A validated collection of mouse monoclonal antibodies to human glycosyltransferases functioning in mucin-type O-glycosylation

• Type of Material: Article
• Authors: Catharina Steentoft, University of Copenhagen; Zhang Yang, University of Copenhagen; Shengjun Wang, University of Copenhagen; Tongzhong Ju, Emory University; Malene B. Vester-Christensen, University of Copenhagen; Maria F. Festari, University of Copenhagen; Sarah L. King, University of Copenhagen; Kelley Moremen, University of Georgia; Ida S. B. Larsen, University of Copenhagen; Christoffer K. Goth, University of Copenhagen; Katrine T. Schjoldager, University of Copenhagen; Lars Hansen, University of Copenhagen; Eric P. Bennett, University of Copenhagen; Ulla Mandel, University of Copenhagen; Yoshiki Narimatsu, University of Copenhagen
• Language: English
• Date: 2019-09-01
• Publisher: Oxford University Press Inc.
• Publication Version: Final Published Version
• Copyright Statement: © The Author(s) 2019. Published by Oxford University Press. All rights reserved.
• License: Creative Commons Attribution 4.0 International
• Final Published Version (URL): http://dx.doi.org/10.1093/glycob/cwz041
• Title of Journal or Parent Work: Glycobiology
• Volume: 29
• Issue: 9
• Start Page: 645
• End Page: 656
• Grant/Funding Information: Lundbeck Foundation [R223-2016-563], Danish Research Councils [DFF – 4004-00397B], Læge Sofus Carl Emil Friis og hustru Olga Doris Friis’ Legat, the Danish National Research Foundation [DNRF107] and National Institutes of Health (USA) [P41GM103390, P01GM107012].
• Keywords: western blot; Immunohistochemistry; Expression; immunocytology; Cells; immunohistology; Linked glycosilaiton; Localization; GalNAc-transferase; Biochemistry & Molecular Biology; Life Sciences & Biomedicine; Cloning; Science & Technology; CRISPR Cas9; Glycoproteome
• Research Categories: Biology, Cell; Chemistry, Biochemistry; Health Sciences, Immunology; Biology, Molecular

Abstract

Complex carbohydrates serve a wide range of biological functions in cells and tissues, and their biosynthesis involves more than 200 distinct glycosyltransferases (GTfs) in human cells. The kinetic properties, cellular expression patterns and subcellular topology of the GTfs direct the glycosylation capacity of a cell. Most GTfs are ER or Golgi resident enzymes, and their specific subcellular localization is believed to be distributed in the secretory pathway according to their sequential role in the glycosylation process, although detailed knowledge for individual enzymes is still highly fragmented. Progress in quantitative transcriptome and proteome analyses has greatly advanced our understanding of the cellular expression of this class of enzymes, but availability of appropriate antibodies for in situ monitoring of expression and subcellular topology have generally been limited. We have previously used catalytically active GTfs produced as recombinant truncated secreted proteins in insect cells for generation of mouse monoclonal antibodies (mAbs) to human enzymes primarily involved in mucin-type O-glycosylation. These mAbs can be used to probe subcellular topology of active GTfs in cells and tissues as well as their presence in body fluids. Here, we present several new mAbs to human GTfs and provide a summary of our entire collection of mAbs, available to the community. Moreover, we present validation of specificity for many of our mAbs using human cell lines with CRISPR/Cas9 or zinc finger nuclease (ZFN) knockout and knockin of relevant GTfs.