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.     

Genetic variants of human red-cell membrane sialoglycoprotein beta. Study of the alterations occurring in the sialoglycoprotein-beta gene.

We have studied the DNA of individuals who express an altered sialoglycoprotein beta on their red cells by using Southern blotting with sialoglycoprotein-beta cDNA probes. Individuals of the Leach phenotype do not express any beta (sialoglycoprotein beta) or gamma (sialoglycoprotein gamma) on their red cells, and we show that about 7 kb of DNA, including the 3' end of the beta gene, is deleted in this DNA. Any protein product of this gene is likely to lack the membrane-associating domain of beta. We have also examined the DNA of two types of other individuals (Yus-type and Gerbich-type) who have red cells that lack beta and gamma, but contain abnormal sialoglycoproteins related to beta. These two types of DNA contain different internal deletions of about 6 kb in the beta gene. We suggest that these deletions result from the presence of two different sets of internal homology in the beta gene, and on this basis we propose structures for the abnormal Yus-type and Gerbich-type sialoglycoproteins which are consistent with the other evidence that is available. We provide evidence that beta and gamma are products of the same gene and suggest a possible mechanism for the origin of gamma based on leaky initiation of translation of beta mRNA.     

Alkali-labile oligosaccharide units of a sialoglycoprotein from rabbit erythrocyte membranes

Two glycoproteins (apparent molecular weights 120,000 and 70,000) were extracted from rabbit erythrocyte membranes, and only one (Mr 120,000), which is a sialoglycoprotein, contained O-glycosidically linked sugar chains. Alkali-labile oligosaccharide units of the sialoglycoprotein were released as reduced oligosaccharides by NaOH-NaB3H4 treatment, and then purified by gel filtration on a Bio-Gel P-4 column followed by ion-exchange chromatography. From the results of methylation analysis, mass spectrometry and chromium trioxide oxidation, the main oligosaccharide unit was determined to be a linear trisaccharide (85% by weight), NeuNGc alpha(2----3)Gal beta(1----3)GalNAcol. In addition, small amounts of a tetrasaccharide (11% by weight) and a disaccharide (4% by weight) were found, which were determined to have the following structures, NeuNGc alpha(2----3)Gal beta(1----3)[NeuNGc alpha(2----6)] GalNAcol and Gal-GalNAcol, respectively.     

A transcortin-binding protein in the plasma membrane of human syncytiotrophoblast.

Using affinity chromatography on immobilized transcortin of 125I-labeled, cholate-solubilized plasma membranes of human syncytiotrophoblast, a transcortin-binding protein with a minimal Mr of about 20 kDa has been isolated. It was found to be a sialoglycoprotein with an isoelectric point at pH 4.4 (about 5.0 after the treatment with neuraminidase). We assume that this protein is a component of membrane recognition system for transcortin-steroid complexes.     

The Dantu erythrocyte phenotype of the NE variety. I. Dodecylsulfate polyacrylamide gel electrophoretic studies

Red cell membranes from patient NE, Mr. Dantu and 16 additional Black individuals, positive for the low-frequency MNSs-system antigen Dantu, were studied by dodecylsulfate polyacrylamide gel electrophoretic techniques. The content of the major, blood group M- or N-active sialoglycoprotein (glycophorin A, GP A) was found to be decreased by about 57%. The blood group S- or s-active sialoglycoprotein (GP B) was decreased by about 51% in membranes from proven Dantu/U heterozygotes and not detectable in those from patient NE and other Dantu+U- individuals. Donor NE was shown to exhibit the genotype Dantu/u. Dantu-positive cells exhibit a proteinase-resistant GP B-GP A hybrid with an apparent molecular mass of 29 KDa whose intramembraneous and cytoplasmic domains were shown to be similar to those of GP A. The molar hybrid: GP A ratio in all cells was found to be about 2.4: 1, indicating that the NE variety of the Dantu phenotype is much more frequent than the Ph or MD types. The significance of an additional minor 'new' component (molecular mass 21 KDa) in Dantu+ membranes and the minor component J (molecular mass 22 KDa) occurring in normal and Dantu+U+ GP preparations, but not in those from Dantu+U- cells, has not been resolved. The apparent molecular mass of the anion channel protein (band 3) in all cells of the NE variety was shown to be decreased by about 3 KDa, due to a shortening of carbohydrate chains. This suggests that the hybrid, just like GP A, might form a complex with band 3.     

Abnormal blood-group-Ss-active sialoglycoproteins in the membrane of Miltenberger class III, IV and V human erythrocytes.

1. We have studied the inherited changes occurring in the sialoglycoproteins of membranes from erythrocytes of type Miltenberger Class III (Mi.III), Miltenberger Class IV (Mi.IV) and Miltenberger Class V (Mi.V) by using sodium dodecyl sulphate/polyacrylamide gel electrophoresis and lactoperoxidase radioiodination. 2. Mi.III erythrocytes lack the normal blood-group-Ss-active sialoglycoprotein but contain an unusual s-active sialoglycoprotein of higher apparent molecular weight. A similar abnormal S-active sialoglycoprotein appears to occur in Mi.IV erythrocytes. 3. The Mi.V condition is associated with the hemizygous absence of both the normal blood-group-MN-active sialoglycoprotein and the normal Ss-active sialoglycorprotein. However, a new sialoglycoprotein component is present in these cells that has properties characteristic of both the MN-active and Ss-active sialoglycoproteins. 4. Our results suggest that the new sialoglycorportein present in Mi.V erythrocytes is a hybrid of the normal MN sialoglycoprotein and an s-active sialoglycoprotein that has properties similar to the s-active sialoglycoprotein found in Mi.III erythrocytes. We suggest that the unusual Mi.V sialoglycoprotein is derived from chromosomal misalignment with unequal crossing-over between the genes for the MN- and Ss-active sialoglycoproteins in a manner similar to that which gives rise to haemoglobin Lepore. 5. Further studies of S-s-erythrocytes confirm that these cells lack normal Ss-active sialoglycoprotein, but contain an unusual component that shows some of the properties of the normal Ss-active sialoglycoprotein. 6. Analysis of erythrocytes of type Mk/Mi.III confirms that, in addition to the known hemizygous lack of the MN-active sialoglycoprotein, the Mk condition is also associated with a loss of the Ss-active sialoglycoprotein. 7. In order to facilitate discussion of the complex changes that occur in these variant erythrocytes, a new unified nomenclature is used for the erythrocyte sialoglycoproteins.     

An electrophoretic and immunochemical characterization of human surfactant-associated proteins

We have prepared an antiserum against a serum-free extract of alveolar proteinosis lavage that recognizes the same proteins as an antiserum to human surfactant. Using one and two-dimensional gel electrophoresis, protein blotting and immunostaining we have found proteins with Mr of approx. 35 and 60 kDa to be present in every source of human surfactant we have examined. These proteins are immunologically related to those found in the lavage from alveolar proteinosis patients, have the same electrophoretic characteristics and are not found in serum. The 35 kDa protein is a group of at least eight isoforms ranging in relative molecular mass Mr from 32 to 36 kDa with isoelectric points between 4.8 and 5.5. Neuraminidase digestion studies have shown that at least part of this charge heterogeneity may be due to sialic acid residues. The less abundant form, with a Mr of about 60 kDa is also a sialoglycoprotein with similar isoelectric points.     

Inheritance of human-erythrocyte Gerbich blood group antigens.

The blood group-antigenic determinant Gerbich was first described greater than 25 years ago, but its mode of inheritance has not been established. We performed protein immunoblotting by means of anti-beta sialoglycoprotein (SGP) and anti-gamma SGP reagents. The anti-beta SGP was a monoclonal antibody that reacts with normal beta SGP and with the abnormal beta-related SGPs associated with Gerbich and Yus types of Ge-negative red cells. In the families studied, we have shown that the products of the Ge alleles are inherited in an autosomal codominant manner.     

Subunit structure and dynamics of the insulin receptor

A model for the minimum subunit composition and stiochiometry of the physiologically relevant insulin receptor has been deduced based on results obtained by affinity labeling of this receptor in a variety of cell types and species. We propose that the receptor is a symmetrical disulfide-linked heterotetramer composed of two alpha (apparent Mr = 125,000) and two beta (apparent Mr = 90,000) glycoprotein subunits in the configuration (beta-S-S-alpha)-S-S-(alpha-S-S-beta). The disulfide or disulfides linking the two (alpha-S-S-beta) halves (class I disulfides) exhibit greater sensitivity to reduction by exogenous reductants than those linking the alpha and beta subunits (class II disulfides). When the class I disulfides are reduced by addition of diothiothreitol to intact cells, the receptor retains its ability to bind insulin and to effect a biological response. The beta subunit contains a site at about the center of its amino acid sequence that is extremely sensitive to proteolytic cleavage by elastaselike proteases, yielding a beta 1 fragment (Mr = 45,000) that remains disulfide linked to the receptor complex and a free beta 2 fragment. Binding of insulin to the receptor complex appears to result in the formation or stabilization of a new receptor conformation as evidenced by an altered susceptibility of the alpha subunit to exogenous trypsin. A receptor structure with high affinity for insulinlike growth factor (IGF) I and low affinity for insulin in fibroblast and placental membranes has also been affinity labeled. It exhibits the same structural features found for the insulin receptor, including two classes of disulfide bridges and beta subunits highly sensitive to proteolytic cleavage. These recent observations identifying the presence of distinct insulin and IGF-I receptors that share similar complex structures suggest that these hormones may also share common mechanisms of transmembrane signaling.     

Characterization of O-glycosidically linked oligosaccharides of rat erythrocyte membrane sialoglycoproteins

The carbohydrate units of the rat erythrocyte membrane sialoglycoprotein rSGP-4 [Edge, A. S. B., & Weber, P. (1981) Arch. Biochem. Biophys. 209, 697-705] have been characterized. All of the carbohydrate of this Mr 19,000 glycoprotein occurs in O-glycosidic linkage to the peptide; following alkaline borohydride treatment and chromatography on Bio-Gel P-2, sialic acid containing oligosaccharides terminating in N-acetylgalactosaminitol were obtained. Their structures were determined by compositional analysis, exoglycosidase digestions, alkaline sulfite degradation, and periodate oxidation. The oligosaccharides were characterized for molecular weight and linkage by direct chemical ionization and gas-liquid chromatography/mass spectrometry, respectively. The structures are proposed to be NeuAc alpha 2----3Gal beta 1----3GalNAc-ol, Gal beta 1----3(NeuAc alpha 2----6)GalNAc-ol, NeuAc alpha 2----3Gal beta 1----3(NeuAc alpha 2----6)GalNAc-ol, and NeuAc alpha 2----3Gal beta 1----3(NeuAc alpha 2----3Gal beta 1----4GlcNAc beta 1----6)GalNAc-ol. Two of the N-acetylglucosamine-containing hexasaccharides were present per molecule of rSGP-4 along with two trisaccharides and seven tetrasaccharides.     

The use of monoclonal antibodies to quantify the levels of sialoglycoproteins alpha and delta and variant sialoglycoproteins in human erythrocyte membranes.

By using radioiodinated monoclonal antibodies we have estimated that there are about 600 000 copies of sialoglycoprotein alpha (synonym glycophorin A) and 80 000 copies of sialoglycoprotein delta (synonym glycophorin B) per normal human erythrocyte. Erythrocytes expressing the product of only one alpha gene contain about 300 000 copies of alpha/cell. Two erythrocyte types containing alpha-delta hybrid molecules were studied. Those with heterozygous expression of the (alpha-delta)Mi.V gene contain about 100 000 alpha-delta copies per cell, whereas those with heterozygous expression of the En(UK) gene contain about 80 000 alpha-delta copies/cell. Erythrocyte types containing delta-alpha hybrid molecules were also studied. About 200 000 copies of (delta-alpha)Dantu were measured in cells with heterozygous expression of the (delta-alpha)Dantu gene (donor M.P.), whereas about 315 000 copies of the putative (delta-alpha)Dantu hybrid were found on the erythrocytes of donor J.O. [which also have heterozygous expression of the putative (delta-alpha)Dantu gene]. The erythrocytes of donor M.P. have normal levels of alpha, whereas those of donor J.O. have only about half-normal levels. It is proposed that the hybrid sialoglycoprotein of donor J.O. is of alpha-delta-alpha composition [(alpha-delta-alpha)Dantu] rather than delta-alpha and results from a double cross-over analogous to that which gives rise to haemoglobin Parchman.     

The VLA protein family. Characterization of five distinct cell surface heterodimers each with a common 130,000 molecular weight beta subunit

The family of human cell surface heterodimers which includes VLA-1 and VLA-2 is now shown to include three additional heterodimers, here called VLA-3, VLA-4, and VLA-5. Each of these separate VLA structures is composed of a distinct alpha subunit (Mr 110,000-200,000 nonreduced) noncovalently associated with a common beta subunit (Mr 110,000 nonreduced). Chemical cross-linking experiments provided evidence that each VLA complex exists predominantly as a 1:1 alpha beta heterodimer. The VLA proteins are widely distributed, with one or more of the heterodimers present on nearly all cell types tested. Evidence for five distinct VLA alpha subunits was obtained from differences observed in antibody recognition, cell distribution patterns, two-dimensional gel analyses, and V8 protease cleavage patterns. On the other hand, the beta subunit present in each heterodimer was immunochemically and electrophoretically indistinguishable, and yielded identical V8 cleavage fragments. Immunoblotting experiments revealed that besides the Mr 110,000 beta normally seen, another beta protein was present that is smaller in size (Mr 90,000 nonreduced), altered or deficient in glycosylation, and not available for cell surface radiolabeling.     

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     

Rearrangements of the red-cell membrane glycophorin C (sialoglycoprotein beta) gene. A further study of alterations in the glycophorin C gene.

We have cloned portions of the glycophorin C (sialoglycoprotein beta) gene from individuals with red cells of normal, Gerbich and Yus phenotypes. The clones contain up to three exons of the glycophorin C gene (designated exons 2, 3 and 4). Analysis by restriction mapping and DNA sequencing confirmed that the deletions causing the Gerbich and Yus phenotypes are located entirely within the glycophorin C gene. Sequencing of the normal gene showed that not only do exon 2 and exon 3 have related DNA sequences, but also that both the 5' and 3' flanking intronic DNA sequences are almost identical. The two variant genes each lack a different exon: the Yus type gene lacks exon 2, whereas the Gerbich-type gene lacks exon 3. We suggest that the observed deletions are due to recombination between the regions of homologous intronic repeats. We also provide evidence that an unequal cross-over mechanism may be responsible for a number of observed glycophorin C gene rearrangements, including an insertion mutation in Lewis II (Lsa)-type red cells that has not previously been reported.     

Quaternary structure of (Na+ + K+)-dependent adenosine triphosphatase.

(Na+ + K+)-dependent adenosine triphosphatase (NaK-ATPase) consists of two polypeptide chains, a large polypeptide with a molecular weight of about 100,000, and a sialoglycoprotein with a molecular weight of about 40,000. Cross-linking of purified NaK-ATPase with the (o-phenanthroline)2-cupric ion complex (CP) results in the reversible formation of dimers, trimers, tetramers, and pentamers of the large polypeptide and loss of NaK-ATPase activity. ATPase activity is partially recovered if NaK-ATPase is incubated with beta-mercaptoethanol after treatment with CP. In contrast to these results, if NaK-ATPase is cross-linked in crude canine kidney microsomes, only a dimer of the large polypeptide is formed. No cross-linking of the sialoglycoprotein to the large polypeptide is detected when NaK-ATPase is cross-linked in purified form. However, when NaK-ATPase is reacted with CP in either purified or microsomal form, the sialoglycoprotein cross-links to itself yielding a high molecular weight aggregate. The results show that the functional subunit structure of NaK-ATPase consists of at least two large polypeptides.     

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.     

Some aspects of structure of murine H-2d antigens

The apparent molecular weight of proteins precipitated by normal rabbit serum from lysates of lymph node cells ranged from 10 000 to 150 000. Mono-specific anti-H-2d serum precipitated molecules composed of heavy and light chains with Mr of 47 000 and 12 000, respectively. Tunicamycin treatment caused a decrease of the molecular weight of heavy chain by 4 000 and a change of pI from 5.2 - 5.9 to 5.4 - 5.8. Non-glycosylated heavy chain exhibited Mr of 43 000. The oligosaccharide side chains were resistant to digestion by endo-beta-N-acetylglucosaminidase H. The Mr and pI of beta 2-microglobulin were not altered by treatment with tunicamycin and endo-beta-N-acetylglucosaminidase H. Tunicamycin caused a 50% decrease of overall synthesis of cellular proteins.     

Antibody to the Filarial Antigen Wb123 Reflects Reduced Transmission and Decreased Exposure in Children Born following Single Mass Drug Administration (MDA)

Background

Antibody (Ab) to the Wuchereria bancrofti (Wb) infective larval (L3) antigen Wb123, using a Luciferase Immunoprecipitation System (LIPS) assay, has been shown to be a species-specific, early marker of infection developed for potential use as a surveillance tool following transmission interruption post mass drug administration. To examine its usefulness in a single filarial-endemic island assessed at two time points with markedly different levels of transmission, Ab to Wb123 was measured in sera collected from subjects from Mauke, Cook Islands in 1975 (no previous treatment) and 1992 (5 years after a one time island-wide treatment with diethylcarbamazine [DEC]).

Findings

Between 1975 and 1992, Wb transmission decreased dramatically as evidenced by reduced prevalences of microfilariae (31% vs. 5%) and circulating Ag (CAg, 49% vs. 16%). Age specific prevalence analysis showed a dramatic reduction in Wb123 Ab positivity from 54% (25/46) in 1975 to 8% (3/38) in 1992 in children 1–5 years (p<0.0001), reflecting the effects of single-dose treatment five years earlier. By 1992, Wb123 Ab prevalence in children 6–10 years had fallen from 75% (42/56) in 1975 to 42% (33/79) consistent with a lower cumulative transmission potential. In the whole population, Wb123 seropositivity decreased from 86% to 60% between 1975 and 1992. In CAg+ subjects the levels of Wb123 Ab were indistinguishable between the 2 time points but differed in those who were CAg− (p<0.0001). In paired sample analysis, individuals who were CAg+ in 1975 but became CAg− in 1992 had significantly lower Ab levels in 1992 (p<0.0001), with 9/40 (23%) becoming seronegative for Wb123.

Conclusions

The relationship between reduction in Wb123 Ab prevalence and the reduction of transmission, seen most clearly in young children, strongly advocates for the continuing assessment and rapid development of Wb123 as a surveillance tool to detect potential transmission of bancroftian filariasis in treated endemic areas.     

Altered membrane sialoglycoproteins in human erythrocytes lacking the Gerbich blood group antigens

The sialoglycoproteins (glycophorins) in human red cell membranes of rare individuals lacking totally (Ge-1,-2,-3 phenotype) or partially (Ge-1,-2,3 phenotype) the Gerbich (Ge) blood group antigens and two Ge-1,-2,-3 heterozygotes were studied by dodecylsulfate polyacrylamide gel electrophoretic techniques. Two sialoglycoproteins (components D and E) were not detectable in the membranes from the homozygotes and found to be decreased by about 50% in those from the heterozygotes. Ge--1,-2,-3 and Ge-1,-2,3 cells were found to contain a 'new' component (mol. masses about 29 and 30 kDa, respectively) possibly representing a D/E hybrid molecule. This sialoglycoprotein was not detectable in membranes from the Ge-1,-2,-3 heterozygotes, suggesting that the Ge-1,-2,-3 phenotype may be caused by at least two different alleles at the Ge blood group antigen locus. Hemagglutination or hemagglutination inhibition tests involving anti-Ge 1,2,3 and -Ge 1,2 as well as native and enzyme-treated normal red cells (phenotype Ge 1,2,3) or membrane and sialoglycoprotein fractions from normal erythrocytes indicate that the receptors of these sera are located within the glycosylated domain(s) of the D and/or E sialoglycoprotein(s). Our data suggest that the Ge locus encodes the polypeptide sequences of the D and E sialoglycoproteins.     

Phospholipid composition and external labeling of aminophospholipids of human En(a--) erythrocyte membranes which lack the major sialoglycorprotein (glycophorin A).

Erythrocytes of the rare human blood group En(a--) lack the major sialoglycoprotein, glycophorin A, and the cell population heterozygous for the En(a) antigen contain half the normal amount of glycophorin A. With such cells we have studied whether glycophorin A influences the phospholipid composition and the availability of aminophospholipids to external labeling reagents. We here demonstrate that the amounts of all phospholipids are closely similar in normal and variant membranes. However, using the amino-reactive reagent trinitrobenzenesulfonate, we show that phosphatidylethanolamine is more easily labeled in intact En(a--) cells as compared to normal cells, whereas phosphatidylethanolamine shows an intermediate labeling in En(a) heterozygous cells.