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

Wataru Hakamata, Kazuki Miura, Takako Hirano, Toshiyuki Nishio

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)73-79
Number of pages7
JournalBioorganic and Medicinal Chemistry
Volume23
Issue number1
DOIs
Publication statusPublished - 2015 Jan 1
Externally publishedYes

Keywords

  • A EM emission wavelength
  • Abbreviations 2MeTG 2-methyl
  • BFA brefeldin
  • ER endoplasmic reticulum
  • EX excitation wavelength Man mannose
  • MS mass spectrometry
  • NMR nuclear magnetic resonance
  • PBS phosphate-buffered saline
  • PTM post-translational modifications
  • QMC quinone methide cleavage
  • TBP tributyl phosphine
  • TFMU 4-Trifluoromethylumbelliferone
  • TokyoGreen

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Medicine
  • Molecular Biology
  • Pharmaceutical Science
  • Drug Discovery
  • Clinical Biochemistry
  • Organic Chemistry

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