Isolation and characterization of rat schwannoma neurite-promoting factor: Evidence that the factor contains laminin

Abstract

Rat RN22 schwannoma cells in vitro release into their growth medium a macromolecular factor that, when bound to polyornithine-coated culture substrata, will stimulate neuritic regeneration from axotomized peripheral and central neurons. During the purification of this factor, the neurite-promoting activity co-purifies with laminin immunoreactivity as measured by an enzyme-linked immunoadsorbant assay. The purified factor has an immunoreactivity per milligram of protein similar to that of purified rat yolk sac tumor laminin. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions, the purified factor exhibits a major band at 200 kilodaltons (dD) and two minor ones at about 130 and 35 kD. The 200-kD band comigrates with the 200 kD band of purified rat laminin. After SDS-PAGE under non-reducing conditions, the rat schwannoma factor and rat laminin both exhibit a band in the 900-kD range with the schwannoma factor band migrating lightly faster than the laminin one. The 200-kD (reducing conditions) and 900-kD (non-reducing conditions) bands of both the schwannoma factor and lamina are stained by immunoblotting with antisera raised against rat and human laminin and against a partially purified preparation of the schwannoma factor. On immunoblots the 400-kD band of laminin (a band not seen in the schwannoma factor preparation) also stains with all three antisera. When the antibodies from each of the three antisera are immobilized on protein A-agarose beads, the beads will completely remove from solution the neurite-promoting activities of both the schwannoma factor and laminin. Antibodies raised against rat laminin fail to block the neurite-promoting activity of the purified schwannoma factor but totally block that of rat laminin. In contrast, antibodies raised against the schwannoma factor will block the neurite-promoting activities of both the schwannoma factor and laminin. By rotatory shadowing electron microscopy the schwannoma factor preparation exhibits cross-shaped images similar but not identical to those previously reported for rat and mouse laminin. In addition, the schwannoma factor preparation contains images resembling proteoglycans. These results suggest that the schwannoma factor may contain a distinct laminin molecule that associates with a proteoglycan.