Journal of Neuroscience, Vol 11, 2443-2452, Copyright © 1991 by Society for Neuroscience
Interaction of ganglioside GM1 with the B subunit of cholera toxin modulates growth and differentiation of neuroblastoma N18 cells
D Masco, M Van de Walle and S Spiegel
Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, Washington, DC 20007.
The present study uses the B subunit of cholera toxin, a protein that binds
specifically to ganglioside GM1, to examine the role of endogenous GM1 in
the process of growth and differentiation of mouse neuroblastoma N18 cells.
Binding of the B subunit to neuroblastoma N18 cells inhibited DNA synthesis
with concomitant induction of differentiation. The B subunit induced
pronounced morphological changes: an increase in neurite outgrowth with
branched neurites and spinelike processes. The distinct morphological
alterations and neuritogenesis in response to the B subunit were also
revealed by immunofluorescence with fluorescein-labeled B subunit. The
mechanism of the B subunit-induced differentiation is different than that
of spontaneous differentiation. Thrombin, a serine protease present in
normal serum, inhibits neurite outgrowth induced by the removal of serum
from the medium. In contrast, thrombin did not cause retraction of the
neurites induced by the B subunit. Thus, thrombin or a thrombin- like
protease is not involved in the process of neurite outgrowth mediated
through endogenous GM1. The biological effects of the B subunit are due to
the binding of the B subunit to ganglioside GM1 and not due to changes in
cAMP levels resulting from contaminating A subunit. We used highly purified
cloned B subunit that cannot contain any A subunit because it was isolated
from a Vibrio cholerae mutant that only expresses the B subunit. Neither
the cloned nor commercial preparations of the B subunit induced increases
of cAMP in these cells. There was a good correlation between the amount of
B subunit bound to the cells and the biological effect. Finally, treatment
with neuraminidase, which caused a fourfold increase in the level of
membrane GM1 as determined by iodinated cholera toxin binding, enhanced the
biological effect of the B subunit. However, neuraminidase treatment alone
did not have significant effects, either on DNA synthesis or on morphology
of the cells, indicating that elevations in the level of GM1 per se are not
sufficient by themselves to cause significant changes in cell growth or
differentiation. It seems most likely that the aggregation of endogenous
GM1 on the cell surface by the B subunit is responsible for these effects
on mouse neuroblastoma N18 cells.
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