Binding Properties and Protease Stability of Recombinant Human Nidogen

Abstract

Recombinant human nidogen was obtained from transfected kidney cell clones as a 150-kDa protein with a three-globule structure. It was modified by sulfation and 0-glycosylation and a lower level of Nglycosylation than mouse nidogen. Recombinant nidogens of both species were, however, indistinguishable in their affinities for laminin-1 and a recombinant laminin y l chain fragment and showed a similar binding to collagen IV and the heparan sulfate proteoglycan perlecan. The two nidogens were also equivalent in the promotion of ternary complex formation between these ligands, indicating that this function has been conserved during mammalian evolution. Fewer zinc-binding sites could be identified in human nidogen and correlated with a lower capacity of zinc to prevent binding to laminin and collagen IV. Most remarkable was the greater sensitivity of human nidogen to endogenous proteolysis in cell culture, yielding fragments of 90-14s kDa. Studies with several exogenous proteases, including thrombin and leucocyte elastase, showed lack of stability of the N-terminal globular domain GI in contrast to what was found for mouse nidogen. Since such degradation could be important for basement membrane remodelling, this difference between human and mouse may be biologically significant.

Cleavage of Nidogen-1 by Cathepsin S Impairs Its Binding to Basement Membrane Partners

“…The half-maximal reactions occurred around 1–2 nM nid-1, in agreement with the apparent dissociation constant, Kd [34]. Previous studies showed that human nid-1 had weaker affinities for collagen IV and perlecan than for laminin [11]. We next examined the binding of a mixture of nid-1 fragments produced by incubation with catS (Fig.…”

“…It has to be noticed that the 150 kDa form of unprocessed nid-1 is known to be highly susceptible to endogeneous proteolysis (particularly within its N-terminal domain G1) during pre-extraction tissue and that N-terminally cleaved form of ∼90 kDa nid-1 was already described in other murine BM tissues extracts. However, G2 and G3 domains are conserved and still bind to BM partners (type IV collagen, laminins, perlecan, fibulins) [11]. Experiments were also performed with an isolated nidogen/laminin complex from EHS.…”

“…Western blot analyses of the catS mediated hydrolysis of recombinant human and mouse nid-1 (Fig. 3E), which is more resistant to proteolysis [11], revealed significant differences. CatS digests of mouse nid-1 contained only four major bands while numerous fragments were detected in the human nid-1.…”

“…They mainly cleave nid-1 at two sites, one is within the flexible linker between the G1 and G2 domains and the other in the G3 globular domain, to generate stable fragments of 110 kDa and 100 kDa that have different laminin-binding activities [48]. Though free nid-1 is highly susceptible to proteolysis, it seems to be less sensitive when it is complexed with other BM [11], [48], [50]. However, our data shown that catS still rapidly hydrolyzes nid-1, even when it is complexed with laminins, type IV collagen or perlecan.…”

Differential expression of entactin-1/nidogen-1 and entactin-2/nidogen-2 in myogenic differentiation

“…Both entactin isoforms are glycosylated proteins that consist of three globular domains (G1 to G3) connected by a flexible link and a rod (Fox et al, 1991;Mayer et al, 1995;Kimura et al, 1998;Kohfeldt et al, 1998).…”