Exposure of major neutral glycolipids in red cells to galactose oxidase. Effect of neuraminidase

The exposure of several major red-cell glycolipids to galactose oxidase was studied by oxidizing the cells with the enzyme and reducing them with NaB2H4. After isolation, the deuterium label was detected by mass fragmentography. 60-70% globoside in human and porcine erythrocytes was exposed as measured by this method. In contrast, asialo-GM2 in guinea-pig erythrocytes and Forssman glycolipid in sheep erythrocytes were mainly in a cryptic state. Neuraminidase treatment increased the incorporation of deuterium label to asialo-GM2 4-8-fold. A similar effect was seen in Forssman glycolipid when sheep red cells were labeled with the neuraminidase/galactose oxidase/NaB3H4 method. In contrast, the increase in labeling was only about 10-40% in porcine and human globosides, which were efficiently exposed to galactose oxidase already in native red cells.     

Receptor distribution and the mechanism of enhanced erythrocyte agglutination by soybean agglutinin

We have examined the role of receptor clustering in intact erythrocyte membranes exhibiting enhanced lectin-mediated cell agglutination by analyzing freeze-fracture and freeze-etch images of human erythrocytes labeled with ferritin-conjugated soybean agglutinin. We find that trypsinization and fixation of intact erythrocytes, in either order, causes no alteration of the random distribution of ferritin-conjugated soybean agglutinin on the surfaces of these cells as compared to their distribution on the surfaces of fixed erythrocytes and untreated erythrocyte ghosts. Furthermore, clustering of the intramembranous particles in the membrane of intact erythrocytes was not found with any of the cells described above.We conclude that clustering of the soybean agglutinin receptors is not a major factor involved in the enhanced agglutination of intact trypsinized erythrocytes. Caution is necessary in transferring information obtained with erythrocyte ghosts, where clustering can be induced, to intact erythrocytes.     

Localization of the binding sites for the Ricinus communis, Agaricus bisporus and wheat germ lectins on human erythrocyte membranes.

Freeze-etch electron microscopy has been utilized to localize the binding sites for the Ricinus communis, Agaricus bisporus and wheat germ lectins on human erythrocyte membranes and to determine the relation of these different glycoprotein receptors to the intramembranous particles. A. bisporus lectin, which could be visualized directly on the surface of erythrocyte membranes, and ferritin conjugates of wheat germ agglutinin showed a distribution that correlates exactly with the intramembranous particles at all lectin concentrations tested. The binding sites for both of these lectins are located on the major sialoglycoprotein of the membrane. The R. communis agglutinin-ferritin conjugate which binds to receptors on membrane glycoproteins that are distinct from the major sialoglycoprotein showed a close correlation with the intramembranous particles at low lectin concentrations and a poor correlation at high lectin concentrations. High concentrations resulted in virtually complete coating of the surface of trypsinized ghosts which displayed marked aggregation of the intramembranous particles. We conclude that the intramembranous particles of erythrocyte membranes contain at least two glycoproteins and that some membrane lectin receptors are not associated with the intramembranous particles.     

ANIONIC SITES OF HUMAN ERYTHROCYTE MEMBRANES : I. Effects of Trypsin, Phospholipase C, and pH on the Topography of Bound Positively Charged Colloidal Particles

The effects of pH, trypsin, and phospholipase C on the topographic distribution of acidic anionic residues on human erythrocytes was investigated using colloidal iron hydroxide labeling of mounted, fixed ghost membranes. After glutaraldehyde fixation at pH 6.5–7.5, the positively charged colloidal particles were bound to the membranes in small randomly distributed clusters. The clusters of anionic sites were reversibly aggregated by incubation at pH 5.5 before fixation at the same pH. These results correlate with the distribution of intramembranous particles found by Pinto da Silva (J. Cell Biol. 53:777), with the exception that the distribution of anionic sites on a majority of the fixed ghosts at pH 4.5 was aggregated instead of dispersed. The randomly distributed clusters could be nonreversibly aggregated by trypsin or phospholipase C treatment of intact ghosts before glutaraldehyde fixation. Previous glutaraldehyde fixation prevented trypsin and pH induced aggregation of the colloidal iron sites. Evidence that N-acetylneuraminic acid groups are the principal acidic residues binding colloidal iron was the elimination of greater than 85% of the colloidal iron labeling to neuraminidase-treated cell membranes. Colloidal iron binding N-acetylneuraminic acid residues may reside on membrane molecules such as glycophorin, a sialoglycoprotein which contains the majority of the N-acetylneuraminic acid found on the human erythrocyte membrane.     

Localization of the binding sites for the Ricinus communis, Agaricus bisporus and wheat germ lectins on human erythrocyte membranes

Freeze-etch electron microscopy has been utilized to localize the binding sites for the Ricinus communis, Agaricus bisporus and wheat germ lectins on human erythrocyte membranes and to determine the relation of these different glycoprotein receptors to the intramembranous particles. A. bisporus lectin, which could be visualized directly on the surface of erythrocyte membranes, and ferritin conjugates of wheat germ agglutinin showed a distribution that correlates exactly with the intramembranous particles at all lectin concentrations tested. The binding sites for both of these lectins are located on the major sialoglycoprotein of the membrane. The R. communis agglutinin-ferritin conjugate which binds to receptors on membrane glycoproteins that are distinct from the major sialoglycoprotein showed a close correlation with the intramembranous particles at low lectin concentrations and a poor correlation at high lectin concentrations. High concentrations resulted in virtually complete coating of the surface of trypsinized ghosts which displayed marked aggregation of the intramembranous particles. We conclude that the intramembranous particles of erythrocyte membranes contain at least two glycoproteins and that some membrane lectin receptors are not associated with the intramembranous particles.     

Interactions of plant lectins with glycolipids in liposomes

A panel of five plant lectins with different binding specificities was used to determine if plant lectins could bind specifically to membrane-associated glycolipids. $\text{Ricinis communis}$ and wheat germ agglutinins both bound specifically to mixed brain gangliosides and globoside I from human erythrocytes. Wheat germ agglutinin also bound to ganglioside G_M1 and human erythrocyte ceramide trihexoside, but not to ceramide dihexoside, mono-, or digalactosyl diglycerides. Concanavalin A bound to liposomes with or without glycolipid substituents, and this binding was partially inhibited by α-methyl mannoside. This study indicates that lectins can specifically recognize and bind to certain glycolipids in membranes.     

Clearance of senescent erythrocytes: wheat germ agglutinin distribution on young and old human erythrocytes

To add an additional aspect to the process of recognition and removal of senescent human erythrocytes from the circulation, the binding of wheat germ agglutinin (WGA) to separated young, old and sialidase-treated human erythrocytes is evaluated with the immune-electron microscopical method. WGA/gold conjugate binding to old erythrocytes was lower (27%) than to young erythrocytes and even lower following treatment with sialidase (82%), exhibiting a clustered, non-continuous labeling pattern in all three erythrocyte populations, thus showing a possible redistribution of WGA binding sites. The decrease in bound WGA/gold particles correlates well with the previously reported decrease in surface sialic acid on old erythrocytes. The binding of WGA/gold are indicative of the changes occurring on erythrocyte membrane surfaces when interacting with different agglutinins.     

Alterations in intramembrane particle distribution during interaction of erythrocyte-bound ligands with immunoprotein receptors

Freeze fracture studies have been performed on rabbit pulmonary alveolar macrophages and a nonphagocytic murine lymphoblastoid cell line, PU-5 Fc+, incubated with sheep erythrocytes, sheep erythrocyte-IgG Forssman antibody complex, sheep erythrocyte-IgG Forssman antibody-C complexes and aggregated IgG. Alveolar macrophages show redistribution of intramembrane particles after interaction with (EIgG) and E(IgM)C. The murine lymphoblastoid cell line shows intramembrane particle redistribution consequential to binding of E(IgG) and aggregated IgG. The results demonstrate that after specific immunoprotein receptor-ligand interaction, there is extensive plasma membrane reorganization which results in a redistribution and loss of intramembrane particles. Changes are observed in the protoplasmic face of the plasma membrane after the binding of ligand to the outer membrane surface. The findings suggest that interaction of erthrocyte-bound ligands with specific lymphoid and macrophage plasma membrane receptors leads to a generalized redistribution of integral membrane components in the membrane.     

Subcellular localization of Forssman glycolipid in epithelial MDCK cells by immuno-electronmicroscopy after freeze-substitution

Forssman antigen, a neutral glycosphingolipid carrying five monosaccharides, was localized in epithelial MDCK cells by the immunogold technique. Labeling with a well defined mAb and protein A-gold after freeze-substitution and low temperature embedding in Lowicryl HM20 of aldehyde-fixed and cryoprotected cells, resulted in high levels of specific labeling and excellent retention of cellular ultrastructure compared to ultra-thin cryosections. No Forssman glycolipid was lost from the cells during freeze-substitution as measured by radio-immunostaining of lipid extracts. Redistribution of the glycolipid between membranes did not occur. Forssman glycolipid, abundantly expressed on the surface of MDCK II cells, did not move to neighboring cell surfaces in cocultures with Forssman negative MDCK I cells, even though they were connected by tight junctions. The labeling density on the apical plasma membrane was 1.4-1.6 times higher than basolateral. Roughly two-thirds of the gold particles were found intracellularly. The Golgi complex was labeled for Forssman as were endosomes, identified by endocytosed albumin-gold, and lysosomes, defined by double labeling for cathepsin D. In most cases, the nuclear envelope was Forssman positive, but the labeling density was 10-fold less than on the plasma membrane. Mitochondria and peroxisomes, the latter identified by catalase, remained free of label, consistent with the notion that they do not receive transport vesicles carrying glycosphingolipids. The present method of lipid immunolabeling holds great potential for the localization of other antigenic lipids.     

An immunochemical study of the human blood group P1, P, and PK glycosphingolipid antigens.

The erythrocyte PK and P blood group antigens have been identified as ceramide trihexoside (CTH), Gal-(alpha, 1 leads to 4)Gal(beta, 1 leads to 4)Glc-Cer, and globoside, GalN-Ac(beta, 1 leads to 3)Gal(alpha, 1 leads to 4)Gal(beta, 1 leads to 4)Glc-Cer, respectively, and the following structure has been proposed for the P1 antigen: Gal(alpha, 1 leads to 4)Gal(beta, 1 leads to 4)GlcNAc(beta, 1 leads to 3)Gal(beta, 1 leads to 4)Glc-Cer. Although the P1 and PK determinants have identical terminal disaccharides, CTH did not inhibit anti-P1. The P1 glycolipid and hydatid cyst glycoprotein inhibited the agglutination of P1K erythrocytes by anti-P1 and unabsorbed anti-P1PPK sera, but neither antigen inhibited a specific anti-PK serum. The P1 and PK glycolipids were equally effective in inhibiting the hemagglutinating activity of a lectin with alpha-galactosyl specificity obtained from ova of Salmo trutta. Anti-P sera were inhibited most effectively by human erythrocyte globoside, and to a lesser extent by Forssman glycolipid and rat kidney globoside. In the latter glycolipid the linkage between the internal galactosyl residues is alpha, 1 leads to 3, rather than alpha, 1 leads to 4, as in erythrocyte globoside. No cross-reactions between P and P1 or PK antigens were detected. New hypotheses are offered to explain the genetic regulation and biosynthesis of the P1, P, and PK antigens.     

Rearrangements of integral membrane components during in vitro aging of sheep erythrocyte membranes

In vitro aged sheep erythrocytes and sheep erythrocyte ghosts spontaneously release vesicles that consist of long protrusions affixed to flattened headlike structures. The intramembranous particles seen on the protoplasmic face of freeze fracture electron micrographs of vesicle protrusions are arranged in paired particle rows. On the equivalent fracture face of headlike structures, the particle density is low; if particles are present, they are clustered along the rim of the flattened headlike structure and at the junction with the protrusion. The released vesicles are depleted of the intramembranous particles seen on the exoplasmic face of ghost but retain almost exclusively particles of the protoplasmic face. Correspondingly, the exoplasmic face of ghosts that have released vesicles reveals a 28 percent higher density of intramembranous particles than that of fresh ghosts. Purified vesicles are depleted of spectrin but retain integral membrane proteins, with one of an apparent mol wt of 160,000 accounting for nearly 50 percent of the total protein (Lutz, H.U.,R. Barber, and R.F. McGuire. 1976. J. Biol. Chem. 251:3500-3510). When vesicles are modified with the cleavable cross-linking reagent [(35)S]dithiobis (succinimidyl propionate)at 0 degrees C, the 160,000 mol wt protein is rapidly converted to disulfide-linked dimers and higher oligomers. Exposure of intact ghosts to the reagent in the same way fails to yield equivalent polymers. A comparison of the morphological and biochemical aspects of ghosts and vesicles suggest that a marked rearrangement of membrane proteins accompanies the supramolecular redistribution of intramembranous particles during spontaneous vesiculation. The results also suggest that the paired particles of the protoplasmic face of vesicle protrusions are arranged in paired helices and contain the 160,000 mol wt protein as dimers.     

Intramembranous particles on freeze-fractured membrane replica and sulfhydryl groups

The correlated distribution of intramembranous particles and sulfhydryl groups was examined in normal adult rabbit erythrocyte ghosts. Ghosts were treated to exhibit characteristic patterns of particle distribution and sulfhydryl groups were stained with Fast Blue BBN. It was shown that particles and sulfhydryl groups were located in corresponding patterns. The results indicate that the intramembranous particles in the erythrocyte membrane consist predominantly of protein.     

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.     

Structural and cytochemical differentiation of membrane elements of the apical membrane of amphibian urinary bladder epithelial cells. A label fracture study

It is now generally accepted that ADH-induced increase in water permeability in responsive epithelia is associated with the insertion of specific structures in the apical membrane of epithelial cells. Up to now, these structures have only been recognized in freeze-fractured preparations and their chemical nature is still unknown. In this study, we used the label-fracture method (Pinto da Silva and Kan, J. Cell Biol., 99, 1156-1161, 1984) to investigate the distribution of wheat germ agglutinin (WGA) on the luminal plasma membrane of freeze-fractured frog urinary bladder epithelial cells. With label-fracture, the cytochemical markers are seen superimposed with the conventional high resolution image of the E face. Label-fracture of tissue treated for 15 min with WGA and subsequently labeled with colloidal gold coated with ovomucoid showed uniform distribution of gold particles along the exoplasmic fracture face. Stereomicrographs show that the gold label is under the fracture face as it is attached to the outer surface of the membrane. Preincubation of the bladder with WGA for 3 hr induced a segregation of the intramembranous particles of the apical plasma membrane. In this condition, we observed a co-distribution of WGA-gold complexes with the segregated particles on the E face. This indicates that WGA-binding sites are located on glycoproteins which probably comprise the large intramembranous particles dispersed on the exoplasmic faces of freeze-fractured luminal membranes. In contrast, the numerous small intramembrane particles observed on P faces remained evenly distributed even after exposure to WGA and are, therefore, unrelated to WGA receptor sites. After WGA treatment, ADH still induced the formation of aggregates inside the smooth domains. A few WGA-binding sites appeared to be associated to these aggregates.     

Intramembranous particles on freeze-fractured membrane replica and sulfhydryl groups

Summary The correlated distribution of intramembranous particles and sulfhydryl groups was examined in normal adult rabbit erythrocyte ghosts. Ghosts were treated to exhibit characteristic patterns of particle distribution and sulfhydryl groups were stained with Fast Blue BBN. It was shown that particles and sulfhydryl groups were located in corresponding patterns. The results indicate that the intramembranous particles in the erythrocyte membrane consist predominantly of protein.Supported by grants from the Japanese Government, Nos. 244016 and 337001     

Isolation, characterization, and biosynthesis of Forssman antigen in human lung and lung carcinoma

A glycolipid was isolated from normal lung and lung carcinoma tissues. This glycolipid was identified as the Forssman antigen of pentaglycosyl ceramide by means of chemical and immunological methods. The presence of this antigenic glycolipid was observed in all the tissues examined of adult and embryo lungs, and of lung tumors irrespective of histological type. The extracts of human lung and lung tumors were capable of catalyzing the synthesis of Forssman antigen from globoside.     

Structures of unique globoside elongation products present in erythrocytes with a rare NOR phenotype

Rare polyagglutinable erythrocytes of NOR phenotype were found to contain two unique glycosphingolipids (designated NOR1 and NOR2). These components (not detected in normal erythrocytes) were reactive with Griffonia simplicifolia isolectin IB4 (GSL-IB4) and commonly present human anti-NOR antibodies. The NOR1 component has been reported to be a globoside containing a single galactose residue linked alpha1,4 to the terminal N-acetylgalactosamine. Here, we report the structural studies on a second glycolipid, NOR2, and a third novel component migrating in high-performance thin-layer chromatography (HPTLC) between NOR1 and NOR2. The structures were determined by a combination of ion trap sequential mass spectrometry (MALDI-QIT-TOF) and step-wise treatment with glycosidases, followed by identification of products on HPTLC plates with lectins and mouse monoclonal anti-NOR antibody. The NOR2 component was found to be a disaccharide extension of NOR1 with the following structure: Galalpha1-4GalNAcbeta1-3Galalpha1-4GalNAcbeta1-3Galalpha1-4Galbeta1-4Glcbeta1-Cer. Treatment of NOR2 with alpha-galactosidase gave a glycolipid migrating between NOR1 and NOR2, which did not react with either GSL-IB4 or anti-NOR antibodies but did react with GalNAc-specific soybean agglutinin. This intermediate glycolipid (now designated NOR(int)) was identified as a relatively abundant component of a neutral glycolipid fraction from NOR erythrocytes, suggesting its presence as a precursor to NOR2. The structure of NOR(int) was also confirmed by sequential mass spectrometry studies. These results indicate that polyagglutination in NOR subjects is due to unique erythrocyte glycolipids that are synthesized by sequential addition of Galalpha1,4 and GalNAcbeta1,3 to globoside.     

Bilayer/cytoskeleton interactions in lipid-symmetric erythrocytes assessed by a photoactivable phospholipid analogue

Two mechanisms have been proposed for maintenance of transbilayer phospholipid asymmetry in the erythrocyte plasma membrane, one involving specific interactions between the aminophospholipids of the inner leaflet of the bilayer and the cytoskeleton, particularly spectrin, and the other involving the aminophospholipid translocase. If the former mechanism is correct, then erythrocytes which have lost their asymmetric distribution of phospholipids should display altered bilayer/cytoskeleton interactions. To test this possibility, normal erythrocytes, erythrocytes from patients with chronic myelogenous leukemia or sickle disease, and lipid-symmetric and -asymmetric erythrocyte ghosts were labeled with the radioactive photoactivable analogue of phosphatidylethanolamine, 2-(2-azido-4-nitrobenzoyl)-1-acyl-sn-glycero-3-phospho[14C]ethanolamine ([14C]AzPE), previously shown to label cytoskeletal proteins from the bilayer. The labeling pattern of cytoskeletal proteins in pathologic erythrocytes and lipid-asymmetric erythrocyte ghosts was indistinguishable from normal erythrocytes, indicating that the probe detects no differences in bilayer/cytoskeleton interactions in these cells. In contrast, in lipid-symmetric erythrocyte ghosts, labeling of bands 4.1 and 4.2 and actin, and to a lesser extent ankyrin, by [14C]AzPE was considerably reduced. Significantly, however, labeling of spectrin was unaltered in the lipid-symmetric ghosts, suggesting that its relationship with the bilayer is normal in these lipid-symmetric cells. These results do not support a model in which spectrin is involved in the maintenance of an asymmetric distribution of phospholipids in erythrocytes.     

Subpopulations of B cells in germinal centers. III. HJ6, a monoclonal antibody, binds globoside and a subpopulation of germinal center B cells.

To identify surface Ag uniquely expressed on human germinal center B cells, we produced a mouse mAb, HJ6. When tonsillar lymphocytes were examined, HJ6 did not label T cells and labeled only about half of PNA+ B cells that were HK23-. HJ6 did not label mononuclear cells from peripheral blood, splenocytes, and any of 29 cell lines including 23 B cell lines. This binding pattern of HJ6 was very similar to that of a mAb named 5B5. It was shown previously that 5B5 bound a glycolipid named CTH (CD77) and its Ag was expressed on HK23- PNA+ tonsillar lymphocytes and Burkitt's lymphoma cell lines. Despite the similarity, HJ6 differed from 5B5: HJ6 did not stain Burkitt's lymphoma cell lines and stained PNA+ tonsillar lymphocytes in the presence of a large concentration of galactose. When its binding to isolated glycolipids was studied, HJ6 was found to bind globoside and Forssman Ag and not to other glycolipids including CTH. When its binding to neutral glycolipids extracted from tonsillar lymphocytes was studied, HJ6 bound only globoside; Forssman Ag was not detected in tonsillar lymphocytes. Taken together, we conclude that globoside is a B cell Ag expressed on a subpopulation of germinal center B cells.     

Correlation between the oscillatory and adhesion activities in erythroid cells

The attachment kinetics of erythroid cells, such as human erythrocytes, their saponin ghosts, and erythroleukemic cells K562 to a glass surface has been studied in the presence of substances inhibiting spontaneous fluctuations of cell membranes. It has been shown that wheat germ agglutinin (WGA) slows down the attachment kinetics of K562 cells, as is the case in intact erythrocytes. Concanavalin A (Con A), which inhibits the attachment of erythrocytes to glass does not affect the adhesion of K562 cells to glass due to the absence of band 3 proteins in the membranes of K562 cells. Both lectins slow down the adhesion rate of saponin ghosts of human erythrocytes, as it takes place in intact erythrocytes. Suramin and the anionic dye ANS bind specifically to the actin protofilaments of the erythrocyte skeleton and also inhibit cell adhesion to glass. At the same time, these substances do not affect the oscillatory and adhesion activities of intact erythrocytes due to the impermeability of erythrocyte membranes for these drugs. The results obtained allow the conclusion that inhibition of erythrocyte adhesion by lectins is due to lectin binding to different constituents of the erythrocyte membrane--sialic acid moieties of glycophorin in the case of WGA and band 3 proteins in the case of Con A. The most probable mechanism of erythrocyte and K562 cell attachment to glass is the formation of the so-called local contacts between cells and the glass surface. It is also suggested that the cell surface oscillations facilitate the formation of cell contacts.