Fig. 5: The second metal site and C-terminal tail influence in vivo O-glycosylation.
a T. gondii cysts in HFF cells probed with GalNAc glyco-epitope-specific anti-CST1 mucin antibody reveal that the glycosylation of the cyst wall is diminished in TxgGalNAc-T3 mutant parasites. GFP expression indicates that the parasites are differentiated into cyst-forming bradyzoites. The lower panels delineate the reduced intensity of cyst wall glycosylation. Quantification of cyst wall glycosylation level by the GalNAc glyco-epitope-specific anti-CST1 mucin antibody. Analysis of variance across all genotypes was performed using a one-way ANOVA, yielding p = 2 × 10−16. *** Further analysis with a pairwise post-hoc analysis Tukey’s HSD test between WT and all other genotypes yielded p = 4.76 × 10−11. The number of observations for genotypes are n = 222, 182, 183, 263, 223, 173, 584, and 190 respectively. The total number of observations was n = 2020. b Immunoblot of TxgGalNAc-T3 mutant parasites expressing a surrogate mucin protein demonstrating the reduction of the degree of glycosylation indicated by a change in their molecular weight. The surrogate mucins were probed with an anti-HA antibody and protein loading was assessed with an anti-GRA1 antibody. Experiments were repeated three times with similar results. c A parallel immunoblot was probed with glyco-epitope-specific antibody to demonstrate the glycosylation of surrogate mucin proteins. High molecular bands bound to glyco-epitope antibody, but not intermediate or low molecular bands. Source data are provided in the Source Data file. The code for image quantification in a is provided as Supplementary Dataset 2.
