Glycobiology Advance Access published online on May 14, 2008
Glycobiology, doi:10.1093/glycob/cwn039
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Ganglioside depletion and EGF Responses of Human GM3 Synthase Deficient Fibroblasts
1 Center for Cancer and Immunology Research, Children's Research Institute, 111 Michigan Avenue, NW, Washington, DC 20010
2 Rainbow Babies & Children's Hospital, 11100 Euclid Avenue, Cleveland, OH 44106
3 Molecular and Cellular Oncology Program, Institute for Biomedical Sciences, the George Washington University, Washington, DC
* to whom correspondence and reprints should be addressed: Center for Cancer and Immunology Research, Children's Research Institute, 111 Michigan Ave, NW, Washington, DC 20010; phone: 202-884-3898, fax: 202-884-3929, e-mail: sladisch{at}cnmc.org
Received on February 29, 2008; accepted on May 11, 2008
Recognition of important roles of gangliosides in normal and abnormal cell function has motivated pharmacological modification of cellular ganglioside content. However, constitutive depletion of gangliosides in untransformed human cells has not been reported. In this context, the recent identification of a kindred carrying a point mutation in the GM3 synthase [ST3Gal5, Siat9] gene (Simpson et al, Nature Genetics 36:1225–9, 2004) provided an opportunity to explore this possibility. We established primary cultures of skin fibroblasts of three patients homozygous for this autosomal recessive defect. They exhibited a 93% reduction in ganglioside content (0.8±0.2 nmol LBSA per 107 cells versus 12.7±1.3 nmol LBSA per 107 normal fibroblasts). Importantly, this marked reduction was not compensated by activation of an alternate pathway of ganglioside synthesis, as occurs in murine GM3 synthase knockout fibroblasts. Cell morphology appeared unaffected, but under stringent conditions EGF-induced proliferation and migration of the mutant fibroblasts were reduced by 80% and 60%, respectively. Probing potential explanations, we found that EGF binding (effective membrane EGF receptor (EGFR) number) was reduced by 52% (to 6.2±1.9 from 12.8±2.0 pmol/108 normal fibroblasts, p<0.01), despite normal total EGFR protein. EGFR activation was likewise reduced as was EGF-induced Rho/Rac1 phosphorylation, which is associated with cell migration. We conclude that this GM3 synthase point mutation almost completely depletes human fibroblast cellular gangliosides, dampens membrane EGFR activation, and modulates related critical cell functions such as proliferation and migration. These cells offer a valuable model for the study of ganglioside modulation of cell function.