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 ingestion of tissue cysts or oocysts) is life-threatening to immunocompromised individuals. Parasite effector molecules transit through the Toxoplasma ER and Golgi to specialized membrane compartments and are secreted during invasion and intracellular growth. We are using metabolic oligosaccharide engineering to profile a subset of these effector proteins. Our approach relies on the incorporation of specific unnatural sugars into Toxoplasma gondii glycoproteins. The sugars are tagged with an abiotic chemical reporter group, azide (N₃; azido sugars), and when fed to cells, the glycan biosynthetic machinery incorporates the 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 a panel of azidosugars, Ac₄ManNAz, Ac₄GalNAz and Ac₄GlcNAz into proteins, although we suspect that the first two sugars are metabolized to Ac₄GlcNAz prior to incorporation. Over 20 labeled bands are visible by SDS PAGE separation of rhodamine-tagged Ac₄GlcNAz-labeled samples. We are currently using cleavable biotin tags to purify the population of Ac₄GlcNAz-labeled proteins for identification with mass spectroscopy.