Glycoprotein Of Human Erythrocyte Membrane Research Articles

Diversity of the human erythrocyte membrane sialic acids in relation with blood groups

The composition of the human erythrocyte membrane (RBC) glycoprotein- and glycolipid-bound sialic acids of A, B, AB and O type donors was studied using a new method (Zanetta et al., Glycobiology 11 (2001) 663–676). In addition to Neu5Ac as the major compound, Kdn, Neu5,9Ac 2, Neu5,7Ac 2, Neu (de- N-acetylated-Neu5Ac), Neu5Ac8Me, Neu5Ac9Lt, Neu4,5Ac 2, Neu5,8Ac 29Lt and Neu5Ac8S were characterised. Among these different compounds, Neu5Ac8Me, Neu5Ac9Lt, Neu4,5Ac 2, Neu5,8Ac 29Lt and Neu5Ac8S have never been described and quantitatively determined before in human tissues or cells. Neu5Gc and its O-alkylated or O-acylated derivatives were not detected.

Structural and functional consequences of an N-glycosylation mutation (HEMPAS) affecting human erythrocyte membrane glycoproteins

Band 3, the human erythrocyte anion exchanger (AE1), and the glucose transporter (GLUT1) proteins each contain a single site of N-glycosylation that is heterogeneously glycosylated. Lectin binding and enzymatic deglycosylation assays showed that the polylactosaminyl oligosaccharide structure of these glycoproteins was altered to a high mannose or hybrid glycan form in three patients with hereditary erythroblastic multinuclearity, with a positive acidified-serum lysis test (HEMPAS). Offspring from one of the HEMPAS patients had intermediate levels of polylactosaminyl oligosaccharide associated with AE1 and GLUT1, suggesting they may have been heterozygous for the genetic defect. The array of polylactosaminyl-containing glycoproteins present in EBV-transformed lymphoblasts derived from fresh blood of HEMPAS patients was similar to control lymphoblasts. HEMPAS lymphoblasts do not therefore express the defect in polylactosamine synthesis found in erythroid cells, indicating that lymphoid cells are not deficient in the processing enzymes or contain an alternative oligosaccharide processing pathway. Purified HEMPAS band 3 had an unaltered oligomeric structure but dimers aggregated more rapidly in detergent solution than normal band 3. The altered oligosaccharide structure did not affect the sensitivity of band 3 to proteolytic digestion in intact red cells but a greater amount of HEMPAS band 3 was associated with the cytoskeleton. The transport activities of AE1 and GLUT1 in HEMPAS erythrocytes were similar to those in normal controls. This shows that the HEMPAS glycosylation defect does not impair the functional accumulation of these two important erythrocyte membrane transporters even though it produces subtle structural changes in band 3 that result in its increased cytoskeletal interaction and self association in detergent solution.Key words: anion exchanger, band 3, erythrocyte, glycoprotein, HEMPAS.

Structural and functional consequences of an N-glycosylation mutation (HEMPAS) affecting human erythrocyte membrane glycoproteins.

Band 3, the human erythrocyte anion exchanger (AE1), and the glucose transporter (GLUT1) proteins each contain a single site of N-glycosylation that is heterogeneously glycosylated. Lectin binding and enzymatic deglycosylation assays showed that the polylactosaminyl oligosaccharide structure of these glycoproteins was altered to a high mannose or hybrid glycan form in three patients with hereditary erythroblastic multinuclearity, with a positive acidified-serum lysis test (HEMPAS). Offspring from one of the HEMPAS patients had intermediate levels of polylactosaminyl oligosaccharide associated with AE1 and GLUT1, suggesting they may have been heterozygous for the genetic defect. The array of polylactosaminyl-containing glycoproteins present in EBV-transformed lymphoblasts derived from fresh blood of HEMPAS patients was similar to control lymphoblasts. HEMPAS lymphoblasts do not therefore express the defect in polylactosamine synthesis found in erythroid cells, indicating that lymphoid cells are not deficient in the processing enzymes or contain an alternative oligosaccharide processing pathway. Purified HEMPAS band 3 had an unaltered oligomeric structure but dimers aggregated more rapidly in detergent solution than normal band 3. The altered oligosaccharide structure did not affect the sensitivity of band 3 to proteolytic digestion in intact red cells but a greater amount of HEMPAS band 3 was associated with the cytoskeleton. The transport activities of AE1 and GLUT1 in HEMPAS erythrocytes were similar to those in normal controls. This shows that the HEMPAS glycosylation defect does not impair the functional accumulation of these two important erythrocyte membrane transporters even though it produces subtle structural changes in band 3 that result in its increased cytoskeletal interaction and self association in detergent solution.

Characterization of lactoferrin-binding proteins of human macrophage membrane: multiple species of lactoferrin-binding proteins with polylactosamine-binding ability.

Human lactoferrin (LF) specifically binds to human monocytic leukemia cell line THP-1 cells differentiated into macrophages, and it has been suggested that the poly-N-acetyllactosaminyl saccharide chains of LF are involved. We partially purified and characterized LF-binding proteins with affinity for polylactosamines from THP-1 cells. LF-binding activity was solubilized by nonionic detergent Triton X-100 from THP-1 cell membrane, and subjected to affinity chromatography using an LF-Sepharose column. LF-binding activity, detected by ligand blotting assay, was eluted and further fractionated by affinity chromatography using a Sepharose column coupled with band 3, a polylactosaminyl chain-containing glycoprotein of human erythrocyte membrane. LF-binding activity was separated into three fractions (frs. B1, B2, and B3). These fractions exhibited band 3-binding activity as detected by ligand blotting assay. Dodecylsulfate-polyacrylamide gel electrophoresis of frs. B1, B2, and B3, followed by detection of LF-binding activity on Western blots, indicated that frs. B1, B2, and B3 contained LF-binding proteins with a molecular mass of 35, 50 and 80, and 35-37 kDa, respectively. Binding of LF to each of the fractions on the dot blots was partially inhibited by LF oligosaccharides, band 3 oligosaccharides and lacto-N-neotetraose, each containing di-N-acetyllactosaminyl or analogous structure, Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4GlcNAc (or Glc). These results suggest that the 35, 50 and/or 80, and 35-37 kDa proteins on THP-1 cells are LF-binding proteins with polylactosamine-binding ability.

Binding characteristics of human lactoferrin to the human monocytic leukemia cell line THP-1 differentiated into macrophages.

The characteristics of the binding of human lactoferrin (LF) to the cells of a human monocytic leukemia cell line, THP-1, were investigated. 125I-Labeled LF (125I-LF) bound to THP-1 cells, and the binding increased markedly as the cells matured into macrophages (THP-1 macrophages) by stimulation with phorbol 12-myristate 13-acetate. Scatchard analysis of the binding of 125I-LF to THP-1 macrophages indicated that high and low affinity receptor sites (Kd = 0.57 x 10(-6) and 3.7 x 10(-6) M, respectively) are present on the cells. The number of these high and low affinity receptor sites were 2.4 x 10(6), and 2.5 x 10(6) per cell, respectively. Removal of iron from 125I-LF did not affect its binding to THP-1 macrophages, indicating that the binding is not dependent on Fe(III) ion. The binding of the labeled LF to THP-1 macrophages was markedly decreased following acetylation, suggesting that the amino residues of the polypeptide portion of LF play a major role in the binding. The binding of labeled LF was partially inhibited by the isolated whole oligosaccharides of LF, and by the isolated whole oligosaccharides of band 3 glycoprotein of human erythrocyte membrane which contain poly-N-acetyllactosaminyl saccharide chains, like the LF oligosaccharides. Their inhibitory activity did not depend on the terminal sialyl residues of the saccharide chains. Lacto-N-fucopentaose III and lacto-N-neotetraose, an analogous structure being present in the poly-N-acetyllactosaminyl chains of LF, also artially inhibited the binding of 125I-LF to the THP-1 macrophages. When poly-N-acetyllactosaminyl saccharide chains of 125I-LF were cleaved by endo beta-galactosidase, the binding of 125I-LF was partially reduced. These results suggest that binding of LF to THP-1 macrophages is primarily mediated by its protein component, but a short oligosaccharide structure, possibly Gal beta 1-4GlcNAc beta 1-3Gal, which is contained in the nonreducing terminal region of poly-N-acetyllactosaminyl saccharide chains of LF and band 3, and in lacto-N-fucopentaose III and lacto-N-neotetraose is also recognized by THP-1 macrophages, and this recognition partly contributes to the binding of LF to cells.

Molecular genetics of the glycophorin gene family, the antigens for MNSs blood groups: Multiple gene rearrangements and modulation of splice site usage result in extensive diversification

The purpose of the review is to describe a system of human erythrocyte membrane glycoproteins exhibiting extensive diversity. Glycophorins A and B (GPA and GPB) are the antigens of the MNSs blood groups; thus individuals bearing variant glycophorins can be readily identified by serological typing. Examination of the wide array of variants of these antigens showed that they include many forms, possibly made evident by lack of constraints due to the apparent dispensability of the parent molecules. This article reviews the molecular genetics of 25 variants of the glycophorin gene family, whose common denominator is that they arise from unequal gene recombinations or gene conversions coupled to splice-site mutations. Most rearrangements occurred within a 2-kb region mainly within GPA and GPB of the gene family and only rarely within the third member, GPE. The key feature is the shuffling of sequences within two specific exons (one of which is silent), homologous in the two parent genes. This has resulted in expression of a mosaic of sequences within this region, leading to polymorphism. The common pattern of recombinations coupled to pre-mRNA splicing was the predominant mechanism of the origin of glycophorin diversity. Thus far this mechanism appears to be unique among human gene families. It could have occurred by chance rearrangements among closely linked genes and been driven by a biological advantage, not as yet identified. This remains to be established. Nevertheless, gene rearrangements observed here are akin to those reported for the major histocompatibility complex (MHC). In the glycophorin family the small size of the region within which gene interactions have occurred and the participation of essentially only two alleles makes this relatively simpler system more focused and easier to dissect and describe molecularly.

Quantitation of the number of molecules of glycophorins C and D on normal red blood cells using radioiodinated Fab fragments of monoclonal antibodies

Two rat monoclonal antibodies (BRAC 1 and BRAC 11) have been produced. BRAC 1 recognizes an epitope common to the human erythrocyte membrane glycoproteins glycophorin C (GPC) and glycophorin D (GPD). BRAC 11 is specific for GPC. Fab fragments of these antibodies and BRIC 10, a murine monoclonal anti-GPC, were radioiodinated and used in quantitative binding assays to measure the number of GPC and GPD molecules on normal erythrocytes. Fab fragments of BRAC 11 and BRIC 10 gave values of 143,000 molecules GPC per red blood cell (RBC). Fab fragments of BRAC 1 gave 225,000 molecules of GPC and GPD per RBC. These results indicate that GPC and GPD together are sufficiently abundant to provide membrane attachment sites for all of the protein 4.1 in normal RBCs.

Quantitation of the Number of Molecules of Glycophorins C and D on Normal Red Blood Cells Using Radioiodinated Fab Fragments of Monoclonal Antibodies

AbstractTwo rat monoclonal antibodies (BRAC 1 and BRAC 11) have been produced. BRAC 1 recognizes an epitope common to the human erythrocyte membrane glycoproteins glycophorin C (GPC) and glycophorin D (GPD). BRAC 11 is specific for GPC. Fab fragments of these antibodies and BRIC 10, a murine monoclonal anti-GPC, were radioiodinated and used in quantitative binding assays to measure the number of GPC and GPD molecules on normal erythrocytes. Fab fragments of BRAC 11 and BRIC 10 gave values of 143,000 molecules GPC per red blood cell (RBC). Fab fragments of BRAC 1 gave 225,000 molecules of GPC and GPD per RBC. These results indicate that GPC and GPD together are sufficiently abundant to provide membrane attachment sites for all of the protein 4.1 in normal RBCs.

Antigenic determinants of senescent antigen of human erythrocytes are located in sialylated carbohydrate chains of Band 3 glycoprotein.

Naturally occurring IgG autoantibody against Band 3 glycoprotein of human erythrocyte membrane (anti-Band 3) recognizes the erythrocytes modified with oxidizing or SH-blocking agents as well as senescent erythrocytes. Location of the antigenic determinants of Band 3 this autoantibody recognizes was investigated by competitive inhibition studies of the antibody binding to the modified cells. Autologous IgG binds to the modified erythrocytes, and purified Band 3 totally inhibits the binding. This inhibitory activity of Band 3 was not affected by digestion of Band 3 with various proteases. Treatment of Band 3 with endo-beta-galactosidase that destroys the poly-N-acetyllactosaminyl sugar chain of Band 3 or with neuraminidase resulted in loss of the inhibitory activity. Oligosaccharides released from Band 3 by hydrazinolysis effectively inhibited the binding of autologous IgG and 125I-labeled purified anti-Band 3 to the modified cells, whereas the oligosaccharides depleted of acidic components did not. Endo-beta-galactosidase and neuraminidase destroyed the activity of the oligosaccharides, but alpha-L-fucosidase did not. Furthermore, human lactoferrin that contains sialylated two N-acetyllactosaminyl units also exhibited potent inhibitory activity, and the activity was destroyed by endo-beta-galactosidase and neuraminidase. These results indicate that the antigenic determinants of Band 3 are located in sialylated poly-N-acetyllactosaminyl sugar chains. Based on this finding, mechanism of appearance of the antigen on senescent erythrocytes recognized by anti-Band 3 (senescent antigen) was discussed.

Contribution of the N-linked carbohydrate of erythrocyte antigen CD59 to its complement-inhibitory activity.

The contribution of N-linked carbohydrate to the complement-inhibitory function of the human erythrocyte membrane glycoprotein, CD59, was investigated. Amino acid sequence analysis of tryptic peptides labeled with [3H]borohydride revealed an N-linked carbohydrate moiety at the Asn18 residue. No O-linked carbohydrate was detected, as judged by the failure of asialo-CD59 to bind peanut agglutinin and by its resistance to digestion by O-glycanase. The apparent molecular mass of CD59 was reduced from 18-20 to 14 kDa upon complete digestion with N-glycanase, with no detectable proteolysis. N-glycanase digestion of CD59 was associated with an 88 +/- 4% loss of the complement-inhibitory activity of the protein, as assessed by its capacity to protect chicken erythrocytes from lysis by the human C5b-9 proteins. By contrast, no change in function was observed after digestion of CD59 with neuraminidase, under conditions that removed greater than 60% of [3H]sialic acid residues. Despite loss of functional activity after N-glycanase digestion, we detected no change in the capacity of the deglycosylated CD59 to incorporate into erythrocyte membranes or to bind specifically and with species selectivity to the C8 and C9 components of the membrane attack complex. In order to alter the branched-chain structure of the N-linked carbohydrate of CD59 without enzymatic digestion, Chinese hamster ovary (CHO) cells transfected with cDNA for human CD59 were grown in the alpha-mannosidase inhibitor, 1-deoxymannojirimycin, resulting in conversion of approximately 70% of the membrane glycoprotein to a high mannose. When grown in the presence of 1-deoxymannojirimycin, the C5b-9-inhibitory activity of CD59 expressed on the surface of the transfected CHO cells was reduced by an amount comparable to that observed for the N-glycanase digested protein. Taken together, these data suggest that normal glycosylation of Asn18 in CD59 is required for the normal expression of its complement-inhibitory activity on membrane surfaces, although these N-linked sugar residues do not contribute to CD59's affinity for the C8 and C9 components of the C5b-9 complex.

Identification and immunochemical characterization of the human erythrocyte membrane glycoproteins that carry the Xga antigen.

When the membrane components of Xg(a+) erythrocytes were separated by electrophoresis and immunoblotted with an anti-Xga of human origin, two diffuse bands of approximate Mr 22,000-25,000 and 26,500-29,000 were stained. These reactive components were not evident in membranes from proteinase-treated Xg(a+) erythrocytes. Neuraminidase treatment of erythrocytes before immunoblotting resulted in one diffuse Xga-reactive band, the leading edge of which had a slightly increased mobility corresponding to a decrease in Mr of approx. 1500. These results suggest that the membrane components that carry Xga are sialoglycoproteins. A genetic relationship exists between Xga and the antigen recognized by the monoclonal antibody 12E7. An immunochemical comparison of the structures that carry the Xga and 12E7 antigens demonstrated that they differ in Mr and in the way in which they are modified by neuraminidase. This is in accord with evidence that Xga and 12E7 are the products of two separate structural loci.

Effect of endoglycosidase F-peptidyl N-glycosidase F preparations on the surface components of the human erythrocyte

Endo- N-acetyl-β- d-glucosaminidase F-Peptidyl N-glycosidase F preparations (abbreviated Endo F) and endo-β- d-galactosidase were used to study the major human erythrocyte membrane glycoproteins and the components carrying the blood group A, B, Rhesus (D), and Duffy (Fy a) antigens. The results are consistent with the known presence of an N-glycosyl-linked oligosaccharide on sialoglycoprotein α and the absence of such an oligosaccharide from sialoglycoprotein δ. Under the conditions used, only a portion of the N-glycosyl-linked oligosaccharides on band 3 molecules were cleaved by Endo F alone or by Endo F in combination with endo-β- d-galactosidase. Immunoblotting experiments showed that treatment of red cells with Endo F alone had little effect on the components carrying blood group A and B antigen activity. However, Endo F used in combination with endo-β- d-galactosidase caused a substantial reduction in the binding of monoclonal anti-A and anti-B antibodies. The results clearly show that sialoglycoproteins α and δ carry little or no blood group A or B activity. Endo F alone, or in combination with endo-β- d-galactosidase, had no effect on the electrophoretic mobility of the Rh(D) polypeptide, supporting previous suggestions that this membrane polypeptide is unusual in not being glycosylated. Endo F had a dramatic effect on the electrophoretic mobility of the component(s) carrying blood group Fy a activity. The diffuse Fy a component of M r 38 500–90 000 was sharpened to a band of M r 26 000. Either endo-β- d-galactosidase or neuraminidase treatment reduced the M r of the Fy a component(s) but did not significantly sharpen the bands, suggesting that the Fy a component contains between 40–50% by mass of N-glycosyl-linked oligosaccharides.

Blood group antigens: synthesis of Ss antigenic peptides related to human glycophorin B.

The human Ss blood group antigens are located on glycophorin B, a minor human erythrocyte membrane glycoprotein. The structural difference in Ss antigens is determined by a Met/Thr polymorphism at position 29. This report describes the first synthesis of the two peptides carrying the Ss specificities, SS: Asn-Gly-Glu-Met-Gly-Gln-Leu-Val and ss: Asn-Gly-Glu-Thr-Gly-Gln-Leu-Val.

Association of human erythrocyte membrane glycoproteins with blood-group Cad specificity.

Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of erythrocyte membranes from a blood-group-B individual with the rare Cad phenotype indicates a lower-than-normal mobility of the main sialoglycoproteins, suggesting an increase in apparent molecular mass of 3kDa and 2kDa respectively for glycoprotein alpha (synonym glycophorin A) and glycoprotein delta (synonym glycophorin B). Since the chief structural determinant of Cad specificity is N-acetylgalactosamine, the membrane receptors have been isolated by affinity binding on immobilized Dolichos biflorus (horse gram) lectin. The predominant species eluted from the gel was the abnormal glycoprotein alpha, whereas in control experiments no material could be recovered from the adsorbent incubated with group-B Cad-negative erythrocyte membranes. After partition of the membranes with organic solvents, the blood-group-Cad activity was found in aqueous phases containing the sialoglycoproteins, but not in the organic phases containing simple or complex glycolipids, which, however, retained the blood-group-B activity. The carbohydrate composition of highly purified lipid-free glycoprotein alpha molecules prepared from Cad and control erythrocytes was determined. Interestingly the molar ratio of N-acetylneuraminic acid to N-acetylgalactosamine was equal to 2:1 in the case of controls and equal to 1:1 in the case of Cad erythrocytes. Taken together these results suggest that Cad specificity is defined by N-acetylgalactosamine residues carried by the alkali-labile oligosaccharide chains attached to the erythrocyte membrane sialo-glycoproteins.

Characterization of an intrinsic human erythrocyte membrane glycoprotein with preferential decay accelerating activity for the C4b2a AS COMPARED with the C3bBb C3-convertase

Characterization of an intrinsic human erythrocyte membrane glycoprotein with preferential decay accelerating activity for the C4b2a AS COMPARED with the C3bBb C3-convertase

Isolation of a human erythrocyte membrane glycoprotein with decay-accelerating activity for C3 convertases of the complement system.

Isolation of a human erythrocyte membrane glycoprotein with decay-accelerating activity for C3 convertases of the complement system.

Identification of the membrane glycoprotein that is the C3b receptor of the human erythrocyte, polymorphonuclear leukocyte, B lymphocyte, and monocyte.

A human erythrocyte membrane glycoprotein of 205,000 mol wt (gp205) has been identified as the C3b receptor of the erythrocyte, polymorphonuclear leukocyte (PMN), B lymphocyte, and monocyte. Initially, gp205 was sought and characterized as a constituent of the human erythrocyte membrane that can impair activation of the alternative complement pathway by inducing loss of function of the properdin-stabilized amplification C3 convertase (C3b,Bb,P) through displacement of Bb from C3b and by promoting cleavage-inactivation of C3b by C3b inactivator. These inhibitory activities of gp205 suggested that this membrane glyeoprotein had an affinity for C3b and prompted an analysis of its possible identity as the C3b receptor of human peripheral blood cells. The F(ab')2 fragment of rabbit IgG anti-gp205 inhibited the formation of rosettes with sheep EC3b of human erythroeytes, B lymphocytes, monocytes and PMN in a dose-response manner; the 50 percent inhibitory doses were 0.13/mug/ml, 0.90 mug/ml, 1.25 mug/ml, and 1.20 mug/ml of F(ab')2, respectively. Anti-gp205 did not impair the formation of rosettes by monocytes and B lymphocytes with sheep EC3bi or with EC3d. Scatchard analysis of the number of specific (125)I-F(ab')(2) anti-gp205 binding sites/cell revealed 950 sites/erythrocyte, 21,000 sites/cell of B lymphocyte preparation, 57,000 sites/PMN, and 48,000 sites/monocyte, indicating that the higher concentrations of antibody that had been required for inhibition of rosette formation by the nucleated cells reflected larger numbers of receptors on these cells. Direct evidence for the identity of gp205 as the C3b receptor of the four cell types was obtained when detergent-solubilized membrane proteins of the surface-radioiodinated cells were reacted with anti- gp205 and the immunoprecipitate was analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. In each instance, the antigenic material reacting with anti-gp205 represented a single protein with an apparent 205,000 mol wt. Thus, gp205 is the C3b receptor of human erythrocytes, PMN, B lymphocytes, and monocytes.

Developmental change and genetic defect in the carbohydrate structure of band 3 glycoprotein of human erythrocyte membrane.

The chemical structure of Band 3 glycopeptide prepared from erythrocytes of normal adult (blood group OI), umbilical cord vessels (Oi), and an i adult variant who fails to develop I antigen (Oi), has been compared. Band 3 glycopeptide of cord erythrocytes gave, on permethylation analysis, predominantly 2,4,6-tri-O-methylgalactose and 3,6-di-O-methyl-2-N-methylacetamido-2-deoxyglucose, whereas the same glycopeptide of normal adult erythrocytes gave much higher amounts of 2,3,4,6-tetra-O-methylgalactose and 2,4-di-O-methylgalactose as compared with that of cord erythrocytes. Band 3 glycopeptide from i adult showed the same methylation pattern as cord-Band 3 glycopeptide. In accordance with these results, Band 3 glycopeptide of cord and i adult erythrocytes were hydrolyzed to mostly small oligosaccharides by endo-beta-galactosidase from Escherichia freundii, whereas that of normal adult produced a number of oligosaccharides with various sizes which was caused by branched structures. Based on these results and structures of released oligosaccharides, the major developmental change of carbohydrate structure in the erythrocyte membrane is the conversion of linear repeating Galbeta1 leads to 4GlcNAcbeta1 leads to 3Gal to a branched Galbeta 1 leads to 4GlcNAcbeta 1 leads to 3 (R leads to 6) Gal structure. i individual may result from the lack of the branching enzyme.

Solubilization of human erythrocyte membrane glycoproteins by triton X-100.

1. The enzymic removal of sialic acid residues from the glycoproteins of the human erythrocyte decreases the solubilization of membrane glycoprotein by Triton X-100. 2. The solubilization of asialoglycoprotein by Triton X-100 may be restored by the addition of borate. 3. Use of this non-ionic detergent in the presence of borate, as a general procedure for the mild solubilization of membrane glycoproteins deficient in sialic acid residues, is discussed.

Investigation into the occurrence and structure of lectin receptors on human and bovine erythrocytes milk fat globule and lymphocyte plasma-membrane glycoproteins.

The reduced disaccharide beta-D-galactosyl(1-3)-N-acetyl-D-galactosaminitol was released, after alkaline borohydride and neuraminidase treatment, from human and bovine erythrocyte membrane and fat globule membrane glycoproteins as well as from human and bovine lymphocyte plasma membranes. This disaccharide was found to be substituted to different extents by sialic acid in the various fractions. beta-D-Galactosyl(1-3)-N-acetyl-D-galactosamine is the immunodominant disaccharide of the Thomsen-Friedenreich antigen (TF) and was detected serologically using specific lectins and antibody-containing sera. Bovine fat globule membrane glycoproteins were the only source of Tn antigen. The Tn antigen was detected by anti-Tn-containing sera and by the lectins from Salvia sclarea and Helix pomatiea as well as gas chromatographic detection of N-acetylgalactosaminitol after alkaline borohydride treatment of the native glycoproteins. Human milk fat globule membrane glycoproteins showed the presence of blood group A substance by reaction with rabbit anti-A serum.