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O-mannose glycosylations influence E-cadherin protein interactions

ABSTRACT: Cadherins are plasma membrane adhesion molecules that play critical roles in maintaining cell-cell adhesion and modulating cell signaling during development. Their functions are mediated by extracellular cadherin (EC) domains, which facilitate adhesive interactions and enable the formation of cis- and trans assemblies at adherens junctions and desmosomes. The EC domains adopt a characteristic immunoglobulin-like fold composed of seven beta-strands (A-G), and are further modified by N-linked and O-linked glycosylation, including alpha-linked mannose monosaccharides (O-Man) on conserved serine and threonine residues of B- and G-strands. EC domain O-Man glycosylation is catalyzed by TMTC enzymes: TMTC3 catalyzes modifications to the G-strands, whereas TMTC2 targets the B-strands. Given the site-specific deposition of O-Man glycans by dedicated enzymes and the central role of EC domains in cadherins functions, we hypothesized that these PTMs may fine-tune cellular adhesion strength and otherwise contribute to diverse physical interactions that involve cadherins. To test these hypotheses, we assayed for changes in protein-protein interactions formed with epithelial (E)-cadherin in model cells where O-Man PTMs were genetically ablated. Herein, we report O-Man-dependent, E-cadherin (CDH1) protein interactions, revealed by affinity proteomics; we orthogonally validate an altered association between CDH1 and CDH3 (P-cadherin), and demonstrate loss-of-function consequences of O-Man ablation on specific positions, highlighting the importance of these PTMs in cadherin-mediated adhesion. These findings provide new insights into how post-translational modifications regulate cadherin-dependent adhesion complexes.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: John LaCava

PROVIDER: MSV000097524 | MassIVE | Fri Apr 04 02:40:00 BST 2025

SECONDARY ACCESSION(S): PXD062594

REPOSITORIES: MassIVE

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Project description:This project was done in collaboration with Dr. Richard Cummings to examine sialylation of CD44v6 and its role in clearance of neutrophils from inflamed intestinal epithelium. Specifically, we have employed a functional approach using membrane preparations from interferon gamma-stimulated intestinal epithelial cells to generate a monoclonal antibody, designated GM35, which blocks neutrophil transepithelial migration through the promotion of neutrophil adhesion at the apical surface of the intestinal epithelium. Protein biochemistry, sequencing, confocal microscopic analysis, and immunoprecipitation studies all identify the protein ligand for this antibody as CD44v6. However, selective inhibition of O- or N-linked glycosylation reveal that the antibody is specific for an O-linked glycotope and glycoarray analysis of the GM35 antibody by Core H of the Consortium for Functional Glycomics reveal that this antibody binds with high affinity and specificity to a carbohydrate epitope consistent in structure with sLeA. Inhibition of O-linked glycosylation attenuated both GM35 binding and its functional effects as did specific cleavage of sialic acid residues from the cell surface, using neuraminidase, although the functional effects of cleavage were smaller and harder to assess. CD44v6 has previously been studied as a marker of inflammation in Inflammatory Bowel Disease, and GM35 staining is clearly upregulated by interferon gamma in T84 and HT29 intestinal epithelial cells. The specific enzymes governing the sialylation of CD44v6 in intestinal epithelium have yet to be determined. Analysis of a panel of intestinal epithelial cells including T84, HT29, and Caco-2 cells revealed that cell-type specific regulation of both splicing and glycosylation is essential for the regulation of the expression of the GM35 epitope. And, forcible expression of CD44 in Caco-2 cells, which are negative for CD44v6 at baseline, results only in upregulation of previously expressed isoforms of CD44, but not in GM35 expression. Based on western blot and immunofluorescent staining, GM35 expression is greatest in interferon gamma-treated T84 cells. Thus, we analyzed RNA samples for the expression of glycosylation specific genes in interferon gamma-stimulated T84 cells relative to that of both non-stimulated T84 cells and GM35-negative Caco-2 cells in order to identify glycosylation-specific targets contributing to the interferon gamma-dependent upregulation of sialylated CD44v6 in intestinal epithelial cells.

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O-mannose glycosylations influence E-cadherin protein interactions

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