Localization of a major nidogen‐binding site to domain III of laminin B2 chain

Self Cite

180

140

A large, low-density form of heparan sulfate proteoglycan was isolated from the EngelbrethHolm-Swarm (EHS) tumor and demonstrated to bind in immobilized-ligand assays to laminin fragment E3, collagen type IV, fibronectin and nidogen. The first three ligands mainly recognize the heparan sulfate chains, as shown by inhibition with heparin and heparan sulfate and by the failure to bind to the proteoglycan protein core. Nidogen, obtained from the EHS tumor or in recombinant form, binds exclusively to the protein core in a heparin-insensitive manner. Studies with other laminin fragments indicate that the fragment E3 possesses a unique binding site of laminin for the proteoglycan. A major binding site of nidogen was localized to its central globular domain G2 by using overlapping fragments. This allows for the formation of ternary complexes between laminin, nidogen and proteoglycan, suggesting a key role for nidogen in basement-membrane assembly.Evidence is provided for a second proteoglycan-binding site in the C-terminal globule G3 of nidogen, but this interaction prevents the formation of such ternary complexes. Therefore, the G3-mediated nidogen binding to laminin and proteoglycan are mutually exclusive.Basement membranes are large, extracellular protein matrices composed of networks of collagen IV and/or laminin [l] with which several more specific components (nidogen, BM-40) are associated. These complex scaffolds are located close to cells and possess a distinct repertoire of biological activities [2, 31. Ubiquitous to these matrices are also heparan sulfate proteoglycans [2, 31 that provide major polyanionic sites implicated in filtration control 141. An abundant proteoglycan of low buoyant density has been isolated from the mouse Engelbreth-Holm-Swarm (EHS) tumor and other sources and characterized [5 -81. It is composed of an approximately 500-kDa polypeptide chain with three heparan sulfate chains attached to one end, as seen by electron microscopy. The protein core is elongated and consists of 5-7 globular domains of variable size connected by short rod-like segments [5]. Some smaller heparan sulfate proteoglycans may also exist in basement membranes [6, 7, 9, 101 but their precise relationship to the large major form has not yet been entirely clarified.The complete amino acid sequence of the protein core has been recently deduced from cDNA clones for the large proteoglycan from mouse [ll, 121 and human [13]. The data agree with the electron microscopical analyses [5] and indicate a complex array of five large domains, each of which is composed of several repeating protein motifs. It was suggested that the N-terminal domain I possesses the heparan-sulfateattachment sites. Domains I1 and I11 consist of cysteine-rich repeats similar to epidermal growth factor (EGF) and of

Section: Discussionmentioning

confidence: 99%

Basement‐membrane heparan sulfate proteoglycan binds to laminin by its heparan sulfate chains and to nidogen by sites in the protein core

Battaglia

Mayer

Aumailley

et al. 1992

Self Cite

180

140

“…The short arms of laminin-1 are contributed by the Nterminal regions of all three chains (Engel, 1993) and appear well preserved in the elastase fragment ElXNd except for the loss of =600 N-terminal residues (fragments E4, E10) in the pl chain (Mann et al, 1988). Pepsin fragments PlX and P1, which also lack substantial distal portions (domains VI and V) of the a1 and y l chain (Gerl et al, 1991), were inactive ligands. Furthermore, the active laminin-1 and the inactive laminin-2 differ only in the a1 and a2 chains.…”

Section: Discussionmentioning

confidence: 99%

Binding of Purified Collagen Receptors (α1β1, α2β1) and RGD‐Dependent Integrins to Laminins and Laminin Fragments

Pfaff

Göhring

Brown

et al. 1994

Self Cite

Integrins alp1 and a2/3l, when purified by collagen affinity chromatography, showed distinct binding to mouse tumor laminin-1, which has the chain composition alplyl. The binding was, however, about 10-fold lower than to collagen IV. Only little (alpl) or no binding (a2pl) was observed to two different laminin isoforms (a2ply1, a2p2yl) from human placenta. Binding to laminin-1 was abolished by EDTA and could be specifically inhibited by antibodies to the respective integrin a subunit. These antibodies also inhibited cell adhesion to collagens. The binding of soluble integrins was weaker than that of immobilized integrins but could be enhanced by an activating anti@ integrin). No enhancement was observed for immobilized integrins. Studies with laminin-1 fragments demonstrated lack of binding to the major cell-adhesive fragment E8 from the long arm, fragments E3 and E4, involved in heparin-binding and self-assembly, respectively, and fragment P1, corresponding to the inner segments of the short arms. A larger short-arm fragment (ElXNd), which lacks the N-terminal pl chain domains V and VI, was as active as laminin. Together, these results, suggested the localization of the binding sites for alp1 and a2pl to the N-terminal region of the laminin a1 chain. Fragment P1 but not intact laminin-1 bound to aVp3 integrin in an EDTA-sensitive and RGD-sensitive manner, underscoring previous data on the cryptic nature of the RGD site in laminin-1. Further analyses by surface plasmon resonance assays demonstrated a KD = 50 nM for a2plllaminin-1 bindmg and a KD = 450 nM for aVp3lfragment P1 binding and confirmed the antipl-mediated increase in affinity for a2pl.

“…It was previously shown that laminin binds by a single high-affinity site in its B2 chain to domain G3 of nidogen (Mann et al, 1988;Gerl et al, 1991;Fox et al, 1991). This site is located within a single EGF-like motif of laminin (Mayer et al, 1993).…”

Section: Discussionmentioning

confidence: 99%

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

Mayer

Mann

Timpl

et al. 1993

Self Cite

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.