Aggregates of human erythrocyte membrane sialoglycoproteins in the presence of deoxycholate and dodecyl sulfate

Gel electrophoresis in the presence of deoxycholate of human erythrocyte membranes solubilized with deoxycholate resolves four glycoprotein zones. Electrophoresis in dodecyl sulfate in a second dimension reveals several components, three of which migrate in the region of PAS-2. One of the zones in deoxycholate gel electrophoresis contains component PAS-3, and this glycoprotein seems to exist as a monomer in deoxycholate, but aggregates partially upon addition of dodecyl sulfate. The major sialoglycoprotein migrates as a diffuse zone in dodecyl sulfate. The major sialoglycoprotein migrates as a diffuse zone in deoxycholate gel electrophoresis, indicating association and dissociation during the electrophoresis. The use of deoxycholate followed by dodecyl sulfate in two-dimentional electrophoresis gave high resolution of membrane proteins and can be used for detection of complexes in one of the detergents.     

Red cell membrane sialoglycoprotein beta in homozygous and heterozygous 4.1(-) hereditary elliptocytosis

Sialoglycoprotein beta, a minor sialoglycoprotein of the red cell membrane, was studied in homozygous and heterozygous 4.1(-) hereditary elliptocytosis, a variety of hereditary elliptocytosis characterized by total or partial absence of protein 4.1. Erythrocytes were treated with the periodic acid-NaB3H4 procedure. Following polyacrylamide gel electrophoresis in the presence of SDS, labelled sialoglycoproteins were revealed by fluorography. (i) In the ghosts from the 4.1(-) homozygote, sialoglycoprotein beta was sharply decreased. It is not sure whether the residual material is sialoglycoprotein beta itself, or a distinct sialoglycoprotein migrating in the same place. In long exposure fluorograms, sialoglycoprotein gamma (a sialoglycoprotein related to sialoglycoprotein beta) also turned out to be reduced. In the homozygote's Triton-shells, sialoglycoprotein beta and gamma appeared completely absent. (ii) In the 4.1(-) heterozygote, sialoglycoprotein beta appeared slightly reduced, whereas sialoglycoprotein gamma appeared normal. Both of these proteins were extracted in seemingly normal amounts in the Triton-shells. These observations bring further support to the view that there is an interaction between skeletal membrane protein 4.1 and sialoglycoprotein beta, that is additional to other interactions between the former protein and the lipid bilayer and/or other transmembrane proteins.     

Major O-glycosylated sialoglycoproteins of human hematopoietic cells: differentiation antigens with poorly understood functions

All human hematopoietic cells seem to contain a major, heavily O-glycosylated sialoglycoprotein. Glycophorin A is specific for the erythroid lineage of cells, and leukocytes have a major sialoglycoprotein, also called leukosialin or sialophorin. Cell differentiation results in patterns of O-glycosylation in these proteins, which reflect the stage of differentiation within a cell lineage as well as lineage specificity. The altered carbohydrate compositions may influence the interactions of the cells with external ligands. Healthy individuals lacking glycophorin A in their red cells are known, whereas a deficiency of the leukocyte sialoglycoprotein may result in immunological disease. Although little is known about the physiological functions of these proteins, they form interesting models for studies on regulation of glycosylation, biosynthesis of O-glycosylated glycoproteins, and function of cell surface receptors.     

A minor sialoglycoprotein of the human erythrocyte membrane

The sialoglycoprotein periodate fuchsin sulfite 2 has about 8% of the sialic acid contained in the sialoglycoproteins of the human erythrocyte membrane. This polypeptide appears to have an apparent monomeric molecular weight of 35,000, somewhat smaller than the monomer of the major sialoglycoprotein (periodate fuchsin sulfite 1) as judged by sodium dodecyl sulfate-polyacry lamide gel electrophoresis, and has frequently been confused with the monomer of the major sialoglycoprotein. Periodate fuchsin sulfite 2 is not labeled by the lactoperoxidase procedure in the intact cell, although it is accessible to neuraminidase and other hydrolases. On the other hand, this component can be labeled by lactoperoxidase on the cytoplasmic surface of open membranes or resealed ghosts. Thus, it is a trans membrane protein. Although most of the other transmembrane proteins of the human erythrocyte membrane are extracted from the membrane by 0.1% Triton X-100 in 7 mm phosphate buffer, pH 7.4, this component is not removed and may be a cytoskeletal component. Trypsin, chymotrypsin, and thermolysin peptides, as well as cyanogen bromide fragments, clearly indicate that the primary sequence of this polypeptide can be distinguished from dimeric or monomeric forms of the major sialoglycoprotein (periodate fuchsin sulfite 1).     

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.     

The size and detergent binding of membrane proteins.

Sucrose density gradient centrifugation has been used to measure the binding of Triton X-100 above its critical micellar concentration to a variety of purified membrane and non-membrane proteins. In addition, binding studies were done on the three proteins below the critical micellar concentration of detergent to distinguish between the interaction of proteins with detergent monomers and detergent micelles. A procedure is described for the calculation of the molecular weight of these Triton X-100 protein complexes and measurements were made for opsin, plasma low density lipoprotein, the (Na-+ plus K-+)-dependent adenosine triphosphatase, the human red blood cell major sialoglycoprotein (PAS-1) and the human red blood cell minor glycoprotein (bandIII). These proteins behave as monomers or dimers in detergent and bind between 0.28 and 1.12 g of detergent per g of protein. A general method is also present for calculating the molecular size and shape of impure membrane proteins in detergent. Finally, Triton X-100 was shown to replace bound Na dodecyl-SO4 on the minor glycoprotein of the red blood cell.     

The major sialoglycoprotein of human T-lympocytes

Human, peripheral-blood T-lymphocytes and human, T-lymphoblastoid cells of a MOLT 4B cell-line were surface-labeled by lactoperoxidase-catalyzed iodination, periodate and sodium borotritide, and galactose oxidase and sodium borotritide, and analyzed by dodecyl sodium sulfate-polyacrylamide gel-electrophoresis. Both types of cells were found to show a major, cell-surface sialoglycoprotein with an apparent mol. wt. of 95 000. After neuraminidase treatment, this glycoprotein showed a higher mol. wt. of 120 000. The major sialoglycoprotein of both types of cells bound to wheat-germ agglutinin and concanavalin A and, after neuraminidase treatment, to Arachis hypogaea agglutinin. The glycopeptides obtained from these glycoproteins by Pronase digestion gave similar elution-profiles on Sephadex G-50 gel filtration. These results suggest that the major sialoglycoprotein of normal T cells and that of MOLT 4B cells are very similar, if not identical.     

Labelling of the intramembranous region of the major sialoglycoprotein of human erythrocytes with a photosensitive hydrophobic probe.

Human erythrocyte membranes were incubated with the photosensitive hydrophobic reagent 1-azido-r-iodo[3H]benzene and the mixture was irradiated. The major sialoglycoprotein was then isolated and the labelled polypeptide subjected to proteolytic dissection. Characterization of the purified tryptic and chymotryptic peptides show that the probe is covalently attached only to the transmembrane region of the protein. This labelling pattern is discussed in relation to the use of such reagents for the identification of segments of membrane proteins exposed to the hydrophobic millieu of the membrane.     

Evolution of glycophorin A in the hominoid primates studied with monoclonal antibodies, and description of a sialoglycoprotein analogous to human glycophorin B in chimpanzee

Comparison of human and primate erythrocyte membrane sialoglycoproteins showed that common chimpanzee, dwarf chimpanzee, gorilla, orangutan, and gibbon have major periodic acid Schiff-positive proteins resembling human glycophorin A (GPA) monomer and dimer in electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels. Immunoperoxidase staining of Western blots with monoclonal antibodies to human GPA showed that these primate bands express some GPA antigenic determinants. A new sialoglycoprotein analogous to human glycophorin B (GPB) was detected in common chimpanzee. Although human MN blood group phenotype results from an amino acid polymorphism of GPA, Western blots showed that in chimpanzee sialoglycoprotein (GPAch) always expresses the M blood group, whereas chimpanzee sialoglycoprotein (GPBch) expresses either the N blood group or a null phenotype. This result explains the detection of M and MN, but not of N, blood group phenotypes in chimpanzee. GPBch has higher apparent m.w. than human GPB, is present in the erythrocyte membrane in greater quantity than human GPB, and contains trypsin cleavage site(s) and the 10F7 determinant (both found on human GPA but not GPB). Expression of human GPA antigenic determinants was consistent with the phylogeny of the hominoid primates; common and dwarf chimpanzee expressed most of the determinants tested, gorilla and orangutan an intermediate number, and gibbon and siamang the least. Of the GPA antigenic determinants examined, the MN blood group determinants were most consistently expressed during evolution of the hominoid primates. The results suggested that variability in expression of GPA antigenic determinants between species was due to both differences in amino acid sequence and glycosylation.     

Abnormal carbohydrate composition of the major penetrating membrane protein of En(a-) human erythrocytes.

The major penetrating membrane glycoprotein (band 3) was isolated from En(a-) and normal human erythrocytes. The two proteins differed only in carbohydrate composition. Band 3 from En(a-) erythrocytes contained greater amounts of galactose and N-acetyl-glucosamine. The loss of the sialoglycoprotein sialotetrasaccharides in the En(a-) cell is not compensated by the appearance of these units in band 3 of En(a-) erythrocytes.     

Sialoglycoproteins of murine RAW117 large cell lymphoma/lymphosarcoma sublines of various metastatic colonization properties

A metastatic model for large-cell lymphoma/lymphosarcoma has been developed by sequential selection in vivo of the murine RAW117 cell line for enhanced liver metastasis or in vitro for loss of lectin-binding properties. The metastatic variants obtained from such selections show alterations in cell surface lectin-binding components, such as the wheat germ agglutinin (WGA)-reactive sialoglycoproteins. Detergent lysates from RAW117 cells were analyzed by polyacrylamide gel electrophoresis (PAGE) followed by reaction with 125I-labeled WGA. The [125I]WGA became bound to a diffuse band of Mr 120 000-200 000 in the gels that overlapped with the major sialoglycoprotein band revealed by the periodate-sodium borotritide labeling. However, the [125I]WGA reactivity diminished when gels were pretreated with mild acid to remove sialic acid in situ. The binding of [125I]WGA to the glycoprotein(s) was greater in the high liver-colonizing RAW117-H10 subline than in the parental RAW117-P line. Another lectin with different saccharide specificity, Ricinus communis agglutinin I (RCAI), became bound to a similar class of sialoglycoproteins, as well as to glycoproteins of lower Mr, but only when the gels were pretreated with mild acid to remove sialic acid. These differences in the relative RCAI-binding intensities after chemical removal of sialic acid were similar to those seen with WGA and indicate that differences in WGA reactivity of this class of sialoglycoproteins were not due to increased sialylation of the carbohydrate chains. Sialic acid was removed from RAW117 cells by neuraminidase treatment, and lysates were analysed for [125I]RCAI reactivity after electrophoresis. The migration of the glycoproteins was not affected by neuraminidase, indicating that the diffuseness of the major sialoglycoprotein band was not due to differences in sialylation. [125I]WGA reactivity to the sialoglycoprotein components, before and after Smith degradation in situ, strongly suggests that the oligosaccharide back-bones are highly branched and asparagine-linked. Only the high Mr portion of the diffuse sialoglycoprotein band was stained with peanut agglutinin (PNA) after in situ removal of sialic acid. To determine whether the expression of the sialoglycoprotein was causally related to liver metastasis, the amounts of sialoglycoproteins in RAW117 cells obtained by in vitro selection for increased or decreased metastasis were examined. Binding of [125I]WGA to intact cells and affinity chromatography of vectorially radiolabeled cell surface proteins on WGA-agarose were performed, and the results indicated that the in vitro selected high liver-colonizing RAW117 variants possesses high WGA r     

Separation and some properties of the major proteins of the human erythrocyte membrane

A fractionation procedure is described which allows the isolation of three major human erythrocyte membrane proteins. Their isolation involves three sequential extraction procedures followed by gel filtration in 1% sodium dodecyl sulphate and preparative gel electrophoresis. All three proteins can be isolated from a single preparation. One of the proteins is the erythrocyte sialoglycoprotein, for which no C- or N-terminal residues were found. The other two proteins, which have not previously been isolated, have subunit molecular weights of 74000 and 93000 and contain 9 and 7% carbohydrate respectively. These glycoproteins have blocked N-terminal residues and show similarities in their chemical properties. Preparations derived from blood-group O erythrocytes contain no N-acetylgalactosamine, but similar preparations from blood-group A erythrocytes do contain this sugar. These three proteins cannot easily be solubilized by gentle aqueous procedures and represent about half of the erythrocyte ;ghost' protein. They carry a large proportion of the cell-surface carbohydrate.     

The organization of the major protein of the human erythrocyte membrane

The enzyme lactoperoxidase was used to catalyse the radioiodination of membrane proteins in intact human erythrocytes and in erythrocyte `ghosts'. Two major proteins of the erythrocyte membrane were isolated after iodination of these two preparations, and the peptide `maps' of each protein so labelled were compared. Peptides from both proteins are labelled in the intact cell. In addition, further mobile peptides derived from one of the proteins are labelled only in the `ghost' preparation. Various sealed `ghost' preparations were also iodinated, lactoperoxidase being present only at either the cytoplasmic or extra-cellular surface of the membrane. The peptide `maps' of protein E (the major membrane protein) labelled in each case were compared. Two discrete sets of labelled peptides were consistently found. One group is obtained when lactoperoxidase is present at the extra-cellular surface and the other group is found when the enzyme is accessible only to the cytoplasmic surface of the membrane. The results support the assumption that the organization of protein E in the membrane of the intact erythrocyte is unaltered on making erythrocyte `ghosts'. They also confirm previous suggestions that both the sialoglycoprotein and protein E extend through the human erythrocyte membrane.     

Cell surface properties of ascites sublines of the 13762 rat mammary adenocarcinoma : Relationship of the major sialoglycoprotein to xenotransplantability

The relationship between cell surface sialoglycoprotein and xenotransplantation has been investigated in ascites sublines of the 13762 rat mammary adenocarcinoma. Two of the five sublines (MAT-C and MAT-C1) can be transplanted into mice. These two sublines also have the greatest amounts of total, trypsin-releasable and neuraminidase-releasable sialic acid. Chemical labeling using periodate treatment followed by [³H]borohydride reduction indicates that most of the protein-bound sialic acid is associated with a single major sialoglycoprotein (or family of glycoproteins) with a low mobility on polyacrylamide gels in dodecyl sulfate (SDS). This glycoprotein, denoted ASGP-1, is also labeled by lactoperoxidase and ¹²⁵I, indicating its presence at the cell surface. Metabolic labeling with [³H]glucosamine shows that ASGP-1 is the major glycosylated protein in both xenotransplantable (MAT-C1) and non-xenotransplantable (MAT-B1) sublines, representing >70% of the protein-bound label in each. The labeling studies indicate that the non-xenotransplantable subline does not have a substantially greater amount of ASGP-1 on its cell surface. Likewise cationized ferritin labeling and transmission electron microscopy (TEM) do not show substantially greater amounts of negatively charged groups distributed along the cell surfaces of MAT-C1 than of MAT-B1 cells. The results indicate that the transplantation differences between these sublines cannot be explained solely by the presence of a major sialoglycoprotein at the cell surface.     

Monoclonal antibodies prepared against PAS-I butyrophilin and GP-55 from guinea-pig milk-fat-globule membrane bind specifically to the apical pole of secretory-epithelial cells in lactating mammary tissue

Monoclonal antibodies to the three major glycoproteins of guinea-pig milk-fat-globule membrane were isolated. The specificity of these antibodies was determined by solid-phase immunoassays and by immunoblotting and autoradiographic techniques after one- and two-dimensional gel electrophoresis. The antibodies bound to PAS-I, a sialoglycoprotein of Mr greater than or equal to 200 000 and the glycoproteins butyrophilin and GP-55, of Mr 63 000 and 55 000, respectively. Immunolocalization studies showed that all three proteins were highly concentrated in the apical pole of secretory-epithelial cells in mammary tissue during lactation. PAS-I, butyrophilin or GP-55, were not detected in either the basal cytoplasm of mammary epithelial cells or in myoepithelial cells, capillary endothelial cells or other cells found in the mammary gland. These proteins were either present in small amounts or were absent from mammary tissue taken in late pregnancy. The monoclonal antibodies characterized in this study will therefore be useful as probes for studies of the biogenesis of apical membrane proteins in mammary epithelial cells during lactation.     

Freeze-fracture electron microscopy of human erythrocytes lacking the major membrane sialoglycoprotein

Human erythrocytes of blood group En (a-), a rare homozygous condition involving a complete lack of the major sialoglycoprotein of the cell membrane (glycophorin A), were compared with erythrocytes from normal (En(a+)) individuals by freeze-fracture electron microscopy. No decrease in number, or variation in morphology, of the intramembranal particles of En (a-) cells was detectable. These results show that the erythrocyte sialoglycoprotein is not essential for the maintenance of the integrity of the intramembranal particles of the human erythrocyte membrane.     

Mechanism of concanavalin A-induced anchorage of the major cell surface glycoproteins to the submembrane cytoskeleton in 13762 ascites mammary adenocarcinoma cells

Concanavalin A (Con A)-induced anchorage of the major cell surface sialoglycoprotein component complex (ASGP-1/ASGP-2) was studied in 13762 rat mammary adenocarcinoma sublines with mobile (MAT-B1 subline) and immobile (MAT-C1 subline) cell surface Con A receptors. Treatment of cells, isolated microvilli, or microvillar membranes with Con A resulted in marked retention of ASGP-1 and ASGP-2, a Con A-binding protein, in cytoskeletal residues of both sublines obtained by extraction with Triton X-100 in PBS. When Con A-treated microvillar membranes were extracted with a buffer containing Triton X-100, the sialoglycoprotein complex was found associated in the residues with a transmembrane complex composed of actin, a 58,000-dalton polypeptide, and a cytoskeleton-associated glycoprotein (CAG), also a Con A-binding protein, in MAT-C1 membranes, and of actin and CAG in MAT-B1 membranes. Untreated membrane Triton residues retained very little ASGP-1/ASGP-2 complex. Association of the sialoglycomembrane complex and the transmembrane complex was also demonstrated in Con A-treated, but not untreated, microvilli by their comigration on CsCl gradients. Association of both complexes with the cytoskeleton of microvilli was shown by sucrose density gradient centrifugation. A fraction of the polymerized actin comigrated with the transmembrane complex alone in the absence of Con A and with both the transmembrane complex and the sialoglycoprotein complex in the presence of Con A. From these results we propose that anchorage of the sialoglycoprotein complex to the cytoskeleton on Con A treatment occurs by cross-linking ASGP-2, the major cell surface Con A-binding component, to CAG of the transmembrane complex, which is natively linked to the cytoskeleton via its actin component. Since Con A-induced anchorage occurs in sublines with mobile and immobile receptors, the anchorage process cannot be responsible for the differences in receptor mobility between the sublines.     

Isolation and characterization of leukosialin, a major sialoglycoprotein on human leukocytes

A major sialoglycoprotein (previously called gp105) on the human erythroleukemic cell line K562 was purified, and specific antibodies were raised in a rabbit. A number of different hematopoietic cell lines belonging to erythroid, myeloid, T-lymphoid, and B-lymphoid cell lineages were found to possess glycoproteins that were immunoprecipitated by these antibodies. However, the apparent molecular weights differed between cell lines, ranging from 113,000 to 150,000. In almost all cases, the immune precipitated molecule corresponded to the major sialoglycoprotein of the respective cell. Pulse-chase experiments showed that all cells produced an early precursor form of the molecule of 54 kDa, which was susceptible to endo-beta-N-acetylglucosaminidase H to give an apoprotein of 52 kDa. Neuraminidase treatment of the mature forms resulted in a characteristic decrease of the mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (apparent molecular weights from 150,000 to 183,000). Amino acid analysis of the glycoprotein isolated from HL-60 cells showed a high content of serine, threonine, and proline, and the carbohydrate composition was compatible with the presence of a large number (approximately 90) of O-linked carbohydrate chains. The name leukosialin is proposed for this sialoglycoprotein, which seems to be widely distributed, but differently glycosylated, on leukocytes with diverse functions. In the following paper (Carlsson, S.R., Sasaki, H., and Fukuda, M. (1986) J. Biol. Chem. 261, 12787-12795), we demonstrate that the structures of O-linked oligosaccharides vary significantly depending on the cells from which leukosialin was isolated.     

Four gel systems for electrophoretic fractionation of membrane proteins using ionic detergents

Membrane proteins were fractionated electrophoretically in polyacrylamide gels containing either anionic or cationic detergent at either pH 2.4 or 8.3. In all four systems, polypeptides migrated as monomers bearing the charge of the detergent ion and semi-logarithmic molecular weight–mobility relationships pertained. Electrophoresis of erythrocyte membrane proteins in these systems yielded very similar protein staining patterns but revealed significant differences in sialoglycoprotein migration.     

Purification and Characterization of the Egg Jelly Macromolecules, Sialoglycoprotein and Fucose Sulfate Glycoconjugate, of the Sea Urchin Hemicentrotus Pulcherrimus

A sialoglycoprotein and a fucose sulfate glycoconjugate (FSG) were purified from egg jelly of the sea urchin Hemicentrotus pulcherrimus . Sialoglycoprotein which consisted of sialic acid (90%, w/w) and protein (10%, w/w) did not cause induction of the acrosome reaction and sperm isoagglutination. FSG which contained one mol sulfate/mol fucose possessed 2.0 times protein to fucose by weight. The proteins in intact FSG were separated to two major (258 kDa and 237 kDa) and one minor (120 kDa) proteins by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in the presence of 2-mercaptoethanol (2-ME) while the proteins could not be separated by HPLC in the presence of 0.1% SDS or SDS-PAGE without 2-ME. However, after carboxymethylation of FSG, two major (260 kDa and 240 kDa) proteins and two minor (140 kDa and 135 kDa) proteins were separated from the fucose sulfate moiety by HPLC in the presence of 0.1% SDS or SDS-PAGE without 2-ME. When FSG was first carboxymethylated with non-radioactive iodoacetic acid and then reduced with 2-ME and finally carboxymethylated with ¹⁴C-iodoacetic acid, the most of radioactivity was detected in 140 kDa and 135 kDa proteins. Carboxymethylted-FSG was less potent than intact FSG in induction of the acrosome reaction. Fucoidan, a fucose sulfate polymer, did not induce the acrosome reaction.