title: Development of isoform-specific sensors of polypeptide GalNAc-transferase activity.
abstract: Humans express up to 20 isoforms of GalNAc-transferase (herein T1-T20) that localize to the 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. Here we describe 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 vitro work. 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.
authors.affiliation: Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213 and.
authors.forename: Lina
authors.initials: L
authors.lastname: Song
authors.affiliation: Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213 and.
authors.forename: 丽娜
authors.initials: 丽
authors.lastname: 宋
authors.affiliation: Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213 and.
authors.forename: Collin
authors.initials: C
authors.lastname: Bachert
authors.affiliation: Departments of Cellular and Molecular Medicine and School of Dentistry, Copenhagen Center for Glycomics, University of Copenhagen, DK-2200 Copenhagen, Denmark.
authors.forename: Katrine T
authors.initials: KT
authors.lastname: Schjoldager
authors.affiliation: Departments of Cellular and Molecular Medicine and School of Dentistry, Copenhagen Center for Glycomics, University of Copenhagen, DK-2200 Copenhagen, Denmark.
authors.forename: Henrik
authors.initials: H
authors.lastname: Clausen
authors.affiliation: Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213 and. Electronic address: linstedt@cmu.edu.
authors.forename: Adam D
authors.initials: AD
authors.lastname: Linstedt
mesh_terms: D000595:Amino Acid Sequence; D000091303:Angiopoietin-Like Protein 3; D000075802:Angiopoietin-like Proteins; D042682:Angiopoietins / Q000737:chemistry; D015374:Biosensing Techniques; D004791:Enzyme Inhibitors / Q000494:pharmacology; D000089703:Fibroblast Growth Factor-23; D005346:Fibroblast Growth Factors / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000502:physiology; D008856:Microscopy, Fluorescence; D017350:N-Acetylgalactosaminyltransferases / Q000037:antagonists & inhibitors / Q000502:physiology*; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D011499:Protein Processing, Post-Translational
chemical_list: C479896:ANGPTL3 protein, human; D000091303:Angiopoietin-Like Protein 3; D000075802:Angiopoietin-like Proteins; D042682:Angiopoietins; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D007527:Isoenzymes; D010446:Peptide Fragments; D005346:Fibroblast Growth Factors; D000089703:Fibroblast Growth Factor-23; D017350:N-Acetylgalactosaminyltransferases
keywords: Biosensor; Fluorescence; Glycosylation; Glycosyltransferase; Golgi; Protein Processing