which are found in other soluble and cell surface eukaryotic
proteins that mediate protein interactions.. Using non-denaturating anion
exchange chromatography and size exclusion chromatography, we identified the
presence of a high molecular weight protein complex formed between EtMIC4 and
EtMIC5, another microneme protein which contains 11 Apple domains. Cell blot
assays showed that EtMIC5 is responsible for binding the complex to cell lines.
We have also used recombinant polypeptides expressed in E. coli to investigate
structural properties of the calcium binding EGF-like domains in EtMIC4.
Circular dichroism analysis and mass spectrometry we show that, in the presence
of calcium, these EGFs mediate resistance to proteolytic degradation and suggest
that this confers on the molecule an elongated shape. This suggests a potential
role for the calcium binding EGF repeats in EtMIC4 in forming an extended stalk
region within the molecule.