Abstract
Backgroundα-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.ResultsWe 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.ConclusionsOur 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.
Highlights
Background αDystroglycan (DG) is the peripheral subunit of the DG complex that lies at the basement membrane/sarcolemma cellular crossroad and that is important for muscle fibers’ stability. α-DG glycosylation pattern is considered crucial for its function in terms of laminin binding affinity, and a disruption of the α-DG/laminin network is at the basis of a group of diseases spanningThe DG complex is composed of two subunits (α and β) that are liberated upon an early processing step of a unique precursor of 895 aa in humans, that is thought to take place within the endoplasmic reticulum right after translation
The mucin-like domain cannot be quantitatively obtained in a glycosylated fashion from eukaryotic cells and we decided to apply our E. coli-based recombinant approach already used for the expression and purification of other DG domains [1,13]
The primary structure of the portion of human α-DG under analysis, including the Arg-Val site that we have found to be sensitive to proteases, is reported in Additional file 1: Table S1
Summary
The DG complex is composed of two subunits (α and β) that are liberated upon an early processing step of a unique precursor of 895 aa in humans (pre-DG), that is thought to take place within the endoplasmic reticulum right after translation. The whole maturation process of α-DG is known to be highly regulated and to take place in the typical subcellular locations, where multiple enzymes are required for the post-translational modification of the α-DG core protein [4]. A long list of nonsense or missense mutations affecting these enzymes seems to lead to muscular dystrophy phenotypes whose major molecular trait is believed to be a reduction of α-DG functionality, depending on the alteration of its glycosylation shell. The general concept is that a hypoglycosylated α-DG would be less active in binding laminin-2, or other matrix binding partners, provoking a reduction of the overall stability of the sarcolemma preceding the onset of inflammation, fibrosis and necrosis typical of many severe muscular dystrophies
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