Toxin A from Clostridium difficile binds to rabbit erythrocyte glycolipids with terminal Gal alpha 1-3Gal beta 1-4GlcNAc sequences

The binding of Toxin A isolated from Clostridium difficile to rabbit erythrocyte glycolipids has been studied. Total lipid extracts from rabbit erythrocytes were subjected to thin-layer chromatography and toxin-binding glycolipids detected by using 125I-labeled Toxin A in a direct binding overlay technique. Two major and several minor toxin-binding glycolipids were detected in rabbit erythrocytes by this method. The results of structural analyses of the major toxin-binding glycolipids were consistent with a pentasaccharide-ceramide (Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-Cer) and a branched decasaccharide-ceramide (Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3[Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-6]Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-Cer) previously identified as the two most abundant glycolipids in rabbit erythrocytes. 125I-Toxin A binding to these glycolipids could be inhibited by bovine thyroglobulin, monospecific antiserum to the toxin, or by treatment of the glycolipids with alpha-galactosidase. The absence of toxin interaction with isoglobotriaosylceramide (Gal alpha 1-3Gal beta 1-4Glc-Cer) isolated from canine intestine suggested that the GlcNAc residue present in the terminal Gal alpha 1-3Gal beta 1-4GLcNAc sequence common to all known toxin binding glycoconjugates is required for carbohydrate-specific recognition by Toxin A. These observations are consistent with the proposed carbohydrate binding specificity of Toxin A for the nonreducing terminal sequence, Gal alpha 1-3Gal beta 1-4GlcNAc.     

Pseudomonas aeruginosa and Pseudomonas cepacia isolated from cystic fibrosis patients bind specifically to gangliotetraosylceramide (asialo GM1) and gangliotriaosylceramide (asialo GM2)

Pseudomonas aeruginosa infection in the lungs is a leading cause of death of patients with cystic fibrosis, yet a specific receptor that mediates adhesion of the bacteria to host tissue has not been identified. To examine the possible role of carbohydrates for bacterial adhesion, two species of Pseudomonas isolated from patients with cystic fibrosis were studied for binding to glycolipids. P. aeruginosa and P. cepacia labeled with 125I were layered on thin-layer chromatograms of separated glycolipids and bound bacteria were detected by autoradiography. Both isolates bound specifically to asialo GM1 (Gal beta 1-3GalNAc beta 1-4Gal beta 1-4Glc beta 1-1Cer) and asialo GM2 (GalNAc beta 1-4Gal beta 1-4Glc beta 1-1Cer) but not to lactosylceramide (Gal beta 1-4Glc beta 1-1Cer), globoside (GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1Cer), paragloboside (Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1Cer), or several other glycolipids that were tested. Asialo GM1 and asialo GM2 bound the bacteria equally well, exhibiting similar binding curves in solid-phase binding assays with a detection limit of 200 ng of either glycolipid. Both isolates also did not bind to GM1, GM2, or GDla suggesting that substitution of the glycolipids with sialosyl residues prevents binding. As the Pseudomonas do not bind to lactosylceramide, the beta-N-acetylgalactosamine residue, positioned internally in asialo GM1 and terminally in asialo GM2, is probably required for binding. beta-N-Acetylgalactosamine itself, however, is not sufficient as the bacteria do not bind to globoside or to the Forssman glycolipid. These data suggest that P. aeruginosa and P. cepacia recognize at least terminal or internal GalNAc beta 1-4Gal sequences in glycolipids which may be receptors for these pathogenic bacteria.     

Tetrodotoxin-insensitive sodium channels. Binding of polypeptide neurotoxins in primary cultures of rat muscle cells

The binding of 125I-labeled derivatives of scorpion toxin and sea anemone toxin to tetrodotoxin-insensitive sodium channels in cultured rat muscle cells has been studied. Specific binding of 125I-labeled scorpion toxin and 125I-labeled sea anemone toxin was each blocked by either native scorpion toxin or native sea anemone toxin. K0.5 for block of binding by several polypeptide toxins was closely correlated with K0.5 for enhancement of sodium channel activation in rat muscle cells. These results directly demonstrate binding of sea anemone toxin and scorpion toxin to a common receptor site on the sodium channel. Binding of both 125I-labeled toxin derivatives is enhanced by the alkaloids aconitine and batrachotoxin due to a decrease in KD for polypeptide toxin. Enhancement of polypeptide toxin binding by aconitine and batrachotoxin is precisely correlated with persistent activation of sodium channels by the alkaloid toxins consistent with the conclusion that there is allosteric coupling between receptor sites for alkaloid and polypeptide toxins on the sodium channel. The binding of both 125I-labeled scorpion toxin and 125I-labeled sea anemone toxin is reduced by depolarization due to a voltage-dependent increase in KD. Scorpion toxin binding is more voltage-sensitive than sea anemone toxin binding. Our results directly demonstrate voltage-dependent binding of both scorpion toxin and sea anemone toxin to a common receptor site on the sodium channel and introduce the 125I-labeled polypeptide toxin derivatives as specific binding probes of tetrodotoxin-insensitive sodium channels in cultured muscle cells.     

Globotetraosylceramide is recognized by the pig edema disease toxin

The pig edema disease toxin has been shown by a tlc glycolipid binding assay to bind specifically to globotetraosylceramide (Gb4, GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4GlcCer.). Binding was reduced for globotriosylceramide (Gb3, Gal alpha 1-4Gal beta 1-4GlcCer) and more markedly for the Forssman antigen (GalNAc alpha 1-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4GlcCer). Paragloboside, blood group A glycolipids, glycolipids terminating in Gal NAc beta 1-4Gal-, and glycolipids in which globoside was present as an internal sequence did not bind the toxin. Isogloboside (GalNAc beta 1-3Gal alpha 1-3Gal beta 1-4GlcCer) was efficiently recognized. This toxin is genetically related to the verotoxin (or Shiga-like) family of toxins for which Gb3 has been shown to be the receptor. The difference in susceptibility of cell lines to the cytotoxicity of the pig edema disease toxin and the Shiga and Shiga-like toxins is consistent with the difference in receptor glycolipid binding.     

Animal glycolipids as attachment sites for microbes

The abundance of carbohydrate at the animal cell surface may explain why microbes have selected primarily carbohydrates as essential attachment sites for colonization or infection. Of the various surface glycoconjugates of interest, primary attention has been given to glycolipids, due in part to an efficient binding assay based on a thin-layer chromatogram with separated glycolipids. In this way the general character of carbohydrate recognition by microbes is being mapped. Mainly two examples are briefly described to illustrate some generalizations: lactosylceramide-recognition by several bacteria, and Gal alpha l----4Gal-binding by Escherichia coli and the Shiga toxin. The unique recognition of internally placed sequences, the often low-affinity binding, and the preference of certain sequences before others are interpreted to be of decisive biological value. The binding to internal parts makes it technically possible to approximate the binding epitope on a receptor glycolipid. For this the binding preferences to glycolipids carrying the binding site in different saccharide environments (isoreceptors) are compared with the computer-calculated preferred conformations (definition of steric hindrances to epitope access). Several binding epitopes dissected with this approach have a common surface character: a nonpolar area of ring hydrogens over one or two sugars, surrounded by polar oxygens or amide. This is in agreement with the recent Lemieux concept for antibody-carbohydrate interaction. This information facilitates a rational synthesis of receptor analogues for potential applications. An outline is finally given of an improved general approach for receptor analysis.     

Interaction of Escherichia coli heat-stable enterotoxin B with rat intestinal epithelial cells and membrane lipids

The binding of 125I-labeled Escherichia coli heat-stable enterotoxin B to rat intestinal epithelial cells was unsaturable and nonspecific, at concentrations well above that required to mediate biological events. Following its interaction with intestinal cells, approximately 50-80% of heat-stable enterotoxin B remained stably associated with the cells, implying that it was partitioned into the membrane and/or internalized by the cell. The toxin bound with different affinities to lipids isolated from intestinal epithelial cells, phospholipids, glycolipids, neutral lipids and to model membrane vesicles containing negatively charged lipids. These results indicate that heat-stable enterotoxin B utilizes the membrane bilayer, rather than a surface protein or glycoprotein in modulating toxin-induced enterotoxicity.     

Toxicity of ligand and antibody-directed ricin A-chain conjugates recognizing the epidermal growth factor receptor

Approximately equal amounts of 125I-mAb 225 (a monoclonal antibody recognizing the human epidermal growth factor receptor) and 125I-labeled epidermal growth factor (125I-EGF) were bound by HeLa cells. However, these two EGF receptor binding moieties had different fates after binding. Sixty percent of cell-associated 125I-EGF was internalized. The majority of internalized 125I was released from the cell within 2 hr. In contrast, whereas only 30% of bound 125I-mAb 225 was internalized by HeLa cells, the internalized radioactivity remained cell-associated. EGF and mAb 225 were used to construct ricin A-chain (RTA) conjugates. The two chimeric molecules, EGF-RTA and mAb 225-RTA, were equally toxic to human HeLa cells. EGF-RTA was also toxic to murine 3T3 cells. In contrast, mAb 225-RTA was not toxic to 3T3 cells, consistent with the human EGF-receptor specificity of mAb 225. Neither conjugate was cytotoxic to EGF receptor-deficient 3T3-NR6 cells. Rapidity and potency of protein synthesis inhibition of HeLa cells were equivalent for the two chimeric conjugates, as was the degree to which colony-forming ability was reduced. However, ammonium chloride enhanced the toxicity of EGF-RTA but not mAb 225-RTA, suggesting that the two toxic chimeric toxins--like the unconjugated receptor-binding moieties--are processed differently by HeLa cells.     

Specific binding of radioiodinated granulocyte-macrophage colony-stimulating factor to hemopoietic cells

The hemopoietic growth factor granulocyte-macrophage colony-stimulating factor, GM-CSF, specifically controls the production of granulocytes and macrophages. This report describes the binding of biologically-active 125I-labeled murine GM-CSF to a range of hemopoietic cells. Specific binding was restricted to murine cells and neither rat nor human bone marrow cells appeared to have surface receptors for 125I-labeled GM-CSF. 125I-Labeled GM-CSF only appeared to bind specifically to cells in the myelomonocytic lineage. The binding of 125I-labeled GM-CSF to both bone marrow cells and WEHI-3B(D+) was rapid (50% maximum binding was attained within 5 min at both 20 degrees C and 37 degrees C). Unlabeled GM-CSF was the only polypeptide hormone which completely inhibited the binding of 125I-labeled GM-CSF to bone marrow cells, however, multi-CSF (also called IL-3) and G-CSF partially reduced the binding of 125I-labeled GM-CSF to bone marrow cells. Interestingly, the binding of 125I-labeled GM-CSF to a myelomonocytic cell line, WEHI-3B(D+), was inhibited by unlabeled GM-CSF but not by multi-CSF or G-CSF. Scatchard analysis of the binding of 125I-labeled GM-CSF to WEHI-3B(D+) cells, bone marrow cells and peritoneal neutrophils indicated that there were two classes of binding sites: one of high affinity (Kd1 = 20 pM) and one of low affinity (Kd2 = 0.8-1.2 nM). Multi-CSF only inhibited the binding of 125I-labeled GM-CSF to the high affinity receptor on bone marrow cells: this inhibition appeared to be a result of down regulation or modification of the GM-CSF receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glycolipid-lectin interactions: reactivity of lectins from Helix pomatia, Wisteria floribunda, and Dolichos biflorus with glycolipids containing N-acetylgalactosamine

The autoradiographic detection of 125I-labeled lectins binding to glycolipids on thin-layer chromatograms can be used to rapidly analyze total glycolipid extracts of cells or tissues for specific oligosaccharide structures. The Helix pomatia lectin which binds with high affinity to terminal alpha-linked GalNAc residues did not bind to globoside (terminal beta 1-3GalNAc) but did bind the ganglioside GM2 and its asialo derivative which have terminal beta 1-4GalNAc residues. The lectin from Dolichos biflorus bound specifically to the Forssman glycolipid with relatively low affinity. The lectin from Wisteria floribunda was bound to Forssman glycolipid, globoside, and the asialo derivative of the ganglioside GM2. The interactions of these lectins with the glycolipid-derived, 3H-labeled oligosaccharides was also analyzed by affinity chromatography. The results indicated that the reactivity of multivalent carbohydrate-binding proteins with polyvalent surfaces of glycolipids is strong enough to permit detection of low-affinity interactions that may not be observed in binding assays that are based on carbohydrate-protein interactions in solution. The autoradiographic analysis of 125I-Helix pomatia lectin binding to thin-layer chromatograms of total lipid extracts from human erythrocyte membranes detected the quantitative differences in the A-active glycolipids from type A1 and A2 cells.     

Escherichia coli K99 binds to N-glycolylsialoparagloboside and N-glycolyl-GM3 found in piglet small intestine

Escherichia coli K12, which possess the K99 plasmid and synthesize K99 fimbriae (E. coli K99), cause severe neonatal diarrhea in piglets, calves, and lambs but not in humans. The organism binds specifically and with high affinity to only two glycolipids in piglet intestinal mucosa as demonstrated by overlaying glycolipid chromatograms with 125I-labeled bacteria. These glycolipids, which are N-glycolyl-GM3 (NeuGc alpha 2-3Gal beta 1-4Glc beta 1-1Cer) and N-glycolylsialoparagloboside (NeuGc alpha 2-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1Cer), occur at about 13 and 0.3 micrograms per gram wet weight of mucosa, respectively. E. coli K99 grown at 18 degrees C, a temperature at which the K99 fimbriae are not expressed, do not bind to these glycolipids. Of the standard glycolipids tested in solid phase binding assays, E. coli K99 binds with highest affinity to N-glycolylsialoparagloboside, with less affinity to N-glycolyl-GM3, and with very low affinity to N-acetylsialoparagloboside. The bacteria do not bind to GM3 (NeuAc alpha 2-3Gal beta 1-4Glc beta 1-1Cer), GM2 (GalNAc beta 1-4[Neu-Ac alpha 2-3]Gal beta 1-4Glc beta 1-1Cer), GM1 (Gal beta 1-3GalNAc beta 1-4[NeuAc alpha 2-3]Gal beta 1-4Glc beta 1-1Cer), or several other N-acetylsialic acid-containing gangliosides and neutral glycolipids at the levels tested. N-Glycolylsialyl residues are found in the glycoproteins and glycolipids of piglets, calves, and lambs but not in the glycoproteins and glycolipids of humans. Possibly this distribution of sialyl derivatives explains the host range of infection by the organism.     

Kinetics of binding of the toxic lectins abrin and ricin to surface receptors of human cells.

Kinetic parameters of the interaction of the toxic lectins abrin and ricin with human erythrocytes and HeLa cells have been measured. The binding of 125I-labeled abrin and ricin to human erythrocytes and to HeLa cells at 37 degrees was maximal around pH 7, whereas at 0 degrees the binding was similar over a broad pH range. The binding occurred at similar rates at 0 degrees and 37 degrees with rate constants in the range 0.9 to 3.0 X 10(5) M-1 s-1. The dissociation was strongly temperature-dependent with rate constants in the range 3.4 to 45 X 10(-4) s-1 at 0 degrees and 3.9 to 18 X 10(-3) s-1 at 37 degrees. The presence of unlabeled lectins as well as lactose increased the rate of dissociation. The association constants measured at equilibrium or calculated from the rate constants were between 0.64 X 10(8) M-1 and 8.2 X 10(8) M-1 for abrus lectins, and between 8.0 X 10(6) M-1 and 4.2 X 10(8) M-1 for ricinus lectins. The association constants for the toxins were lower at 37 degrees than at 0 degrees. Isolated ricin B chain appeared to bind with similar affinity as intact ricin. The number of binding sites was estimated to be 2 to 3 X 10(6) per erythrocyte and 1 to 3 X 10(7) per HeLa cell. The binding sites of HeLa cells all displayed a uniform affinity towards abrin and ricin, both at 0 degrees and at 37 degrees. The same was the case with the binding sites of erythrocytes at 0 degrees. However, the data indicated that at 20 degrees erythrocytes possessed binding sites with two different affinities. Only a fraction of the cell-bound toxin appeared to be irreversibly bound and could not be removed by washing with 0.1 M lactose. The fraction of the total amount of bound toxin which became irreversibly bound to HeLa cells was for both toxins about 2 X 10(-3)/min at 37 degrees, whereas no toxin was irreversibly bound at 0 degrees. In the case of erythrocytes no toxin became irreversibly bound, either at 0 degrees or 37 degrees, indicating that the toxins are unable to penetrate into these cells.     

Initial characterization of a chlamydial receptor on mammalian cells

We have examined characteristics of the binding of eukaryotic cells to chlamydial elementary body (EB)-specific proteins. A wide variety of eukaryotic cell lines bound to representatives of both Chlamydia trachomatis lymphogranuloma venereum (LGV) and trachoma biovars and a C. psittaci strain meningopneumonitis (Mn) suggesting the presence of a common host cell receptor. Neither tunicamycin nor neuraminidase treatment of HeLa cells impaired binding to C. trachomatis EB, implying that host cell N-linked carbohydrate domains and sialic acid moieties, respectively, are not involved in attachment. However, trypsinized HeLa cells do not bind to EB, suggestive of a proteinaceous host cell receptor. The trypsin sensitivity of two EB-specific binding proteins Mr = 18,000 and 31,000) was also examined, and the finding that 125I-labeled HeLa cells bind both the 18,000 and 31,000-dalton proteins after chlamydial trypsinization corroborates our earlier observation that these EB binding proteins mediate attachment.     

Carbohydrate specificity of the receptor sites of mistletoe toxic lectin-I.

The carbohydrate specificity of mistletoe toxic lectin-I (ML-I) was studied by haemagglutination-inhibition assay. The results indicated that ML-I has a broad range of affinity for Gal alpha,beta linked sequences. The galabiose (E, Gal alpha 1----4Gal) sequence, a receptor of the uropathogenic E. coli ligand, was one of the best disaccharide inhibitors tested. The lectin also exhibits affinity for Lac(Gal beta 1----4Glc), T(Gal beta 1----3GalNAc), I/II(Gal beta 1----3/4GlcNAc) and B(Gal alpha 1----3Gal) sequences. Gal alpha 1----4Gal and Gal beta 1----4Glc are frequently occurring sequences of many glycosphingolipids located at the mammalian cell membranes, such as intestinal and red blood cell membranes, for ligand binding and toxin attachment. This finding provides important information concerning the possible mechanism of intoxication of cells by the mistletoe preparation.     

Studies on the binding of staphylococcal 125I-labeled alpha-toxin to rabbit erythrocytes.

Staphylococcal alpha-toxin, a hemolytic exotoxin, can be iodinated using the lactoperoxidase method. 125 I-Labeled alpha-toxin binds to rabbit erythrocytes in an apparently irreversible and highly specific manner. The binding of 125 I-labeled alpha-toxin to erythrocytes of rabbit and human reflects the species specificity of native alpha-toxin. Binding of 125I-labeled alpha-toxin is blocked by the presence of native alpha-toxin, 127I-labeled alpha-toxin, or anti-alpha-toxin antibody. Simultaneous assays of 125I-labeled alpha-toxin binding and leakage of intracellular 86Rb+ suggest that toxin binding and membrane damage are separate, sequential functions. Both the rate and extent of binding are temperature dependent. Rabbit erythrocytes possess 5 X 10(3) binding sites/cell, while human erythrocytes possess no detectable binding sites. Treatment of rabbit erythrocytes with 125I-labeled alpha-toxin appears to decrease the number of unoccupied binding sites. Chaotropic ions can inhibit 125I-labeled alpha-toxin binding and cause bound 125I-labeled alpha-toxin to dissociate from rabbit erythrocyte membranes. Treatment of intact rabbit erythrocytes with pronase reduces both the binding capacity of the cells for 125I-labeled alpha-toxin, and the cells' sensitivity to hemolysis by native alpha-toxin. It is proposed that the primary binding site for alpha-toxin in biomembranes is a surface membrane protein.     

Nicotinic acetylcholine receptor of mammalian brain: naja toxin binding to subcellular fractions of rat brain

Abstract— Subcellular fractions of rat brain cortex bound 6.8 ± 0.4 fmol of ¹²⁵I-labeled α-naja toxin per milligram of protein. Toxin binding was saturable and specific e.g. nicotinic agonist and antagonist blockable. Regions in rat brain varied in saturable, specific toxin binding with hippocampus, olfactory area and corpus striatum displaying greatest toxin binding. We conclude that ¹²⁵I-α-naja toxin is a suitable probe for nicotinic cholinergic receptor in brain.     

Apolipoprotein B: its role in the control of fibroblast cholesterol biosynthesis and in the regulation of its own binding to cellular receptors.

Apolipoprotein B transports cholesterol in plasma as low density lipoprotein (LDL) and targets its delivery to cells by binding to a specific plasma membrane receptor. The cellular consequences of apoB binding to its receptor were investigated to determine whether it suppresses cholesterol biosynthesis and reduces the number of cellular receptors for the apoprotein. Upon preincubation of fibroblasts with lipoprotein-deficient medium alone or supplemented with either LDL or apoB complexed to BSA (apoB-BSA), LDL suppressed cholesterol biosynthesis, but apoB enhanced it. Similarly, fibroblasts preincubated in medium supplemented with LDL bound decreased amounts of either (125)I-labeled LDL or (125)I-labeled apoB-BSA to their receptors, while preincubation with apoB-BSA increased the binding relative to the controls. These latter results occurred in association with a decrease in cellular cholesterol content, indicating that apoB in the medium bound cholesterol and removed it from the cells, thus stimulating both cholesterol synthesis and cellular binding of apoB. Accordingly, fibroblast cholesterol synthesis and the number of functional LDL receptors are not suppressed by the binding of the apoprotein to the receptor, and the known role of apoB remains that of transporting cholesterol in plasma and delivering it to the cell. A possible physiologic role for apoB in depleting cells of cholesterol is presently unknown since apoB is not known to exist free in plasma; however, these findings demonstrate such a functional capability for this apoprotein.-Shireman, R. B., and W. R. Fisher. Apolipoprotein B: its role in the control of fibroblast cholesterol biosynthesis and in the regulation of its own binding to cellular receptors.     

Antipeptide antibody against the human low-density-lipoprotein receptor. Characterization and cross-reactivity with bovine lymphocytes.

A dodecapeptide corresponding to the external N-terminal sequence of the human low-density-lipoprotein (LDL) receptor was synthesized. Antibodies raised in rabbits against the peptide were purified and were shown to react specifically with the peptide and with human LDL receptor of fibroblasts, HeLa cells and lymphocytes using binding studies and immunoblotting. By indirect immunogold analysis, antibodies bound to the LDL receptor of human lymphocytes could be revealed as clusters. Anti-receptor peptide immunoglobulins specifically bound to the human HeLa cell's LDL receptor with a lower affinity than LDL (Kd x 3). The anti-receptor peptide immunoglobulins and 125I-labelled-LDL competed with each other for the LDL-receptor sites. Antibodies failed to react with lymphocytes of subjects with the homozygous form of familial hypercholesterolaemia. Cross-reactivity with the dodecapeptide of the bovine LDL receptor was limited, but this cross-reactivity was confirmed by the binding of anti-receptor peptide immunoglobulins to the LDL receptor from bovine lymphocytes.     

Chlamydia trachomatis and Chlamydia pneumoniae bind specifically to phosphatidylethanolamine in HeLa cells and to GalNAc beta 1-4Gal beta 1-4GLC sequences-found in asialo-GM1 and asial-GM2

To examine the possible role of lipids as adhesion receptors for infection, Chlamydia trachomatis and Chlamydia pneumoniae were labeled with 125I and layered on thin-layer chromatograms (tlc) of separated lipids isolated from target cells, and bound bacteria were detected by autoradiography. Elementary bodies from both species bound specifically and with high affinity to one lipid in HeLa 229 cells. Purification of this receptor by column chromatography on DEAE Sepharose followed by continuous preparative tlc, and structural analysis by 500-MHz 1H-NMR spectroscopy and fast atom bombardment mass spectrometry confirmed the HeLa cell chlamydial receptor to be phosphatidylethanolamine (PE). The chlamydiae also bound strongly to purified asialo-GM1 and asialo-GM2, but not to other neutral or acidic lipids tested. The relative binding of chlamydiae to human PE and asialo-GM1 was modified in the presence divalent cations, suggesting that chlamydiae have two interrelated receptor binding sites.     

A novel method for the detection of receptors and membrane proteins by scintillation proximity radioassay

A rapid and convenient binding assay for receptors and membrane proteins has been developed. It is based on the binding of 125I-labeled ligands to membrane proteins adsorbed to polyvinyltoluene plastic scintillation microspheres. Membranes or isolated membrane proteins adsorb to the beads upon mixing, and addition of 125I-labeled ligand induces photon emission which is proportional to the amount of added receptor or membrane protein. The interaction of acetylcholine receptor with 125I-labeled alpha-bungarotoxin and antigens with 125I-labeled antibodies or protein A were used as models to test the system. As little as 1 ng of acetylcholine receptor is detected by the assay and a linear relationship with receptor concentration is observed up to 50 ng of receptor per 250 microliter reaction medium. The effects of detergents, salts, soluble proteins, and neutral membranes were studied. Inclusion of bovine serum albumin up to 1 mg/ml, sodium chloride up to 0.5 M, and membranes up to 10 micrograms/ml cause little or no effect on the assay. Detergents at 10-fold below their critical micelle concentrations had little or no effect on the assay. The pharmacological effects of agonists such as acetylcholine were conveniently studied by following the displacement of the 125I-labeled ligand. Similarly, the amount of toxin in crude snake venom can be assayed by measuring competition with the labeled toxin. Only a few seconds are required to perform each binding assay.     

Endocytosis and intracellular transport of the glycolipid-binding ligand Shiga toxin in polarized MDCK cells

The glycolipid-binding cytotoxin produced by Shigella dysenteriae 1, Shiga toxin, binds to MDCK cells (strain 1) only after treatment with short-chain fatty acids like butyric acid or with the tumor promoter 12-O-tetradecanoylphorbol 13-acetate. The induced binding sites were found to be functional with respect to endocytosis and translocation of toxin to the cytosol. Glycolipids that bind Shiga toxin appeared at both the apical and the basolateral surface of polarized MDCK cells grown on filters, and Shiga toxin was found to be endocytosed from both sides of the cells. This was demonstrated by EM of cells incubated with Shiga-HRP and by subcellular fractionation of cells incubated with 125I-labeled Shiga toxin. The data indicated that toxin molecules are endocytosed from coated pits, and that some internalized Shiga toxin is transported to the Golgi apparatus. Fractionation of polarized cells incubated with 125I-Shiga toxin showed that the transport of toxin to the Golgi apparatus was equally efficient from both poles of the cells. After 1-h incubation at 37 degrees C approximately 10% of the internalized toxin was found in the Golgi fractions. The results thus suggest that glycolipids can be efficiently transported to the Golgi apparatus from both sides of polarized MDCK cell monolayers.