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ABSTRACT: Dystroglycan (DG) is a cell surface receptor consisting of two subunits: alpha-dystroglycan, extracellular and highly glycosylated, and beta-dystroglycan, spanning the cell membrane. It is a pivotal member of the dystrophin-glycoprotein complex and is involved in a wide variety of important cellular processes such as the stabilization of the muscle fiber sarcolemma or the clustering of acetylcholine receptors. We report the 2.3-A resolution crystal structure of the murine skeletal muscle N-terminal alpha-DG region, which confirms the presence of two autonomous domains; the first finally identified as an Ig-like and the second resembling ribosomal RNA-binding proteins. Solid-phase laminin binding assays show the occurrence of protein-protein type of interactions involving the Ig-like domain of alpha-DG.
Journal of Biological Chemistry 11/2004; 279(43):44812-6. · 4.77 Impact Factor
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ABSTRACT: alpha-dystroglycan is a cell surface receptor that is expressed in many tissues including the nervous system. The study shows that a recombinant, non-glycosylated N-terminal fragment of alpha-dystroglycan comprising residues 30 to 315 [alphaDG (30-315)] bound to laminin-2/-4 and laminin-1, fibronectin and fibrinogen, all molecules highly upregulated in the regenerating peripheral nerve. The interaction was concentration dependent and saturable and could not be inhibited by heparin suggesting only minor involvement of sulfated carbohydrate moieties. In contrast to published data, addition of bivalent cations increased the binding affinity by only ten fold.alphaDG (30-315) promotes neurite extension of PC12 cells in a similar amount as described for laminin isoforms and could be inhibited in a concentration dependent manner by alphaDG (30-315) itself, soluble laminin-1, partially by heparin, EDTA, and an RGD-peptide. Furthermore, co-immunoprecipitations between alpha-dystroglycan and beta1-integrin from PC12 cell surfaces suggested complex interactions between neuronal dystroglycan, integrins, and the ECM that induce neurite extension in vitro.
Molecular and Cellular Neuroscience 01/2004; 24(4):1062-73. · 3.66 Impact Factor
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ABSTRACT: Death effector domains (DEDs) are protein-protein interaction domains found in the death inducing signaling complex (DISC). Performing a structure-based alignment of all DED sequences we identified a region of high diversity in alpha-helix 3 and propose a classification of DEDs into class I DEDs typically containing a stretch of basic residues in the alpha-helix 3 region whereas DEDs of class II do not. Functional assays using mutants of Fas-associated death domain revealed that this basic region influences binding and recruitment of caspase-8 and cellular FLICE inhibitor protein to the DISC.
FEBS Letters 10/2002; 527(1-3):250-4. · 3.54 Impact Factor
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ABSTRACT: Abstract : Several studies indicate that cell adhesion molecules have to be clustered on the cell surface to engage in adhesive functions. We investigated adhesive functions of clustered versus monomeric L1 extracellular parts in vitro to distinguish how clustering affects ligand binding and promotion of neurite outgrowth. Trimeric L1 was recombinantly expressed and covalently assembled by the cartilage matrix protein's coiled-coil domain. Trimeric L1 has an apparent molecular mass of ~380 kDa in the nonreduced form and ~130 kDa in the reduced form. Rotary shadowing electron micrographs of trimeric L1 revealed a rod-like shape terminating in three globular domains. Monomeric L1 assumes a horseshoe shape of domains Ig I-IV followed by a rod-like structure consisting of Ig V and VI and fibronectin type III 1-5. Circular dichroism measurements showed that the secondary structure consists of β-sheets. Trimeric L1 binds to itself, to monomeric L1, to laminin-1, and to α5β1 integrin in a concentration-dependent manner. In contrast, binding of monomeric L1 could only be saturated with itself but not with laminin-1 and with α5β1 integrin. Promotion of neurite outgrowth from PC12 cells cultured on adsorbed trimeric L1 was increased by 100%, whereas on monomeric L1 the increase was only 50% over the control value. Promotion of neurite outgrowth from PC12 cells was specifically inhibited in a concentration-dependent manner by a polyclonal antibody against L1. These findings show that clustering of only three extracellular domains increases considerably L1's binding affinity to different ligands and enhances neurite outgrowth, suggesting that adhesive functions of L1 on the cell surface depend on cluster formation.
Journal of Neurochemistry 12/2001; 75(1):336 - 346. · 4.06 Impact Factor
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ABSTRACT: Recently, we demonstrated that the N-terminal region of mouse α-dystroglycan represents an autonomously folding globular domain, organized into at least two subdomains (Brancaccio et al., Eur. J. Biochem. 246, 166–172, 1997). We have now found a similarity between a part of the α-dystroglycan N-terminal sequence (approximately from position 80 to 180) and several protein sequences belonging to the immunoglobulin κ family. Moreover, we have recombinantly expressed and purified a 31 kDa protein fragment which matches the entire α-dystroglycan N-terminal globular domain. To prevent the action of bacterial endogenous proteases and/or thrombin, which cleaves the protein into two fragments at an Arg-Ala trypsin-sensitive site in positions 168–169, we have introduced a single mutation (Arg168 → His), thus making the whole domain more stable and suitable for crystallization. Crystals of this mutant protein were obtained by vapor diffusion using the hanging drop technique, and they diffract to 0.28 nm Bragg spacing.
Matrix Biology.
