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PubMed1986

Con A induction of IgG secretion from a B-lymphoid tumour cell line, A20.

We have examined the effect of concanavalin A (Con A), a conventionally known T-cell mitogen, on the induction of Ig secretion from BALB/c B lymphoid cell lines expressing membrane IgM (X16C8.5, BALENTL 17.7.2 and BCL1) or membrane IgG (A20 and M12.4). A20 tumour cells, but not other tumour cells, responded to Con A by secreting Ig as measured by the reverse plaque-forming cell (PFC) assay. Another typical T-cell mitogen, phytohaemagglutinin (PHA), did not have any effect on the differentiation of A20 tumour cells. The addition of NaN3 abolished the PFC formation, showing that IgG PFC formation of A20 tumour cells was due to the active synthesis of secreted IgG and not due to the shedding of mIgG. The increase in the biosynthesis of secreted IgG from A20 tumour cells by Con A was also observed by polyacrylamide gel electrophoresis. The ability of Con A to induce IgG secretion from A20 tumour cells was abolished by the addition of alpha-methylmannoside, confirming that Con A itself, and not some contaminating material in Con A, induced the maturation of A20 tumour cells. Con A was also shown to induce Ig secretion, but not proliferation, in a subset of normal B cells. Thus, we conclude that Con A does give a differentiation signal to a subset of B cells.

Animals↗

PubMed1985

There are two forms of androgen binding protein in human testes. Comparison of their protomeric variants with serum testosterone-estradiol binding globulin.

To determine how the androgen binding protein in human testes (hABP) is related to the serum protein, testosterone-estradiol binding globulin (hTeBG), both proteins were isolated and compared. The hABP in extracts of human testes was composed of two molecular species based on concanavalin A (ConA)-Sepharose chromatography. Form I hABP did not interact with ConA while Form II hABP bound to ConA and eluted with alpha-methylmannoside. Form I and Form II hABP from five batches of testes were then purified approximately 30,500- and 30,000-fold to apparent homogeneity by high-performance liquid chromatography and compared with hTeBG isolated from human pregnancy serum. Fractionation of both forms of hABP and hTeBG by polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulfate suggested that the native forms of these proteins were indistinguishable. However, analysis of the purified proteins on sodium dodecyl sulfate-containing polyacrylamide gels indicated that all three were dimers and that each was composed of monomers of at least two sizes which were not present in equimolar concentrations. Two distinctive monomers or protomers of each protein were designated as heavy (H) and light (L) according to their electrophoretic mobilities on sodium dodecyl sulfate-polyacrylamide gels. The H and L protomers of Form I hABP showed apparent molecular weights of 55,000 and 52,000, respectively, in all preparations and were usually present in a 4:5 ratio (H:L). The two components of Form II hABP had apparent molecular weights of 53,000 and 48,000, respectively, and existed in a ratio of approximately 20:1. These two components could not be distinguished in some preparations where Form II hABP migrated as a broad band rather than as distinct protomers. By contrast, hTeBG, which was similar to Form II hABP with respect to ConA binding, always exhibited discrete H and L protomers in a 10:1 ratio. Photolysis of these highly purified proteins with delta 6-[3H]testosterone resulted in specific covalent labeling of their binding sites, confirming that the products identified by silver staining and immunoblotting were indeed steroid binding proteins. The H and L protomers of Form I hABP and hTeBG were separated and examined by peptide mapping using Staphylococcus aureus protease V8 and chymotrypsin. The comparison of the respective fragmentation patterns of protomers indicated that Form I hABP and hTeBG contained distinctive peptides.(ABSTRACT TRUNCATED AT 400 WORDS)

Androgen-Binding Protein↗

PubMed1979

Synthetic glycolipids and the lectin-mediated aggregation of liposomes.

Synthetic mannose-containing glycolipids utilizing the cholesterol nucleus as a lipid anchor, and either the 6-aminohexyl- or the 6-(6-aminohexanamido)hexyl-1-thio-alpha-D-mannopyranosides as the carbohydrate ligands, have been synthesized and incorporated into small unilamellar liposomes. Incorporation of these cholesterol-mannoside derivatives at concentrations up to 14 mol% apparently does not affect the physical characteristics of the liposomes. Addition of concanavalin A to a suspension of liposomes containing the long chain cholesterol-mannose derivative causes an increase in light-scattering at 360 nm. As the increase in absorbance is completely reversed by the addition of alpha-methylmannoside, aggregation rather than fusion of the liposomes appears to be occurring. Liposomes containing 14 mol % of the short chain (6-aminohexyl-) derivative are aggregated by concanavalin A indicating that the lectin can approach to within 10 A of the lipid bilayer. Preliminary results suggest that the aggregation of vesicles containing either the long or short chain derivatives is highly dependent on the density of the sugar in the membrane.

Chemical Phenomena↗

PubMed1980

Isolation and properties of an NAD- and guanidine-dependent ADP-ribosyltransferase from turkey erythrocytes.

An NAD- and guanidine-dependent ADP-ribosyltransferase has been purified more than 500,000-fold from turkey erythrocytes with an 18% yield. The enzyme in the 100,000 X g supernatant fraction was bound to phenyl-Sepharose, eluted with 50% propylene glycol, and further purified by sequential chromatographic steps on carboxymethylcellulose, NAD-agarose and concanavalin A-agarose. The transferase was specifically eluted from concanavalin A-agarose with alpha-methylmannoside. The enzymatic activity was extremely labile following the first purification step. Both propylene glycol and NaCl stabilized the transferase; significant increases in enzyme recovery were obtained by conducting the NAD- and concanavalin A-agarose chromatography in buffer containing propylene glycol. The purified protein exhibits one predominant protein band on SDS-polyacrylamide gels with an estimated molecular weight of 28,300. On Ultrogel AcA54 chromatography, single coincident peaks of ADP-ribosyltransferase activity and protein were observed. Enzyme activity was independent of DNA; the highly purified transferase was inhibited by thymidine, nicotinamide, and theophylline. The specific activity of the purified enzyme (350 mumol of ADP-ribose transferred from NAD to arginine methyl estermin-1mg-1) is comparable to that reported for purified NAD glycohydrolases and poly(ADP-ribosyl)transferases.

Animals↗

PubMed1983

Prolactin molecular heterogeneity. Response to thyrotropin-releasing hormone stimulation of concanavalin A-bound and -unbound immunoassayable prolactin during human pregnancy.

Since the milieu of pregnancy stimulates physiologic hyperprolactinemia, we questioned whether prolactin secreted during normal pregnancy contains a large-molecular weight component that binds to concanavalin A and whether this large-molecular weight prolactin contributes to the thyrotropin-releasing hormone (TRH)-releasable pool. Serum was collected from pregnant patients (n = 28) undergoing TRH stimulation tests. This serum was passed through a concanavalin A column and eluted with 0.2M alpha-methylmannoside. Concanavalin A-bound prolactin, as determined by radioimmunoassay, ranged from 10% to 30% of the total immunoassayable prolactin. An increase in the basal serum concentration of both concanavalin A-bound and -unbound prolactin occurred as pregnancy progressed. However, throughout gestation, only the concanavalin A-unbound prolactin increased after TRH stimulation. The concanavalin A-bound prolactin was found to have a molecular weight of 60,000 by means of Sephadex G-100 permeation chromatography.

Concanavalin A↗

PubMed1980

T suppressor cells and suppressor factor which act at the efferent stage of the contact sensitivity skin reaction: their production by mice injected with water-soluble, chemically reactive derivatives of oxazolone and picryl chloride.

The water soluble, chemically reactive thioglycollic acid thioether derivatives of oxazolone and picryl chloride were synthesized and tested for their ability to prevent the development of contact sensitivity. Mice given two injections of these agents showed partial or complete unresponsiveness when subsequently sensitized and challenged with oxazolone and picryl chloride, respectively. This unresponsiveness was associated with T suppressor cells, Ts-eff(cs), which blocked the efferent stage of the contact sensitivity reaction, i.e. the passive transfer of contact sensitivity. These Ts-eff(cs) were entirely specific when tested with the corresponding antigen. However, the suppression which they caused had a non-specific final common pathway. Cell from mice injected with the oxazolone and picryl thioethers and painted with the corresponding contact sensitizer produced a suppressor factor in vitro. This factor specifically blocked passive transfer by immune cells incubated in it. It also armed macrophages which then caused suppression. These macrophages were most effective when injected intraperitoneally. The suppressor factor had a molecular weight between 30,000 and 100,000 and the alpha-oxazolone factor was absorbed by oxazolone-albumin Sepharose and could be eluted with oxazolone-epsilon-aminocaproic acid. It was also absorbed by concanavalin-A-sepharose and could be eluted with alpha-methylmannoside. It is proposed that the ability of water soluble, chemically reactive haptenes to evoke a Ts-eff(cs) population may be relevent to the rarity of severe drug reactions following the injection of chemically reactive drugs.

Absorption↗

PubMed1983

Decrease in human lymphocyte surface glycoconjugates in leukemia as demonstrated by lectin binding.

Qualitative variations in the glycoconjugates which make up the lectin receptor sites on the membranes of leukemic lymphocytes, compared with those of normal cells, have been studied by the use of three tritiated lectins: Robinia pseudoacacia lectin, Concanavalin A and Ricinus communis (var. Sanquineus) agglutinin (RCA 120). The binding specificity of these lectins has been demonstrated using specific determinants: alpha-methylmannoside and galactose for Concanavalin A and Ricinus communis agglutinin respectively. For the Robinia lectin this specificity was determined by saturation of the receptor sites with the unlabeled Robinia lectin before the addition of isotopically labeled Robinia lectin. The results show a decrease in the number of receptor sites on the leukemia cells, especially in chronic lymphoid leukemia, relative to that on normal cells. The apparent affinity constants of leukemic cells in all cases remain higher than those of normal cells.

Cell Membrane↗

PubMed1983

Lysis of fresh natural killer-resistant tumor cells by lectin-activated syngeneic and allogeneic murine splenocytes.

This paper demonstrates that lectin-activated lymphocytes of selected mouse strains can lyse fresh autologous or allogeneic tumor cells but not the fresh normal cells tested in short-term 51Cr release assays. Murine splenocytes, incubated with concanavalin A for 3 days, lysed tumor cells from fresh syngeneic P815 mastocytoma, 102 methylcholanthrene sarcoma, and FBL3 lymphoma; fresh allogeneic 3LL lung carcinoma and MethA sarcoma; and tissue-cultured YAK cells in 18-hr51Cr release assays. Natural killer cells in fresh splenocyte preparations only lysed tissue-cultured YAK cells and not the other targets. Syngeneic and allogeneic lymphoblasts, lung, or liver cells were not lysed by the concanavalin A-activated killer (CAK) cells. The induction of cytotoxicity by concanavalin A incubation was abrogated by alpha-methylmannoside in the 3-day incubation, but not in cytotoxicity assay. Radiosensitive cells and adherent cells were necessary for the generation of CAK cells. The CAK effectors themselves were radioresistant, nonadherent, and mostly Thy 1+ and Ly 2+. The CAK phenomenon may be mediated by lymphokine production by an Ly 1+ cell, since depletion of Ly 1+ cells prior to activation abrogates CAK induction, and the ability of numerous mouse strains (and nude mice) to generate CAK cells correlated with their ability to produce Interleukin 2. The biological and therapeutic role of these cells is currently being investigated in murine syngeneic primary and metastatic tumor models.

Animals↗

PubMed1983

Acetyl CoA:alpha-glucosaminide N-acetyl transferase: partial purification from human liver.

The lysosomal enzyme acetyl CoA:alpha-glucosaminide N-acetyltransferase (GNAT) was shown to be an integral membrane protein requiring high concentrations of the detergent Triton X-100 for maximal solubilization. Using a concentration dependent Triton X-100 solubilization procedure and Concanavalin A-Sepharose affinity chromatography, GNAT was purified 50-fold with a yield of 45%. GNAT activity was separated from N-acetyltransferase activity toward glucosamine 6-phosphate, an alternative non-lysosomal pathway for glucosamine metabolism. GNAT was different from other lysosomal enzymes which bound to Concanavalin A-Sepharose in that both alpha-methylmannoside and Triton X-100 were required for elution of enzyme activity. GNAT activity, which bound to Concanavalin A-Sepharose, required at least one other component which did not bind for maximal expression of enzyme activity and for storage stability. Phospholipids and glycolipids, such as phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol, sphingomyelin and gangliosides, and bovine serum albumin allowed expression of enzyme activity and storage stability similar to the component(s) which did not bind to Concanavalin A-Sepharose.

Acetyltransferases↗

PubMed1982

Adsorptive pinocytosis of phosphorylated oligosaccharides by human fibroblasts.

Adsorptive pinocytosis of lysosomal enzymes by human fibroblasts depends on phosphomannosyl recognition markers on the enzymes and on high affinity receptors on the cell surface. To define the role of phosphorylated oligosaccharides in enzyme recognition, we studied the pinocytosis of [2-3H]mannose-labeled oligosaccharides purified from glycoproteins secreted by fibroblasts. Uptake of the oligosaccharides was inhibited 97% by 2 mM mannose-6-phosphate, 33% by 2 mM glucose 6-phosphate, and 5% or less by 2 mM alpha-methylmannoside, mannose, galactose, or L-fucose. The oligosaccharides were separated into neutral and five anionic species by chromatography on quaternary aminoethyl-Sephadex, characterized, and compared for susceptibility to pinocytosis. Treatment of the phosphorylated oligosaccharides with alkaline phosphatase before or after mild acid hydrolysis demonstrated that they contained one or two phosphates in phosphodiester linkage (covered) or phosphomonoester linkage (uncovered), or two phosphates, one in monoester linkage and one in diester linkage. Neutral oligosaccharides and those with one covered phosphate were not taken up by fibroblasts. Species with one uncovered phosphate or two covered phosphates showed low but detectable uptake. Oligosaccharides isolated as species with two uncovered phosphates, or those converted to this form by mild acid hydrolysis, were taken up 30-fold greater than the lower uptake forms during a 12-h incubation. Thus, oligosaccharides with two uncovered phosphates were far better ligands for the phosphomannosyl receptor than other oligosaccharides on acid hydrolases secreted by fibroblasts and initial rates of uptake of these oligosaccharides were comparable to those reported for several "high uptake" lysosomal enzymes.

Cells, Cultured↗

PubMed1981

Lactoferrin: a promoter of polymorphonuclear leukocyte adhesiveness.

Polymorphonuclear leukocytes (PMN) degranulate, adhere to vascular endothelium, or aggregate to each other following exposure of the cells to high concentrations of chemotactic stimuli such as formyl-methionyl-leucyl phenylalanine (FMLP). PMN released the specific granule product lactoferrin more readily in response to chemotactic stimuli, which correlated with promotion of PMN aggregation as measured by light transmission and enhanced PMN adherence. Both concanavalin A (Con-A) and phorbol myristate acetate (PMA), agents that lead to specific granule discharge, induced and sustained human PMN aggregation. Similarly, supernatants, generated from Con-A-treated PMN, aggregated fresh PMN in the presence of alpha-methylmannoside, a competitive inhibitor of the lectin. Anti-human lactoferrin IgG but not normal goat IgG blunted the aggregation elicited by both PMA and FMLP. Both human milk lactoferrin and rabbit PMN lactoferrin aggregated human lactoferrin promoted PMN adherence to endothelial cells. The enhanced PMN stickiness was correlated with the early phase of degranulation. Thus, PMN lactoferrin serves an autoregulatory role to retain PMN at inflammatory sites to amplify the inflammatory response.

Animals↗

PubMed1980

Synthesis and secretion of colonic glycoproteins: evidence for shedding in vivo of low molecular weight membrane components.

In vivo glycoprotein synthesis and secretion was studied in rat colonic epithelial cells using precursor labelling with radiolabelled glucosamine. Sepharose 4B gel filtration of radiolabelled glycoproteins obtained from isolated colonic epithelial cells revealed two major fractions: (1) high molecular weight mucus in the excluded fraction and (2) lower molecular weight glycoproteins in the included volume. These glycoproteins were further fractionated by affinity chromatography on concanavalin A-Sepharose. The low molecular weight [3H]glucosamine-labelled glycoproteins contained a major subfraction which specifically adhered to concanavalin A, and could be eluted with 0.2 M alpha-methylmannoside. Fractionation of the concanavalin A-reactive glycoproteins on Sephadex G-100 revealed a major peak with a molecular weight of 15 000. In contrast, high molecular weight mucus glycoprotein did not adhere appreciably to concanavalin A-Sepharose. Perfusion experiments indicated that colonic secretions contained both mucus and concanavalin A-reactive glycoproteins. The major concanavalin A-reactive glycoprotein in the colonic perfusate was not derived from serum, but was released directly from the colonic membrane into the lumen.

Animals↗

PubMed1981

A taurodeoxycholate-activated galactosylceramidase in the murine intestine.

Highly active enzymatic hydrolysis of galactosylceramide was detected in the murine intestine in confirmation of an earlier report in the rat intestine (Brady, R. O., Gal, A. E., Kanfer, J. N., and Bradley, R. M. (1965) J. Biol. Chem. 240, 3766-3770). Unlike the classical galactosylceramidase (EC 3.2.1.46), which is present in other organs, as well as also in the intestine, this intestinal enzyme was not activated by sodium taurocholate and was inhibited by oleic acid. It was effectively activated by sodium taurodeoxycholate and had a pH optimum of 5.2. This taurodeoxycholate-activated galactosylceramidase did not appear to be present in the brain, liver, kidney, and spleen. Its activity was not deficient in affected twitcher mice, a newly discovered mutant caused by a genetic deficiency of the taurocholate-activated galactosylceramidase. Although it showed a relatively neutral pH optimum, the taurodeoxycholate-activated galactosylceramidase is not a nonlysosomal "neutral" beta-galactosidase, because unlike the latter, it was adsorbed to Concanavalin A-Sepharose after solubilization with 0.5% sodium taurodeoxycholate and was eluted by alpha-methylmannoside or alpha-methylglucoside. The taurodeoxycholate-activated galactosylceramidase could be completely separated from the taurocholate-activated galactosylceramidase and GM1-ganglioside beta-galactosidase (EC 3.2.1.23) by octyl-Sepharose hydrophobic chromatography. Thus, the taurodeoxycholate-activated galactosylceramidase localized in the intestine is distinct from the two known glycosphingolipid beta-galactosidases and the neutral beta-galactosidase.

Animals↗

PubMed1994

Surfactant protein A amino acids Glu195 and Arg197 are essential for receptor binding, phospholipid aggregation, regulation of secretion, and the facilitated uptake of phospholipid by type II cells.

Pulmonary surfactant protein A (SP-A) is a mammalian lectin that regulates the uptake and secretion of surfactant by alveolar type II cells and is an important component of surfactant complexes. The domains of SP-A which mediate these functions have not been fully mapped. The binding of SP-A to its high affinity receptor on alveolar type II cells is thought to be dependent on a carbohydrate recognition domain (CRD), while the interaction with lipids has been attributed to the hydrophobic neck region of the molecule. To explore the role of the CRD in the interactions of SP-A with type II cells and lipids, we introduced mutations into the cDNA to encode for the substitutions Glu195-->Gln and Arg197-->Asp (SP-Ahyp,Gln195,Asp197) and expressed the mutant protein in insect (Sf9) cells using recombinant baculoviruses. Similar mutations introduced into mannose-binding protein A have been shown to switch the carbohydrate binding specificity from mannose > galactose to the converse. Wild type SP-A produced in Sf9 cells does not contain hydroxyproline (SP-Ahyp), but like rat SP-A it binds to carbohydrate affinity columns, lipids, and the SP-A receptor and is a potent inhibitor of the secretion of surfactant from type II cells (IC50 = 0.5-1.0 micrograms/ml). The SP-Ahyp,Gln195,Asp197 also bound to affinity matrices of galactose-Sepharose and mannose-Sepharose but the indicated mutations rendered the binding at least 100 times more susceptible than SP-Ahyp to competition by free galactose. The SP-Ahyp,Gln195,Asp197 did not compete with rat SP-A for occupancy of its high affinity receptor on type II cells and the mutant protein was 25-50-fold less potent as an inhibitor of the secretion of surfactant from type II cells (IC50 = 26.0 micrograms/ml). Unlike SP-Ahyp, the inhibition of secretion of surfactant by SP-Ahyp,Gln195,Asp197 was reversed by 0.25 M alpha-methylmannoside or galactose. In addition, the SP-Ahyp,Gln195,Asp197 bound avidly to phospholipid but did not aggregate vesicles or augment the uptake of phospholipid into type II cells. We conclude that the binding of SP-A to its receptor and the inhibition of surfactant secretion are critically dependent on the carbohydrate binding specificity of the CRD. Furthermore, phospholipid aggregation and augmentation of phospholipid uptake into type II cells are mediated by the COOH-terminal region of SP-A by a mechanism that is distinct from phospholipid binding.

Animals↗

PubMed1976

Heterogeneity of concanavalin A receptors on Ehrlich tumor cells.

Concanavalin A (Con A)-binding glycoproteins have been purified from Ehrlich ascites tumor cells by precipitation of cell extracts with Con A in agarose gels and by lithium diiodosalicylate (LIS) extraction. Antisera prepared against these two glycoprotein fractions indicate the presence of a minimum of five different antigens in the Con A-cell extract precipitate and a minimum of three different antigens in the LIS-extracted preparation. That both kinds of antisera are directed towards surface membrane antigens is strongly suggested by the fact that they give precipitin bands with purified plasma membrane from Ehrlich cells, they agglutinate whole cells, and they no longer give precipitin bands after absorption by whole cells. Cell extract material which was specifically eluted from a Con A-Sepharose 4B column with alpha-methylmannoside was recognized by antisera to both glycoprotein preparation, demonstrating that the antigens were Con A-binding components; material which did not bind the column failed to react with either antisera. Immunochemical analysis indicated that one of the antigens present in the LIS-extracted material was distinct from the five antigens prepared by lectin precipitation in agarose gels suggesting that a minimum of six individual Con A receptors are present on the surface of Ehrlich tumor cells. Immunochromatography was found to be a convenient method to purify the antigens obtained by the LIS extraction of Ehrlich cells.

Animals↗

PubMed1995

Binding of Galanthus nivalis lectin to Chlamydia trachomatis and inhibition of in vitro infection.

A glycoprotein present in Chlamydia trachomatis, serotype L1, elementary bodies (EBs) was earlier found to bind the lectin from Galanthus nivalis (GNA). In the present paper we investigate the interaction of GNA with chlamydial EBs and its effect on in vitro infectivity. The binding affinity was studied with 125I-GNA lectin. Within 15 min about 80% maximal binding was obtained. The chlamydia-GNA interaction was inhibited by alpha-methylmannoside, causing a decrease of about 50% at 1 mM. Curve fit analyses indicated two types of binding sites for GNA on the EBs. The affinity to these differed by a factor of 15. The influence of the lectin on the ability of C. trachomatis to infect McCoy cells was also investigated. There was a GNA-dependent inhibition with a 50% reduction in the number of intracellular inclusions at 0.2 microM of the lectin. The findings indicate the presence of terminal mannose structures on the chlamydial surface at or in the proximity of the cell-binding domains. Mannose-binding proteins of eukaryotic cells could be important for the initial uptake of EBs.

Animals↗

PubMed1996

Analysis of the sugar specificity and molecular location of the beta-glucan-binding lectin site of complement receptor type 3 (CD11b/CD18).

Zymosan, the cell wall from Saccharomyces cerevisiae, was reported to be a macrophage activator through its beta-glucan over 30 yr ago. Nevertheless, the identity of the beta-glucan receptor has been controversial. This study showed that the alpha M beta 2-integrin, CR3 (Mac-1, CD11b/CD18) served as the beta-glucan receptor through one or more lectin sites located outside of the CD11b I-domain that contains the binding sites for iC3b, ICAM-1, and fibrinogen. Sugar specificity, analyzed with FITC-labeled soluble polysaccharides and flow cytometry, showed CR3-specific staining with several pure beta-glucans but not with alpha-mannan. However, a 10-kDa soluble zymosan polysaccharide (SZP) with high affinity (6.7 x 10(-8) M) for CR3 consisted largely of mannose and approximately 5% glucose. Binding of either SZP-FITC or beta-glucan-FITC to CR3 was blocked not only by pure beta-glucans from yeast, mushroom, seaweed, or barley, but also by N-acetyl-D-glucosamine (NADG), alpha- or beta-methylmannoside, and alpha- or beta-methyl-glucoside. SZP-FITC and beta-glucan-FITC stained all leukocyte types similarly to anti-CR3-FITC, and polysaccharide-FITC staining was inhibited > or = 95% by unlabeled anti-CR3. SZP-FITC staining of cells expressing recombinant chimeras between CR3 and CR4 (p150,95, CD11c/CD18) suggested that both the divalent cation-binding region of CD11b and the region C-terminal to it may regulate binding of polysaccharides to CR3. Unlabeled SZP or beta-glucan also blocked CR3 staining by 11 mAb to C-terminal domain epitopes of CD11b but had no effect on staining by mAb directed to the I-domain. In conclusion, CR3 serves as the leukocyte beta-glucan receptor through a cation-independent lectin site located C-terminal to the I-domain of CD11b. Its sugar specificity is broader than originally appreciated, allowing it to react with certain polysaccharides containing mannose or NADG, as well as glucose.

Animals↗

PubMed1999

The beta-glucan-binding lectin site of mouse CR3 (CD11b/CD18) and its function in generating a primed state of the receptor that mediates cytotoxic activation in response to iC3b-opsonized target cells.

Mouse leukocyte CR3 (Mac-1, alphaMbeta2 integrin) was shown to function as a receptor for beta-glucans in the same way as human CR3. Soluble zymosan polysaccharide (SZP) or pure beta-glucans labeled with FITC or 125I bound in a saturable and reversible manner to neutrophils, macrophages, and NK cells. This lectin activity was blocked by anti-CD11b mAb M1/70 or 5C6 and did not occur with leukocytes from CR3-/- (CD11b-deficient) mice. SZP preparations containing primarily mannose or glucose bound to CR3, and the binding of 125I-labeled beta-glucan to CR3 was competitively inhibited by beta-glucans from barley or seaweed, but not by yeast alpha-mannan. Also, as with human CR3, the lectin site of mouse CR3 was inhibited by alpha- or beta-methylglucoside (but not D-glucose), alpha- or beta-methylmannoside, and N-acetyl-D-glucosamine. Phagocytosis of zymosan and serum-opsonized zymosan was partially inhibited by anti-CR3 and was reduced to <40% of normal with leukocytes from CR3-/- mice. As with neutrophils from patients with CD18 deficiency, neutrophils from CR3-/- mice exhibited no phagocytosis of particulate beta-glucan. SZP or beta-glucans primed CR3 of neutrophils, macrophages, and NK cells for cytotoxicity of iC3b-opsonized tumor cells that otherwise did not trigger killing. beta-Glucan priming for cytotoxicity was inhibited by anti-CR3 and did not occur with leukocytes from CR3-/- mice. The primed state of macrophage and NK cell CR3 remained detectable for 18 to 24 h after pulsing with beta-glucans. The similarity of mouse and human CR3 in response to beta-glucans highlights the utility of mouse tumor models for development of therapeutic beta-glucans.

Animals↗