Protein O-mannosylation is crucial for E-cadherin-mediated cell adhesion

Abstract

In recent years protein O-mannosylation has become a focus of attention as a pathomechanism underlying severe congenital muscular dystrophies associated with neuronal migration defects. A key feature of these disorders is the lack of O-mannosyl glycans on α-dystroglycan, resulting in abnormal basement membrane formation. Additional functions of O-mannosylation are still largely unknown. Here, we identify the essential cell-cell adhesion glycoprotein epithelial (E)-cadherin as an O-mannosylated protein and establish a functional link between O-mannosyl glycans and cadherin-mediated cell-cell adhesion. By genetically and pharmacologically blocking protein O-mannosyltransferases, we found that this posttranslational modification is essential for preimplantation development of the mouse embryo. O-mannosylation-deficient embryos failed to proceed from the morula to the blastocyst stage because of defects in the molecular architecture of cell-cell contact sites, including the adherens and tight junctions. Using mass spectrometry, we demonstrate that O-mannosyl glycans are present on E-cadherin, the major cell-adhesion molecule of blastomeres, and present evidence that this modification is generally conserved in cadherins. Further, the use of newly raised antibodies specific for an O-mannosyl-conjugated epitope revealed that these glycans are present on early mouse embryos. Finally, our cell-aggregation assays demonstrated that O-mannosyl glycans are crucial for cadherin-based cell adhesion. Our results redefine the significance of O-mannosylation in humans and other mammals, showing the immense impact of cadherins on normal as well as pathogenic cell behavior.

Keywords: O-glycans; POMT1; POMT2; mouse preimplantation development; protein-glycosylation.

Fig. 1.

Loss of O-mannosyl glycan biosynthesis in mouse embryos. (A) Schematic representation of the Pomt2 genomic locus, targeting construct, and mutant Pomt2 allele after homologous recombination. Arrowheads indicate PCR primers. NEO, neomycin resistance gene; TK, Herpes simplex virus thymidine kinase gene. (B) Southern blot analysis of EcoRI/AscI-digested genomic DNA from targeted ES cells. Pomt2 WT (5.6 kb) and mutant (1.7 kb) alleles were detected. (C) PCR-based genotyping of preimplantation embryos. PCR products of Pomt2 WT (+/+), heterozygous (+/−), and homozygous (−/−) embryos are shown. (D and E) Genotypes and developmental stages/states of embryos (D) and in vitro-cultured embryos (E) derived from heterozygous Pomt2^+/− intercrosses. (F) Relative levels of the Pomt1 and Pomt2 mRNAs during early development, as determined by quantitative RT-PCR and normalized to Ppia. Maternal Pomt1 and Pomt2 transcripts were present at high levels in oocytes but were degraded in zygotes and two-cell embryos. (G) Effect of increasing levels of R3A-5a on in vitro activity of POMT. Mouse-liver membranes were used as the source of enzyme. Reactions were supplemented with DMSO or 25 µM, 50 µM, 100 µM, 200 µM, and 400 µM R3A-5a. Mean values of three independent experiments are shown. (H) Western blot of α-DG isolated from MDCK cells cultured in the presence of 12.5 µM and 50 µM R3A-5a. The VIA4-1 monoclonal antibody was used to determine the O-mannosylation state of α-DG. β-DG levels also were assessed to confirm that protein expression and loading were equal across samples. (I) Developmental progress of WT embryos cultivated in the presence of 50 µM of the inhibitor R3A-5a.

Fig. 2.

Occurrence of O-mannosyl glycans in preimplantation embryos. (A–C) Characterization of anti–T[α1-Man] antibodies. In A and B, Western blots were probed with anti–T[α1-Man] or antibodies directed against the protein core of α-DG (core αDG) as indicated. (A) α-DG–enriched glycoprotein fractions were treated with glycosidases as indicated. N-linked glycans were removed using PNGase F, and sialylated core 1 and core 3 O-linked oligosaccharides were removed by combined treatment with sialidase A and O-glucanase. Further trimming of the O-mannosyl glycan core structure Galβ1–4GlcNAcβ1–2Man-Ser/Thr was achieved by treatment with β-galactosidase and N-acetyl-β-glucosaminidase. For further details, see Fig. S3A. (B) α-DG–enriched glycoprotein fractions were treated with nonaqueous TFMS to remove attached glycans fully. Identical blots were probed. (C) Immunofluorescence staining of skeletal muscle cross-sections from an unaffected control (Upper) and a POMT1-deficient patient with Walker–Warburg syndrome (Lower) (21). Anti–T[α1-Man] immunoreactivity coinciding with the localization of O-mannosylated α-DG is observed at the sarcolemma of control muscle cells but not in the sarcolemma of the patient. (D) Detection of O-mannosyl glycans during preimplantation development. Embryos at different developmental stages were analyzed by whole-mount immunofluorescence with anti–T[α1-Man]. O-mannosyl glycans emerge at the blastomere surface at the four-cell stage. The blastomere surface was visualized using PNA, and cortical actin was visualized by staining with phalloidin.

Fig. 3.

Characterization of O-mannosylation impaired embryos. (A) Whole-mount immunofluorescence analyses of embryos from intercrosses of Pomt2^+/− mice and of WT embryos treated with R3A-5a. Morula- and blastocyst-stage embryos were analyzed with the T[α1-Man]–specific antibody and phalloidin. O-mannosyl glycans were not detectable in Pomt2^−/− or inhibitor-treated embryos. Arrowheads indicate sites of impaired blastomere adhesion. Genotypes of the individual embryos shown were determined by PCR analysis as in Fig. 1, following microscopy. (B) Whole-mount immunofluorescent analysis of adherens and tight junctions in WT embryos after R3A-5a or mock (DMSO) treatment. Morula-stage embryos stained with E-cad– or ZO-1–directed antibodies or with phalloidin are shown. Arrowheads indicate diminished E-cad staining at sites of reduced blastomere attachment.

Fig. 4.

Detection of O-mannosyl glycans on E-cad. (A and B) E-cad–derived glycopeptides were analyzed by a combination of LC-MS/MS sequencing after CID and specific demannosylation using α-mannosidase as recently described (ref. and SI Materials and Methods). The EC4-derived peptide TAQEPDTFMEQK was found to be modified with a single O-linked mannose residue. (A) MS3 analysis of the hexosylated peptide TAQEPDTFMEQK. (Inset) CID of a doubly charged peptide (m/z = 793.9) identified a dominant fragment ion (m/z = 712.9) formed by a neutral loss of the mass of a hexose. This fragment ion was selected for additional fragmentation leading to β and γ ions caused by backbone fragmentation of a peptide, allowing its identification. (B) Enzymatic demannosylation of the E-cad peptide TAQEPDTFMEQK (lanes 1 and 2) and a synthetic O-mannosylated control peptide LSDAGT(α1-Man)VVSGQIR (lanes 3 and 4). Peptides were treated without (dashed line) and with (solid line) α-mannosidase and were analyzed by LC-MS. Extracted-ion chromatograms of the mannosylated peptides (lanes 1 and 3) show that intensity decreased significantly upon α-mannosidase treatment. Consequently, signal intensities of the demannosylated peptides (lanes 2 and 4) increased. NL, normalized intensity level (counts per second). (C) Western blotting of affinity-purified E-cad following treatment of MDCK cells with 50 µM R3A-5a or mock treatment (DMSO), using the E-cad antibody DECMA-1 and the T[α1-Man]–specific antibody. Glycans were removed by treatment with glycosidases as indicated (for details see Fig. 2A).

Fig. 5.

O-mannosyl glycans affect cadherin-based cell–cell adhesion. (A) Slow-aggregation assays. MDCK cells were aggregated overnight in the presence of an E-cad antibody (DECMA-1), a T[α1-Man]–specific antibody, or antibodies directed against carbohydrate epitopes that are O-mannosidically linked to α-DG (IIH6 and VIA4-1), as indicated. EGTA was used to block Ca²⁺-dependent, cadherin-mediated adhesion. (B) Quantitative summary of data from fast-aggregation assays. MDCK cells were aggregated in the presence of EGTA, DMSO, or increasing concentrations of R3A-5a (6.25–50 µM). The percentage of nonaggregated cells (fewer than four cells) was determined after a 30-min incubation. Mean values of three independent experiments are shown. Protein extracts were analyzed by Western blotting using the E-cad–specific antibody DECMA-1 (E-cad) and a K-cad–specific antibody (K-cad). O-mannosyl glycans on purified E-cad were detected by the T[α1-Man]–specific antibody. Tubulin served as the loading control.