A SINGLE EGF-LIKE MOTIF OF LAMININ IS RESPONSIBLE FOR HIGH-AFFINITY NIDOGEN BINDING

A major nidogen binding site of mouse laminin was previously localized to about three EGF-like repeats (Nos 3-5) of its B2 chain domain III [M.Gerl et al. (1991) Eur. J. Biochem., 202, 167]. The corresponding c DNA was amplified by polymerase chain reaction and inserted into a euk aryotic expression vector tagged with a signal peptide. Stably transfe cted human kidney cell clones were shown to process and secrete the re sulting fragment B2III3-5 in substantial quantities. It possessed high binding activity for recombinant nidogen in ligand assays, with an af finity comparable with that of authentic laminin fragments. In additio n, complexes of B2III3-5 and nidogen could be efficiently converted in to a covalent complex by cross-linking reagents. Proteolytic degradati on of the covalent complex demonstrated the association of B2III3-5 wi th a approximately 80 residue segment of nidogen domain G3 to which la minin binding has previously been attributed. The correct formation of most of the 12 disulfide bridges in B2III3-5 was indicated from its p rotease resistance and the complete loss of cross-reacting epitopes as well as of nidogen-binding activity after reduction and alkylation. S maller fragments were prepared by the same recombinant procedure and s howed that combinations of EGF-like repeats 3-4 and 4-5 and the single repeat 4 but not repeats 3 or 5 possess full nidogen-binding activity . This identifies repeat 4 as the only binding structure. The sequence of repeat 4 is well conserved in the human and in part in the Drosoph ila laminin B2 chain. It was further shown that antibodies against B2I II3-5 inhibit laminin binding to nidogen, indicating that repeat 4 rep resents the only high affinity binding site of laminin.