SELF-ASSEMBLY AND CALCIUM-BINDING SITES IN LAMININ - A 3-ARM INTERACTION-MODEL

Laminin, a four-arm glycoprotein, polymerizes in vitro into networks s imilar to those found in basement membranes. We have dissected this ca lcium-dependent assembly by analyzing proteolytic fragment binding usi ng equilibrium gel filtration, ultracentrifugation, and electron micro scopy. The cathepsin G fragment C1-4 (three short arms) was found to a ggregate with a critical concentration similar to that for laminin. Li ke laminin polymerization, this assembly was inhibited by the smaller elastase short arm fragments E4 (B1 domains VI and V) and E1' (A-B2 sh ort arm complex). Fragment E4 bound directly to E1' in a calcium-depen dent manner (K(D) = 1.4 muM). In contrast, homologous self-interaction s of short arm fragments and all interactions of long arm fragments we re considerably weaker or nonexistent. While electron micrographs of E 1' or E4 alone contained mostly monomers, those of E1'/E4 mixtures con tained dimers and oligomers with E1' dimers connected to each other th rough their A and B2 arms, often with visible E4 molecules at their ju nctions. Ca-45(2+) bound principally to fragment E1' with localization to the end of the B2 chain. These data support a model in which polym erization requires the interaction of all three ligands, one from each short arm, with calcium activating assembly by binding to the B2 shor t arm.