Isolation of membrane glycoproteins by affinity chromatography in the presence of detergents

Wheat germ agglutinin has been used in a one-step preparative method to isolate the major sialoglycoprotein (glycophorin A) from the human erythrocyte membrane. The conditions for isolation and purification of the sialoglycopeptide included low concentration of sodium dodecyl sulfate in the presence of relatively high salt concentration. This medium caused complete solubilization of the membrane but still allowed almost quantitative binding of the sialoglycopeptide to wheat germ agglutinin-Sepharose. The eluted protein from such affinity systems was found to be chemically comparable to glycophorin A, as prepared by other procedures.     

Preliminary X-ray diffraction results on co-crystals of wheat germ agglutinin with a sialoglycopeptide from the red cell receptor glycophorin A

Diffraction-quality crystals have been obtained for complexes of each of the major wheat germ agglutinin (WGA) isolectins with the tryptic sialoglycopeptide T-5 from the WGA red cell receptor glycophorin A. This octa-glycopeptide possesses a Thr-linked carbohydrate moiety (GalNAc(NeuNAc)-Gal-NeuNAc) with specificity for the WGA binding site. The crystals belong to the orthorhombic space group P2(1)2(1)2 and have unit cell dimensions: a = 112.2 A, b = 51.0 A, c = 63.5 A (isolectin 1); a = 109.0 A, b = 52.3 A, c = 62.4 A (isolectin 2). There are two monomer complexes in each asymmetric unit.     

Purification and characterization of anti-N lectin from Vicia unijuga leaves

1. An anti-N lectin was extracted from Vicia unijuga leaves with phosphate-buffered saline (PBS). Purification of the lectin was achieved, after pretreatment of the PBS extract by ammonium sulfate fractionation and absorption with human M erythrocytes, by using a combination of conventional chromatographic techniques with asialoglycophorin AN-Sepharose CL-4B affinity chromatography. Purification steps were followed by increase of specific activity. 2. Homogeneity of the purified lectin was demonstrated by HPLC and SDS-PAGE. The purified lectin was a glycoprotein with 11.4% carbohydrate and relatively high percentages of serine, threonine and aspartic acid residues and had a Mw of 120,000 Da. 3. This lectin agglutinated human N and MN erythrocytes, but did not agglutinate M erythrocytes. Hemagglutination of the lectin was inhibited by glycophorin AN and N-active sialoglycopeptide released from human N erythrocytes by treatment with Pronase or trypsin. However, it was not inhibited by any of mono- and di-saccharides, ABH-active glycoproteins, glycophorin AM and M-active sialoglycopeptide liberated from human M erythrocytes by treatment with Pronase or trypsin.     

Sialoglycopeptides from bovine milk fat globule membrane.

Milk fat globule membrane was shown to contain sialic acid, all of which could be released without disruption of the fat globule. Sialoglycopeptides were cleaved from the surface of intact fat globules by Pronase and fractionated on Sephadex G-50. Further fractionation of the major sialoglycopeptide peak on DEAE-Sephadex gave two groups of sialoglycopeptides eluted with 0.1 M NaCl (Group A) and 0.5 M NaCl (Group B), respectively. Refractionation gave a major sialoglycopeptide from each of the two groups together with a total of three minor sialoglycopeptides. All five sialoglycopeptides eluted as single peaks using shallow salt gradients on DEAE-Sephadex and contained a hydrophilic peptide chain together with galactose, mannose, N-acetylgalactosamine, N-acetylglucosamine, and sialic acid. Glycopeptides of Group A but not Group B contained fucose. The major sialoglycopeptide of Group B released 35% of its hexose and hexosamine on treatment with alkaline borohydride leaving a sialoglycopeptide which had reduced serine and threonine and elevated alanine levels and in addition contained 2-aminobutyric acid. An oligosaccharide fraction containing N-acetylgalactosaminitol, galactose and sialic acid in a molar ratio of 1:1:2 was partially characterised from the clevage mixture. The major sialoglycopeptide of Group A had a more complex carbohydrate structure and showed no released carbohydrate on treatment with alkaline borohydride. The sialoglycopeptides of milk fat globule membrane show many similarities with those of erythrocyte membrane and have a potential use in comparative and structural studies.     

Identification of a sialoglycopeptide released by self-digestion from human erythrocyte membranes.

Membranes from human O Rhesus-positive erythrocyte 'ghosts' were tested in vitro for their ability to digest their own glycoproteins. 'Ghost' membranes incubated in Tris/HCl buffer, pH 7.4, release a sialoglycopeptide, which contains glucosamine, galactosamine, galactose and mainly polar amino acids. Chemical composition, molecular size and aggregation properties suggest that this glycopeptide may be a fragment of glycophorin.     

Interactions between a membrane sialoglycoprotein and planar lipid bilayers

Summary Bilayer membranes formed from lipids dissolved in decane were exposed to glycophorin, a sialoglycoprotein which had been extracted from human red cell membranes. The interaction with the bilayer produced an increase in the steady state electrical conductance of the membrane proportional to the amount added. Fluctuations in membrane current when the electrical potential difference was constant were observed concommitantly with this increase in membrane conductance. The minimum size of the fluctuations corresponds to a conductance of 10^–10 mho. The increase in conductance as well as the current fluctuations persisted after extensive washout of the chamber containing the protein (cisside). Subsequent addition of lectins (wheat germ agglutinin and phytohemoagglutinin) to the cis-side produced rupture of the membranes, whilst these hemoagglutinins added to the trans-side failed to produce an effect. Measurements of changes in surface potential using K⁺ nonactin as a probe indicated that glycophorin induces a negative surface charge. At high protein concentrations, the magnitude of the induced surface potential became independent of glycophorin concentration. The maximum number of charges introduced onto the membrane under these conditions was 1.4×10⁵/m². Cis (but not trans)-side addition of neuraminidase abolished these charges, indicating that they can be ascribed to the sialic acid residues that the protein bears. These results suggest that glycophorin incorporates into bilayer membranes with its N-terminal end (where the sialic acid and carbohydrates are located) facing the cis-side. Spectrin reversibly lowered the glycophorin-induced membrane conductance when added to the trans-side. Cis-side additions failed to produce an effect. Trypsin present on the trans-side irreversibly lowered the membrane conductance. These results indicate that parts of the glycophorin molecule, probably the C-terminal end, are accessible to reagents in the solution bathing the trans-side of the membrane. Thus glycophorin spans the planar bilayer in much the same way as it spans the red cell membrane.     

Purification and Characterization of a Bovine Cerebral Cortex Cell Surface Sialoglycopeptide that Inhibits Cell Proliferation and Metabolism

A sialoglycopeptide from bovine cerebral cortex cells was purified to apparent homogeneity by a procedure that included chloroform/methanol extraction, diethylaminoethyl ion exchange chromatography, wheat germ agglutinin affinity chromatography, size-exclusion HPLC, and hydrophobic interaction chromatography. The cell surface inhibitor had a molecular weight of approximately 18,000, no subunit composition was detectable on reduction and polyacrylamide gel electrophoresis analysis, and the glycopeptide apparently contained sialic acid, as illustrated by its ability to bind to Limulus polyhemus lectin. Deglycosylation of the molecule, however, did not reduce its protein synthesis inhibitory activity. As little as 20 ng of the sialoglycopeptide was capable of inhibiting protein synthesis in a wide variety of fibroblast cell lines but not in transformed cells. Mice immunized with the sialoglycopeptide produced antibodies that, when bound to protein A-agarose gel, removed the inhibitory activity from solution. The antibodies were used to identify a single isoelectric focused band and to establish the pI of 3.0 for the molecule.     

Sialoglycopeptides from bovine milk fat globule membrane

Milk fat globule membrane was shown to contain sialic acid, all of which could be released without disruption of the fat globule. Sialoglycopeptides were cleaved from the surface of intact fat globules by Pronase and fractionated on Sephadex G-50. Further fractionation of the major sialoglycopeptide peak on DEAE-Sephadex gave two groups of sialoglycopeptides eluted with 0.1 M NaCI (Group A) and 0.5 M NaCI (Group B), respectively. Refractionation gave a major sialoglycopeptide from each of the two groups together with a total of three minor sialoglycopeptides. All five sialoglycopeptides eluted as single peaks using shallow salt gradients on DEAE-Sephadex and contained a hydrophilic peptide chain together with galactose, mannose, N-acetylgalactosamine, N-acetylglucosamine, and sialic acid. Glycopeptides of Group A but not Group B contained fucose.The major sialoglycopeptide of Group B released 35 % of its hexose and hexosamine on treatment with alkaline borohydride leaving a sialoglycopeptide which had reduced serine and threonine and elevated alanine levels and in addition contained 2-aminobutyric acid. An oligosaccharide fraction containing N-acetylgalactosaminitol galactose and sialic acid in a molar ratio of 1 : 1 : 2 was partially characterised from the cleavage mixture.The major sialoglycopeptide of Group A had a more complex carbohydrate structure and showed no released carbohydrate on treatment with alkaline borohydride.The sialoglycopeptides of milk fat globule membrane show many similarities with those of erythrocyte membrane and have a potential use in comparative and structural studies.     

Isolation of the major intrinsic transmembrane protein of the human erythrocyte membrane.

The major intrinsic protein of the human erythrocyte membrane commonly referred to as "Band 3", was isolated by a multi-step procedure. Extraction of ghost membranes in dilute solutions of lithium diiodosalicylate removed most of the proteins considered to be extrinsic to the membrane. The resulting membrane fragments were solubilized in sodium dodecyl sulfate, and the major sialoglycoprotein (glycophorin A) was removed by wheat germ agglutinin-Sepharose affinity chromatography. Gel filtration in sodium dodecyl sulfate was used as the final step to yield the band 3 polypeptide in electrophoretically homogeneous form.     

Interaction of sialoglycoproteins with wheat germ agglutinin-sepharose of varying ratio of lectin to Sepharose. Use for the purification of mucin glycoproteins from membrane extracts

The influence of varying the amount of wheat germ agglutinin immobilized on Sepharose beads on the binding of glycoproteins to these beads was investigated. A series of wheat germ agglutinin-Sepharose gels containing between 0.10 and 10.0 mg of lectin/ml of gel was prepared, and the actual lectin content was established by acid hydrolysis of the gel followed by analysis of glycine, a major amino acid in wheat germ agglutinin. Affinity chromatography of labeled glycoproteins indicated that glycophorin bound to all the wheat germ agglutinin-Sepharose preparations. Fetuin, ovomucoid, and alpha 1-acid glycoprotein bound not at all or very poorly to gels with a low content of wheat germ agglutinin (less than 0.95 mg/ml). The specific binding of these glycoproteins increased with increasing lectin content on the gels, and on gels of high content (greater than 3 mg/ml) the binding was virtually quantitative. On chromatographing a mixture of glycophorin, alpha 1-acid glycoprotein, fetuin, and ovomucoid on wheat germ agglutinin-Sepharose, containing 0.08 mg of lectin/ml of gel, glycophorin was selectively retained on the gel. It was possible to purify glycophorin from an extract of human erythrocyte membranes in one step by chromatography on the above gel. By using the series of gels, it was demonstrated that Morris hepatoma 7777 membranes contained at least 4-fold more sialoglycoproteins which bound to low density wheat germ agglutinin-Sepharose compared to rat liver membranes. These hepatoma sialoglycoproteins were isolated, purified, and partially characterized as having a high proportion of O-linked sialyloligosaccharides. Our studies illustrate the use of low density wheat germ agglutinin-Sepharose gels both for the detection and for easy isolation of mucin-type glycoproteins from crude extracts of cells or membranes.     

Influence of glycophorin incorporation on Ca2+-induced fusion of phosphatidylserine vesicles

The effect of incorporation of glycophorin, the major integral sialoglycoprotein of the erythrocyte membrane, into bovine brain phosphatidylserine (PS) vesicles on the Ca2+-induced fusion of these vesicles has been investigated. Fusion was monitored by the terbium-dipicolinic acid fluorescence assay for the mixing of aqueous contents of the vesicles and by a resonance energy transfer assay that follows the intermixing of membrane lipids. The Ca2+-induced fusion of PS vesicles is completely prevented by incorporation of glycophorin (molar ratio of PS/glycophorin = 400-500:1) for Ca2+ concentrations up to 50 mM. The ability to fuse is partially restored after treating the glycophorin-containing vesicles with neuraminidase, which removes the negatively charged sialic acid residues of glycophorin. Fusion is further facilitated by trypsin treatment, removing the entire extravesicular glycosylated head group of glycophorin. However, Ca2+-induced fusion of enzyme-treated glycophorin-PS vesicles proceeds at a slower rate and to a smaller extent than fusion of protein-free PS vesicles. The influence of the aggregation state of the glycophorin molecules on fusion has been investigated in experiments using wheat germ agglutinin (WGA). Addition of WGA to the glycophorin-PS vesicles does not induce fusion. However, upon subsequent addition of Ca2+, distinct fusion occurs concomitantly with release of vesicle contents. The inhibition of Ca2+-induced fusion of PS vesicles by incorporation of glycophorin is explained by a combination of steric hindrance and electrostatic repulsion between the vesicles by the glycosylated head group of glycophorin and a direct bilayer stabilization by the intramembranous hydrophobic part of the glycophorin molecule.     

Lectin-mediated agglutination of liposomes containing glycophorin: Effects of acyl chain length

Glycophorin from human erythrocytes has been incorporated into liposomes of dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC) and distearoylphosphatidylcholine (DSPC). The thermal properties of unsonicated liposomes with glycophorin/lipid molar ratios up to 4·10^?3 have been studied by differential scanning calorimetry and the numbers of lipids withdrawn from participation in the gel-to-lamellar phase transition were found to be 42±22 (DMPC), 197±28 (DPPC) and 240±64 (DSPC). The initial rates of agglutination of sonicated liposomes with glycophorin/lipid molar ratios up to 4·10^?3 by wheat germ agglutinin in the concentration range 0–7 μM have been measured over a range of temperature. Below the gel-to-lamellar phase transition (T_c) the rates of agglutination increase with acyl chain length in the sequence DMPC < DPPC < DSPC. Agglutination is found to be second order in liposome concentration and is completely reversed on saturation of the wheat germ agglutinin-binding sites by N-acetylglucosamine. Agglutination rates decrease with increasing temperature below T_c and are largely independent of temperature above T_c. The results are discussed in relation to the clustering of glycophorin in the phospholipid bilayers and its effect on binding and subsequent interliposomal bridge formation by wheat germ agglutinin.     

Control of high affinity lectin binding to an integral membrane glycoprotein in lipid bilayers

High affinity binding of wheat germ agglutinin to glycophorin is demonstrated to be potently affected by non-specific interaction of the receptor with other protein and oligosaccharide structures present at the membrane surface. It is suggested that this may represent a significant general mechanism of receptor control.     

Modification of glycophorin A during oxidation of erythrocyte membrane

Human erythrocyte ghosts were oxidized with tert-butyl hydroperoxide and subsequently treated with tritiated borohydride to label the membrane proteins modified during the membrane oxidation. From the ghosts, oxidized-and-tritiated glycophorin A was isolated and characterized. No intermolecular cross-links were observed as analyzed by sodium dodecylsulfate gel electrophoresis. But, the number of lysine residues was significantly reduced and susceptibility to proteinases such as trypsin, chymotrypsin and pronase was lower than that of control glycophorin A. Trypsinization of the oxidized-and-tritiated glycophorin A gave insoluble and soluble trypsin fragments. After dansylation, N-terminal amino acids of the trypsin-fragments were determined. Dansyl amino acids from the insoluble trypsin fragments were not identical with those from control insoluble counterparts in the membrane-spanning region of glycophorin A molecule. Fractionation by gel filtration of dansyl-soluble trypsin fragments, and the N-terminal amino acid analysis of the fractionated peptides indicated that the peptides derived from the glycosylated region located in the outside of the membrane matrix were identical with those from control soluble counterparts. The results suggest that the glycosylated outside region of glycophorin A was modified only slightly but the hydrophobic membrane-spanning region was extensively modified during membrane oxidation, most likely by oxidized lipids.     

Schistosomula of Schistosoma mansoni use lysophosphatidylcholine to lyse adherent human red blood cells and immobilize red cell membrane components

Human red blood cells (RBCs) adhere to and are lysed by schistosomula of Schistosoma mansoni. We have investigated the mechanism of RBC lysis by comparing the dynamic properties of transmembrane protein and lipid probes in adherent ghost membranes with those in control RBCs and in RBCs treated with various membrane perturbants. Fluorescence photobleaching recovery was used to measure the lateral mobility of two integral membrane proteins, glycophorin and band 3, and two lipid analogues, fluorescein phosphatidylethanolamine (Fl-PE) and carbocyanine dyes, in RBCs and ghosts adherent to schistosomula. Adherent ghosts manifested 95-100% immobilization of both membrane proteins and 45-55% immobilization of both lipid probes. In separate experiments, diamide-induced cross-linking of RBC cytoskeletal proteins slowed transmembrane protein diffusion by 30-40%, without affecting either transmembrane protein fractional mobility or lipid probe lateral mobility. Wheat germ agglutinin- and polylysine-induced cross-linking of glycophorin at the extracellular surface caused 80-95% immobilization of the transmembrane proteins, without affecting the fractional mobility of the lipid probe. Egg lysophosphatidylcholine (lysoPC) induced both lysis of RBCs and a concentration-dependent decrease in the lateral mobility of glycophorin, band 3, and Fl-PE in ghost membranes. At a concentration of 8.4 micrograms/ml, lysoPC caused a pattern of protein and lipid immobilization in RBC ghosts identical to that in ghosts adherent to schistosomula. Schistosomula incubated with labeled palmitate released lysoPC into the culture medium at a rate of 1.5 fmol/h per 10(3) organisms. These data suggest that lysoPC is transferred from schistosomula to adherent RBCs, causing their lysis.     

A re-examination of the effects of chymotrypsin and trypsin on the erythrocyte membrane surface topology

Silver/Coomassie blue staining of human erythrocyte membrane electrophoretograms permits simultaneous visualization and color differentiation of asialoproteins, sialoglycoproteins and lipids in the same gel. Using this technique evidence is provided that chymotrypsin cleaves glycophorin A as well as band 3. The chymotryptic fragmentation pattern of glycophorin A in situ intact cells was different from that generated by trypsin treatment. Chymotryptic cleavage of band 3 generated two Coomassie blue stained fragments at 62,000 and 38,000 Mr, whereas simultaneous cleavage of glycophorin A dimer and glycophorin A B heterodimer yielded yellow silver stained fragments at 68,000 and 47,000 Mr. Trypsin cleaved glycophorin A dimer (88,000 Mr) and monomer (38,000 Mr) to form membrane associated fragments of Mr = 40,000 and 18,000 respectively.     

Crystal structure of a wheat germ agglutinin/glycophorin-sialoglycopeptide receptor complex. Structural basis for cooperative lectin-cell binding.

The crystal structure of wheat germ agglutinin isolectin 1 (WGA1) complexed with a tryptic sialoglycopeptide fragment (T-5) from its erythrocyte receptor glycophorin A, which contains the O-linked tetrasaccharide NeuNAc-alpha 2,3-Gal-beta 1,3-(alpha 2,6-NeuNAc) Gal-NAc-alpha 1-O-Thr, has been determined by molecular replacement techniques and refined at 2.0-A resolution (R = 18.1%). The structure reveals that association between WGA1 dimers, composed of two identical four-domain (A-D) monomers, and T-5 is asymmetric and involves sialic acid binding at three nonequivalent aromatic residue-rich sites. Two independent binding modes are observed. In the dominant ("major") binding mode, the two highest affinity sites are utilized to cross-link neighboring crystallographically related WGA1 dimers. The branched tetrasaccharide has an extended rigid conformation, and its terminal alpha 2,6-NeuNAc and alpha 2,3-NeuNAc residues occupy specificity sites in domains B1 (monomer 1) and C2 (monomer 2) on opposing dimers, respectively. This asymmetric selection of binding sites leads to infinite open-ended arrays of interlinked lectin molecules. In the subsidiary "minor" binding mode, only the terminal alpha 2,6-NeuNAc, anchored to the aromatic residue-rich binding site in domain A2, is clearly visible. The remaining portion of T-5 is disordered. This structure presents the first evidence for NeuNAc binding in the aromatic residue-rich sites of domains A and C and suggests a preference of WGA for alpha 2,6-linked NeuNAc. Moreover, the unusual asymmetric WGA1-tetrasaccharide association, involving domain binding sites that differ in their binding affinities for NeuNAc, offers explanations for the widely observed cooperative cell binding behavior of WGA.     

Structural analysis of glycophorin A from Miltenberger class VIII erythrocytes

The major human erythrocyte membrane sialoglycoprotein (glycophorin A or MN glycoprotein) was purified from the erythrocytes of two individuals heterozygous for the Mi-VIII gene in the Miltenberger subsystem of the MNSs blood-group system. The complete structure of a tryptic glycopetide from glycophorin A comprising the residues 40-61 was deduced from automated and manual sequence analyses. The Mi-VIII-specific glycophorin A was found to exhibit an arginine----threonine exchange at position 49. The threonine residue was found to be glycosylated. Hemagglutination and hemagglutination inhibition assays demonstrated that one of the Mi-VIII-characteristic antigenic determinants (Anek) is located within the residues 40-61 of glycophorin A. Furthermore, erythrocytes from the two Mi-VIII heterozygotes reacted only weakly with anti-EnaKTsera, suggesting that the Mi-VIII-specific glycophorin A does not express the EnaKT antigen that is located within the positions 46-56 of normal glycophorin A. Our data suggest that the Mi-VIII-specific glycophorin A represents the evolutionary link between normal glycophorin A and the Mi-VIII-specific molecule which exhibits arginine----threonine and tyrosine----serine exchanges at the positions 49 and 52, respectively. Our data also provide an explanation for the close serological similarity between Mi-VII and Mi-VIII erythrocytes.     

Loss of rotational mobility of band 3 proteins in human erythrocyte membranes induced by antibodies to glycophorin A.

The effect of antibodies to glycophorin A on the rotational diffusion of band 3 in human erythrocyte membranes was investigated by transient dichrosim. Three antibodies that recognize different epitopes on the exofacial domain of glycophorin A all strongly reduce the rotational mobility of band 3. The effect is at most only weakly dependent on the distance of the epitope from the membrane surface. The degree of immobilization obtained with two of the antibodies, BRIC14 and R18, is very similar to that produced by antibodies to band 3 itself. Similar results were obtained with membranes stripped of skeletal proteins. Fab fragments and an antibody to glycophorin C had no effect on band 3 rotational mobility. These results rule out a mechanism whereby band 3 rotational immobilization results from enhanced interactions with the membrane skeleton that are mediated by a conformational change in glycophorin A. Rather, they strongly indicate that the antibodies to glycophorin A cross-link existing band 3-glycophorin A complexes that have lifetimes that are long compared with the millisecond time scale of the transient dichroism measurements.     

Force profiles of protein pulling with or without cytoskeletal links studied by AFM

To test the capability of the atomic force microscope for distinguishing membrane proteins with/without cytoskeletal associations, we studied the pull-out mechanics of lipid tethers from the red blood cell (RBC). When wheat germ agglutinin, a glycophorin A (GLA) specific lectin, was used to pull out tethers from RBC, characteristic force curves for tether elongation having a long plateau force were observed but without force peaks which are usually attributed to the forced unbinding of membrane components from the cytoskeleton. The result was in agreement with the reports that GLA is substantially free of cytoskeletal interactions. On the contrary, when the Band 3 specific lectin, concanavalin A, was used, the force peaks were indeed observed together with a plateau supporting its reported cytoskeletal association. Based on these observations, we postulate that the state of cytoskeletal association of particular membrane proteins can be identified from the force profiles of their pull-out mechanics.