Glycobiology Advance Access originally published online on May 14, 2008
Glycobiology 2008 18(8):593-601; doi:10.1093/glycob/cwn039
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Ganglioside depletion and EGF responses of human GM3 synthase-deficient fibroblasts
2 Center for Cancer and Immunology Research, Children's Research Institute, 111 Michigan Avenue, NW, Washington, DC 20010
3 Rainbow Babies and Children's Hospital, 11100 Euclid Avenue, Cleveland, OH 44106
4 Molecular and Cellular Oncology Program, Institute for Biomedical Sciences, the George Washington University, Washington, DC, USA
1 To whom correspondence should be addressed: Tel: +1-202-884-3898; Fax: +1-202-884-3929; e-mail: sladisch{at}cnmc.org
Received on February 29, 2008; revised on May 7, 2008; accepted on May 11, 2008
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Abstract |
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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 MA, Cross H, Proukakis C, Priestman DA, Neville DC, Reinkensmeier G, Wang H, Wiznitzer M, Gurtz K, Verganelaki A, Pryde A, Patton MA, Dwek RA, Butters TD, Platt FM, Crosby AH. 2004. Infantile-onset symptomatic epilepsy syndrome caused by a homozygous loss-of-function mutation of GM3 synthase. Nat Genet. 36:1225–1229) 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 lipid-bound sialic acid per 107 cells versus 12.7 ± 1.3 nmol per 107 normal fibroblasts). Importantly, this marked reduction was not compensated by the 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.
Key words: GM3 synthase / ganglioside / EGF signaling / cellmigration
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary DataConflict of interest statementReferences |
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As a class of molecules, the membrane glycosphingolipids, gangliosides, are suspected of having multiple biological functions in addition to their roles as epitopes/cell surface markers, and some have been identified. These roles include both intercellular and intracellular signaling functions (Hakomori et al. 1998
; Walkley et al. 2000; Proia 2003). As a class of molecules, gangliosides are ubiquitous in mammalian cells, present in high concentrations in brain tissue, and frequently altered in tumor cells, all providing reasons to probe their biological activity.
Until recently, however, it has been difficult to clearly delineate the impact of gangliosides on cell function. One approach has been the pharmacological alteration of ganglioside content, either inhibition of synthesis or addition of exogenous gangliosides, and then assessing cell function. These studies have been very helpful, but always pose the question of the relationship between constitutive expression of these molecules and cell function. The study of transgenic murine models has been also helpful in this direction, because genetic manipulation has allowed depletion or augmentation of certain gangliosides and then observation of the effects of these constitutive alterations in vivo (Sheikh et al. 1999; Kawai et al. 2001; Handa et al. 2005). Such studies have greatly contributed to understanding of the effects of modification of ganglioside synthesis and expression, particularly on normal brain development, as well as on cell signaling (Yamashita et al. 2003, 2005; Hashiramoto et al. 2006).
To understand the role of gangliosides in human physiology and biology is more difficult, however. On the one hand, mutations or absent activity of a number of enzymes in ganglioside catabolism, resulting in ganglioside accumulation, have been identified and result in severe disease, including cognitive deficiency and developmental regression. On the other hand, it had been thought that the absence of any recognized disorders of ganglioside biosynthesis, resulting in ganglioside depletion, is a reflection of incompatibility with life (Ozkara 2004). Until recently there had only been a single case report of a patient with a genetic defect in ganglioside anabolism, GM2 synthase deficiency (Fishman et al. 1975), and that resulted in an accumulation of GM3 ganglioside. There has been no report of genetic ganglioside depletion in human cells, analogous to that of murine model knockouts. Until now, studies of human cells have had to rely on the use of pharmacological inhibitors, generally in vitro, to effect ganglioside depletion.
This picture has changed with the recent discovery of a defect in ganglioside synthesis due to a mutation in the GM3 synthase gene in a cohort of Mennonite children (Simpson et al. 2004). Because GM3 synthase is the entry enzyme to normal ganglioside synthesis in humans and could theoretically result in ganglioside depletion, the discovery of this defect offered the possibility for the first time to probe a human constitutive ganglioside deficiency and its effects, both in vitro and in vivo. Delineation of the biochemical effects of this enzyme deficiency was limited to characterization of the circulating gangliosides in these patients. GM3 and its downstream biosynthetic derivatives were absent in the patient plasma, levels of the immediate GM3 precursor, lactosylceramide, were elevated, and there was evidence of increased flux through the globoside and paragloboside pathways (Simpson et al. 2004).
Studying fibroblasts of three patients homozygous for this mutation in the gene encoding GM3 synthase, here we report the first characterization of ganglioside synthesis by cells of patients with this genetic defect in GM3 ganglioside synthase. Cultured skin fibroblasts from three patients showed a striking (93%) reduction in total sialic acid-containing glycosphingolipids, gangliosides. This ganglioside deficiency was associated with certain phenotypic changes of the fibroblasts related to altered (diminished) proliferation and migration in response to EGF and associated with diminished activation and signaling of the EGF receptor.
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Results |
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Genotype and ganglioside characterization of human GM3 synthase mutant fibroblasts
Primary fibroblast cultures were established from three patients and from normal controls (neonatal human foreskin and adult human dermal fibroblasts). We confirmed the genotypes of patient and control fibroblasts by PCR and sequence analysis and documented the point mutation of 694C
T in exon 8 of SIAT9 in all three patient fibroblast cultures. A representative PCR analysis (patient 1) is shown in Figure 1.
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Total gangliosides were isolated from fibroblasts of all three patients and from normal controls and were analyzed by HPTLC and quantified by densitometry. A representative HPTLC of total gangliosides (Figure 2A, resorcinol stain) shows that normal human fibroblasts contain predominantly GM3 ganglioside, with GD3 as a second major ganglioside, as is known (Tatsumi et al. 2001
). The patient fibroblast ganglioside HPTLC pattern, in contrast, was devoid of essentially all resorcinol-positive (sialic acid-containing) glycosphingolipids, including the ganglio series gangliosides. The total ganglioside content of fibroblasts of the three patients (0.8 ± 0.2 nmol/107 cells, mean ± SD) constitutes a 93% reduction versus the total ganglioside content of normal fibroblasts (12.7 ± 1.3 nmol/107 cells, Table I).
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We confirmed an almost complete absence of ganglioside synthesis in these fibroblasts by a second approach, metabolic radiolabeling. Fibroblasts were incubated with D-[1-14C]-glucosamine and D-[1-14C]-galactose, and the radiolabeled gangliosides developed by HPTLC. The autoradiogram (Figure 2B) of the same HPTLC plate that was stained with resorcinol in Figure 2A shows essentially no ganglioside synthesis as detectable by radiolabeled sugar incorporation.
The morphology of the patient and normal fibroblasts cultured in complete medium was also examined (Figure 3). When subconfluent, the cells all had multiple polar bodies or bipolar shapes and spread well on the culture surface, with no differences apparent in comparing normal and ganglioside-deficient patient fibroblasts. At confluence, both produced typical and indistinguishable spindle-shaped fibroblast patterns, suggesting that the virtual absence of gangliosides in these cells did not alter their morphology.
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EGF-induced cell proliferation of human GM3 synthase mutant fibroblasts
To begin to evaluate the cell biology of these ganglioside-depleted fibroblasts, we assessed cell proliferation. Our previous studies with normal human and murine fibroblasts had shown that the modification of membrane ganglioside content modifies fibroblast proliferative responses to EGF, with membrane ganglioside enrichment generally associated with enhanced proliferation and pharmacological ganglioside depletion associated with decreased proliferation (Li et al. 2001
). Here we examined responsiveness of the patient fibroblasts to EGF in proliferation assays. Under standard culture conditions (FGM-2) cell proliferation was mildly, although not significantly, reduced (from 47 ± 29 to 37 ± 18 x 103 cells/cm2; Figure 4). However, under stringent, suboptimal, culture conditions, cell proliferation was markedly decreased in the mutant fibroblasts: in medium containing 1% FBS the cell count was reduced to 3 ± 1 x 103 from 17 ± 8 x 103 cells/cm2 for normal fibroblasts (P = 0.04) and under intermediate conditions (1% FBS + 0.5 ng/mL EGF) to 5 ± 2 from 29 ± 18 x 103 cells/cm2 (P = 0.08). Quantification of 3H-thymidine uptake confirmed these findings—in 1% FBS-containing medium patient fibroblast thymidine uptake was decreased from 1.7 ± 0.5 to 0.8 ± 0.2 x 103 cpm/well (P = 0.03) and in EGF-supplemented medium from 12.0 ± 7.0 to 2.0 ± 0.4 x 103 cpm/well (P = 0.006). These results show a particular sensitivity of the ganglioside-deficient patient fibroblasts to suboptimal culture conditions.
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normal; 
patients. Key: *P = 0.04, **P = 0.08.EGF-induced cell migration of human GM3 synthase mutant fibroblasts
In a second series of experiments to probe the effect of constitutive ganglioside depletion on growth factor-mediated human fibroblast function, we assessed cell migration, which is stimulated by EGF (Tarnawski and Jones 1998
). We first used the wound scratch assay and observed combined fibroblast migration and proliferation in basal medium with 1% FBS and 2 ng/mL EGF. Fibroblasts of all three patients acted similarly in this wound-scratch assay (Figure 5A), with migration and proliferation of the mutant fibroblasts markedly inhibited. Quantified as the number of cells found in the wound area (Figure 5B), there was an approximate 60% reduction from the mean control of 180 cells (P = 0.006). An apparent aggregation or "pile-up" of cells at the wound border, seen particularly well in mutant fibroblast samples P1 and P2, suggests that while these fibroblasts were able to undergo some proliferation, they were unable to migrate in response to EGF stimulation.
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To further distinguish between separate effects on migration and fibroblast proliferation, in one set of wells we added 0.001 ng/mL actinomycin D, which completely inhibits fibroblast proliferation, to the culture medium. Normal fibroblasts continued to undergo variable degrees of EGF-induced migration under these conditions (Figure 5C), demonstrating a proliferation-independent induction of fibroblast migration by EGF. The mutant fibroblasts, however, were unable to migrate into the scratch under these conditions, pinpointing a specific inhibitory effect of ganglioside depletion on migration (Figure 5C and D, P = 0.04).
We confirmed the specific effect on migration in a second assay system, a Transwell assay using EGF as the attractant to induce migration across the membrane (Figure 6). The GM3 synthase-deficient fibroblasts did not show a statistically significant decrease in migration under optimal conditions, i.e., in the FGM-2 medium. However, under suboptimal culture conditions (FBM with 1% FBS and 2 ng EGF/mL), cell migration was decreased by 40% (P = 0.045), confirming the findings of the wound-scratch assay.
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Taken together, the composite of these data suggest that both EGF-dependent cell proliferation and cell migration were affected by the genetic ganglioside depletion.
EGFR membrane expression and activation in human GM3 synthase mutant fibroblasts
These blunted responses in proliferation and migration to EGF stimulation, together with our previous studies implicating ganglioside enrichment in enhancing fibroblast cell signaling and proliferation (Li et al. 2000, 2001) suggested that the mutant, ganglioside-deficient human fibroblasts might have defective EGF-induced signaling. While total EGFR levels were similar in five of the six patient and normal fibroblast cultures (Figure 7A), binding of 125I-EGF by the mutant fibroblasts was clearly decreased (P = 0.03; Figure 7B) and the calculated maximal binding (Scatchard analysis) by patient fibroblasts (6.2 ± 1.9 pmol/108 cells) was less than half of that of normal fibroblasts (12.8 ± 2.0 pmol/108 cells; P < 0.01, Table II).
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The reduced effective EGFR expression and reduced downstream cellular responses by the mutant fibroblasts suggested that receptor activation might also be affected by the ganglioside deficiency caused by the GM3 synthase mutation. To this end, we assessed fibroblast EGFR phosphorylation in response to either optimal (5 ng/mL) or suboptimal (0.5 ng/mL) concentrations of EGF (Figure 8). Absolute amounts of receptor phosphorylation (equivalent amounts of cell protein) were markedly reduced in fibroblasts of two of the three patients, while one (patient 2) retained significant receptor activity (Figure 8A), possibly due to the confirmed but unexplained twice-normal level of total fibroblast EGFR. Including all samples, the mean fold increase in EGF-induced EGFR phosphorylation by the patient fibroblasts, normalized for the amount of total EGFR, was significantly less than that of normal fibroblasts, reduced by 81% (P = 0.04) at suboptimal (0.5 ng/mL) and by 50% (P = 0.05) at optimal (5 ng/mL) EGF concentrations (Figure 8B). These findings confirm that these ganglioside-deficient mutant human fibroblasts have reduced EGFR activation.
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We conducted experiments to determine whether the blunted response to EGF could be reversed by the addition of GM3 to the cells (supplemental Figure 1). Neither exogenous GM3, the major ganglioside of human fibroblasts, nor GD1a, which we had previously shown to be stimulatory to normal murine and human fibroblasts (Li et al. 2001
), caused recovery of the depressed EGFR activation in the mutant fibroblasts, even though the control (add back to normal fibroblasts) confirmed the previously observed enhancement of EGFR activation caused by the enrichment of the cell membranes by exogenous gangliosides.
Rho/Rac1 signaling in human GM3 synthase mutant fibroblasts
As a possible explanation for the significant defect in EGF-induced fibroblast migration, we assessed EGF-induced activation of the Rho/Rac1 GTPase pathway. This signaling pathway is one that both links EGF stimulation to cell migration (Maddala et al. 2003) and has been shown to be modulated by gangliosides (Iwabuchi et al. 1998; Satoh et al. 2000). Representative results of fibroblasts of patient 1 are shown in Figure 9A while the composite results of all three patients and normals are found in Figure 9B and Table III. Exposure of normal fibroblasts to EGF (2 ng/mL) caused an expected increase in Rho-GTP and Rac1-GTP, to approximately 2–2.5 times the unstimulated level (Figure 9B). Significantly, however, despite similar total Rac1 and Rho content to that of the normal fibroblasts, the mutant fibroblasts showed no detectable EGF-induced increase in either Rho-GTP (P = 0.01) or Rac1-GTP (P = 0.04). The complete absence of EGF-induced activation of the Rho/Rac1 pathway therefore constitutes a possible explanation for the decrease in EGF-induced cell migration.
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In summary, the constitutive depletion in membrane gangliosides caused by a point mutation in the gene encoding GM3 synthase in human fibroblasts results in a constellation of effects—decreased EGF-induced fibroblast proliferation, markedly blunted EGF-induced fibroblast migration, altered membrane EGFR expression, reduced ligand-induced EGFR activation and inhibition of the migration-related Rho/Rac1 signaling pathway.
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Discussion |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary DataConflict of interest statementReferences |
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This study concerns cellular gangliosides of the first identified genetic human defect in ganglioside biosynthesis resulting in ganglioside depletion. The defect affects GM3 synthase, the entry enzyme for the normal major ganglioside biosynthetic pathways in humans. The point mutation on chromosome 2p12-p11.2 (694C
T in exon 8 of SIAT9) was predicted to result in the premature termination of the GM3 synthase enzyme protein (Simpson et al. 2004). This autosomal recessive gene defect resulted in the absence of the expected downstream ganglioside products of this enzyme—GM3, GD3, and the ganglio series gangliosides—in the plasma of these patients. However, the authors did detect other sialic acid-containing glycosphingolipids, those of the neolacto series (SPG and diSPG), in part replacing the normally present GM3 and GD3 in the bloodstream. What could not be known from these studies is whether cellular gangliosides would also be depleted, or whether alternate pathways of synthesis would be activated, as we observed in GM3 synthase knockout mice (Shevchuk et al. 2007).
To begin to address this, we developed fibroblast cultures from previously obtained skin biopsies of three patients with this GM3 synthase gene mutation. Both major gangliosides of normal human dermal fibroblasts, GM3 and GD3, were virtually undetectable in the patients’ fibroblasts. Furthermore, sensitive metabolic radiolabeling of glycosphingolipids did not reveal evidence of synthesis of any other sialic acid-containing glycosphingolipids. This was surprising because the almost complete absence of all sialic acid-containing glycosphingolipids is in sharp contrast to what is seen GM3 synthase knockout in mice—synthesis of "0" pathway gangliosides in the brain (Yamashita et al. 2003) and striking activation of the "0" pathway of ganglioside synthesis in fibroblasts (Shevchuk et al. 2007). The absence of detectable activation of an alternate pathway of sialic acid-containing glycosphingolipid synthesis in the human GM3 synthase-deficient fibroblasts is important because other sialic acid-containing glycosphingolipids could possibly substitute for gangliosides (which are normally ubiquitously present in essentially all cells in mammalian species) in some functions in the cell membrane such as maintaining membrane fluidity, organization of microdomains, and particularly in modulating cell signaling (Hakomori 2002; Proia 2003).
The present study specifically addressed EGF-induced cellular responses, based on our previous observations and consequent understanding of how fibroblast membrane ganglioside modifications affect normal fibroblasts. We had found that EGFR signaling and related fibroblast proliferation is substantially enhanced by membrane enrichment with exogenous complex gangliosides such as GD1a or GD3 (Li et al. 2001; Liu et al. 2004). In contrast, complete inhibition of ganglioside synthesis by inhibition of glucosylceramide synthase with D-1-threo-1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol-HCl reduced cell proliferation (Li et al. 2000) and signaling (unpublished data). Thus, constitutively, ganglioside-depleted (otherwise "normal") human fibroblasts might provide a unique window into the role of natural ganglioside depletion on fibroblast function.
We probed the EGF-induced cellular functions of proliferation and migration and associated signaling pathways, including EGFR activation and the Rho/Rac1 signaling pathway, which regulate EGF-induced cell migration in fibroblasts (Malliri and Collard 2003; del Pozo et al. 2004).
Our findings of decreased EGF-induced fibroblast signaling and function associated with the ganglioside-depleted state of these cells are consistent with a number of previous studies that have suggested a positive effect of GM3 on cell function. Examples include restoration by GM3 of fibroblast morphology distorted by inhibition of glycolipid synthesis (Meivar-Levy et al. 1997), suppression of metastasis by silencing of GM3 synthase in a tumor model (Gu et al. 2008), and effects on Rho/Rac signaling (Iwabuchi et al. 1998). While we do not know why ganglioside addback did not reverse the depressed EGFR activation and signaling, one could speculate that an intrinsic absence of cellular gangliosides, versus depletion (e.g., pharmacological) of gangliosides once present in cells, could have different effects on cell function. Finally, since here we specifically studied EGF-induced responses, our findings cannot be easily or directly compared to those of other investigations based on different models, such as those (Hashiramoto et al. 2006) in which complete medium (rather than EGF administered to starved cells) was used, and resultant less-specific cellular responses (e.g., MAPK phosphorylation) were assessed.
It is interesting that cellular proliferation and migration, and the associated EGF-induced cell signaling were more affected under stringent than under optimal culture conditions. This sensitivity to changes in membrane ganglioside content, particularly evident at suboptimal growth factor concentrations, supports a developing picture, of cell sensitivity to ganglioside changes under conditions of stress (here, reduced growth factor availability). As conditions in vivo may be much less "optimal" than those that we construct in vitro, the results underscore the potential biological relevance of studies under "suboptimal conditions." One possible explanation for this is that their presence in the membrane may facilitate cellular responses by acting as an "amplification mechanism," especially for suboptimal growth factor concentrations. This possibility is consistent with our observations here as well as with our previous studies: membrane ganglioside enrichment of vascular endothelial cells caused significant enhancement of VEGF-induced VEGF receptor signaling particularly under the stringent conditions of reduced serum or suboptimal and even subthreshold VEGF concentrations (Liu et al. 2006).
How these findings may relate to the clinical observations in patients with GM3 synthase deficiency, the first known ganglioside anabolic metabolic defect causing ganglioside depletion in humans, is both of ultimate interest and less apparent. This defect in ganglioside biosynthesis has a characteristic phenotype: these children, now numbering more than 20, have no apparent abnormality at birth. Shortly thereafter they develop hypotonia, poor feeding and irritability, seizures (only partially controllable), and evidence of poor developmental progression (Wiznitzer et al. 2005). It is difficult to know whether this severe neurological impairment is due to lack of GM3 and its derivatives, or to accumulation of precursors, or to another explanation especially since in some studies pharmacological depletion of cellular gangliosides did not impede stimulated neurite outgrowth (Li and Ladisch 1997) or neuronal differentiation (Liour and Yu 2002) of cultured cells.
Although studies of dermal fibroblasts may identify potential cellular mechanisms, it will ultimately be critical to determine (a) whether the same fundamental mechanisms affecting fibroblasts affect other types of cells and (b) whether the cellular defects in migration and proliferation could underlie problems in normal brain development. For example, a migration defect could cause potentially poor localization of cells during development, but this would have to be at the microscopic level, since there are no gross lesions. Or could it prevent functional synaptic formation and eventually lead to the clinical syndrome? Also, what would happen in the case of a physiological or pathophysiological stress, such as that mimicked by the suboptimal growth factor stimulation we used in our experiments? Would wound healing be inhibited? It is not possible to say, although until now there are no reports of defective wound healing in these children.
Some clues about the central role of gangliosides in these problems are suggested by observations in the GM3 synthase knockout mouse, which despite the same enzyme, GM3 synthase, being affected have no obvious severe phenotypic consequences (Yamashita et al. 2003). Possibly this is explained by robust "0" pathway ganglioside synthesis in coexistence with the absence of GM3 and downstream ganglioside biosynthesis. Does this compensatory synthesis by alternate pathway activation prevent more severe damage? Supporting this idea is the fact that the double knockout (GM3 synthase/GM2 synthase) mouse, in which synthesis of all brain gangliosides, including the "0" series, is prevented, develops severe neurodegenerative disease that results in early death (Yamashita et al. 2005).
The absence of alternate pathways of ganglioside synthesis, if confirmed in other cells and tissues of patients with GM3 synthase deficiency, may provide an explanation for the striking phenotypic difference between the human mutation and the knockout mouse—absent or inadequate compensatory sialic acid-containing glycosphingolipid synthesis in the former versus striking alternate pathway activation in the latter. Overall, our results support an evolving picture of normal biological roles of gangliosides. Important in membrane structure among other functions, they modulate growth factor-induced cell signaling, cell proliferation, and cell migration. The almost complete absence of gangliosides in these morphologically human normal cells, which are the first genetically determined, nontransformed human cells that are constitutively highly ganglioside deficient, should provide an ideal human model in vitro system to further delineate the role of gangliosides in cell membrane organization and cell biology.
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Materials and methods |
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Materials
EGF (mouse submaxillary glands, receptor grade) and actinomycin D were purchased from Sigma (St. Louis, MO), the Rho/RAC1/Cdc42 Activation Assay Kit from Cell BIOLABS, Inc. (San Diego, CA), anti-EGFR (sheep polyclonal IgG), rabbit anti-sheep IgG (HRP conjugate), and anti-phosphotyrosine antibody, p-Tyr (PY99) from Santa Cruz Biotechnology (Santa Cruz, CA), 125I-EGF, D-[1-14C]-glucosamine and D-[1-14C]-galactose from NEN (Boston, MA), and DNA molecular size standards from Promega (Madison, WI). Purified gangliosides were obtained from Matreya (Pleasant Gap, PA).
Cell culture
Fibroblasts were cultured with the IRB approval from de-identified existing skin biopsies of three patients homozygous for the GM3 synthase mutation and from normal healthy donors (discarded human foreskin, and adult normal human dermal fibroblasts). The cells were cultured in complete growth medium (FGM-2, Clonetics, Walkersville, MD), which contains 2% FCS, 10 ng EGF/ml, and insulin. Experiments were performed using subconfluent cultures of passages 8 to 15. Cell viability was assessed by trypan blue dye exclusion. Serum-free culture was performed in a fibroblast basal medium (FBM, Clonetics), containing no serum or growth factors (Li, Liu, et al. 2001). Genotypes were confirmed by PCR and also by sequencing as described (Simpson et al. 2004). Fibroblast morphology was assessed by phase contrast microscopy (Zeiss, Axiovert 135) of living cells.
Ganglioside purification and analysis
Gangliosides were purified by partitioning the dried total lipid extract of fibroblast pellets in di-isopropyl ether/1-butanol/ 17 mM aqueous NaCl (Ladisch and Gillard 1985) followed by Sephadex G-50 gel filtration and lyophilization. HPTLC analysis of gangliosides was performed using 10 x 20 cm precoated silica gel 60 HPTLC plates (Merck, Darmstadt) that were developed in chloroform/methanol/0.25% aqueous CaCl2·2H2O (60:40:9, by volume). Gangliosides were visualized as purple bands by resorcinol staining.
To assess ganglioside synthesis by metabolic radiolabeling, fibroblasts were incubated with 1.0 µCi/mL D-[1-14C]-glucosamine hydrochloride and D-[1-14C]-galactose for 24 h and the gangliosides purified and analyzed by HPTLC autoradiography (Chang et al. 1997).
Cell proliferation and migration assays
Cell proliferation was assessed by seeding fibroblasts in six-well culture plates at 1 x 105 cells/well in FGM-2. They were then cultured for 4 days in FGM-2 (standard culture conditions) or in FBM with 1% FBS ± 2 ng EGF/mL and then harvested, and the cell number quantified as cells/cm2. As a second approach, thymidine incorporation was assessed by seeding the cells in 96-well culture plates at 5 x 103 cells in 200 µL FBM with 1% FBS ± 2 ng EGF/mL for 24 h and to 0.5 µCi 3H-thymidine/well during the last 3 h. Thymidine uptake was quantified by β-scintillation counting.
To assess cell migration in the wound-scratch assay, fibroblasts were cultured to near confluence and then starved in serum-free FBM for 6 h. Then the wells were scratched with a 1000 µL pipet tip and photographed (inverted phase-contrast microscope). After an additional 48-h incubation in FBM with 1% FBS and 2 ng EGF/mL, cells in the scratched area were re-photographed. Because the scratch test may be influenced by proliferation, which would also occur during the 48-h culture period, in a parallel set of experiments we added actinomycin D in the scratch assay to inhibit proliferation. The cell number in six representative scratch fields/well was quantified (Etscheid et al. 2005).
A second migration assay utilized the Transwell system as described (Ronnov-Jessen et al. 2002) to study the effect of EGF on GM3 synthase-deficient fibroblast migration. The CMTM 24-well colorimetric cell migration assay kit (Chemicon Int. Temecula, CA) with an 8-µm pore size was used for quantitative assay EGF-induced cell migration. Fibroblasts were washed and resuspended in either FGM-2 (regular medium) or in the 1% FBS/FBM medium and then seeded in the upper chamber at a density of 5 x 104/200 µL/well. The lower chambers were filled with 500 µL 1% FBS/FBM + 2 ng/mL EGF. The chambers were incubated for 24 h at 37°C in 5% CO2 to allow the cells to migrate from the upper well toward the EGF gradient in the lower chamber. Then, cell stain was added for 20 min at room temperature, the chamber washed three times with fresh water, and the cells in the upper chamber were gently removed by scraping with a cotton-tipped swab. The membrane was air dried and transferred to a clean well containing 200 µL of extraction buffer for 15 min at room temperature, and then 100 µL of the resulting dye mixture was quantified by spectrophotometry (O.D. = 560 nm).
125I-EGF binding
Binding of 125I-EGF to intact cells was assessed as in our previous studies (Liu et al. 2004). Briefly, three parallel sets of 104 fibroblasts were cultured for 24 h in a 96-well plate in FGM-2. After starvation in FBM for 18 h, two sets of cells were incubated at 4°C for 2 h with 0.01 to 50 ng/mL 125I-labeled human recombinant EGF in FBM, with or without 300 ng/mL unlabelled EGF, to determine the specific binding. The third set of cells was trypsinized and the cell number determined. The binding curves and Scatchard analysis (Scatchard 1949) were performed using GraphPad Prism 3.03 software.
EGF-induced EGFR, Rho, and Rac1 activity
1 x 105 fibroblasts were seeded per well in six-well plates. When subconfluent, the cells were starved in FBM for 6 h. In the addback experiments, the cells were incubated in 10 uM ganglioside in FBM during the same time. Then the cells were washed and exposed to EGF (0.5 and 5 ng/mL) in FBM for 5 min at 37°C. Then the cells were lysed for 20 min in a 300 µL lysis buffer. EGFR autophosphorylation was assayed as in our previous studies (Liu et al. 2004). Briefly, 200 µL of cell lysate (
100 µg total protein) were used for each sample. After immunoprecipitation with anti-EGFR IgG, phosphorylated EGFR was detected by western blot using the anti-phosphotyrosine antibody p-Tyr (PY99), and the total EGFR was detected by an anti-EGFR antibody after stripping the film. The expression of Rho-GTP and Rac1-GTP was assessed on 500 µg lysate protein in a final volume of 1 mL. Rho-GTP and Rac1-GTP were separately concentrated by Rhotekin RBD and PAK PBD agarose beads, following the protocol of the kit (STA-405, Cell Biolabs, Inc., San Diego, CA) and detected by western blot using anti-Rho and -Rac1 antibodies.
Data analysis
All western blots shown are representative results of three separate experiments. The optical density of protein bands on the western blots was quantified using software from the Scion Corporation. Bars on all graphs indicate mean ± SD values, normalized to control levels. The fold induction or percentage increase was calculated by comparing the differences of the means between control and treatment groups. Statistical significance was evaluated by Student's t-test, two-tailed.
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Supplementary Data |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary DataConflict of interest statementReferences |
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Supplementary data for this article is available online at http://glycob.oxfordjournals.org/.
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Conflict of interest statement |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary DataConflict of interest statementReferences |
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This study was presented in part at the Society for Glycobiology annual meeting, November 11–14, 2007, Boston, MA (Liu et al. 2007
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