Mucin sialylation and characterization.

A) Mucins (or any glycoconjugate bearing a terminal β-galactose) may be tagged by the TS using a Neu5Az donor such as Neu5Azα(2–3)LacβOMe. Then the Azide group may be coupled via the Staudinger-Bertozzi chemistry or the Cu2+-free click chemistry to obtain a FLAG or biotin tag ready for detection. B) Western blot of Neu5Az trans-sialylated trypomastigote lysates revealing the relative molecular mass distribution of acceptor molecules. A line profile of the blot is also plotted. Neg Ctrl: Negative control. C) Neu5Az trans-sialylated parasites were submitted to organic solvents extraction as described in [20] to determine their mucin nature. Extracted material was subjected to Western blot. F1, F2 and F3 refer to the different purification fractions (for details see M&M). D) Neu5Az trans-sialylated trypomastigotes (900x106) were lysed and sialylated proteins pulled-down with anti-FLAG antibodies. Western blots of this material were revealed with anti-TcMUC II antibodies. E-F) Confocal images displaying partial colocalization of anti-FLAG and anti-TcMUC II (E) or anti-αGal (F) labeling at the parasite surface.