Oral Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

Isoform-specific functions of polypeptide GalNAc-transferases probed with glycoengineered human skin organoids and mass spectrometry (#138)

Ieva Bagdonaite 1 , Emil MH Pallesen 1 , Zilu Ye 1 , Sergey Y Vakhrushev 1 , Irina N Marinova 1 , Mathias I Nielsen 1 , Signe H Kramer 2 , Stine F Pedersen 2 , Hiren J Joshi 1 , Eric P Bennett 3 , Sally Dabelsteen 3 , Hans H Wandall 1
  1. Copenhagen Center for Glycomics, Department of Cellular and Molecular Medicine, University of Copenhagen, Copenhagen N, Denmark
  2. Cell Biology and Physiology, Department of Science, University of Copenhagen, Copenhagen N, Denmark
  3. Department of Odontology, University of Copenhagen, Copenhagen N, Denmark

Post-translational modifications (PTMs) greatly expand the function and potential for regulation of protein activity, and O-glycosylation is among the most abundant and diverse PTMs. Initiation of O-GalNAc glycosylation is regulated by 20 distinct GalNAc-transferases (GalNAc-Ts), and deficiencies in individual GalNAc-Ts are associated with human disease, causing subtle but distinct phenotypes in model organisms. Here, we generated a set of isogenic keratinocyte cell lines with and without the three dominant and differentially expressed GalNAc-Ts. Through the ability of keratinocytes to form epithelia, we investigated the phenotypic consequences of the loss of individual GalNAc-Ts. We also used isogenic keratinocyte cell lines to probe cellular responses to the ablation of GalNAc-Ts through global transcriptomic, differential glycoproteomic, and differential phosphoproteomic analyses. We demonstrate that loss of individual GalNAc-T isoforms causes distinct epithelial phenotypes through their effect on distinct biological pathways; GalNAc-T1 targets are associated with components of the endomembrane system, GalNAc-T2 targets – with cell-ECM adhesion, and GalNAc-T3 targets – with epithelial differentiation. Thus, GalNAc-T isoforms serve specific roles during human epithelial tissue formation.

  1. Radhakrishnan P, Dabelsteen S, et al. Immature truncated O-glycophenotype of cancer directly induces oncogenic features. (2014) Proc Natl Acad Sci USA 111(39):E4066-75.
  2. Lavrsen, K, et al. De novo expression of human polypeptide N-acetylgalactosaminyltransferase 6 (GalNAc-T6) in colon adenocarcinoma inhibits the differentiation of colonic epithelium. (2018) The Journal of biological chemistry 293, 1298-1314.
  3. Schjoldager KT et al. Deconstruction of O-glycosylation--GalNAc-T isoforms direct distinct subsets of the O-glycoproteome. (2015) EMBO Rep 16: 1713-22.