Terminal short arm domains of basement membrane laminin are critical for its self-assembly.
Laminin self-assembles into large polymers by a cooperative two-step calcium-dependent mechanism (Yurchenco, P. D., E. C. Tsilibary, A. S. Charonis, and H. Furthmayr. 1985. J. Biol. Chem. 260:7636-7644). The domain specificity of this process was investigated using defined proteolytically generated fragments corresponding to the NH2-terminal globule and adjacent stem of the short arm of the B1 chain (E4), a complex of the two short arms of the A and B2 chains attached to the proximal stem of a third short arm (E1'), a similar complex lacking the globular domains (P1'), and the distal half of the long arm attached to the adjacent portion of the large globule (E8). Polymerization, followed by an increase of turbidity at 360 nm in neutral isotonic TBS containing CaCl2 at 35 degrees C, was quantitatively inhibited in a concentration-dependent manner with laminin fragments E4 and E1' but not with fragments E8 and P1'. Affinity retardation chromatography was used for further characterization of the binding of laminin domains. The migration of fragment E4, but not of fragments E8 and P1', was retarded in a temperature- and calcium-dependent fashion on a laminin affinity column but not on a similar BSA column. These data are evidence that laminin fragments E4 and E1' possess essential terminal binding domains for the self-aggregation of laminin, while fragments E8 and P1' do not. Furthermore, the individual domain-specific interactions that contribute to assembly are calcium dependent and of low affinity.
Schittny
J C
JC
Department of Pathology, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854-5635.
Yurchenco
P D
PD
D001485:Basement Membrane / Q000378:metabolism; D002846:Chromatography, Affinity; D007700:Kinetics; D007797:Laminin / Q000378:metabolism* / Q000648:ultrastructure; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D008958:Models, Molecular; D010446:Peptide Fragments / Q000302:isolation & purification; D011487:Protein Conformation
D007797:Laminin; D046911:Macromolecular Substances; D010446:Peptide Fragments