Purification and characterization of the major sialoglycoproteins of the rat erythrocyte membrane

The major sialoglycoproteins of the rat erythrocyte membrane were purified by hot phenol partitioning followed by cation-exchange chromatography on SP-Sephadex. Further purification was obtained by extraction with n-butanol and anion-exchange chromatography on DEAE-cellulose. The resulting sialoglycoprotein fraction was free of lipids and nonsialylated glycoproteins and gave rise to four major periodic acid-Schiff staining bands when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The fastest migrating protein on these gels with an apparent molecular weight of 19,000 was purified to homogeneity by gel filtration. The amino acid and sugar compositions of these materials are reported. The protein moiety is rich in serine, threonine, and hydrophobic amino acids and the carbohydrate moiety is high in sialic acid and N-acetylgalactosamine. Most of the carbohydrate is linked O-glycosidically to serine and threonine residues, as shown by susceptibility to base-catalyzed β-elimination and concomitant reduction of serine and threonine to alanine and α-aminobutyric acid and of N-acetylgalactosamine to N-acetylgalactosaminitol in the presence of reducing agents. The significance of these data in light of the known role of the rat erythrocyte membrane sialoglycoproteins in erythropoiesis is discussed. The properties of the rat erythrocyte membrane sialoglycoproteins are compared to those of other species.     

Isolation and partial characterization of the sialoglycoprotein fraction of murine erythrocyte ghosts

With the lithium diiodosalicylate (LIS1) extraction-phenol partition method, we have isolated a sialoglycoprotein fraction from DBA/2 mouse erythrocyte ghosts. We have demonstrated that the Laemmeli system for SDS PAGE can resolve this fraction into four monomers of which two (gp- 2.1 and gp-3.1) appear to be authentic, whereas the other two (gp-2.2 and gp-3.2) are probably generated from gp-2.1 and gp-3.1, by limited proteolysis during the isolation procedure. All four components contain O-acetylated neuraminic acid residues, can be stained with Periodic acid-Schiff reagent (PAS) and with Coomassie Brilliant Blue (CB), and can be radioiodinated with the lactoperoxidase-glucose oxidase (LPO-GO) method. All monomers but especially gp-2.1 and gp-3.1 generate characteristic aggregates during solubilization in SDS. The aggregation is enhanced by boiling at high concentrations, and can be reversed by boiling at low concentrations. In addition, the fraction contains a diffuse component present also in ghosts which stains poorly with CB and with PAS and cannot be radioiodinated by the LPO-GO technique. SDS PAGE in the Steck and Yu gel system does not give an accurate separation of the sialoglycoprotein monomers.     

Selective radioactive labeling of cell surface sialoglycoproteins by periodate-tritiated borohydride.

Low concentrations of sodium metaperiodate induce specific oxidative cleavage of sialic acids between carbon 7 and carbon 8 or carbon 8 and carbon 9. The aldehydes formed can easily be reduced with NaB3H4 to tritiated 5-acetamido-3,5-dideoxy-L-arabino-2-heptulosonic acid or 5-acetamido-3,5-dideoxy-L-arabino-2-octulosonic acid. At 0 degrees, the periodate anion penetrates the cell plasma membrane very slowly and only externally exposed sialic acids are oxidized. This was shown by (a) limited labeling of the sialoglycoproteins in a preparation of inside-out erythrocyte vesicles; (b) trapping 14C-labeled fetuin within resealed erythrocyte ghosts; fetuin was then poorly labeled, whereas the erythrocyte sialoglycoproteins were highly labeled; (c) comparison of labeled glycoproteins of mouse lymphoid cells before and after treatment with neuraminidase. This simple method of specifically introducing a radioactive label into cell surface sialic acids is useful in the study of cell surface sialic acid-containing glycoproteins.     

Analytical detection of 9(4)-O-acetylated sialoglycoproteins and gangliosides using influenza C virus.

The unique glycoprotein of influenza C virus, designated hemagglutinin (HEF), exhibits three functions: hemagglutination, esterase activity, and fusion factor. As the virus uses 9-O-acetylated sialic acid as a high-affinity receptor determinant for attachment to cells, its binding activity was used to reveal O-acetylated sialic acid residues after polyacrylamide gel electrophoresis and transfer onto nitrocellulose sheets of proteins and thin-layer chromatography of lipids. The specificity of the binding for O-acetylated sialoglycoconjugates was investigated. Our results showed that influenza C virus could detect the different forms of the two murine glycophorins which are known to be O-acetylated sialoglycoconjugates. The virus also bound to O-acetylated gangliosides isolated from embryonic chicken brain such as purified O-acetylated NeuAc alpha (2-8)NeuAc alpha (2-8)NeuAc alpha (2-3)Gal beta (1-4)Glc beta (1-1)ceramide (GT3). The esterase activity of the HEF protein of influenza C virus was used to unmask the sialic acid. After its deacetylation by the virus enzyme, the O-acetylated GT3 was recognized by a monoclonal antibody which binds only to the nonacetylated derivative. The results presented here show that influenza C virus is a discriminating analytical probe for identifying O-acetylated sialoglycoconjugates directly after Western blotting of proteins and thin-layer chromatography of lipids, thus providing a new analytical tool.     

In vitro labeling of the sialic acid moiety of glycoconjugates with carbon-14

Labeling of sialoglycoproteins with carbon-14 in vitro was performed by reacting the aldehyde groups, generated by mild periodate oxidation of the terminal sialyl groups, with 14C-labeled sodium cyanide to produce the labeled cyanohydrin derivatives (Kiliani reaction). Labeling with tritium was carried out by reduction of the aldehyde groups generated on the sialyl residues with 3H-labeled sodium borohydride following standard procedures. The behavior of both types of labeled specimens of fetuin and ovine submaxillary mucin, individually and in mixtures, was investigated by gel-filtration chromatography, gel electrophoresis, and cesium bromide gradient ultracentrifugation. The labeled sialyl residues were subjected to partial characterization: color yield with the resorcinol and thiobarbituric acid reagents, behavior on ion-exchange chromatography, and susceptibility to mild acid and enzymatic hydrolyses. In addition to these model glycoproteins, this procedure was also utilized to label the sialoglycoproteins present in human tracheobronchial secretions collected from normal subjects and patients with chronic bronchitis. The potential uses of this approach for comparative studies of normal and pathological sialoglycoconjugates available in minute amounts is described. The extension of this approach to the labeling of the galactosyl and N-acetylgalactosaminyl moieties of glycoconjugates following treatment with galactose oxidase is outlined.     

Isolation of murine sialoglycoprotein using consecutive chromatography

Affinity columns and high performance liquid chromatography were employed consecutively to obtain 89, 65, 46 and 29 kilodalton sialoglycoproteins from mouse erythrocyte ghosts free of the Band 3 protein which traditionally co-purifies with these proteins. The purification scheme involves Concanavalin A, Wheat Germ Agglutinin and/or Limulus lectin Sepharose 4B columns. We have designated these glycophorin-like proteins Sialoglycoproteins 1, 2, 3, and 4, respectively. Sialoglycoprotein 2 can be isolated independently using a Limulus column combination, while Sialoglycoproteins 3 and 4 were isolated separately during high performance liquid chromatography, demonstrating heterogeneity in binding properties between these sialoglycoproteins.     

Solubilization of the binding protein from Ehrlich ascites cells and erythrocytes to Pseudomonas aeruginosa cytotoxin.

The binding protein for pore-forming Pseudomonas aeruginosa cytotoxin was solubilized from Ehrlich ascites cell plasma membranes and rabbit and bovine erythrocyte ghosts using nonionic and zwittergent detergents. Analysis of solubilized plasma membranes from Ehrlich cells by a ligand-blot technique after separation by SDS-PAGE/electrophoretic transfer to nitrocellulose or affinity chromatography showed a protein of 70 kDa molecular mass, which binds to cytotoxin. The binding protein solubilized from rabbit erythrocyte ghosts showed a molecular mass of 50 kDa and that from bovine ghosts 55 kDa according to the former test. The binding proteins could be characterized as acidic. They contain a glycan moiety which is, however, not involved in the interaction of cytotoxin with the binding site.     

Cell-free synthesis of myosin by cardiac myofibrillar ribosomes

Human erythrocyte ghosts were treated with a bifunctional cross-linking reagent, dimethyl adipimidate dihydrochloride. On SDS-polyacrylamide electrophoresis of the cross-linked membrane proteins after solubilization, sialoglycoproteins and the proteins disappeared from the original band positions and appeared in a new band of aggregates.     

Lectin-mediated agglutination of erythrocyte ghost membranes following depletion of membrane protein and intramembrane particles

Profound digestion of unsealed human erythrocyte ghosts with high concentrations of Pronase results in a near complete loss of intramembrane particles while trypsin digestion is less effective. The small vesicles formed by proteolysis are agglutinable by soybean agglutinin (SBA), wheat germ agglutinin (WGA), and phytohemagglutinin (PHA), but not concanavalin A (ConA). Densitometer tracings of Pronase-treated vesicles analyzed on SDS-polyacrylamide gels demonstrated no detectable protein or glycoprotein migrating slower than the marking dye. The vesicles showed a loss of 90% Lowry positive material (the remainder may be non-protein chromogens), near depletion of sialyl residues, no significant change in lipid composition, and equal amounts of phospholipid phosphorus compared to an equal volume of ghosts. The lipid material extracted from Pronase-derived vesicles or intact ghosts inhibited hemagglutination with SBA and WGA but not ConA. SBA but not ConA was found to specifically bind to Pronase-derived vesicles while both lectins bound to native ghosts. These observations suggest that neither the integrity of the intramembrane particles nor the presence of membrane glycoprotein appears essential for SBA-, WGA-, and PHA-mediated agglutination. Furthermore, it appears that native membrane glycolipids (and perhaps glycopeptides) can bind SBA, WGA and PHA. The membrane glycolipids may play a larger role than heretofore realized in lectin-mediated agglutination of cells.     

Identification of differences between the surface proteins and glycoproteins of normal mouse (Balb/c) and human erythrocytes

Summary The topography of the external surface of the Balb/c mouse erythrocyte has been investigated and compared to the human erythrocyte by using a series of protein radiolabeling probes. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the pattern of Coomassie Blue stained proteins was very similar for mouse and human erythrocyte ghosts, as was the distribution of radioactivity in protein bands after lactoperoxidase catalyzed radioiodination. The mouse erythrocyte glycoproteins identified by periodic-acid-Schiff and Stains-All reagents, sialic acid analysis of gel slices, binding of¹²⁵I-wheat germ agglutinin and¹²⁵I-concanavalin A to the gels, and glycoprotein radiolabeling techniques, differed markedly from the sets of proteins labeled by radioiodination, and also differed from the human erythrocyte glycoproteins. Instead of the PAS I to PAS IV series of sialoglycoproteins characteristic of human erythrocytes, the mouse erythrocyte possesses a broad band of sialoglycoproteins with several peaks ranging in mol wt from 65,000 to 32,000. The same group of sialoglycoproteins were labeled by the periodate/B³H₄ ^– technique specific for terminal sialic acid, and the galactose oxidase/B³H₄ ^– method (plus neuraminidase) specific for galactosyl/N-acetylgalactosaminyl residues penultimate to sialic acid. These results emphasize the necessity to employ a variety of protein radiolabeling probes based on different labeling specificities, to study the membrane topography of cells which are poorly understood compared to the human erythrocyte membrane.     

Distribution of sialic acid between sialoglycoproteins and other membrane components of different erythrocyte phenotypes.

The sialic acid content of erythrocytes of three different AB0 blood groups have been studied. The sialic acid contents of erythrocyte membranes containing 300 mg protein were determined and compared. Groups 0 (Rhesus negative), AB (both Rhesus negative and positive), and B (Rhesus negative) blood differed significantly (p less than 0.05) in total sialic acid content and in the distribution of sialic acid between sialoglycoproteins and other membrane components. Membrane materials containing 300 mg total protein showed sialic acid contents of 52.73 +/- 2.2 mumol sialic acid for group 0 (Rhesus negative) 34.77 +/- 1.16 mumol for group AB (Rh negative), 32.88 +/- 1.52 mumol for AB (Rh positive) and 21.23 +/- 0.84 mumol for B (Rh negative). In group 0 (Rh. neg.) membranes 39.4 +/- 1.4% of the total sialic acid was associated with the sialoglycoproteins. The percentage of sialic acids associated with sialoglycoproteins in other erythrocyte membranes were 77.7 +/- 1.3% for group B, and 55.6 +/- 1.0% and 56.4 +/- 1.8% for group AB (Rh. negative) and (Rh. positive) respectively. The changes appear to be independent of the Rhesus grouping but dependent on the AB0 grouping since membranes of the two Rhesus types of group AB had identical total sialic acid and sialoglycoproteins sialic acids. The sialic acid densities in sialoglycoproteins also differed from one erythrocyte type to another. Group 0 (Rh. negative) membrane sialoglycoproteins had sialic acid density of 140.5 +/- 3.1 nmol/mg compared to 71.7 +/- 1.2 nmol/mg for group B and 128.1 +/- 2.2 and 124.5 +/- 4.0 nmol/mg for group AB Rhesus negative and Rhesus positive respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

The structure of glycophorins of animal erythrocytes

Glycophorins are red cell membrane sialoglycoproteins, which contain multipleO-linked oligosacchride chains and carry most of the cell surface sialic acid. Due to this high content of sialic acid the glycophorins are strongly stained with periodic acid-Schiff (PAS) reagent after sodium sodicylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The term glycophorin was proposed initially for human red cell sialoglycoproteins [1,2] and now it is also used for sialoglycoproteins in animal red cell membranes. Furthermore, similar glycoproteins of non-erythrocyte origin have also been identified and given the same name [3], although the terms leukosialin and sialophorin were proposed for a major sialoglycoproteins of human leukocytes [4,5]. In this article the term glycophorin will be used only for sialoglycoproteins existing in the erythrocyte membrane.Glycophorins of human erythrocytes, carrying blood group MN, Ss and other determinants, have been thoroughly studied and their properties described in several review articles [3,6,7,8]. The aim of this article is to summarize studies carriedout on the structure of non-human glycophorins, although some data concerning human glycophorins are included for comparative purposes.Abbreviations SDS-PAGE sodium dodecylsulphate-polycrylamide gel electrophoresis - PAS periodic acid-Schiff - LIS lithium diiodosalicylate     

Differential phosphorylation of band 3 and glycophorin in intact and extracted erythrocyte membranes

This report presents an analysis of the phosphorylation of human and rabbit erythrocyte membrane proteins which migrate in NaDodSO₄-polyacrylamide gels in the area of the Coomassie Blue-stained proteins generally known as band 3. The phosphorylation of these proteins is of interest as band 3 has been implicated in transport processes. This study shows that there are at least three distinct phosphoproteins associated with the band 3 region of human erythrocyte membranes. These are band 2.9, the major band 3, and PAS-1. The phosphorylation of these proteins is differentially catalyzed by solubilized membrane and cytoplasmic cyclic AMP-dependent and -independent erythrocyte protein kinases. Band 2.9 is present and phosphorylated in unfractionated human and rabbit erythrocyte ghosts but not in NaI- or dimethylmaleic anhydride (DMMA)-extracted membranes. These latter membrane preparations are enriched in band 3 and in sialoglycoproteins. The NaI-extracted ghosts contain residual protein kinase activity which can catalyze the autophosphorylation of band 3 whereas the DMMA-extracted ghosts are usually devoid of any kinase activity. However, both NaI- and DMMA-extracted ghosts, as well as Triton X-100 extracts of the DMMA-extracted ghosts, can be phosphorylated by various erythrocyte protein kinases. The kinases which preferentially phosphorylate the major band 3 protein are inactive towards PAS-1 while the kinases active towards PAS-1 are less active towards band 3. The band 3 protein in the DMMA-extracted ghosts can be cross-linked with the Cu²⁺ -σ-phenanthroline complex. The cross-linking of band 3 does not affect its capacity to serve as a phosphoryl acceptor nor does phosphorylation affect the capacity of band 3 to form cross-links. In addition to band 2.9, the major band 3 and PAS-1, another minor protein component appears to be present in the band 3 region in human erythrocyte membranes. This protein is specifically phosphorylated by the cyclic AMP-dependent protein kinases isolated from the cytoplasm of rabbit erythrocytes. The rabbit erythrocyte membranes lack PAS-1 and the cyclic AMP-dependent protein kinase substrate.     

Glycophorins B and C from human erythrocyte membranes. Purification and sequence analysis

We have developed methods for the preparative purification of two sialoglycoproteins (glycophorins B and C) from human erythrocyte membranes by high-performance ion exchange and gel permeation chromatography in the presence of Triton X-100. Glycophorin B was obtained without any detectable contaminants, and glycophorin C exhibited a purity of about 90-95%. The amino acid sequence of the intramembranous domain (residues 36-71) of glycophorin B was determined and found to be similar to that of the hydrophobic region of the major sialoglycoprotein (glycophorin A). The amino acid sequence of the hydrophobic domain (residues 49-88) of glycophorin C, that was also determined, agreed completely with the structure recently deduced from cDNA sequencing.     

A histochemical study on the snout tentacles and snout skin of bristlenose catfish Ancistrus triradiatus

Histological sections of the tentacles of Ancistrus triradiatus revealed that they contain many goblet cells with granule-like sections distributed along the edge of the tentacles (mean 302 cells mm⁻¹). Various histochemical methods were used to characterize the contents of the goblet cells. The results indicated that the contents were periodic acid-Schiff (PAS) positive, glycogen negative and no sulphated mucins could be found. The contents of the goblet cells were acid mucins and O-acetylated sialomucins which had high energy content. The snout skin of the male also contained goblet cells with higher density (mean 755 cells mm⁻¹) than that of the tentacles. Snout skin of female A. triradiatus , on the contrary, did not contain any goblet cells. Instead, many minute spines could be found. Based on the acid mucins and O-acetylated sialomucins secretions of goblet cells which are of high nutritional values, it is hypothesized that snout tentacle secretions are used to provide nutrients to fish larvae when they are under paternal care.     

External labeling of human erythrocyte glycoproteins. Studies with galactose oxidase and fluorography.

Glycoproteins of the human erythrocyte membrane were labeled with tritiated sodium borohydride after oxidation of terminal galactosyl and N-acetylgalactosaminyl residues with galactose oxidase. After separation of the polypeptides on polyacrylamide slab gels, a scintillator was introduced into the gel, and the radioactive proteins were visualed by autoradiography (fluorography). The following results were obtained. (a) The erythrocyte membrane contains at least 20 glycoproteins, many of which are minor components. (b) The carbohydrate of all the labeled glycoproteins is exposed only to the outside, since no additional glycoproteins can be labeled in isolated unsealed ghosts. (c) The membrane contains two major groups of glycoproteins. The first group of proteins contains sialic acids linked to the penultimate galactosyl/N-acetylgalactosaminyl residues, which are efficiently labeled only after pretreatment with neuraminidase. The second group has terminal galactosyl/N-acetylgalactosaminyl residues which can be easily labeled without neuraminidase treatment. The glycoproteins from fetal erythrocytes all belong to the first group, whereas only five glycoproteins of erythrocytes from adults belong. (d) Trypsin cleaves the proteins containing sialic acids, and fragments containing carbohydrate remain tightly bound and exposed in the membrane. (e) Pronase cleaves Band 3 in addition to the sialic acid containing glycoproteins, but most of the glycoproteins still remain unmodified in the membrane. (f) No difference is seen between membrane glycoproteins from cells of different ABH blood groups.     

Rat erythrocyte glycophorins can be isolated by the lithium diiodosalicylate method used for other glycophorins.

1. The lithium diiodosalicylate/phenol method, widely employed for the isolation of membrane sialoglycoproteins (glycophorins) from mammalian erythrocytes, was applied for the first time to the purification of homologous glycoproteins from rat erythrocyte membranes. 2. The resulting preparations showed to be composed of four components, fractionated on SDS-PAGE. All four were positive for periodic acid-Schiff's reagent stain, the two largest of them being major. 3. Isolated rat glycophorins accounted for 60% of the ghost sialic acid and 1.5% of their protein. The presence of O-acetyl groups was confirmed in one-third of the sialic acid residues. 4. The molecular masses of the four glycophorin components were determined by a method which takes into account the anomalous mobility of glycoproteins on SDS-electrophoresis. Estimated values thus obtained for the actual molecular masses were 74, 32, 25 and 17 kDa.     

O-acetylated sialic acids in gangliosides from pig spleen lymphocytes

The sialic acid content of gangliosides from pig spleen lymphocytes was studied by thin-layer chromatography. N-glycolylneuraminic acid and N-acetylneuraminic acid were detected for the first time in this material as the major sialic acids. In addition, two other sialic acids, tentatively designated O-acetylated sialic acids, according to their RF values on cellulose plates, were also found. We have detected several gangliosides showing a retarded migration pattern in two dimensional thin-layer chromatography with an intermediate ammonia treatment. One of these gangliosides could be an O-acetylated derivative of the disialoganglioside GD3, since after de-O-acetyation it co-migrates with GD3. Another ganglioside co-migrated with GM2 before the alkaline treatment; however, after the treatment it was also retarded and co-migrates with GD3.     

Abundance of the Ca2+-pumping ATPase in pig erythrocyte membranes.

The Ca2+-pumping ATPase (Ca2+-ATPase) was purified from human and pig erythrocyte membranes by calmodulin affinity chromatography in the presence of phosphatidylcholine. The amount of enzyme present in pig erythrocytes is at least 7 times greater than that isolated from human erythrocyte ghosts. However, the properties of the enzyme from the two species are similar in many respects.     

Atypical sialylated N-glycan structures are attached to neuronal voltage-gated potassium channels

Mammalian brains contain relatively high amounts of common and uncommon sialylated N-glycan structures. Sialic acid linkages were identified for voltage-gated potassium channels, Kv3.1, 3.3, 3.4, 1.1, 1.2 and 1.4, by evaluating their electrophoretic migration patterns in adult rat brain membranes digested with various glycosidases. Additionally, their electrophoretic migration patterns were compared with those of NCAM (neural cell adhesion molecule), transferrin and the Kv3.1 protein heterologously expressed in B35 neuroblastoma cells. Metabolic labelling of the carbohydrates combined with glycosidase digestion reactions were utilized to show that the N-glycan of recombinant Kv3.1 protein was capped with an oligo/poly-sialyl unit. All three brain Kv3 glycoproteins, like NCAM, were terminated with alpha2,3-linked sialyl residues, as well as atypical alpha2,8-linked sialyl residues. Additionally, at least one of their antennae was terminated with an oligo/poly-sialyl unit, similar to recombinant Kv3.1 and NCAM. In contrast, brain Kv1 glycoproteins consisted of sialyl residues with alpha2,8-linkage, as well as sialyl residues linked to internal carbohydrate residues of the carbohydrate chains of the N-glycans. This type of linkage was also supported for Kv3 glycoproteins. To date, such a sialyl linkage has only been identified in gangliosides, not N-linked glycoproteins. We conclude that all six Kv channels (voltage-gated K+ channels) contribute to the alpha2,8-linked sialylated N-glycan pool in mammalian brain and furthermore that their N-glycan structures contain branched sialyl residues. Identification of these novel and unique sialylated N-glycan structures implicate a connection between potassium channel activity and atypical sialylated N-glycans in modulating and fine-tuning the excitable properties of neurons in the nervous system.