Galactocerebroside Research Articles

Secretory IgA specific for a conserved epitope on gp41 envelope glycoprotein inhibits epithelial transcytosis of HIV-1.

As one of the initial mucosal transmission pathways of HIV (HIV-1), epithelial cells translocate HIV-1 from apical to basolateral surface by nondegradative transcytosis. Transcytosis is initiated when HIV-1 envelope glycoproteins bind to the epithelial cell membrane. Here we show that the transmembrane gp41 subunit of the viral envelope binds to the epithelial glycosphingolipid galactosyl ceramide (Gal Cer), an alternative receptor for HIV-1, at a site involving the conserved ELDKWA epitope. Disrupting the raft organization of the Gal Cer-containing microdomains at the apical surface inhibited HIV-1 transcytosis. Immunological studies confirmed the critical role of the conserved ELDKWA hexapeptide in HIV-1 transcytosis. Mucosal IgA, but not IgG, from seropositive subjects targeted the conserved peptide, neutralized gp41 binding to Gal Cer, and blocked HIV-1 transcytosis. These results underscore the important role of secretory IgA in designing strategies for mucosal protection against HIV-1 infection.

Cervical and prostate primary epithelial cells are not productively infected but sequester human immunodeficiency virus type 1.

Primary prostate and cervical epithelial cells and epithelial cell lines were examined for human immunodeficiency virus type 1 (HIV-1) infection or transmission to peripheral blood mononuclear cells (PBMC). Neither cell-free nor cell-associated HIV-1 infected primary epithelial cells or cell lines. Pretreatment of HIV-1 to enhance CD4-independent entry did not augment infection. Cell surface expression was detected for galactosyl ceramide but not for CC-chemokine receptor 5, CXC-chemokine receptor 4, or CD4. The ability to transfer HIV-1 to resting or activated PBMC was tested by culturing with rinsed or trypsinized and replated HIV-1-exposed epithelial cells. Virus was not recovered from the rinsed or replated cocultures with resting PBMC; however, activated PBMC recovered HIV-1 from rinsed epithelial cells and rarely from replated epithelial cells. Although urogenital epithelial cells are not infected, these data suggest that they can transfer virus to activated immune cells and have implications for sexual transmission of HIV-1.

Molecular biology of NK T cell specificity and development

NK T cells recognize a glycosphingolipid, α -galactosyl ceramide, presented by CD1d. CD1d is capable of binding a variety of lipids, however, and the hydrophilic groups of the antigen contribute relatively little to CD1d binding. Amino acids in the CD1d groove and the top of the α helices are involved in lipid antigen presentation, suggesting a conventional mode of presentation and antigen recognition. NK T cells also have unique requirements for their differentiation, as suggested by the analysis of a number of mouse germline mutations. For example, the development of NK T cells, unlike conventional T lymphocytes, is highly lymphotoxin dependent.

Assembly of myelin by association of proteolipid protein with cholesterol- and galactosylceramide-rich membrane domains.

Myelin is a specialized membrane enriched in glycosphingolipids and cholesterol that contains a limited spectrum of proteins. We investigated the assembly of myelin components by oligodendrocytes and analyzed the role of lipid-protein interactions in this process. Proteolipid protein (PLP), the major myelin protein, was recovered from cultured oligodendrocytes from a low-density CHAPS-insoluble membrane fraction (CIMF) enriched in myelin lipids. PLP associated with the CIMF after leaving the endoplasmic reticulum but before exiting the Golgi apparatus, suggesting that myelin lipid and protein components assemble in the Golgi complex. The specific association of PLP with myelin lipids in CIMF was supported by the finding that it was efficiently cross-linked to photoactivable cholesterol, but not to phosphatidylcholine, which is underrepresented in both myelin and CIMF. Furthermore, depletion of cholesterol or inhibition of sphingolipid synthesis in oligodendrocytes abolished the association of PLP with CIMF. Thus, PLP may be recruited to myelin rafts, represented by CIMF, via lipid-protein interactions. In contrast to oligodendrocytes, after transfection in BHK cells, PLP is absent from isolated CIMF, suggesting that PLP requires specific lipids for raft association. In mice deficient in the enzyme ceramide galactosyl transferase, which cannot synthesize the main myelin glycosphingolipids, a large fraction of PLP no longer associates with rafts. Formation of a cholesterol- and galactosylceramide-rich membrane domain (myelin rafts) may be critical for the sorting of PLP and assembly of myelin in oligodendrocytes.

Atypical glial cells in demyelinated and hypomyelinated mouse brains

Bis-cyclohexanone oxalyldihydrazone (cuprizone) was administered to young adult mice in order to investigate the hypothesis that the differentiation of oligodendrocyte progenitors or precursors, or of immature oligodendrocytes, might be interrupted during cuprizone intoxication. Vibratome sections were prepared from brains from control mice, mice that were fed cuprizone for 27 days and mice that were fed cuprizone for 25 days, followed by normal diet for 2 days, and the sections were immunostained with monoclonal antibodies: MAbO4, which is directed against galactocerebroside sulfate (sulfatide); and RMAb, which is directed against galactocerebroside (GC). Process-bearing RMAb+/O4− cells were abundant in the brains of mice that had consumed cuprizone for 27 days, and the numbers of O4-positive cells were subnormal. Two days after refeeding the normal diet the RMAb+/O4− structures were less abundant and O4-positive cells more numerous. Moreover, the numbers of O4-positive cells were only ∼20% of normal in the brains of hypomyelinating jimpy mutant mice, while the numbers of RMAb-positive cells were ∼80% of normal, and the processes of the latter were associated with axons. It is suggested that RMAb+/O4− cells in the affected brains may be process-bearing oligodendrocyte precursors containing unsulfated GC or a toxic galactolipid.

Oxidation of glycosphingolipids under basic conditions: synthesis of glycosyl "serine acids" as opposed to "ceramide acids". Precursors for neoglycoconjugates with increased ligand binding affinity.

Two types of oxidative cleavage of the double bond of glycosphingolipids (GSLs) are described. Oxidation of peracetylated GSL precursors with stoichiometric proportions of KMnO4 and an excess of NaIO4, in a neutral aqueous tert-butanol solvent system, gave nearly quantitative yields of the glycosyl ceramide acid, 2-hydroxy-3-(N-acyl)-4-(O-glycosyl)oxybutyric acid [Mylvaganam, M., and Lingwood, C. A. (1999) J. Biol. Chem. 274, 20725-20732]. However, if the reaction medium was made alkaline, the hydroxyallylic function of the sphingolipid, as a whole, was oxidized and the glycosyl serine acid, 2-(N-acyl)-3-(O-glycosyl)oxypropionic acid, was obtained in good yield. This represents a new type of oxidation reaction. Optimized conditions gave glycosyl ceramide or serine acids with greater than 90% selectivity and in good yields (90%). Oxidation of dGSLs gave serine and ceramide oligosaccharides, devoid of hydrocarbon chains. An intriguing glycosyl species containing 5-hydroxy-4-oxo-3-hydroxy-2-(N-acyl)sphingosine (hydroxy-acyl intermediate) was identified via ESMS analyses. We propose that further oxidation of this intermediate is pH-dependent and will be oxidized to either serine or ceramide acids. On the basis of MS-MS analysis of specific homologues of serine and ceramide acids, two types of collision-induced dissociation (CID) patterns have been established. These CID patterns were then used in the identification of serine and ceramide acids synthesized from natural GSL samples. Also, on a qualitative basis, this oxidation protocol, in conjunction with ESMS, provides a novel method for characterizing the aglycone composition (acyl chain length, unsaturation position, dihydrosphingosine content, etc.) of natural GSLs. A novel class of neohydrocarbon conjugates were synthesized by coupling the acids to rigid hydrocarbon frames such as 2-aminoadamantane. Preliminary studies with conjugates derived from globotriaosyl ceramide (Gb3C), lactosyl ceramide (LC), and galactosyl ceramide (GalC) bound verotoxin with the expected specificity but with affinities much greater than that of the natural glycolipid. Also, the ceramide acid-based conjugates were better ligands than serine acid conjugates.

Reconstitution of the glycosylphosphatidylinositol-anchored protein Thy-1: interaction with membrane phospholipids and galactosylceramide

Glycosylphosphatidylinositol (GPI)-anchored membrane proteins are proposed to interact preferentially with glycosphingolipids and cholesterol to form microdomains, which may play an important role in apical targeting and signal transduction. The objective of the present study was to investigate the interaction of the GPI-anchored protein Thy-1 with phospholipids and a glycosphingolipid. Purified Thy-1 was reconstituted into lipid bilayer vesicles of dimyristoyl-phosphatidylcholine (DMPC) alone or in combination with galactosylceramide (GC). The ability of Thy-1 to perturb the gel to a liquid-crystalline phase transition of DMPC was examined by differential scanning calorimetry. As the mole fraction of Thy-1 increased, the phase transition enthalpy, deltaH, declined. Analysis indicated that each molecule of Thy-1 perturbed over 50 phospholipids, suggesting that, in addition to the anchor insertion into the bilayer, the protein itself may interact with the membrane surface. Inclusion of 5% w/w GC in the bilayer resulted in a striking change in the interaction of Thy-1 with phospholipids. At low Thy-1 content, there was a reduction in the phase transition temperature and an increase in phospholipid cooperativity, suggesting the formation of Thy-1/GC-enriched domains. DeltaH initially decreased with increasing Thy-1 content of the bilayer; however, at higher Thy-1 mole ratios, deltaH rose again. These results are interpreted in terms of a model whereby, at low protein:lipid mole ratios, Thy-1 preferentially sequesters GC to form enriched microdomains. At high protein:lipid mole ratios, Thy-1 may alter its conformation in response to steric crowding within these domains such that its interaction with the bilayer surface is reduced.

Spatial and temporal expression of UDP-galactose: ceramide galactosyl transferase mRNA during rat brain development.

We studied the expression of UDP-galactose: ceramide galactosyl transferase (CGT) mRNA in postnatal rat brains using an in situ hybridization technique. From P0 to P16, there was a defined temporal and spatial pattern to the earliest acquisition of CGT mRNA expression. In the forebrain, CGT mRNA-expressing (CGT+) cells were first detected in regions outside the subventricular zone around the lateral ventricle at P2. Cells in the external capsule, internal capsule and corpus callosum were later found to be CGT-positive. At P8 to P16, CGT+ cells were found in the thalamus, striatum, occipital and frontal cortex. In the case of midbrain and hindbrain, the first CGT+ signals were detected in the medullary raphe of the medulla oblongata at P0. CGT+ cells were subsequently located in the cerebellum, midbrain and pons from P4 to P16. That is, in regions closer to the areas in which CGT+ cells were first found, CGT mRNA expression was observed much earlier. These findings support the notion that there are at least two discrete waves of CGT mRNA signal expression in the forebrain and hindbrain.

Mucosal HIV Infection

The gastrointestinal tract is not only a major site of clinical manifestations of the acquired immune deficiency syndrome but also an important compartment for HIV infection at all stages of the disease. In intestinal biopsies HIV has been detected so far mainly in mononuclear cells of the lamina propria but also in epithelial cells. Primary and permanent epithelial cell lines can be productively infected by HIV in vitro and epithelial cell monolayers can rapidly transport HIV by transcytosis. Both phenomena are most effective for cell-associated virus and do not require CD4 expression but seem to involve the interaction of gp120 with galactosyl ceramide. In vivo, productive HIV infection of epithelial cells appears to be rare if it occurs at all. Mucosal HIV infection is highly active at all stages of HIV infection and the gastrointestinal tract is probably a major source of HIV in the body. Highest mucosal HIV production is found rather early in HIV disease and is associated with histological abnormalities and gastrointestinal symptoms. The excessive production of HIV in the intestine appears to be due to transcriptional or translational upregulation and local variations in HIV production correlate with local cytokine levels.

On antibody level to brain galactoc erebrosides in blood serum in healthy children of early age and women

The results of autoantibodies determination to glycolipid antigens of brain cells - galactocerebrosides in blood serum of healthy neonates and their mothers, healthy children up to three y.o. and healthy women are presented. The results were obtained with the help of micromodification of complement fixation reaction. It was determind that in healthy women at intergeneric period these antibodies are absent or are present in vestige quantities. For the first time presence of counter - galactocerebroside antibodies was determined in neonates and their mothers in high titers up to 1:512. It was also established, that counter - galactocerebroside antibodies circulate in healthy neonates blood for not less than 3 months, then their level begins to descrease, and in 5-12 months it approximates to the adults norm border.

Myelin Galactolipids Are Essential for Proper Node of Ranvier Formation in the CNS

The vertebrate myelin sheath is greatly enriched in the galactolipids galactocerebroside (GalC) and sulfatide. Mice with a disruption in the gene that encodes the biosynthetic enzyme UDP-galactose:ceramide galactosyl transferase (CGT) are incapable of synthesizing these lipids yet form myelin sheaths that exhibit major and minor dense lines with spacing comparable to controls. These CGT mutant mice exhibit a severe tremor that is accompanied by hindlimb paralysis. Furthermore, electrophysiological studies reveal nerve conduction deficits in the spinal cord of these mutants. Here, using electron microscopic techniques, we demonstrate ultrastructural myelin abnormalities in the CNS that are consistent with the electrophysiological deficits. These abnormalities include altered nodal lengths, an abundance of heminodes, an absence of transverse bands, and the presence of reversed lateral loops. In contrast to the CNS, no ultrastructural abnormalities and only modest electrophysiological deficits were observed in the peripheral nervous system. Taken together, the data presented here indicate that GalC and sulfatide are essential in proper CNS node and paranode formation and that these lipids are important in ensuring proper axo-oligodendrocyte interactions.

Transport of proteolipid protein to the plasma membrane does not depend on glycosphingolipid cotransport in oligodendrocyte cultures.

The possibility that transport of proteolipid protein (PLP) from its site of synthesis to the plasma membrane is dependent on cotransport with (sulfo)galacto-cerebrosides was investigated in primary cultured oligodendrocytes and Chinese hamster ovary (CHO) cells expressing PLP. Sulfation was inhibited by growing oligodendrocytes in the presence of a competitive inhibitor of this process, sodium chlorate. Under these circumstances, sulfatide synthesis was inhibited by 85%. Nevertheless, PLP was still delivered to the plasma membrane in quantitative amounts. Furthermore, when PLP was expressed in CHO cells, which normally synthesize very low amounts of galactosyl ceramide (GalCer) and no sulfatide, PLP was transported to the plasma membrane. Moreover, in CHO cells coexpressing PLP and ceramide galactosyl transferase, PLP cell surface labeling was unaltered. Noting that it has been demonstrated that proteins destined for the apical surface of epithelial cells colocalize with glycolipid-enriched microdomains, we isolated detergent-insoluble membrane complexes from cultured oligodendrocytes. We found, however, that most of the PLP is present in the detergent-soluble fraction and, furthermore, that PLP could not be chased into or out of the insoluble fraction. Taken together, these data make it very likely that in oligodendrocytes PLP transport takes place irrespective of the presence of glycosphingolipids GalCer and sulfatide.

Cells of the oligodendrocyte lineage proliferate following cortical stab wounds: an in vitro analysis.

We have previously shown that a cortical stab wound induces the proliferation of microglia and astrocytes in situ, but no evidence was obtained for proliferation of cells of the oligodendrocyte lineage (Amat et al., 1996). To study further the properties of cells involved in repair following brain injury, groups of adult rats received either sham operations or bilateral stab wounds. Proliferating cells were labeled in vivo 3 days later with [3H]-thymidine (Thy) and sacrificed the same day. Oligodendrocyte-enriched preparations were isolated, cultured, and analyzed. The fate and antigenic phenotype of the proliferating cells was analyzed using three-color immunofluorescence combined with autoradiography at 1, 2, 3, 5, and 10 days in vitro (DIV). Cells were immunostained for ganglioside GD3 (glial stem cells), O4 antigen (cells of the oligodendrocyte lineage), galactosyl ceramide (GC, differentiated oligodendrocytes), and GFAP (astrocytes). Thymidine-labeled O4+/GC- cells were found only in cultures from wounded animals and most of them differentiated in vitro as mature oligodendrocytes, but no Thy+/O4+/GC+ oligodendrocytes were seen at 1, 2, or 3 DIV. There was also a marked increase in the number of Thy+/GD3+ cells in the experimental cultures. In both experimental and control groups the total number of Thy+ and Thy- GD3+ cells declined with time in culture concomitant with an increase in total number of both Thy+ and Thy- GFAP+ astrocytes, and without any significant change in the Thy+ cell fraction of O4+ oligodendrocytes in the experimental cultures. Therefore most of the GD3+/O4- cells apparently differentiated as GFAP+ astrocytes, not as oligodendrocytes. We conclude that O4+/GC- oligodendrocyte precursor cells, but not differentiated oligodendrocytes, proliferate in response to brain injury. These cells proliferate slowly or not at all in normal adult animals and constitute a phenotypically and kinetically distinct group from the GD3+ glial precursors. This result is consistent with the existence within the adult CNS of a quiescent premyelinating oligodendrocyte. We propose that these immature committed oligodendrocytes are induced to proliferate at the wound site and serve as a source of new oligodendrocytes.

Effects of cyclic AMP on expression of myelin genes in the N20.1 oligodendroglial cell line.

The N20.1 immortalized cell line has several characteristics of differentiating oligodendrocytes (OLs), including expression of the glycolipids galactocerebroside (GalC) and sulfatide, and the myelin proteins CNPase and myelin basic protein (MBP) (1,2). Addition of 1-100 microM forskolin to elevate cyclic AMP (cAMP) levels changed cell morphology from irregular and flattened to a more rounded birefringent cell with multiple branched processes. GalC and sulfatide were detected immunocytochemically after permeabilization in the untreated cells and levels appeared to increase slightly following exposure to forskolin. Further analysis showed that most of the glycolipid was internal, with virtually no detectable levels on the cell surface in untreated cells and a very slight change following treatment with forskolin. Synthesis of the two lipids as measured by [H3]galactose incorporation doubled within 24 hours of treatment with forskolin. Levels of message for UDP-galactose: ceramide galactosyl transferase (CGT), a key enzyme in the synthesis of GalC and sulfatide, were compared with those of MBP and proteolipid protein (PLP), before and after elevation of cAMP. No changes were observed in levels of mRNA for CGT and PLP after 24 hours, with a possible increase by 48 hours. In contrast, levels of MBP message dropped precipitously by 24 hours; this was accompanied by an increase in levels of message for suppressed cAMP-inducible POU (SCIP). Thus CGT transcription is regulated independently of MBP and SCIP in N20.1 cells. Analysis of MBP levels by immunocytochemistry and Western blot showed little or no change in protein levels at 24 and 48 hours, in contrast to the sharp decrease in message levels by 24 hours, indicating a relatively long half life for MBP in this cell line. Thus, the N20.1 cells are an informative model for examining regulation of expression of myelinotypic proteins and GalC, as well as the transport of this lipid to the plasma membrane.

HIV infection of human fetal neural cells is mediated by gp120 binding to a cell membrane-associated molecule that is not CD4 nor galactocerebroside

HIV infection of central nervous system (CNS) tissue is a common finding in both adult and pediatric AIDS. Because most children are believed to be infected perinatally, we have developed a model of HIV CNS infection that utilizes explant organotypic cultures of human fetal CNS tissue. Using this model we previously reported that both lymphocytotropic and monocytotropic HIV isolates infect microglia and astrocytes. However, the mechanism by which HIV infects these cells remains to be elucidated. We have observed that neural cell infection in these cultures may be the result of receptor-mediated endocytosis. In order to confirm this observation and to determine the ligand responsible for this process, organotypic cultures were exposed to untreated HIV, HIV pretreated with soluble CD4 (sCD4) or, as a control, heat-inactivated HIV. To address the question of a putative receptor for HIV infection, CNS cultures were either untreated or pretreated with gp120 or with the deglycosylated form of this protein. Other cultures were treated with antibodies to CD4 (anti-T4A) or to galactocerebroside (GC). Results demonstrate that pretreatment of either HIV with sCD4 or CNS cultures with gp120 significantly inhibits HIV infection. The inhibition of infection was demonstrated by a reduction in the number of cells positive for HIV proteins and by decreases in HIV proviral DNA and p24 production. Pretreatment of CNS cultures with deglycosylated gp120, anti-T4A or anti-GC antibodies did not inhibit HIV infection. These data suggest that HIV gp120 is needed for binding to a surface molecule on CNS cells that is not CD4 nor GC and that this molecule may function as a receptor and lead to infection of neural cells.

Toward a transgenic mouse model of remyelination

The molecular mechanisms necessary for remyelination by oligodendrocytes remain unexplored. We previously characterized a myelin basic protein promoter-lacZ (MBP-lacZ) transgene whose expression is regulated uniquely during development, and also in pathological situations, suggesting that it may be a useful reporter of molecular mechanisms during remyelination. As a first step toward creating a transgenic mouse model of remyelination, we cultured oligodendrocytes from these transgenic mice and showed that expression of MBP-lacZ appeared in parallel with a marker of oligodendrocyte maturation, galactocerebroside (GC). In addition, basic fibroblast growth factor blocked the expression of both MBP-lacZ and GC in these cells. Therefore, expression of MBP-lacZ reflects not only the developmental stage of oligodendrocytes, but also extrinsic influences on oligodendrocytes. These data suggest that MBP-lacZ may be a useful marker in transgenic mouse models of remyelination.

Morphological alterations associated with HIV infection of CD4−/GalCer+ human intestinal epithelial cells

The ultrastructural alterations induced by human immunodeficiency virus type 1 (HIV-1) in the human colon epithelial cell line HT-29 infected in vitro have been evaluated. These cells express high levels of galactosyl ceramide (GalCer), an alternative receptor for HIV-1 surface envelope glycoprotein gp120. Using immunolabelling for electron microscopy and immunofluorescence techniques, we detected the GalCer receptor in both the apical and basolateral membranes of differentiated HT-29 subclones. This nonpolar distribution is consistent with our previous observation that HIV infects both the apical and basolateral surfaces of epithelial cells in vitro. A transmission electron microscopy study demonstrated two major ultrastructural perturbations in the HIV-producing cells: (i) an unusual number of secretory granules; and (ii) the appearance of intracellular lumina with disorganized microvilli, indicating a defect in striated border assembly and differentiation. In addition to these morphological alterations, our study revealed the presence of fat vacuoles, concentric membranous bodies, tubuloreticular inclusions and giant mitochondria in the cytoplasm of HIV-infected cells. Taken together, these abnormalities could account for HIV-induced enteropathy, consisting of chronic diarrhea and malabsorption in the absence of enteric pathogens.

Differentiation of cerebroside isomers and study of fragmentation by liquid secondary ion mass spectrometry and mass spectrometry/mass spectrometry of selected derivatives

The possibility of distinguishing two cerebroside isomers, whose structural variation is in the sugar rings, was investigated by liquid secondary ion mass spectrometry (LSIMS) and tandem mass spectrometry (MS/MS). In addition to the native materials, four types of derivatives of these cerebroside isomers were prepared and studied using these techniques. A first level of comparison between isomers consisted of seeking differences in the conventional LSIMS spectra. Native compounds, galactosyl and glucosyl ceramides, did not yield consistent and meaningful elements of comparison and a few nanomoles of material were required to produce significant spectra. Permethylated cerebrosides gave rise to abundant ceramide ions that do not reveal information about sugar structure; relative abundances of low-mass sugar-related ions allowed isomeric discrimination to a limited extent. Peracetylated and perbenzoylated derivatives of both cerebrosides yielded several sugar-related ions whose relative abundances in the spectra varied systematically between species and allowed distinction of glucosyl- from galacto-cerebrosides. At a second level of comparison, collision-induced dissociation (CID) MS/MS spectra of selected ions containing the hexosyl residues and observed in the LSIMS spectra were recorded to study the extent and pathways of fragmentation for each isomer. Native and permethylated compounds led to disappointing results; on the other hand, peracetylated and perbenzoylated derivatives yielded informative spectra where isomeric differentiation was possible, mostly owing to the production of carbohydrate-related product ions, whereas native and permethylated compounds only produced ceramide-related ions upon CID MS/MS. Key words: glycosphingolipids, cerebrosides, derivatization, mass spectrometry, MS/MS, LSIMS.

Sulphatide binds to human and animal influenza A viruses, and inhibits the viral infection.

We found, by using a virus overlay assay, that influenza A virus isolates bind to sulphatide (HSO3-Gal beta 1-->1'Cer), which has no sialic acid residue, and that the infection of Madin-Darby canine kidney cells with the human influenza virus A/Memphis/1/71 (H3N2) is inhibited by sulphatide. A/Memphis/1/71 (H3N2) causes obvious haemagglutination and low-pH haemolysis of asialoerythrocytes reconstituted with sulphatide. All influenza A virus isolates from the species of animals so far tested bound to sulphatide. The sulphatide-binding specificity of the isolates was different from the viral sialyl-linkage specificity. Influenza A virus isolates also bound to galactosyl ceramide (GalCer; Gal beta 1-->1'Cer), as well as sulphatide, in the virus overlay assays. In contrast, the influenza virus did not bind to N-deacyl, a derivative of sulphatide, glucosyl ceramide or the other neutral glycolipids tested. These results indicate that the linkage of galactose, or sulphated galactose, to ceramide is important for viral binding.

Single Amino Acid Residues in the E- and P-selectin Epidermal Growth Factor Domains Can Determine Carbohydrate Binding Specificity

E-selectin and P-selectin are two closely related vascular cell adhesion proteins. Each selectin has an amino-terminal C-type lectin domain that is thought to possess the carbohydrate binding site that binds the sialylated Lewisx antigen (sLex or CD15s) (Neu5Acalpha2-3Galbeta1-4(Fucalpha1-3)GlcNAc). In addition to the sLex carbohydrate, P-selectin binds sulfated proteoglycan, 3-sulfated galactosyl ceramide (sulfatide), and heparin. Both E- and P-selectin have an EGF-like (EGF) domain that is immediately adjacent to and COOH-terminal to the lectin domain. We report that mutagenic substitution of single amino acid residues in either the P- or E-selectin EGF domain can dramatically alter selectin binding to sLex, heparin, or sulfatide. Substitution of E- and P-selectin EGF domain residue Ser128 with an arginine results in E- and P-selectin proteins that have lost the requirement for alpha1-3-linked fucose and are thus able to bind to sialyllactosamine. A similar phenotype is reported for an E-selectin mutation within the lectin domain. Additionally, we have determined that conservative substitution of EGF domain residues 124 and 128 can alter E-selectin binding such that it is able to adhere to heparin or sulfatide and can reduce P-selectin adherence to these ligands. The distance between the substituted EGF domain amino acid residues and the primary carbohydrate binding site within the lectin domain and their relative positioning as determined by the three-dimensional crystal structure of the E-selectin lectin and EGF domains (Graves, B. J., Crowther, R. L., Chandran, C., Rumberger, J. B., Li, S., Huang, D.-S., Presky, D. H., Familletti, P. C., Wolitzky, B. A., and Burns, D. K. (1994) Nature 367, 532-538) suggest that there is little direct contact between the two domains. However, we report mutant binding characteristics which indicate that selectin oligosaccharide binding may be modulated by both domains and that wild-type E- and P-selectin/sLex binding interactions may be significantly different from those previously hypothesized.