Metabolism, topology, and possible mechanisms for regulation of the ganglioside GM3 content in the cell are reviewed. Under consideration are biological functions of GM3, such as involvement in cell differentiation, proliferation, oncogenesis, and apoptosis.
An ultradian oscillation of protein synthesis was detected by synchronization of metabolic activity in rat hepatocyte cultures. This oscillation occurs in dense cultures in fresh medium, but not in sparse ones. Metabolic synchronization of sparse cultures, however, was initiated by conditioned medium or addition of 0.3-0.5 microm of a mixture of bovine brain gangliosides to fresh culture medium along with either 0.06-0.2 microm GM1 or 0.1-0.2 microm GDIa. GTIb and GDIb did not produce oscillations, nor did human liver ganglioside GM3. High expression of GM1 ganglioside determinants in hepatocytes maintained in the conditioned medium purified polyclonal antibodies to GM1 was coupled with protein synthetic oscillatory activity, i.e. metabolic synchronization. Incubation of dense cultures with GM1-antibodies for 24 h decreased the amplitude of these oscillations. In sparse cultures maintained in fresh medium where protein synthesis showed no oscillatory pattern, GM1 expression was low.
We found that GM3 levels in human peripheral blood monocytes and cultured monocyte-derived macrophages were 0.37 and 2.7 microg per million cells, respectively. GM3 synthase of monocytes and to a greater extent of monocyte-derived macrophages was shown to be able to sialylate endogenous substrate, lactosylceramide (LacCer), to form GM3. With exogenously added LacCer, GM3 synthase activity was 57.1 and 563 pmol/h per mg protein in monocytes and monocyte-derived macrophages, respectively. The revealed changes in ganglioside GM3 biosynthesis are specific as the activity of some other sialyltransferases under these conditions was not altered. Human anti-GM3 synthase antibody detected in monocytes a main protein with molecular weight of 60 kD and minor proteins with molecular masses of 52 and 64 kD. In monocyte-derived macrophages the amounts of 60 kD protein and especially 64 kD protein sharply rose. Thus, the increase in ganglioside GM3 levels, GM3 synthase activity, and the enzyme amounts during culturing of monocyte/macrophages may be one of the mechanisms of in vivo increased ganglioside GM3 levels in arterial atherosclerotic lesions.
Immunohistochemical examination showed that sections of intimal atherosclerotic plaques contained cells and cell clusters as well as areas of extracellular matrix specifically stained with antibodies against ganglioside GM3. No immunohistochemical staining was observed in areas bordering the plaques where there was no histological evidence of atherosclerosis. To determine whether the ganglioside GM3 deposits in the intimal plaques derived directly from plasma or were synthesised by intimal cells. intimal plaque and plasma LDL were assayed for ganglioside GM3 fatty acid composition. This assay showed that more than 50% of the fatty acids of GM3 isolated from both atherosclerotic and normal intima are either minor fatty acids or those absent from LDL GM3. We conclude that the GM3 deposits present in intimal plaque arise in intimal cells and do not derive from plasma LDL.
In previous studies, we showed that ganglioside levels (GM3 being the main ganglioside) in human aortic intima isolated from atherosclerotic lesions were 5 times greater compared to intima from non-diseased vascular areas. Recently, we found that GM3 and GM3 synthase levels in differentiated in vitro macrophages were five and ten times higher, respectively, compared to freshly isolated human monocytes. In this article, we report that GM3 synthase mRNA levels were significantly higher in differentiated human monocyte-derived macrophages compared to monocytes and in atherosclerotic aorta compared to normal aorta. The depletion of GM3 synthesis in cultured monocyte-derived macrophages with DL-threo-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol, an inhibitor of ganglioside synthesis, delayed the acquisition of CD206 antigen, prevented the loss of CD163 antigen and enhanced anti-inflammatory cytokine (CCL18) secretion. In the current study, we performed purification of CMP-N-acetylneuraminic acid:lactosylceramide alpha2,3-sialyltransferase (GM3 synthase) from Triton X-100 extract of human blood mononuclear cells by immunoaffinity chromatography on Sepharose coupled with anti-GM3 synthase antibody. Comparison with several glycolipid substrates showed high specificity of the purified enzyme for lactosylceramide. The apparent K(M) for lactosylceramide and CMP-NeuAc were 101 and 180 muM, respectively. Analysis of the purified enzyme by SDS-PAGE followed by the anti-GM3 synthase antibody probing detected two bands with apparent molecular masses of 60 and 64 kDa. There were no other protein bands as revealed by Coomassie Blue staining. Thus, ganglioside GM3 may be considered as a physiological modulator of macrophage differentiation in human atherosclerotic aorta. The presented data suggest that up-regulation of GM3 levels is an element of monocyte/macrophage differentiation that provides a tool for control of macrophage accumulation in inflammatory loci.
Earlier we reported that atherosclerotic plaques contain cells which were specifically and very intensively stained with anti-GM3 antibodies although no GM3 positive cells were detected in the normal non-diseased arterial intima. Because of their lipid inclusions, GM3 positive cells in atherosclerotic lesions seemed to be foam cells but their origin needed clarification. Using an immunohistochemical technique in the present work, we showed that some of these foam cells contained CD68 antigen. However, the most intense accumulation of GM3 occurred in the areas composed of foam cells which did not stain with any cell type-specific antibodies, including antibodies to macrophages (anti-CD68) and smooth muscle cells (anti-smooth muscle alpha-actin), perhaps, because the cell type-specific antigens were lost during the transformation of intimal cells into foam cells. Ultrastructural analysis of the areas where foam cells overexpressed GM3 demonstrated that some foam cells lacked both a basal membrane and myofilaments but contained a large number of secondary lysosomes and phagolysosomes, morphological features which might indicate their macrophage origin. Other foam cells contained a few myofilaments and fragments of basal membrane around their plasmalemmal membrane, suggesting a smooth muscle cell origin. These observations indicate that accumulation of excessive amounts of GM3 occurs in different cell types transforming into foam cells. We suggest that up-regulation of GM3 synthesis in intimal cells might be an essential event in foam cell formation. Shedding of a large number of membrane-bound microvesicles from the cell surface of foam cells was observed in areas of atherosclerotic lesions corresponding to extracellular GM3 accumulation. We speculate that extracellularly localised GM3 might affect the differentiation and modification of intimal cells in atherosclerotic lesions.
Sialyltransferase activity has been determined in membrane preparations containing the Golgi apparatus that were isolated from atherosclerotic and normal human aortic intima as well as in plasma of patients with documented atherosclerosis and healthy donors by measuring the transfer of N-acetylneuraminic acid (NeuAc) from CMP-NeuAc to asialofetuin. The asialofetuin sialyltransferase activity was found to be 2 times higher in the atherosclerotic intima as compared to the normal intima and 2-fold higher in patients' plasma than in that from healthy donors. The mean values of the apparent Michaelis constant (K(m)) for the sialylating enzyme for both tissues did not differ and were close for the intima and plasma. In contrast, the maximal velocity (V(max)) was 2 times higher for the atherosclerotic intima than for the normal intima and 3 times higher for patients' plasma than for that of the donors. These results suggest that the activity of asialofetuin sialyltransferases of aortal intima is enhanced in atherosclerosis as is the secretion of their soluble forms into patients' plasma.
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