Mucin sialylation and characterization.
Andrés B. Lantos
Giannina Carlevaro
Beatriz Araoz
Pablo Ruiz Diaz
María de los Milagros Camara
Carlos A. Buscaglia
Mariano Bossi
Hai Yu
Xi Chen
Carolyn R. Bertozzi
Juan Mucci
Oscar Campetella
10.1371/journal.ppat.1005559.g001
https://plos.figshare.com/articles/figure/Mucin_sialylation_and_characterization_/3951852
<p>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 Cu<sup>2+</sup>-free click chemistry to obtain a FLAG or biotin tag ready for detection. B) Western blot of Neu5Az <i>trans</i>-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 <i>trans</i>-sialylated parasites were submitted to organic solvents extraction as described in [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005559#ppat.1005559.ref020" target="_blank">20</a>] 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 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005559#sec010" target="_blank">M&M</a>). D) Neu5Az <i>trans</i>-sialylated trypomastigotes (900x10<sup>6</sup>) 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.</p>
2016-04-08 23:05:59
membrane
trypomastigote
residue
scavenges sialic acid
bioorthogonal chemical reporters
Advanced microscopy techniques
Main sialyl acceptors
cruzi life cycle
protein
TS
parasite surface
Glycobiology Unveils Trypanosoma cruzi Trypomastigote Membrane Physiology Trypanosoma cruzi
sialylated mucins