The requirement for cell surface galactosyl residues during oxidative activation of normal and chronic lymphocytic leukemia lymphocytes.

Galactose oxidase stimulated normal and leukemic lymphocytes to undergo DNA synthesis and cell division. Although the response of normal lymphocytes to galactose oxidase was enhanced with neuraminidase pretreatment, substantial activation of leukemic lymphocytes required pretreatment with neuraminidase. Leukemic lymphocytes exhibited maximal response to neuraminidase-galactose oxidase later than that observed in normal lymphocytes. Treatment of lymphocytes with trypsin diminished their response to galactose oxidase. When lymphocytes were pretreated with β-galactosidase to specifically remove cell surface galactosyl residues, the response to galactose oxidase was prevented. The response of normal and leukemic lymphocytes to sodium periodate was also reduced after treatment with galactose oxidase. These data support the concept that oxidation of cell surface galactosyl residues is critical during lymphocyte activation.     

Mechanism of human interferon-gamma production: involvement of beta-2-microglobulin

The ability of several monoclonal antibodies (MoAbs) against beta-2-microglobulin (beta 2m) to inhibit interferon-gamma (IFN) production was assayed in peripheral blood mononuclear cells (PBMC). All of them strongly reduce IFN-gamma induction by galactose oxidase (GO), a well-characterized enzyme capable of activating T lymphocytes through mediation of macrophages. In contrast, many MoAbs directed against HLA class I (heavy chain) and class II antigens do not inhibit IFN induction by GO. On the other hand, anti-beta 2m MoAbs do not effectively reduce IFN-gamma induction by A23187, a calcium ionophore that acts on T cells in the absence of accessory cells. Competition experiments demonstrate that (i) the inhibition of anti-beta 2m antibodies was specific for beta 2m protein, and (ii) beta 2m is not itself the site of action of GO. Moreover, it is demonstrated that the addition of beta 2m to oxidated PBMC strongly enhances subsequent IFN-gamma production. Oxidation of galactose residues on glycoproteins of macrophage membrane is an obligate step for IFN-gamma induction whatever the inducer, thus our results suggest that beta 2m is involved in the mechanism of induction of IFN-gamma.     

Membrane events leading to interferon-gamma induction by antigens

Mitogenic induction of interferon-gamma in human peripheral blood mononuclear cells (PBMC) is prevented by enzymatic cleavage of galactose residues on the cell membrane, and by calcium depletion, suggesting that oxidation of galactose on the membrane glycoproteins and activation of a calcium flux across the membrane are critical events for interferon-gamma induction in nonspecifically stimulated human PBMC. The same experimental design has been applied to human PBMC cultures enriched of specifically sensitized lymphocytes and stimulated with the respective antigens. The results of these experiments show that also antigenic induction of interferon-gamma by purified protein derivative, tetanus toxoid, and MLR requires integrity of galactose residues and calcium intake suggesting that alteration of membrane-bound galactose and activation of a calcium flow are critical triggering events for both specific and nonspecific lymphocyte activation.     

A macrophage-derived factor different from interleukin 1 and able to induce interferon-gamma and lymphoproliferation in resting T lymphocytes

A novel monokine different from interleukin 1 (IL-1) is secreted from human or murine macrophages stimulated with galactose oxidase, a well-characterized enzyme able to induce marked polyclonal activation of lymphocytes. This monokine, preliminarly termed macrophage-derived blastogenic factor (MBF), stimulates resting T lymphocytes to produce interferon (IFN)-gamma and to proliferate. The apparent MW of MBF ranges between 29,000 and 35,000, although some active fractions show smaller MW ranging from 2000 to 7000, as demonstrated by gel filtration. The biological relationship between MBF and IL-1 was examined and it was found that MBF (1) is able to induce IFN-gamma production and proliferation by T lymphocytes in the absence of other inducers, (2) is not able to activate PHA-stimulated C3H mouse thymocytes, (3) is not able to induce production of IFN-beta by fibroblast cultures, (4) is resistant to proteolytic enzymes, and (5) is not neutralized by antibodies to IL-1. MBF was released in the macrophage supernatants in two waves after the oxidation process, namely, after 15 min and 2.5 hr, and each wave was capable of inducing lymphocyte activation at dilutions up to 1:32. Because the oxidation of galactose residues on cell surface structures is considered a general feature of lymphocyte activation whatever the inducer, it seems that MBF may be a mediator involved in mitogenic activation of T cells leading to IFN-gamma production and proliferation.     

Mechanism of production of interferon-gamma: role of arachidonic acid metabolites

The effects of arachidonic acid metabolites on mitogen-induced interferon (IFN)-gamma production by human peripheral blood mononuclear cells (PBMC) were examined. Both prostaglandins E2 (PGE2) and leukotrienes B4 (LTB4) were produced after macrophage activation stimulated by galactose oxidase (GO) and Staphylococcal enterotoxin B (SEB), two well known inducers of IFN-gamma. To test the involvement of PGE2 and LTB4 in IFN-gamma production, GO- and SEB-activated PBMC were treated with two inhibitors of cyclooxygenase (aspirin and indomethacin) and with an inhibitor of lipoxygenase [nordihydroguaiaretic acid (NDGA)]. The results of these experiments showed that aspirin and indomethacin cause a marked increase of IFN-gamma production by GO- and SEB-activated PBMC. On the contrary, NDGA treatment reduced IFN-gamma production induced by the same agents. Moreover, whereas the addition of exogenous PGE2 reduces IFN gamma production, the addition of exogenous LTB4 does not affect IFN-gamma production. Taken together these findings indicate that arachidonic acid metabolites, produced during mitogenic activation, are involved in the regulation of IFN-gamma production and suggest that, in our system, LTB4 exerts a positive modulating signal while PGE2 represents a negative signal.     

Role of carbohydrates in rat leukemia cell-liver macrophage cell contacts

The mechanism by which macrophages recognize tumor cells is still unknown. We have studied interactions between rat liver macrophages and rat L 5222 leukemia cells. These tumor cells, but not normal leukocytes or erythrocytes, adhere to freshly isolated macrophages in vitro. Binding of tumor cells by macrophages can be inhibited by N-acetyl-D-galactosamine, D-galactose and more potently by glycoproteins with terminal N-acetyl-D-galactosamine or D-galactose residues. Tumor cell adhesion is calcium-dependent. The relevant leukemia cell membrane structures which bear terminal beta-D-galactosyl or related residues have been determined as trypsin- and pronase-sensitive, and hence may presumably be glycoproteins. The tumor cell receptor on liver macrophages appears to be a lectin with the carbohydrate specificity N-acetyl-D-galactosamine greater than D-galactose greater than L-fucose.     

Role of carbohydrates in rat leukemia cell-liver macrophage cell contacts

The mechanism by which macrophages recognize tumor cells is still unknown. We have studied interactions between rat liver macrophages and rat L 5222 leukemia cells. These tumor cells, but not normal leukocytes or erythrocytes, adhere to freshly isolated macrophages in vitro. Binding of tumor cells by macrophages can be inhibited by N-acetyl-D-galactosamine, D-galactose and more potently by glycoproteins with terminal N-acetyl-D-galactosamine or D-galactose residues. Tumor cell adhesion is calcium-dependent. The relevant leukemia cell membrane structures which bear terminal beta-D-galactosyl or related residues have been determined as trypsin- and pronase-sensitive, and hence may presumably be glycoproteins. The tumor cell receptor on liver macrophages appears to be a lectin with the carbohydrate specificity N-acetyl-D-galactosamine greater than D-galactose greater than L-fucose.     

In vitro labeling of the sialic acid moiety of glycoconjugates with carbon-14

Labeling of sialoglycoproteins with carbon-14 in vitro was performed by reacting the aldehyde groups, generated by mild periodate oxidation of the terminal sialyl groups, with 14C-labeled sodium cyanide to produce the labeled cyanohydrin derivatives (Kiliani reaction). Labeling with tritium was carried out by reduction of the aldehyde groups generated on the sialyl residues with 3H-labeled sodium borohydride following standard procedures. The behavior of both types of labeled specimens of fetuin and ovine submaxillary mucin, individually and in mixtures, was investigated by gel-filtration chromatography, gel electrophoresis, and cesium bromide gradient ultracentrifugation. The labeled sialyl residues were subjected to partial characterization: color yield with the resorcinol and thiobarbituric acid reagents, behavior on ion-exchange chromatography, and susceptibility to mild acid and enzymatic hydrolyses. In addition to these model glycoproteins, this procedure was also utilized to label the sialoglycoproteins present in human tracheobronchial secretions collected from normal subjects and patients with chronic bronchitis. The potential uses of this approach for comparative studies of normal and pathological sialoglycoconjugates available in minute amounts is described. The extension of this approach to the labeling of the galactosyl and N-acetylgalactosaminyl moieties of glycoconjugates following treatment with galactose oxidase is outlined.     

Content and accessibility of sialic acid on the surface of rat hepatocytes during primary culture

The content and accessibility of terminal sialic acid and galactose residues of rat hepatocytes in primary culture were determined by in situ labeling using either periodate or sialidase/galactose oxidase treatment followed by sodium borotritiide reduction. Rat erythrocytes which were used for comparison showed a strongly enhanced tritium incorporation into galactose after sialidase treatment. In contrast, with freshly prepared rat hepatocytes only a small amount of galactose labeling was achieved after sialidase treatment. The amount of galactose labeled following sialidase treatment increased with time in culture up to day 6 and roughly paralleled the increase of the total sialic acid content. Major changes of sialic acid-containing glycoconjugates were restricted to the gangliosides. There was a transient drop in surface labeling of ganglioside-associated sialic acid on the first day in culture. The specific radioactivity of the in situ-tritiated ganglioside-sialic acid also fell by 50% in this period. Between day 2 and 4, there was an increase in gangliosidesialic acid labeling but the specific radioactivity of the sialic acid remained constant. This indicates that newly synthesized gangliosides but not the preexisting ones were accessible to periodate oxidation. The data allow conclusions about turnover and topology of the sialic acid-containing glycolipids.     

The structure of a sulfated glycoprotein of chick allantoic fluid: methylation and periodate oxidation.

The structure of an antigenic, sulfated glycoprotein from chick chorioallantoic fluid has been investigated by exogalactosidase digestion, methylation and mass spectral analyses, periodate oxidation, and Smith degradation. The main carbohydrate chains are composed of D-galactosyl residues linked at C-3 and 2-acetamido-2-deoxyglucose residues linked at C-4. Fucose and N-acetylneuraminic acid residues are nonreducing terminal groups, and the N-acetylneuraminic acid groups are linked to the D-galactose residues at C-3. Most of the sulfate groups (91% of the sulfate) are located on C-6 of the 2-acetamido-2-deoxyglucose residues, and the rest on C-6 of the D-galactose residues. A large number of the D-galactose residues (36.9% of the total) are present as nonreducing terminal groups and another 21.7% of the D-galactose residues are in penultimate position to the nonreducing terminal N-acetylneuraminic acid residues. Although mild periodate oxidation indicates the presence of D-galactose in furanoside form (5.5% of total D-galactose), no 5-O-methyl derivative of D-galactose was observed on methylation.     

Analysis of the envelope of Rauscher murine oncornavirus: in vitro labeling of glycopeptides.

The identity and localization of the oligosaccharides of Rauscher murine type C viral glycoproteins have been examined by techniques of in vitro labeling. Terminal sialic acid was labeled with tritium by borohydride reduction after selective periodate oxidation, and galactose was labeled by borohydride reduction after specific enzymatic oxidation of the nonreducing terminal of the sugar. The results were compared with those of protein surface labeling with pyridoxal phosphate or lactoperoxidase catalyzed radioiodination. Examination of the labeled reaction products by polyacrylamide gel electrophoresis in sodium dodecyl sulfate showed that in every case the major component labeled was a glycoprotein of about 70,000 daltons. The identity of this glycoprotein as the virion envelope component was confirmed by immunoprecipitation with mono-specific antiserum prepared against purified Rauscher virus glycopeptides of 69,000 and 71,000 daltons. No other protein or glycoprotein on the surface of the virion was detected, and disruption of virions-before labeling did not reveal additional distinctive glycoproteins. There was minor labeling of sugar residues of other components, but these remain to be characterized and are not now identified as other viral proteins. Studies of the structural organization of virion proteins using the cross-linking reagent methyl-4-mercaptobutyrimidate showed only linkage of the virion envelope or core proteins to themselves. These results indicate that most, if not all, of the oligosaccharides at the surface of Rauscher virus are entities of the 69,000- and 71,000-dalton glycopeptides and that they contain a terminal sialic acid and galactose and a subterminal galactose.     

Localization of penultimate carbohydrate residues in zona pellucida and acrosomes by means of lectin cytochemistry and enzymatic treatments

Lectins from peanuts (PNA) and soy beans (SBA) bind terminal residues of galactose (Gal) and N-acetyl-galactosamine (GalNAc) respectively. Galactose oxidase oxidizes the hydroxyl group at C-6 of terminal Gal and GalNAc blocking the binding of PNA and SBA. Binding of these lectins to sugar residues is also severely limited by the existence of terminal residues of sialic acid. In the present study, lectin cytochemistry in combination with enzymatic treatments and quantitative analysis has been applied at light and electron microscopical levels to develop a simple methodology allowing the in situ discrimination between penultimate and terminal Gal/GalNAc residues. The areas selected for the demonstration of the method included rat zona pellucida and acrosomes of rat spermatids, which contain abundant glycoproteins with terminal Gal/GalNAc residues. Zona pellucida was labelled by LFA, PNA and SBA. After galactose oxidase treatment, terminal Gal/GalNAc residues are oxidized, and reactivity to PNA/SBA is abolished. The sequential application of galactose oxidase, neuraminidase and PNA/ SBA has the following effects: (i) oxidation of terminal Gal/GalNAc residues; (ii) elimination of terminal sialic acid residues rendering accessible to the lectins preterminal Gal/GalNAc residues; and (iii) binding of the lectins to the sugar residues. Acrosomes were reactive to PNA and SBA. No LFA reactivity was detected, thus indicating the absence of terminal sialic acid residues. Therefore, no labelling was observed after both galactose oxidase--PNA/SBA and galactose oxidase--neuraminidase--PNA/SBA sequences. In conclusion, the combined application of galactose oxidase, neuraminidase and PNA/SBA cytochemistry is a useful technique for the demonstration of penultimate carbohydrate residues with affinity for these lectins. This revised version was published online in November 2006 with corrections to the Cover Date.     

A macrophage derived blastogenic factor -MBF- induces cytokines and cytotoxic effectors in human peripheral blood mononuclear cells.

A soluble macrophage-derived blastogenic factor, previously reported as MBF, is secreted from macrophages activated with galactose oxidase. It was previously shown that MBF is able to induce IFN-gamma production and proliferation of T lymphocytes. In this study we found that MBF is able to induce in human peripheral blood mononuclear cells (PBMC) production of interleukin 1 (IL-1) beta, interleukin 2 (IL-2) and tumor necrosis factor (TNF) alpha and generation of MHC-unrestricted cytotoxic activity. The induction of killer cells is likely to rely on IFN-gamma production in that in PBMC treated with a monoclonal antibody (Mab) against IFN-gamma, the MBF induced cytotoxic activity was drastically reduced. A comparison of MBF induced cytotoxic effectors with those induced by IL-2 showed that both cytotoxic effectors pertain to NK lineage, in that they were CD3- and CD16+. On the contrary, the precursors of MBF and IL-2 induced killer cells were different; MBF cytotoxic precursor cells were highly sensitive to L-Leucine methyl ester (Leu-OME), a drug able to eliminate monocytes and NK cells, whereas IL-2 cytotoxic precursors were unaffected by this drug.     

Interaction of rat peritoneal macrophages with homologous, sialidase-treated lymphocytes in vitro

The interaction in vitro between rat peritoneal macrophages and homologous, sialidase-treated lymphocytes was investigated. Lymphocytes were isolated from blood, thymus, and spleen on a density gradient. Total sialic acids obtained by acid hydrolysis were 10 nmol/10(8) lymphocytes, composed of 29% N-acetyl-neuraminic acid and 71% N-glycoloylneuraminic acid. Sialidase treatment released maximally 33% of membrane sialic acids. Lymphocytes were bound to peritoneal macrophages to an extent which increased in parallel with the amount of sialic acids released, whereas binding of untreated lymphocytes was not significant. This interaction was inhibited by free galactose and substances containing terminal galactose residues. Asialoorosomucoid with its oligoantennary sugar chains proved to be a 10(5) times more potent inhibitor of the interaction than lactose. The addition of homologous serum had no influence on binding. Electron microscopy revealed that vital lymphocytes were tightly bound to macrophages and only damaged lymphocytes appeared to be phagocytozed. The experiments demonstrate that the interaction between rat peritoneal macrophages and sialidase-treated lymphocytes is mediated by a macrophage receptor specific for galactose. This sugar is demasked on the surface of lymphocytes after the removal of terminal sialic acids. The role of this mechanism in cell recognition, elimination and homing of lymphocytes is discussed.     

The structure of a glycopeptide purified from porcine thyroglobulin

1. The structure of a purified glycopeptide isolated from porcine thyroglobulin was studied by sequential hydrolysis with specific glycosidases, by periodate oxidation and by treatment with galactose oxidase. 2. Sequential hydrolysis with several combinations of neuraminidase, alpha-l-fucosidase, beta-d-galactosidase, beta-N-acetyl-d-glucosaminidase and alpha-d-mannosidase presented the evidence for the following structure. 3. The monosaccharide sequence of the peripheral moiety of the heteropolysaccharide chain was sialic acid-->galactose-->N-acetylglucosamine. Some of the galactose residues were non-reducing end-groups with the sequence galactose-->N-acetylglucosamine. 4. After removal of the peripheral moiety composed of sialic acid, fucose, galactose and N-acetylglucosamine, alpha-mannosidase released 1.4mol of mannose/mol of glycopeptide, indicating that two of the three mannose residues were located between peripheral N-acetylglucosamine and internal N-acetylglucosamine or mannose. 5. Periodate oxidation and sodium borohydride reduction confirmed the results obtained by enzymic degradation and gave information concerning the position of substitution. 6. Based on the results obtained by enzymic hydrolysis and periodate oxidation together with the treatment with galactose oxidase, a structure is proposed for the glycopeptide.     

Differences between the carbohydrate units of cell-surface glycoproteins of moust B- and T-lymphocytes.

Carbohydrate units of cell-surface glycoproteins of mouse B- and T-lymphocytes, labelled in their sialic acid residues by the periodate/NaB3H4 method and in their galactose residues by the galactose oxidase/NaB3H4 method after neuraminidase treatment, have been studied. Glycopeptides were prepared from the labelled cells by Pronase digestion and fractionated by concanavalin A affinity chromatography into two fractions (A and B). Alkali-labile oligosaccharides were isolated after mild NaOH/NaBH4 treatment by gel filtration. The alkali-labile oligosaccharides were further analysed by t.l.c. To study the relative proportion of neutral mannose-rich carbohydrate units (fraction C) in lymphocyte glycoproteins, glycopeptides were also prepared from unlabelled cells and subjected to concanavalin A affinity chromatography after N-[3H]acetylation of their peptide moiety. The major alkali-labile oligosaccharide component of both cell types was identified as galactosyl-(beta 1 leads to 3)-N-acetylgalactosaminitol. T-Lymphocytes were characterized by a high proportion of this oligosaccharide and a lower proportion of alkali-stable fraction A glycopeptides, whereas the opposite was observed for B-lymphocytes. The relative proportions of the concanavalin A-binding fractions B and C were similar in both cell types. The differences observed may correlate with the different surface properties of B- and T-lymphocytes.     

Topography of glycoproteins in the chick synaptosomal plasma membrane

Chick brain synaptosomes or synaptic subfractions were treated with neuraminidase (EC 3.2.1.18) and/or galactose oxidase (EC 1.1.3.9) preparations in which proteolytic activity was inhibited with phenylmethanesulfonyl fluoride followed, after washing, by reductive incorporation of sodium boro[³H]hydride to identify galactose residues exposed on the synaptosomal external surface. Control experiments to demonstrate restriction of labeling to the external surface involved comparing the radioactivity in synaptoplasmic, soluble polypeptides isolated after labeling with labeled, isolated synaptoplasm and examining incorporation into fractions incubated without enzymes. Intactness of the synaptic plasma membrane after labeling was shown by trypsin digestion studies. Polypeptides were separated on sodium dodecyl sulfate polyacrylamide gels and were detected by a liquid scintillation counting procedure. Eleven major radioactive peaks were found after galactose oxidase treatment and reduction of isolated synaptic membranes. When intact synaptosomes were labeled, the same components were detected. When isolated synaptic membranes or intact synaptosomes were treated with neuraminidase before galactose oxidase treatment, three additional components were labeled. These results suggest that (a) chick synaptic membranes have a complex mixture of glycoproteins, (b) all major chick synaptic membrane glycoproteins labeled by galactose oxidase have most or all carbohydrate groups exposed at the exterior surface of the synaptosome, (c) all major, externally-disposed polypeptides of these synaptic membranes are glycoproteins.     

The use of galactosyltransferase to probe nitrocellulose-immobilized glycoproteins for nonreducing terminal N-acetylglucosamine residues

We report the use of UDPgalactose:N-acetyl-D-glucosaminyl-glycopeptide 4-beta-D-galactosyl-transferase (EC 2.4.1.38), purified from bovine milk, to detect nonreducing terminal N-acetylglucosamine residues on glycoproteins immobilized on nitrocellulose by electrophoretic transfer from sodium dodecyl sulfate-polyacrylamide gels. Soluble galactosyltransferase incorporates radiolabeled galactose from the substrate UDP-[6-3H]galactose into the appropriate immobilized acceptor with high specificity. Incorporation is proportional to substrate amount and is saturable with time. The kinetics of labeling are independent of substrate amount. Half-maximal incorporation occurs by 4 h and saturation occurs by 16 h. We have used galactosyltransferase as a probe (i) to verify the presence of nonreducing terminal N-acetylglucosamine residues in bovine rod outer segment membrane rhodopsin and in several glycoproteins in F9 murine teratocarcinoma cells and (ii) to detect previously reported endo-beta-N-acetylglucosaminidase activity in a commercial preparation of endoglycosidase F.     

Fluorometric determination of mucin-type glycoproteins by the galactose oxidase-peroxidase method

We developed a convenient and specific method for the determination of mucin-type glycoproteins using galactose oxidase and horseradish peroxidase on the basis of the contents of galactosyl and N-acetylgalactosaminyl residues in glycoproteins. Galactose and galactosamine residues released from glycoproteins after hydrolysis were oxidized with galactose oxidase and subsequently the resultant hydrogen peroxide was determined by a combination of horseradish peroxidase and 3-(p-hydroxyphenyl) propionic acid as a fluorogenic substrate. The contents of galactose/galactosamine residues in N- and O-glycans, as determined by the galactose oxidase-peroxidase method, were in good agreement with those described in the previous reports. We applied the present method to determine mucin-type glycoproteins secreted from rat gastric mucosa by stimulation with misoprostol, a prostaglandin E(1) analogue in vivo. Thus, the galactose oxidase-peroxidase method is useful for the determination of mucin-type glycoproteins in biological materials.     

The requirement for macrophage-lymphocyte interaction in T lymphocyte proliferation induced by generation of aldehydes on cell membranes.

Guinea pig T lymphocyte proliferation induced by sodium periodate (NaIO4) or neuraminidase-galactose oxidase (NG) occurs when lymphocytes and macrophages are cultured together after treatment of either purified T lymphocytes or macrophages with these agents. Regardless of which cell initially bears the modified surface carbohydrate, lymphocyte proliferation requires the presence of viable homologous macrophages and fails to occur when they are replaced with fibroblasts, erythrocytes, L2C leukemia cells, thymocytes, PMN, line I hepatoma cells, or murine macrophages. Lymphocyte proliferation resulting from NaIO4 or NG treatment of lymphocytes is diminished when these cells are treated with proteolytic enzymes or aged in in vitro culture for 48 hr. By contrast, proteolytic enzyme treatment or in vitro aging has no effect on the ability of NaIO4 or NG-treated macrophages to induce lymphocyte proliferation. The requirement for macrophage-lymphocyte interaction in NaIO4 or NG-induced lymphocyte proliferation is indicative of a central role for the macrophage in the initiation of T lymphocyte proliferation.