STRUCTURAL REQUIREMENT FOR CELL-ADHESION TO KALININ (LAMININ-5)

Laminin-5 (kalinin) was purified from spent cell culture media (SCC25 cells) by affinity chromatography on monoclonal antibody BM165. The pr otein was recovered as a mixture of the typical polypeptides of 165-15 5, 140, and 105 kDa as judged by SDS-polyacrylamide gel electrophoresi s analysis under reducing conditions. The amino acid composition of pu rified laminin-5 was in agreement with that compiled from the recently published cDNA sequences of the alpha(3)-, beta(3)-, and gamma(2)-lam inin chains. Moreover, the content of half-cystine residues in laminin -5 was about two-thirds that in laminin-1, which confirms the predicti on of a smaller number of epidermal growth factor-like repeats in the amino-terminal portion of the three chains. The content of coiled-coil alpha-helices (27%) determined by CD spectroscopy was comparable to t hat reported for laminin-1, which indicates that the long arm portion of laminin-5 is equivalent to that of other laminin isoforms. The melt ing temperature was recorded at 72 degrees C by CD monitoring of unfol ding and refolding of the coiled-coil structures during thermal denatu ration and renaturation, respectively. The thermal stability of lamini n-5 is therefore significantly higher than that of laminin-1 or alpha( 2)-chain-containing laminins, which suggests higher ionic interactions between the three polypeptide chains of laminin-5. Cell adhesion-prom oting activity of laminin 5 was found to be strictly and entirely depe ndent on the presence of coiled-coil structures, It decreased graduall y after heat denaturation of the protein above 65 degrees C and was to tally abrogated at 75 degrees C. This is in contrast to laminin-1, whi ch contains both conformation-dependent and -independent cell-binding sites on the long and short arm domains, respectively.