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Enhancing of GM3 synthase expression during differentiation of human blood monocytes into macrophages as in vitro model of GM3 accumulation in atherosclerotic lesion

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Abstract

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 α2,3-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 KM for lactosylceramide and CMP-NeuAc were 101 and 180 μM, 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.

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Abbreviations

CMP:

Cytidine 5′-monophosphate

GalCer:

Galactosylceramide

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

LacCer:

Lactosylceramide

NeuAc:

N-acetylneuraminic acid

PPPP:

D-threo-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol

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Acknowledgments

This study was supported by a Russian Foundation for Basic Research Grant (06-04-48277-a).

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Authors and Affiliations

  1. Russian Cardiology Research Center, Ministry of Health of Russian Federation, 3rd Cherepkovskaya Street 15A, 121552, Moscow, Russia

    Elena V. Gracheva, Nelya N. Samovilova, Natalia K. Golovanova, Svetlana V. Kashirina, Alexander Shevelev, Igor Rybalkin, Tat’yana Gurskaya, Tat’yana N. Vlasik, Elena R. Andreeva & Nina V. Prokazova

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  1. Elena V. Gracheva
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  2. Nelya N. Samovilova
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  3. Natalia K. Golovanova
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  10. Nina V. Prokazova
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Correspondence to Nina V. Prokazova.

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Gracheva, E.V., Samovilova, N.N., Golovanova, N.K. et al. Enhancing of GM3 synthase expression during differentiation of human blood monocytes into macrophages as in vitro model of GM3 accumulation in atherosclerotic lesion. Mol Cell Biochem 330, 121–129 (2009). https://doi.org/10.1007/s11010-009-0125-2

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  • DOI: https://doi.org/10.1007/s11010-009-0125-2

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