Synchrotron Radiation as a tool for the investigation of the structural basis of primary dystroglycanopathies.

Dystroglycan (DG) is a glycoprotein complex that links the cytoskeleton with the extracellular matrix and it is composed of two subunits: the extracellular ?-DG and the transmembrane ?-DG. ?-DG is a highly glycosylated protein and its hypoglycosylation weakens its interaction with the extracellular matrix proteins, such as laminins, resulting in a functionally compromised protein. The DG complex undergoes a still poorly understood molecular maturation/targeting pathway, where a precursor is cleaved into ? and ? subunits. Furthermore, the ?-DG subunit is decorated with complex glycans in a multistep glycosylation process. Most of the dystroglycanopathies currently identified are due to an impaired functional state of the enzymes involved in ?-DG maturation (secondary dystroglycanopathies). More recently, missense mutations of the dystroglycan core protein has been identified as the cause of distroglycanopathies (primary dystroglycanopathies) of diverse severity. Indeed, a set of point mutations on the N-terminal region of ?-DG (a.a. 50-313 in mouse) determine the hypoglycosylation of the DG complex, due to the impairment of a key step in ?-DG glycosylation operated by the bifunctional glycosyltransferase LARGE1. Moreover, a point mutation affecting ?-DG, namely the mutation of cysteine 667 to phenylalanine results in a pathological retention of ?-DG at the endoplasmic reticulum level leading to a severe pathological state. With the aim of elucidating the structural implications of the pathological mutations leading to primary distroglycanopathies, we have undertaken a multi-technique study, involving biochemical and biophysical methods as well. Indeed, synchrotron radiation (SR) provides an invaluable tool for the elucidation of the molecular structure of biological macromolecules in both crystals and solution. We have used SR-based X-ray crystallography in order to determine the molecular structure, at the atomic resolution, of ?-DG point mutants. Moreover, SR-based Small Angle X-ray Scattering (SAXS) has been used as a low-resolution probe for the structural organization of ? and ? DG mutants at near-physiological conditions. The results of the SR-based experiments, combined with biochemical and microscopic analysis allowed to shed light on the molecular and structural basis of primary dystroglycanopathies.