A single EGF‐like motif of laminin is responsible for high affinity nidogen binding

• Published in: The EMBO journal Vol. 12; no. 5; pp. 1879 - 1885
• Main Authors: Mayer, U., Nischt, R., Pöschl, E., Mann, K., Fukuda, K., Gerl, M., Yamada, Y., Timpl, R.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group 01.05.1993
• Subjects: Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Animals; Antibodies; Base Sequence; Binding Sites; Biological and medical sciences; Cross-Linking Reagents; Epidermal Growth Factor - chemistry; Epidermal Growth Factor - metabolism; Fundamental and applied biological sciences. Psychology; Genetic Vectors; Glycoproteins; Laminin - chemistry; Laminin - metabolism; Membrane Glycoproteins - metabolism; Mice; Molecular Sequence Data; Oligodeoxyribonucleotides; Peptide Fragments - chemistry; Protein Binding; Protein Conformation; Proteins; Recombinant Proteins - metabolism; Transfection; Proteins; Vertebrata; Mammalia; Laminin; Mouse; Rodentia; Molecular interaction; Glycoproteins; Binding site; Recombinant protein; In vitro
• ISSN: 0261-4189; 1460-2075
• DOI: 10.1002/j.1460-2075.1993.tb05836.x

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 cDNA was amplified by polymerase chain reaction and inserted into a eukaryotic expression vector tagged with a signal peptide. Stably transfected human kidney cell clones were shown to process and secrete the resulting fragment B2III3‐5 in substantial quantities. It possessed high binding activity for recombinant nidogen in ligand assays, with an affinity comparable with that of authentic laminin fragments. In addition, complexes of B2III3‐5 and nidogen could be efficiently converted into a covalent complex by cross‐linking reagents. Proteolytic degradation of the covalent complex demonstrated the association of B2III3‐5 with a approximately 80 residue segment of nidogen domain G3 to which laminin binding has previously been attributed. The correct formation of most of the 12 disulfide bridges in B2III3‐5 was indicated from its protease resistance and the complete loss of cross‐reacting epitopes as well as of nidogen‐binding activity after reduction and alkylation. Smaller fragments were prepared by the same recombinant procedure and showed 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 Drosophila laminin B2 chain.