Glycoconjugate Journal 19, 181–186, 2003 C  2003 Kluwer Academic Publishers. Manufactured in The Netherlands. Changes of the ganglioside pattern and content in human fibroblasts by high density cell population subculture progression Mariateresa Sciannamblo 1 , Vanna Chigorno 1 , Alberto Passi 2 , Rea Valaperta 3 , Ileana Zucchi 3 and Sandro Sonnino 1∗ 1 Study Center for the Biochemistry and Biotechnology of Glycolipids, Center of Excellence on Neurodegenerative Diseases, Department of Medical Chemistry, Biochemistry and Biotechnology, University of Milan, Segrate, Italy, 2 Department of Experimental and Clinical Biomedical Science, University of Insubria, Varese, 3 Institute of Advanced Biomedical Technologies – CNR, Segrate, Italy In this study we show that the ganglioside content and pattern of human skin fibroblasts change along the process of cell subculture progression by varying the cell density. GM3, GD3 and GD1a were components of the total cell ganglioside mixtures extracted from cells, but GD1a was in all the extracts a minor component or very scant. Other gangliosides present in traces were not characterised. The fibroblast ganglioside content of 52 pools of cells obtained from 5 different cell lines cultured at variable cell density ranged from 2.0 to 13.1 nmoles per mg of cell protein. The molar ratio between GM3 and GD3 varied from 418 to 0.6 in the ganglioside mixtures, as determined by densitometric quantitative analysis after thin layer chromatographic separation. Both the ganglioside content and the GM3/GD3 molar ratio were constant along several passages of subculture pro- gression performed by plating cells collected at confluence. Instead, when the subculture progression was performed by plating cells collected at a few days after reaching confluence, a progressive increase of the ganglioside content was observed. GD3 increased proportionally more than GM3 so that a progressive decrease of the ratio between GM3 and GD3 was observed. In some experiments, GD3 was very scant at the beginning of the progression, while it was near 30% after 5 passages under these conditions. The progressive increase of GD3 along the high density cell population subculture progression was associated to a moderate increase of the mRNA GD3 synthase. Published in 2003. Keywords: gangliosides, GD3, biosynthesis, cell density, GD3 synthase, over-expression Abbreviations: Ganglioside and glycosphingolipid nomenclature is in accordance with Svennerholm [29], and the IUPAC- IUBMB recommendations [30]. LacCer, ß-Gal-(1-4)-ß-Glc-(1-1)-Cer; GM3, II 3 Neu5AcLacCer, α-Neu5Ac-(2-3)-ß-Gal-(1-4)-ß-Glc-(1-1)-Cer; GD3, II 3 Neu5Ac 2 - LacCer, α-Neu5Ac-(2-8)-α-Neu5Ac-(2-3)-ß-Gal-(1-4)-ß-Glc-(1-1)-Cer; GM1, II 3 Neu5AcGgOse 4 Cer, ß-Gal-(1-3)-ß-GalNAc- (1-4)-[α-Neu5Ac-(2-3)]-ß-Gal-(1-4)-ß-Glc-(1-1)-Cer; GD1a, IV 3 Neu5AcII 3 Neu5AcGgOse 4 Cer, α-Neu5Ac-(2-3)-ß-Gal-(1-3)-ß- GalNAc-(1-4)-[α-Neu5Ac-(2-3)]-ß-Gal-(1-4)-ß-Glc-(1-1)-Cer; Neu5Ac, N-acetylneuraminic acid; Cer, ceramide, N-acyl- sphingosine; Sph, sphingosine, (2S,3 R,4 E)-2-amino-1,3-dihydroxy-octadecene. EMEM, Eagle’s minimum essential medium; FCS, fetal calf serum; PBS, phosphate-buffered saline. Introduction Gangliosides [1], sialic acid containing glycosphingolipids, are inserted with the lipid moiety in the outer layer of the plasma ∗ To whom correspondence should be addressed: Prof. Sandro Sonnino, Dipartimento di Chimica, Biochimica e Biotecnologie per la Medicina—LITA-Segrate, Via Fratelli Cervi 93, 20090 Segrate (Milano), Italy. Tel./Fax: +390250330365; E-mail: Sandro.Sonnino@unimi.it membranes of vertebrate cells. Their oligosaccharide moiety faces the external medium; this makes them free to interact with soluble extracellular molecules and with the hydrophilic portion of the same or other cell membrane components. Gan- gliosides in the cell membranes are organized in domains [2,3] containing proteins involved in signal transduction [4,5], where it is believed that they play an important role in modulating biological functions.