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

Abstract

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

Binding Properties and Protease Stability of Recombinant Human Nidogen

“…4). These three niclogen interactions, which are mediated by different globular domains (G2, G3), have also been shown to mediate the formation of ternary complexes between the ligands using mouse nidogen as a connecting protein (Fox et al, 1991 ;Battaglia et al, 1992;Aumailley et al, 1993). Similar assays showed an activity for human nidogen comparable to that of mouse nidogen (Fig.…”

“…Further binding sites of mouse nidogen for collagen IV and perlecan were found on its G2 domain (Battaglia et al, 1992;Reinhardt et al, 1993). Similar binding sites exist on human nidogen, although their affinities are somewhat lower when examined with mouse collagen IV and perlecan (Fig.…”

“…4). The second type of assay was used to study the interactions with mouse collagen IV and the heparan sulfate proteoglycan perlecan Battaglia et al, 1992). Here again significant binding could be demonstrated for human nidogen, but the concentrations required to obtain the same level of binding seen with mouse nidogen were approxiniately 5 -10-fold higher (Fig.…”

“…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.…”

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

“…This was particularly indicated from the binding properties of nidogen which can mediate the formation of ternary complexes between laminin, collagen IV and heparan-sul€ate proteoglycan (Fox et a]., 1991 ; Battaglia et al, 1992). Structural studies and cDNA sequencing have shown that mouse and human nidogen consist of a single, approximately 1200-residue polypeptide chain (Paulsson et al, 1986;Mann et al, 1989;Nagayoshi et al, 1989), and that nidogen and entactin, a protein obtained from a cellculture matrix (Carlin et al, 1981;Durkin et al, 1988), are identical.…”