Structural and Genetic Analysis of Laminin-Nidogen Interaction

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Abstract

ABSTRACT:

High-affinity binding of nidogen to laminins involves a single binding site on the laminin γ1 chain and is thus a property shared by almost all laminin isoforms. This binding mediates the connection of laminins to the collagen IV network, perlecan and other proteins and is considered to be an essential step in the stabilization of basement membranes. Nidogen binding has been located to a single LE module (γ1III4) by recombinant analysis. Site-directed mutagenesis and X-ray crystallography demonstrated that three amino acids (Asp, Asn, Val) in loop a of γ1III4 are crucial for binding and are supported by some other residues. A restricted complementary binding region seems to exist on nidogen domain G3. A mutant laminin γ1 chain gene that lacks the region encoding γ1III4 was prepared in mouse embryonic stem (ES) cells by homologous recombination. ES cells homozygous for this defect were shown to assemble laminin-1 into a cruciform structure and to secrete it properly. Yet the mutant laminin failed to associate with nidogen. The mutant ES cells were still able to form embryoid bodies with a similar differentiated histology as the wild type. Immunofluorescence, however, indicated an impaired deposition of nidogen into basement membrane-like structures.

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