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1.  Insights from Molecular Dynamics Simulations: Structural Basis for the V567D Mutation-Induced Instability of Zebrafish Alpha-Dystroglycan and Comparison with the Murine Model 
PLoS ONE  2014;9(7):e103866.
A missense amino acid mutation of valine to aspartic acid in 567 position of alpha-dystroglycan (DG), identified in dag1-mutated zebrafish, results in a reduced transcription and a complete absence of the protein. Lacking experimental structural data for zebrafish DG domains, the detailed mechanism for the observed mutation-induced destabilization of the DG complex and membrane damage, remained unclear. With the aim to contribute to a better clarification of the structure-function relationships featuring the DG complex, three-dimensional structural models of wild-type and mutant (V567D) C-terminal domain of alpha-DG from zebrafish were constructed by a template-based modelling approach. We then ran extensive molecular dynamics (MD) simulations to reveal the structural and dynamic properties of the C-terminal domain and to evaluate the effect of the single mutation on alpha-DG stability. A comparative study has been also carried out on our previously generated model of murine alpha-DG C-terminal domain including the I591D mutation, which is topologically equivalent to the V567D mutation found in zebrafish. Trajectories from MD simulations were analyzed in detail, revealing extensive structural disorder involving multiple beta-strands in the mutated variant of the zebrafish protein whereas local effects have been detected in the murine protein. A biochemical analysis of the murine alpha-DG mutant I591D confirmed a pronounced instability of the protein. Taken together, the computational and biochemical analysis suggest that the V567D/I591D mutation, belonging to the G beta-strand, plays a key role in inducing a destabilization of the alpha-DG C-terminal Ig-like domain that could possibly affect and propagate to the entire DG complex. The structural features herein identified may be of crucial help to understand the molecular basis of primary dystroglycanopathies.
doi:10.1371/journal.pone.0103866
PMCID: PMC4117597  PMID: 25078606
2.  Probing the stability of the “naked” mucin-like domain of human α-dystroglycan 
BMC Biochemistry  2013;14:15.
Background
α-Dystroglycan (α-DG) is heavily glycosylated within its central mucin-like domain. The glycosylation shell of α-dystroglycan is known to largely influence its functional properties toward extracellular ligands. The structural features of this α-dystroglycan domain have been poorly studied so far. For the first time, we have attempted a recombinant expression approach in E. coli cells, in order to analyze by biochemical and biophysical techniques this important domain of the α-dystroglycan core protein.
Results
We expressed the recombinant mucin-like domain of human α-dystroglycan in E. coli cells, and purified it as a soluble peptide of 174 aa. A cleavage event, that progressively emerges under repeated cycles of freeze/thaw, occurs at the carboxy side of Arg461, liberating a 151 aa fragment as revealed by mass spectrometry analysis. The mucin-like peptide lacks any particular fold, as confirmed by its hydrodynamic properties and its fluorescence behavior under guanidine hydrochloride denaturation. Dynamic light scattering has been used to demonstrate that this mucin-like peptide is arranged in a conformation that is prone to aggregation at room temperature, with a melting temperature of ~40°C, which indicates a pronounced instability. Such a conclusion has been corroborated by trypsin limited proteolysis, upon which the protein has been fully degraded in less than 60 min.
Conclusions
Our analysis indirectly confirms the idea that the mucin-like domain of α-dystroglycan needs to be extensively glycosylated in order to reach a stable conformation. The absence/reduction of glycosylation by itself may greatly reduce the stability of the dystroglycan complex. Although an altered pattern of α-dystroglycan O-mannosylation, that is not significantly changing its overall glycosylation fraction, represents the primary molecular clue behind currently known dystroglycanopathies, it cannot be ruled out that still unidentified forms of αDG-related dystrophy might originate by a more substantial reduction of α-dystroglycan glycosylation and by its consequent destabilization.
doi:10.1186/1471-2091-14-15
PMCID: PMC3704865  PMID: 23815856
Dystroglycan; Dynamic light scattering; Capillary electrophoresis; Mass spectrometry
3.  Dystroglycan is associated to the disulfide isomerase ERp57 
Experimental Cell Research  2012;318(19):2460-2469.
Dystroglycan (DG) is an extracellular receptor composed of two subunits, α-DG and β-DG, connected through the α-DG C-terminal domain and the β-DG N-terminal domain. We report an alanine scanning of all DG cysteine residues performed on DG-GFP constructs overexpressed in 293-Ebna cells, demonstrating that Cys-669 and Cys-713, both located within the β-DG N-terminal domain, are key residues for the DG precursor cleavage and trafficking, but not for the interaction between the two DG subunits. In addition, we have used immunprecipitation and confocal microscopy showing that ERp57, a member of the disulfide isomerase family involved in glycoprotein folding, is associated and colocalizes immunohistochemically with β-DG in the ER and at the plasma membrane of 293-Ebna cells. The β-DG–ERp57 complex also included α-DG. DG mutants, unable to undergo the precursor cleavage, were still associated to ERp57. β-DG and ERp57 were also co-immunoprecipitated in rat heart and kidney tissues. In vitro, a mutant ERp57, mimicking the reduced form of the wild-type protein, interacts directly with the recombinant N-terminal domain of both α-DG and β-DG with apparent dissociation constant values in the micromolar range. ERp57 is likely to be involved in the DG processing/maturation pathway, but its association to the mature DG complex might also suggest some further functional role that needs to be investigated.
Highlights
► Cys-669 and Cys-713 are key residues for dystroglycan precursor cleavage. ► ERp57 is co-immunopurified with dystroglycan. ► ERp57 co-localizes with dystroglycan in the ER and at the plasma membrane. ► Recombinant ERp57 binds directly to dystroglycan recombinant domains.
doi:10.1016/j.yexcr.2012.07.006
PMCID: PMC3459099  PMID: 22814252
DG, dystroglycan; pre-DG, dystroglycan precursor; NEM, N-ethylmaleimide; DTT, dithiothreitol; sWGL, succinylated wheat germ lectin; Dystroglycan; ERp57; Immunoprecipitation; Fluorescence microscopy; Solid-phase binding assay
4.  Insertion of a myc-tag within α-dystroglycan domains improves its biochemical and microscopic detection 
BMC Biochemistry  2012;13:14.
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 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.
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.
doi:10.1186/1471-2091-13-14
PMCID: PMC3432625  PMID: 22835149
5.  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.
doi:10.2174/1874205X01105010068
PMCID: PMC3204415  PMID: 22046204
Dystroglycan; limb-girdle muscular dystrophy; distal myopathy; Miyoshi myopathy; secondary dystroglycanopathies; dystrophin-glycoprotein complex.

Results 1-5 (5)