THE N-TERMINAL REGION OF ALPHA-DYSTROGLYCAN IS AN AUTONOMOUS GLOBULARDOMAIN

The structure of the N-terminal region of mouse alpha-dystroglycan (DG N) was investigated by expression of two protein fragments (residues 3 0-180 and 30-438) in Escherichia coli cells. Trypsin susceptibility ex periments show the presence of a stable alpha-dystroglycan N-terminal region (approximately from residue 30 to 315). In addition, guanidiniu m hydrochloride (Gdn/HC1) denaturation of DGN-(30-438)-peptide, monito red by means of tryptophan fluorescence, produces a cooperative transi tion typical of folded protein structures. These results strongly sugg est that the alpha-dystroglycan N-terminal is an autonomous folding un it preluding a flexible mucin like region and that its folding is not influenced by the absence of glycosylation. In order to obtain more in formation on the structural features of the N-terminal domain we have also used circular dichroism, analytical sedimentation and electron mi croscopy analysis. Circular dichroic spectra show the absence of typic al secondary structure (e.g. alpha-helix or beta-sheet) and closely re semble 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 e lectron microscopy analysis reveal that the N-terminal domain has a gl obular structure. DGN-(30-438)-peptide does not bind in the nanomolar ranee to an iodinated agrin fragment which binds with high affinity to tissue purified alpha-dystroglycan. No binding was detected also to l aminin. This result suggests that the alpha-dystroglycan N-terminal do main does not contain the binding site to its extracellular matrix bin ding partners. It is less likely than the lack of glycosylation reduce s its binding affinity, because the N-terminal globular domain only co ntains two glycosylation sites.