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

Abstract

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)

Binding Properties and Protease Stability of Recombinant Human Nidogen

“…Edman degradation of the major 150-kDa band revealed a single N-terminal sequence (Table 1) corresponding to that predicted from the cDNA construct. The first four residues (APLA) originated from the necessity to introduce a signal peptide into the cDNA (Mayer et al, 1993a). The following sequence SRQE corresponds to human nidogen, starting at position 2 (Nagayoshi et al, 1989).…”

“…The radioligand binding assay for nidogen binding to '251-labelled laminin fragments was that used previously (Mann et al, 1988;Mayer et al, 1993a). Binding of soluble ligands to immobilized ligands followed by a colorimetric enzyme-linked antibody assay was according to an established procedure .…”

“…Binding of soluble ligands to immobilized ligands followed by a colorimetric enzyme-linked antibody assay was according to an established procedure . The recombinant mouse nidogen, laminin-1 fragments P1 and ylIII3-5 (previously B21113-5), mouse collagen IV and the proteoglycan perlecan were those used previously (Fox et al, 1991 ;Battaglia et al, 1992;Mayer et al, 1993a). Here, we use a recently adopted nomenclature for the laminins (Burgeson et al, 1994) in which y l refers to the previous B2 chain and laminin-1 to the previous mouse tumor laminin isoform.…”

“…Mouse nidogen, when obtained as a recombinant product from human cell clones, consists of three globular domains (G1 -G3) which are connected by a rod and a more flexible link (Fox et al, 1991). It shows a high affinity for laminin with a dissociation constant (KJ of approximately 0.5 nM and associates via the G3 domain with a single epidermal-growthfactor-(EGF)-like repeat in the short arm structure of the laminin y l chain (Mayer et al, 1993a). Significant binding was also observed to collagen IV and the basement membrane heparan sulfate proteoglycan perlecan (Battaglia et al, 1992) and attributed to the G2 domain of nidogen .…”

High-affinity binding of the NC1 domain of collagen VII to laminin 5 and collagen IV

“…The way in which laminin 5 connects with collagen IV within the lamina densa, a layer of the basement membrane facing the papillary dermis, is not fully understood, but it has been proposed that there is no direct connection between these two molecules. Even though laminin 5 does not bind to collagen IV directly it has been postulated that these molecules interact indirectly via the laminin 6/nidogen complex, which binds to collagen IV through interaction with nidogen, a 150-kDa glycoprotein [9][10][11].…”