TY - JOUR
T1 - Identification of a novel glycan processing enzyme with exo-acting β-allosidase activity in the Golgi apparatus using a new platform for the synthesis of fluorescent substrates
AU - Hakamata, Wataru
AU - Miura, Kazuki
AU - Hirano, Takako
AU - Nishio, Toshiyuki
N1 - Funding Information:
The authors wish to express their sincere thanks to Mrs. T. Kiuchi of the General Research Institute of the College of Bioresource Sciences of Nihon University for performing FABMS measurements and Mrs. A. Sato of A-Rabbit-Science Japan Co., Ltd for performing the elemental analyses. This work was partly supported by JSPS KAKENHI Grant Number 26460157 .
Publisher Copyright:
© 2014 Elsevier Ltd. All rights reserved.
PY - 2015/1/1
Y1 - 2015/1/1
N2 - The majority of eukaryotic proteins undergo post-translational modifications (PTMs) involving the attachment of complex glycans, predominantly through N-glycosylation and O-glycosylation. PTMs play important roles in virtually all cellular processes, and aberrant regulation of protein glycosylation and glycan processing has been implicated in various diseases. However, glycan processing on proteins in various cellular contexts has not been visualized. We had previously developed a quinone methide cleavage (QMC) platform for enhanced substrate design. This platform was applied here to screen for novel glycan-processing enzymes. We designed and synthesized fluorescent substrates with β-allopyranoside residues using the QMC platform. When applied in cell-based assays, the fluorescent substrates allowed rapid and clear visualization of β-allosidase activity in the Golgi apparatus of human cultured cells. The QMC platform will likely find broad applications in visualizing the activities of glycan processing enzymes in living cells and in studying PTMs.
AB - The majority of eukaryotic proteins undergo post-translational modifications (PTMs) involving the attachment of complex glycans, predominantly through N-glycosylation and O-glycosylation. PTMs play important roles in virtually all cellular processes, and aberrant regulation of protein glycosylation and glycan processing has been implicated in various diseases. However, glycan processing on proteins in various cellular contexts has not been visualized. We had previously developed a quinone methide cleavage (QMC) platform for enhanced substrate design. This platform was applied here to screen for novel glycan-processing enzymes. We designed and synthesized fluorescent substrates with β-allopyranoside residues using the QMC platform. When applied in cell-based assays, the fluorescent substrates allowed rapid and clear visualization of β-allosidase activity in the Golgi apparatus of human cultured cells. The QMC platform will likely find broad applications in visualizing the activities of glycan processing enzymes in living cells and in studying PTMs.
KW - A EM emission wavelength
KW - Abbreviations 2MeTG 2-methyl
KW - BFA brefeldin
KW - ER endoplasmic reticulum
KW - EX excitation wavelength Man mannose
KW - MS mass spectrometry
KW - NMR nuclear magnetic resonance
KW - PBS phosphate-buffered saline
KW - PTM post-translational modifications
KW - QMC quinone methide cleavage
KW - TBP tributyl phosphine
KW - TFMU 4-Trifluoromethylumbelliferone
KW - TokyoGreen
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U2 - 10.1016/j.bmc.2014.11.023
DO - 10.1016/j.bmc.2014.11.023
M3 - Article
C2 - 25497961
AN - SCOPUS:84918820034
SN - 0968-0896
VL - 23
SP - 73
EP - 79
JO - Bioorganic and Medicinal Chemistry
JF - Bioorganic and Medicinal Chemistry
IS - 1
ER -
