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Gp120 Binds Cooperatively to Several Biologically Relevant Glycosphingolipids: Quantitative Measurements at Equilibrium by Total Internal Reflection Fluorescence Microscopy This research was supported by the NIH (AI40359-02), the NSF (CHE-9726132 and CHE-9623583), Eli Lilly (JGH), and the Alfred P. Sloan Foundation (J.G.H.). K.D.M. gratefully acknowledges receipt of the University of Arizona Dean's Fellowship and the Department of Chemistry Carl S. Marvel Fellowship. We thank Ying-Mei Gu for per
Angewandte Chemie International Edition
... Later it was also shown that HIV particles prefer to bind at the edges of cholesterol-rich lipid domains that were reconstituted in supported bilayers [86,87]; however, observing this preference in live cells due to the small scale and dynamism of lipid rafts is difficult, illustrating the power of using an in vitro system for such studies. Focusing further on the receptors themselves, TIRF microscopy has also been used to measure the affinity of HIV glycoprotein 120 for the glycosphingolipids galactosyl ceramide, glucosylceramide, lactosylceramide and α-hydroxy glucosylceramide in SLBs [88,89]. The affinity of glycoprotein 120 for these lipids is roughly 5 times lower than its affinity for CD4 [90]. ...
The most widely-used assays for studying viral entry, including infectivity, cofloatation, and cell-cell fusion assays, yield functional information but provide low resolution of individual entry steps. Structural characterization provides high-resolution conformational information, but on its own is unable to address the functional significance of these conformations. Single virion tracking microscopy techniques provide more detail on the intermediate entry steps than infection assays and more functional information than structural methods, bridging the gap between these methods. In addition, single virion approaches also provide dynamic information about the kinetics of entry processes. This chapter reviews single virion tracking techniques and describes how they can be applied to study specific virus entry steps. These techniques provide information complementary to traditional ensemble approaches. Single virion techniques may either probe virion behavior in live cells or in biomimetic platforms. Synthesizing information from ensemble, structural, and single virion techniques ultimately yields a more complete understanding of the viral entry process than can be achieved by any single method alone.
... this context to demonstrate the binding ability of hyperbranched β-galceramide-containing dendritic polymers 7 and 8 toward HIV-1 rgp120 IIIB, a kinetic analysis was performed using surface plasmon resonance (SPR). The rate and affinity constants for hyperbranched-rgp120 IIIB interactions are shown inTable 2. This data suggests that rgp120 IIIB exhibits similar binding to hyperbranched β-galceramide-containing dendritic polymers 7 and 8. Additionally, this results are close to those obtained with the glycodendrimers galcer (GC-32mer with an average of 21 sugars) (entry 3,Table 2), and lower than sulfated galcer (SGal-32mer with an average of 25 sugars).[22][23][24]Table 2. Rate and equilibrium constants for the interaction of rgp120 IIIB with hyperbranched glycodendritic β-galceramide-containing polymers 7 and 8.On the other hand, the ability of hyperbranched glycodendritic polymers to inhibit HIV-1 BaL (R5-tropic) infection of U373-MAGI-CCR5 cells and their effects on cell viability was tested (see Supporting Information). ...
We report the design, synthesis, and characterization of a series of water-soluble hyperbranched β-galceramide-containing dendritic. Polymers showing useful binding ability to HIV-1 rgp120 as demonstrated with surface plasmon resonance.
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Mucosal epithelial cell surface galactosylceramide (Galcer) has been postulated to be a receptor for HIV-1 envelope (Env) interactions with mucosal epithelial cells. Disruption of the HIV-1 Env interaction with such alternate receptors could be one strategy to prevent HIV-1 entry through the mucosal barrier. To study antibody modulation of HIV-1 Env-Galcer interactions, we used Galcer-containing liposomes to assess whether natural- and vaccine-induced monoclonal antibodies can block HIV-1 Env binding to Galcer. HIV-1 Env gp140 proteins bound to Galcer liposomes with Kds (dissociation constants) in the nanomolar range. Several HIV-1 ALVAC/AIDSVAX vaccinee-derived monoclonal antibodies (MAbs) specific for the gp120 first constant (C1) region blocked Galcer binding of a transmitted/founder HIV-1 Env gp140. Among the C1-specific MAbs that showed Galcer blocking, the antibody-dependent cellular cytotoxicity-mediating CH38 IgG and its natural IgA isotype were the most potent blocking antibodies. C1-specific IgG monoclonal antibodies that blocked Env binding to Galcer induced upregulation of the gp120 CD4-inducible (CD4i) epitope bound by MAb 17B, demonstrating that a conformational change in gp120 may be required for Galcer blocking. However, the MAb 17B itself did not block Env-Galcer binding, suggesting that the C1 antibody-induced gp120 conformational changes resulted in alteration in a Galcer binding site distant from the CD4i 17B MAb binding site.
Importance:
Galactosyl ceramide, a glycosphingolipid, has been postulated to be a receptor for the HIV-1 envelope glycoprotein (Env) interaction with mucosal epithelial cells. Here, we have mimicked this interaction by using an artificial membrane containing synthetic Galcer and recombinant HIV-1 Env proteins to identify antibodies that would block the HIV-1 Env-Galcer interaction. Our study revealed that a class of vaccine-induced human antibodies potently blocks HIV-1 Env-Galcer binding by perturbing the HIV-1 Env conformation.
A sticky situation: Domain-dependent recognition of the glycosphingolipid galactosylceramide by norovirus-like particles (see picture; red/yellow) is shown using supported lipid bilayers (purple) as model membranes. Optimal ligand presentation is found to promote strong binding to GalCer. This presentation can be found at the edges of the glycosphingolipid-enriched domains (green) and binding is repressed in the absence of these domains.
As part of our continuing efforts to study the molecular events involved in HIV recognition of mucosal membrane cells, we are studying the interactions of the HIV-associated glycoprotein gp120, with the cellular receptor GalCer. In this work, we report investigations of multivalent interactions of Au glyconanoparticles containing galactosyl and glucosyl headgroups with recombinant gp120. These particles were prepared from disulfides containing C-glycosides linked to triethylene glycol via an amide bond using a modification of the Brust method.1 Results from transmission electron microscopy, atomic force microscopy, and UV−vis absorption spectroscopy data are consistent with an average particle diameter of 2 nm. Elemental analyses indicate that the average composition of the particles is 1.6:1 Au:carbohydrate. A biotin−NeutrAvidin adhesion assay was used to evaluate the relative ability of carbohydrate disulfides and Au glyconanopraticles to displace rgp120 from plate-bound GalCer. The data indicate that divalent disulfides were less than 12% as active as biotinylated GalCer, a water-soluble surrogate of GalCer. However, when these same carbohydrates were presented in a polyvalent display on gold, they were greater than 300 times more active than the disulfides and at least 20 times more active than biotinylated GalCer. These results collectively demonstrate the potential utility of polyvalent ligand arrays on nanoplatforms.
Interactions of recombinant gp120 (rgp120) with non-natural glycosphingolipids (GSLs) and structurally simpler analogues have been studied using a competitive adhesion assay. Conjugates of cellobiosyl ceramide and melibiosyl ceramide were synthetically prepared as water-soluble GSL analogues. These ligands were screened against a panel of biologically relevant analogues, and the results show that their interactions with rgp120 are comparable to natural cellular receptors. Glycolipid interactions with rgp120 were probed further by the synthesis and testing of structurally simpler analogues that were obtained by reductive amination of lactose, cellobiose, and melibiose with a biotinylated amino ethylene glycol moiety. RGp120 did not recognize conjugates lacking a lipid component. However, palmitoylation of the secondary amino alditols yielded compounds with comparable rgp120 affinity to the natural cellular receptor, galactosyl ceramide (GalCer). Taken together, the SAR showed that both a hydrophobic and a hydrophilic component are required for rgp120 recognition. Moreover, structural variability in the carbohydrate headgroup did not significantly alter rgp120 recognition indicating that this interaction is not highly specific.
The interaction of recombinant HIV-1 surface glycoprotein gp120 (rgp120) with natural isolates of lactosylceramide (LacCer), glucosylceramide (GlcCer), and galactosylceramide (GalCer) has been quantitatively measured under equilibrium conditions using total internal reflection fluorescence (TIRF) spectroscopy. The binding affinity (K(a)) of rgp120 to these glycosphingolipids (GSLs), reconstituted at 5 mol % in supported planar lipid bilayers composed of 95 mol % POPC, is ca. 10(6) M(-1) for dissolved rgp120 concentrations greater than 25 nM. In contrast, at concentrations of rgp120 between 0.2 and 15 nM, rgp120 does not bind significantly to LacCer and GlcCer, but has a high affinity for GalCer with a measured K(a) value of 1.6 x 10(9) M(-1). However, protein surface coverage measurements show that this strong binding process accounts for very little of the total protein adsorbed over the entire concentration range studied. At a protein concentration of ca. 20 nM, the surface coverage is only 3% of that achieved at apparent saturation (i.e., when the protein concentration is ca. 220 nM). Thus the "high affinity" binding sites comprise only a small fraction of the total number of binding sites. Several other variables were investigated. Rgp120 binding behavior at membranes doped with alpha-hydroxygalactosylceramide (alpha-GalCer) was very similar to that observed with GalCer, showing that the presence/absence of an alpha-hydroxy moiety does not significantly affect galactosylceramide recognition. Phase segregation of GalCer, which occurs when the mole fraction of this GSL in a POPC bilayer exceeds ca. 0.1, was also investigated and showed no effect on binding affinity at low rgp120 concentrations. To investigate the influence of fatty acid chain length, GSLs with monodisperse C(18) and C(24) chain lengths, both with and without an alpha-hydroxy moiety, were synthesized, and their binding affinity to rgp120 was examined. Relative to the natural isolates (which contain a mixture of chain lengths), minimal differences were observed; thus among the compounds tested, fatty acid chain length does not affect GSL recognition. The results of this work should aid efforts to design anti-HIV-1 agents based on membrane-tethered, carbohydrate-based receptors for rgp120.
The behavior of methylene blue (MB) molecules at silica/water and silylated silica/water interfaces was examined using visible attenuated total reflection spectroscopy with a slab optical waveguide (SOWG). An alkyl silane coating changed the adsorbed form of MB on the surface from a dimer (lambda max = 600 nm, bare silica surface) to a monomer (lambda max = 670 nm), and the carbon number of the silylation reagent influenced the amount of adsorption and the orientation of the molecule. Moreover, the addition of an anionic surfactant, dodecylbenzenesulfonate (DBS), caused the deposition of MB/DBS ion pairs, which gave an identical attenuated total reflection (ATR) spectrum to that of the dimer. Linear dichroism measurements revealed that the ion pairs were adsorbed onto the silylated silica surface randomly in terms of the orientation angle of MB, while the MB monomer was strongly oriented, i.e., the direction of the transition moment of MB roughly parallels the surface plane. This difference in the orientation angles of the adsorbed species can be utilized for their selective detection using polarization SOWG measurements.
The synthesis of bipharmacophore anti-HIV compounds which, in a single molecule, combine two ligands, that is, the bicyclam AMD3100 and a GalCer analogue, that might inhibit several steps of the complex virus/cell cascade interactions has been performed. The 'double-drug' Gal-AMD3100 conjugates elicited inhibitory effects on T (or X4)-tropic HIV-1 replication in all CXCR4 expressing cell lines with EC(50) values ranging from 0.25 to 6.0 microM which were however approximately 40- to 125-fold lower than that of AMD3100. Concerning the mechanism of inhibition of the Gal-AMD3100 conjugates, experiments performed with X4 or R5HIV-1 strains and GHOST cells genetically modified to express CD4 and CXCR4 or CCR5 indicated clearly that the conjugates interact with CXCR4 and not with CCR5.
[reaction: see text] A highly efficient protocol for making Man(3) and Man(5) oligosaccharides with use of orthogonally protected glycosyl iodide donors has been developed. Glycosylation of a C-2-O-acetyl mannosyl iodide donor in the presence of silver triflate at -40 degrees C initially gave a mixture of the desired alpha-linked mannoside and an orthoacetate resulting from attack at the C-2 acetate. However, upon warming to room temperature the orthoacetate quantitatively rearranged to the desired oligosaccharide. Employing a 3,6-dihydroxy acceptor and subjecting it to double glycosidation quickly afforded high mannose sugars in nearly quantitative yields. Glycosyl iodide donors offer advantages over previously reported chloride donors as the reactions are faster, proceed in higher yields, and are not diminished in higher order constructs. These studies continue to dispel the notion that glycosyl iodides are too reactive to be of synthetic utility.
Our previous studies show that the depletion of cholesterol or sphingolipids (raft-associated lipids) from receptor-bearing adherent cell lines blocks HIV-1 entry and HIV-1 Env-mediated membrane fusion. Here we have evaluated the mechanism(s) by which these lipids contribute to the HIV-1 Env-mediated membrane fusion. We report the following: (1) GSL depletion from a suspension T lymphocyte cell line (Sup-T1) reduced subsequent fusion with HIV-1IIIB-expressing cells by 70%. (2) Cholesterol depletion from NIH3T3 cells bearing HIV-1 receptors (NIH3T3CD4R5/NIH3T3CD4X4) did not impair subsequent fusion with HeLa cells expressing the corresponding HIV-1 Envs. In contrast GSL depletion from these targets reduced fusion by 50% suggesting that GSL facilitate fusion in different ways. (3) GSL-deficient GM95 cells bearing high receptors fused with HIV-1 Env-expressing cells at 37 degrees C with kinetics similar to that of GSL + NIH3T3 targets. Based on these observations, we propose that the plasma membrane cholesterol is required to maintain the integrity of receptor pools whereas GSLs are involved in stabilizing the coupling of inter-receptor pools.
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A sterically encumbered [N(2)S(6)] macropentacycle (5) related to diazamacrobicycles and cryptands has been synthesized in 53% yield by the [1+1] condensation reaction between functionalized macrocyclic and macrotricyclic precursors. A macrononacycle (18) resulting from the corresponding [2+2] condensation was isolated in 7% yield from the reaction mixture. Both compounds showed broad features in their room-temperature (1)H NMR spectra, but their maximal average symmetry (D(3h) and D(2h), respectively) was achieved at high temperature (380 K). At low temperature (200 K, CD(2)Cl(2) solution), the macropentacycle is "frozen" to a single asymmetric (C1) conformation on the (1)H NMR time scale, which has also the molecular structure observed in the solid state by X-ray crystallography: pseudo-triple helical ( not equalC(3)) shape, io (in, out) form resulting from the endo/exo configuration at the nitrogen bridgehead atoms, and similar orientations of the tosyl substituents. The solution dynamics of the molecule can be described by coupled bridgehead nitrogen inversion, triple helix symmetrization, and reversal of triple helix handedness, with DeltaGc = 54.2 kJ mol(-1) in CD(2)Cl(2) at 300 K. Adoption of the io form by macropentacycle 5 in the crystal and in solution at low-temperature most probably results from the steric crowding and strain introduced by the [15]ane-N(2)S(2) macrocyclic bridging subunits.
Cellular glycosphingolipids mediate the fusion between some viruses and the plasma membrane of target cells. In the present study, we have analyzed the interaction of human immunodeficiency virus (HIV)-1 and HIV-2 surface envelope glycoproteins from distinct viral isolates with monolayers of various glycosphingolipids at the air-water interface. The penetration of the viral glycoproteins into glycosphingolipid monolayers was detected as an increase in the surface pressure. We found that HIV-1 recombinant gp120 (IIIB isolate) could penetrate into a monomolecular film of alpha-hydroxylated galactosylceramide (GalCer-HFA), while ceramides, GluCer, and nonhydroxylated GalCer were totally inactive. The glycoproteins isolated from HIV-1 isolates LAI and NDK and from HIV-2(ROD) could also interact with a GalCer-HFA monolayer, whereas gp120 from HIV-1(SEN) and HIV-1(89.6) did not react. These data correlated with the ability of the corresponding viruses to gain entry into the CD4(-)/GalCer+ cell line HT-29, demonstrating the determinant role of GalCer-HFA in this CD4-independent pathway of HIV-1 and HIV-2 infection. In contrast, all HIV-1 and HIV-2 glycoproteins tested were found to interact with a monolayer of GM3, a ganglioside abundantly expressed in the plasma membrane of CD4(+) lymphocytes and macrophages. A V3 loop-derived synthetic peptide inhibitor of HIV-1 and HIV-2 infection in both CD4(-) and CD4(+) cells could penetrate into various glycosphingolipid monolayers, including GalCer-HFA and GM3. Taken together, these data suggest that the adsorption of human immunodeficiency viruses to the surface of target cells involves an interaction between the V3 domain of the surface envelope glycoprotein and specific glycosphingolipids, i.e. GalCer-HFA for CD4(-) cells and GM3 for CD4(+) cells.
The gastrointestinal tract is considered to be a major route of infection for human immunodeficiency virus (HIV). Infection of human colon epithelial cells by HIV is not blocked by anti-CD4 antibodies known to block infection of lymphoid cells (J. Fantini, N. Yahi, and J. C. Chermann, Proc. Natl. Acad. Sci. USA 88:9297-9301, 1991), suggesting the presence of an alternate receptor for HIV on these cells. In this report, we show that (i) a monoclonal antibody specifically directed against galactosyl ceramide inhibited the infection of HT29 cells by two markedly different strains of HIV-1, as assessed by polymerase chain reaction amplification and reverse transcriptase assay; (ii) this antibody strongly labeled the surface of HT29 cells by immunofluorescence and electron microscopic immunolocalization; (iii) the labeling was preferentially but not totally restricted to the basolateral membrane domain of differentiated colonic cells, in agreement with the ability of HIV to infect both the apical and basolateral surfaces of these epithelial cells; and (iv) in thin-layer chromatography-immunostaining experiments with neutral glycolipids prepared from HT29 cells, the antibody specifically reacted with a ceramide monoglycoside fraction corresponding to galactosyl ceramide. We did not detect this glycolipid in lymphoid cells, and anti-galactosyl ceramide antibodies consistently failed to inhibit HIV infection of these cells. These data suggest that galactosyl ceramide (or a derivative) is an essential component of the receptor for HIV on the surface of HT29 cells.
This report demonstrates that galactosyl ceramide (GalCer) or a molecule derived from it may serve as an alternative receptor for human immunodeficiency virus in the nervous system. Recombinant gp120, an envelope glycoprotein of human immunodeficiency virus type 1, specifically binds to GalCer and its derivatives. This specificity was studied by inhibiting binding of radioiodinated gp120 to GalCer with antibodies to GalCer, antibodies to gp120, and an excess of unlabeled gp120. Binding activity was also removed by absorbing gp120 with liposomes containing GalCer. In addition, studies using natural and semisynthetic lipids indicate that the linkage between galactose and ceramide is essential for binding. The significance of an alternative receptor for human immunodeficiency virus in the nervous system is discussed.
A number of studies have indicated that central nervous system-derived cells can be infected with human immunodeficiency virus type 1 (HIV-1). To determine whether CD4, the receptor for HIV-1 in lymphoid cells, was responsible for infection of neural cells, we characterized infectable human central nervous system tumor lines and primary fetal neural cells and did not detect either CD4 protein or mRNA. We then attempted to block infection with anti-CD4 antibodies known to block infection of lymphoid cells; we noted no effect on any of these cultured cells. The results indicate that CD4 is not the receptor for HIV-1 infection of the glioblastoma line U373-MG, medulloblastoma line MED 217, or primary human fetal neural cells.
Fluorescence photobleaching recovery with total internal reflection illumination (TIR-FPR) has been used to measure the dissociation kinetics of a fluorescein-labeled anti-dinitrophenyl monoclonal Fab specifically bound to supported monolayers composed of a mixture of dipalmitoylphosphatidylcholine and dinitrophenyl-conjugated dipalmitoylphosphatidylethanolamine. The fluorescence recovery curves were not monoexponential; when analyzed as a sum of two exponentials, the rates and fractional recoveries were approximately 1 s-1 (approximately 50%) and approximately 0.1 s-1 (approximately 30%). The data did not change as a function of the Fab solution concentration, indicating that the fluorescence recovery curves were not influenced by the rate of diffusion in bulk solution. Also, the recovery curves were independent of the size of the illuminated area, indicating that surface diffusion did not significantly contribute to the rate and shape of the fluorescence recovery. The measured off rates and apparent association constant (1.6 x 10(5) M-1) were analyzed with the theoretical formalism for a proposed mechanism that accounts for the nonmonoexponential kinetics.
Although the CD4 molecule is the principal cellular receptor for the human immunodeficiency virus (HIV), several CD4-negative
cell lines are susceptible to infection with one or more HIV strains. These findings indicate that there are alternate modes
of viral entry, perhaps involving one or more receptor molecules. Antibodies against galactosyl ceramide (galactocerebroside,
or GalC) inhibited viral internalization and infection in two CD4-negative cell lines derived from the nervous system: U373-MG
and SK-N-MC. Furthermore, recombinant HIV surface glycoprotein gp120 bound to GalC but not to other glycolipids. These results
suggest a role for GalC or a highly related molecule in HIV entry into neural cells.
The association of a fluorescently labelled anti-dinitrophenyl monoclonal antibody (ANO2) with Langmuir-Blodgett monolayers composed of three different binary mixtures of phosphatidylcholine and dinitrophenyl-conjugated phosphatidylethanolamine has been characterized. Quantitative fluorescence microscopy measurements demonstrated that measurable amounts of antibodies bound to the monolayers only at high molar fractions of dinitrophenyl-conjugated lipid (greater than or equal to 5 mol%). Fluorescence pattern photobleaching recovery measurements showed that the apparent translational diffusion coefficients and mobile fractions of a fluorescent lipid were high for all monolayer compositions and that the antibody translational mobility was measurable but slow and depended on the two-dimensional antibody density. The results demonstrate that the ANO2-binding characteristics of Langmuir-Blodgett monolayers containing dinitrophenyl-conjugated phospholipids are substantially different from those of similar model systems but that the ANO2 antibodies, when bound, display similar diffusive behavior.
The association of an anti-dinitrophenyl monoclonal antibody and its Fab fragment with supported phospholipid monolayers composed of a mixture of dipalmitoylphosphatidylcholine and dinitrophenyl-conjugated dipalmitoylphosphatidylethanolamine has been characterized with total internal reflection fluorescence microscopy. The surface densities of bound antibodies were measured as a function of the antibody and Fab solution concentrations, and as a function of the solution concentration of dinitrophenylglycine. The apparent association constant of Fab fragments with surface-associated haptens was approximately 10-fold lower than the association constant for haptens in solution, and the apparent surface association constant for intact antibodies was only approximately 10-fold higher than the constant for Fab fragments. Data analysis with simple theoretical models indicated that, at most antibody surface densities, 50-90% of membrane-associated intact antibodies were attached to the surface by two antigen binding sites.
A new quantitative technique for measuring the binding of proteins to membranes is described. The method is based on a combination of total internal reflection fluorescence microscopy and the preparation of supported planar bilayers. Specific and reversible binding of a fluorescence-labeled monoclonal antibody to lipid haptens that were embedded in supported bilayers has been measured by this technique and compared to binding experiments that were conducted on membrane vesicles in solution. Equilibrium binding constants and kinetic parameters have been determined and used to expand the picture of the antibody-lipid hapten reaction. Estimates demonstrate that this technique is capable of measuring a broad range of binding constants (down to about 10(4) M-1) using only small amounts of ligand and receptor.
Helper T cells recognize foreign antigen displayed on antigenpresenting cells which also express self-molecules of the major histocompatibility complex (MHC). A single T-cell receptor mediates recognition of both MHC and foreign antigen. A proposed ternary complex between T-cell receptor, foreign antigen and MHC antigen has not yet been demonstrated (see ref. 1 for review). Here, we show that a fluorescein-labelled synthetic peptide, together with Texas red-labelled class II MHC antigen, I-Ad, stimulates the production of interleukin-2 by a peptide-specific I-Ad-restricted T-cell hybridoma when reconstituted in a lipid membrane on a glass substrate. Under the same conditions, resonance-energy transfer from donor peptide to acceptor I-A can be stimulated in an evanescent wave-field only in the presence of the specific T-hybrid. Our results show that the T cell stabilizes an association between peptide antigen and class II MHC protein to within a distance of about 40 A.
The envelope of the human immunodeficiency virus type 1 (HIV-1) plays a central role in the process of virus entry into the host cell and in the cytopathicity of the virus for lymphocytes bearing the CD4 molecule. Mutations that affect the ability of the envelope glycoprotein to form syncytia in CD4+ cells can be divided into five groups: those that decrease the binding of the envelope protein to the CD4 molecule, those that prevent a post-binding fusion reaction, those that disrupt the anchorage of the envelope glycoprotein in the membrane, those that affect the association of the two subunits of the envelope glycoprotein, and those that affect post-translational proteolytic processing of the envelope precursor protein. These findings provide a functional model of the HIV envelope glycoprotein.
Sexual transmission is a major mode of spread of HIV-1 although the mechanisms involved remain to be elucidated. The role of spermatozoa as carriers of the HIV is supported by recent publications, while the expression of the CD4 on the membrane of the sperm has not yet been demonstrated. The data reported in this paper show that a glycolipid molecule, most likely the galactosyl-alkyl-acylglycerol, structurally similar to galactosylceramides, is present on the surface membrane of the spermatozoa. Consistent with a structure similar to galactosylceramide, the sperm glycolipid is capable of binding the gp120 as demonstrated utilizing an improved ELISA assay which favors sensitivity and specificity. Immunocytochemistry of testicular tissue shows the presence of this glycolipid on the membrane of immature germ cells, preferentially in the spermatogonia. These data indicate that human sperm express a glycolipid similar in structure to the receptor for HIV described on the CD4- neural and colonic epithelial cell lines, and moreover suggest that this glycolipid could also function as HIV receptor and possibly be implied in its transmission. The demonstration that this molecule is also expressed by the spermatogonia suggests its involvement in the interaction of the HIV with spermatogonia, as recently reported, and could explain the inhibition of spermatogenesis observed in AIDS patients.
We examined the binding of the gp120 envelope glycoprotein (gp120) of the human immunodeficiency virus (HIV-1) to sulfatide (GalS), galactocerebroside (GalC), and GM1-ganglioside (GM1). The gp120 glycoprotein bound to GalS but not to GalC or GM1 by enzyme-linked immunosorbent assay (ELISA) and by an immunospot assay on nitrocellulose paper. However, it bound to all three glycolipids by an immunospot assay on thin layer chromatography (TLC) plates. In studies to determine whether GalS could be a receptor for gp120 on the surface of cells, gp120 bound to GalS incorporated into the plasma membrane of lymphoid cells as determined by cytofluorometric analysis and immunofluorescence microscopy. These studies indicate that GalS may function as a receptor for gp120 and HIV-1.
Human immunodeficiency virus type 1 (HIV-1) infects some cell types which lack CD4, demonstrating that one or more alternative viral receptors exist. One such receptor is galactosylceramide (GalCer), a glycosphingolipid distributed widely in the nervous system and in colonic epithelial cells. Using a liposome flotation assay, we found that the HIV-1 surface glycoprotein, gp120, quantitatively bound to liposomes containing GalCer but not to liposomes containing phospholipids and cholesterol alone. Binding was saturable and was inhibited by preincubating liposomes with anti-GalCer antibodies. We observed less efficient binding of gp120 to liposomes containing lactosylceramide, glucosylceramide, and galactosylsulfate, whereas no binding to liposomes containing mixed gangliosides, psychosine, or sphingomyelin was detected. Binding to GalCer was rapid, largely independent of temperature and pH, and stable to conditions which remove most peripheral membrane proteins. By contrast, gp120 bound to lactosylceramide could be removed by 2 M potassium chloride or 3 M potassium thiocyanate, demonstrating a less stable interaction. Removal of N-linked oligosaccharides on gp120 did not affect binding efficiency. However, as previously observed for CD4 binding, heat denaturation of gp120 prevented binding to GalCer. Finally, binding was critically dependent on the concentration of GalCer in the target membrane, suggesting that binding to glycolipid-rich domains occurs and that GalCer conformation may be important for gp120 recognition.
The lentivirus human immunodeficiency virus (HIV) causes AIDS by interacting with a large number of different cells in the body and escaping the host immune response against it. HIV is transmitted primarily through blood and genital fluids and to newborn infants from infected mothers. The steps occurring in infection involve an interaction of HIV not only with the CD4 molecule on cells but also with other cellular receptors recently identified. Virus-cell fusion and HIV entry subsequently take place. Following virus infection, a variety of intracellular mechanisms determine the relative expression of viral regulatory and accessory genes leading to productive or latent infection. With CD4+ lymphocytes, HIV replication can cause syncytium formation and cell death; with other cells, such as macrophages, persistent infection can occur, creating reservoirs for the virus in many cells and tissues. HIV strains are highly heterogeneous, and certain biologic and serologic properties determined by specific genetic sequences can be linked to pathogenic pathways and resistance to the immune response. The host reaction against HIV, through neutralizing antibodies and particularly through strong cellular immune responses, can keep the virus suppressed for many years. Long-term survival appears to involve infection with a relatively low-virulence strain that remains sensitive to the immune response, particularly to control by CD8+ cell antiviral activity. Several therapeutic approaches have been attempted, and others are under investigation. Vaccine development has provided some encouraging results, but the observations indicate the major challenge of preventing infection by HIV. Ongoing research is necessary to find a solution to this devastating worldwide epidemic.
The expression of a molecule recognized by anti-galactosyl ceramide antibodies (MAb) O1 on the surface membrane of human spermatozoa was investigated by biochemical and immunochemical methods. Indirect immunofluorescence shows that this molecule is preferentially localized on the middle piece of the sperm tail. Immuno-thin-layer chromatography has identified it as a glycolipid related but not identical to galactosylceramide. Consistent with a structure similar to galactosylceramide, the sperm glycolipid is capable of binding gp120. An improved ELISA has been utilized to demonstrate the specificity of binding of the antibodies and gp120 to the isolated lipid fraction. Identity of the binding site of the two ligands to the glycolipid is suggested by competition assays. On the basis of preliminary biochemical analysis this glycolipid was tentatively classified as a galactosylalkylacylglycerolipid (GalAAG), the nonsulfated form of the seminolipid, a glycolipid known to be present in the testis and germ cells of mammals. These data indicate that human sperm express a glycolipid similar in structure to the receptor for HIV described on the CD4- neural and colonic epithelial cell lines, and moreover suggest that this glycolipid could also function as HIV receptor and possibly be implicated in its transmission.
The envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) consists of a complex of gp120 and gp41. gp120 determines viral tropism by binding to target-cell receptors, while gp41 mediates fusion between viral and cellular membranes. Previous studies identified an alpha-helical domain within gp41 composed of a trimer of two interacting peptides. The crystal structure of this complex, composed of the peptides N36 and C34, is a six-helical bundle. Three N36 helices form an interior, parallel coiled-coil trimer, while three C34 helices pack in an oblique, antiparallel manner into highly conserved, hydrophobic grooves on the surface of this trimer. This structure shows striking similarity to the low-pH-induced conformation of influenza hemagglutinin and likely represents the core of fusion-active gp41. Avenues for the design/discovery of small-molecule inhibitors of HIV infection are directly suggested by this structure.
Cell adhesion and function depend upon the formation of adhesive contacts between the cell and substrate. Determination of the cell substrate contact area is necessary in order to understand how biomaterial properties influence cell adhesion. In this review we describe the development and application of total internal reflection fluorescence microscopy (TIRFM) to quantify the separation distance of cells from a biomaterial surface. An approximate theory is presented for the straightforward calculation of separation distances when a fluor is placed in the cell membrane. The validity of this approach is discussed. TIRFM is compared to interference reflection microscopy and related techniques that measure cell/substrate separation distances. This approach is then applied to a number of important problems in cell substrate interactions, including changes in contact area and adhesion strength on biomaterial surfaces, analysis of bond strength, and real-time measurement of cell/substrate separation distances following exposure to flow.
As part of our program directed toward the design and synthesis of high-affinity ligands for the GalCer-binding site on the HIV cell surface glycoprotein, gp120, we required a reliable method for qualitatively assessing relative binding affinities for related analogues. Due to the hydrophilic nature of these synthetic conjugates, difficulties were encountered with typical ELISA methods, which rely upon hydrophobic interactions to anchor the ligand to a microtiter plate. Other types of assays were also problematic due to nonspecific binding of gp120. Therefore, we developed a general method for plating water-soluble ligands on microtiter plates using biotin/NeutrAvidin recognition for adhesion. A water-soluble GalCer analogue was prepared by conjugating psychosine to biotin using a novel tetraethylene glycol linker. In a similar manner, LacCer and GlcCer analogues were prepared and these conjugates were plated into microtiter wells containing NeutrAvidin. Unoccupied sites were blocked using biotin functionalized as a primary amide. Gp120 binding to galactosyl sphingosine, GalSph (19), GlcSph (22), and LacSph (23) conjugates was assessed through incubation with recombinant HRP-gp120. It was determined that LacSph has the strongest interaction with gp120. The binding affinities of GalSph and GlcSph were similar to each other and less strong than LacSph. These data contradict earlier studies where HPTLC showed that LacCer and GlcCer do not significantly bind gp120. They also contradict liposome-based assays that reported psychosine is not recognized by gp120. The extent of plating for each biotinylated molecule was quantified using HRP-biotin, allowing direct comparison of ligand plating efficiencies for the first time. Several other synthetic biotin conjugates were prepared and tested, demonstrating the feasibility of performing ELISA on water-soluble ligands.