Francesca Sciandra

Activation of Muscle-specific Receptor Tyrosine Kinase and Binding to Dystroglycan Are Regulated by Alternative mRNA Splicing of Agrin

Agrin induces the aggregation of postsynaptic proteins at the neuromuscular junction (NMJ). This activity requires the receptor-tyrosine kinase MuSK. Agrin isoforms differ in short amino acid stretches at two sites, called A and B, that are localized in the two most C-terminal laminin G (LG) domains. Importantly, agrin isoforms greatly differ in their activities of inducing MuSK phosphorylation and of binding to α-dystroglycan. By using site-directed mutagenesis, we characterized the amino acids important for these activities of agrin. We find that the conserved tripeptide asparagineglutamate-isoleucine in the eight-amino acid long insert at the B-site is necessary and sufficient for full MuSK phosphorylation activity. However, even if all eight amino acids were replaced by alanines, this agrin mutant still has significantly higher MuSK phosphorylation activity than the splice version lacking any insert. We also show that binding to α-dystroglycan requires at least two LG domains and that amino acid inserts at the A and the B splice sites negatively affect binding.

α-Dystroglycan does not play a major pathogenic role in autosomal recessive hereditary inclusion-body myopathy

Mutations of the GNE gene are responsible for autosomal recessive hereditary inclusion-body myopathy (HIBM). In this study we searched for the presence of any significant abnormality of alpha-dystroglycan (alpha-DG), a highly glycosylated component of the dystrophin-glycoprotein complex, in 5 HIBM patients which were previously clinically and genetically characterized. Immunocytochemical and immunoblot analysis showed that alpha-DG extracted from muscle biopsies was normally expressed and displayed its typical molecular mass. Immunoblot analysis on the wheat germ lectin-enriched glycoprotein fraction of muscles and primary myotubes showed a reduced amount of alpha-DG in 4 out of 5 HIBM patients, compared to normal and other diseased muscles. However, such altered lectin-binding behaviour, possibly reflecting a partial hyposialylation of alpha-DG, did not affect the laminin binding properties of alpha-DG. Therefore, the subtle changes within the alpha-DG glycosylation pattern, detected in HIBM muscles, likely do not play a key pathogenic role in this disorder.

A synthetic peptide corresponding to the 550–585 region of α-dystroglycan binds β-dystroglycan as revealed by NMR spectroscopy

We have probed the binding of a synthetic peptide corresponding to the region 550–585 of the α subunit of dystroglycan with a recombinant protein fragment corresponding to the N‐terminal extracellular region of β‐dystroglycan (654–750), using NMR in solution. In a 30:1 molar ratio, the peptide binds to the recombinant protein fragment in the fast/intermediate exchange regime. By monitoring the peptide intra‐residue HN–Hα peak volumes of the 2D TOCSY NMR spectra, both in the absence and in the presence of the recombinant fragment, we determined the differential binding affinities of each amino acid. We found that the residues in the region 550–565 (SWVQFNSNSQLMYGLP) are more influenced by the presence of the protein, whereas the C‐terminal portion is marginally involved. These NMR results have been confirmed by solid‐phase binding assays.

Plasticity of secondary structure in the N-terminal region of β-dystroglycan

The secondary structure content of the N-terminal extracellular domain of beta-dystroglycan (a recombinant fragment extending from positions 654 to 750) has been quantitatively determined by means of CD and FTIR spectroscopies. The elements of secondary structure, namely an 8-10 residue long alpha-helix (10%) and two beta-strands (24%) have been assigned to specific amino acid sequences by means of a GOR constrained prediction method. The remaining 66% of the whole sequence is classified as turns or unordered. The temperature dependence of CD and FTIR spectra has been investigated in detail. A reversible, non-cooperative thermal transition is observed with both CD and FTIR spectroscopies up to 95 degrees C. The profile of the transition is typical of the unfolding of isolated peptides and corresponds to the progressive loss of the secondary structure elements of the protein with no evidence for collapsing phenomena, typical of globular proteins, upon heating.

A second Ig-like domain identified in dystroglycan by molecular modelling and dynamics

Dystroglycan (DG) is a cell surface receptor which is composed of two subunits that interact noncovalently, namely α- and β-DG. In skeletal muscle, DG is the central component of the dystrophin-glycoprotein complex (DGC) that anchors the actin cytoskeleton to the extracellular matrix. To date only the three-dimensional structure of the N-terminal region of α-DG has been solved by X-ray crystallography. To expand such a structural analysis, a theoretical molecular model of the murine α-DG C-terminal region was built based on folding recognition/threading techniques. Although there is no a significant (<30%) sequence homology with the N-terminal region of α-DG, protein fold recognition methods found a significant resemblance to the α-DG N-terminal crystallographic structure. Our in silico structural prediction identified two subdomains in this region. Amino acid residues ∼ 500-600 of α-DG were predicted to adopt an immunoglobulin-like (Ig-like) β-sandwich fold. Such modeled domain includes the β-DG binding epitope of α-DG and, confirming our previous experimental results, suggests that the linear epitope (residues 550-565) assumes a β-strand conformation. The remaining segment of the α-DG C-terminal region (residues 601-653) is organized in a coil-helix-coil motif. A 20-ns molecular dynamics simulation in explicit water solvent provided support to the predicted Ig-like model structure. The identification of a second Ig-like domain in DG represents another important step towards a full structural and functional description of the α/β DG interface. Preliminary characterization of a novel recombinant peptide (505-600) encompassing this second Ig-like domain demonstrates that it is soluble and stable, further corroborating our in silico analysis.

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.

Enzymatic processing by MMP-2 and MMP-9 of wild-type and mutated mouse β-dystroglycan

Dystroglycan (DG) is a membrane‐associated protein complex formed by two noncovalently linked subunits, α‐DG, a highly glycosylated extracellular protein, and β‐DG, a transmembrane protein. The interface between the two DG subunits, which is crucial to maintain the integrity of the plasma membrane, involves the C‐terminal domain of α‐DG and the N‐terminal extracellular domain of β‐DG. It is well known that under both, physiological and pathological conditions, gelatinases (i.e. MMP‐9 and/or MMP‐2) can degrade DG, disrupting the connection between the extracellular matrix and the cytoskeleton. However, the molecular mechanisms and the exact cleavage sites underlying these events are still largely unknown. In a previous study, we have characterized the enzymatic digestion of the murine β‐DG ectodomain by gelatinases, identifying a main cleavage site on the β‐DG ectodomain produced by MMP‐9. In this article, we have deepened the pattern of the β‐DG ectodomain digestion by MMP‐2 by using a combined approach based on SDS‐PAGE, Orbitrap, and HPLC‐ESI‐IT mass spectrometry. Furthermore, we have characterized the kineticparameters of the digestion of some β‐DG ectodomain mutants by gelatinases.

Insertion of a myc-tag within α-dystroglycan domains improves its biochemical and microscopic detection

BackgroundEpitope tags and fluorescent fusion proteins have become indispensable molecular tools for studies in the fields of biochemistry and cell biology. The knowledge collected on the subdomain organization of the two subunits of the adhesion complex dystroglycan (DG) enabled us to insert the 10 amino acids myc-tag at different locations along the α-subunit, in order to better visualize and investigate the DG complex in eukaryotic cells.ResultsWe have generated two forms of DG polypeptides via the insertion of the myc-tag 1) within a flexible loop (between a.a. 170 and 171) that separates two autonomous subdomains, and 2) within the C-terminal domain in position 500. Their analysis showed that double-tagging (the β-subunit is linked to GFP) does not significantly interfere with the correct processing of the DG precursor (pre-DG) and confirmed that the α-DG N-terminal domain is processed in the cell before α-DG reaches its plasma membrane localization. In addition, myc insertion in position 500, right before the second Ig-like domain of α-DG, proved to be an efficient tool for the detection and pulling-down of glycosylated α-DG molecules targeted at the membrane.ConclusionsFurther characterization of these and other myc-permissive site(s) will represent a valid support for the study of the maturation process of pre-DG and could result in the creation of a new class of intrinsic doubly-fluorescent DG molecules that would allow the monitoring of the two DG subunits, or of pre-DG, in cells without the need of antibodies.

Mutagenesis at the α–β interface impairs the cleavage of the dystroglycan precursor

The interaction between α‐dystroglycan (α‐DG) and β‐dystroglycan (β‐DG), the two constituent subunits of the adhesion complex dystroglycan, is crucial in maintaining the integrity of the dystrophin–glycoprotein complex. The importance of the α–β interface can be seen in the skeletal muscle of humans affected by severe conditions, such as Duchenne muscular dystrophy, where the α–β interaction can be secondarily weakened or completely lost, causing sarcolemmal instability and muscular necrosis. The reciprocal binding epitopes of the two subunits reside within the C‐terminus of α‐DG and the ectodomain of β‐DG. As no ultimate structural data are yet available on the α–β interface, site‐directed mutagenesis was used to identify which specific amino acids are involved in the interaction. A previous alanine‐scanning analysis of the recombinant β‐DG ectodomain allowed the identification of two phenylalanines important for α‐DG binding, namely F692 and F718. In this article, similar experiments performed on the α‐DG C‐terminal domain pinpointed two residues, G563 and P565, as possible binding counterparts of the two β‐DG phenylalanines. In 293‐Ebna cells, the introduction of alanine residues instead of F692, F718, G563 and P565 prevented the cleavage of the DG precursor that liberates α‐ and β‐DG, generating a pre‐DG of about 160 kDa. This uncleaved pre‐DG tetramutant is properly targeted at the cell membrane, is partially glycosylated and still binds laminin in pull‐down assays. These data reinforce the notion that DG processing and its membrane targeting are two independent processes, and shed new light on the molecular mechanism that drives the maturation of the DG precursor.

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.