Characterization of proteolytic fragments of the laminin‐nidogen complex and their activity in ligand‐binding assays

Abstract

Some 12 new nidogen and laminin fragments were purified from elastase, thrombin and trypsin digests and characterized by their sizes (22 kDa to > 300 kDa), subunit patterns on electrophoresis, partial amino acid sequences, content of specific epitopes and their binding to laminin or nidogen structures in radioligand assays. This permitted the various fragments to be ordered along the dumbbell-shaped structure of nidogen and to compare them with previously described nidogen fragments arising by endogenous proteolysis. Two nidogen fragments (E-50, E-90; 50 kDa and 90 kDa) remain associated with a large laminin fragment in elastase digests of the complex and could be dissociated with 2 M guanidine · HCl. Recombination studies demonstrated K_d= 10–20 nM for this interaction. Nidogen fragments devoid of binding activity included the tryptic peptide T-40 (40 kDa) corresponding to the rod-like domain and several larger fragments extending more to the N-terminus of nidogen. An N-terminal thrombin fragment of about 50 kDa was also inactive. Together the data show a lack of laminin binding to the N-terminal globule and rod of nidogen and provide indirect evidence that this activity is located within or close to its C-terminal globular domain. Nidogen-binding structures of laminin were obtained as two large fragments (> 300 kDa), P1X and E1X. They correspond to the short arm structure of laminin with one (E1X) or two (P1X) arms decreased in size to the inner rod-like segment. Shortening in E1X is mainly due to the B1 chain segment including the central globular domain which was identified as a new laminin fragment E10. Binding of E1X and P1X to nidogen was comparable to that of laminin while much lower activity was found for other laminin fragments. A 10-fold lower binding potential was also observed for the laminin-nidogen complex whose structure can now be defined in more precise molecular terms.