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 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Max-Planck-Institut für Biochemie, Martinsried, Germany.
U
U
Mayer
R
R
Nischt
E
E
Pöschl
K
K
Mann
K
K
Fukuda
M
M
Gerl
Y
Y
Yamada
R
R
Timpl
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001483:Base Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents; D004815:Epidermal Growth Factor / Q000737:chemistry / Q000378:metabolism*; D005822:Genetic Vectors; D007797:Laminin / Q000737:chemistry / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection
D000906:Antibodies; D003432:Cross-Linking Reagents; D007797:Laminin; D008562:Membrane Glycoproteins; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D011994:Recombinant Proteins; C029967:nidogen; D004815:Epidermal Growth Factor