Self-assembly and calcium-binding sites in laminin. A three-arm interaction model.
Laminin, a four-arm glycoprotein, polymerizes in vitro into networks similar to those found in basement membranes. We have dissected this calcium-dependent assembly by analyzing proteolytic fragment binding using equilibrium gel filtration, ultracentrifugation, and electron microscopy. The cathepsin G fragment C1-4 (three short arms) was found to aggregate with a critical concentration similar to that for laminin. Like laminin polymerization, this assembly was inhibited by the smaller elastase short arm fragments E4 (B1 domains VI and V) and E1' (A-B2 short arm complex). Fragment E4 bound directly to E1' in a calcium-dependent manner (KD = 1.4 microM). In contrast, homologous self-interactions of short arm fragments and all interactions of long arm fragments were considerably weaker or nonexistent. While electron micrographs of E1' or E4 alone contained mostly monomers, those of E1'/E4 mixtures contained dimers and oligomers with E1' dimers connected to each other through their A and B2 arms, often with visible E4 molecules at their junctions. 45Ca2+ bound principally to fragment E1' with localization to the end of the B2 chain. These data support a model in which polymerization requires the interaction of all three ligands, one from each short arm, with calcium activating assembly by binding to the B2 short arm.
Yurchenco
P D
PD
Department of Pathology, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854.
Cheng
Y S
YS
D000595:Amino Acid Sequence; D001665:Binding Sites; D001704:Biopolymers; D002118:Calcium / Q000378:metabolism*; D056649:Cathepsin G; D002403:Cathepsins / Q000378:metabolism; D002850:Chromatography, Gel; D007797:Laminin / Q000378:metabolism* / Q000648:ultrastructure; D008854:Microscopy, Electron; D008954:Models, Biological; D008969:Molecular Sequence Data; D012697:Serine Endopeptidases; D014461:Ultracentrifugation
D001704:Biopolymers; D007797:Laminin; D002403:Cathepsins; D012697:Serine Endopeptidases; D056649:Cathepsin G; D002118:Calcium