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Title
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Recombinant nidogen consists of three globular domains and mediates binding of laminin to collagen type IV.
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Authors
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J.W.Fox,
U.Mayer,
R.Nischt,
M.Aumailley,
D.Reinhardt,
H.Wiedemann,
K.Mann,
R.Timpl,
T.Krieg,
J.Engel.
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Ref.
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Embo J, 1991,
10,
3137-3146.
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PubMed id
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Abstract
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Recombinant mouse nidogen and two fragments were produced in mammalian cells and
purified from culture medium without resorting to denaturing conditions. The
truncated products were fragments Nd-I (positions 1-905) comprising the
N-terminal globule and rod-like domain and Nd-II corresponding mainly to the
C-terminal globule (position 906-1217). Recombinant nidogen was
indistinguishable from authentic nidogen obtained by guanidine dissociation from
tumor tissue with respect to size, N-terminal sequence, CD spectra and
immunochemical properties. They differed in protease stability and shape
indicating that the N-terminal domain of the more native, recombinant protein
consists of two globules connected by a flexible segment. This established a new
model for the shape of nidogen consisting of three globes of variable mass
(31-56 kDa) connected by either a rod-like or a thin segment. Recombinant
nidogen formed stable complexes (Kd less than or equal to 1 nM) with laminin and
collagen IV in binding assays with soluble and immobilized ligands and as shown
by electron microscopy. Inhibition assays demonstrated different binding sites
on nidogen for both ligands with different specificities. This was confirmed in
studies with fragment Nd-I binding to collagen IV and fragment Nd-II binding to
laminin fragment P1. In addition, recombinant nidogen but not Nd-I was able to
bridge between laminin or P1 and collagen IV. Formation of such ternary
complexes implicates a similar role for nidogen in the supramolecular
organization of basement membranes.
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