Basement-membrane heparan sulfate proteoglycan binds to laminin by its heparan sulfate chains and to nidogen by sites in the protein core (original) (raw)
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Binding of nidogen and the laminin-nidogen complex to basement membrane collagen type IV
European Journal of Biochemistry, 1989
The laminin-nidogen complex and purified nidogen both bind collagen IV but not other collagens, as shown by solid-state ligand-binding and inhibition assays. Laminin purified from the dissociated complex and a variety of laminin proteolytic fragments failed to bind collagen IV. Complexes formed in solution between nidogen or laminin-nidogen and collagen IV were visualized by rotary shadowing which identified one major binding site about 80 nm away from the C-terminus of the collagen triple helix. A second, weaker binding site may exist closer to its N-terminus. Binding sites of nidogen were assigned to its C-terminal globular domain which also possesses laminin-binding structures. A more diverse collagen-IV-binding pattern was observed for the laminin-nidogen complex, whereby interactions may involve both nidogen and short-arm structures of laminin.
Neurochemical Research, 1987
The present studies were undertaken to confirm the presence and identity of a putative proteoglycan associated with laminin in neurite-promoting factor complexes isolated from rat schwannoma cell conditioned medium. Sucrose density gradient centrifugation of the complex resolved two laminin-associated Na2135S]Oa-labeled peaks which were termed Pools A and B. Both pools had nearly all their [35S] cpms associated with glycosaminoglycan, contained heparan sulfate-proteoglycan core protein antigen and displayed a similarly high neurite promoting potency relative to their laminin contents. However, Pool A contained about twice as many [35S] cpms and twice as much proteoglycan core protein per laminin than Pool B. Seventy percent of Pool A cpms was associated with heparan sulfate and 30% with chondroitin sulfate whereas the inverse was true for Pool B. Treatment with heparitinase and/or chondroitinase ABC caused laminin in either pool to elute at lower salt concentrations from DEAE cellulose. In SDS-PAGE the [35S] cpms of both pools ran with the same mobility as laminin but could be separated from laminin under reducing conditions. The Pool A cpms remained at 900 KD and the Pool B cpms spread over the 200-900 KD range. By rotary shadowing electron microscopy, Pool B fractions contained primarily cross-shaped laminin images, often associated with proteoglycan-like images. Pool A fractions contained i) dense, aggregated images including intact laminin from which emanated proteoglycan-like strands, ii) circular images bearing globular domains and less commonly, iii) distorted cross-shaped laminin-like images. These studies support the existence of at least two forms of laminin-proteoglycan complexes which differ in biochemical, immunochemical and ultrastructural characteristics.
The EMBO Journal, 1999
The C-terminal G domain of the mouse laminin α2 chain consists of five lamin-type G domain (LG) modules (α2LG1 to α2LG5) and was obtained as several recombinant fragments, corresponding to either individual modules or the tandem arrays α2LG1-3 and α2LG4-5. These fragments were compared with similar modules from the laminin α1 chain and from the C-terminal region of perlecan (PGV) in several binding studies. Major heparin-binding sites were located on the two tandem fragments and the individual α2LG1, α2LG3 and α2LG5 modules. The binding epitope on α2LG5 could be localized to a cluster of lysines by site-directed mutagenesis. In the α1 chain, however, strong heparin binding was found on α1LG4 and not on α1LG5. Binding to sulfatides correlated to heparin binding in most but not all cases. Fragments α2LG1-3 and α2LG4-5 also bound to fibulin-1, fibulin-2 and nidogen-2 with K d ⍧ 13-150 nM. Both tandem fragments, but not the individual modules, bound strongly to α-dystroglycan and this interaction was abolished by EDTA but not by high concentrations of heparin and NaCl. The binding of perlecan fragment PGV to α-dystroglycan was even stronger and was also not sensitive to heparin. This demonstrated similar binding repertoires for the LG modules of three basement membrane proteins involved in cell-matrix interactions and supramolecular assembly.
1999
Working with Mel-85 (a human melanoma cell line), we have been able to detect a laminin-binding molecule with an apparent molecular mass of 100/110 kDa (Mel-85-LBM). Reduction with β-mercaptoethanol decreases its molecular mass but does not affect its ability to bind laminin. This laminin interaction seems to be very specific since Mel-85-LBM binds laminin, but not fibronectin, vitronectin or type I collagen in affinity chromatography experiments. The molecule has a negative net charge at physiological pH and binds laminin in a divalent cation dependent way. Mel-85-LBM was metabolically radiolabeled with sodium [ 35 S]-sulfate and chemical β-elimination of purified Mel-85-LBM releases chondroitin sulfate chains. Mel-85-LBM is also sensitive to chondroitinase ABC digestion. These findings show that this molecule is a chondroitin sulfate proteoglycan. The location of this proteoglycan at the cell surface is evidenced by experiments using a polyclonal antiserum raised against purified Mel-85LBM, that specifically reacts with just one molecule by western blotting among Mel-85 total cell extract as well as produces a positive signal by flow cytometry and a fluorescence profile of Mel-85 cells adhered on laminin. (Mol Cell Biochem 197: 39-48, 1999) Key words: laminin receptors, proteoglycan, cell adhesion Abbreviations: BCIP -bromochloroindolyl phosphate; ECM -extracellular matrix; EHS -Engelbreth-Holm-Swarm; LBMlaminin binding molecule; NBT -nitro blue tetrazolium; NaCl/Pi -phosphate-buffered saline; PMSF -phenylmethylsulfonylfluoride; SDS-PAGE -sodium dodecyl sulfate polyacrylamide gel electrophoresis; Tris/NaCl -tris-buffered saline; Tris/Ca 2+ /Mg 2+ -tris-buffered containing Ca 2+ and Mg 2+ ; TX-100 -Triton R X 100
The Journal of Cell Biology, 1991
The large carboxy-terminal globular domain (G domain ; residues 2,110-3,060) of the A chain of murine-derived laminin has been shown to promote heparin binding, cell adhesion, and neurite outgrowth . This study was conducted to define the potential sequence(s) originating from the G domain of laminin with any of these functional activities . A series of peptides were synthesized from the G domain, termed GD peptides, each -20 amino acids long and containing multiple positively charged amino acids . In direct 3 H-heparin binding assays, peptides GD-1 and GD-2 bound high levels of 3H-heparin, while peptides GD-3 and GD-4 bound lower levels of 3 H-heparin, and GD-5 bound essentially no 3H-heparin . The binding of 3 H-heparin to peptides GD-1 and GD-2 appeared to be of high affinity, since significant binding of 3 H-heparin to these two peptides was still observed even when the NaCl concentration was raised to 1 .0 M . Four of the T HE basement membrane glycoprotein laminin isolated from the murine Engelbreth-Holm-Swarm (EHS) tumor consists ofone 400-kD A chain and two B chains, each of ti 200 kD. Laminin promotes the adhesion and spreading of a variety of cells and binds many different types of molecules, such as glycosaminoglycans or proteoglycans . Studies using enzymatic digests of laminin or monoclonal antibodies raised against laminin have defined some of the biologically active regions ofthe 400-kD A chain of murine laminin . The large carboxy-terminal globular domain (G domain) on the long arm of the A chain of laminin is reported to be the major heparin binding domain of laminin . In early studies, enzymatic digests oflaminin were passed over heparin-affinity columns and proteolytic fragments containing the G domain were found to adhere . Later studies using monoclonal antibodies in combination with heparin-affinity chromatography and a solid-phase radioligand binding assay showed that the G domain, also termed This work previously appeared in part in abstract form (
Matrix, 1989
A panel of nine monoclonal antibodies has been characterized, all of which have reactivity with the core protein of a large heparan sulfate proteoglycan derived from the murine EHS tumor matrix. These rat monoclonal antibodies stained mouse basement membranes intensely, including those of all muscle, endothelia, peripheral nerve fibers and epithelia so far examined. In addition, two of the monoclonal antibodies show cross-species reactivity, staining bovine and human basement membranes, and immunoprecipitating proteoglycans from human endothelial cell cultures. These antibodies do not, however, cross-react with avian tissues. These results show the ubiquitous distribution of a heparan sulfate proteoglycan in mammalian tissues, which will be useful in vitro and in vivo for studies on the biology of basement membrane proteoglycans and investigations of possible roles of these molecules in human disease processes.