CDw17: a neutrophil glycolipid antigen regulated by activation

The plasma membrane and intracellular granules of human polymorphonuclear neutrophils (PMN) contain large amounts of the glycolipid, lactosylceramide (LacCer; Gal beta 1----4Glc beta 1----1Cer). Despite its abundance, novel subcellular distribution, and lineage-restricted expression, nothing of PMN LacCer function is known. We examined the relationship between LacCer and PMN activation by assessing binding of anti-LacCer mAb (T5A7; anti-CDw17) to PMN during and after cell stimulation. CDw17 expression markedly decreased after treatment with PMA, dioctanoylglycerol, calcium ionophore, FMLP (with or without cytochalasin B or added Ca2+), TNF-alpha, or lymphotoxin. Depending on the stimulus, CDw17 declined to levels ranging from 70% (TNF, lymphotoxin) to less than 5% (phorbol ester, dioctanoylglycerol) of levels detected on untreated PMN. Loss of CDw17 from PMA-treated PMN followed dose- and temperature-dependent kinetics, with loss being detected after PMA treatment for 1 min. Membrane internalization explained PMA-induced loss of CDw17, as cell-associated 125I-anti-CDw17 became inaccessible to fluorescent anti-Ig after PMA treatment. CDw17 on PMN cytoplasts or retinoic acid-induced HL-60 cells was only slightly affected by stimulation, suggesting that down-regulation of the epitope is associated with granule exocytosis rather than superoxide production. Results with PMN from a patient with chronic granulomatous disease confirmed that normal superoxide production is not required for CDw17 loss induced by PMA or FMLP treatment. The data collectively demonstrate that reduced levels of cell-surface CDw17 are associated with granule exocytosis after PMN activation.     

The nature of large noncovalent complexes containing glycosyl-phosphatidylinositol-anchored membrane glycoproteins and protein tyrosine kinases.

A significant fraction of human glycosyl-phosphatidylinositol-anchored Ag CD59, CD55, CD48, and CDw52 is present in several cell lines tested (HPB-ALL, Jurkat, HL-60, Raji) in very large noncovalent complexes relatively resistant to dissociation by detergents. These complexes also contain some (glyco)lipids, such as these bearing the CD15, CDw17, and CDw65 determinants, and several intracellular components including protein tyrosine kinases and probably several of their potential substrates. Preclearing of the detergent lysates with different antibodies indicated that all these components are present jointly in a common single type of complexes the size of which is around 100 nm (molecular mass in the range of at least tens of thousands kilodaltons) as determined by ultrafiltration and gel chromatography. These results indicate the existence of cell-surface domains, specifically enriched in the above listed components, that may play a critical role in the so far poorly understood phenomenon of cell activation mediated through many different glycosyl-phosphatidylinositol-anchored (glyco)proteins and glycolipids.     

The HB-6, CDw75, and CD76 differentiation antigens are unique cell- surface carbohydrate determinants generated by the beta-galactoside alpha 2,6-sialyltransferase

Expression of the beta-galactoside alpha 2,6-sialyltransferase (alpha 2,6-ST) was shown to regulate the generation of multiple cell-surface differentiation antigens (Ags) that may be necessary for lymphocyte function. A new mAb was produced, termed HB-6, that was shown to identify a novel neuraminidase-sensitive cell-surface Ag expressed by subpopulations of human lymphocytes and erythrocytes. In attempting to isolate a cDNA encoding the HB-6 antigen by expression cloning, a cDNA encoding the alpha 2,6-ST (EC 2.4.99.1) was obtained. Since expression of the alpha 2,6-ST protein was shown to be limited to the Golgi apparatus, the cell-surface HB-6 Ag was demonstrated to be the product of alpha 2,6-ST activity. Interestingly, alpha 2,6-ST expression also generated two other neuraminidase-sensitive lymphocyte cell-surface differentiation Ags, CDw75, and CD76. The HB-6, CDw75, and CD76 mAb identified distinct Ags that were differentially expressed by different B cell lines and exhibited different patterns of expression in tissue sections. These results indicate that alpha 2,6-ST expression is a critical regulatory step in the formation of the Ags that are recognized by these mAb, and that an alpha 2,6-linked sialic acid residue is an essential component of each Ag. Thus, expression of a single ST can result in the generation of multiple distinct antigenic determinants on the cell surface which can be distinguished by mAb and may have regulatory roles in lymphocyte function.     

Flow cytometric assay for the measurement of human bone marrow phenotype, function and cell cycle

A flow cytometric assay for the measurement of human bone marrow and blood leukocyte antigen expression, phagocytosis, and proliferation is described. Subpopulations of leukocytes were identified by their light scatter characteristics, and the expression of a myeloid differentiation antigen (designated CDw65) determined following incubation with CDw65 specific fluorescein-isothiocyanate (FITC) conjugated monoclonal antibodies (VIM2). Incubation of leukocytes with ethidium monoazide (EMA) labeled Candida albicans followed by staining with FITC conjugated VIM2 allowed the combined determination of cellular CDw65 expression and phagocytic capacity. In addition, immunostained leukocytes were fixed, and their DNA labeled with propidium iodide (PI), before CDw65 expression was measured for cells in different phases of the cell cycle. The method allows evaluation of phenotypic and functional heterogeneity, as well as cell cycle parameters, within subpopulations of cells during hematopoietic differentiation.     

The Epstein-Barr virus encoded membrane protein (LMP) induces phenotypic changes in epithelial cells

The Epstein-Barr virus (EBV)-encoded membrane protein, LMP, is expressed in a proportion of undifferentiated nasopharyngeal carcinomas (NPC). Previous studies have shown that the transfection of the gene encoding LMP into a human keratinocyte line, RHEK-1, induces morphological alterations and a reduced expression of cytokeratins. We have analyzed immunophenotypic changes in the RHEK-1 line following LMP-transfection and compared these changes with the phenotype of NPC biopsies. We demonstrate a downregulation of two epithelial markers, an epithelial glycoprotein (EGP) defined by the monoclonal antibody Ber-EP4 and the epithelial membrane antigen (EMA). Furthermore, a lymphocyte activation-associated antigen, CDw70 antigen, which was absent from the parental line was expressed in virtually all LMP-transfected cells, whereas no similar effect was seen with respect to the CD30 activation antigen. Nine EBV-positive human NPCs, six of which were LMP-positive expressed the EGP and EMA. The CDw70 antigen, which is not normally present in epithelial cells, was expressed in eight biopsies, whereas the CD30 antigen was not detectable. Our findings are in keeping with the notion that LMP expression may contribute to the immunophenotype of human NPCs.     

Immunocytochemical localization of CDw60 antigens on human peripheral T cells

About 25-35% of human T cells display the CDw60 ganglioside (9-O-acetyl-GD3) antigen at the cell surface [E.P. Rieber, in W. Knapp, B. D?rken, W.R. Gilks, E.P. Rieber, R.E. Schmidt, H. Stein, A.E.G.K. von dem Borne (Eds.), Leucocyte Typing IV, Oxford University, Oxford, 1989, p. 361.]. Other leucocytes do not express this antigen on the cell surface. This led us to investigate its presence by flow cytometry and immunoelectron microscopy (IEM). Flow cytometric analysis of isolated peripheral T cells showed 26% of the cell population to have the CDw60 antigen expressed on the cell surface whereas 74% did not. Similarly, IEM analysis of 262 random T cells by the preembedding immunogold labeling technique revealed CDw60 surface expression to be tetrapartite: (a) the majority of 63.7% of the T cells did not show any surface associated gold label; (b) 19.5% were of low CDw60 surface exposition, corresponding to a linear density of 0.05-2.0 gold markers per microm; (c) about 13.4% showed a medium surface exposition with a linear density of 2.1-4.5 gold markers per microm; and (d) a high exposition, ranging from 4.6 to 9.0 gold markers per microm, was seen at 3.4% of the T cells. From postembedding label experiments, which additionally make access to the antigen localized within the cytoplasm, it was found that nearly all T cells contained low levels of intracellular CDw60. Most of it was found to be associated with the cytoplasmic membrane or vesicles, derived from the Golgi. Immunogold conjugates associated with the cytoplasmic membrane showed a linear density up to 0.6 gold markers per microm. The asymmetric expression of the CDw60 antigen on human T cells and its occurrence in nearly all T cells suggests that its surface presentation is tightly regulated.     

CDw60: an antigen expressed in many normal tissues and in some tumours

CDw60 is a recently described T-cell antigen, which functionally delivers a costimulatory signal in T-cell activation. In addition, CDw60 has been regarded as a melanoma-associated antigen. To date, only limited information exists on the distribution of CDw60 in other normal and pathologically altered tissues in human. In the present study, the expression of CDw60 was analysed immunohistologically in a large panel of formalin-fixed and paraffin-embedded normal and pathological human tissues. The antigen was detected in several normal tissues, such as epithelia of the reproductive system, exocrine and endocrine glands, glial cells and neurons of the central and peripheral nervous systems, and lymphoid cells. These showed different subcellular distribution patterns, i.e. (1) cell surface labelling of peripheral lymphocytes and lymphocytes of the lymph node and thymus, (2) diffuse cytosolic staining in lymphocytes, subpial glial processes, and the outer plexiform layer of the retina, (3) granular cytoplasmic staining associated with the Golgi apparatus in epithelial cells of certain endocrine and exocrine glands, of the ductus epididymis and deferens, neurons of the peripheral and central nervous system, and lymphocytes and megakaryocytes of the bone marrow.In exocrine glands, e.g. of the prostate and uterine corpus, CDw60-positive Golgi fields were located in the juxtaluminal cell compartment, thus reflecting a polarized distribution. In some malignant tumours, the neoplastic cells contained CDw60-immunolabelled Golgi complexes, which were disorderly distributed throughout the cytoplasm, thus reflecting a loss of epithelial polarity. Only in mammary carcinomas was abnormal cell surface labelling detected. A putative de novo expression of CDw60 was observed in pleomorphic adenoma and mucoepidermoid carcinoma of the parotid gland, seminoma, embryonal and teratocarcinoma of the testis, small cell carcinoma of the lung, and malignant melanoma. These results define the CDw60 determinant as a broadly distributed antigen within a large panel of normal human tissues. The antigen is also detectable in some previously undescribed benign and malignant tumours, which may give importance to CDw60 as a possible diagnostic marker.     

Effect of protein kinase C activators on the phosphorylation and the surface expression of the CDw50 leukocyte antigen.

The CDw50 antigen is a constitutively non-phosphorylated leukocyte surface molecule which becomes highly phosphorylated in all the normal and lymphoblastoid cells analyzed (peripheral blood mononuclear cells, Molt 4, CEM, 8402, Namalwa), after stimulation with tumor promoter agents (phorbol 12-myristate 13-acetate, phorbol 12,13-dibutyrate, mezerein). This phosphorylation is rapid (within 1-5 min), dose-dependent and results in the incorporation of PO(3-)4 groups on serine residues. Furthermore, the level of CDw50 phosphorylation induced by tumor promoter agents is decreased by the protein kinase C inhibitors staurosporine and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine. Activation of peripheral lymphocytes with concanavalin A, phytohemagglutinin and cross-linking of CD3 molecules also induces CDw50 phosphorylation, but the response is delayed and less intense than when tumor promoting agents are used. Treatment with any of the aforementioned agents is not accompanied by quantitative changes in the CDw50 surface expression. We therefore conclude that protein-kinase-C-mediated mechanisms are involved in phosphorylation, but not in regulation of the surface expression of the CDw50 leukocyte antigen.     

Membrane microdomains in immunity: glycosphingolipid-enriched domain-mediated innate immune responses

Over the last 30 years, many studies have indicated that glycosphingolipids (GSLs) expressed on the cell surface may act as binding sites for microorganisms. Based on their physicochemical characteristics, GSLs form membrane microdomains with cholesterol, sphingomyelin, glycosylphosphatidylinositol (GPI)-anchored proteins, and various signaling molecules, and GSL-enriched domains have been shown to be involved in these defense responses. Among the GSLs, lactosylceramide (LacCer, CDw17) can bind to various microorganisms. LacCer is expressed at high levels on the plasma membrane of human neutrophils, and forms membrane microdomains associated with the Src family tyrosine kinase Lyn. LacCer-enriched membrane microdomains mediate superoxide generation, chemotaxis, and non-opsonic phagocytosis. Therefore, LacCer-enriched membrane microdomains are thought to function as pattern recognition receptors (PRRs) to recognize pathogen-associated molecular patterns (PAMPs) expressed on microorganisms. In contrast, several pathogens have developed infection mechanisms using membrane microdomains. In addition, some pathogens have the ability to avoid degradation by escaping from the vacuolar compartment or preventing phagosome maturation, utilizing membrane microdomains, such as LacCer-enriched domains, of host cells. The detailed molecular mechanisms of these membrane microdomain-associated host-pathogen interactions remain to be elucidated.     

A novel role of lactosylceramide in the regulation of tumor necrosis factor alpha-mediated proliferation of rat primary astrocytes. Implications for astrogliosis following neurotrauma

The present study describes the role of glycosphingolipids in neuroinflammatory disease and investigates tumor necrosis factor alpha (TNFalpha)-induced astrogliosis following spinal cord injury. Astrogliosis is the hallmark of neuroinflammation and is characterized by proliferation of astrocytes and increased glial fibrillary acidic protein (GFAP) gene expression. In primary astrocytes, TNFalpha stimulation increased the intracellular levels of lactosylceramide (LacCer) and induced GFAP expression and astrocyte proliferation. D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol.HCl (PDMP), a glucosylceramide synthase and LacCer synthase (GalT-2) inhibitor, inhibited astrocyte proliferation and GFAP expression, which were reversed by exogenous supplementation of LacCer but not by other glycosphingolipids. TNFalpha caused a rapid increase in the activity of GalT-2 and synthesis of LacCer. Silencing of GalT-2 gene using antisense oligonucleotides also attenuated the proliferation of astrocytes and GFAP expression. The PDMP and antisense-mediated inhibition of proliferation and GFAP expression was well correlated with decreased Ras/ERK1/2 pathway activation. Furthermore, TNFalpha-mediated astrocyte proliferation and GFAP expression was also inhibited by LY294002, a phosphatidylinositol 3-kinase inhibitor, which was reversed by exogenous LacCer. LY294002 also inhibited TNFalpha-induced GalT-2 activation and LacCer synthesis, suggesting a phosphatidylinositol 3-kinase-mediated regulation of GalT-2. In vivo, PDMP treatment attenuated chronic ERK1/2 activation and spinal cord injury (SCI)-induced astrocyte proliferation with improved functional recovery post-SCI. Therefore, the in vivo studies support the conclusions drawn from cell culture studies and provide evidence for the role of LacCer in TNFalpha-induced astrogliosis in a rat model of SCI. To our knowledge, this is the first report demonstrating the role of LacCer in the regulation of TNFalpha-induced proliferation and reactivity of primary astrocytes.     

A lymphocyte molecule implicated in lymph node homing is a member of the cartilage link protein family

Monoclonal antibodies in the Hermes family recognize a lymphocyte structure that participates in lymphocyte adhesion to endothelium and has been suggested to be the human homolog of the murine Mel-14 lymph node homing receptor. Recently, antibodies against the Hermes antigen, the polymorphic glycoprotein Pgp-1 antigen, and the broadly expressed CDw44 antigen have been shown to recognize the same structure. In this work, cDNA clones encoding the CDw44 antigen were isolated and expressed in COS cells. Two forms were identified: a lymphoid form expressed in hematopoietic cells, and an epithelial form weakly expressed in normal epithelium but highly expressed in carcinomas. The extracellular domain of CDw44 bears homology to cartilage link proteins and a related segment of proteoglycan core protein. However, comparison with the recently identified sequence of the Mel-14 antigen shows that CDw44 and Mel-14 are unrelated.     

Intracellular localization of lactosylceramide, the major human neutrophil glycosphingolipid

The abundance of lactosylceramide (LacCer; Gal beta 1-4Glc beta 1-1Cer) in human polymorphonuclear neutrophils (PMN) (about 10(9) molecules/cell) seemed inconsistent with an exclusive plasma membrane LacCer localization in these cells. Therefore, the distribution of LacCer between plasma membrane and intracellular compartments was analyzed. Binding of 125I-labeled monoclonal anti-LacCer antibody (T5A7) to intact cells indicated that only 0.1-0.2% of total LacCer was accessible to antibody. Chemical and immunochemical comparisons of organic extracts prepared from PMN cytoplasts (i.e. PMN depleted of nucleus and granules) and intact PMN demonstrated that less than 25% of PMN LacCer was plasma membrane-derived. Simultaneous particle volume and surface staining analyses suggested that selective LacCer loss from cytoplasts could not explain this result. Intracellular LacCer was demonstrated by intense staining of PMN frozen thin sections with T5A7 in indirect immunofluorescence tests. Two-color fluorescence studies using frozen thin sections of neutrophils previously surface-stained with saturating concentrations of T5A7 indicated that this staining did not reflect section artifact. Organic extracts of density gradient-fractionated PMN cavitates were analyzed by radioimmunoassay to determine whether LacCer associates with known populations of PMN granules. Antigen predominantly cosedimented with enzyme markers for primary and secondary granules rather than with plasma membrane marker. Thin layer chromatography of glycolipids extracted from these density gradient fractions identified LacCer as the only antigenic species and demonstrated that chemically detectable LacCer was primarily in granule-enriched rather than plasma membrane fractions. These data indicate that human PMN LacCer is predominantly intracellular and that the glycolipid may be a constituent of PMN lysosomal granules.     

Activation of human phagocytes through carbohydrate antigens (CD15, sialyl-CD15, CDw17, and CDw65).

The leukocyte carbohydrate (CHO) Ag CD15, sialyl-CD15, and CDw65 have recently been found to function as ligands for CD62 and ELAM-1 cell adhesion molecules on platelets and endothelium, respectively. Cell adhesion ligands also may act as receptors capable of signal transduction. We therefore investigated the possibility that these CHO Ag and CDw17, a glycolipid Ag whose expression is regulated by leukocyte activation, may have receptor-like characteristics. The effects of antibody cross-linking of CHO Ag on phagocyte activation were measured by using flow cytometry and fluorescent indicators for cytoplasmic calcium ions, oxidative burst, and the granule-associated proteins CD11b and CD67. Cross-linking of CD15, sialyl-CD15, CDw65, or CDw17 induced a moderate release of calcium ions into the cytoplasm of granulocytes, a strong activation of oxidative burst, and a low up-regulation of CD11b and CD67 compared to the effects of treatment with 4 microM FMLP. The results suggest a role for CHO Ag in leukocyte signal transduction and support the view that these molecules are involved in phagocyte activation.     

Mutational analysis of antigen receptor regulation of B lymphocyte growth. Evidence for involvement of the phosphoinositide signaling pathway.

Stimulation of the antigen receptor of WEHI-231 B lymphoma cells with anti-receptor antibodies (anti-IgM) induces irreversible growth arrest. Anti-IgM stimulates two kinds of transmembrane signaling events, phosphorylation of proteins on tyrosyl residues and breakdown of inositol phospholipids, which results in increases of inositol phosphates, diacylglycerol, and calcium. The roles of these reactions in mediating the growth arrest of the B lymphoma cells have not been established. To examine this issue, we took a genetic approach. Mutants of WEHI-231 cells were isolated that were resistant to anti-IgM-induced growth arrest. Five out of seven independent mutants analyzed had normal cell-surface expression of antigen receptors. Although each of these five mutants had tyrosine protein phosphorylation patterns comparable to wild-type cells, they exhibited alterations in the phosphoinositide signaling pathway. Four of the mutants had decreased phosphoinositide breakdown, probably due to an alteration in phospholipase C. Decreased second messenger production may be responsible for the growth-resistant phenotype. Full growth arrest was restored upon addition of the calcium ionophore ionomycin, suggesting that the limiting second messenger was intracellular free calcium. The final mutant appeared to be altered in a component(s) that responds to diacylglycerol and calcium. Taken together, these results provide further evidence that the phosphoinositide pathway is at least partly responsible for mediating antigen receptor regulation of B lymphoma cell growth.     

Human memory T lymphocytes express increased levels of three cell adhesion molecules (LFA-3, CD2, and LFA-1) and three other molecules (UCHL1, CDw29, and Pgp-1) and have enhanced IFN-gamma production

Studies of cell-surface molecules involved in human T cell interaction reveal that differential expression of each of three adhesion molecules (LFA-3, CD2, and LFA-1) subdivides human peripheral blood T cells into major subpopulations. Systematic analysis of the relationship between expression of these and other markers of T cell subsets demonstrates a single major subset of human peripheral blood T lymphocytes distinguished by enhanced expression of LFA-3, CD2, LFA-1, and three other markers (CDw29 [4B4], UCHL1, and Pgp-1). Large differences in relative expression are observed for UCHL1 (29-fold) and LFA-3 (greater than 8-fold), and smaller differences (2- to 4-fold) are seen for CDw29, CD2, LFA-1, and Pgp-1. Bimodal distribution of LFA-3 is found on both CD4+ cells and on CD8+ cells as well as on B lymphocytes (CD19+). Neonatal T cells (CD3+) are comprised almost exclusively of the subset expressing low LFA-3, CD2, LFA-1, CDw29, and UCHL1. Activation of cord peripheral blood mononuclear leukocytes with PHA leads to uniform enhanced expression of each of these molecules on CD3+ cells. Functional analyses of these T cell subsets were performed after sorting of adult T cells based on differential LFA-3 expression. Only the LFA-3+ subset proliferated in response to the Ag tetanus toxoid, even though the LFA-3- subset proliferated more strongly to PHA. Furthermore, the LFA-3+ subset made greater than fivefold more IFN-gamma than the LFA-3- subset in response to PHA, despite the fact that both subsets made equivalent amounts of IL-2. This phenotypic and functional analysis of resting and activated newborn and adult T cells indicates that human memory T cells express enhanced levels of LFA-3, CD2, LFA-1, UCHL1, CDw29, and Pgp-1; we speculate that the increase in expression of T cell adhesion molecules LFA-3, CD2, and LFA-1 on memory cells is functionally important in their enhanced responsiveness.     

Studies on the human blood group P system: an existence of UDP-Gal:lactosylceramide alpha 1----4 galactosyltransferase in the small p type cells

Human red blood cells and other tissues with the rare small p type lack all P antigens, and are assumed to be missing key glycosyltransferases in the synthetic pathway of P antigens. Galactosyltransferase activities of the P1 and small p cell extracts were measured using lactosylceramide and GlcNAc as galactose acceptors. The two transferase activities of the small p lymphoblastoid cell extract were comparable to that of the P1 cell extract. The anomeric configuration of the galactosylated lactosylceramide was established by digestion with alpha- and beta-galactosidases, by identification of methylated products, and by staining with the monoclonal antibody against globotriaosyl ceramide (Pk antigen). The results indicate that UDP-Gal:LacCer alpha 1----4 Gal transferase, which produces the Pk antigen from the precursor LacCer, exists in the small p cells. However, intact small p cells could not produce the Pk antigen, and, instead, LacCer was accumulated in the cells. The Pk enzyme appears to be not functional in the small p cells in vivo.     

Identification of a cell-surface antigen produced by a gene on human chromosome 3 (cen-q22) and not expressed by Rhnull cells.

A monoclonal antibody, 1D8, which recognizes a cell-surface antigen expressed by human chromosome 3 in Chinese hamster-human somatic-cell hybrids, has been produced. Testing of hybrids containing various deletions of chromosome 3 determines that the gene encoding the antigen is regionally localized to 3q (cen-22). This regional mapping is distinct from that elsewhere reported for two other cell-surface antigens assigned to chromosome 3--namely, the human transferrin receptor and the p97 melanoma-associated antigen. In addition, biochemical characterization is different from that elsewhere reported for other chromosome 3-encoded cell-surface antigens. When tested against a panel of rare-phenotype red blood cells, the only cells that failed to react were those of the Rhnull phenotype. The antibody reacts only weakly with homozygous -D- and fetal red cells, in contrast with a previously described antibody, R6A, which does not react with Rhnull cells. Furthermore, R6A does not recognize a cell-surface antigen expressed by chromosome 3 in Chinese hamster-human somatic-cell hybrids. Thus, the monoclonal antibody 1D8 recognizes a previously undescribed cell-surface antigen encoded by human chromosome 3 and not expressed on Rhnull cells. The gene on chromosome 3 regulating expression of this antigen may be that defective in Rhnull disease or may require the normal allele at an unlinked Rhnull locus for expression. Linkage studies will be required to further elucidate this matter.     

Cell adhesion, spreading, and motility of GM3-expressing cells based on glycolipid-glycolipid interaction

Cell lines expressing varying levels of ganglioside GM3 at the cell surface show different degrees of adhesion and spreading on solid phase coated with such glycosphingolipids (GSLs) as Gg3 (GalNAc beta 1----4Gal beta 1----4Glc beta 1----1Cer), LacCer (Gal beta 1----4Glc beta 1----1Cer), or Gb4 (GalNAc beta 1----3Gal alpha 1----4Gal beta 1----4Glc beta 1----1Cer) (where Cer is ceramide), which may have structures complementary to GM3, but not on solid phase coated with various other GSLs. The degree of cell adhesion and spreading on Gg3 was correlated with the degree of cell-surface GM3 expression, as defined by reactivity with anti-GM3 monoclonal antibody (mAb) DH2. Only cells with high GM3 expression adhered on solid phase coated with LacCer or Gb4. Adhesion of GM3-expressing cells on Gg3-, LacCer-, and Gb4-coated solid phase is based on interaction of GM3 with Gg3 and, to a lesser extent, with LacCer and Gb4, as demonstrated by: (i) the interaction of the GM3 liposome with solid phase coated with Gg3, LacCer, and Gb4, respectively; (ii) the abolition of cell adhesion on each GSL-coated solid phase by treatment of cells with mAb DH2 or sialidase; and (iii) the inhibition of cell adhesion by treatment of GSL-coated solid phase with mAb specific to each GSL. Sialosyllactosyl-lysyllysine conjugate was bound to Gg3 adsorbed on a C18 silica gel column in the presence of bivalent cation, suggesting that the carbohydrate moiety of GM3 is involved in GM3-Gg3 interaction. Not only the adhesion and spreading of GM3-expressing cells, but also their cell motility was greatly enhanced on Gg3-coated solid phase, as determined by Transwell assay and phagokinetic track assay on a gold sol-coated surface. Spreading and motility of GM3-expressing cells on Gg3-coated solid phase were both inhibited by treatment of cells with mAb DH2 or sialidase. These results provide evidence that not only cell adhesion, but also spreading and motility in these cell lines are controlled by complementary GSL-GSL interaction.