Journal of the American Chemical Society
Page 8 of 9
is unnecessary and does not lead to more efficient labeling in cells
1
2
3
4
(Supplementary Figure S8). Accordingly, we expect that this
approach will prove useful in a wide range of cells to explore the
physiological roles and regulation of O-GlcNAc in different
cellular models.
REFERENCES
(1) Torres, C. R.; Hart, G. W., J Biol Chem 1984, 259, 3308-17.
(2) Zachara, N.; Akimoto, Y.; Hart, G. W., The O-GlcNAc Modification.
In Essentials of Glycobiology, 3rd ed.; Varki, A.; Cummings, R. D.; Esko,
J. D.; Stanley, P.; Hart, G. W.; Aebi, M.; Darvill, A. G.; Kinoshita, T.;
Packer, N. H.; Prestegard, J. H.; Schnaar, R. L.; Seeberger, P. H., Eds.
Cold Spring Harbor (NY), 2015.
(3) Yang, X.; Qian, K., Nat Rev Mol Cell Biol 2017, 18, 452-465.
(4) Hart, G. W.; Slawson, C.; Ramirez-Correa, G.; Lagerlof, O., Annu.
Rev. Biochem. 2011, 80, 825-858.
(5) Hardivillé, S.; Hart, G. W., Cell Metab. 2014, 20, 208-213.
(6) Lagerlof, O.; Slocomb, J. E.; Hong, I.; Aponte, Y.; Blackshaw, S.;
Hart, G. W.; Huganir, R. L., Science 2016, 351, 1293-6.
(7) Groves, J. A.; Lee, A.; Yildirir, G.; Zachara, N. E., Cell Stress
Chaperones 2013, 18, 535-58.
(8) Zachara, N. E.; O'Donnell, N.; Cheung, W. D.; Mercer, J. J.; Marth, J.
D.; Hart, G. W., J. Biol. Chem. 2004, 279, 30133-30142.
(9) Ferrer, C. M.; Sodi, V. L.; Reginato, M. J., J. Mol. Biol. 2016, 428,
3282-3294.
(10) Hanover, J. A.; Chen, W.; Bond, M. R., J Bioenerg Biomembr 2018,
50, 155-173.
(11) Wright, J. N.; Collins, H. E.; Wende, A. R.; Chatham, J. C., Biochem
Soc Trans 2017, 45, 545-553.
(12) Dassanayaka, S.; Jones, S. P., Pharmacol Ther 2014, 142, 62-71.
(13) Zhu, Y.; Shan, X.; Yuzwa, S. A.; Vocadlo, D. J., J. Biol. Chem.
2014, 289, 34472-34481.
(14) Gong, C. X.; Liu, F.; Iqbal, K., Alzheimers Dement 2016, 12, 1078-
1089.
(15) Haltiwanger, R. S.; Blomberg, M. A.; Hart, G. W., J. Biol. Chem.
1992, 267, 9005-9013.
(16) Kreppel, L. K.; Blomberg, M. A.; Hart, G. W., J Biol Chem 1997,
272, 9308-15.
(17) Dong, D.; Hart, G. W., J. Biol. Chem. 1994, 269, 19321-19330.
(18) Gao, Y.; Wells, L.; Comer, F. I.; Parker, G. J.; Hart, G. W., J. Biol.
Chem. 2001, 276, 9838-9845.
(19) Levine, Z. G.; Fan, C.; Melicher, M. S.; Orman, M.; Benjamin, T.;
Walker, S., J Am Chem Soc 2018, 140, 3510-3513.
(20) Rafie, K.; Raimi, O.; Ferenbach, A. T.; Borodkin, V. S.; Kapuria, V.;
van Aalten, D. M. F., Open Biol 2017, 7.
(21) Levine, Z. G.; Walker, S., Annu Rev Biochem 2016, 85, 631-57.
(22) Shen, D. L.; Gloster, T. M.; Yuzwa, S. A.; Vocadlo, D. J., J. Biol.
Chem. 2012, 287, 15395-15408.
(23) Cecioni, S.; Vocadlo, D. J., Curr. Opin. Chem. Biol. 2013, 17, 719-
728.
(24) Worth, M.; Li, H.; Jiang, J., ACS Chem Biol 2017, 12, 326-335.
(25) Hu, C.-W.; Worth, M.; Fan, D.; Li, B.; Li, H.; Lu, L.; Zhong, X.;
Lin, Z.; Wei, L.; Ge, Y., Nat. Chem. Biol. 2017, 13, 1267.
(26) Mayer, A.; Gloster, T. M.; Chou, W. K.; Vocadlo, D. J.; Tanner, M.
E., Bioorg. Med. Chem. Lett. 2011, 21, 1199-1201.
(27) Macauley, M. S.; Chan, J.; Zandberg, W. F.; He, Y.; Whitworth, G.
E.; Stubbs, K. A.; Yuzwa, S. A.; Bennet, A. J.; Varki, A.; Davies, G. J.;
Vocadlo, D. J., J Biol Chem 2012, 287, 28882-97.
Though we find that OGT is clearly the principle transferase
mediating the labeling of cells with GlcN-NBD, it is possible that
other transferases may tolerate this unnatural donor sugar, though
given our observations this would likely be at a lower level of
efficiency than OGT-catalyzed transfer. An obvious limitation to
this one-step approach, shared with two-step metabolic labeling
strategies, is that the kinetics of OGT-catalyzed transfer of these
unnatural GlcNAc analogues do not match the kinetics of transfer
of GlcNAc itself. Nevertheless, such one step metabolic in cell
labeling presents the possibility of performing a range of
experiments.
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
One can envision for example, generating additional probes that
incorporate photophysically distinct fluorochromes that could
enable two colour pulse-chase experiments. Additionally, we
expect that engineering these metabolic precursors should permit
delivery of other moieties of interest, such as biotin, which could
enable simple proteomic strategies to studying O-GlcNAcylation.
Perhaps most notably, this one step strategy may be used in
conjunction with approaches described recently in which
fluorescence lifetime imaging (FLIM) to monitor the extent of
modification of specific proteins of interest directly within cells.30,
38
These FLIM studies made use of two-step chemoselective
ligation chemistry, which does not permit continuous imaging of
protein modification in cells. Accordingly, this one-step metabolic
feeding strategy, when used in conjunction with genetically
engineered proteins of interest fused to suitable fluorescent
proteins, could permit monitoring the spatiotemporal action of
OGT activity on specific recombinant target proteins of interest
within live cells in real time using FLIM.
ASSOCIATED-CONTENT
Supporting figures, Experimental methods, NMR characterization,
and kinetic data figures. This material is available free of charge
AUTHOR INFORMATION
Corresponding Author
*E-mail: dvocadlo@sfu.ca
ORCID:
David J. Vocadlo: 0000-0001-6897-5558
Hong Yee Tan: 0000-0003-3530-9350
Notes:
(28) Vocadlo, D. J.; Hang, H. C.; Kim, E.-J.; Hanover, J. A.; Bertozzi, C.
R., Proc. Natl. Acad. Sci. 2003, 100, 9116-9121.
(29) Zaro, B. W.; Yang, Y.-Y.; Hang, H. C.; Pratt, M. R., Proc. Natl.
Acad. Sci. U S A 2011, 108, 8146-8151.
(30) Doll, F.; Buntz, A.; Späte, A. K.; Schart, V. F.; Timper, A.;
Schrimpf, W.; Hauck, C. R.; Zumbusch, A.; Wittmann, V., Angew. Chem.
Int. Ed. 2016, 55, 2262-2266.
(31) Bateman, L. A.; Zaro, B. W.; Chuh, K. N.; Pratt, M. R., Chem.
Commun. 2013, 49, 4328-4330.
(32) Chuh, K. N.; Zaro, B. W.; Piller, F.; Piller, V. r.; Pratt, M. R., J. Am.
Chem. Soc. 2014, 136, 12283-12295.
(33) Chuh, K. N.; Batt, A. R.; Zaro, B. W.; Darabedian, N.; Marotta, N.
P.; Brennan, C. K.; Amirhekmat, A.; Pratt, M. R., J. Am. Chem. Soc. 2017,
139, 7872-7885.
(34) Li, J.; Wang, J.; Wen, L.; Zhu, H.; Li, S.; Huang, K.; Jiang, K.; Li,
X.; Ma, C.; Qu, J.; Parameswaran, A.; Song, J.; Zhao, W.; Wang, P. G.,
ACS Chem Biol 2016, 11, 3002-3006.
The authors declare no competing financial interest.
ACKNOWLEDGEMENTS
Financial support through a Discovery grant from the Natural
Sciences and Engineering Research (D.V., NSERC, RGPIN/-2015-
05426) and Project grant from the Canadian Institutes of Health
Research (D.V., CIHR PJT-148732) is gratefully acknowledged.
H.Y.T. was supported by the NSERC CREATE program ChemNet.
The authors thank J. Kohler (UT Southwestern) for the AGX
mutant cell line and N. Zachara (Johns Hopkins) for the inducible
OGT KO MEF cells. D.V. acknowledges the kind support of the
Canada Research Chairs program for a Tier I Canada Research
Chair in Chemical Glycobiology and NSERC for support as an
E.W.R. Steacie Memorial Fellow.
ACS Paragon Plus Environment