O‐glycan initiation directs distinct biological pathways and controls epithelial differentiation

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.

Genetic glycoengineering in mammalian cells

“…However, targeting isoenzymes also present unique opportunities to uncover their nonredundant functions. For isoenzymes that function in pathway specific steps, such as the many polypeptide GalNAc-transferases (GALNTs), selective KO/KI of individual isoenzyme genes ( GALNT1-20 ) in cell models was very useful to dissect non-redundant functions using differential O-glycoproteomics ( 145 , 146 , 147 ). This strategy provides deeper and more unbiased insights into substrates for GALNTs compared with in vitro enzyme assays with short peptides and has revealed important isoform-specific targets such as the O-glycosylation of the ligand-binding region of the low density lipoprotein receptor-related receptors directed exclusively by GALNT11 ( 148 , 149 , 150 ).…”

“…Studies in these isogenic cell model systems further provide opportunities to apply wider multiomics approaches to explore changes in the transcriptome and, e.g. , phosphoproteome for discovery and dissection of mechanism ( 147 , 206 ).

Figure 4 Dissecting glycan functions using glycoengineered cell lines and organotypic tissue models.

…”

“…Loss of complex N-linked glycans (KO MGAT1 ) affects transport and secretion of select proteins, loss of elongated glycosphingolipids (KO B4GALT5 ) affects tyrosine kinase regulation and produces barrier defects, and loss of elongated O-glycans (KO C1GALT1 ) affects differentiation and cell–cell interactions ( 103 ). Importantly, global proteomics and genomics can be integrated in the workflow to identify the molecular pathways affected by the loss/gain of individual glycoconjugates ( 147 ). …”

“…Similarly, the characteristic expression of aberrant truncated O-glycans (Tn [GalNAcα1-O-Ser/Thr] and STn [Neu5Acα2-6GalNAcα1-O-Ser/Thr] in cancer was replicated in human keratinocyte cell (HaCaT) human skin models by KO of COSMC (private chaperone for the core1 synthase C1GALT1 that controls elongation of O-glycans) and used to demonstrate that the truncation of O-glycans induced oncogenic features including hyperproliferation and invasive growth (206). Another human 3D skin model (N/TERT-1) was used to address contributions of elaborated glycans on distinct types of glycoconjugates (glycolipids, N-glycans, O-GalNAc, O-Fuc, O-Glc) by KO targeting of earliest core extension steps (103), as well as probe nonredundant functions of GALNTs isoenzymes (147). Studies in these isogenic cell model systems further provide opportunities to apply wider multiomics approaches to explore changes in the Libraries of such isogenic cell lines constitute the cell-based glycan arrays (34), and sublibraries can be designed to dissect select glycosylation steps or glycosylation pathways by combinatorial engineering of GTs (and other genes such as sulfotransferase genes modifying glycans).…”

“…This is thus different from studies with traditional glycan arrays that directly report glycan features and structures involved in binding to defined glycans. transcriptome and, e.g., phosphoproteome for discovery and dissection of mechanism (147,206).…”