title: The N-terminal region of alpha-dystroglycan is an autonomous globular domain.
abstract: The structure of the N-terminal region of mouse alpha-dystroglycan (DGN) was investigated by expression of two protein fragments (residues 30-180 and 30-438) in Escherichia coli cells. Trypsin susceptibility experiments show the presence of a stable alpha-dystroglycan N-terminal region (approximately from residue 30 to 315). In addition, guanidinium hydrochloride (Gdn/HCl) denaturation of DGN-(30-438)-peptide, monitored by means of tryptophan fluorescence, produces a cooperative transition typical of folded protein structures. These results strongly suggest that the alpha-dystroglycan N-terminal is an autonomous folding unit preluding a flexible mucin-like region and that its folding is not influenced by the absence of glycosylation. In order to obtain more information on the structural features of the N-terminal domain we have also used circular dichroism, analytical sedimentation and electron microscopy analysis. Circular dichroic spectra show the absence of typical secondary structure (e.g. alpha-helix or beta-sheet) and closely resemble those recorded for loop-containing proteins. This is consistent with a sequence similarity of the alpha-dystroglycan domain with the loop-containing protein elastase. Analytical ultracentrifugation and electron microscopy analysis reveal that the N-terminal domain has a globular structure. DGN-(30-438)-peptide does not bind in the nanomolar range to an iodinated agrin fragment which binds with high affinity to tissue purified alpha-dystroglycan. No binding was detected also to laminin. This result suggests that the alpha-dystroglycan N-terminal domain does not contain the binding site to its extracellular matrix binding partners. It is less likely than the lack of glycosylation reduces its binding affinity, because the N-terminal globular domain only contains two glycosylation sites.
authors.affiliation: Department of Biophysical Chemistry, Biozentrum, University of Basel, Switzerland.
authors.forename: A
authors.initials: A
authors.lastname: Brancaccio
authors.forename: T
authors.initials: T
authors.lastname: Schulthess
authors.forename: M
authors.initials: M
authors.lastname: Gesemann
authors.forename: J
authors.initials: J
authors.lastname: Engel
mesh_terms: D018171:Agrin / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002645:Chickens; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D003598:Cytoskeletal Proteins / Q000737:chemistry* / Q000378:metabolism*; D049030:Dystroglycans; D016189:Dystrophin / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006031:Glycosylation; D019791:Guanidine; D006146:Guanidines; D007797:Laminin / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000378:metabolism*; D051379:Mice; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D013050:Spectrometry, Fluorescence; D014357:Trypsin / Q000378:metabolism; D014461:Ultracentrifugation
chemical_list: D018171:Agrin; D003598:Cytoskeletal Proteins; D016189:Dystrophin; D006146:Guanidines; D007797:Laminin; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D049030:Dystroglycans; D014357:Trypsin; D019791:Guanidine