A small-molecule switch for Golgi sulfotransferases

Abstract

The study of glycan function is a major frontier in biology that could benefit from small molecules capable of perturbing carbohydrate structures on cells. The widespread role of sulfotransferases in modulating glycan function makes them prime targets for small-molecule modulators. Here, we report a system for conditional activation of Golgi-resident sulfotransferases using a chemical inducer of dimerization. Our approach capitalizes on two features shared by these enzymes: their requirement of Golgi localization for activity on cellular substrates and the modularity of their catalytic and localization domains. Fusion of these domains to the proteins FRB and FKBP enabled their induced assembly by the natural product rapamycin. We applied this strategy to the GlcNAc-6-sulfotransferases GlcNAc6ST-1 and GlcNAc6ST-2, which collaborate in the sulfation of L-selectin ligands. Both the activity and specificity of the inducible enzymes were indistinguishable from their WT counterparts. We further generated rapamycin-inducible chimeric enzymes comprising the localization domain of a sulfotransferase and the catalytic domain of a glycosyltransferase, demonstrating the generality of the system among other Golgi enzymes. The approach provides a means for studying sulfate-dependent processes in cellular systems and, potentially, in vivo.

Imaging the glycome

“…Nevertheless, applications to immunofluorescence microscopy of cultured cells or tissue sections abound. Antibodies have been used for glycan visualization on fixed cells (52), as well as sections from chick cornea (53), bovine mammary gland (54), rabbit appendix (55), and human thymus (56), among others.…”

A Dual Small‐Molecule Rheostat for Precise Control of Protein Concentration in Mammalian Cells

“…Taken together, these data support a model where rapamycin-induced co-localization of the ubiquitin fragments (i.e., UbN and UbC) is not absolutely required for their complementation and GFP release. To test if this is the case, TTShld-GFP expressing HEK cells were treated with the same concentration of doxycycline (0, 50, 1000 ng/mL) and three different concentrations of FK-506 (0, 50, 500 nM), which will bind-to and stabilize FKBP* but not recruit FRB [13,14] . Analysis by western blotting (Figure 2C) showed that GFP was released in a similar dose-dependent manner compared to the cells that were treated with rapamycin (Figure 2B).…”

Chemical intervention in signalling networks: recent advances and applications

“…This provided a powerful method for co-localising membrane-bound signalling proteins by incorporating a small-molecule binding domain; binding to a divalent chemical tool then allowed dimerisation to occur, and a signal to be read out to a reporter gene (Figure 4). This seminal work was later used to create conditional membrane co-localisation systems [42], adapted to heterodimeric systems [43], and continues to be used today for the switching of signalling and other membrane bound processes [44,45].…”