Immunochemical studies on the interaction between synthetic glycoconjugates and alpha-L-fucosyl binding lectins

Evonymus europaea lectin precipitated with alpha DGal(1----3) beta DGal(1----4)beta DGlcNAc-bovine serum albumin (BSA), alpha LFuc(1----2)beta DGal(1----3)beta DGlcNAc-BSA, alpha LFuc(1----2)beta DGal(1----4)DGlcNAc, and alpha DGal(1----3)[alpha LFuc(1----2)]beta DGal-BSA. However, the lectin neither precipitated with alpha LFuc(1----2)-beta DGal-BSA, alpha DGal(1----3)beta DGal-BSA, or beta DGal(1----4)beta DGlcNAc-BSA nor agglutinated erythrocytes of Oh phenotype having multiple terminal beta DGal(1----4)beta DGlcNAc residues. These results indicate that the minimal structural requirement for glycoprotein precipitation or cell agglutination by the lectin includes any of the three trisaccharides (fucosylated or nonfucosylated) derived from the blood group B tetrasaccharide. The monosaccharides linked to the beta-D-galactosyl residue in the blood group B tetrasaccharide, namely, alpha-D-galactose, alpha-L-fucose, and N-acetyl-beta-D-glucosamine, participate almost equally in binding to the lectin in as much as removal of any one of these sugars reduces the inhibiting potency of the resulting trisaccharide. alpha LFuc(1----2)beta DGal(1----3)beta DGlcNAc-BSA (H type 1) and alpha LFuc(1----2)beta DGal(1----4)beta DGlcNAc (H type 2) were precipitated to the same extent. The E. europaea lectin neither precipitated alpha DGal(1----4)-beta DGal(1----4)beta DGlcNAc-BSA, Lea-BSA, Leb-BSA, or beta DGlcNAc(1----4)[alpha LFuc(1----6)]beta DGlcNAc-BSA nor agglutinated Oh,Lea and Oh,Leb erythrocytes, demonstrating that terminal D-galactose linked alpha-(1----4) to subterminal beta-D-galactose, or alpha-L-fucose linked to N-acetylglucosamine, prevents lectin binding. Corey-Pauling-Koltun molecular models, built on the basis of data from 1H NMR and hard-sphere exo-anomeric (HSEA) calculations provided by Lemieux and co-workers [Lemieux, R. U., Bock, K., Delbaere, L. T. J., Koto, S., & Rao, V. S. (1980) Can. J. Chem. 58, 631-653], show that these alpha-D-galactosyl and alpha-L-fucosyl groups act to sterically hinder lectin binding to these oligosaccharides; these observations also suggest that the lectin binds to the beta-side of these oligosaccharides. These sides, on both blood group H type 1 and blood group H type 2 oligosaccharides, provide a similar contour which can fully account for their equal reactivity with E. europaea lectin. The only difference found between Lotus and Ulex I lectins in precipitating ability was that only Lotus precipitated with beta DGlcNAc(1----4)[alpha LFuc(1----6)]beta DGlcNAc-BSA.(ABSTRACT TRUNCATED AT 400 WORDS)     

Isolectins I-A and I-B of Griffonia (Bandeiraea) simplicifolia. Crystal structure of metal-free GS I-B(4) and molecular basis for metal binding and monosaccharide specificity.

Seeds from the African legume shrub Griffonia simplicifolia contain several lectins. Among them the tetrameric lectin GS I-B(4) has strict specificity for terminal alpha Gal residues, whereas the closely related lectin GS I-A(4) can also bind to alpha GalNAc. These two lectins are commonly used as markers in histology or for research in xenotransplantation. To elucidate the basis for the fine difference in specificity, the amino acid sequences of both lectins have been determined and show 89% identity. The crystal structure of GS I-B(4), determined at 2.5-A resolution, reveals a new quaternary structure that has never been observed in other legume lectins. An unexpected loss of both Ca(2+) and Mn(2+) ions, which are necessary for carbohydrate binding in legume lectins, may be related to a particular amino acid sequence Pro-Glu-Pro in the metal binding loop. Comparison with demetallized concanavalin A reveals a different process for the loss of metal ions and for the subsequent loss of carbohydrate binding activity. The GS I-A x alpha GalNAc and GS I-B x alpha Gal complexes were constructed using homology modeling and docking approaches. The unusual presence of an aromatic amino acid at position 47 (Tyr in I-A and Trp in I-B) explains the strong preference for alpha-anomeric sugars in both isolectins. Alteration at one amino acid position, Ala(106) in I-A versus Glu(106) in I-B, is the basis for the observed specificities toward alpha GalNAc and alpha Gal.     

alpha-D-galactose-bearing glycoproteins on the surface of stimulated murine peritoneal macrophages. Biochemical and immunochemical characterization of purified glycoproteins.

Two glycoproteins were isolated from lysates of thioglycollate-stimulated, murine peritoneal macrophages by affinity chromatography on immobilized Griffonia simplicifolia I lectin and by preparative SDS/PAGE. The glycoproteins were readily labeled on the surface of intact macrophages with 3H and 125I. The labeled glycoproteins migrated as broad bands of molecular mass 92-109 kDa and 115-125 kDa. The mobility of the glycoproteins decreased only slightly after reduction with dithiothreitol, indicating the absence of intersubunit disulfide bridges. The 92-kDa and 115-kDa glycoproteins had pI 5.2-5.4 and pI less than or equal to 4, respectively. Digestion of both glycoproteins with alpha-galactosidase released 23% of their 3H content and abolished their ability to bind to the G. simplicifolia I lectin, showing that they contain terminal alpha-D-galactosyl groups. After reduction with 2-mercaptoethanol, each glycoprotein fraction was sensitive to N-glycanase; the 115-kDa glycoproteins produced a smear with the front at approximately 67 kDa, whereas the 92-kDa glycoprotein gave two bands of 61 kDa and 75 kDa. Unreduced glycoproteins were insensitive to N-glycanase, suggesting the presence of intramolecular disulfide bonds. Although each glycoprotein fraction was sensitive to endoglycosidase H, this enzyme produced only slight changes in molecular mass when compared with N-glycanase. From these results as well as from the specificity of the enzymes involved, it is concluded that each glycoprotein fraction contains complex-type oligosaccharides and a small amount of high-mannose and/or hybrid-type oligosaccharides. While each glycoprotein fraction was bound to Datura stramonium lectin, they failed to react with anti-[i-(Den)] serum and their digestion with endo-beta-galactosidase did not cause a band shift in SDS/PAGE. Taken together, these results suggest the presence of N-acetyllactosamine units which are not arrayed in linear form but occur as single units, bound either to C2 and C6, or to C2 and C4, or both, of outer mannosyl residues on complex-type oligosaccharides. The glycoprotein(s) fraction precipitated with anti-[I (Step)] serum, suggesting the presence of branched lactosaminoglycans. Digestion of both glycoprotein fractions with a mixture of sialidase and O-glycanase did not alter their mobility in SDS/PAGE, suggesting a lack or low content of O-linked trisaccharides and tetrasaccharides. Each glycoprotein fraction was bound specifically to Sambucus nigra and Maackia amurensis immobilized lectins, indicating the presence of sialic acid linked alpha 2,6 to subterminal D-galactose or N-acetylgalactosamine residues, and alpha 2,3 to N-acetyllactosamine residues, respectively.     

Binding of Griffonia simplicifolia I lectin to rat pulmonary alveolar macrophages and its use in purifying type II alveolar epithelial cells

We report that the isolectin Griffonia simplicifolia I-B4 isolated from G. simplicifolia seeds binds to rat alveolar macrophages present in frozen sections of lung tissue or bronchoalveolar lavage fluid. G. simplicifolia I-B4 does not bind to alveolar epithelial cells. We established that G. simplicifolia I-B4 binds to the macrophages via interaction with terminal alpha-D-galactopyranosyl residues present on these cells. This was substantiated by demonstrating that binding is inhibited either by the haptenic sugar alpha-D-galactopyranoside or by treating the cells with coffee bean alpha-galactosidase. Because murine laminin is known to contain terminal alpha-D-galactopyranosyl end-groups, and because we found that an anti-laminin antiserum binds to rat alveolar macrophages, we suspect that G. simplicifolia I-B4 may be binding to laminin present on the macrophages. To isolate alveolar type II epithelial cells from rat lungs, we developed a method that utilizes the lectin G. simplicifolia I. When proteinase-derived suspensions of pulmonary cells are incubated with G. simplicifolia I, the macrophages agglutinate and can be removed by filtration through nylon mesh. After incubating the resulting cellular suspension in tissue culture, the adherent cells are 94 +/- 2% (S.D.) type II cells. When compared to cells isolated by repeated differential adherence, the lectin-prepared type II cells have similar morphology and staining characteristics, form domes in monolayers and incorporate similar amounts of palmitate into disaturated phosphatidylcholine. We believe that the procedure outlined in this report provides a simple and effective method to isolate type II alveolar epithelial cells from rat lungs.     

Microglial cells in the central nervous system of the rabbit and rat: cytochemical identification using two different lectins

Microglial cells were selectively demonstrated in the central nervous system of adult rabbits and rats using lectin histochemistry. Biotinylated Ricinus communis agglutinin-120 (RCA-1) and biotinylated Griffonia simplicifolia B4 isolectin (GSA I-B4) were used as histochemical markers on sections of Bouin-fixed paraffin-embedded cerebrum and cerebellum. Results were quite similar using both lectins and both species. GSA I-B4 resulted in a better staining in the rat, while RCA-1 labelling was superior in the rabbit. Neither neurons nor glial cells other than microglia were stained with our technique. Lectin histochemistry applied for the detection of microglial cells appears to be of sufficient selectivity and may be considered as an important tool in the morphological and neurobiological study of these cells.     

Glycoconjugate with terminal galactose. A selective property of macrophages in developing rat lung

Pulmonary macrophages in pre- and postnatal rats were examined histochemically with a battery of peroxidase labeled lectins. Among them, Griffonia simplicifolia agglutinin I-B4 (GSA I-B4) which binds specifically to terminal alpha-galactose showed selective affinity in lung for the monocyte-macrophage line. These cells were demonstrable with GSA I-B4 from the 14th day of gestation through the adult. Extension to the ultrastructural level showed strong selective binding of this lectin to the surface of the plasmalemma and inner face of membranes limiting phagosomes in macrophages. At day 14 of gestation, monocyte-like cells positive with GSA I-B4 were scattered in various organs including lung. The lectin reactive cells in lung increased in number and size with development, infiltrating the interstitium through day 20 of gestation and then also entering the alveolar space. These findings suggest that GSA I-B4 recognizes a surface glycoconjugate characteristic of the pulmonary monocyte-macrophage line. Such selective lectin affinity offers a marker for detecting the pulmonary macrophages and examining their kinetics by light and electron microscopy.     

Lectin binding sites on head structures of the spermatid and spermatozoon of the mosquito Culex quinquefasciatus (Diptera, Culicidae).

The presence of intranuclear and acrosomal lectin binding sites in spermatids and spermatozoa of the mosquito Culex quinquefasciatus was analysed. Direct and indirect lectin-gold techniques were used on LR White-embedded cells. The nuclear compartment was the structure most intensely labelled. Early spermatid nucleus showed moderate labelling for peanut agglutinin (PNA), Griffonia simplicifolia IB4 (GS-IB4) and Ricinus communis agglutinin (RCA), and light labelling for the other lectins tested. The sperm nucleus was intensely labelled by all lectins. The acrosome, an enzyme-containing structure, was labelled by some lectins. The anterior acrosomal region was labelled by PNA, while the proximal acrosomal region was labelled by PNA and G. simplicifolia II (GS II) lectins, and showed the presence of fucose residues with the use of Ulex europaeus I (UEA-I) lectin. The spermatozoa stored in the spermatheca showed the same pattern of labelling as that observed in spermatozoa localized in testis and seminal vesicles for all lectins tested. Carbohydrate residues in the nuclear compartment may be involved with the process of chromatin condensation. In the acrosomal region these residues may play a role in the process of sperm-oocyte interaction.     

ESR and fluorescence studies on the adenine binding site of lectins using a spin-labeled analogue

The techniques of electron spin resonance (ESR) and fluorescence spectroscopy have been used to study the interaction of a spin-labeled analogue of adenine, N6-(2,2,6,6-tetramethyl-1-oxypiperidin-4-yl)adenine (I), with several plant lectins. While most adenine derivatives enhanced lectin-induced fluorescence of 1,8-anilinonaphthalenesulfonic acid by binding to a separate, adenine-specific site [Roberts, D.D., & Goldstein, I.J. (1982) J. Biol. Chem. 257, 11274-11277], the spin label I caused a decrease in this fluorescence with certain lectins. ESR showed the ligand to interact strongly with lectins from lima bean (Phaseolus lunatus), Dolichos biflorus, and Phaseolus vulgaris (PHA); however, no binding was observed with Griffonia simplicifolia isolectins A4 and B4, soybean agglutinin, or Amphicarpaea bracteata lectins. The spin label was highly immobilized by each of these proteins (2T magnitude of = 68 G). Apparent affinities of the spin label for the lectins decreased in the order lima bean lectin greater than PHA erythroagglutinin greater than PHA leukoagglutinin greater than D. biflorus. Spin-labeled adenine appeared to bind specifically to the adenine binding site of D. biflorus and PHA leukoagglutinin, as demonstrated by total abolition of the ESR spectrum of bound spin label by adenine. PHA erythroagglutinin and lima bean lectin bound the analogue with apparent dissociation constants of 5 X 10(-5) and 3.2 X 10(-5) M, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and characterization of an N-acetylgalactosamine specific lectin from Salvia sclarea seeds

Crude extracts from Salvia sclarea seeds were known to contain a lectin which specifically agglutinates Tn erythrocytes (Bird, G. W. G., and Wingham, G. (1974) Vox Sang. 26, 163-166). We have purified the lectin to homogeneity by ion-exchange chromatography and affinity chromatography. The agglutinin was found to be a glycoprotein of Mr = 50,000, composed of two identical subunits of Mr = 35,000 linked together by disulfide bonds. The purified lectin agglutinates specifically Tn erythrocytes and, at higher concentrations, also Cad erythrocytes. Native A, B, or O red blood cells are not agglutinated by the lectin and, even after treatment with sialidase or papain, these cells are not recognized. Tn red cells present 1.45 X 10(6) accessible sites to the lectin which binds to these erythrocytes with an association constant of 1.8 X 10(6) M-1. On Cad red cells, 1.73 X 10(6) sites are accessible to the lectin which binds with an association constant of 1.0 X 10(6) M-1. The carbohydrate specificity of the S. sclarea lectin has been determined in detail, using well defined monosaccharide, oligosaccharide, and glycopeptide structures. The lectin was found to be specific for terminal N-acetylgalactosamine (GalNAc) residues. It binds preferentially alpha GalNAc determinants either linked to Ser or Thr (as in Tn structures) or linked in 1-3 to a beta GalNAc or to an unsubstituted beta Gal. Although more weakly, the lectin binds beta GalNAc residues linked in 1-4 to a beta Gal (as in Cad structures). It does not recognize beta GalNAc determinants linked in 1-3 to a Gal (as in globoside) or the alpha GalNAc residues of blood group A structures.     

Heterogeneity of the blood group ABH antigens and variation in the expression of these antigens of secretory granules in human cervical glands. An electron microscopic observation using lectins and monoclonal antibodies

Cytochemical localization of blood group ABH antigens was examined in secretory cells of human cervical glands by application of a post-embedding lectin-gold as well as immuno-gold labeling procedure using monoclonal antibodies. Blood group specific lectins such as Dolichos biflorus agglutinin (DBA), Helix pomatia agglutinin (HPA), Griffonia simplicifolia agglutinin I-B4 (GSAI-B4) and Ulex europaeus agglutinin-I (UEA-I) reacted with secretory granules but not with other cytoplasmic organellae such as nucleus and cell membrane. The reactivity of secretory granules with these lectins showed strict dependence on the blood group and secretor status of tissue donors. The binding patterns with these lectins were not homogeneous, but exhibited marked cellular and subcellular heterogeneity. Thus, for example, in blood group A individuals, some granules were stained strongly with DBA and others were weakly or not at all with the lectin. Such a heterogenous labeling with the lectin was observed even in the same cells. Similar results were obtained with UEA-I and GSAI-B4 staining in blood group O and B secretor individuals, respectively. Monoclonal antibodies likewise reacted specifically with the granules but they occasionally bound to some nucleus. The labeling pattern of the antibodies with the granules was essentially the same as those of lectins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biosynthesis of bi-, tri-, and tetraantennary oligosaccharides containing alpha-D-galactosyl residues at their nonreducing termini. Branch specificity of the Ehrlich tumor cell alpha(1,3)-galactosyltransferase

The ability of Ehrlich tumor cell alpha(1,3)-galactosyltransferase to catalyze the incorporation of alpha-D-Gal residues into a specific branch of bi-, tri-, and tetraantennary oligosaccharides has been investigated by acetolysis followed by gel filtration of the fragments on Bio-Gel P-4. Taking advantage of the carbohydrate specificity of the Griffonia simplicifolia I-B4 isolectin, the mono-[14C]alpha-D-Gal derivatives were isolated by affinity chromatography. Analysis of the acetolysis fragments generated by cleavage of the multiantennary substrates indicates that the Ehrlich cell alpha(1,3)-galactosyltransferase acts preferentially on the alpha-D-Man(1,6) arm. This branch is preferred 2.5 times in bi-, 5.6-8.5 times in tri-, and 12.7 times in tetraantennary structures over the alpha-D-Man(1,3) arm. Within the alpha-D-Man(1,6) branch, in turn, there is a 1.3-1.9-fold consistently higher frequency of galactosylation of the beta-D-GlcNAc(1,2) as compared to the beta-D-GlcNAc(1,6) antenna.     

Alteration of cell surface carbohydrates associated with ordered and disordered proliferation of oral epithelia: a lectin histochemical study in oral leukoplakias, papillomas and carcinomas

Cell surface carbohydrates in healthy oral mucosa (n = 15), leukoplakias without (n = 48) and with (n = 62) dysplasia, oral papillomas (n = 6) and squamous cell carcinomas (SCCs) (n = 40) were examined using the lectins peanut agglutinin (PNA), Ulex europaeus agglutinin I (UEA I), soybean agglutinin (SBA), Helix pomatia agglutinin (HPA), and Griffonia simplicifolia agglutinin I (GS I-B4). Binding of these lectins in formalin-fixed, paraffin-embedded tissues was demonstrated using either the peroxidase-anti-peroxidase (PAP) method or the avidin-biotin method. Healthy oral epithelia revealed binding sites for these lectins mostly in the suprabasal keratinocytes with occasional PNA binding also in their basal cells. Unlike healthy mucosa, a number of leukoplakias without and with dysplasia revealed receptor sites for UEA I also in their basal layer. Only those keratinocytes undergoing squamoidal differentiation exhibited SBA binding. Staining patterns of UEA I and SBA did not vary significantly between either leukoplakias without and with dysplasia or papillomas and SCCs. Conversely, a reduction or lack of binding sites for PNA (Gal beta 1-3GalNAc), HPA (D-GalNAc alpha) and GS I-B4 (alpha D-Gal) was observed more frequently in leukoplakias with dysplasia and SCCs contrasting their counterparts lacking epithelial dysplasia. Cell surface glycosyl residues play an important role in the regulation of cell proliferation and epithelial growth. Aberrant glycosylation in oral dysplastic leukoplakias and carcinomas leading to the lack of the relevant terminal sugar residues from their cell surface carbohydrates is probably a major reason for the hyper-/disordered proliferation.     

Modification of cell surface glycoprotein: addition of fucosyl residues during epidermal differentiation

When cutaneous sections from the newborn rat were treated with alpha- fucosidase, Ulex europeus agglutinin I (UEA) binding to the cell surface of the differentiated cells in the epidermis was diminished and there was an appearance in these cell layers of binding by Bandeiraea simplicifolia I-B4 lectin (BS I-B4), which normally is specific for the basal cells. A similar treatment with alpha-galactosidase resulted in a loss of BS I-B4 binding, but had no effect on UEA binding. Glycoproteins isolated from the membranes of epidermal cells showed a threefold increase in the ratio of binding to UEA versus BS I-B4 affinity columns as the proteins were derived from the more differentiated cell populations. These data suggest that alpha-fucosyl residues are added to the glycoproteins on the cell surfaces of differentiated cells, thus blocking alpha-galactosyl residues and changing the lectin binding specificity as epidermal cells move out of the basal cell layer.     

Carbohydrate binding properties of banana (Musa acuminata) lectin I. Novel recognition of internal alpha1,3-linked glucosyl residues.

Examination of lectins of banana (Musa acuminata) and the closely related plantain (Musa spp.) by the techniques of quantitative precipitation, hapten inhibition of precipitation, and isothermal titration calorimetry showed that they are mannose/glucose binding proteins with a preference for the alpha-anomeric form of these sugars. Both generate precipitin curves with branched chain alpha-mannans (yeast mannans) and alpha-glucans (glycogens, dextrans, and starches), but not with linear alpha-glucans containing only alpha1,4- and alpha1,6-glucosidic bonds (isolichenan and pullulan). The novel observation was made that banana and plantain lectins recognize internal alpha1,3-linked glucosyl residues, which occur in the linear polysaccharides elsinan and nigeran. Concanavalin A and lectins from pea and lentil, also mannose/glucose binding lectins, did not precipitate with any of these linear alpha-glucans. This is, the authors believe, the first report of the recognition of internal alpha1,3-glucosidic bonds by a plant lectin. It is possible that these lectins are present in the pulp of their respective fruit, complexed with starch.     

In vivo formation of leukotriene E5 by murine peritoneal cells

Resident mouse peritoneal cells, stimulated in vivo with opsonized zymosan, produced leukotriene C4 and E4, with LTE4 being the major (80-90%) product. When mice were placed on diets containing increasing amounts of fish oil, four additional sulfidopeptide leukotrienes (SP-LT), LTC5, LTE5, 11-trans LTC5 and 11-trans LTE5, were identified. The identity of LTE5 was confirmed by spectrophotometric, chromatographic and enzymatic methods. When equivalent amounts of n-6 and n-3 polyunsaturated fatty acids (PUFA) were included in the diet, the stimulated peritoneal cells (in vivo) produced higher quantities of LTE5 (30.2 +/- 5.4 ng/10(6) cells) than LTE4 (22.8 +/- 7.3 ng/10(6) cells). In addition, in vitro studies demonstrated a 60% reduction in LTC4 (42.0 +/- 10.8 ng/10(6) cells to 16.7 +/- 6.2 ng/10(6) cells) and the appearance of LTC5 (2.1 +/- 0.9 ng/10(6) cells) in resident macrophages (stimulated with A23187) from mice maintained on a fish oil diet compared to mice fed the control diet. This study demonstrated that formation of the pentaenyl SP-LT in vivo, in particular LTE5, by peritoneal cells can significantly contribute to the endogenous SP-LT pool in response to an inflammatory stimulus following a dietary regimen containing fish oil.     

Inhibition of yeast binding to mouse peritoneal macrophages by wheat germ agglutinin: a novel effect of the lectin on phagocytic cells

The ability of wheat germ agglutinin (WGA) to enhance the binding of bacteria and tumor cells to phagocytic cells, and to induce the killing of tumor cells by macrophages and monocytes, is well established. We observed, however, that WGA inhibits the binding to and phagocytosis of yeast cells by thioglycolate-elicited murine peritoneal macrophages. In order to follow these processes rapidly, the yeasts were labeled with Congo-red and their binding to the macrophages was measured spectrophotometrically after treatment with sodium dodecylsulfate. Phagocytosis was also followed by light microscopy. Binding of the yeasts was inhibited by about 80% after pretreating the macrophages with 150 micrograms/ml of WGA. This effect was reversed by subsequent incubation with N-acetyl-D-glucosamine, chitobiose or chitotriose, but was unaffected by methyl alpha-D-mannoside, N-acetyl-D-mannosamine, D-mannose or D-galactose. Pretreatment of the Congo-red yeasts with WGA did not inhibit their binding by the macrophages. Of a variety of lectins tested, only WGA and Datura stramonium lectin had this effect. Pretreating the macrophages with sialidase prevented the inhibition induced by WGA. Our findings suggest the presence on the macrophages of a class of WGA receptors not previously reported.     

Phagocytic killing of Candida albicans by different murine effector cells

Three major phagocytic populations in the mouse were tested in vitro for killing of Candida albicans by means of 51Cr release assay: early inflammatory peritoneal polymorphonuclear cells (PMN), unfractionated or adherent spleen cells and resident peritoneal macrophages (PEC). Considerable candidacidal activity was found in the early inflammatory neutrophil and adherent spleen cell populations. On the contrary, only limited activity was found to be associated with resident peritoneal macrophages. The phagocytic killing apparently involved multiple mechanisms.     

Comparative studies of the haemagglutination of adult and umbilical cord erythrocytes by animal lectins

1. The sugar specificities of four lactose-binding lectins were studied through the agglutination of adult and/or umbilical cord human erythrocytes (AHRBC and/or CHRBC). 2. Rana catesbeiana egg lectin specifically agglutinated both intact blood group A-AHRBC and intact blood group A-CHRBC. 3. Rana catesbeiana liver lectin agglutinated intact A-AHRBC much more strongly than intact A-CHRBC. 4. Xenopus laevis skin lectin nonspecifically agglutinated AHRBC and CHRBC. 5. Plecoglossus altivelis egg lectin specifically agglutinated intact B-AHRBC, but weakly agglutinated intact B-CHRBC. 6. Comparative studies of lectin-induced AHRBC or CHRBC agglutination clarified the sugar-binding specificities of these lectins.     

Glycolipid-lectin interactions: detection by direct binding of 125I-lectins to thin layer chromatograms

Glycolipids that bind 125I-labeled lectins are detected by autoradiography after thin layer chromatography of glycolipid standards or crude lipid extracts. Soybean agglutinin, Bandeiraea simplicifolia I isolectins A4 and B4, and Helix pomatia lectin are used to detect corresponding cell surface, glycolipid receptors in human and bovine erythrocytes. When lipid extracts from A and AB erythrocyte stroma are analyzed with Helix pomatia lectin, a polymorphic expression of blood group A glycolipid determinants is detected. The Bandeiraea simplicifolia isolectins react weakly with human erythrocyte glycolipids but bind at least 4 glycolipids in bovine stroma extracts. Soybean agglutinin reacts with glycolipids in all erythrocytes analyzed. This technique extends lectin specificity studies from inhibition analyses in aqueous systems using available, known structures to identification of specific, lectin-binding glycolipids in crude lipid extracts of cell membranes.     

Circular dichroism studies on the alpha-D-galactopyranosyl binding lectin isolated from the seeds of Bandeiraea simplicifolia.

The conformation of the alpha-D-galactopyranosyl binding lectin isolated from Bandeiraea simplicifolia seeds has been investigated over a broad range of pH in the presence of various solvents by circular dichroism (CD) spectroscopy in the region 200-300 nm. Analyses of the spectra obtained on the native protein show the lectin to contain a considerable proportion of beta structure (30-40%). The native conformation was found to be largely insensitive to changes in pH, but was influenced by sodium dodecyl sulfate or trifluoroethanol. Alterations in conformation in the presence of these agents were reflected in the CD spectra and show the presence of alpha helix under these conditions. These changes in conformation are accompanied by a loss in polysaccharide-precipitating activity. The protein is irreversibly denatured in 8 M urea. Neither removal of the intrinsic calcium ions from the protein nor addition of methyl alpha-D-galactopyranoside induces any appreciable change in the CD spectra of the protein although the former treatment abolishes the polysaccharide-precipitating capacity of the lectin. The conformational data obtained in the present study are compared with data available from conformational studies of other lectins and leads to the hypothesis that most lectins probably contain beta structure as the predominant conformational feature.