Sites of nidogen cleavage by proteases involved in tissue homeostasis and remodelling

Abstract

The cleavage of recombinant mouse nidogen in its native form was examined with granulestored proteases (leucocyte elastase, mast-cell chymase), blood proteases (thrombin, plasmin, kallikrein), matrix metalloproteinases (stromelysin, matrilysin, collagenases) and, for comparison, with trypsin and the endoproteinase Glu-C. More than 50 major cleavage sites were identified by Edman degradation of several large fragments and smaller peptides. The data show an almost exclusive localization of protease-sensitive sites to the flexible segment, connecting the N-terminal globular domains G1 and G2, and within the C-terminal, laminin-binding domain G3. Domains G1, G2 and the rod-like segment were much more stable against proteolysis. Kinetic analysis indicated a fast cleavage of several different sites in the link region followed by destruction of G3 but this was to some extent variable depending on the particular protease. Leucocyte elastase was identified as the most active protease in the cleavage of nidogen whilst stromelysin, matrilysin, plasmin and kallikrein were of distinctly lower activity. No cleavage could be detected with interstitial collagenase and gelatinase A. The peptide analyses also allowed the location of two disulfide bridges within the G3 domain. Complex formation between nidogen and laminin fragments caused some protection against cleavage by thrombin, leucocyte elastase and stromelysin particularly in domain G3. The data indicate a relatively uniform cleavage pattern of nidogen which may be relevant in the context of proteidligand-binding activities associated with domains G2 and G3. The proteolytic processes involved in remodelling and the cellular penetration of basement membranes could therefore be essential for the modulation of the mediator function of nidogen.

Binding Properties and Protease Stability of Recombinant Human Nidogen

“…4). The release of these fragments was previously shown to be due to an extensive degradation of mouse nidogen G3 (Mayer et al, 1993b). A further difference was the partial formation of disulfide-linked dimers in recombinant human nidogen.…”

“…2). They shared similar 80-100-kDa fragments, shown previously to correspond to central and C-terminal domains of mouse nidogen (Mayer et al, 1993b). Human nidogen, however, lacked stable N-terminal fragments, which have a size of 30-65 kDa in mouse nidogen (Fig.…”

“…Digestion with endoproteinase Glu-C converted the initial N-terminal65-kDa fragment of human nidogen to a 20-kDa component starting at sequence position 64 (Table 1). A further 30-kDa fragment started at position 348 in front of domain G2 (Table l), indicating cleavage between G2 and the rod domain, which was not observed with mouse nidogen (Mayer et al, 1993b).…”

“…Nidogen dissolved in 0.05 M Tris/HCl, pH 7.4, 0.1 M NaCl and 2 mM CaC1, (0.2 mg/ ml) was digested at 37°C for various periods at an enzyme/ substrate ratio of 3 : 100, except for leucocyte elastase which was used at an enzyme/substrate ratio of 1 : 1000. Preparative thrombin digestion and purification of peptides followed previous protocols (Mayer et al, 1993b). Zinc-binding peptides were identified by passing a trypsin digest of reduced and alkylated nidogen over a zinc-loaded chelating column (HR 10/2, Pharmacia) followed by reverse-phase chromatography .…”

Gelatinase-mediated migration and invasion of cancer cells

“…Indeed, some substrates show over 200-fold selectivity towards MMP-2 Kridel et al, 2001). (Morodomi et al, 1992;Okada et al, 1990) Native collagen type: I yes no (Aimes and Quigley, 1995) III yes no (Berton et al, 2000) IV yes yes (Morodomi et al, 1992) V yes yes (Morodomi et al, 1992) VII yes (Seltzer et al, 1989) X yes (Cole et al, 1993) XI yes (Smith et al, 1991) XVIII yes (Ferreras et al, 2000) Aggrecan yes yes (Fosang et al, 1992) BM-40/SPARC/ Osteonectin yes yes (Sasaki et al, 1997) Brevican yes no Decorin yes no (Imai et al, 1997) Elastin yes yes Entactin/nidogen no yes (Mayer et al, 1993;Sires et al, 1993) Fibrillin yes yes (Ashworth et al, 1999) Fibrin yes (Lelongt et al, 2001) Fibrinogen yes yes (Bini et al, 1996;Lelongt et al, 2001) Fibronectin yes no (Okada et al, 1990) Laminin yes yes (Giannelli et al, 1997;Morodomi et al, 1992) Link protein yes yes (Nguyen et al, 1993) NG2 proteoglycan yes (Larsen et al, 2003) Neurocan yes (Turk et al, 2001) Tenascin yes no (Siri et al, 1995) Vitronectin yes yes (Imai et al, 1995) α2-macroglobulin yes yes (Arbelaez et al, 1997) αB-crystallin yes (Starckx et al, 2003) Amyloid protein precursor yes (LePage et al, 1995) Big endothelin-1 yes yes (Fernandez-Patron et al, 2002) Calcitonin gene-related peptide (CGRP) yes (Fernandez-Patron et al, 2000) Complement protein C1q yes yes (Ruiz et al, 1999) Connective tissue-activating peptide-III (CTAP-III) yes (Van den Eph B1 tyrosine kinase receptor yes no Epithelial-cell derived neutrophil activating pept...…”