Muscle-eye-brain disease (MEB) is an autosomal recessive disorder characterized by congenital muscular dystrophy, ocular abnormalities, and lissencephaly. Mammalian O-mannosyl glycosylation is a rare type of protein modification that is observed in a limited number of glycoproteins of brain, nerve, and skeletal muscle. Here we isolated a human cDNA for protein O-mannose beta-1,2-N-acetylglucosaminyltransferase (POMGnT1), which participates in O-mannosyl glycan synthesis. We also identified six independent mutations of the POMGnT1 gene in six patients with MEB. Expression of most frequent mutation revealed a great loss of the enzymatic activity. These findings suggest that interference in O-mannosyl glycosylation is a new pathomechanism for muscular dystrophy as well as neuronal migration disorder.

Fibronectin (FN) is secreted as a disulfide-bonded FN dimer. Each subunit contains three types of repeating modules: FN-I, FN-II, and FN-III. The interactions of α5β1 or αv integrins with the RGD motif of FN-III repeat 10 (FN-III10) are considered an essential step in the assembly of FN fibrils. To test this hypothesis in vivo, we replaced the RGD motif with the inactive RGE in mice. FN-RGE homozygous embryos die at embryonic day 10 with shortened posterior trunk, absent tail bud–derived somites, and severe vascular defects resembling the phenotype of α5 integrin–deficient mice. Surprisingly, the absence of a functional RGD motif in FN did not compromise assembly of an FN matrix in mutant embryos or on mutant cells. Matrix assembly assays and solid-phase binding assays reveal that αvβ3 integrin assembles FN-RGE by binding an isoDGR motif in FN-I5, which is generated by the nonenzymatic rearrangement of asparagines (N) into an iso-aspartate (iso-D). Our findings demonstrate that FN contains a novel motif for integrin binding and fibril formation whose activity is controlled by amino acid modification.

SUMMARYFibronectin (FN) is a major component of the extracellular matrix and functions in cell adhesion, cell spreading and cell migration. In the retina, FN is transiently expressed and assembled on astrocytes (ACs), which guide sprouting tip cells and deposit a provisional matrix for sprouting angiogenesis. The precise function of FN in retinal angiogenesis is largely unknown. Using genetic tools, we show that astrocytes are the major source of cellular FN during angiogenesis in the mouse retina. Deletion of astrocytic FN reduces radial endothelial migration during vascular plexus formation in a gene dose-dependent manner. This effect correlates with reduced VEGF receptor 2 and PI3K/AKT signalling, and can be mimicked by selectively inhibiting VEGF-A binding to FN through intraocular injection of blocking peptides. By contrast, AC-specific replacement of the integrin-binding RGD sequence with FN-RGE or endothelial deletion of itga5 shows little effect on migration and PI3K/AKT signalling, but impairs filopodial alignment along AC processes, suggesting that FN-integrin 51 interaction is involved in filopodial adhesion to the astrocytic matrix. AC FN shares its VEGF-binding function and cell-surface distribution with heparan-sulfate (HS), and genetic deletion of both FN and HS together greatly enhances the migration defect, indicating a synergistic function of FN and HS in VEGF binding. We propose that in vivo the VEGF-binding properties of FN and HS promote directional tip cell migration, whereas FN integrin-binding functions to support filopodia adhesion to the astrocytic migration template.

Newly deposited microfibrils strongly colocalise with fibronectin in primary fibroblasts. Microfibril formation is grossly inhibited by fibronectin depletion, but rescued by supplementation with exogenous cellular fibronectin. As integrin receptors are key determinants of fibronectin assembly, we investigated whether they also influenced microfibril deposition. Analysis of β1-integrin-receptor-null fibroblasts, blockage of cell surface integrin receptors that regulate fibronectin assembly and disruption of Rho kinase all result in suppressed deposition of both fibronectin and microfibrils. Antibody activation of β1 integrins in fibronectin-depleted cultures is insufficient to rescue microfibril assembly. In fibronectinRGE/RGE mutant mouse fibroblast cultures, which do not engage α5β1 integrin, extracellular assembly of both fibronectin and microfibrils is markedly reduced. Thus, pericellular microfibril assembly is regulated by fibronectin fibrillogenesis.

Eukaryotic cell adhesion is a fundamental process in tissue development, homeostasis, and disease and is mediated by specific interactions of cell surface receptors with extracellular matrix (ECM) 2 proteins (1-5). The ECM is a meshwork of fibrillar and nonfibrillar components assembled into complex structures such as basement membranes. The latter provide a scaffold for cell adhesion, spreading, and migration. ECM regulates numerous cell functions by activating multiple signaling pathways at the adhesion sites. ECMs, composed of collagens, laminins, and other glycoproteins such as fibronectin (FN), serve as substrates for different adhesion molecules including the integrin family of transmembrane receptors. The assembly of ECM components into functional supramolecular modules is highly regulated (3-7). FN matrix assembly alone is a dynamic cell-driven process in which the soluble FN molecules assemble into insoluble fibrillar polymeric ECM structures (8).FN and integrin receptors play crucial roles in a variety of morphogenetic processes, which are regulated by processes termed outside-in and inside-out signaling cascades (3-5). Deregulation of integrin and FN functions associates with disease development including chronic inflammation, heart failure, cancer, and metastasis (7, 9 -11). The outside-in signaling triggered by ligation of integrin receptors with FN and other ECM components results in the reorganization of cytoskeletal and signaling molecules into complexes of more than 90 proteins (9 -13). This occurs by synergistic processes dependent on integrin aggregation and occupancy, as well as tyrosine phosphorylation. Integrins also cooperate with growth factor receptors such as epidermal growth factor receptor (EGFR) to enhance signaling (14).FN consists of multiple domains (classified types I-III) that show binding specificities for specific cell membrane receptors, collagen, fibrin, and heparin. FN alone is sufficient to induce highly efficient spreading of many mammalian cell types including fibroblast and epithelial cells in vitro. An important functional unit of FN is its RGD tripeptide motif, which acts in