Detecting Complex Carbohydrate Modification in Toxoplasma gondii with Unnatural Sugars

Toxoplasma gondii is an obligate intracellular parasite that infects all nucleated cell types in diverse warm-blooded organisms. Infection (which occurs by accidental ingestion of tissue cysts or oocysts) is life-threatening to immunocompromised individuals and the developing fetus. Parasite effector molecules transit through the Toxoplasma ER and Golgi apparatus to specialized membrane compartments and are secreted during invasion and intracellular growth. We are using metabolic oligosaccharide engineering to profile the subset of effector proteins that are glycosylated. Our approach relies on the metabolic incorporation of specific unnatural sugars into Toxoplasma gondii glycoproteins. The sugars are tagged with an abiotic chemical reporter group, organic azide (Az), and when fed to cells, the glycan biosynthetic machinery incorporates these unnatural sugars into glycoconjugates.  Metabolically incorporated azido sugars can be detected by covalent reaction with complementary probes amenable to detection by flow cytometry, microscopy or affinity enrichment. Extracellular Toxoplasma tachyzoites in an “Endo” buffer that mimics intracellular conditions incorporate the azidosugars Ac₄ManNAz, Ac₄GalNAz and Ac₄GlcNAz into proteins. Over 20 labeled bands are visible by SDS PAGE separation of rhodamine-tagged Ac₄GlcNAz-labeled samples. We used biotin tags to purify the population of Ac₄GlcNAz-labeled proteins, and identified a number of glycosylated proteins by mass spectrometry. Some of the proteins identified by this approach were known to be glycosylated while others are novel, previously uncharacterized proteins. Current experiments are focused on confirming that the proteins are glycosylated and defining the sites of modification. These results will provide a more comprehensive understanding of protein glycosylation in Toxoplasma. Additionally, the identification of glycosylated proteins that are destined for secretion is likely to reveal novel components that are critically important to parasite-host cell interactions and parasite virulence.