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1.  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
2.  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
3.  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

Results 1-3 (3)