Peroxidase catalysed cross-linking of an intrinsically unstructured protein via dityrosine bonds in the oocyst wall of the apicomplexan parasite, Eimeria maxima

Mai, Kelly, Smith, Nicholas C., Feng, Zhi-Ping, Katrib, Marilyn, Šlapeta, Jan, Šlapetova, Iveta, Wallach, Michael G., Luxford, Catherine, Davies, Michael J., Zhang, Xuecheng, Norton, Raymond S., and Belli, Sabina I. (2011) Peroxidase catalysed cross-linking of an intrinsically unstructured protein via dityrosine bonds in the oocyst wall of the apicomplexan parasite, Eimeria maxima. International Journal for Parasitology, 41 (11). pp. 1157-1164.

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DOI: 10.1016/j.ijpara.2011.07.001

View at Publisher Website: http://dx.doi.org/10.1016/j.ijpara.2011....

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 42 kDa 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.


Item ID:	19287
Item Type:	Article (Research - C1)
ISSN:	1879-0135
Keywords:	Eimeria, Coccidia, Apicomplexa, oocyst, macrogamete, dityrosine bonds, intrinsically unstructured protein
Additional Information:	This work was supported by a grant from the Australian Research Council (ARC) through the ARC Linkage Grant scheme (in partnership with Abic Ltd., Israel; Project No. LP0454145) and an ARC Discovery Grant (DP0664013) to N.C.S. and S.I.B., and an ARC Centre of Excellence Grant (CE0561607) and fellowship (DP0988311) to MJD. Additional support was provided by the National Health and Medical Research Council (NHMRC), Australia through a Fellowship and an NHMRC IRIISS grant to RSN and by a Victorian State Government, Australia, OIS grant to RSN. KM was supported by a postgraduate scholarship from the Rural Industries Research and Development Corporation (RIRDC), Australia. None of the funding organisations played any role in study design, data collection, analysis, interpretation or in the writing of this manuscript. The ARC, NHMRC, RIRDC and the Victorian State Government played no role in the decision to publish; Abic Ltd. approve submission as per contractual agreements in place for the ARC Linkage Grant LP0454145. We gratefully acknowledge the assistance of the staff of the Ernst Facility, for care of the animals used in this research, in accordance with University of Technology, Sydney, Animal Care and Ethics Committee policies and procedures under approval number UTS ACEC 2008-096. We also gratefully acknowledge the expert assistance of Lisa Jones for preparation of the figures. Supplemental material available at http://dx.doi.org/10.1016/j.ijpara.2011.07.001
Date Deposited:	06 Dec 2011 02:45
FoR Codes:	11 MEDICAL AND HEALTH SCIENCES > 1108 Medical Microbiology > 110803 Medical Parasitology @ 100%
SEO Codes:	92 HEALTH > 9201 Clinical Health (Organs, Diseases and Abnormal Conditions) > 920109 Infectious Diseases @ 100%
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