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Title
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A single EGF-like motif of laminin is responsible for high affinity nidogen binding.
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Authors
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U.Mayer,
R.Nischt,
E.Pöschl,
K.Mann,
K.Fukuda,
M.Gerl,
Y.Yamada,
R.Timpl.
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Ref.
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Embo J, 1993,
12,
1879-1885.
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PubMed id
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Abstract
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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)
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