Uptake and metabolism of gangliosides in transformed mouse fibroblasts. Relationship of ganglioside structure to choleragen response.

Abstract

NCTC 2071 cells, transformed mouse fibroblasts, when grown in chemically defined medium, are deficient in gangliosides and do not respond to choleragen. The cells lack two biosynthetic enzymes, CMP-sialic acid:lactosylceramide sialyltransferase and UDP-galactose:GM2 (GalNAc-[AcNeu]-Gal-Glc-ceramide) galactosyltransferase, which are required for ganglioside synthesis. Following uptake of ganglioside GM1 (Gal-GalNAc-[AcNeu]-Gal-Glc-ceramide) from the medium, the cells respond to choleragen; however, they remain unresponsive following uptake of gangliosides GM2 (approximately 6 X 10(6) molecules/cell) and GM3 (AcNeu-Gal-Glc-ceramide) (approximately 2 X 10(6) molecules/cell). A response was observed when the cells had bound approximately 2 X 10(7) molecules of GM2/cell. After binding GD1a (AcNeu-Gal-GalNAc-[AcNeu]-Gal-Glc ceramide) (approximately 2 X 10(5) molecules/cell), cells exhibit some response to the toxin which can be attributed to enzymatic conversion of GD1a to GM1. A second line of NCTC 2071 cells which have 2.5 X 10(7) molecules of endogenous GM2/cell is slightly responsive to choleragen; adenosine 3':5'-monophosphate (cyclic AMP) levels rise 150%. However, when these cells have bound 4.4 X 10(4) molecules of GM1 per cell, cyclic AMP levels rise 7-fold following toxin treatment. GM1, which becomes functionally integrated into the cells, appears to be the natural receptor for choleragen and is 50 to 1000 times more effective than other gangliosides in eliciting a choleragen response.