Differential Labeling of Glycoproteins with Alkynyl Fucose Analogs.

Chenyu Ma,Tetsuya Okajima,Yasuhiko Kizuka,Masamichi Nagae,Chizuko Yonekawa,Hideyuki Takeuchi,Huilin Hao,Kazuki Nakajima,Robert S. Haltiwanger

doi:10.3390/ijms21176007

Abstract

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.

Highlights

Glycosylation is the most common form of post-translational modifications in mammals, providing vast functional diversity to proteins [1,2]
We reported that 7-Alk-Fuc labeled cellular glycans with higher efficiency than 6-Alk-Fuc in mouse embryonic fibroblast (MEF) and HEK293 cells [42]
To examine whether this finding is commonly observed in various cell types, we first treated several cells with peracetylated 6-Alk-Fuc or 7-Alk-Fuc, and the labeled glycans on cellular proteins were detected by click chemistry (Figure 2A)

Summary

Introduction

Glycosylation is the most common form of post-translational modifications in mammals, providing vast functional diversity to proteins [1,2]. Loss of FUT8 in mice results in severe phenotypes, including early death [10], emphysema [10], schizophrenia-like behavior [11], and immune dysfunctions [12,13], demonstrating the physiological importance of core Fuc for various glycoproteins. The emergence of cancer-specific Le antigens is clinically used as a cancer biomarker, such as sialyl Lea (CA19-9) [22], demonstrating the physiological and pathological significance of Le type Fuc residues. Genetic deletion of Pofut in mice results in embryonic lethality with gastrulation defects mainly due to the loss of secretion of ADAMTS9 matrix protease [30,31] These studies underscore the biological significance of fucosylated glycans, increasing the need for novel methods to detect and modulate specific fucosylated glycans

Methods

Results

Conclusion