Development of isoform-specific sensors of polypeptide GalNAc-transferase activity

Lina Song, Collin Bachert, Katrine T. Schjoldager, Henrik Clausen, Adam D. Linstedt

Research output: Contribution to journalArticlepeer-review

12 Scopus citations

Abstract

Humans express up to 20 isoforms of GalNAc-transferase (herein T1-T20) that localizetothe Golgi apparatus and initiate O-glycosylation. Regulation of this enzyme family affects a vast array of proteins transiting the secretory pathway and diseases arise upon misregulation of specific isoforms. Surprisingly, molecular probes to monitor GalNAc-transferase activity are lacking and there exist no effective global or isoform-specific inhibitors. Herewedescribe the development of T2- and T3-isoform specific fluorescence sensors that traffic in the secretory pathway. Each sensor yielded little signal when glycosylated but was strongly activated in the absence of its glycosylation. Specificity of each sensor was assessed in HEK cells with either the T2 or T3 enzymes deleted. Although the sensors are based on specific substrates of the T2 and T3 enzymes, elements in or near the enzyme recognition sequence influenced their activity and required modification, which we carried out based on previous in vitrowork. Significantly, the modified T2 and T3 sensors were activated only in cells lacking their corresponding isozymes. Thus, we have developed T2- and T3-specific sensors that will be valuable in both the study of GalNAc-transferase regulation and in high-throughput screening for potential therapeutic regulators of specific GalNAc-transferases.

Original languageEnglish (US)
Pages (from-to)30556-30566
Number of pages11
JournalJournal of Biological Chemistry
Volume289
Issue number44
DOIs
StatePublished - Oct 31 2014
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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