The major sialoglycoprotein of the human erythrocyte membrane. Release with a non-ionic detergent and purification.

The major sialoglycoprotein of the human erythrocyte membrane has been selectively released by the non-ionic detergent Tween 20 and further purified in detergent-free buffers by hydroxyapatite chromatography and, finally, by hydrophobic interaction chromatography on pentyl-Sepharose. The purified glycoprotein shows one main zone, PAS-1, and up to three minor zones after staining both for protein and carbohydrate in polyacrylamide gel electrophoresis in the presence of dodecyl sulfate. The relative staining intensities are concentration dependent. When the purified glycoprotein has been heated to 100 degrees C in dodecyl sulfate, more stain appears in the most rapid zone, PAS-2, and less in the slower zones, indicating a disaggregation of oligomeric forms of this glycoprotein, including a dimer, PAS-1.     

The membrane change in En(a-) human erythrocytes. Absence of the major erythrocyte sialoglycoprotein.

We investigated the membrane of En(a-) human erythrocytes as part of a study of the structure and biochemical function of the surface glycoproteins of the mammalian cell. 2. En(a-) erythrocytes were selected because they have more extensive changes at the cell surface than any other known erythrocyte variant. 3. Our results show that in En(a-) erythrocytes: (a) the major membrane sialoglycoprotein is lacking; (b) the other major membrane-penetrating glycoprotein (band 3) has an altered electrophoretic mobility. 4. The apparent clinical normality of En(a-) cells suggests that the change in band 3 may compensate for the loss of the membrane sialoglycoproteins. It is clear that a viable erythrocyte can exist despite the absence of one of its major surface components.     

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).     

Structural analysis of the major human erythrocyte membrane sialoglycoprotein from Miltenberger class VII cells

The major human erythrocyte membrane sialoglycoprotein (glycophorin A or MN glycoprotein) was purified from the red blood cells of an individual, homozygous for the Mi-VII gene in the Miltenberger subsystem of the MNSs blood-group system. The complete structure of a tryptic peptide comprising the residues 40-61 of glycophorin A was deduced from manual sequence analyses. The Mi-VII-specific glycophorin A was shown to exhibit an arginine----threonine and a tyrosine----serine exchange at the positions 49 and 52 respectively. The threonine-49 residue was found to be glycosylated. Inhibition assays demonstrated that one of the Mi-VII-specific antigen determinants (Anek) is located within the residues 40-61 of glycophorin A and comprises sialic acid residue(s) attached to O-glycosidically linked oligosaccharide(s). Our data contribute to an understanding of the Miltenberger system and provide an explanation at the molecular level for the previous finding that the erythrocytes from the Mi-VII homozygote lack a high-frequency antigen (EnaKT), located within the residues 46-56 of normal glycophorin A.     

Glycophorin-enriched vesicles obtained by a selective extraction of human erythrocyte membranes with a non-ionic detergent.

A method is described for isolating glycophorin-enriched vesicles from human erythrocytes by extracting membranes that were incubated for 30 min at 37 degrees C at pH 4.5 and washed at low and high ionic strength with the nonionic detergent Triton X-100. The extracts were 11.8 +/- 2.4 fold enriched in glycophorin and contained 325 +/- 69 microgram sialic acid/mg protein, which represented 61 +/- 16% of the total sialic acid. Upon removal of Triton X-100 one third of the total glycophorin forms glycophorin-enriched vesicles with coextracted, endogenous lipids as shown sedimintation, dextran-density gradient centrifugation, and electron microscopy. Addition of exogenous lipids increased the fraction of glycophorin-enriched vesicles up to 87%. The incorporation of glycophorin in the membrane was shown by hemagglutination inhibition assays using anti-M sera and by the accessibility of glycophorin to trypsin. Freeze-fractured vesicles did not reveal intramembranous particles. The selectivity of the extraction procedure is not simply due to chemical constraints introduced by disulfide cross-linkage of protein component 3, because only 20% of this protein undergo disulfide cross-linking. The selective extraction of glycophorin implies that glycophorin is segregated from protein component 3 and thus from intramembranous particles when erythrocyte membranes have been incubated at pH 4.5. This segregation may precede aggregation of intramembranous particles.     

Solubilisation of human erythrocyte band 4.1 protein in the non-ionic detergent Tween 20

Extraction of spectrin-depleted erythrocyte membranes with the non-ionic detergent Tween 20, in a 0.1 M glycine-NaOH buffer (pH 9.8) leads to the solubilization of band 4.1 and the sialoglycoproteins. The comigration of band 4.1 with the sialoglycoproteins in gel filtration and detergent-free electrophoresis indicated that these proteins may be associated as complexes of high molecular weight. Although treatment of intact membranes with Tween 20 under the same conditions does not lead to direct solubilization of proteins, severe disruption of the membranes was observed under phase contrast microscopy. Suspension of the treated membranes in 5 mM phosphate buffer (pH 8.0) leads to the solubilization of band 4.1, spectrin, actin and the sialoglycoproteins. High molecular weight complexes of band 4.1 and the sialoglycoproteins were isolated from these extracts, suggesting a possible interaction between band 4.1 and sialoglycoproteins which may be important for linking the cytoskeleton to the membrane.     

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.     

The phosphorylation of the major proteins of the human erythrocyte membrane.

Both the major sialoglycoprotein (PAS-1) and the component designated by Fairbanks et al. (G. Fairbanks, T. L. Steck, and D. F. H. Wallach, 1971, Biochemistry10, 2606–2617) as Band 3 are shown to be bonafide phosphoproteins by virtue of the presence of covalently bound serine and threonine phosphate residues. In agreement with the findings of others, PAS-1 does not seem to be phosphorylated when ghosts are incubated with [γ-³²P]ATP, but the phosphorylation is significant (about 0.15 mol/mol) when the cells are incubated in the presence of ³²P_i. Band 3 is phosphorylated to the extent of 0.90 mol/mol, and these sites are apparently distributed in several places along the polypeptide chain. Spectrin is also a phosphoprotein containing approximately four molecules of phosphate per 450,000 daltons of protein. The phosphorylation of these three polypeptides is not stimulated by the presence of cAMP.     

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.     

The association between major sialoglycoprotein and spectrin of human erythrocyte membranes as detected by fluorescence resonance energy transfer.

The interactions among the major sialoglycoprotein and peripheral proteins of human erythrocyte membranes were investigated by the long range resonance energy transfer between different fluorescent moieties separately conjugated to proteins. Consequently, direct association between the major sialoglycoprotein and spectrin was observed and divalent cations were required for their association.     

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.     

Automated purification of human protein band 3, the major integral protein of the erythrocyte membrane

Human erythrocyte protein band 3 was purified from a Triton X-100 extract of white ghosts. This purification, including an ion-exchange chromatography and a group-affinity chromatography, was automated. The apparatus was assembled from commercially available elements and allowed for the recovery of 2 to 3 mg pure band 3 in 2 hr. The purification could be repeated several times a day. The advantages of automation are discussed.     

Some observations on the choice of detergent for solubilization of the human erythrocyte membrane.

Solubilization of the human erythrocyte membrane by seven detergents is described. Components released into the supernatant or retained in the residue were identified by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Two non-ionic detergents exhibiting little u.v. absorption were more efficient than u.v.-absorbing Triton X-100. Evidence is presented of an interchange between protein PAS 1 and protein PAS 2.     

Characteristics of n-octyl beta-D-thioglucopyranoside, a new non-ionic detergent useful for membrane biochemistry.

n-Octyl beta-D-thioglucopyranoside, a new non-ionic detergent, was synthesized. Properties, and applicability to membrane proteins, of this detergent were investigated. The detergent was easily removed by dialysis. The solubilizing power of this detergent for Escherichia coli membrane proteins was similar to that of n-octyl beta-D-glucopyranoside, which has been widely used in membrane biochemistry. No inactivation of proteins was observed after the solubilization. n-Octyl beta-D-thioglucopyranoside was superior to n-octyl beta-D-glucopyranoside in that it was much more stable and could be synthesized at much lower cost.     

Crossed immunoelectrophoresis of bovine milk fat globule membrane protein solubilized with non-ionic detergent.

Detergent solubilized bovine milk fat globule membrane material studied by crossed immunoelectrophoresis combined with histochemical techniques revealed four major protein complexes. All four were found to bind to concanavalin A and three were identified as sialoglycoproteins. Xanthine oxidase activity was associated with the non-sialoglycoprotein precipitate. Immunoabsorption with intact milk fat globules showed an internal location of the xanthine oxidase, whereas the three other main proteins plus Mg2+-ATPase and 5'-nucleotidase were disposed on the outer membrane surface. The major proteins from milk fat globule membrane and membrane material isolated from skim milk showed immunochemical identity.     

A new human erythrocyte variant (Ph) containing an abnormal membrane sialoglycoprotein

1. A new human erythrocyte variant (Ph) is described. The variant contains an unusual sialic acid-rich glycoprotein in addition to the blood-group-MN([unk])- and blood-group-Ss(δ)-active sialoglycoproteins found in normal erythrocytes. 2. The unusual component Ph has an apparent mol.wt. of 32000 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The Ph component is not degraded during trypsin treatment of intact erythrocytes. 3. The Ph component was labelled by lacto-peroxidase-mediated radioiodination of intact erythrocytes and was found to be present in amounts approximately equimolar to α-sialoglycoprotein in the variant erythrocytes. 4. The Ph component had receptors for the lectins from Maclura aurantiaca (osage orange) and Triticum vulgaris (wheat-germ), but lacked a receptor for the Phaseolus vulgaris (red kidney bean) lectin, suggesting that it carries only O-linked oligosaccharides. 5. The presence of the Ph component in these erythrocytes does not correspond to any of the known blood-group-MNSs-related antigens examined. 6. We suggest that this component may be a hybrid polypeptide containing the N-terminal portion derived from normal δ-sialoglycoprotein, and the C-terminal portion from normal α-sialoglycoprotein, in a manner similar to the anti-Lepore haemoglobin.