RICERCA ITALIANA     

Functional basis and structural aspects of the extracellular domain of beta-dystroglycan, a "natively unfolded" protein, involved in neuromuscular pathologies.

Università degli Studi di Roma "Tor Vergata"

Abstract

Dystroglycan is an adhesion molecule constituted by two subunits, alpha and beta, that forms a linkage between the cytoskeleton and the extracellular matrix. The high affinity interaction between the two subunits of dystroglycan is not covalent and is to be considered an important factor that influence the stability of the entire complex of dystroglycan. From a biomedical point of view, the importance of dystroglycan dramatically emerges in several forms of muscular dystrophy, where abnormalities in dystroglycan targeting to the sarcolemma lead to instability and progressive weakness of the muscle fibers. The main characteristic of the extracellular domain of beta-dystroglycan is to be a protein belonging to the family of the so called "natively unfolded". Although this class of proteins does not present a classical secondary and three-dimensional structure, it maintains a high binding selectivity. The main goal of the present project is the biochemical and biophysical characterization of a natively unfolded proteic domain in order to identify the regions involved in its functions. Using the structural characterization of the extracellular domain of beta-dystroglycan, obtained by high field NMR spectroscopy, a structural study of such domain bound to alpha-dystroglycan will be carried out. Further, point-mutations of both, beta and alpha subunits, will be produced in order to identify with higher detail the residues directly involved in the interation, using techniques >>>

Principal Investigator

Maurizio PACI Università degli Studi di ROMA "Tor Vergata"

Research Objectives

_Aims
The main goal of the present project is to obtain information on the structure and function of a peptide belonging to the class of natively unfolded proteins, the extracellular domain of beta-DG. An approach based on techniques from biochemistry (recombinant expression and mutagenesis), structural biology (NMR), and cellular biology (transfection and immunofluorescence of cells and myoblasts in colture) will be used. As DG is involved in a number of biological and pathologic process, biological samples from patients affected by muscular and metabolic pathologies will be analysed, in order to study the role of the natively unfolded domain of beta-DG in the stability of the entire DG complex and in the possible ethiology of diseases.
_Strategy and methodology
Using the backbone assignment of beta-DG(654-750) already obtained [1], a series of spectra will be carried out in order to assign the resonances of beta-DG(654-750), in complex with a synthetic peptide representing the binding epitope of alpha-DG [2-3]. Afterwards, the three-dimensional solution structure of the complex will be tackled, through NOEs collection and molecular dynamic simulations (Unit 1, University of "Tor Vergata"). The quantitative production of recombinant proteins, doubly labeled with 15N and 13C, will involve the use of expression and purification techniques already developed in the laboratory of the Istituto di Biochimica e Biochimica Clinica of the Catholic University >>>

First Results

First year
1) identification of the natively unfolded beta-DG subunit amino acids which are crucial for the alpha- and beta-subunits interaction.
2) DG complex characterization in human patients affected by neuromuscular and metabolic diseases.

Second year
1) identification of the single-nucleotide polymorphisms in the DG gene, extracted by genotyped patients.
2) determination of the three-dimensional structure of the beta-DG(654-750) and alfa-DG(550-585) complex.Second year:

a) Identification of the regions and conformation in the interaction between a-DG and b-DG
b) Solution structure of the complex between the two proteins
c) Identification of the sequential and structural characteristics of b-DG in the frame of "natively unfolded" protein studies.
d) The identification of the a-DG binding epitope of b-DG will represent a remarkable advancement in the knowledge of the molecular interface between DG subunits and it could be a crucial step to achieve results useful for a future rationale design of efficient therapies for either muscular dystrophies, or also to prevent those infections depending on DG targeting.

Timescale

24 months

National and international background

_PURIFICATION AND CLONING OF DG
Dystroglycan (DG) was identified and successively cloned from microsomes of rabbit skeletal muscle in the laboratories of Kevin Campbell in Iowa City (USA) in 1992 [4]. DG (895 amino acids) is formed by two subunits, alpha (a-DG) and beta (b-DG), and it is an important component of the glycoprotein complex associated to distrophin (DGC), containing sarcoglycans, sarcospan, syntrophins and dystrobrevins [4-5]. It is interesting that in 1987 Smalheiser and Schwartz had already identified from the ship brain a protein of about 120 kDa (cranin), that is able to bind to the laminin, and that corresponds to alpha-DG [6].
_SUBUNITS OF DG
DG is encoded by a single gene [4] and is highly conserved in higher vertebrates. Recently, DG orthologues have also been identified in invertebrates [7-8]. DG is produced as a unique polipeptide chain that is successively cut in two subunits a and beta-DG, at the level of endoplasmatic reticulum [9]. alpha-DG is a heavily glycosylated peripheral membrane protein which binds a number of extracellular molecules such as laminin, agrin, perlecan, neurexin and biglycan [5].
DG is expressed in a wide variety of tissues other than muscle: epithelia and the central and peripheral nervous systems [10]. The predicted molecular weight of alpha-DG core protein is about 72 kDa but when the protein is isolated from different tissues it shows size heterogeneity.
These differences are probably >>>