Ernesto Pavoni

O mannosylation of α-dystroglycan is essential for lymphocytic choriomeningitis virus receptor function

ABSTRACT α-Dystroglycan (α-DG) was identified as a common receptor for lymphocytic choriomeningitis virus (LCMV) and several other arenaviruses including the human pathogenic Lassa fever virus. Initial work postulated that interactions between arenavirus glycoproteins and α-DG are based on protein-protein interactions. We found, however, that susceptibility toward LCMV infection differed in various cell lines despite them expressing comparable levels of DG, suggesting that posttranslational modifications of α-DG would be involved in viral receptor function. Here, we demonstrate that glycosylation of α-DG, and in particular, O mannosylation, which is a rare type of O-linked glycosylation in mammals, is essential for LCMV receptor function. Cells that are defective in components of the O-mannosylation pathway showed strikingly reduced LCMV infectibility. As defective O mannosylation is associated with severe clinical symptoms in mammals such as congenital muscular dystrophies, it is likely that LCMV and potentially other arenaviruses may have selected this conserved and crucial posttranslational modification as the primary target structure for cell entry and infection.

MMP2-9 cleavage of dystroglycan alters the size and molecular composition of Schwann cell domains.

Myelinating glial cells exhibit a spectacular cytoarchitecture, because they polarize on multiple axes and domains. How this occurs is essentially unknown. The dystroglycan–dystrophin complex is required for the function of myelin-forming Schwann cells. Similar to other tissues, the dystroglycan complex in Schwann cells localizes with different dystrophin family members in specific domains, thus promoting polarization. We show here that cleavage of dystroglycan by matrix metalloproteinases 2 and 9, an event that is considered pathological in most tissues, is finely and dynamically regulated in normal nerves and modulates dystroglycan complex composition and the size of Schwann cell compartments. In contrast, in nerves of Dy2j/2j mice, a model of laminin 211 deficiency, metalloproteinases 2 and 9 are increased, causing excessive dystroglycan cleavage and abnormal compartments. Pharmacological inhibition of cleavage rescues the cytoplasmic defects of Dy2j/2j Schwann cells. Thus, regulated cleavage may be a general mechanism to regulate protein complex composition in physiological conditions, whereas unregulated processing is pathogenic and a target for treatment in disease.

Increased expression of dystroglycan inhibits the growth and tumorigenicity of human mammary epithelial cells.

Dystroglycan (DG) is an adhesion molecule formed by two subunits, _ (extracellular) and _ (transmembrane) DG, which are codified by a single gene and form a continuous link from the extracellular matrix to the intracellular cytoskeleton. Reduction or loss of expression of DG has been observed in human cancer cell lines and primary tumors and has been suggested to promote tumor development and invasiveness. In this study, the human breast epithelial non-tumorigenic MCF10F and the breast cancer MCF7 cell lines were engineered to stably express an exogenous DG cDNA and the effects on the phenotype of both cell lines were evaluated. The MCF10F transfected cells displayed an increased expression of both DG subunits which was associated with inhibition of the anchorage-dependent growth, accumulation of cells in the G0/G1 phase of the cell cycle and increased adhesion to a substratum. The MCF7 transfected cells were unable to restore _-DG despite an increased expression of the _-DG subunit. Anchorage-dependent and independent growth and the in vivo tumorigenicity were reduced in these derivatives that also displayed a reduced adhesion to a substratum and were shown to release _-DG in the culture medium. These findings confirm and extend previous evidence that transformation of mammary epithelial cells is associated with loss of their ability to retain _-DG on the cell membrane. Moreover, they indicate that DG is involved in cell functions other than cell adhesion to the extracellular matrix, and that its loss of function might predispose to tumor progression by compromising regulatory controls over cell growth and proliferation.

Altered expression of alpha-dystroglycan subunit in human gliomas

Dystroglycan (DG) is an integral membrane receptor of extracellular matrix proteins, composed of two subunits alpha and beta derived from a common precursor. In brain DG is expressed in neurons, glia limitans, astrocytic endfeet around vessels and endothelial cells. We investigate whether DG may play a role in brain tumors. Western blot and immunofluorescence analysis showed that, while beta-DG subunit was present, the highly glycosylated alpha-DG subunit was strongly reduced in surgically derived human glioblastoma biopsies, in low passage patient-derived cultures and in glioma cell lines, U87MG and A172MG, but not in all glioma cell lines tested. Immunohistochemistry of tumor frozen sections revealed that the loss of alpha-DG was confined in the tumor area but not around blood vessels. Overexpression of DG decreased the growth rate of the glioma cell lines lacking the highly glycosylated a-DG subunit and the colony-forming efficiency. Clonogenic assay in presence of temozolomide showed an additive effect between DG overexpression and drug treatment. Our data suggest that DG may be involved in the progression of primary brain tumors.

Perlecan is recruited by dystroglycan to nodes of Ranvier and binds the clustering molecule gliomedin

Dystroglycan promotes nodogenesis in part through recruitment of perlecan to nodes of Ranvier, where it binds to gliomedin and may thereby promote sodium channel clustering.

Enzymatic processing of beta-dystroglycan recombinant ectodomain by MMP-9: identification of the main cleavage site.

Dystroglycan (DG) is a membrane receptor belonging to the complex of glycoproteins associated to dystrophin. DG is formed by two subunits, α‐DG, a highly glycosylated extracellular matrix protein, and β‐DG, a transmembrane protein. The two DG subunits interact through the C‐terminal domain of α‐DG and the N‐terminal extracellular domain of β‐DG in a noncovalent way. Such interaction is crucial to maintain the integrity of the plasma membrane. In some pathological conditions, the interaction between the two DG subunits may be disrupted by the proteolytic activity of gelatinases (i.e. MMP‐9 and/or MMP‐2) that removes a portion or the whole β‐DG ectodomain producing a 30 kDa truncated form of β‐DG. However, the molecular mechanism underlying this event is still unknown. In this study, we carried out proteolysis of the recombinant extracellular domain of β‐DG, β‐DG(654‐750) with human MMP‐9, characterizing the catalytic parameters of its cleavage. Furthermore, using a combined approach based on SDS‐PAGE, MALDI‐TOF and HPLC‐ESI‐IT mass spectrometry, we were able to identify one main MMP‐9 cleavage site that is localized between the amino acids His‐715 and Leu‐716 of β‐DG, and we analysed the proteolytic fragments of β‐DG(654‐750) produced by MMP‐9 enzymatic activity.

Concerted mutation of Phe residues belonging to the β‐dystroglycan ectodomain strongly inhibits the interaction with α‐dystroglycan in vitro

The dystroglycan adhesion complex consists of two noncovalently interacting proteins: α‐dystroglycan, a peripheral extracellular subunit that is extensively glycosylated, and the transmembrane β‐dystroglycan, whose cytosolic tail interacts with dystrophin, thus linking the F‐actin cytoskeleton to the extracellular matrix. Dystroglycan is thought to play a crucial role in the stability of the plasmalemma, and forms strong contacts between the extracellular matrix and the cytoskeleton in a wide variety of tissues. Abnormal membrane targeting of dystroglycan subunits and/or their aberrant post‐translational modification are often associated with several pathologic conditions, ranging from neuromuscular disorders to carcinomas. A putative functional hotspot of dystroglycan is represented by its intersubunit surface, which is contributed by two amino acid stretches: approximately 30 amino acids of β‐dystroglycan (691–719), and approximately 15 amino acids of α‐dystroglycan (550–565). Exploiting alanine scanning, we have produced a panel of site‐directed mutants of our two consolidated recombinant peptides β‐dystroglycan (654–750), corresponding to the ectodomain of β‐dystroglycan, and α‐dystroglycan (485–630), spanning the C‐terminal domain of α‐dystroglycan. By solid‐phase binding assays and surface plasmon resonance, we have determined the binding affinities of mutated peptides in comparison to those of wild‐type α‐dystroglycan and β‐dystroglycan, and shown the crucial role of two β‐dystroglycan phenylalanines, namely Phe692 and Phe718, for the α–β interaction. Substitution of the α‐dystroglycan residues Trp551, Phe554 and Asn555 by Ala does not affect the interaction between dystroglycan subunits in vitro. As a preliminary analysis of the possible effects of the aforementioned mutations in vivo, detection through immunofluorescence and western blot of the two dystroglycan subunits was pursued in dystroglycan‐transfected 293‐Ebna cells.

Immunodetection of partially glycosylated isoforms of α-dystroglycan by a new monoclonal antibody against its β-dystroglycan-binding epitope

The α/β dystroglycan (DG) complex links the extracellular matrix to the actin cytoskeleton. The extensive glycosylation of α‐DG is believed to be crucial for the interaction with its extracellular matrix‐binding partners. We characterized a monoclonal antibody, directed against the β‐DG‐binding epitope (≈positions 550–565), which recognizes preferentially hypoglycosylated α‐DG. In Western blot, the antibody was able to detect a number of partially glycosylated α‐DG isoforms from rat brain and chicken skeletal muscle tissue samples. In addition, we demonstrated its inhibitory effect on the interaction between α‐ and β‐DG in vitro and preliminary immunostaining experiments suggest that such hypoglycosylated α‐DG isoforms could play a role within cells.

Duplication of the dystroglycan gene in most branches of teleost fish.

BackgroundThe dystroglycan (DG) complex is a major non-integrin cell adhesion system whose multiple biological roles involve, among others, skeletal muscle stability, embryonic development and synapse maturation. DG is composed of two subunits: α-DG, extracellular and highly glycosylated, and the transmembrane β-DG, linking the cytoskeleton to the surrounding basement membrane in a wide variety of tissues. A single copy of the DG gene (DAG1) has been identified so far in humans and other mammals, encoding for a precursor protein which is post-translationally cleaved to liberate the two DG subunits. Similarly, D. rerio (zebrafish) seems to have a single copy of DAG1, whose removal was shown to cause a severe dystrophic phenotype in adult animals, although it is known that during evolution, due to a whole genome duplication (WGD) event, many teleost fish acquired multiple copies of several genes (paralogues).ResultsData mining of pufferfish (T. nigroviridis and T. rubripes) and other teleost fish (O. latipes and G. aculeatus) available nucleotide sequences revealed the presence of two functional paralogous DG sequences. RT-PCR analysis proved that both the DG sequences are transcribed in T. nigroviridis. One of the two DG sequences harbours an additional mini-intronic sequence, 137 bp long, interrupting the uncomplicated exon-intron-exon pattern displayed by DAG1 in mammals and D. rerio. A similar scenario emerged also in D. labrax (sea bass), from whose genome we have cloned and sequenced a new DG sequence that also harbours a shorter additional intronic sequence of 116 bp. Western blot analysis confirmed the presence of DG protein products in all the species analysed including two teleost Antarctic species (T. bernacchii and C. hamatus).ConclusionOur evolutionary analysis has shown that the whole-genome duplication event in the Class Actinopterygii (ray-finned fish) involved also DAG1. We unravelled new important molecular genetic details about fish orthologous DGs, which might help to increase the current knowledge on DG expression, maturation and targeting and on its physiopathological role in higher organisms.

An Immunological Analysis of Dystroglycan Subunits: Lessons Learned from a Small Cohort of Non-Congenital Dystrophic Patients

The dystroglycan (DG) expression pattern can be altered in severe muscular dystrophies. In fact, some congenital muscular dystrophies (CMDs) and limb-girdle muscular dystrophies (LGMDs) are caused by point mutations identified in six glycosyltransferase genes which are likely to target different steps along the posttranslational “O-glycosylation route” leading to a fully decorated and functional α-DG subunit. Indeed, hypoglycosylation of α-DG is thought to represent a major pathological event, in that it could reduce the DG’s ability to bind the basement membrane components, thus leading to sarcolemmal instability and necrosis. In order to set up an efficient standard immunological protocol, taking advantage of a wide panel of antibodies, we have analyzed the two DG subunits in a small cohort of adult dystrophic patients, whom an extensive medical examination had already clinically classified as affected by LGMD (5), Miyoshi (1) or distal (1) myopathy. Immunofluorescence analysis of skeletal muscle tissue sections revealed a proper sarcolemmal localization of the DG subunits in all the patients analyzed. However, Western blot analysis of lectin enriched skeletal muscle samples revealed an abnormal glycosylation of α-DG in two patients. Our work reinforces the notion that a careful immunological and biochemical analysis of the two DG subunits should be always considered as a prerequisite for the identification of new putative cases of dystroglycanopathy.