Wang, K, Peng, ED, Huang, AS, Xia, D, Vermont, SJ, Lentini, G, Lebrun, M, Wastling, JM and Bradley, PJ (2016) Identification of Novel O-Linked Glycosylated Toxoplasma Proteins by Vicia villosa Lectin Chromatography. PLoS One, 11 (3). e0150561 -?. ISSN 1932-6203

[thumbnail of wastling_plos1_mar_2016.pdf]
wastling_plos1_mar_2016.pdf - Published Version
Available under License Creative Commons Attribution.

Download (1MB) | Preview


Toxoplasma gondii maintains its intracellular life cycle using an extraordinary arsenal of parasite-specific organelles including the inner membrane complex (IMC), rhoptries, micronemes, and dense granules. While these unique compartments play critical roles in pathogenesis, many of their protein constituents have yet to be identified. We exploited the Vicia villosa lectin (VVL) to identify new glycosylated proteins that are present in these organelles. Purification of VVL-binding proteins by lectin affinity chromatography yielded a number of novel proteins that were subjected to further study, resulting in the identification of proteins from the dense granules, micronemes, rhoptries and IMC. We then chose to focus on three proteins identified by this approach, the SAG1 repeat containing protein SRS44, the rhoptry neck protein RON11 as well as a novel IMC protein we named IMC25. To assess function, we disrupted their genes by homologous recombination or CRISPR/Cas9. The knockouts were all successful, demonstrating that these proteins are not essential for invasion or intracellular survival. We also show that IMC25 undergoes substantial proteolytic processing that separates the C-terminal domain from the predicted glycosylation site. Together, we have demonstrated that lectin affinity chromatography is an efficient method of identifying new glycosylated parasite-specific proteins.

Item Type: Article
Uncontrolled Keywords: Parasitic diseases, Toxoplasma, Lectins, Glycosylation, Vacuoles, Toxoplasma gondii, Host cells, Cloning
Subjects: Q Science > QH Natural history > QH301 Biology
R Medicine > R Medicine (General)
Divisions: Faculty of Natural Sciences > School of Life Sciences
Related URLs:
Depositing User: Symplectic
Date Deposited: 31 Mar 2016 09:05
Last Modified: 30 Nov 2016 14:06

Actions (login required)

View Item
View Item