Modification of the erythrocyte surface in rats bearing Yoshida ascites sarcoma is brought about by a tumour variant of alpha2-macroglobulin.

Erythrocytes from the circulation of rats bearing Yoshida ascites sarcoma exhibit higher concanavalin A (ConA)-mediated agglutinability than those from normal animals. A tetrameric glycoprotein of subunit molecular mass 170 kDa, purified from the cell-free ascites fluid, was found to confer higher ConA-mediated agglutinability on erythrocytes in vitro. An antiserum to this tumour-derived protein failed to detect any cross-reactive component in normal rat plasma or in any of the normal tissues examined. An immunoreactive protein was, however, detected in blood plasma when the acute-phase reaction was stimulated by injection of turpentine. The cross-reactive acute-phase protein was purified by ConA-affinity, gel-filtration and ion-exchange chromatography, and identified as alpha2-macroglobulin. The acute-phase protein and the protein obtained from the ascites fluid have identical or very similar native and subunit molecular masses, subunit arrangement and pI. They both are able to inhibit trypsin and, as a consequence, acquire greater mobility in native PAGE. In addition, the two proteins bind to rat erythrocytes non-specifically, and in similar amounts. However, despite these similarities, the acute-phase protein is unable to enhance the agglutinability of erythrocytes. The two proteins differ in their carbohydrate content, but this differential glycosylation is not the cause of the difference in their surface modification activity. The chemically deglycosylated proteins show a small but consistent difference in the size of their polypeptides. Their tryptic peptide maps, although largely similar, show some differences, as do their amino acid compositions. It is probable that the proteins are independent members of the same (alpha-macroglobulin) family. The rat embryo is also found to express a soluble protein consisting of a 170 kDa polypeptide that cross-reacts with the antibody to the tumour-derived protein. The purified embryo protein is able to alter the ConA-mediated agglutinability of erythrocytes in vitro, and also yields a tryptic peptide map that is identical to that of the tumour-derived protein. The modification of the host cell surface in the tumour-bearing rats is thus caused by what appears to be a tumour (oncofetal?) variant of alpha2-macroglobulin.

Effect of antibodies to membrane skeletal proteins on the shape of erythrocytes and their ability to respond to shape-modulating agents. Important role of 4.1 protein in the determination/maintenance of the discoid shape of erythrocytes.

Monospecific anti-spectrin, anti-4.1 protein, anti-ankyrin, and anti-band 3 cytoplasmic domain antibodies were individually internalized in human erythrocytes by hypotonic lysis in the presence of 2 mM Mg-ATP. The concentrations of the polyclonal antibodies, or their Fab fragments, varied from 0.5 to 4.0 mg/ml. After resealing and glutaraldehyde fixation, the shape of the erythrocytes was examined by phase-contrast or scanning electron microscopy. The antibody-internalized cells were also treated, prior to fixation, with dinitrophenol, chlorpromazine (membrane-penetrable crenation- and cup-forming agents, respectively), or serum albumin (membrane nonpenetrating cup-forming agent). Anti-spectrin antibodies produced a cup shape in the cells at low concentrations (0.5 mg/ml), with a few cells showing multiple cavities. The proportion of the latter increased with increasing concentration of the antibody. The Fab fragments of the antibody were without any effect on the shape. At lower concentrations of anti-4.1 antibodies (0.5 and 1 mg/ml), crenations were observed in antibody-internalized cells. At higher antibody concentrations, the cells underwent sphering. These looked dense under the phase-contrast microscope and often were found to project vesicles from their surface via long stalks. These were generally absent on cells when viewed under the electron microscope, probably due to their breakage during processing. The Fab fragments of the anti-4.1, at equimolar concentrations, were as effective as the intact antibody in altering the cell shape. Anti-ankyrin antibodies or the Fab fragments produced no effect on the shape of the cell. The intact anti-band 3 cytoplasmic domain antibodies, but not their Fab fragments, produced cups at low concentrations and multiple cavities at higher concentrations and multiple cavities at higher concentrations. The anti-spectrin- and anti-band 3 cytoplasmic domain-internalized cells resisted the shape-modulating actions of dinitrophenol, chlorpromazine, and serum albumin. The anti-4.1 antibody-internalized cells also did not respond to dinitrophenol and serum albumin; however, they were converted to contracted cups in the presence of high concentration of chlorpromazine. The cells in which anti-ankyrin antibodies were internalized responded to the actions of the shape-changing agents similarly to normal cells. The results show that the 4.1 protein plays an important role in the determination or maintenance of the normal shape of the erythrocyte and that the cells possessing antibody-modified membrane skeleton do not respond to amphiphilic ions in the manner that normal cells do.

Erythrocytes from Yoshida ascites sarcoma-bearing rats have reduced surface charge: the effect of tumor products.

Erythrocytes from the circulation of rats bearing intraperitoneal Yoshida ascites sarcoma, as well as the red cells contaminating the tumor fluid, exhibit higher concanavalin A (Con A)-mediated agglutination than the cells from normal animals. Two proteins have been purified earlier from the ascites fluid that can impart high agglutinability on the cells in vitro. We report here that the erythrocytes from the tumor-bearing rats exhibit reduced electrophoretic mobility compared to the cells from normal animals (P < 0.001), which correlates with the increase in their Con A-agglutinability (r = 0.998). The two purified proteins also cause reduction in the electrophoretic mobility of erythrocytes (P < 0.01). Thus the modification of the host cell surface observed in tumor-bearing rats arises due to non-specific binding of tumor products that reduce the host cell surface charge.

Neutral polymers elicit, and antibodies to spectrin, band 4.1 protein and cytoplasmic domain of band 3 protein inhibit the concanavalin A-mediated agglutination of human erythrocytes.

Concanavalin A (Con A) is known to agglutinate human erythrocytes if the cells are pre-treated with a proteinase or neuraminidase. We report that untreated cells can also be made to agglutinate with the lectin if the lectin-bound cells are treated with anti-Con A antibodies, or if a neutral polymer such as serum albumin, polyvinylpyrrolidone or Ficoll is added. Thus, Con A falls in the category of 'incomplete' lectins. The polymer induces Con A-agglutinability without altering the receptor number, or deformability of the cells. If the polymer is sequestered within erythrocyte ghosts, Con A is unable to agglutinate them; but the presence of the polymer only on the outer surface (as in intact cells) or on both the surfaces permits agglutinability. Thus, the site of the polymer effect resides on the outer surface of the membrane. The polymer, however, is unable to induce agglutinability in erythrocyte vesicles, whose membrane lacks skeletal proteins. The result suggests a positive role for the membrane skeleton in the process of agglutination brought about by the polymer, as is true also for the agglutination of proteinase-treated cells. In order to obtain detailed information on the proteins participating in agglutination, monospecific antibodies to spectrins, band 4.1 protein, ankyrin and the cytoplasmic domain of band 3 protein were internalized in erythrocytes. It is found that anti-spectrin and anti-band 3 cytoplasmic domain, but not their Fab's, inhibit the Con A-mediated agglutinability partially, and anti-4.1 antibodies, as well as the Fab's, inhibit the agglutinability substantially. Anti-ankyrin, however, was without any effect. The results confirm a positive role for the membrane skeleton in the Con A-mediated agglutination of normal erythrocytes in the presence of a neutral polymer, or in proteinase treated cells. We also provide evidence for requirement of Mg-ATP in the agglutination process.

Isolation of a glycoprotein responsible for the enhanced concanavalin A agglutinability of erythrocytes in Yoshida-ascites-sarcoma-bearing rats: the mechanism of paraneoplastic syndromes.

As a model for the development of paraneoplastic syndromes, we have studied the mechanism by which erythrocytes in the circulation of rats bearing intraperitoneal Yoshida ascites sarcoma acquire higher agglutinability with concanavalin A (Con A). The in vitro incubation of erythrocytes from normal animals with the cell-free ascites fluid or the plasma of tumour-bearing animals is able to confer an enhanced agglutinability on the cells. Fractionation of the ascites fluid has yielded three subfractions that are active in vitro. Two of these, occurring in small amounts, are a particulate fraction rich in plasma-membrane markers and a soluble fraction containing protein of molecular mass equal to or less than 50 kDa. These two are, however, unable to affect the agglutinability of erythrocytes in vivo, i.e. when injected intraperitoneally into normal rats. The third, and major, fraction consists of proteins of molecular mass equal to or greater than 680 kDa, and is able to modify the erythrocyte agglutinability in vivo. From this fraction, by using a combination of Con A affinity chromatography, gel filtration, (NH4)2SO4 fractionation and DEAE-Sephadex chromatography, an active protein has been purified to apparent homogeneity. It yields a subunit of 310 kDa in the presence of SDS and further breaks down into a polypeptide of 170 kDa when reduced with 2-mercaptoethanol. It has a pI of 5.35. The protein is rich in Glx, and appears to contain hybrid-type N-linked oligosaccharides. The protein is also present in the blood plasma of tumour-bearing, but not normal, rats. The radioiodinated protein binds to the erythrocyte surface adding about 7400 molecules/cell. The study unequivocally demonstrates that a protein from the tumour fluid can appear in the circulation, interact with host cells that are not in contact with the tumour and modify their properties.

Relationship between the concanavalin A-agglutinability and deformability of human erythrocytes.

Cellular deformability has been proposed in the past as a major determinant of lectin-mediated agglutination of cells. In this paper we have evaluated the correlation between deformability and Con A-agglutinability of human erythrocytes by subjecting them to agents that alter either one of the properties and evaluating the effect on the other property. The following results have been obtained: (i) Treatment with pronase or trypsin, which makes the Con A-nonagglutinable normal red cells highly agglutinable, has practically no effect on deformability; while neuraminidase treatment, with a similar effect on agglutinability, produces a small but statistically significant reduction in deformability. (ii) Diamide treatment, on the other hand, produces a drastic reduction in the deformability of pronase-treated erythrocytes but has no effect on the Con A-agglutinability of the cells. Dinitrophenol also reduces deformability but without altering the agglutinability, (iii) Chlorpromazine, at 2 x 10(-5) M, does not have any effect on the deformability of trypsinized cells, but increases the agglutinability substantially. When the Con A-agglutinability of the cells and their deformability after these treatments are compared, a correlation coefficient r = -0.353 (P greater than 0.1) is obtained. This indicates the lack of any direct correlation between the two parameters, and rules out any significant role of deformability in the determination of Con A-agglutinability of erythrocytes. The agglutination with the lectin is completely reversed by methyl alpha-D-mannoside, the specific inhibitory sugar for Con A, also ruling out any secondary role for deformability in the non-lectin-mediated stabilization of clumps. Upon incubation of normal erythrocytes with Con A. a dose-dependent decrease in deformability is observed, with the deformability index falling to almost 25% of the normal value with 500 microgram/ml Con A. This indicates that Con A binding to its receptor produces changes in the membrane probably by altering properties of the membrane skeleton.

Mechanism of host cell membrane modification by tumour: a model for paraneoplastic syndromes.

A not well-appreciated but clinically important aspect of malignant tumours is their effects on distantly located host cells. The effects, termed paraneoplastic syndromes, also pose an intriguing mechanistic problem: how do malignant cells influence properties of host cells not in contact with them? Erythrocytes from the circulation of rats bearing intraperitoneal Yoshida ascites sarcoma exhibit higher agglutinability with concanavalin A (Con A) than the cells from normal animals. Since the tumour and the red cells are not in contact, the enhanced agglutinability of the latter is a paraneoplastic effect. The mechanism by which the tumour brings about this effect is investigated as a model for paraneoplastic syndromes. The cell-free ascites fluid is able to impart high agglutinability on cells from normal animals in vitro. Also, when injected intraperitoneally in normal animals, the ascites fluid is able to enhance the agglutinability of erythrocytes in circulation. Apparently the tumour produces a substance(s) that appears in the ascites fluid and is able to diffuse into circulation, explaining the mechanism by which it can reach distant sites. From the cell-free ascites fluid three fractions have been isolated that are active in vitro. Of these, only one showed activity in vivo. From this fraction, a glycoprotein has been purified to homogeneity that confers maximal Con A-agglutinability on normal erythrocytes at 8 x 10(-7)M, at which concentration 6,400 molecules bind per cell. The protein has a molecular weight of 600 kDa in the native state and a pI of 5.35. It is made up of 4 identical subunits of Mr 170,000. It is detected in the plasma of tumour-bearing but not normal rats.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of the membrane skeleton in concanavalin A-mediated agglutination of human erythrocytes.

In the erythrocyte membrane, the mobility of band 3 protein, the receptor for concanavalin A (Con A), is drastically reduced by the membrane skeleton. Yet, the vesicles free of membrane skeletal proteins, isolated from the highly agglutinable proteinase-treated cells, are found to be devoid of Con A agglutinability. The vesicles bind Con A in normal amounts, and remain agglutinable with the wheat germ and Ricinus agglutinins. Intracellular entrapment of monospecific antibodies to spectrin and 4.1 protein (two of the major skeletal components of the membrane) is also found to inhibit agglutination by 30-50%. Thus the membrane skeleton appears to play a positive role in the agglutination of the cells with Con A. The anti-ankyrin antibodies are found to be without any effect. The anti-band 3 (cytoplasmic domain) antibodies are also inhibitory to agglutination. Since Con A binding to cells alters the shape responses and deformability of the cells, and the cells resist fragmentation at 49 degrees C, the properties of the whole skeleton, especially spectrin, appear to be changed. The Con A-bound membranes also do not release the complex of spectrin-band 4.1-actin when extracted with a hypotonic medium. It appears that Con A binding leads to interaction of the cytoplasmic domain of the receptor with a skeletal component, possibly spectrin. Subsequent to this, the receptor molecules and the skeletal proteins undergo aggregation in the membrane, which is detected by their crosslinking by an 8.6-A span bifunctional reagent. The contractility believed to be associated with the membrane skeleton may be responsible for the aggregation.

Concanavalin A-agglutinability of membrane-skeleton-free vesicles and aged cellular remnants derived from human erythrocytes. Is the membrane skeleton required for agglutination?

Vesicles and cell remnants have been obtained by aging of erythrocytes in vitro. The vesicles lacking the membrane skeletal proteins and the remnants known to possess a rigid skeleton have been used to assess the role of membrane skeletal proteins in the process of Con A (concanavalin A)-mediated agglutination of erythrocytes. Both the vesicles and the remnants were found to bind Con A at the same density as did intact cells. The vesicles, isolated from normal as well as from the Con A-agglutinable trypsin- and Pronase-treated cells, failed to agglutinate with Con A. They were, however, well agglutinated by WGA (wheat-germ agglutinin) and RCA [Ricinus communis (castor bean) agglutinin], indicating that the vesicles are not defective in agglutination. Large, cytoskeleton-free, vesicles prepared by another procedure also gave the same results. The aged remnants from trypsin- and Pronase-treated erythrocytes showed significantly decreased agglutination with Con A, but were agglutinated as well as the fresh cells by WGA and RCA. The agglutination with Con A is thus abolished when the membrane skeleton is absent, and reduced when it is rigid, suggesting that the skeleton may play an important role in the agglutination of erythrocytes by Con A.

Glycophorin A interferes in the agglutination of human erythrocytes by concanavalin A. Explanation of the requirement for enzymic predigestion.

Human erythrocytes become agglutinable with concanavalin A (Con A) after treatment with various proteinases or neuraminidase. The extent of agglutinability achieved with different enzymes is, however, different: Pronase, papain, trypsin, neuraminidase and chymotrypsin enhance the agglutinability in decreasing order, the last being barely effective. The actions of the enzymes on band 3, the Con A receptor, do not correlate with their abilities to increase the agglutinability: Pronase, papain and chymotrypsin cleave the protein, but not trypsin or neuraminidase. No significant differences are found in the number of Con A-binding sites or the affinities for the lectin between the normal and trypsin- or Pronase-treated cells. Thus the receptor does not seem to play a role in determining the Con A-agglutinability of erythrocytes. On the other hand, the cleavage of glycophorins, especially glycophorin A, and the release of sialic acid (in the peptide-bound form) are well-correlated with the enhancement in agglutination after the action of proteinases. The release of sialic acid by graded neuraminidase digestion and the increase in Con A-agglutinability show a correlation coefficient of 0.88. The major inhibitory role of glycophorin A in the process is indicated by the agglutination of En(a) heterozygous erythrocytes; the cells, known to bear about 50% glycophorin A molecules in their membrane, are agglutinated approximately half as well without proteolysis as are the trypsin-treated cells. Possible mechanisms by which glycophorin A could affect Con A-mediated agglutination are discussed.

Concanavalin A binding to human erythrocytes leads to alterations in properties of the membrane skeleton.

Three properties related to the erythrocyte membrane skeleton are found to be altered after the binding of concanavalin A (Con A) to erythrocytes or their isolated membranes. Con A binding to normal erythrocytes imparts resistance to heat (49 degrees C)-induced fragmentation of the cells. The fragmentation, due to denaturation of spectrin at 49 degrees C, is prevented by Con A in a dose-dependent manner, but levels off at concentrations of Con A in excess of 100 micrograms/ml. The binding of Con A to ghosts isolated from normal, trypsin- or Pronase-treated cells prevents (completely or substantially) the elution of the skeletal protein complex when the membranes are extracted under low-ionic-strength conditions in the cold. The Con A-agglutinated membranes of trypsin- and Pronase-treated, but not normal, cells show cross-linking of skeletal proteins and band 3 with dimethyl adipimidate, a 0.86 nm (8.6 A)-span bifunctional reagent. The extent of cross-linking is greater in the Pronase-treated membrane than in the less-agglutinable trypsin-treated membranes. The results show that, after Con A has bound, rearrangements occur in the membrane that alter properties of the skeletal proteins. Additionally, redistribution of the skeletal proteins and the Con A receptor occurs in the lectin-agglutinated membranes.

N-ethylmaleimide causes mechanical fragility and accumulation of spectrin dimers in the rat erythrocyte membrane.

Treatment of rat erythrocytes with N-ethylmaleimide is found to render them mechanically fragile. Membranes of the lysed cells show degradation of band 3 and, to a lesser extent, of spectrin; as well as considerable accumulation of dimeric spectrin. The predominant action of N-ethylmaleimide on isolated membranes, however, is the conversion of spectrin to its dimeric form.

Role of membrane integral proteins in the modulation of red cell shape by albumin, dinitrophenol and the glass effect.

Defatted serum albumin is found to induce a cup shape in erythrocytes. At 40 mg/ml of albumin, approx. 80% of washed erythrocytes possess this morphology, which can be reversed to disc shape by dinitrophenol. Erythrocytes treated with trypsin, papain, pronase or neuraminidase show enhanced susceptibility to cup formation by albumin; however, chymotrypsinized erythrocytes exhibit a normal response. Red cells treated with concanavalin A (but not its succinylated derivative) show resistance to the cupping effect of albumin as well as the crenating effects of dinitrophenol and glass. The resistance develops after about 20 min following the exposure of cells to the lectin, and is rapidly abrogated on removal of the bound lectin by alpha-methylmannoside. Incubation of the concanavalin A-exposed cells at low temperature leads to prolongation of the time required to achieve the resistance. These results indicate an involvement of membrane integral proteins in mediating the shape modulating effects of albumin, dinitrophenol and exposure to glass.

ABO(H) blood group antigens of the human erythrocyte membrane: contribution of glycoprotein and glycolipid.

Formaldehyde-fixed human erythrocytes were extracted with sodium dodecyl sulfate and with three other solvent systems, at least two of which are known to remove glycolipids quantitatively. The extracted cells possessed the ability to absorb the ABO blood group-specific antibody at about one-third the level of unextracted cells. Treatment of fresh cells with pronase also reduced the ability of the cells to absorb the antibody, further supporting the presence of ABO blood group active glycoprotein in the membrane. Trypsinization of red cells, while removing PAS-1 and partly PAS-2, did not lead to any decrease in the activity. Papainization also did not diminish the activity, although PAS-1, PAS-2, and PAS-3 were removed from the cells. Thus, both glycolipid and glycoprotein contribute to ABO antigens of erythrocytes. Also, none of the three major glycoproteins of the membrane bears this activity.