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.

Fig. 1.

A chemical approach for modulating Golgi sulfotransferase activity. (A) The domain structure of the carbohydrate sulfotransferases. The enzymes are type II transmembrane proteins with an N-terminal cytosolic tail, single pass transmembrane domain, luminal stem region, and a C-terminal catalytic domain. The tail, transmembrane domain, and stem (abbreviated Loc) are usually sufficient to confer Golgi localization. (B) Small-molecule-mediated domain assembly as a means to control sulfotransferase activity in cells. The Cat and Loc domains of the enzymes are separated and fused to FRB and FKBP, respectively. Rapamycin induces association of FKBP and FRB, thereby localizing the Cat domain in the Golgi where it can encounter its substrates. (C) The structure of sulfoadhesin. Two antibodies used in this study bind to discrete components of sulfoadhesin. G72 recognizes 6-sulfo sLe^x, shown in blue, whereas MECA-79 recognizes a sulfated extension of the lower core 1 branch, shown in green.

Fig. 2.

Initial characterization of the inducible sulfotransferases. (A) Flow cytometry analysis of FucT7-CHO cells transfected with 1Cat-FRB₃ and 1Loc-FKBP. (Left) Cells were analyzed with G72. (Upper Left) Cells were treated with 200 nM rapamycin (Rap) (gray), or they were untreated (white). (Lower Left) Cells were treated with 200 nM rapamycin (Rap) (gray), or they were transfected with full-length GlcNAc6ST-1 and treated with 200 nM rapamycin (white). (Right) Cells were cotransfected with Core1-β3GlcNAcT and analyzed with MECA-79. (Upper Right) Cells were treated with 200 nM rapamycin (Rap) (gray), or they were untreated (white). (Lower Right) Cells were treated with 200 nM rapamycin (Rap) (gray), or they were transfected with full-length GlcNAc6ST-1 and treated with 200 nM rapamycin (white). (B) Flow cytometry analysis of FucT7-CHO cells transfected with 2Cat-FRB₃ and 2Loc-FKBP. Panels are represented as in A, with comparison to full-length GlcNAc6ST-2 in the lower left and right. (C) Mean fluorescence intensities (MFI) from flow cytometry data obtained as in A. Error bars represent the standard deviation of triplicate data points. (D) Mean fluorescence intensities (MFI) from flow cytometry data obtained as in B. Error bars are as in C.(E) Effects of increasing rapamycin on the activity of inducible sulfotransferases. (F) Effect of ascomycin on rapamycin-dependent sulfotransferase activity.

Fig. 3.

Effect of rapamycin (Rap) on Cat domain localization and sulfotransferase activity. (A) FucT7-CHO cells were transfected with the GlcNAc6ST-1 and –2 Cat and Loc domains in the presence or absence of 200 nM rapamycin. The cells were fixed, permeabilized, and stained with an anti-HA antibody followed by an Alexa546-conjugated secondary antibody (red). The cells were also stained with DAPI to highlight the nucleus (blue). (Scale bar = 10 μm.) (B) FucT7-CHO cells were transfected with either 1Cat-FRB₃ and 1Loc-FKBP (denoted 1 in rows 1 and 3) or 2Cat-FRB₃ and 2Loc-FKBP (denoted 2 in rows 2 and 4) in the presence or absence of 200 nM rapamycin. The cells were fixed, permeabilized, and stained with anti-HA antibody and anti-MannII sera, followed by Alexa546-conjugated (against anti-HA) and Alexa647-conjugated secondary antibodies. Panels in the top two rows show single sections of a deconvolved data set with the signal from the HA tag and MannII shown in monochrome in the first and second columns. The third column shows three color overlays with the HA tag in green, MannII in red, and the nuclear stain DAPI in blue. Panels in the bottom two rows are 3D projections containing the maximum pixel intensities of a deconvolved data set. The DAPI-stained nucleus is shown for the purpose of orientation. The color scheme is identical to that in the top two rows. (Scale bar = 5 μm.) (C) FucT7-CHO cells were transfected with 2Cat-FRB₃ and 2Loc-FKBP in the presence (Upper) or absence (Lower) of 200 nM rapamycin. The cells were fixed, permeabilized, and stained with anti-HA, G72, and anti-Giantin, followed by the corresponding secondary antibodies (Alexa546-, Alexa488-, and Alexa647-conjugated secondary antibodies, respectively). (Left) HA signal is shown in red, the G72 signal is shown in green, and the DAPI signal is shown in blue. (Right) The G72 signal is shown in green, the Giantin signal is shown in red, and the DAPI signal is shown in blue. (Scale bar = 10 μm.)

Fig. 4.

Tunable activity of GlcNAc6ST-2 on the secreted glycoprotein GlyCAM-Ig. CHO cells were transfected with GlyCAM-Ig, 2Cat-FRB₃, 2Loc-FKBP, and Core1-β3GlcNAcT. The cells were then incubated with rapamycin for 4 d. The secreted GlyCAM-Ig was captured on Protein A-agarose and analyzed by Western blot probing with MECA-79 (Upper) or anti-human IgG (Lower). Far right lane shows GlyCAM-Ig isolated from CHO cells transfected with full-length GlcNAc6ST-2. Molecular mass marker in both blots equals 64 kDa.

Fig. 5.

Chimeric enzymes comprising Cat and Loc domains of different proteins are functionally assembled by rapamycin (rap). (A) CHO cells were cotransfected with FT7Cat-FRB₃ and one of the following Loc domains: FT7Loc-FKBP (Left), 1Loc-FKBP (Center), or 2Loc-FKBP (Right). (Upper) The cells were treated with 200 nM rapamycin (gray) or untreated (white). (Lower) Comparison of the rapamycin-induced enzyme (gray) with full-length FT7 (white). The cells were stained with mAb HECA-452 and analyzed by flow cytometry. (B) Graphical representation of the mean fluorescence intensities (MFI) from flow cytometry data generated as in A. Error bars represent the standard deviation of triplicate data points. (C) Cells transfected as in A were lysed, and the expression levels of the different Loc domains were compared by Western blot probing with an anti-myc mAb (Upper). The blot was stripped and reprobed with an anti-actin antibody to verify equivalent protein loading (Lower).