Reconstitution of the masking effect of sialic acid groups on sialidase-treated erythrocytes by the action of sialyltransferases

Glutardialdehyde-fixed or native rat erythrocytes were partially desialylated by the action of Vibrio cholerae sialidase, resulting in the binding of these cells to homologous peritoneal macrophages. Resialylation of these erythrocytes by purified alpha-(2----3)- or alpha-(2----6)-sialyltransferases with CMP-N-acetylneuraminic acid led to the incorporation of 60-80% of the enzymically released sialic acid. Binding of the resialylated erythrocytes to peritoneal macrophages was reduced when compared with corresponding, partially desialylated erythrocytes. Thus, the amount of transferred sialic acid was sufficient to demonstrate reconstitution of the masking effect of sialic acids.     

Recognition and invasion of human erythrocytes by malarial parasites: contribution of sialoglycoproteins to attachment and host specificity

The receptivity of human erythrocytes to invasion by Plasmodium falciparum merozoites can be decreased by neuraminidase or trypsin treatment, an observation that supports a role for the erythrocyte sialoglycoproteins (glycophorins) in invasion. We have found that alpha 1-acid glycoprotein (AGP), added to in vitro cultures, can restore invasion of enzyme-treated human erythrocytes. AGP is structurally different from the glycophorins although it does carry 12% sialic acid. Its ability to restore receptivity to desialylated cells is dependent on its sialic acid complement, its concentration, and its binding to the erythrocyte surface. We present evidence that AGP forms a bridge between the merozoite and the enzyme-treated erythrocyte that allows the stronger and more complex interactions of invasion to proceed. We suggest that the glycophorins play the same role on the surface of the intact erythrocyte.     

Rosetting of human T lymphocytes with sheep and human erythrocytes. Comparison of human and sheep ligand binding using purified E receptor

Previous studies have shown that the purified T lymphocyte glycoprotein, cluster differentiation 2 (CD2) (also known as T11, lymphocyte function-associated antigen (LFA)-2, and the erythrocyte (E) rosette receptor) interacts with the LFA-3 molecule on human E. We have examined the interaction of the purified CD2 molecule with the T11 target structure (T11TS) molecule on sheep E, and compared the two interactions. Purified, 125I-labeled CD2 bound to sheep E and the binding was inhibited by anti-T11TS monoclonal antibody (mAb). Reciprocally, the binding of T11TS mAb to sheep E was inhibited by pretreatment of sheep E with purified CD2. High concentrations of purified CD2 aggregated sheep E, possibly by inserting into the membrane, and the aggregation was inhibited by T11TS mAb. The affinity and number of binding sites for purified CD2 on sheep and human E was found to be similar, with Ka of 9 X 10(7)/M and 6 X 10(7)/M and 9800 and 8300 CD2 binding sites/E, respectively. Thus, the human T lymphocyte CD2 molecule is a receptor that cross-reacts between LFA-3 on human E and T11TS on sheep E, suggesting that LFA-3 and T11TS are functionally homologous ligands. As measured by saturation mAb binding, there are 8100 and 3900 ligand molecules/sheep and human E, respectively. Human and sheep E have surface areas of 145 and 54 micron 2, respectively. The 3.2- to 5.6-fold higher ligand density on sheep E appears to account for the ability of sheep but not human E to rosette with certain types of human T lymphocytes.     

Neu5Acalpha3Gal is part of the Helicobacter pylori binding epitope in polyglycosylceramides of human erythrocytes.

The sialic acid dependent binding by the human pathogen Helicobacter pylori to polyglycosylceramides of human erythrocytes was investigated. Polyglycosylceramides, complex glycosphingolipids with a branched N-acetyllactosamine core, were isolated from human erythrocytes, blood group O, and subfractionated after peracetylation by anion-exchange chromatography. Three subfractions were deacetylated, analysed by matrix-assisted laser desorption ionization-time of flight MS and 2D 1H NMR spectroscopy. The observed mass ranges were m/z = 3093-7622, 3968-7255 and 3459-7987 in the mass spectra of the first, second and third fractions, respectively. The observed ions agreed with the general formula Hex(x+2)HexNAcxFucyNeu5AczCer. Two-dimensional 1H total correlation spectra of the mixtures showed that the first fraction contained 3-linked sialic acid and the second and third fractions contained both 3-linked and 6-linked sialic acid. Thin-layer chromatogram binding assays using the lectins from Maackia amurensis, specific for Neu5Acalpha3Galbeta4GlcNAc, and Sambucus nigra, specific for Neu5Acalpha6Gal/GalNAc, were used to confirm this distribution. H. pylori recognized all three fractions in the binding assay, indicating that the 3-linked, rather than 6-linked, sialic acid is essential for binding.     

Characterization of normal, glutathione-deficient and arginase-deficient sheep erythrocytes by 1H-NMR spectroscopy

Normal sheep erythrocytes as well as glutathione- (GSH-) deficient and arginase-deficient sheep erythrocytes have been characterized by 1H nuclear magnetic resonance spectroscopy. The GSH deficiency is a result of defective amino acid transport (lesion 1), diminished gamma-glutamylcysteine synthetase activity (lesion 2), or both (lesions (1 + 2)). 1H-NMR spectra of normal sheep erythrocytes are similar to those for human erythrocytes, and consist of resonances from a number of small intracellular molecules, including GSH. In contrast, the resonances for GSH in the GSH-deficient erythrocytes are much weaker, and strong resonances are observed for lysine, threonine and ornithine or arginine, depending on the arginase activity, in erythrocytes with lesion 1 and lesions (1 + 2). A comparison of the intensity of GSH resonances in spectra for normal and GSH-deficient erythrocytes with GSH levels determined spectrophotometrically following reaction with the nonspecific thiol reagent 5,5'-dithiobis(2-nitrobenzoate) (DTNB) indicates that either not all of the GSH determined with Ellman's reagent is free and observable by 1H-NMR or that not all of the thiol determined by Ellman's reagent is GSH. If the latter is the case, the GSH levels determined with Ellman's reagent for erythrocytes with lesions (1 + 2) are most affected, which might account for their high susceptibility to oxidative stress.     

Serologic cross-reactivity of T11 target structure and lymphocyte function-associated antigen 3. Evidence for structural homology of the sheep and human ligands of CD2

T11 target structure (T11TS) and lymphocyte function-associated antigen (LFA) 3 are the cell-surface glycoproteins on sheep and human erythrocytes (E) binding to cluster differentiation 2 (the E-receptor) on T cells in E rosette formation. Here we show that this functional cross-reactivity is most likely due to a structural homology of these molecules. A rabbit antiserum to sheep T11TS is shown to cross-react with LFA-3 in several independent assays: (a) rabbit anti-T11TS antiserum blocks the formation of E rosettes by human T cells with both autologous and xenogeneic (sheep) E by binding to the respective E; (b) the antiserum blocks the binding of anti-LFA-3 monoclonal antibody to human E; and (c) it reacts with purified LFA-3 in Western blotting. Together, these findings demonstrate that T11TS on sheep E and LFA-3 on human E are serologically related, providing further support for the notion that T11TS and LFA-3 are the sheep and human forms of the same cell interaction molecule.     

Identification of N-glycolylneuraminic acid-containing gangliosides of cat and sheep erythrocytes. 252Cf fission fragment ionization mass spectrometry in the analysis of glycosphingolipids

A number of gangliosides were isolated from cat and sheep erythrocytes for use in analyzing the specificity of a panel of human anti-heterophile monoclonal antibodies. The structures of these compounds were determined by a combination of different procedures, including sugar analysis, glycosidase treatment, periodate oxidation, TLC immunostaining, methylation analysis, and mass spectrometry. These methods identified the cat erythrocytes gangliosides (C1 and C2) as N-glycolylneuraminic acid (NeuGc)-containing hematosides; C1 was shown to be NeuGc alpha 2----8NeuGc alpha 2----3Gal beta I----4Glc-Cer [NeuGc)2GD3) and C2 to be NeuAc alpha 2----8NeuGc alpha 2----3Gal beta 1----4Glc-Cer [NeuAc-NeuGc-)GD3). The two sheep gangliosides (S1 and S2) were found to be novel glycolipids based on the paragloboside sequence; S1 was identified as NeuGc alpha 2----8NeuGc alpha 2----3Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4Glc-Cer [NeuGc)2-disialylparagloboside) and S2 as NeuAc alpha 2----8NeuGc alpha 2----3Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4Glc-Cer [NeuAc-NeuGc-)-disialylparagloboside). Structural analysis of these compounds was aided by the use of 252Cf fission fragment ionization time-of-flight mass spectrometry. This method provided easily interpretable spectra on methylated derivatives which were particularly useful in determining the sialic acid composition of the gangliosides and the sequence of their disialosyl side chains.     

Role of linkage specific 9-O-acetylated sialoglycoconjugates in activation of the alternate complement pathway in mammalian erythrocytes

Substitution of the -OH group at C-9 of sialic acid by an O-acetyl ester has been suggested to modify various biological phenomena that are regulated by sialic acids. Amongst them, enhancement of erythrocyte lysis by 9-O-acetylated sialic acid determinants through modulation of the alternate pathway of complement has been extensively studied on murine erythrocytes [1]. A variable expression of linkage specific 9-O-acetylated sialoglycoconjugates as defined by the lectinogenic epitope of Achatinin-H namely 9-O-acetylated sialic acid 26Gal NAc was identified on rabbit, guinea pig, hamster, rat, mouse and human erythrocytes. This differential expression of linkage specific 9-O-acetylated sialoglycoconjugates strongly correlated with the susceptibility of mammalian erythrocytes to lysis by the alternate pathway of complement. Additionally, low levels of antibodies directed against O-acetylated sialic acids in these mammalian species suggested that these constitutively present determinants have low immunogenicity. Taken together, our results indicate that complement mediated hemolysis depends not simply upon the extent of surface 9-O-acetylated sialic acids present but more importantly upon the specific linkage.     

An unique specificity of a sialic acid binding lectin AchatininH, from the hemolymph of Achatina fulica snail

A sialic acid-binding lectin, AchatininH, from the hemolymph of Achatina fulica snail is found to be highly specific for 9-0-acetyl sialic acid. The binding specificity of AchatininH distinguishes it from other known sialic-acid specific lectins which usually show a broader range of specificity for sialic acid. It is even better than crab lectin which shows specificity for both 4- and 9-0-acetylated derivatives of sialic acid. This limited specificity of AchatininH appear to account for the fact that it agglutinates only rabbit, rat and guinea pig erythrocytes which contain 9-0-acetylated sialic acid but not horse (mainly contain 4-0-acetylated sialic acid), human, monkey, sheep, goat and chicken erythrocytes which contain either N-acetyl or N-glycolyl neuraminic acid but no 0-acetylated derivatives. This finding was further supported by the potent inhibition of hemagglutination by free 9-0-acetylated neuraminic acid and by several glyco shingolipids of human origin having 0-acetylated sialic acid.     

Nucleoside transport in human and sheep erythrocytes. Evidence that nitrobenzylthioinosine binds specifically to functional nucleoside-transport sites.

Nitrobenzyl[35S]thioinosine binding and nitro[3H]benzylthioinosine binding to nucleoside-permeable and nucleoside-impermeable sheep erythrocyte membranes was investigated, and compared with that found for human erythrocytes. High-affinity nitrobenzylthioinosine-binding sites (apparent KD congruent to 1 nM) were present on human and nucleoside-permeable but not nucleoside-impermeable sheep erythrocyte membranes (8400 and 18 sites/cell for human and sheep nucleoside-permeable sheep erythrocytes was displaced by nitrobenzylthioguanosine and dipyridamole. Uridine, inosine and adenosine inhibited binding. The smaller number of nitrobenzylthioinosine sites on nucleoside-permeable cells compared with human erythrocytes corresponded to a considerably lower Vmax. for uridine influx in these cells (0.53 X 10(-20) mol/cell per s at 25 degrees C compared with 254 X 10(-20) mol/cell per s). It is suggested that high-affinity nitrobenzylthioinosine binding represents a specific interaction with functional nucleoside-transport sites. The uridine-translocation capacity for each transport site at 25 degrees C is 180 molecules/site per s for both nucleoside-permeable sheep cells and human erythrocytes (assuming a 1:1 interaction between nitrobenzylthioinosine and the nucleoside-transport system).     

Substrate specificity of the Trypanosoma cruzi trans-sialidase.

Trypanosoma cruzi trypomastigotes acquire sialic acid (SA) from host glycoconjugates by means of a plasma membrane-associated trans-sialidase (TS). Here we study the substrate specificity of TS, which differs from all known sialyltransferases in that it does not require cytidine monophosphate (CMP)-SA as donor. The T. cruzi TS reversibly transfers SA to saccharides with terminal beta-Gal (but not alpha-Gal) residues. Donors are saccharides with SA linked to terminal beta-Gal residues by (alpha 2-3), but not (alpha 2-6) bonds. The type of beta-linkage of the terminal Gal residue is of minor importance (beta 1-4 and beta 1-6 are slightly better than beta 1-3), whereas chain length and the structure of additional vicinal sugar residues are not relevant. SA on the surface of living trypomastigotes of T. cruzi is transferred back and forth between the parasite surface and acceptor molecules with terminal beta-Gal, either in solution or on the surface of neighbouring mammalian cells. Addition of fucose residue on or close to the terminal galactose impairs TS activity. As a consequence, the enzyme acts poorly on the E-selectin ligand sialyl-Lewisx and its precursor Lewisx, and in vitro adhesion of TS-treated neutrophils to L-cells expressing L-selectin is not affected. Modifications in the structure of the (alpha 2-3)-linked N-acetyl-neuraminic acid (Neu5Ac) (deoxy or methoxy) of the donor molecules do not impair transfer if the changes are at C9, whereas changes at C4, C7 and C8 impair the ability to donate the modified SA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of carbohydrates on the pharmacokinetics and biological activity of equine chorionic gonadotropin in vivo.

The sialylation of eCG was examined to determine its influence on the in vivo metabolism and biological activity of the molecule. Sialic acid was decrementally removed from eCG by incubation with agarose-linked neuraminidase for varying time periods. Pharmacokinetic parameters for the disappearance of 4,000 IU (267 micrograms) of three desialylated eCG preparations (20%, 53%, and 80% sialic acid removed) and control eCG were determined in sheep. The clearance rate of eCG increased (p less than 0.05) with each decrement of sialic acid. The removal of 53% sialic acid enhanced the distribution of eCG into the tissues, compared to control and 20% desialylated eCG (p less than 0.05), presumably because of increased lipid solubility and decreased molecular size. Desialylation to 53% did not alter the elimination half-life of eCG. The removal of 80% sialic acid resulted in the disappearance of eCG from the serum within 1 h, whereas control eCG was still present at 120 h. In vivo trials in rats disclosed that the control eCG preparation increased ovulatory rate at doses of 10-100 IU and ovarian weight at doses of 10-300 IU relative to saline-treated rats (p less than 0.01). The 20% desialylated eCG induced superovulatory and ovarian weight responses, but 100-500 IU were required to achieve the same result as that produced by control eCG. The 53% and 80% desialylated eCG preparations induced a mild superovulatory response (p less than 0.01) but no ovarian weight response. It was concluded that sialic acid was significant to the distribution and disappearance of eCG. The effects of carbohydrate removal on biological activity (e.g., superovulation) are primarily a function of clearance rate rather than tissue-specific phenomena.     

Sequestration of neuraminidase-treated erythrocytes

Summary Scintigraphic experiments and radioactivity measurements of tissues have shown that the radioactivity of ⁵¹Cr-labelled and neuraminidase-treated rabbit erythrocytes is rapidly accumulated in liver and spleen. Sequestration of these erythrocytes by liver and spleen was demonstrated by light and electron microscopy of these tissues after perfusion of the rabbits with solutions for tissue fixation. In liver the phagocytic activity of Kupffer cells was increased after injection of desialylated erythrocytes, while in spleen a significantly enhanced number of erythrocytes was found attached to the sinusoidal walls and in the reticulum of the red pulp.It was shown by scanning electron microscopy that neuraminidasetreatment did not influence the shape of erythrocytes.Desialylated and ⁵¹Cr-labelled erythrocytes from the cow are rapidly cleared from the blood-stream with a half-life time of about 3 h.It was shown in an in-vitro test that they adsorb to surviving slices from liver and spleen derived from the same animal. The amount of radioactivity adsorbed is appreciably enhanced in the presence of homologous serum when compared with buffer only.Human neuraminidase-treated erythrocytes are agglutinated in the direct and especially in the indirect Coombs-tests. The involvement of T-antigen in this phenomenon was largely excluded.The in vitro experiments and antibody consumption tests suggest that immunoglobulins (IgG) and complement from serum may be involved in recognition and sequestration of desialylated erythrocytes by macrophages in vivo.Part of this work has been reported at the 3. Int. Symposium on Glycoconjugates: Functions in Animals, Brighton, Great Britain, July 6–12, 1975Part of a doctoral thesis, Abteilung für Naturwissenschaftliche Medizin, Ruhr-Universität BochumWe are greatly indebted to Mrs. A. Chen Stute for her help with the Scintigraphic studies. We also thank Prof. Dr. W. Opferkuch, Universität Bochum, for discussion, Prof. Dr. W. Kaufmann, Institut für Milchforschung, Kiel-Schaedtbek, for valuable advices, and Mr. H.G. Richter, Universität Bochum, for the scanning electron microscopic studies of erythrocytes. A gift of human -globulin from Dr. W. Schneider from the Blutspendezentrale Hagen and a sample of desialylated N-blood-group substance from Prof. Dr. G. Uhlenbruck, Universität Köln, are gratefully acknowledged. This work was financially supported by the Deutsche Forschungsgemeinschaft (grants Scha 202/3 and 202/5) and by the Fonds der chemischen Industrie     

Isolation of atherogenic modified (desialylated) low density lipoprotein from blood of atherosclerotic patients: separation from native lipoprotein by affinity chromatography

A part of low density lipoproteins (LDL) isolated from the blood of healthy subjects and patients with coronary atherosclerosis bind to a Sepharose-linked Ricinus communis agglutinin, a lectin that interacts specifically with galactose residues. Bound LDL can be replaced by galactose, but not other saccharide constituents of the LDL molecule (mannose, glucose, N-acetylglucosamine, sialic acid). Bound LDL subfraction has a 2-3-fold lower content of sialic acid as compared with unbound LDL. The blood content of desialylated LDL in atherosclerotic patients was about 3-fold higher (1.5- to 6-fold) than in healthy subjects. Desialylated LDL induced a 2- to 4-fold more intensive accumulation of total cholesterol in cultured human aortic intimal cells. Unbound LDL had no effect on intracellular deposition of lipids. It is suggested that the subfraction of desialylated LDL may be responsible for the atherogenicity of LDL isolated from blood of atherosclerotic patients.     

The binding of calcium ions by erythrocytes and `ghost''-cell membranes

1. Washed human erythrocytes, suspended in iso-osmotic sucrose containing 2.5mm-calcium chloride, bind about 400mug-atoms of calcium/litre of packed cells. Sucrose may be replaced by other sugars. 2. Partial replacement of sucrose by iso-osmotic potassium chloride diminishes the uptake of calcium, 50% inhibition occurring at about 50mm-potassium chloride. 3. Other univalent cations behave like potassium, whereas bivalent cations are much more inhibitory. The tervalent cations, yttrium and lanthanum, however, are the most effective inhibitors of calcium uptake. 4. An approximate correlation exists between the calcium uptake and the sialic acid content of erythrocytes of various species and of human erythrocytes that have been partially depleted of sialic acid by treatment with neuraminidase. However, even after complete removal of sialic acid, human erythrocytes still bind about 140mug-atoms of calcium/litre of packed cells. 5. A Scatchard (1949) plot of calcium uptake at various Ca(2+) concentrations in the suspending media shows the presence of three different binding sites on the external surface of the human erythrocyte membrane. 6. Erythrocyte ;ghost' cells, the membranes of which appear to be permeable to Ca(2+) ions, can bind about 1000mug-atoms of calcium per ;ghost'-cell equivalent of 1 litre of packed erythrocytes. This indicates that there are also binding sites for calcium on the internal surface of the erythrocyte membrane.     

Roles of plasma proteins and surface negative charge of erythrocytes in erythrocyte aggregation

The effect of plasma proteins (and IgG fragments) and sialic acid content of erythrocytes on the aggregation of human erythrocytes was quantitatively examined by using a rheoscope combined with a television image analyser and a computer. (1) The velocity of erythrocyte aggregation by plasma proteins was increased with increasing in their molecular weight, i.e., IgG less than IgA less than fibrinogen less than IgM. F(ab')2. Fab and Fc could not induce the aggregation. (2) The aggregation induced by fibrinogen was accelerated by IgG and its peptic fragment, F(ab')2, but was unaffected by the plasmic fragments, Fab and Fc. The accelerating effect by IgG and F(ab')2 was inhibited by Fab and Fc. (3) The aggregation of erythrocytes was accelerated by decreasing the sialic acid content (due to the reduction of the electrostatic repulsive force among erythrocytes), and the effect of desialylation on the IgG-induced aggregation was greater than that of desialylation on the fibrinogen-induced aggregation. (4) The roles of plasma proteins and of sialic acid content of erythrocytes on the aggregation of erythrocytes were discussed.     

GSH biosynthesis in glutathione deficient erythrocytes from Finnish landrace and Tasmanian merino sheep.

1. The maximum activities of the enzymes for the biosynthesis of GSH (gamma-glutamyl-cysteine synthetase and GSH synthetase) have been assayed in high GSH and low GSH erythrocytes from Tasmanian Merino and Finnish Landrace sheep. 2. For the Merinos, the activities (mumol product/g haemoglobin per min +/- S.E.M. (n)) in the high and low GSH erythrocytes respectively were: gamma-glutamyl-cysteine synthetase: 0.776 +/- 0.065 (11) and 0.375 +/- 0.063 (13); and GSH synthetase: 0.069 +/- 0.003 (11) and 0.066 +/- 0.002 (13). 3. For the Finnish Landrace sheep the activities in the high and low GSH erythrocytes respectively were: gamma-glutamyl-cysteine synthetase: 0.595 +/- 0.063 (12) and 0.555 +/- 0.033 (10) and gamma-glutamyl-cysteine synthetase: 0.073 +/- 0.002 (12) and 0.070 +/- 0.002 (10). 4. gamma-Glutamyl-cysteine synthetase was markedly inhibited by physiological GSH concentrations. No evidence was found for the presence of an inhibitor of GSH biosynthesis (other than GSH) in low GSH erythrocytes from Finnish Landrace sheep. 5. Although for the Merinos the low GSH trait can be explained in terms of a diminished activity of gamma-glutamyl-cysteine synthetase, no such explanation is tenable for the Finnish Landrace sheep.     

Trans-sialidase recombinant protein mixed with CpG motif-containing oligodeoxynucleotide induces protective mucosal and systemic trypanosoma cruzi immunity involving CD8+ CTL and B cell-mediated cross-priming

The Trypanosoma cruzi trans-sialidase (TS) is a unique enzyme with neuraminidase and sialic acid transfer activities important for parasite infectivity. The T. cruzi genome contains a large family of TS homologous genes, and it has been suggested that TS homologues provide a mechanism of immune escape important for chronic infection. We have investigated whether the consensus TS enzymatic domain could induce immunity protective against acute and chronic, as well as mucosal and systemic, T. cruzi infection. We have shown that: 1) TS-specific immunity can protect against acute T. cruzi infection; 2) effective TS-specific immunity is maintained during chronic T. cruzi infection despite the expression of numerous related TS superfamily genes encoding altered peptide ligands that in theory could promote immune tolerization; and 3) the practical intranasal delivery of recombinant TS protein combined with a ssDNA oligodeoxynucleotide (ODN) adjuvant containing unmethylated CpG motifs can induce both mucosal and systemic protective immunity. We have further demonstrated that the intranasal delivery of soluble TS recombinant Ag combined with CpG ODN induces both TS-specific CD4(+) and CD8(+) T cells associated with vaccine-induced protective immunity. In addition, optimal protection induced by intranasal TS Ag combined with CpG ODN requires B cells, which, after treatment with CpG ODN, have the ability to induce TS-specific CD8(+) T cell cross-priming. Our results support the development of TS vaccines for human use, suggest surrogate markers for use in future human vaccine trials, and mechanistically identify B cells as important APC targets for vaccines designed to induce CD8(+) CTL responses.     

Total sialic acid content of glycophorins during senescence of human red blood cells.

Glycophorins extracted from membranes of young and old human red blood cells have within an error of +/- 1.5% the same sialic acid content when referred to a relative measure of the number of glycophorins. The degree of surface iodination in glycophorins, which was shown to be the same in young and old cells, served as this relative measure. This finding implies that senescent human red blood cells hardly reveal desialylated surface proteins (less than or equal to 3%). However, the sialic acid content per cell was repeatedly reported to be 10 to 15% lower in old than in young cells. Therefore, we conclude 1) that human red blood cells lose intact glycophorin together with membrane during red blood cell senescence, and 2) that removal of desialylated and senescent red blood cells from the circulation proceeds by different routes.     

The effects of neuraminidase on concanavalin a agglutination of erythrocytes: Evidence for adsorption of neuraminidase to erythrocyte membrane

Neuraminidase-treated human erythrocytes, but not untreated erythrocytes, were agglutinated by concanavalin A. The degree of concanavalin A agglutinability was not directly related to sialic acid removal by neuraminidase. While maximal sialic acid release was obtained with 5 units neuraminidase/2 × 10⁹ erythrocytes, maximal concanavalin A agglutination was only obtained after exposure to 20 units neuraminidase. Binding of ³H-concanavalin A by erythrocytes was 10-fold higher with rabbit compared to human red cells.Neuraminidase treatment of human erythrocytes caused a relative increase in ³H-concanavalin binding, but the absolute amount was still 10-fold less than that bound to rabbit erythrocytes. Specific adherence of neuraminidase to Con A-Agarose could not be demonstrated. There was no evidence for contamination of the neuraminidase preparation with proteases using a sensitive assay. These studies suggest that neuraminidase adsorbs to erythrocyte membranes and leads to concanavalin A agglutination of human erythrocytes by a mechanism other than removal of sialic acid.