Evaluation of glycoconjugates on the K562 cell surface by means of lectin binding — comparison with human erythrocytes

The binding of five radiolabelled lectins (Vicia graminea, peanut,Phaseolus vulgaris isolectins E-PHA and L-PHA,Evonymus europaeus) to untreated and desialylated K562 cells and human erythrocytes was compared. The number of glycophorin A receptors recognized on the K562 cells by anti-blood group NV. graminea lectin was comparable to that found on the MN or NN erythrocyte surface. However, K562 cells had a severalfold higher number of oligosaccharide chains (presumablyO-glycosidic) which after desialylation became high-affinity receptors for peanut agglutinin, and of complex typeN-glycosidic chains available for the reaction with E-PHA and also with L-PHA (the latter lectin was not bound to erythrocytes). Moreover, K562 cells not treated with neuraminidase had a significant amount of extremely low affinity receptors for peanut agglutinin, whereas binding of this lectin to untreated erythrocytes was undetectable. On the other hand, the untreated K562 cells did not bind anti-blood group B and HE. europaeus lectin, but a small amount of binding by the desialylated cells was observed. Some other differences observed in the mode of lectin binding to K562 cells and erythrocytes are discussed.     

Association of human erythrocyte membrane glycoproteins with blood-group Cad specificity.

Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of erythrocyte membranes from a blood-group-B individual with the rare Cad phenotype indicates a lower-than-normal mobility of the main sialoglycoproteins, suggesting an increase in apparent molecular mass of 3kDa and 2kDa respectively for glycoprotein alpha (synonym glycophorin A) and glycoprotein delta (synonym glycophorin B). Since the chief structural determinant of Cad specificity is N-acetylgalactosamine, the membrane receptors have been isolated by affinity binding on immobilized Dolichos biflorus (horse gram) lectin. The predominant species eluted from the gel was the abnormal glycoprotein alpha, whereas in control experiments no material could be recovered from the adsorbent incubated with group-B Cad-negative erythrocyte membranes. After partition of the membranes with organic solvents, the blood-group-Cad activity was found in aqueous phases containing the sialoglycoproteins, but not in the organic phases containing simple or complex glycolipids, which, however, retained the blood-group-B activity. The carbohydrate composition of highly purified lipid-free glycoprotein alpha molecules prepared from Cad and control erythrocytes was determined. Interestingly the molar ratio of N-acetylneuraminic acid to N-acetylgalactosamine was equal to 2:1 in the case of controls and equal to 1:1 in the case of Cad erythrocytes. Taken together these results suggest that Cad specificity is defined by N-acetylgalactosamine residues carried by the alkali-labile oligosaccharide chains attached to the erythrocyte membrane sialo-glycoproteins.     

Co-localization of the immunoreactivities of corticotropin-releasing factor and arginine vasotocin in the brain and pituitary system of the teleost Catostomus commersoni

Summary Using the label-fracture technique, an ultrastructural comparison was made of the number and distribution of wheat germ agglutinin (WGA)-binding sites between human normal and sickle red blood cells. The WGA was adsorbed to colloidal gold, and quantitative analysis of the electron micrographs revealed that more binding sites were present on the sickle erythrocytes than on the normal erythrocytes. Moreover, the sites were more clustered on the sickle red cells than on the normal red cells. Use of another lectin, Bandieraea simplicifolia-II, revealed that it did not bind to normal or sickle red cells. Because of the affinity of the WGA for sialic acid residues, it is probable that the WGA is binding to a transmembrane sialoglycoprotein, glycophorin A. The conformation and/or distribution of the glycophorin A molecules may be altered by the sickle hemoglobin that binds to the red cell membrane. Hence, as detected by WGA, new surface receptors, which could play a role in the adhesion of sickle cells to endothelium may be exposed.     

Membrane receptors for Vicia graminea anti-N lectin and its binding to native and neuraminidase-treated human erythrocytes

Membrane receptors for Vicia graminea (Vg) lectin on human red cells were analyzed using deoxycholate lysates obtained from ¹²⁵I-erythrocyte membranes incubated with a purified lectin immobilized on Sepharose 4B. The glycoproteins (GP) specifically bound to the gel were eluted and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Using native erythrocytes the results obtained demonstrate that N red cells have exposed Vg receptors located on GPα (synonym glycophorin A) and GPδ (synonym glycophorin B) whereas on M erythrocytes the Vg receptors are restricted to GPδ. The presence of Vg receptors was also found on the hybrid glycoprotein (made of the N-ter of GPδ and C-ter of GPα) carried by St(a+) erythrocytes. A similar amount of radioactivity was bound to Vg-Sepharose incubated with neuraminidase-treated N or M membranes. The material eluted was tentatively identified as asialo GPα and asialo GPδ, suggesting that numerous receptors have been uncovered mainly on asialo GPα species from M erythrocytes. No glycoprotein component could be identified from the material eluted from Vg Sepharose incubated with native or neuraminidase-treated membrane from a Tn(+) individual. Scatchard plot analysis obtained from binding experiments at equilibrium with M, N, and St(a+) cells revealed the existence of at least two classes of receptors both on native and neuraminidase-treated erythrocytes. Desialylation of the M, N, and St(a+) erythrocytes resulted in an increase in the number of low- and high-affinity binding sites but had no significant effect on the association constants. However, high-affinity binding constants were about six times higher with N (7.07 × 10⁷ and 6.61 × 10⁷m^?1 for native and neuraminidase-treated N cells, respectively) as compared to M erythrocytes (1.13 × 10⁷ and 1.17 × 10⁷m^?1 for native and neuraminidase-treated M cells, respectively) whereas the low-affinity binding constants were similar for all types of cells (in the range of 0.1 to 0.3 × 10⁷m^?1). The number of Vg binding sites increases from 0.085 × 10⁵ to 0.8 × 10⁵ (high affinity) and from 2.10 × 10⁵ to 6.25 × 10⁵ (low affinity) per native and neuraminidase-treated N cell, respectively. On native and neuraminidase-treated M cells the number of Vg receptors increases from 0.011 × 10⁵ to 0.51 × 10⁵ (high affinity) and 0.13 × 10⁵ (low affinity), respectively. The large increase in the number of Vg receptors on neuraminidase-treated M cells is correlated with a large increase in agglutinability. Under similar treatment St(a+) cells behave like N erythrocytes whereas only 0.16 × 10⁵ Vg receptors of low affinity could be detected on neuraminidase-treated Tn erythrocytes. The results demonstrate that sialic acid is not required for binding and favor the view that the binding site of V. graminea lectin accommodates with two types of erythrocyte membrane receptors, one including both a contribution of polypeptide and oligosaccharide chains and a second which involves a simple interaction with sugar sequence Galβ1–3GalNAc available only when sialic acids are removed. The latter disaccharide is recognized by the Arachis hypogea lectin which therefore inhibits further binding of the V. graminea to neuraminidase-treated erythrocytes.     

Some properties of the human erythrocyte receptors for Neisseria gonorrhoeae

The nature of the receptors for T1 and T4 Neisseria gonorrhoeae on erythrocytes and other cells was investigated. In general, cells of nonprimate origin contained few receptors for gonococci. Receptors for T4 gonococci were only uncovered when host cells were pretreated with trypsin. Trypsinization, while unnecessary for T1 adherence to erythrocytes, enhanced attachment in inverse proportion to original erythrocyte sensitivity. Receptors for T1 and T4 organisms on trypsinized and trypsin-neuraminidase-treated erythrocytes were blocked by concanavalin A and peanut lectins, respectively, but a distinction could be made between them with wheat germ lectin and galactose oxidase. Of a number of sugars tested as inhibitors, only D-galactose blocked adherence of T4 but was without effect on T1. While the identity of erythrocyte receptors is uncertain, likely candidates are "band 3" protein and glycophorin, by virtue of their galactose content, lectin binding capacity, and partial exposure on the outer surface of the erythrocyte.     

Purification and characterization of anti-N lectin from Vicia unijuga leaves

1. An anti-N lectin was extracted from Vicia unijuga leaves with phosphate-buffered saline (PBS). Purification of the lectin was achieved, after pretreatment of the PBS extract by ammonium sulfate fractionation and absorption with human M erythrocytes, by using a combination of conventional chromatographic techniques with asialoglycophorin AN-Sepharose CL-4B affinity chromatography. Purification steps were followed by increase of specific activity. 2. Homogeneity of the purified lectin was demonstrated by HPLC and SDS-PAGE. The purified lectin was a glycoprotein with 11.4% carbohydrate and relatively high percentages of serine, threonine and aspartic acid residues and had a Mw of 120,000 Da. 3. This lectin agglutinated human N and MN erythrocytes, but did not agglutinate M erythrocytes. Hemagglutination of the lectin was inhibited by glycophorin AN and N-active sialoglycopeptide released from human N erythrocytes by treatment with Pronase or trypsin. However, it was not inhibited by any of mono- and di-saccharides, ABH-active glycoproteins, glycophorin AM and M-active sialoglycopeptide liberated from human M erythrocytes by treatment with Pronase or trypsin.     

Interaction of human erythrocyte Band 3 with Ricinus communis agglutinin and other lectins

Agglutination and competition studies suggest that human erythrocyte Band 3 can interact with both mannose/glucose- and galactose-specific lectins. Purified Band 3 reconstituted into lipid vesicles binds concanavalin A, but the nonspecific binding component, measured in the presence of alpha-methylmannoside, is very high. This glycoprotein also carries binding sites for the galactose-specific lectin Ricinus communis agglutinin. Binding was inhibited poorly by lactose, but much more effectively by desialylated fetuin glycopeptides, suggesting that the lectin recognizes a complex oligosaccharide sequence on Band 3. The glycoprotein bears two separate classes of binding sites for R. communis agglutinin. High-affinity binding sites exist which show strong positive cooperativity and correspond in number to the outward-facing Band 3 molecules. A low-affinity binding mode is abolished by 40% ethyleneglycol, suggesting the involvement of hydrophobic lectin-glycoprotein interactions. Studies on binding of R. communis agglutinin to human erythrocytes indicate positively cooperative binding to 7 X 10(5) very-high-affinity sites per cell, and lectin binding is completely inhibitable by lactose. Based on its binding characteristics in vesicles, it seems likely that Band 3 forms the major receptor for this lectin in human erythrocytes. Properties such as positive cooperativity thus appear to be a common feature of the interaction of Band 3 with a variety of lectins of different specificity, both in erythrocytes and lipid bilayers.     

Use of carbohydrate probes in conjunction with fluorescence-activated cell sorting to select mutant cell lines of Chlamydomonas with defects in cell surface glycoproteins

Two carbohydrate-binding probes (the lectin concanavalin A and the anti-carbohydrate monoclonal antibody FMG-1) have been utilized in conjunction with fluorescence-activated cell sorting to select cell lines of Chlamydomonas reinhardtii that contain defects in cell surface-exposed glycoproteins. Two very different selection strategies (sorting cells with the lowest binding for the FMG-1 monoclonal antibody or the highest binding of concanavalin A) yield a class of mutant cells that exhibit a total lack of binding of the monoclonal antibody to cell wall and plasma membrane glycoproteins along with an increased affinity for concanavalin A. Detailed characterization of one such mutant cell line, designated L-23, is provided. The subtle glycosylation defect exhibited by this cell line does not alter the ability of the affected glycoproteins to be targeted to the flagellar membrane and does not affect the expression of flagellar surface motility, a phenomenon that appears to involve the major concanavalin A-binding glycoprotein of the flagellar membrane. This approach has general applicability for dissecting the role of carbohydrate epitopes in the targeting and function of any cell surface glycoprotein for which suitable carbohydrate probes are available.     

The B4 lectin from Vicia villosa seeds interacts with N-acetylgalactosamine residues on erythrocytes with blood group Cad specificity

We have previously shown that the B4 lectin from Vicia villosa seeds interacts with N-acetylgalactosamine alpha-linked to serine or threonine in cell surface glycoproteins. In the present study, we show that the lectin also binds to Cad erythrocytes (0.44-2.78 X 10(6) sites/cell) with an association constant of 0.61-0.84 X 10(7)M-1. Variability in the number of B4 lectin binding sites in Cad erythrocytes from different individuals parallels reactivity of these erythrocytes with other N-acetylgalactosamine-binding lectins. Agglutination of Cad erythrocytes with B4 lectin is inhibited by urinary Tamm-Horsfall Sda-active glycoprotein. Since the Cad and Sda determinants share the terminal GalNAc beta 1.4----Gal sequence, our results indicate that Vicia villosa B4 lectin can also interact with terminal beta-linked N-acetylgalactosamine in closely-spaced oligosaccharide units of cell surface glycoproteins.     

Simultaneous interaction of monoclonal antibody-targeted liposomes with two receptors on K562 cells

We have investigated the interaction of targeted liposomes with human erythrocytes, and K562 cells, a human leukemic line which expresses both glycophorin A and Fc receptors. Liposomes conjugated to monoclonal anti-human glycophorin A bind to human erythrocytes in 80-fold greater amounts than liposomes conjugated to a non-specific monoclonal antibody. Binding is inhibited by soluble anti-glycophorin but not by its Fab fragment. In contrast, binding of antibody-conjugated liposomes to K562 cells is very high irrespective of the specificity of the antibody. Liposomes conjugated to a nonspecific monoclonal antibody interact with K562 cells via an Fc receptor, and binding is inhibited by soluble human IgG. Liposomes conjugated to anti-human glycophorin A interact with K562 cells via an Fc receptor and glycophorin A. Binding is not inhibited by either human IgG or anti-glycophorin Fab alone. Binding is only partially inhibited by anti-glycophorin, or by human IgG in the presence of anti-glycophorin Fab, and completely inhibited only by human IgG in the presence of anti-glycophorin. Simultaneous binding of targeted liposomes to two cell membrane antigens is therefore partially resistant to inhibition by single soluble ligands even when they are present in large excess. We conclude that simultaneous binding to more than one receptor may be of considerable advantage for in vivo applications of targeted liposomes.     

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

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

81 Recent introduction of polysiphonia morrowii (ceramiales, rhodophyta) to punta arenas, chile

In red algae, fertilization begins with gamete-gamete contact between the trichogyne cell wall of the female carpogonium and spermatial coverings. During the fertilization in Aglaothamnion oosumiense , reproductive cells interact with each other through sex specific adhesion molecules on the surface of spermatia and trichogyne. The gamete binding is highly selective suggesting the presence of recognition factors along their surfaces. In the previous studies, we have reported that spermatial binding to trichogynes of a red alga, Aglaothamnion oosumiense is mediated by a lectin-carbohydrate complementary system. Spermatial binding to trichogynes was inhibited by pre-incubation of trichogynes with N-acetyl-D-galactosamine and D-glucose and hence lectins specific to these sugars were expected to present on the surfaces of trichogyne cell wall. We have isolated a new lectin from Aglaothamnion oosumiense by the use of agarose bound N-acetyl-D-galactosamine affinity chromatography and named it as rhodobindin. Rhodobindin agglutinated human erythrocytes as well as spermatia of Aglaothmanion oosumiense . The agglutinating activity of this lectin was inhibited by N-acetyl-D-galactosamine and N-acetyl-D-glucosamine. SDS-PAGE results showed that this lectin may be monomeric. The molecular weight was determined as 21,876 dalton by matrix-assisted laser desorption ionization (MALDI) mass-spectrometry. N-terminal amino acid sequence of the lectin was analyzed and revealed to have no identity with those of known proteins. The complementary male glycoprotein was also isolated and purified by the use of SBA-agarose affinity chromatography. The subtractive cloning was carried out to characterize the recognition molecules.     

Two monoclonal antibodies highly specific for the blood group N determinant

Two monoclonal IgM antibodies, 179K and 35/5F, obtained following immunization of mice with A₂,MN or O,MN human erythrocytes, agglutinate NN and MN red cells strongly, and MM erythrocytes weakly. As shown by hemagglutination inhibition and solid phase ELISA, both antibodies are highly specific for the blood group N determinant. They react with N glycoprotein, its amino-terminal glycopeptides and with Ss glycoprotein (glycophorin B), which carries the blood group N determinant. They fail to react with M glycoprotein, M glycoprotein-derived glycopeptides, or with internal glycopeptides derived from N glycoprotein. Reaction of the antibodies with N glycoprotein is abolished by desialylation, periodate oxidation/borohydride reduction, orN-acetylation of the glycoprotein. Thus, the antibodies are specific for an epitope which includes sialylated oligosaccharide chain(s) and is located in the region of the amino-terminal leucine residue of N glycoprotein. MMU^– erythrocytes, lacking both blood group N and Ss glycophorin are non-reactive. Agglutination of MMU⁺ erythrocytes by the anti-N antibodies occursvia interaction with glycophorin B and correlates with the Ss phenotype of red cells MM,S erythrocytes are usually more strongly, agglutinated than MM,ss cells. The agglutination of MM erythrocytes decreases markedly as the pH is increased from 6 to 8, while agglutination of NN red cells is much less affected by shifts in pH over this range. As a result, both monoclonal antibodies are highly anti-N specific typing reagents when the agglutination assay is carried out at pH 8.     

Effect of covalent modification on the binding of cholera toxin B subunit to ileal brush border surfaces.

A competitive binding assay has been developed to determine how modifications to the B subunit of cholera toxin affect the binding affinity of the subunit for an ileal brush border membrane surface. The Ricinus communis120 agglutinin (RCA120) specifically binds to terminal beta-D-galactosyl residues such as those found in oligosaccharide side chains of glycoproteins and ganglioside GM1. Conditions were designed to produce binding competition between the B subunit of cholera toxin and the RCA120 agglutinin. Displacement of RCA120 from brush border surfaces was proportional to the concentration of B subunit added. This assay was used to study the effect of modification of B subunit on competitive binding affinity for the ileal brush border surface. The B subunit of cholera toxin was modified by coupling an average of five sulfhydryl groups to each B subunit molecule and by reaction of the SH-modified B subunit with liposomes containing a surface maleimide group attached to phosphatidylethanolamine. SH-modified B subunit was approximately 200-fold more effective than native B subunit in displacing lectin from brush border surfaces in the competitive binding assay. The enhanced binding activity was retained on covalent attachment of the modified B subunit to the liposome surface. We conclude that the B subunit of cholera toxin may be a useful targeting agent for directing liposomes to cell surfaces that contain a ganglioside GM1 ligand.     

Neo-mannosylated liposomes: synthesis and interaction with mouse Kupffer cells and resident peritoneal macrophages

In order to target liposomes to cells expressing at their surface mannose receptors, e.g. mouse Kupffer cells and peritoneal macrophages, we have developed a new synthetic strategy which allows a chemically well defined preparation of neo-mannosylated vesicles. alpha-D-Thiomannopyranoside residues, substituted with a hydrophilic spacer arm and functionalized with a sulfhydryl group, were covalently coupled to preformed large unilamellar vesicles containing 4-(p-maleimidophenyl)butyryl phosphatidylethanolamine. Liposomes, containing 15 mol% of mannosyl residues, were specifically aggregated with concanavalin A; this aggregation could be reversed by an excess of free methyl alpha-D-mannopyranoside indicating that the surface ligands were freely accessible to the lectin. The neo-mannosylated liposomes presented in vitro an increased binding to cells possessing alpha-D-mannose specific binding sites. At 37 degrees C a specific binding, up to 9-fold compared to control vesicles, was observed. These neo-mannosylated vesicles represent attractive tools for targeting bio-active molecules to macrophage-associated diseases.     

80 Purification and characterization of male and female gamete recognition molecules of a marine red alga aglaothamnion oosumiense

In red algae, fertilization begins with gamete‐gamete contact between the trichogyne cell wall of the female carpogonium and spermatial coverings. During the fertilization in Aglaothamnion oosumiense, reproductive cells interact with each other through sex specific adhesion molecules on the surface of spermatia and trichogyne. The gamete binding is highly selective suggesting the presence of recognition factors along their surfaces. In the previous studies, we have reported that spermatial binding to trichogynes of a red alga, Aglaothamnion oosumiense is mediated by a lectin‐carbohydrate complementary system. Spermatial binding to trichogynes was inhibited by pre‐incubation of trichogynes with N‐acetyl‐D‐galactosamine and D‐glucose and hence lectins specific to these sugars were expected to present on the surfaces of trichogyne cell wall. We have isolated a new lectin from Aglaothamnion oosumiense by the use of agarose bound N‐acetyl‐D‐galactosamine affinity chromatography and named it as rhodobindin. Rhodobindin agglutinated human erythrocytes as well as spermatia of Aglaothmanion oosumiense. The agglutinating activity of this lectin was inhibited by N‐acetyl‐D‐galactosamine and N‐acetyl‐D‐glucosamine. SDS‐PAGE results showed that this lectin may be monomeric. The molecular weight was determined as 21,876 dalton by matrix‐assisted laser desorption ionization (MALDI) mass‐spectrometry. N‐terminal amino acid sequence of the lectin was analyzed and revealed to have no identity with those of known proteins. The complementary male glycoprotein was also isolated and purified by the use of SBA‐agarose affinity chromatography. The subtractive cloning was carried out to characterize the recognition molecules.     

Lectin-mediated targeting of liposomes to a model surface. An ELISA method

Wheat germ agglutinin has been conjugated to the surfaces of sonicated phospholipid liposomes by reacting the protein derivatised with N-succinimidyl-S-acetylthioacetate (SATA) with the m-maleimidobenzoyl-N-hydroxysuccinimide (MBS) derivative of dipalmitoylphosphatidylethanolamine (DPPE) incorporated into the liposomal bilayers. The liposomes as characterised by photon correlation spectroscopy had a weight-average radius of 44 +/- 10 nm and the number of WGA molecules per liposome was in the range up to approx. 120. An ELISA method has been developed to assess the efficiency of targeting the conjugated liposomes to the antigenic determinants on a surface coated with glycophorin A (blood group B). For liposomes in which the degree of conjugation was controlled by varying the mol% DPPE-MBS from 3 to 27% targeting efficiency as assessed from the extent of inhibition of the ELISA increased by a factor of 10.     

Effect of lipid composition on HVJ-mediated fusion of glycophorin liposomes to erythrocytes

We previously reported that liposomes containing glycophorin or gangliosides, both of which were isolated from human erythrocytes, are efficiently fused to erythrocyte membranes in the presence of HVJ (Umeda, M. et al., J. Biochem. 94, 1955-1966 (1983), and Virology 133, 172-182 (1984]. In the present work, the effect of lipid composition in glycophorin liposomes on their sensitivity to fusion with erythrocytes was studied. Very little fusion occurred when glycophorin liposomes composed of dipalmitoylphosphatidylcholine-dicetylphosphate (9:1), dimyristoylphosphatidylcholine-dicetylphosphate (9:1), or egg yolk phosphatidylcholine-dicetylphosphate (9:1) were incubated with human erythrocytes in the presence of HVJ at 37 degrees C. Addition of cholesterol into these liposomal membranes greatly enhanced the sensitivity of the liposomes to fusion. The presence of phosphatidic acid and phosphatidylethanolamine in liposomes also enhanced the sensitivity, whereas the presence of lysophosphatidylcholine had no significant effect on the ability of the liposomes to fuse. The fusion efficiency of liposomes was also enhanced by the presence of glucosylceramide. Change of lipid composition in liposomes had, however, no appreciable influence on the HVJ-mediated binding of liposomes to erythrocytes, suggesting that the interaction between HANA protein of HVJ and glycophorin in liposomes was not affected by the lipid composition of the liposomes.     

Automatic identification and representation of protein binding sites for molecular docking.

Molecular docking is a popular way to screen for novel drug compounds. The method involves aligning small molecules to a protein structure and estimating their binding affinity. To do this rapidly for tens of thousands of molecules requires an effective representation of the binding region of the target protein. This paper presents an algorithm for representing a protein's binding site in a way that is specifically suited to molecular docking applications. Initially the protein's surface is coated with a collection of molecular fragments that could potentially interact with the protein. Each fragment, or probe, serves as a potential alignment point for atoms in a ligand, and is scored to represent that probe's affinity for the protein. Probes are then clustered by accumulating their affinities, where high affinity clusters are identified as being the "stickiest" portions of the protein surface. The stickiest cluster is used as a computational binding "pocket" for docking. This method of site identification was tested on a number of ligand-protein complexes; in each case the pocket constructed by the algorithm coincided with the known ligand binding site. Successful docking experiments demonstrated the effectiveness of the probe representation.     

Computational analysis of multivalency in lectins: structures of garlic lectin-oligosaccharide complexes and their aggregates

Multivalency in lectins is a phenomenon that has been discussed at considerable length. The structural basis for the role of multivalency in garlic lectin has been investigated here through computational studies. Biochemical studies have shown that the binding affinity of garlic lectin for high mannose oligosaccharides is orders of magnitude greater than that for mannose. Modeling and energy calculations clearly indicate that such increase in affinity cannot be accounted for by binding of these oligosaccharides at any of the six sites of a garlic lectin dimer. These studies also indicate that a given oligosaccharide cannot bind simultaneously to more than one binding site on a lectin dimer. The possibility of a given oligosaccharide simultaneously binding to and hence linking two or more lectin molecules was therefore explored. This study showed that trimannosides and higher oligomers can cross-link lectin dimers, amplifying the protein-oligosaccharide interactions severalfold, thus explaining the role of multivalency in enhancing affinity. A comprehensive exploration of all possible cross-links posed a formidable computational problem. Even a partial exploration involving a carefully chosen region of the conformational space clearly showed that a given dimer pair can be cross-linked not only by a single oligosaccharide molecule but also simultaneously by two oligosaccharides. The number of such possible double cross-links, including those forming interesting tetrameric structures, generally increases with the size of the oligosaccharide, correlating with the biochemical data. In addition to their immediate relevance to garlic lectin, these studies are of general interest in relation to lectin-oligosaccharide interactions.