title: Peroxidase catalysed cross-linking of an intrinsically unstructured protein via dityrosine bonds in the oocyst wall of the apicomplexan parasite, Eimeria maxima.
abstract: Apicomplexan parasites such as Eimeria maxima possess a resilient oocyst wall that protects them upon excretion in host faeces and in the outside world, allowing them to survive between hosts. The wall is formed from the contents of specialised organelles - wall-forming bodies - found in macrogametes of the parasites. The presence of dityrosine in the oocyst wall suggests that peroxidase-catalysed dityrosine cross-linking of tyrosine-rich proteins from wall-forming bodies forms a matrix that is a crucial component of oocyst walls. Bioinformatic analyses showed that one of these tyrosine-rich proteins, EmGAM56, is an intrinsically unstructured protein, dominated by random coil (52-70%), with some α-helix (28-43%) but a relatively low percentage of β-sheet (1-11%); this was confirmed by nuclear magnetic resonance and circular dichroism. Furthermore, the structural integrity of EmGAM56 under extreme temperatures and pH indicated its disordered nature. The intrinsic lack of structure in EmGAM56 could facilitate its incorporation into the oocyst wall in two ways: first, intrinsically unstructured proteins are highly susceptible to proteolysis, explaining the several differently-sized oocyst wall proteins derived from EmGAM56; and, second, its flexibility could facilitate cross-linking between these tyrosine-rich derivatives. An in vitro cross-linking assay was developed using a recombinant 42kDa truncation of EmGAM56. Peroxides, in combination with plant or fungal peroxidases, catalysed the rapid formation of dityrosine cross-linked polymers of the truncated EmGAM56, as determined by western blotting and HPLC, confirming this protein's propensity to form dityrosine bonds.
authors.affiliation: Institute for the Biotechnology of Infectious Diseases, University of Technology, Sydney, NSW 2007, Australia.
authors.forename: Kelly
authors.initials: K
authors.lastname: Mai
authors.forename: Nicholas C
authors.initials: NC
authors.lastname: Smith
authors.forename: Zhi-Ping
authors.initials: ZP
authors.lastname: Feng
authors.forename: Marilyn
authors.initials: M
authors.lastname: Katrib
authors.forename: Jan
authors.initials: J
authors.lastname: Slapeta
authors.forename: Iveta
authors.initials: I
authors.lastname: Slapetova
authors.forename: Michael G
authors.initials: MG
authors.lastname: Wallach
authors.forename: Catherine
authors.initials: C
authors.lastname: Luxford
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Davies
authors.forename: Xuecheng
authors.initials: X
authors.lastname: Zhang
authors.forename: Raymond S
authors.initials: RS
authors.lastname: Norton
authors.forename: Sabina I
authors.initials: SI
authors.lastname: Belli
mesh_terms: D055162:Biocatalysis; D002473:Cell Wall / Q000737:chemistry* / Q000201:enzymology / Q000235:genetics; D003432:Cross-Linking Reagents / Q000378:metabolism*; D004539:Eimeria / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D033661:Oocysts / Q000737:chemistry* / Q000201:enzymology; D009195:Peroxidase / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism
chemical_list: D003432:Cross-Linking Reagents; D015800:Protozoan Proteins; D014443:Tyrosine; C007543:dityrosine; D009195:Peroxidase