Glycosyltransferase POMGNT1 deficiency strengthens N-cadherin-mediated cell–cell adhesion

Abstract

Defects in protein O -mannosylation lead to severe congenital muscular dystrophies collectively known as α-dystroglycanopathy. A hallmark of these diseases is the loss of the O -mannose-bound matriglycan on α-dystroglycan, which reduces 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, have mainly been 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, specific molecular consequences of POMGNT1 deficiency on cell–cell adhesion are largely unknown. We used POMGNT1 knockout HEK293T cells and fibroblasts from an MEB patient to gain deeper insight into the molecular changes in POMGNT1 deficiency. Biochemical and molecular biological techniques combined 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. Moreover, in POMGNT1-deficient cells, ERK1/2 and p38 signaling pathways are activated and transcriptional changes that are comparable with 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 that the impact of changes in O -mannosylation on N -glycosylation has been underestimated.

A Bacterial Mannose Binding Lectin as a Tool for the Enrichment of C- and O-Mannosylated Peptides

“…N-Cadherin is modified by Omannoses and N-glycans on distinct peptides. 40 An Nglycosylation analysis by LC-MS/MS of N-cadherin-derived tryptic peptides revealed only a few N-glycosylated peptides in bound and unbound fractions of the lectin affinity (Table S3). However, peptides with oligomannose-type N-glycans were only identified in the bound ConA and BC2L-A fractions.…”

“…Full-length Mus musculus N-cadherin (CDH1) was cloned in a modified pSecTag B vector containing a C-terminal Strep-and His8-tag as described. 40 Preparation of Lectin Columns. For lectin purification, the BC2L-A plasmid was transformed in Escherichia coli BL21-Gold and cultured in power broth medium with carbenicillin.…”

“…A C-terminally His6-tagged human thrombospondin-1 (THBS1) fragment containing the 3 TSRs (UniProt-P07996 amino acid 277–548) was cloned via Hin dIII/XbaI in pSecTag B (Invitrogen), and a V5-tag was introduced before the His-tag to allow detection by Western blot (Figure S1). Full-length Mus musculus N-cadherin (CDH1) was cloned in a modified pSecTag B vector containing a C-terminal Strep- and His8-tag as described …”

Involvement of abnormal dystroglycan expression and matriglycan levels in cancer pathogenesis

“…Conversely, POMGNT1 silencing decreases cell growth and invasion in human glioblastoma cell lines [114]. In this context, it has been recently found that POMGNT1 knockout in the human HEK293T cell line also leads to a lower proliferation and migration rates, in a fashion associated with loss of celllaminin interaction and increase of cell-cell adhesion mediated by N-cadherin [160]. This was attributable to an upregulation of this protein and its encoding mRNA (CDH2 gene) found together with a quantitatively and qualitatively altered N-cadherin N-glycosylation, possibly related to a mildly-decreased MGAT5 expression, encoding the GnT-Va enzyme.…”

“…This was attributable to an upregulation of this protein and its encoding mRNA (CDH2 gene) found together with a quantitatively and qualitatively altered N-cadherin N-glycosylation, possibly related to a mildly-decreased MGAT5 expression, encoding the GnT-Va enzyme. Also, a sustained activation of the ERK/MAPK pathway was demonstrated, reflected by increased levels of phosphorylated ERK1/2, p38 MAPK and MEK, together with overexpression of a set of EMT-promoting transcription factors, including Snail [160]. These and other molecular events indicative of the occurrence of an EMT-like transition were also evidenced in skin fibroblasts from a MEB patient, harboring a POMGNT1 D179Rfs*11 mutation, this contributing to explain some of his/her clinical DGP symptoms.…”