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Mutagenesis at the α–β interface impairs the cleavage of the dystroglycan precursor
Author(s) -
Sciandra Francesca,
Bozzi Manuela,
Morlacchi Simona,
Galtieri Antonio,
Giardina Bruno,
Brancaccio Andrea
Publication year - 2009
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2009.07196.x
Subject(s) - ectodomain , dystroglycan , beta (programming language) , laminin , alanine scanning , microbiology and biotechnology , biochemistry , biology , chemistry , mutagenesis , mutation , cell , receptor , computer science , gene , programming language
The interaction between α‐dystroglycan (α‐DG) and β‐dystroglycan (β‐DG), the two constituent subunits of the adhesion complex dystroglycan, is crucial in maintaining the integrity of the dystrophin–glycoprotein complex. The importance of the α–β interface can be seen in the skeletal muscle of humans affected by severe conditions, such as Duchenne muscular dystrophy, where the α–β interaction can be secondarily weakened or completely lost, causing sarcolemmal instability and muscular necrosis. The reciprocal binding epitopes of the two subunits reside within the C‐terminus of α‐DG and the ectodomain of β‐DG. As no ultimate structural data are yet available on the α–β interface, site‐directed mutagenesis was used to identify which specific amino acids are involved in the interaction. A previous alanine‐scanning analysis of the recombinant β‐DG ectodomain allowed the identification of two phenylalanines important for α‐DG binding, namely F692 and F718. In this article, similar experiments performed on the α‐DG C‐terminal domain pinpointed two residues, G563 and P565, as possible binding counterparts of the two β‐DG phenylalanines. In 293‐Ebna cells, the introduction of alanine residues instead of F692, F718, G563 and P565 prevented the cleavage of the DG precursor that liberates α‐ and β‐DG, generating a pre‐DG of about 160 kDa. This uncleaved pre‐DG tetramutant is properly targeted at the cell membrane, is partially glycosylated and still binds laminin in pull‐down assays. These data reinforce the notion that DG processing and its membrane targeting are two independent processes, and shed new light on the molecular mechanism that drives the maturation of the DG precursor. Structured digital abstract• MINT‐7214494 : alpha DG (uniprotkb: Q62165 ) binds ( MI:0407 ) to beta DG (uniprotkb: Q62165 ) by solid phase assay ( MI:0892 ) • MINT‐7214516 : laminin (uniprotkb: P19137 ) binds ( MI:0407 ) to beta DG (uniprotkb: Q62165 ) by pull down ( MI:0096 )