Crystallization of the lectin IV of Griffonia simplicifolia and its complexes with the Lewis b and Y human blood group determinants

Single crystals of the lectin IV from Griffonia simplicifolia have been grown in the tetragonal crystal system. The space group is P4(2)2(1)2 with a = 78.95(5) A and c = 89.01(2) A, and there is one subunit of the dimeric glycoprotein in the crystallographic asymmetric unit. The crystals diffract to at least 2.5 A d spacings and are stable in the x-ray beam for 3 weeks. Crystals of the complex with the Lewis b (Leb) and Y human blood group determinants as the methyl glycosides, alpha-L-Fuc(1----2)beta-D-Gal(1----3)[alpha-L-Fuc(1----4)]beta-D-GlcNAc-OMe (where Fuc is fucose and -OMe is methoxy) and alpha-L-Fuc(1----2)beta-D-Gal(1----4)[alpha-L-Fuc(1----3)]-beta-D-GlcNAc- OMe, respectively, have also been grown and found to be isomorphous with the native lectin. Crystals have also been obtained with several derivatives of the Lewis b-OMe tetrasaccharide including that which has the 6-hydroxyl of the beta-D-GlcNAc unit replaced by iodine. In the latter case, the presence of the iodine atoms was established.     

A structural comparison of the A and B subunits of Griffonia simplicifolia I isolectins

A structural comparison between the A and B subunits of the five tetrameric Griffonia simplicifolia I isolectins (A4, A3B, A2B2, AB3, B4) was undertaken to determine the extent of homology between the subunits. The first 25 N-terminal amino acids of both A and B subunits were determined following the enzymatic removal of N-terminal pyroglutamate blocking groups with pyroglutamate aminopeptidase. Although 21 amino acids were common to both subunits, there were four unique amino acids in the N-terminal sequence of A and B. Residues 8, 9, 17, and 19 were asparagine, leucine, lysine, and asparagine in subunit A and threonine, phenylalanine, glutamic acid, and serine in subunit B. The last six C-terminal amino acids, released by digestion with carboxypeptidase Y, were the same for both subunits: Arg-(Phe, Val)-Leu-Thr-Ser-COOH. Subunit B, which contains one methionyl residue, was cleaved by cyanogen bromide into two fragments, a large (Mr = 31,000) and a small (Mr = 2700) polypeptide. Failure of the small fragment to undergo manual Edman degradation indicated an N-terminal blocking group, presumably pyroglutamate. Both subunits were digested with trypsin and the tryptic peptides were analyzed using reverse-phase HPLC. Tryptic glycopeptides were identified by labeling the carbohydrate moiety of the A and B subunit using sodium [3H] borohydride. Cysteine-containing tryptic peptides were similarly identified by using [1-14C]iodoacetamide. Approximately 30% of the tryptic peptides were common to both subunits. Thus, although the N- and C-terminal regions of A and B are similar, the subunits each possess unique sequences.     

Comparative analysis by frontal affinity chromatography of oligosaccharide specificity of GlcNAc-binding lectins, Griffonia simplicifolia lectin-II (GSL-II) and Boletopsis leucomelas lectin (BLL)

Lectin-based structural glycomics requires a search for useful lectins and their biochemical characterization to profile complex features of glycans. In this paper, two GlcNAc-binding lectins are reported with their detailed oligosaccharide specificity. One is a classic plant lectin, Griffonia simplicifolia lectin-II (GSL-II), and the other is a novel fungal lectin, Boletopsis leucomelas lectin (BLL). Their sugar-binding specificity was analyzed by frontal affinity chromatography using 146 glycans (125 pyridylaminated and 21 p-nitrophenyl saccharides). As a result, it was found that both GSL-II and BLL showed significant affinity toward complex-type N-glycans, which are either partially or completely agalactosylated. However, their branch-specific features differed significantly: GSL-II strongly bound to agalacto-type, tri- or tetra-antennary N-glycans with its primary recognition of a GlcNAc residue transferred by GlcNAc-transferase IV, while BLL preferred N-glycans with fewer branches. In fact, the presence of a GlcNAc residue transferred by GlcNAc-transferase V abolishes the binding of BLL. Thus, GSL-II and BLL forms a pair of complementally probes to profile a series of agalacto-type N-glycans.     

Calcium modulates protease resistance and carbohydrate binding of a plant defense legume lectin,Griffonia simplicifolia lectin II (GSII)

Site-directed mutagenesis previously identified the residues responsible for the biological activity of the plant defense legume lectin, Griffonia simplicifolia lectin II (GSII) [Proc. Natl. Acad. Sci. USA 95, (1998) 15123-15128]. However, these results were inconclusive as to whether these residues function as direct defense determinants through carbohydrate binding, or whether substantial changes of the protein structure had occurred in mutated proteins, with this structural disruption actually causing the loss of biochemical and biological functions. Evidence shown here supports the former explanation: circular dichroism and fluorescence spectra showed that mutations at carbohydrate-binding residues of GSII do not render it dysfunctional because of substantial secondary or tertiary structure modifications; and trypsin treatment confirmed that rGSII structural integrity is retained in these mutants. Reduced biochemical stability was observed through papain digestion and urea denaturation in mutant versions that had lost carbohydrate-binding ability, and this was correlated with lower Ca(2+) content. Accordingly, the re-addition of Ca(2+) to demetalized proteins could recover resistance to papain in the carbohydrate-binding mutant, but not in the non-binding mutant. Thus, both carbohydrate binding (presumably to targets in the insect gut) and biochemical stability to proteolytic degradation in situ indeed contribute to anti-insect activity, and these activities are Ca(2+)-dependent.     

Cell type-specific lectin staining of the tracheobronchial epithelium of the rat: quantitative studies with Griffonia simplicifolia I isolectin B4.

We compared lectin staining patterns to cell population densities, as determined by morphological criteria in rat airways. Eight lectins were studied: Griffonia simplicifolia I isolectin B4 (GSI-B4), Arachis hypogaea (PNA), Wisteria floribunda (WFA), Glycine maximus (SBA), Dolichos biflorus (DBA), Helix pomatia (HPA), Ulex europaeus (UEA-1), and Maclura pomifera (MPA). Two of the lectins strongly stained morphologically distinct cell subpopulations. GSI-B4 stained basal cells, and MPA stained non-ciliated bronchiolar (Clara) cells. The specificity and sensitivity of GSI-B4 as a marker for basal cells was examined. In the trachea, 35% of all cells were GSI-B4 positive; 84% of these were basal cells, 7% were unidentified cells, 5% were serous/mucous cells, and 4% were ciliated, brush, or inflammatory cells. Comparison to cell population density data strongly suggested that all basal cells were GSI-B4 positive. The segmental bronchus was a transitional area; GSI-B4 positive basal cells were present in the region closest to the lobar bronchus but were absent in the distal region; instead, MPA-positive Clara cells appeared. When dissociated tracheal cells were obtained by pronase digestion, 43% were GSI-B4 positive. These results show that GSI-B4 is a sensitive and relatively specific marker for basal cells in the rat trachea which can be used to study dissociated epithelial cells.     

Heterogeneous histochemical reaction pattern of the lectin Bandeiraea (Griffonia) simplicifolia with blood vessels of human full-term placenta

Bandeiraea simplicifolia lectin (BS-I) stains vascular endothelium in various species. In humans, less than 10% of the specimens studied exhibit a reaction with BS-I. In the present histochemical study, the reactivity of BS-I with placental blood vessels and its correlation with the blood group from mother and newborn child was investigated. Acetone-fixed cryosections of representative tissue segments of human full-term placenta and umbilical cord were stained with BS-I. The staining pattern of tissues from patients with different blood groups was identical, although the reaction of BS-I in the placenta was heterogeneous. BS-I did not react with the umbilical cord. Vascular smooth muscle cells at the insertion site of the umbilical cord into the chorionic plate, and endothelium deeper in the chorionic plate, became progressively stained. The endothelial cells and tunica muscularis of smaller arteries and veins in stem villi lost their reactivity in parallel with decreasing vessel size. Arterioles and venules reacted heterogeneously. Capillaries, trophoblastic basement membranes, especially epithelial plates, and sometimes the syncytiotrophoblast were labelled in several terminal villi. The data indicate that 1) the placenta binds BS-I to fetal endothelium independent of the blood group, 2) cell-surface antigens on placental endothelial cells are expressed heterogeneously and 3) cell-surface glycans are constituted in an organ-specific manner on human endothelial cells.     

Facile preparation of the alpha-Gal-recognizing Griffonia simplicifolia I-B4 isolectin

The B4 isolectin from Griffonia simplicifolia is of great utility as a reagent for the identification of alpha-D-galactopyranosyl end groups. Its separation from isolectins containing A subunits has been greatly improved by a simple, rapid procedure using a column of N-acetylgalactosamine coupled to vinyl sulfone-activated Sepharose 4B to selectively retain the A subunit-containing isolectins. The procedure has the advantages over previous affinity procedures of speed (the isolation of B4 isolectin can be achieved in one day), simplicity, and high degree of resolution of the B4 isolectin.     

An improved staining method for rat microglial cells using the lectin from Griffonia simplicifolia (GSA I-B4)

A simple method for the lectin histochemical visualization of rat microglial cells is described. Advantages include ease of fixation of brain tissue using paraformaldehyde, and rapidity of tissue processing by vibratome sectioning. Furthermore, in addition to providing good structural preservation, the method achieves improved lectin binding, resulting in complete labeling of all microglial cells and in superior visualization of cellular processes. The lectin histochemical technique for rat microglia has the potential to be adapted to any mammalian species, and should prove valuable for neuroscientists interested in studying this glial cell type.     

Functional mechanics of the plant defensive Griffonia simplicifolia lectin II: resistance to proteolysis is independent of glycoconjugate binding in the insect gut.

Griffonia simplicifolia lectin II (GSII) is a plant defensive protein that significantly delays development of the cowpea bruchid Callosobruchus maculatus (F.). Previous structure/function analysis by site-directed mutagenesis indicated that carbohydrate binding and resistance to insect gut proteolysis are required for the anti-insect activity of this lectin. However, whether there is a causal link between carbohydrate binding and resistance to insect metabolism remains unknown. Two proteases principally responsible for digestive proteolysis in third and fourth instar larvae of C. maculatus were purified by activated thiol sepharose chromatography and resolved as cathepsin L-like proteases, based on N-terminal amino acid sequence analysis. Digestion of bacterially expressed recombinant GSII (rGSII) and its mutant protein variants with the purified gut proteases indicates that carbohydrate binding, presumably to a target ligand in insect gut, and proteolytic resistance are independent properties of rGSII, and that both facilitate its efficacy as a plant defensive molecule.     

2-Substituted methyl alpha-D-galactopyranosides: synthesis and binding affinity for the A and B subunits of the Griffonia simplicifolia I isolectins

The binding affinities of the N-acetyl, N-trifluoroacetyl, N-propionyl, N-formyl, N-benzoyl, N-p-nitrobenzoyl, N-p-aminobenzoyl, and N-methyl derivatives of methyl 2-amino-2-deoxy-alpha-D-galactopyranoside and the 2-O-acetyl, -benzoyl, -benzyl, and -methyl derivatives of methyl alpha-D-galactopyranoside for the A and B subunits of the Griffonia simplicifolia I isolectins have been determined by hapten inhibition analysis of a galactomannan-isolectin precipitation system. Models for these carbohydrate-protein interactions are presented together with an interpretation of the results on the basis of electronic and steric effects.     

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.     

Purification and immunohistochemistry of Griffonia simplicifolia agglutinin-II-binding mucus glycoprotein in rat stomach

Gastric mucus is thought to protect the gastric wall from mechanicaltrauma, desiccation, pathogenic microorganisms, acid and proteases.We purified Griffonia simp1icifolia agglutinin-II (GSA-II)-bindingmucus glycoprotein (GMG) from rat gastric mucosa by solubilizationin a guanidine- containing buffer, gel permeation chromatography,Ricinus communis agglutinin-I (RCA-I)-affinity chromatographyand GSA-II-affinity chromatography. Rat GMG showed high molecularweight on a Sephacryl S-1000 column, and a single band in 0.5%agarose-2% polyacrylamide composite gels and blots. A proteinof {small tilde}60 kDa was contained in the GMG preparation.GMG was deglycosylated with trifluoromethanesulphonic acid treatment.An antibody was raised against deglycosylated GMG (deGMG). Theantibody recognized deGMG, GMG, periodic acid-treated deGMGand O-glycanase-digested deGMG, but did not react to trypsin-digesteddeGMG. These results suggest that the antibody recognizes proteinase-sensitiveregion or peptide backbone of GMG. In immunohistochemistry,the mucous gel layer of the stomach luminal surface was stainedwith antibody. The antibody recognized not only gastric mucousneck cells and pyloric gland cells, but also gastric surfacemucous cells, mucous cells in the duodenal gland, and gobletcells in the small intestine and colon. These results indicatethat GMG is a component of rat gastric mucus, and that the antibodyrecognizes mucous-secreting cells in rat stomach and intestine. antibody immunohistochemistry lectin-affinity gel chromatography mucus glycoprotein rat stomach     

Comparative study of procedures for histological detection of lectin binding by use of Griffonia simplicifolia agglutinin I and gastrointestinal mucosa of the rat

The histological localisation of alpha-D-galactopyranosyl residues in glycoconjugates of rat stomach and duodenal mucosae was studied by use of Griffonia simplicifolia agglutinin I, i.e. the isolectin mixture (A + B) and the isolectin B4 (B4). Cryostat sections which were either unfixed or acetone fixed and paraffin sections from both ethanol-acetic acid and formaldehyde fixed tissue blocks were compared. Cellular details were better preserved in paraffin than in cryostat sections. Reactivity of cells binding GS I was less sensitive after formaldehyde than after ethanol-acetic acid fixation inasmuch as higher concentrations of lectins were needed. This drawback could be overcome by trypsinisation of the sections. The binding pattern of GS I (A + B) corresponded with that of GS I (B4) in either cryostat or paraffin sections. GS I was detected in the cytoplasm of parietal cells and in Brunner's gland cells. In duodenal crypts and villi, lectin was bound to supranuclear regions in the cytoplasm of columnar and goblet cells. The staining efficiency of fluorescein (FITC), horseradish peroxidase (HRP) and colloidal gold particle (CGP) labels in both direct and indirect lectin stainings was compared. Under all experimental conditions, indirect methods required lower concentrations of lectins than direct ones; indirect procedures increased sensitivity about 5-10 fold. CGP labels were always of highest sensitivity when gold particles were further developed by a silver precipitation method. HRP was not as efficient in lectin localisation as CGP, but cytochemical staining was more convenient in routine work. Direct FITC labellings proved to be of lowest sensitivity.     

Comparative study of procedures for histological detection of lectin binding by use of Griffonia simplicifolia agglutinin I and gastrointestinal mucosa of the rat

Summary The histological localisation of -D-galactopyranosyl residues in glycoconjugates of rat stomach and duodenal mucosae was studied by use of Griffonia simplicifolia agglutinin I, i.e. the isolectin mixture (A+B) and the isolectin B₄ (B₄). Cryostat sections which were either unfixed or acetone fixed and paraffin sections from both ethanolacetic acid and formaldehyde fixed tissue blocks were compared. Cellular details were better preserved in paraffin than in cryostat sections. Reactivity of cells binding GS I was less sensitive after formaldehyde than after ethanol-acetic acid fixation inasmuch as higher concentrations of lectins were needed. This drawback could be overcome by trypsinisation of the sections. The binding pattern of GS I (A+B) corresponded with that of GS I (B₄) in either cryostat or paraffin sections. GS I was detected in the cytoplasm of parietal cells and in Brunner's gland cells. In duodenal crypts and villi, lectin was bound to supranuclear regions in the cytoplasm of columnar and goblet cells. The staining efficiency of fluorescein (FITC), horseradish peroxidase (HRP) and colloidal gold particle (CGP) labels in both direct and indirect lectin stainings was compared. Under all experimental conditions, indirect methods required lower concentrations of lectins than direct ones; indirect procedures increased sensitivity about 5–10 fold. CGP labels were always of highest sensitivity when gold particles were further developed by a silver precipitation method. HRP was not as efficient in lectin localisation as CGP, but cytochemical staining was more convenient in routine work. Direct FITC labellings proved to be of lowest sensitivity.     

The xenograft antigen bound to Griffonia simplicifolia lectin 1-B(4). X-ray crystal structure of the complex and molecular dynamics characterization of the binding site.

The shortage of organs for transplantation into human patients continues to be a driving force behind research into the use of tissues from non-human donors, particularly pig. The primary barrier to such xenotransplantation is the reaction between natural antibodies present in humans and Old World monkeys and the Gal alpha(1-3)Gal epitope (xenograft antigen, xenoantigen) found on the cell surfaces of the donor organ. This hyperacute immune response leads ultimately to graft rejection. Because of its high specificity for the xenograft antigen, isolectin 1-B(4) from Griffonia simplicifolia (GS-1-B(4)) has been used as an immunodiagnostic reagent. Furthermore, haptens that inhibit natural antibodies also inhibit GS-1-B(4) from binding to the xenoantigen. Here we report the first x-ray crystal structure of the xenograft antigen bound to a protein (GS-1-B(4)). The three-dimensional structure was determined from orthorhombic crystals at a resolution of 2.3 A. To probe the influence of binding on ligand properties, we report also the results of molecular dynamics (MD) simulations on this complex as well as on the free ligand. The MD simulations were performed with the AMBER force-field for proteins augmented with the GLYCAM parameters for glycosides and glycoproteins. The simulations were performed for up to 10 ns in the presence of explicit solvent. Through comparison with MD simulations performed for the free ligand, it has been determined that GS-1-B(4) recognizes the lowest energy conformation of the disaccharide. In addition, the x-ray and modeling data provide clear explanations for the reported specificities of the GS-1-B(4) lectin. It is anticipated that a further understanding of the interactions involving the xenograft antigen will help in the development of therapeutic agents for application in the prevention of hyperacute xenograft rejection.     

Different fates of the oligosaccharide moieties of lipid intermediates.

We have previously described that the N-glycosylation process was accompanied by the release of oligosaccharide-phosphates and neutral oligosaccharides. The relationship between oligosaccharide-P-P-dolichol and its metabolic products (glycoproteins, oligosaccharide-phosphates and neutral oligosaccharides) was investigated by analysing the structure of the oligosaccharide moieties and the kinetic behaviour of the various species in pulse and pulse/chase experiments. For these studies, a glycosylation mutant of Chinese hamster ovary cells (B3F7) which does not synthesize mannosylphosphoryldolichol was utilized. Evidence was obtained for the presence of two pools of oligosaccharide-P-P-dolichol which have different fates. One pool is not glucosylated, is rapidly labelled and immediately chased by mannose, and generates the oligosaccharide-phosphate species. The second pool is glucosylated, exhibits a lag time (5-10 min) prior to being labelled, and is utilized in the glycosylation of proteins and in the production of neutral oligosaccharides. We postulate that the cleavage of non-glycosylated lipid intermediates generating oligosaccharide-phosphates represents a 'bypass' in the dolichol cycle which allows direct regeneration of dolichyl phosphate. The other metabolic fate of non-glucosylated oligosaccharide-lipids, glucosylation, results in their use as effective substrates for the glycosylation of proteins or in the generation of neutral oligosaccharides.     

Glycoconjugate histochemistry of the rat fundic gland using Griffonia simplicifolia agglutinin-II during the development

The development and maturation of fundic glands of Wistar rats were studied using Griffonia simplicifolia agglutinin-II (GSA-II) histochemistry at the light microscopic and electron microscopic levels. In adult rats, mucous neck cells and cells intermediate between mucous neck cells and chief cells were specifically labeled with GSA-II, whereas other fundic gland cells were virtually negative. Ontogenetic studies revealed that GSA-II positive cells appeared at the bottom of the gland by 21 days of gestation. With differentiation and aging, the elongation of the fundic gland continued, and the labeling intensity of the mucous neck cells increased by 3 weeks after birth. Cells intermediate between mucous neck cells and chief cells were discernible from 3 days after birth. Typical mucous neck cells appeared at 3 weeks after birth, when their labeling intensity with colloidal gold (CG) particles approximated that of adults. On the other hand, the reactive cell population gradually moved from the bottom toward the middle portion of the gland. Finally, the reactive cells were localized at the neck portion of the fundic gland. These results suggest that GSA-II is a valuable marker for studying mucous neck cells and both their precursor cells and their derivatives.     

Lectin from Griffonia simplicifolia identifies an immature-appearing subpopulation of sensory hair cells in the avian utricle

Brain Cell Biology - The sensory hair cells of the inner ear are coated with a variety of glycoproteins and glycolipids which can be identified by the binding of specific lectins. The present study...     

Study of the transformation of amoeboid microglial cells into microglia labelled with the isolectin Griffonia simplicifolia in postnatal rats.

The transformation of amoeboid microglial cells into ramified microglial cells in the brain of postnatal rats has been studied by labeling the cells with the isolectin Griffonia simplicifolia (GSA1-B4). The latter served as a specific membrane marker of the cell type. Thus, at the light-microscopic level, the amoeboid microglial cells in 1- to 5-day-old rats were intensely stained with GSA1-B4. All the stained cells appeared round. In 10-day-old rats, while most of the stained cells were round, some had assumed an oval appearance. In older rats, i.e. 15-22 days, all the stained cells became flattened or fusiform with long cytoplasmic processes. The present electron-microscopic study confirmed the above features but also added the fact that the reaction for GSA1-B4 was localized at the plasma membrane in the amoeboid microglial cells in all the age groups studied. The reaction for the isolectin was also detected in some vacuoles in the cytoplasm of the round cells. It was concluded from this study that the round amoeboid microglial cells differentiate to become the ramified microglia with age. In the course of this transformation, they retained specific membrane receptors for the isolectin which distinguished them from other glial cell types.     

Macrophage membrane glycoprotein binding of Griffonia simplicifolia I-B4 induces TNF-alpha production and a tumoricidal response.

Thioglycollate-elicited macrophages (m phi), upon binding the lectin Griffonia simplicifolia IB4 (GSIB4) at the plasma membrane, are induced to secrete several low molecular weight proteins. In this investigation, results from specific ELISA and immunoprecipitation analysis of these molecules confirmed that the cytokine, tumor necrosis factor-alpha (TNF-alpha), belongs to the group of elicited proteins. This specific m phi response is directly influenced by the dose of GSIB4 used and the time in contact with the cells. At 40 micrograms/ml GSIB4, the maximum dose of lectin used, the m phi activity was equal to that achieved when the cells were incubated with an interferon-gamma/lipopolysaccharide (IFN/LPS) stimulus alone. Moreover, the data showed that TNF-mediated tumoricidal activity was significantly influenced by GSIB4 binding to the m phi membrane. When the lectin was incubated alone or in sequence with IFN/LPS, this ligand-receptor binding promoted the lysis of WEHI 164 tumor target cells. However, concurrent incubation of both IFN/LPS and GSIB4 with m phi significantly diminished the tumoricidal response. This suggested that one of the metabolic pathways utilized subsequent to receptor-ligand binding was altered by these interactions. When cyclic AMP (cAMP) and inositol triphosphate (IP3) levels were examined, the results showed that the concentration of cAMP was unchanged despite the fact that IP3 levels were significantly enhanced upon m phi-GSIB4 binding. Collectively, the data show that GSIB4 binding to specific glycoproteins in the m phi membrane induces TNF-alpha production and facilitates TNF-alpha dependent tumoricidal responses. It also appears that the transduction of the signal, in part, at least utilizes the phosphatidyl inositol pathway. Finally, it is noteworthy that m phi activity is influenced by the sequence in which GSIB4 is presented to the m phi relative to the IFN/LPS treatment.