Glycosyltransferase POMGNT1 deficiency affects N-cadherin-mediated cell-cell adhesion

Defects in protein O -mannosylation lead to severe congenital muscular dystrophies known as α-dystroglycanopathy. A hallmark of these diseases is the loss of the O -mannose-bound matriglycan on α-dystroglycan, which leads to a reduction in cell adhesion to the extracellular matrix. Mutations in protein O -mannose β1,2-N-acetylglucosaminyltransferase 1 (POMGNT1), which is crucial for the elongation of O -mannosyl glycans, are mainly associated with muscle-eye-brain (MEB) disease. In addition to defects in cell-extracellular matrix adhesion, aberrant cell-cell adhesion has occasionally been observed in response to defects in POMGNT1. However, direct molecular mechanisms are largely unknown. We used POMGNT1 knock-out HEK293T cells and fibroblasts from a MEB patient to gain a deeper insight into the molecular changes in POMGNT1 deficiency. A combination of biochemical and molecular biological techniques with proteomics, glycoproteomics and glycomics revealed that a lack of POMGNT1 activity strengthens cell-cell adhesion. We demonstrate that the altered intrinsic adhesion properties are due to an increased abundance of N-cadherin (N-Cdh). In addition, site-specific changes in the N -glycan structures in the extracellular domain of N-Cdh were detected, which positively impact on homotypic interactions. We found that in POMGNT1 deficient cells ERK1/2 and p38 signaling pathways are activated and transcriptional changes that are comparable to the epithelial-mesenchymal transition (EMT) are triggered, defining a possible molecular mechanism underlying the observed phenotype. Our study indicates that changes in cadherin-mediated cell-cell adhesion and other EMT-related processes may contribute to the complex clinical symptoms of MEB or α-dystroglycanopathy in general, and suggests a previously underestimated impact of changes in O -mannosylation on N -glycosylation. ### Competing Interest Statement The authors have declared no competing interest. * α/β-DG : α/β-Dystroglycan E/N-Cdh : E(epithelial)/N(neural)-Cadherin ECM : Extracellular Matrix EMT : Epithelial-Mesenchymal Transition ER : Endoplasmic Reticulum HILIC SPE : Hydrophilic Interaction Liquid Chromatography Solid Phase Extraction HPRT : Hypoxanthine-guanine Phosphoribosyltransferase MEB disease : Muscle-Eye-Brain disease MMPs : Matrix Metallopeptidases qRT-PCR : Quantitative Real-Time PCR RFLP : Restriction Fragment Length Polymorphism RP-LC-ESI-OT MS/MS : Reverse-Phase Liquid Chromatography Coupled Online to Electrospray Ionization Orbitrap Tandem Mass Spectrometer PCA : Principle Component Analysis TALENs : Transcription Activator-Like Effector Nucleases WGA : Wheat Germ Agglutinin xCGE-LIF : Multiplexed Capillary Gel Electrophoresis with Laser Induced Fluorescence Detection