Novel sulfated gangliosides, high-affinity ligands for neural siglecs, inhibit NADase activity of leukocyte cell surface antigen CD38

Three kinds of novel sulfated gangliosides structurally related to the Chol-1 (alpha-series) ganglioside GQ1balpha were synthesized. These sulfated gangliosides were potent inhibitors of NADase activity of leukocyte cell surface antigen CD38. Among the synthetic gangliosides, GSC-338 (II(3)III(6)-disulfate of iso-GM1b) was surprisingly found to be the most potent structure in both the NADase inhibition and MAG-binding activity. The present study indicates that the sulfated gangliosides are useful to study the recognition of the internal tandem sialic acid residues alpha2-3-linked to Gal(II(3)) as well as the siglec-dependent recognition including a terminal sialic acid residue.     

Nicotinamide adenine dinucleotide (NAD) and its metabolites inhibit T lymphocyte proliferation: role of cell surface NAD glycohydrolase and pyrophosphatase activities

The presence of NAD-metabolizing enzymes (e.g., ADP-ribosyltransferase (ART)2) on the surface of immune cells suggests a potential immunomodulatory activity for ecto-NAD or its metabolites at sites of inflammation and cell lysis where extracellular levels of NAD may be high. In vitro, NAD inhibits mitogen-stimulated rat T cell proliferation. To investigate the mechanism of inhibition, the effects of NAD and its metabolites on T cell proliferation were studied using ART2a+ and ART2b+ rat T cells. NAD and ADP-ribose, but not nicotinamide, inhibited proliferation of mitogen-activated T cells independent of ART2 allele-specific expression. Inhibition by P2 purinergic receptor agonists was comparable to that induced by NAD and ADP-ribose; these compounds were more potent than P1 agonists. Analysis of the NAD-metabolizing activity of intact rat T cells demonstrated that ADP-ribose was the predominant metabolite, consistent with the presence of cell surface NAD glycohydrolase (NADase) activities. Treatment of T cells with phosphatidylinositol-specific phospholipase C removed much of the NADase activity, consistent with at least one NADase having a GPI anchor; ART2- T cell subsets contained NADase activity that was not releasable by phosphatidylinositol-specific phospholipase C treatment. Formation of AMP from NAD and ADP-ribose also occurred, a result of cell surface pyrophosphatase activity. Because AMP and its metabolite, adenosine, were less inhibitory to rat T cell proliferation than was NAD or ADP-ribose, pyrophosphatases may serve a regulatory role in modifying the inhibitory effect of ecto-NAD on T cell activation. These data suggest that T cells express multiple NAD and adenine nucleotide-metabolizing activities that together modulate immune function.     

ATRA-induced HL-60 myeloid leukemia cell differentiation depends on the CD38 cytosolic tail needed for membrane localization, but CD38 enzymatic activity is unnecessary

Leukocyte antigen CD38 expression is an early marker of all-trans retinoic acid (ATRA) stimulated differentiation in the leukemic cell line HL-60. It promotes induced myeloid maturation when overexpressed, whereas knocking it down is inhibitory. It is a type II membrane protein with an extracellular C-terminal enzymatic domain with NADase/NADPase and ADPR cyclase activity and a short cytoplasmic N-terminal tail. Here we determined whether CD38 enzymatic activity or the cytoplasmic tail is required for ATRA-induced differentiation. Neither a specific CD38 ectoenzyme inhibitor nor a point mutation that cripples enzymatic activity (CD38 E226Q) diminishes ATRA-induced differentiation or G1/0 arrest. In contrast a cytosolic deletion mutation (CD38 Δ11–20) prevents membrane expression and inhibits differentiation and G1/0 arrest. These results may be consistent with disrupting the function of critical molecules necessary for membrane-expressed CD38 signal transduction. One candidate molecule is the Src family kinase Fgr, which failed to undergo ATRA-induced upregulation in CD38 Δ11–20 expressing cells. Another is Vav1, which also showed only basal expression after ATRA treatment in CD38 Δ11–20 expressing cells. Therefore, the ability of CD38 to propel ATRA-induced myeloid differentiation and G1/0 arrest is unimpaired by loss of its ectoenzyme activity. However a cytosolic tail deletion mutation disrupted membrane localization and inhibited differentiation. ATRA-induced differentiation thus does not require the CD38 ectoenzyme function, but is dependent on a membrane receptor function.     

Gangliosides act as co-receptors for Salmonella enteritidis FliC and promote FliC induction of human beta-defensin-2 expression in Caco-2 cells

Antimicrobial peptides such as defensins are crucial for host defense at mucosal surfaces. We reported previously that Salmonella enteritidis flagellin (FliC) induced human beta-defensin-2 (hBD-2) mRNA expression in Caco-2 cells via NF-kappaB activation (Ogushi, K., Wada, A., Niidome, T., Mori, N., Oishi, K., Nagatake, T., Takahashi, A., Asakura, H., Makino, S., Hojo, H., Nakahara, Y., Ohsaki, M., Hatakeyama, T., Aoyagi, H., Kurazono, H., Moss, J., and Hirayama, T. (2001) J. Biol. Chem. 276, 30521-30526). In this study, we examined the role of ganglioside as co-receptors with Toll-like receptor 5 (TLR5) on FliC induction of hBD-2 expression in Caco-2 cells. Exogenous gangliosides suppressed FliC induction of hBD-2 promoter activity and binding of FliC to Caco-2 cells. Incorporation of exogenous ganglioside GD1a into Caco-2 cell membranes increased the effect of FliC on hBD-2 promoter activity. In support of a role for endogenous gangliosides, incubation of Caco-2 cells with dl-threo-2-hexadecanoylamino-3-morpholino-1-phenylpropanol, a glucosylceramide synthase inhibitor, reduced FliC induction of hBD-2 promoter activity. GD1a-loaded CHO-K1-expressing TLR5 cells had a higher potential for hBD-2 induction following FliC stimulation than GD1a-loaded CHO-K1 cells not expressing TLR5. FliC increased phosphorylation of mitogen-activated protein kinase, p38, and ERK1/2. Exogenous gangliosides GD1a, GD1b, and GT1b each suppressed FliC induction of p38 and ERK1/2 phosphorylation. Furthermore, FliC did not enhance luciferase activity in Caco-2 cells transfected with a plasmid containing a mutated activator protein 1-binding site. These results suggest that gangliosides act as co-receptors with TLR5 for FliC and promote hBD-2 expression via mitogen-activated protein kinase.     

Tumor gangliosides inhibit the tumor-specific immune response.

Tumor gangliosides are highly immunosuppressive membrane glycosphingolipids that are shed into the tumor cell microenvironment. We directly tested the impact of shed gangliosides on the in vivo antitumor immune response in a syngeneic fully autochthonous system (FBL-3 erythroleukemia cells, C57BL/6 mice, and highly purified FBL-3 cell gangliosides). The major FBL-3 ganglioside was identified as GM1b by mass spectrometry. Substantial ganglioside shedding (90 pmol/108 cells/h), a requisite for their inhibition of the immune function of tumor-infiltrating leukocytes, was detected. Immunosuppression by FBL-3 gangliosides was potent; 5-20 microM inhibited the tumor-specific secondary proliferative response (80-100%) and suppressed the generation of tumor-specific CTLs (97% reduction of FBL-3 cell lysis at an E:T ratio of 100:1). In vivo, coinjection of 10 nmol of FBL-3 gangliosides with a primary FBL-3 cell immunization led to a reduced response to a secondary challenge (the increase in the draining popliteal lymph node mass, cell number, and lymphocyte thymidine incorporation were lowered by 70, 69, and 72%, respectively). Coinjection of gangliosides with a secondary tumor challenge led to a 61, 74, and 42% reduction of the increase in lymph node mass, cell number, and thymidine uptake and a 63-74% inhibition of the increase of draining lymph node T cells (CD3+), B cells (CD19+), and dendritic cells/macrophages (Mac-3+). Overall, the clear conclusion that tumor-derived gangliosides inhibit syngeneic antitumor immune responses implicates these molecules as a potent factor in promoting tumor formation and progression.     

A ligand for CD5 is CD5

Recognition by scavenger receptor cysteine-rich domains on membrane proteins regulates innate and adaptive immune responses. Two receptors expressed primarily on T cells, CD5 and CD6, are linked genetically and are structurally similar, both containing three scavenger receptor cysteine-rich domains in their extracellular regions. A specific cell surface interaction for CD5 has been difficult to define at the molecular level because of the susceptibility of CD5 protein to denaturation. By using soluble CD5 purified at neutral pH to preserve biological activity, we show that CD5 mediates species-specific homophilic interactions. CD5 domain 1 only is involved in the interaction. CD5 mAbs that have functional effects in humans, rats, and mice block homophilic binding. Ag-specific responses by mouse T cells in vitro were increased when engagement of human CD5 domain 1 was inhibited by mutation or by IgG or Fab fragment from a CD5 mAb. This showed that homophilic binding results in productive engagement. Enhancement of polyclonal immune responses of rat lymph node cells by a Fab fragment from a CD5 mAb shown to block homophilic interactions provided evidence that the extracellular region of CD5 regulates inhibition in normal cells. These biochemical and in vitro functional assays provide evidence that the extracellular region of CD5 regulates immunity through species-specific homophilic interactions.     

Myelin-associated glycoprotein interacts with ganglioside GT1b. A mechanism for neurite outgrowth inhibition

Myelin-associated glycoprotein (MAG) is expressed on myelinating glia and inhibits neurite outgrowth from post-natal neurons. MAG has a sialic acid binding site in its N-terminal domain and binds to specific sialylated glycans and gangliosides present on the surface of neurons, but the significance of these interactions in the effect of MAG on neurite outgrowth is unclear. Here we present evidence to suggest that recognition of sialylated glycans is essential for inhibition of neurite outgrowth by MAG. Arginine 118 on MAG is known to make a key contact with sialic acid. We show that mutation of this residue reduces the potency of MAG inhibitory activity but that residual activity is also a result of carbohydrate recognition. We then go on to investigate gangliosides GT1b and GD1a as candidate MAG receptors. We show that MAG specifically binds both gangliosides and that both are expressed on the surface of MAG-responsive neurons. Furthermore, antibody cross-linking of cell surface GT1b, but not GD1a, mimics the effect of MAG, in that neurite outgrowth is inhibited through activation of Rho kinase. These data strongly suggest that interaction with GT1b on the neuronal cell surface is a potential mechanism for inhibition of neurite outgrowth by MAG.     

SHP-1 and SHP-2 associate with immunoreceptor tyrosine-based switch motif of programmed death 1 upon primary human T cell stimulation, but only receptor ligation prevents T cell activation

To study the cis- and trans-acting factors that mediate programmed death 1 (PD-1) signaling in primary human CD4 T cells, we constructed a chimeric molecule consisting of the murine CD28 extracellular domain and human PD-1 cytoplasmic tail. When introduced into CD4 T cells, this construct mimics the activity of endogenous PD-1 in terms of its ability to suppress T cell expansion and cytokine production. The cytoplasmic tail of PD-1 contains two structural motifs, an ITIM and an immunoreceptor tyrosine-based switch motif (ITSM). Mutation of the ITIM had little effect on PD-1 signaling or functional activity. In contrast, mutation of the ITSM abrogated the ability of PD-1 to block cytokine synthesis and to limit T cell expansion. Further biochemical analyses revealed that the ability of PD-1 to block T cell activation correlated with recruitment of Src homology region 2 domain-containing phosphatase-1 (SHP-1) and SHP-2, and not the adaptor Src homology 2 domain-containing molecule 1A, to the ITSM domain. In TCR-stimulated T cells, SHP-2 associated with PD-1, even in the absence of PD-1 engagement. Despite this interaction, the ability of PD-1 to block T cell activation required receptor ligation, suggesting that colocalization of PD-1 with CD3 and/or CD28 may be necessary for inhibition of T cell activation.     

Neolacto-series gangliosides induce granulocytic differentiation of human promyelocytic leukemia cell line HL-60

Neolacto-series gangliosides having linear poly-N-acetyl-lactosaminyl oligosaccharide structure have been demonstrated to be increased characteristically during granulocytic differentiation of human promyelocytic leukemia cell line HL-60 cells induced by dimethyl sulfoxide or retinoic acid (Nojiri, H., Takaku, F., Tetsuka, T., Motoyoshi, K., Miura, Y., and Saito, M. (1984) Blood 64, 534-541). When HL-60 cells were cultured in the presence of neolacto-series gangliosides prepared from mature granulocytes, the cells were found to be differentiated into mature granulocytes on the basis of the changes of morphology, surface membrane antigens, nonspecific esterase activity, and the activity of phagocytosis and respiratory burst. The differentiation of cells was dependent on the concentration of gangliosides and accompanied with inhibition of cell growth. These findings suggest that the particular ganglioside molecules play an important role in regulation of cell differentiation and that the appearance of neolacto-series gangliosides on cell surface membrane not only triggers the differentiation but also determines the direction of differentiation in HL-60 cells.     

Soluble CD26/dipeptidyl peptidase IV enhances transendothelial migration via its interaction with mannose 6-phosphate/insulin-like growth factor II receptor

CD26 is a T cell surface molecule with dipeptidyl peptidase IV (DPPIV) enzyme activity in its extracellular region. In addition to its membrane form, CD26 exists in plasma as a soluble form (sCD26), which is the extracellular domain of the molecule thought to be cleaved from the cell surface. In this paper, we demonstrate that sCD26 mediates enhanced transendothelial T cell migration, an effect that requires its intrinsic DPPIV enzyme activity. We also show that sCD26 directly targets endothelial cells and that mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGFIIR) on the endothelial cell surface acts as a receptor for sCD26. Our findings therefore suggest that sCD26 influences T cell migration through its interaction with M6P/IGFIIR.     

Interaction of the extracellular domain of the epidermal growth factor receptor with gangliosides.

Ganglioside GM3 inhibits epidermal growth factor (EGF)-dependent cell proliferation in a variety of cell lines. Both in vitro and in vivo, this glycosphingolipid inhibits the kinase activity of the EGF receptor (EGFR). Furthermore, membrane preparations containing EGFR can bind to GM3-coated surfaces. These data suggest that GM3 may interact directly with the EGFR. In this study, the interaction of gangliosides with the extracellular domain (ECD) of the EGFR was investigated. The purified human recombinant ECD from insect cells bound directly to ganglioside GM3. The ganglioside interaction site appears to be distinct from the EGF-binding site. In agreement with previous reports on the effects of specific gangliosides on EGFR kinase activity, the ECD preferentially interacted with GM3. The order of relative binding of other gangliosides investigated was as follows: GM3 GM2, GD3, GM4 > GM1, GD1a, GD1b, GT1b, GD2, GQ1b > lactosylceramide. These data suggest that NeuAc-lactose is essential for binding and that any sugar substitution reduces binding. In agreement with the specificity of soluble ECD binding to gangliosides, GM3 specifically inhibited EGFR autophosphorylation. Identification of a ganglioside interaction site on the ECD of the EGFR is consistent with the hypothesis that endogenous GM3 may function as a direct modulator of EGFR activity.     

Brain gangliosides: functional ligands for myelin stability and the control of nerve regeneration

Gangliosides, sialylated glycosphingolipids which are the predominant glycans on vertebrate nerve cell surfaces, are emerging as components of membrane rafts, where they can mediate important physiological functions. Myelin associated glycoprotein (MAG), a minor constituent of myelin, is a sialic acid binding lectin with two established physiological functions: it is involved in myelin-axon stability and cytoarchitecture, and controls nerve regeneration. MAG is found selectively on the myelin membranes directly apposed to the axon surface, where it has been proposed to mediate myelin-axon interactions. Although the nerve cell surface ligands for MAG remain to be established, evidence supports a functional role for sialylated glycoconjugates. Here we review recent studies that reflect on the role of gangliosides, sialylated glycosphingolipids, as functional MAG ligands. MAG binds to gangliosides with the terminal sequence 'NeuAc alpha 3Gal beta 3GalNAc' which is found on the major nerve gangliosides GD1a and GT1b. Gangliosides lacking that terminus (e.g., GM1 or GD1b), or having any biochemical modification of the terminal NeuAc residue fail to support MAG binding. Genetically engineered mice lacking the GalNAc transferase required for biosynthesis of the 'NeuAc alpha 3Gal beta 3GalNAc' terminus have grossly impaired myelination and progressive neurodegeneration. Notably the MAG level in these animals is dysregulated. Furthermore, removal of NeuAc residues from nerve cells reverses MAG-mediated inhibition of neuritogenesis, and neurons from mice lacking the 'NeuAc alpha 3 Gal beta 3GalNAc' terminus have an attenuated response to MAG. Cross-linking nerve cell surface gangliosides can mimic MAG-mediated inhibition of nerve regeneration. Taken together these observations implicate gangliosides as functional MAG ligands.     

Enhancement of epidermal growth factor signaling and activation of SRC kinase by gangliosides

In a recent study, inhibition of cellular ganglioside synthesis blocked growth factor-induced fibroblast proliferation. Conversely, enrichment of cell membrane gangliosides by ganglioside preincubation enhanced growth factor-elicited cell proliferation. In the absence of serum and growth factors, NeuNAcalpha2-3Galbeta1-3GalNAcbeta1-4(NeuNAcalpha2-3)Galbeta1-4Glcbeta1-1Cer (G(D1a)) acted like a growth factor when cells were pretreated with the ganglioside, stimulating proliferation of normal human dermal fibroblasts and Swiss 3T3 fibroblasts. In contrast, growth inhibition was observed when high concentrations of gangliosides were continuously present in the culture medium during incubation of fibroblasts with growth factors (Li, R., Manela, J., Kong, Y., and Ladisch, S. (2000) J. Biol. Chem. 275, 34213-34223). Here, we investigated the mechanisms whereby gangliosides elicit proliferation-coupled signaling in normal human dermal fibroblasts. Incubation of the fibroblasts with G(D1a) enhanced epidermal growth factor (EGF) receptor autophosphorylation and Ras and MAPK activation in a dose-dependent manner. Exposure of the cells to G(D1a) also enhanced the phosphorylation of Elk-1 by the activated MAPK. Brief pretreatment of the cells with PD98059 blocked the enhancing effect of gangliosides on EGF-induced MAPK activation. In the absence of serum and growth factors, G(D1a) incubation induced phosphorylation of Src kinase, Ras activation, and phosphorylation of MAPK and Elk-1 in a dose-dependent manner. The activation of Src kinase was confirmed by enhanced Src kinase activity. Brief treatment of the cells with PP1 blocked the activation of Src kinase and MAPK. Again, PD98059 treatment inhibited ganglioside-elicited MAPK phosphorylation. Among the gangliosides tested, G(D1a), was the most active molecule, whereas lactosylceramide was the least active one, indicating relative structural specificity of the ganglioside action. In conclusion, gangliosides promote fibroblast proliferation through enhancement of growth factor signaling and activation of Src kinase.     

Water-soluble LYNX1 Residues Important for Interaction with Muscle-type and/or Neuronal Nicotinic Receptors

Human LYNX1, belonging to the Ly6/neurotoxin family of three-finger proteins, is membrane-tethered with a glycosylphosphatidylinositol anchor and modulates the activity of nicotinic acetylcholine receptors (nAChR). Recent preparation of LYNX1 as an individual protein in the form of water-soluble domain lacking glycosylphosphatidylinositol anchor (ws-LYNX1; Lyukmanova, E. N., Shenkarev, Z. O., Shulepko, M. A., Mineev, K. S., D''Hoedt, D., Kasheverov, I. E., Filkin, S. Y., Krivolapova, A. P., Janickova, H., Dolezal, V., Dolgikh, D. A., Arseniev, A. S., Bertrand, D., Tsetlin, V. I., and Kirpichnikov, M. P. (2011) NMR structure and action on nicotinic acetylcholine receptors of water-soluble domain of human LYNX1. J. Biol. Chem. 286, 10618–10627) revealed the attachment at the agonist-binding site in the acetylcholine-binding protein (AChBP) and muscle nAChR but outside it, in the neuronal nAChRs. Here, we obtained a series of ws-LYNX1 mutants (T35A, P36A, T37A, R38A, K40A, Y54A, Y57A, K59A) and examined by radioligand analysis or patch clamp technique their interaction with the AChBP, Torpedo californica nAChR and chimeric receptor composed of the α7 nAChR extracellular ligand-binding domain and the transmembrane domain of α1 glycine receptor (α7-GlyR). Against AChBP, there was either no change in activity (T35A, T37A), slight decrease (K40A, K59A), and even enhancement for the rest mutants (most pronounced for P36A and R38A). With both receptors, many mutants lost inhibitory activity, but the increased inhibition was observed for P36A at α7-GlyR. Thus, there are subtype-specific and common ws-LYNX1 residues recognizing distinct targets. Because ws-LYNX1 was inactive against glycine receptor, its “non-classical” binding sites on α7 nAChR should be within the extracellular domain. Micromolar affinities and fast washout rates measured for ws-LYNX1 and its mutants are in contrast to nanomolar affinities and irreversibility of binding for α-bungarotoxin and similar snake α-neurotoxins also targeting α7 nAChR. This distinction may underlie their different actions, i.e. nAChRs modulation versus irreversible inhibition, for these two types of three-finger proteins.     

PECAM-1/CD31 trans-homophilic binding at the intercellular junctions is independent of its cytoplasmic domain; evidence for heterophilic interaction with integrin alphavbeta3 in Cis

PECAM-1/CD31 is a cell adhesion and signaling molecule that is enriched at the endothelial cell junctions. Alternative splicing generates multiple PECAM-1 splice variants, which differ in their cytoplasmic domains. It has been suggested that the extracellular ligand-binding property, homophilic versus heterophilic, of these isoforms is controlled by their cytoplasmic tails. To determine whether the cytoplasmic domains also regulate the cell surface distribution of PECAM-1 splice variants, we examined the distribution of CD31-EGFPs (PECAM-1 isoforms tagged with the enhanced green fluorescent protein) in living Chinese hamster ovary cells and in PECAM-1-deficient endothelial cells. Our results indicate that the extracellular, rather than the cytoplasmic domain, directs PECAM-1 to the cell-cell borders. Furthermore, coculturing PECAM-1 expressing and deficient cells along with transfection of CD31-EGFP cDNAs into PECAM-1 deficient cells reveal that this PECAM-1 localization is mediated by homophilic interactions. Although the integrin alphavbeta3 has been shown to interact with PECAM-1, this trans-heterophilic interaction was not detected at the borders of endothelial cells. However, based on cocapping experiments performed on proT cells, we provide evidence that the integrin alphavbeta3 associates with PECAM-1 on the same cell surface as in a cis manner.     

Incorporation of exogenous ganglioside GM1 into neuroblastoma membranes: Inhibition by calcium ion and dependence upon membrane protein

Since exogenous gangliosides are known to promote neuritogenesis, the incorporation of exogenous GM1 into neuroblastoma membranes was examined. Neuro-2A cells, synchronized in the G1/G0 phase, were suspended in HEPES buffered saline containing 10^–4 M [³H]GM1, and membrane incorporation was measured as radioactivity remaining with the cell pellet following incubation with serum-containing medium and trypsin. Calcium ion (0.01 to 10 mM) reduced incorporation of exogenous GM1, due to its interaction with GM1 micelles in solution. When cells were treated with proteases prior to incubation with GM1, the inhibitory effect of Ca²⁺ was lost and total incorporation into membranes was lowered by approximately one order of magnitude. Pretreatment of cells with 0.05% trypsin resulted in an inhibition of GM1 incorporation within 5 minutes. When trypsinized cells were resuspended in complete growth medium, the cells recovered the ability to incorporate GM1 with time, and this paralleled labeling of cellular protein with [³H]leucine. The role of membrane protein in the incorporation of exogenous GM1 could not be explained by the lytic release of cytosolic transfer proteins nor the artifactual coating of the cell surface by serum proteins. These results suggest that the incorporation of exogenous gangliosides into cellular membrane lipid bilayers cannot be fully explained by considerations of lipophilicity alone, and leads us to propose that initial recognition by membrane protein(s) is necessary.Abbreviations used GM1 H³NeuAc-GgOse₄Cer - HBS HEPES buffered saline - DMEM Dulbecco's modified Eagle's medium - FCS fetal calf serum     

Gangliosides induce selective modulation of CD4 from helper T lymphocytes

The cluster designation (CD)4 molecule is one of several nonpolymorphic T lymphocyte surface proteins that have been implicated in T cell-target cell interactions, and is thought to play an important role in regulating T helper cell function. Previously, we found that gangliosides inhibited the function of rat T helper cell lines, and simultaneously inhibited the expression of the rat CD4 molecule identified by the W3/25 antibody. We have now evaluated the generality and mechanism(s) of ganglioside-induced modulation of CD4 expressed by mouse, rat, and human T helper lymphocytes. Ganglioside pretreatment induced rapid and selective disappearance of the CD4 molecule from T helper cells of all three species. The ganglioside effect was temperature- and dose-dependent, reversible within 24 hr of ganglioside removal, azide-insensitive, and was neutralized completely by 10% serum. CD4 modulation appeared to be a general property of gangliosides since the effect could be induced similarly by highly purified individual gangliosides with varying amounts of sialic acid, or by mixed gangliosides. The activity of gangliosides appeared to require both the lipid and sialated oligosaccharide moieties. Gangliosides did not inactivate antibody function, but prevented binding at the cell surface by 12 different monoclonal antibodies specific for a variety of different CD4 epitopes. Preclearance of CD4 by antibody-mediated capping reduced binding of 3H-GM1 to T helper cells. Labeled GM1 bound to several detergent-extracted and transblotted lymphocyte-associated proteins, but apparently did not bind directly to the CD4 molecule under these conditions. These results indicate that gangliosides induce a profound change in the molecular orientation of CD4 within the T helper cell membrane which renders epitopes on the CD4 molecule inaccessible to antibody. This ganglioside effect represents a novel pathway which may contribute to the understanding of the role of CD4 as a regulatory molecule and as a specific receptor for the acquired immune deficiency syndrome virus.     

Crystallization and preliminary X-ray diffraction analysis of the extracellular domain of the cell surface antigen CD38 complexed with ganglioside

The cell surface antigen CD38 is a multifunctional ectoenzyme that acts as an NAD(+) glycohydrolase, an ADP-ribosyl cyclase, and also a cyclic ADP-ribose hydrolase. The extracellular catalytic domain of CD38 was expressed as a fusion protein with maltose-binding protein, and was crystallized in the complex with a ganglioside, G(T1b), one of the possible physiological inhibitors of this ectoenzyme. Two different crystal forms were obtained using the hanging-drop vapor diffusion method with PEG 10,000 as the precipitant. One form diffracted up to 2.4 A resolution with synchrotron radiation at 100 K, but suffered serious X-ray damage. It belongs to the space group P2(1)2(1)2(1) with unit-cell parameters of a = 47.9, b = 94.9, c = 125.2 A. The other form is a thin plate, but the data sets were successfully collected up to 2.4 A resolution by use of synchrotron radiation at 100 K. The crystals belong to the space group P2(1) with unit-cell parameters of a = 57.4, b = 51.2, c = 101.1 A, and beta = 97.9 degrees, and contain one molecule per asymmetric unit with a VM value of 2.05 A(3)/Da.