O-glycan initiation directs distinct biological pathways and controls epithelial differentiation.

Ieva Bagdonaite,Irina N Marinova,Signe H Kramer,Hiren J Joshi,Emil Mh Pallesen,Mathias I Nielsen,Hans H Wandall,Sergey Y Vakhrushev,Stine F Pedersen,Sally Dabelsteen,Zilu Ye,Eric P Bennett

doi:10.15252/embr.201948885

Ieva Bagdonaite, Irina N Marinova + Show 10 more

Open Access

https://doi.org/10.15252/embr.201948885

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Journal: EMBO Reports	Publication Date: Apr 23, 2020
Citations: 38	License type: CC BY 4.0

Affiliation: University of Copenhagen

Abstract

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 generate a set of isogenic keratinocyte cell lines lacking either of the three dominant and differentially expressed GalNAc‐Ts. Through the ability of keratinocytes to form epithelia, we investigate the phenotypic consequences of the loss of individual GalNAc‐Ts. Moreover, we probe the cellular responses 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 specific 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.

Highlights

Glycosylation is an abundant post-translational modification of both intracellular and extracellular proteins [1]
The model provides a strong tool to analyze the phenotypic consequences of losing individual GalNAc-Ts and the effects on defined biological functions, including proliferation, differentiation, cell–cell interactions, cell–matrix interactions, and signaling
Immunocytochemistry showed the localization of GalNAc-T1, GalNAc-T2, and GalNAc-T3; human skin and HaCaT 3D models expressed GalNAcTs in a similar expression pattern, with GalNAc-T2 primarily expressed in basal cells and broader expression of GalNAc-T1 and GalNAc-T3 in all epithelial layers (Fig 1D)

Summary

Introduction

Glycosylation is an abundant post-translational modification of both intracellular and extracellular proteins [1]. We characterized the GalNAc-T repertoire in the HaCaT cell line by RNA sequencing and immunocytochemistry, demonstrating that the expression profiles of GALNT genes are comparable to human skin (Fig 1C and D). Organotypic cultures generated from wild-type (WT) HaCaT keratinocytes had multiple layers of differentiating cells, with proliferation restricted to the single basal layer of K10-negative cells, though with low levels of terminal keratinization as described previously (Fig 1E).

Results

Conclusion