Concerted mutation of Phe residues belonging to the beta-dystroglycan ectodomain strongly inhibits the interaction with alpha-dystroglycan in vitro.
The dystroglycan adhesion complex consists of two noncovalently interacting proteins: alpha-dystroglycan, a peripheral extracellular subunit that is extensively glycosylated, and the transmembrane beta-dystroglycan, whose cytosolic tail interacts with dystrophin, thus linking the F-actin cytoskeleton to the extracellular matrix. Dystroglycan is thought to play a crucial role in the stability of the plasmalemma, and forms strong contacts between the extracellular matrix and the cytoskeleton in a wide variety of tissues. Abnormal membrane targeting of dystroglycan subunits and/or their aberrant post-translational modification are often associated with several pathologic conditions, ranging from neuromuscular disorders to carcinomas. A putative functional hotspot of dystroglycan is represented by its intersubunit surface, which is contributed by two amino acid stretches: approximately 30 amino acids of beta-dystroglycan (691-719), and approximately 15 amino acids of alpha-dystroglycan (550-565). Exploiting alanine scanning, we have produced a panel of site-directed mutants of our two consolidated recombinant peptides beta-dystroglycan (654-750), corresponding to the ectodomain of beta-dystroglycan, and alpha-dystroglycan (485-630), spanning the C-terminal domain of alpha-dystroglycan. By solid-phase binding assays and surface plasmon resonance, we have determined the binding affinities of mutated peptides in comparison to those of wild-type alpha-dystroglycan and beta-dystroglycan, and shown the crucial role of two beta-dystroglycan phenylalanines, namely Phe692 and Phe718, for the alpha-beta interaction. Substitution of the alpha-dystroglycan residues Trp551, Phe554 and Asn555 by Ala does not affect the interaction between dystroglycan subunits in vitro. As a preliminary analysis of the possible effects of the aforementioned mutations in vivo, detection through immunofluorescence and western blot of the two dystroglycan subunits was pursued in dystroglycan-transfected 293-Ebna cells.
Bozzi
Manuela
M
Istituto di Biochimica e Biochimica Clinica, Università Cattolica del Sacro Cuore, Largo Francesco Vito 1, 00168 Rome, Italy.
Sciandra
Francesca
F
Ferri
Lorenzo
L
Torreri
Paola
P
Pavoni
Ernesto
E
Petrucci
Tamara C
TC
Giardina
Bruno
B
Brancaccio
Andrea
A
D002460:Cell Line; D049030:Dystroglycans / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D010649:Phenylalanine / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D010446:Peptide Fragments; D021122:Protein Subunits; D011994:Recombinant Proteins; D049030:Dystroglycans; D010649:Phenylalanine