g(m1)-ganglioside and 6-carboxyfluorescein

Exogenously added gangliosides enhance sprouting, neurite outgrowth, and other neuronal activities; this effect may be initiated when a ganglioside binds to a membrane protein or when a ganglioside intercalates into the plasma membrane. To test whether binding to membrane proteins is sufficient for ganglioside-mediated activity, anti-idiotypic antibodies were generated that mimic the functional binding sites of the ganglioside GM1 as described by M. J. Riggott and W. D. Matthew (1996, Glycobiology, 6, 581-589). These anti-idiotypic antibodies are proteinaceous probes that model the biochemical and biological effects of gangliosides. Those anti-idiotypic ganglioside (AIG) monoclonal antibodies (mAb's) were selected based on their ability to bind a known GM1 binding protein, the beta-subunit of cholera toxin. These studies described neuronal cell surface proteins that were identified by immunocytochemistry and Western blotting using these AIG mAb's. Here we show that AIG mAb's mimic the functional properties of GM1 in that they facilitate neurite outgrowth from central and peripheral nervous system neurons in in vitro bioassays. In addition, AIG mAb binding modulates second messenger activity, suggesting that membrane protein binding alone is sufficient to invoke intracellular activation. The similarity in the pattern of protein tyrosine phosphorylation evoked by GM1 and the anti-idiotypic ganglioside antibodies suggests that the AIG mAb's modulate neurite outgrowth in a manner similar to that of GM1. Because antibodies cannot intercalate into the plasma membrane, these results suggest that the ganglioside GM1 can mediate neuronal cellular activity by binding to cell surface proteins.

Topics: Animals; Antibodies, Monoclonal; Antigen-Antibody Reactions; Cells, Cultured; Denervation; Female; Fluoresceins; Fluorescent Dyes; G(M1) Ganglioside; Ganglia, Spinal; Hippocampus; Nerve Regeneration; Neurites; Phosphorylation; Pregnancy; Rats; Rats, Sprague-Dawley; Sciatic Nerve; Second Messenger Systems; Tyrosine

In this report we have investigated the differences in the uptake and metabolization of exogenous GM1 by human fibroblasts, as a function of its supramolecular organization in solution. For this we used a tritium labelled GM1, given alone or inserted in dispersions of phosphatidylcholine (PC) or sulphatide. The addition of fetal calf serum (FCS) to these dispersions was also studied. With respect to GM1 pure micelles, the presence in the medium of a sulphatide/GM1, 10:1 molar ratio, greatly increased the incorporation of GM1-associated radioactivity by the cultured cells. Conversely, the presence of PC dramatically diminished the GM1 incorporation values. The metabolization of exogenous GM1 was favoured by the presence of FCS, regardless of the presence of sulphatide. The obtained data provide useful information on the appropriate procedure for feeding cultured fibroblasts with gangliosides.

Topics: Animals; Cattle; Cells, Cultured; Chromatography, Gel; Fibroblasts; Fluoresceins; G(M1) Ganglioside; Humans; Liposomes; Macromolecular Substances; Phosphatidylcholines; Sulfoglycosphingolipids