Glycoconjugate Journal 20, 63–70, 2004 C  2004 Kluwer Academic Publishers. Manufactured in The Netherlands. Role of GM3-enriched microdomains in signal transduction regulation in T lymphocytes Maurizio Sorice 1 , Agostina Longo 1 , Tina Garofalo 1 , Vincenzo Mattei 1 , Roberta Misasi 1 and Antonio Pavan 2 1 Dipartimento Medicina Sperimentale e Patologia, Universit ` a “La Sapienza”, Roma, 2 Dipartimento Medicina Sperimentale, Universit` a di L’Aquila, L’Aquila, Italy Gangliosides, sialic acid containing glycosphigolipids, are ubiquitous constituents of cell plasma membranes. Each cell type shows a peculiar ganglioside expression pattern. In human T lymphocytes monosialoganglioside GM3 represents the main ganglioside constituent of cell plasma membrane where it is concentrated in glycosphingolipid-enriched mi- crodomains (GEM). The presence of tyrosine kinase receptors, mono- (Ras, Rap) and heterotrimeric G proteins, Src-like tyrosine kinases (lck, lyn, fyn), PKC isozymes, glycosylphosphatidylinositol (GPI)-anchored proteins and, after T cell acti- vation, the Syk-family kinase Zap-70, prompts these portions of the plasma membrane to be considered as “glycosignaling domains.” In particular, during T cell activation and/or other dynamic functions of the cell, such as apoptosis, key signal- ing molecules are recruited to these microdomains, where they strictly interact with GM3. The association of transducer proteins with GM3 in microdomains suggests that this ganglioside is the main marker of GEM in human lymphocytes and is a component of a cell plasma membrane multimolecular signaling complex involved in cell-cell interaction, signal transduction, and cell activation. Published in 2004. Keywords: microdomains, rafts, GM3, gangliosides, T lymphocytes Introduction Gangliosides, sialic acid containing glycosphingolipids, are membrane constituents of all cell types, including lymphocytes, and the pattern is peculiar in different cells. These molecules consist of a double chain hydrophobic part (ceramide) and a hydrophilic head and are implicated in a variety of cell sur- face phenomena [1]. In addition to their well known func- tions as antigens and as receptors for different molecules and for bacterial toxins [2], gangliosides play an important role in cell adhesion, protein trafficking and transmembrane signaling [3–6]. Gangliosides are synthesized by virtually all the cells of pe- ripheral blood. However, patterns of ganglioside cell expression depend on the species, cell type and age of the individual. In human peripheral blood lymphocytes (PBL) monosialoganglio- side GM3 is the major ganglioside constituent (about 72% of total ganglioside content) [7], although not correlated with a particular lymphocyte subpopulation; both CD4+ and CD8+ To whom correspondence should be addressed: Prof. Antonio Pavan, Dipartimento di Medicina Sperimentale, Universit ` a degli Studi di L’Aquila, Via Vetoio, Coppito 2, 67100 L’Aquila, Italy. Tel.: 39-862- 433683; Fax: 39-862-433523; E-mail: pavan@univaq.it cells express a similar amount of GM3 [8]. The GM3 content, determined as lipid-bound sialic acid, was 17.5 ± 1.4 μg/mg of protein in GEM fraction, as compared to 0.864 ± 0.1 μg/mg of protein in total lymphocytes. The identity of the GM3 comigrating band was verified by gas liquid chromatographic (GLC) analysis. In the hydrophilic head galactose, glucose and N-acetylneuraminic acid in a molar ratio of 1:1:1 were found; in the hydrophobic part the main fatty acids were C16:0, C18:0, C18:1, and C15:0. Long-chain bases, analyzed by GLC as O-trimethysilyl derivatives, revealed ex- clusively the presence of n-C18-sphingenine that accounted for more than 95% of long-chain bases. The mass spectroscopy analysis of GM3 from GEM fraction ganglioside extract con- clusively confirmed the identity of the molecule [9]. Minor ganglioside constituents of human PBL include sialosyl paragloboside (neuAcnLc4Cer) (about 14%) and monosialo-lactohexaosyl-ceramide (about 7%). Disialogan- glioside GD3 is also a minor component on a small subset of human peripheral blood T cells, where it has been given the cluster designation CDw60, a surface marker of T cell ac- tivation [10]. Relatively little is known about the expression of these gangliosides in human lymphocyte subpopulations. GD3 is expressed predominantly on activated memory CD4+