Contributed Papers: Oral Presentations Cell Biology |
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