The C-terminal domain V of perlecan promotes beta1 integrin-mediated cell adhesion, binds heparin, nidogen and fibulin-2 and can be modified by glycosaminoglycans.
Domain V of the major basement-membrane proteoglycan perlecan, a domain which consists of three laminin type G (LG) and four epidermal-growth-factor-like (EG) modules, was obtained in recombinant form by transfecting embryonic kidney cells with an episomal expression vector. A major 90-kDa fragment V was obtained together with fragments Va (74 kDa) and Vb (26 kDa) which were generated by endogenous proteolysis in front of the most C-terminal LG module. All three fragments bound to a heparin affinity column and could be displaced at a moderate (0.2 M) NaCl concentration. Rotary-shadowing electron microscopy demonstrated a three-globule structure for fragment V. Fragment V also showed a strong immunological cross-reaction with tissue-derived perlecan, indicating that it was folded into a native structure. A further, larger fragment, Vc, was apparently substituted with heparan sulphate and/or chondroitin sulphate chains and failed to bind to heparin. Fragment V but not fragment Vc promoted a distinct adhesion of several cell lines and this could be blocked by antibodies against the integrin beta1 chain. This domain may, however, represent only one of several cell-adhesive sites of perlecan. The recombinant perlecan fragment V bound in surface plasmon resonance assays to fibulin-2, laminin-nidogen complex, nidogen and two nidogen fragments. This indicated two different nidogen-binding epitopes on perlecan domain V with about a 10-fold difference in their affinities (Kd = 0.05-0.2 microM and about 2 microM). Perlecan domain V therefore seems to participate in the supramolecular assembly and cell connections of basement membranes.
Brown
J C
JC
Max-Planck-Institut für Biochemie, Martinsried, Germany.
Sasaki
T
T
Göhring
W
W
Yamada
Y
Y
Timpl
R
R
D000818:Animals; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002448:Cell Adhesion / Q000502:physiology*; D002846:Chromatography, Affinity; D016326:Extracellular Matrix Proteins / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism; D006025:Glycosaminoglycans / Q000737:chemistry / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000378:metabolism; D019812:Heparan Sulfate Proteoglycans; D006493:Heparin / Q000378:metabolism; D006497:Heparan Sulfate / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D006595:Hexosamines / Q000032:analysis; D006801:Humans; D007150:Immunohistochemistry; D019012:Integrin beta1 / Q000276:immunology / Q000378:metabolism*; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000378:metabolism; D051379:Mice; D008854:Microscopy, Electron; D011485:Protein Binding; D011509:Proteoglycans / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011863:Radioimmunoassay; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014407:Tumor Cells, Cultured
D002135:Calcium-Binding Proteins; D016326:Extracellular Matrix Proteins; D005353:Fibronectins; D006025:Glycosaminoglycans; D006026:Glycoside Hydrolases; D019812:Heparan Sulfate Proteoglycans; D006493:Heparin; D006497:Heparan Sulfate; D006595:Hexosamines; D019012:Integrin beta1; D008562:Membrane Glycoproteins; D011509:Proteoglycans; D011994:Recombinant Proteins; C084612:fibulin 2; C029967:nidogen; C071927:perlecan; C020668:glycanase