TY - JOUR
T1 - Differential labeling of glycoproteins with alkynyl fucose analogs
AU - Ma, Chenyu
AU - Takeuchi, Hideyuki
AU - Hao, Huilin
AU - Yonekawa, Chizuko
AU - Nakajima, Kazuki
AU - Nagae, Masamichi
AU - Okajima, Tetsuya
AU - Haltiwanger, Robert S.
AU - Kizuka, Yasuhiko
N1 - Funding Information:
This research was partially supported by the project for utilizing glycans in the development of innovative drug discovery technologies from AMED to YK (grant number 16809274), a Leading Initiative for Excellent Young Researchers (LEADER) project from JSPS to YK (grant number 16811705), Grant-in-Aid for Scientific Research (B) from JSPS to YK (grant number JP20H03207), HT (JP19H03176), and TO (JP19H03416), Grant-in-Aid for Fund for the Promotion of Joint International Research (Fostering Joint International Research (B)) from JSPS to HT (JP19KK0195), grants from the Takeda Science Foundation to YK and HT, a grant from the National Institutes of General Medical Sciences to RSH (GM061126), and a grant from the Tokyo Biochemical Research Foundation to YK. Acknowledgments: We would like to thank Hidenori Tanaka (Gifu University) for drawing chemical structures. We would also like to thank Kentaro Taki at the Division for Medical Research Engineering, Nagoya University Graduate School of Medicine, for the technical help for the mass spectral analysis of products from POFUTs? reactions.
Publisher Copyright:
© 2020 by the authors. Licensee MDPI, Basel, Switzerland.
PY - 2020/9/1
Y1 - 2020/9/1
N2 - Fucosylated glycans critically regulate the physiological functions of proteins and cells. Alterations in levels of fucosylated glycans are associated with various diseases. For detection and functional modulation of fucosylated glycans, chemical biology approaches using fucose (Fuc) analogs are useful. However, little is known about how efficiently each unnatural Fuc analog is utilized by enzymes in the biosynthetic pathway of fucosylated glycans. We show here that three clickable Fuc analogs with similar but distinct structures labeled cellular glycans with different efficiency and protein specificity. For instance, 6-alkynyl (Alk)-Fuc modified O-Fuc glycans much more efficiently than 7-Alk-Fuc. The level of GDP-6-Alk-Fuc produced in cells was also higher than that of GDP-7-Alk-Fuc. Comprehensive in vitro fucosyltransferase assays revealed that 7-Alk-Fuc is commonly tolerated by most fucosyltransferases. Surprisingly, both protein O-fucosyltransferases (POFUTs) could transfer all Fuc analogs in vitro, likely because POFUT structures have a larger space around their Fuc binding sites. These findings demonstrate that labeling and detection of fucosylated glycans with Fuc analogs depend on multiple cellular steps, including conversion to GDP form, transport into the ER or Golgi, and utilization by each fucosyltransferase, providing insights into design of novel sugar analogs for specific detection of target glycans or inhibition of their functions.
AB - Fucosylated glycans critically regulate the physiological functions of proteins and cells. Alterations in levels of fucosylated glycans are associated with various diseases. For detection and functional modulation of fucosylated glycans, chemical biology approaches using fucose (Fuc) analogs are useful. However, little is known about how efficiently each unnatural Fuc analog is utilized by enzymes in the biosynthetic pathway of fucosylated glycans. We show here that three clickable Fuc analogs with similar but distinct structures labeled cellular glycans with different efficiency and protein specificity. For instance, 6-alkynyl (Alk)-Fuc modified O-Fuc glycans much more efficiently than 7-Alk-Fuc. The level of GDP-6-Alk-Fuc produced in cells was also higher than that of GDP-7-Alk-Fuc. Comprehensive in vitro fucosyltransferase assays revealed that 7-Alk-Fuc is commonly tolerated by most fucosyltransferases. Surprisingly, both protein O-fucosyltransferases (POFUTs) could transfer all Fuc analogs in vitro, likely because POFUT structures have a larger space around their Fuc binding sites. These findings demonstrate that labeling and detection of fucosylated glycans with Fuc analogs depend on multiple cellular steps, including conversion to GDP form, transport into the ER or Golgi, and utilization by each fucosyltransferase, providing insights into design of novel sugar analogs for specific detection of target glycans or inhibition of their functions.
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U2 - 10.3390/ijms21176007
DO - 10.3390/ijms21176007
M3 - Article
C2 - 32825463
AN - SCOPUS:85089687641
VL - 21
SP - 1
EP - 18
JO - International Journal of Molecular Sciences
JF - International Journal of Molecular Sciences
SN - 1661-6596
IS - 17
M1 - 6007
ER -