Protein binding and cell adhesion properties of two laminin isoforms (AmB1eB2e, AmB1sB2e) from human placenta.
Two isoforms of laminin were extracted from human placenta by neutral buffer containing EDTA, copurified through several steps and finally separated by Mono Q anion exchange chromatography. One variant consisted of disulphide-linked 340, 230 and 190 kDa subunits, which were identified by immunoblotting as Am, B1e and B2e chains. In the other variant, the B1e chain was replaced by B1s of 180 kDa. After rotary shadowing, both variants showed a similar cross-shaped structure. The nidogen content of these laminins was substoichiometric and variable (3-70%), indicating loss by endogenous proteolysis. Yet both human isoforms were able to bind mouse nidogen with an affinity (Kd approximately 0.5 nM) comparable to that of AeB1eB2e laminin from a mouse tumour. Since the binding site is known to be contributed by a single EGF-like motif of the B2e chain, this demonstrates that activity of this site is independent of chain assembly. Binding activity of both isoforms to collagen IV and the heparan sulphate proteoglycan perlecan was correlated to the nidogen content and could be enhanced by adding nidogen. Binding to heparin was only partial and heparin did not inhibit perlecan binding. This indicated a crucial role for nidogen in mediating the integration of these laminin isoforms into basement membranes. Variant AmB1sB2e showed calcium-dependent binding to fibulin-1, while only a little activity was found for AmB1eB2e. Both isoforms promoted adhesion and spreading of several cell lines. Adhesion could be completely inhibited by antibodies to the integrin beta 1 subunit but not, or only weakly, by antibodies against beta 3, alpha 2, alpha 3, alpha 5 and alpha 6 subunits. No inhibition was observed with an Arg-Gly-Asp-containing peptide.
Brown
J C
JC
Max-Planck-Institut für Biochemie, Martinsried, Germany.
Wiedemann
H
H
Timpl
R
R
D000818:Animals; D001485:Basement Membrane; D002448:Cell Adhesion; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D007797:Laminin / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins / Q000378:metabolism; D051379:Mice; D008854:Microscopy, Electron; D008970:Molecular Weight; D010920:Placenta / Q000378:metabolism*; D011247:Pregnancy; D011485:Protein Binding
D007797:Laminin; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins; C029967:nidogen