Corrigendum: From adhesion complex to signaling hub: the dual role of dystroglycan.

[This corrects the article DOI: 10.3389/fmolb.2023.1325284.].

The α-dystroglycan N-terminus is a broad-spectrum antiviral agent against SARS-CoV-2 and enveloped viruses.

The COVID-19 pandemic has shown the need to develop effective therapeutics in preparedness for further epidemics of virus infections that pose a significant threat to human health. As a natural compound antiviral candidate, we focused on α-dystroglycan, a highly glycosylated basement membrane protein that links the extracellular matrix to the intracellular cytoskeleton. Here we show that the N-terminal fragment of α-dystroglycan (α-DGN), as produced in E. coli in the absence of post-translational modifications, blocks infection of SARS-CoV-2 in cell culture, human primary gut organoids and the lungs of transgenic mice expressing the human receptor angiotensin I-converting enzyme 2 (hACE2). Prophylactic and therapeutic administration of α-DGN reduced SARS-CoV-2 lung titres and protected the mice from respiratory symptoms and death. Recombinant α-DGN also blocked infection of a wide range of enveloped viruses including the four Dengue virus serotypes, influenza A virus, respiratory syncytial virus, tick-borne encephalitis virus, but not human adenovirus, a non-enveloped virus in vitro. This study establishes soluble recombinant α-DGN as a broad-band, natural compound candidate therapeutic against enveloped viruses.

From adhesion complex to signaling hub: the dual role of dystroglycan.

Dystroglycan (DG) is a transmembrane protein widely expressed in multiple cells and tissues. It is formed by two subunits, α- and ß-DG, and represents a molecular bridge between the outside and the inside of the cell, which is essential for the mechanical and structural stability of the plasma membrane. The α-subunit is a cell-surface protein that binds to the extracellular matrix (ECM) and is tightly associated with the plasma membrane via a non-covalent interaction with the ß-subunit, which, in turn, is a transmembrane protein that binds to the cytoskeletal actin. DG is a versatile molecule acting not only as a mechanical building block but also as a modulator of outside-inside signaling events. The cytoplasmic domain of ß-DG interacts with different adaptor and cytoskeletal proteins that function as molecular switches for the transmission of ECM signals inside the cells. These interactions can modulate the involvement of DG in different biological processes, ranging from cell growth and survival to differentiation and proliferation/regeneration. Although the molecular events that characterize signaling through the ECM-DG-cytoskeleton axis are still largely unknown, in recent years, a growing list of evidence has started to fill the gaps in our understanding of the role of DG in signal transduction. This mini-review represents an update of recent developments, uncovering the dual role of DG as an adhesion and signaling molecule that might inspire new ideas for the design of novel therapeutic strategies for pathologies such as muscular dystrophy, cardiomyopathy, and cancer, where the DG signaling hub plays important roles.

A Segmental Approach from Molecular Profiling to Medical Imaging to Study Bicuspid Aortic Valve Aortopathy.

Bicuspid aortic valve (BAV) patients develop ascending aortic (AAo) dilation. The pathogenesis of BAV aortopathy (genetic vs. haemodynamic) remains unclear. This study aims to identify regional changes around the AAo wall in BAV patients with aortopathy, integrating molecular data and clinical imaging. BAV patients with aortopathy (n = 15) were prospectively recruited to surgically collect aortic tissue and measure molecular markers across the AAo circumference. Dilated (anterior/right) vs. non-dilated (posterior/left) circumferential segments were profiled for whole-genomic microRNAs (next-generation RNA sequencing, miRCURY LNA PCR), protein content (tandem mass spectrometry), and elastin fragmentation and degeneration (histomorphometric analysis). Integrated bioinformatic analyses of RNA sequencing and proteomic datasets identified five microRNAs (miR-128-3p, miR-210-3p, miR-150-5p, miR-199b-5p, and miR-21-5p) differentially expressed across the AAo circumference. Among them, three miRNAs (miR-128-3p, miR-150-5p, and miR-199b-5p) were predicted to have an effect on eight common target genes, whose expression was dysregulated, according to proteomic analyses, and involved in the vascular-endothelial growth-factor signalling, Hippo signalling, and arachidonic acid pathways. Decreased elastic fibre levels and elastic layer thickness were observed in the dilated segments. Additionally, in a subset of patients n = 6/15, a four-dimensional cardiac magnetic resonance (CMR) scan was performed. Interestingly, an increase in wall shear stress (WSS) was observed at the anterior/right wall segments, concomitantly with the differentially expressed miRNAs and decreased elastic fibres. This study identified new miRNAs involved in the BAV aortic wall and revealed the concomitant expressional dysregulation of miRNAs, proteins, and elastic fibres on the anterior/right wall in dilated BAV patients, corresponding to regions of elevated WSS.

Determination of Agrin and Related Proteins Levels as a Function of Age in Human Hearts.

Background: Mature cardiomyocytes are unable to proliferate, preventing the injured adult heart from repairing itself. Studies in rodents have suggested that the extracellular matrix protein agrin promotes cardiomyocyte proliferation in the developing heart and that agrin expression is downregulated shortly after birth, resulting in the cessation of proliferation. Agrin based therapies have proven successful at inducing repair in animal models of cardiac injury, however whether similar pathways exist in the human heart is unknown. Methods: Right ventricular (RV) biopsies were collected from 40 patients undergoing surgery for congenital heart disease and the expression of agrin and associated proteins was investigated. Results: Agrin transcripts were found in all samples and their levels were significantly negatively correlated to age (p = 0.026), as were laminin transcripts (p = 0.023), whereas no such correlation was found for the other proteins analyzed. No significant correlations for any of the proteins were found when grouping patients by their gender or pathology. Immunohistochemistry and western blots to detect and localize agrin and the other proteins under analysis in RV tissue, confirmed their presence in patients of all ages. Conclusions: We show that agrin is progressively downregulated with age in human RV tissue but not as dramatically as has been demonstrated in mice; highlighting both similarities and differences to findings in rodents. Our results lay the groundwork for future studies exploring the potential of agrin-based therapies in the repair of damaged human hearts.

High degree of conservation of the enzymes synthesizing the laminin-binding glycoepitope of α-dystroglycan.

The dystroglycan (DG) complex plays a pivotal role for the stabilization of muscles in Metazoa. It is formed by two subunits, extracellular α-DG and transmembrane ß-DG, originating from a unique precursor via a complex post-translational maturation process. The α-DG subunit is extensively glycosylated in sequential steps by several specific enzymes and employs such glycan scaffold to tightly bind basement membrane molecules. Mutations of several of these enzymes cause an alteration of the carbohydrate structure of α-DG, resulting in severe neuromuscular disorders collectively named dystroglycanopathies. Given the fundamental role played by DG in muscle stability, it is biochemically and clinically relevant to investigate these post-translational modifying enzymes from an evolutionary perspective. A first phylogenetic history of the thirteen enzymes involved in the fabrication of the so-called 'M3 core' laminin-binding epitope has been traced by an overall sequence comparison approach, and interesting details on the primordial enzyme set have emerged, as well as substantial conservation in Metazoa. The optimization along with the evolution of a well-conserved enzymatic set responsible for the glycosylation of α-DG indicate the importance of the glycosylation shell in modulating the connection between sarcolemma and surrounding basement membranes to increase skeletal muscle stability, and eventually support movement and locomotion.

Corrigendum: Agrin-Mediated Cardiac Regeneration: Some Open Questions.

[This corrects the article on p. 594 in vol. 8, PMID: 32612983.].

Agrin-Mediated Cardiac Regeneration: Some Open Questions.

After cardiac injury, the mammalian adult heart has a very limited capacity to regenerate, due to the inability of fully differentiated cardiomyocytes (CMs) to efficiently proliferate. This has been directly linked to the extracellular matrix (ECM) surrounding and connecting cardiomyocytes, as its increasing rigidity during heart maturation has a crucial impact over the proliferative capacity of CMs. Very recent studies using mouse models have demonstrated how the ECM protein agrin might promote heart regeneration through CMs de-differentiation and proliferation. In maturing CMs, this proteoglycan would act as an inducer of a specific molecular pathway involving ECM receptor(s) within the transmembrane dystrophin-glycoprotein complex (DGC) as well as intracellular Yap, an effector of the Hippo pathway involved in the replication/regeneration program of CMs. According to the mechanism proposed, during mice heart development agrin gets progressively downregulated and ultimately replaced by other ECM proteins eventually leading to loss of proliferation/ regenerative capacity in mature CMs. Although the role played by the agrin-DGC-YAP axis during human heart development remains still largely to be defined, this scenario opens up fascinating and promising therapeutic avenues. Herein, we discuss the currently available relevant information on this system, with a view to explore how the fundamental understanding of the regenerative potential of this cellular program can be translated into therapeutic treatment of injured human hearts.

Identification and Modeling of a GT-A Fold in the α-Dystroglycan Glycosylating Enzyme LARGE1.

The acetylglucosaminyltransferase-like protein LARGE1 is an enzyme that is responsible for the final steps of the post-translational modifications of dystroglycan (DG), a membrane receptor that links the cytoskeleton with the extracellular matrix in the skeletal muscle and in a variety of other tissues. LARGE1 acts by adding the repeating disaccharide unit [-3Xyl-α1,3GlcAß1-] to the extracellular portion of the DG complex (α-DG); defects in the LARGE1 gene result in an aberrant glycosylation of α-DG and consequent impairment of its binding to laminin, eventually affecting the connection between the cell and the extracellular environment. In the skeletal muscle, this leads to degeneration of the muscular tissue and muscular dystrophy. So far, a few missense mutations have been identified within the LARGE1 protein and linked to congenital muscular dystrophy, and because no structural information is available on this enzyme, our understanding of the molecular mechanisms underlying these pathologies is still very limited. Here, we generated a 3D model structure of the two catalytic domains of LARGE1, combining different molecular modeling approaches. Furthermore, by using molecular dynamics simulations, we analyzed the effect on the structure and stability of the first catalytic domain of the pathological missense mutation S331F that gives rise to a severe form of muscle-eye-brain disease.

Analysis of α-Dystroglycan/LG Domain Binding Modes: Investigating Protein Motifs That Regulate the Affinity of Isolated LG Domains.

Dystroglycan (DG) is an adhesion complex that links the cytoskeleton to the surrounding extracellular matrix in skeletal muscle and a wide variety of other tissues. It is composed of a highly glycosylated extracellular α-DG associated noncovalently with a transmembrane ß-DG whose cytodomain interacts with dystrophin and its isoforms. Alpha-dystroglycan (α-DG) binds tightly and in a calcium-dependent fashion to multiple extracellular proteins and proteoglycans, each of which harbors at least one, or, more frequently, tandem arrays of laminin-globular (LG) domains. Considerable biochemical and structural work has accumulated on the α-DG-binding LG domains, highlighting a significant heterogeneity in ligand-binding properties of domains from different proteins as well as between single and multiple LG domains within the same protein. Here we review biochemical, structural, and functional information on the LG domains reported to bind α-dystroglycan. In addition, we have incorporated bioinformatics and modeling to explore whether specific motifs responsible for α-dystroglycan recognition can be identified within isolated LG domains. In particular, we analyzed the LG domains of slits and agrin as well as those of paradigmatic α-DG non-binders such as laminin-α3. While some stretches of basic residues may be important, no universally conserved motifs could be identified. However, the data confirm that the coordinated calcium atom within the LG domain is needed to establish an interaction with the sugars of α-DG, although it appears that this alone is insufficient to mediate significant α-DG binding. We develop a scenario involving different binding modes of a single LG domain unit, or tandemly repeated units, with α-DG. A variability of binding modes might be important to generate a range of affinities to allow physiological regulation of this interaction, reflecting its crucial biological importance.

Intraring allostery controls the function and assembly of a hetero-oligomeric class II chaperonin.

Class II chaperonins are essential multisubunit complexes that aid the folding of nonnative proteins in the cytosol of archaea and eukarya. They use energy derived from ATP to drive a series of structural rearrangements that enable polypeptides to fold within their central cavity. These events are regulated by an elaborate allosteric mechanism in need of elucidation. We employed mutagenesis and experimental analysis in concert with in silico molecular dynamics simulations and interface-binding energy calculations to investigate the class II chaperonin from Thermoplasma acidophilum. Here we describe the effects on the asymmetric allosteric mechanism and on hetero-oligomeric complex formation in a panel of mutants in the ATP-binding pocket of the α and ß subunits. Our observations reveal a potential model for a nonconcerted folding mechanism optimized for protecting and refolding a range of nonnative substrates under different environmental conditions, starting to unravel the role of subunit heterogeneity in this folding machine and establishing important links with the behavior of the most complex eukaryotic chaperonins.-Shoemark, D. K., Sessions, R. B., Brancaccio, A., Bigotti, M. G. Intraring allostery controls the function and assembly of a hetero-oligomeric class II chaperonin.

Evaluation of the effect of a floxed Neo cassette within the dystroglycan (Dag1) gene.

OBJECTIVE: Dystroglycan (DG) is an adhesion complex formed by two subunits, α-DG and ß-DG. In skeletal muscle, DG is part of the dystrophin-glycoprotein complex that is crucial for sarcolemma stability and it is involved in a plethora of muscular dystrophy phenotypes. Due to the important role played by DG in skeletal muscle stability as well as in a wide variety of other tissues including brain and the peripheral nervous system, it is essential to investigate its genetic assembly and transcriptional regulation. RESULTS: Herein, we analyze the effect of the insertion of a floxed neomycin (Neo) cassette within the 3' portion of the universally conserved IG1-intron of the DG gene (Dag1). We analyzed the transcription level of Dag1 and the expression of the DG protein in skeletal muscle of targeted mice compared to wild-type and we did not find any alterations that might be attributed to the gene targeting. However, we found an increase of the cross-sectional areas of tibialis anterior that might have some physiological significance that needs to be assessed in the future. Moreover, in targeted mice the skeletal muscle morphology and its regeneration capacity after injury did not show any evident alterations. We confirmed that the targeting of Dag1 with a floxed Neo-cassette did not produce any gross undesired effects.

The effect of the pathological V72I, D109N and T190M missense mutations on the molecular structure of α-dystroglycan.

Dystroglycan (DG) is a highly glycosylated protein complex that links the cytoskeleton with the extracellular matrix, mediating fundamental physiological functions such as mechanical stability of tissues, matrix organization and cell polarity. A crucial role in the glycosylation of the DG α subunit is played by its own N-terminal region that is required by the glycosyltransferase LARGE. Alteration in this O-glycosylation deeply impairs the high affinity binding to other extracellular matrix proteins such as laminins. Recently, three missense mutations in the gene encoding DG, mapped in the α-DG N-terminal region, were found to be responsible for hypoglycosylated states, causing congenital diseases of different severity referred as primary dystroglycanopaties.To gain insight on the molecular basis of these disorders, we investigated the crystallographic and solution structures of these pathological point mutants, namely V72I, D109N and T190M. Small Angle X-ray Scattering analysis reveals that these mutations affect the structures in solution, altering the distribution between compact and more elongated conformations. These results, supported by biochemical and biophysical assays, point to an altered structural flexibility of the mutant α-DG N-terminal region that may have repercussions on its interaction with LARGE and/or other DG-modifying enzymes, eventually reducing their catalytic efficiency.

Structural flexibility of human α-dystroglycan.

Dystroglycan (DG), composed of α and ß subunits, belongs to the dystrophin-associated glycoprotein complex. α-DG is an extracellular matrix protein that undergoes a complex post-translational glycosylation process. The bifunctional glycosyltransferase like-acetylglucosaminyltransferase (LARGE) plays a crucial role in the maturation of α-DG, enabling its binding to laminin. We have already structurally analyzed the N-terminal region of murine α-DG (α-DG-Nt) and of a pathological single point mutant that may affect recognition of LARGE, although the structural features of the potential interaction between LARGE and DG remain elusive. We now report on the crystal structure of the wild-type human α-DG-Nt that has allowed us to assess the reliability of our murine crystallographic structure as a α-DG-Nt general model. Moreover, we address for the first time both structures in solution. Interestingly, small-angle X-ray scattering (SAXS) reveals the existence of two main protein conformations ensembles. The predominant species is reminiscent of the crystal structure, while the less populated one assumes a more extended fold. A comparative analysis of the human and murine α-DG-Nt solution structures reveals that the two proteins share a common interdomain flexibility and population distribution of the two conformers. This is confirmed by the very similar stability displayed by the two orthologs as assessed by biochemical and biophysical experiments. These results highlight the need to take into account the molecular plasticity of α-DG-Nt in solution, as it can play an important role in the functional interactions with other binding partners.

Internal (His)6-tagging delivers a fully functional hetero-oligomeric class II chaperonin in high yield.

Group II chaperonins are ATP-ases indispensable for the folding of many proteins that play a crucial role in Archaea and Eukarya. They display a conserved two-ringed assembly enclosing an internal chamber where newly translated or misfolded polypeptides can fold to their native structure. They are mainly hexadecamers, with each eight-membered ring composed of one or two (in Archaea) or eight (in Eukarya) different subunits. A major recurring problem within group II chaperonin research, especially with the hetero-oligomeric forms, is to establish an efficient recombinant system for the expression of large amounts of wild-type as well as mutated variants. Herein we show how we can produce, in E. coli cells, unprecedented amounts of correctly assembled and active αß-thermosome, the class II chaperonin from Thermoplasma acidophilum, by introducing a (His)6-tag within a loop in the α subunit of the complex. The specific location was identified via a rational approach and proved not to disturb the structure of the chaperonin, as demonstrated by size-exclusion chromatography, native gel electrophoresis and electron microscopy. Likewise, the tagged protein showed an ATP-ase activity and an ability to refold substrates identical to the wild type. This tagging strategy might be employed for the overexpression of other recombinant chaperonins.

Genetic Engineering of Dystroglycan in Animal Models of Muscular Dystrophy.

In skeletal muscle, dystroglycan (DG) is the central component of the dystrophin-glycoprotein complex (DGC), a multimeric protein complex that ensures a strong mechanical link between the extracellular matrix and the cytoskeleton. Several muscular dystrophies arise from mutations hitting most of the components of the DGC. Mutations within the DG gene (DAG1) have been recently associated with two forms of muscular dystrophy, one displaying a milder and one a more severe phenotype. This review focuses specifically on the animal (murine and others) model systems that have been developed with the aim of directly engineering DAG1 in order to study the DG function in skeletal muscle as well as in other tissues. In the last years, conditional animal models overcoming the embryonic lethality of the DG knock-out in mouse have been generated and helped clarifying the crucial role of DG in skeletal muscle, while an increasing number of studies on knock-in mice are aimed at understanding the contribution of single amino acids to the stability of DG and to the possible development of muscular dystrophy.

The Structure of the T190M Mutant of Murine α-Dystroglycan at High Resolution: Insight into the Molecular Basis of a Primary Dystroglycanopathy.

The severe dystroglycanopathy known as a form of limb-girdle muscular dystrophy (LGMD2P) is an autosomal recessive disease caused by the point mutation T192M in α-dystroglycan. Functional expression analysis in vitro and in vivo indicated that the mutation was responsible for a decrease in posttranslational glycosylation of dystroglycan, eventually interfering with its extracellular-matrix receptor function and laminin binding in skeletal muscle and brain. The X-ray crystal structure of the missense variant T190M of the murine N-terminal domain of α-dystroglycan (50-313) has been determined, and showed an overall topology (Ig-like domain followed by a basket-shaped domain reminiscent of the small subunit ribosomal protein S6) very similar to that of the wild-type structure. The crystallographic analysis revealed a change of the conformation assumed by the highly flexible loop encompassing residues 159-180. Moreover, a solvent shell reorganization around Met190 affects the interaction between the B1-B5 anti-parallel strands forming part of the floor of the basket-shaped domain, with likely repercussions on the folding stability of the protein domain(s) and on the overall molecular flexibility. Chemical denaturation and limited proteolysis experiments point to a decreased stability of the T190M variant with respect to its wild-type counterpart. This mutation may render the entire L-shaped protein architecture less flexible. The overall reduced flexibility and stability may affect the functional properties of α-dystroglycan via negatively influencing its binding behavior to factors needed for dystroglycan maturation, and may lay the molecular basis of the T190M-driven primary dystroglycanopathy.

The multiple affinities of α-dystroglycan.

The dystroglycan (DG) adhesion complex is formed by the peripheral α-DG and the transmembrane ß-DG, both originating from the same precursor. α-DG plays a crucial role for tissue stability since it binds with high affinity a variety of proteins and proteoglycans in many different cell types. One common molecular feature of most of the α-DG ligands is the presence of laminin globular (LG) domains that are likely to interact with some of the carbohydrates protruding from the mucin-like region of α-DG. Every tissue is supposed to produce a specific α-DG harboring a particular sugar moiety that will enable it to bind a specific ligand, but often several α-DG ligands are co-expressed within the same tissue. It is therefore very important to assess all these different interactions, ultimately measuring the affinity constants (KDs) underlying them. Herein, we present an updated list of α-DG interactors, including non LG-domains containing ligands, offering both a historic perspective on the original contributions made by several laboratories and an update on the different techniques used and the KD values obtained so far. For the cure of some muscular dystrophies, the reinstatement of a prominent affinity between α-DG and one of its vicarious ligands is becoming an increasingly popular choice for strengthening the basement membrane-tissue connection. An update on the current available information about α- DG's multiple, and often "concomitant" affinities, may be of interest for those wishing to better direct their molecular therapy approaches. A final paragraph is dedicated to comment on the evidence that an increase in affinity is not always advantageous.

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

BACKGROUND: Epitope 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. RESULTS: We 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 ß-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. CONCLUSIONS: Further 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.

Functional diversity of dystroglycan.

During the last 15 years, following its identification and first detailed molecular characterization, the dystroglycan (DG) complex has taken centre stage in biology and biomedicine. Functions in different cells and tissues have been identified for this complex, ranging from its typical role in skeletal muscle as a sarcolemmal stabilizer, highlighted by the recently identified "secondary dystroglycanopathies", to a variety of very diverse functions including embryogenesis, cancer progression, virus particle entry and cell signalling. Such functional promiscuity can be in part explained when considering the multiple domain organization of the two DG subunits, the extracellular alpha-DG and the transmembrane beta-DG, that has been largely scrutinized, but only in part unraveled, exploiting a variety of recombinant and transgenic approaches. Herein, while rapidly recapitulating some of the functions that nowadays can be assigned safely to each DG domain, we also try to envisage a sort of worry list featuring and dwelling on some of the most compelling "mysteries" that should be solved to finally understand DG's functional diversity.