It has been reported that the immune response due to alpha-Gal epitopes is an important factor in tissue valve failure. The elimination of the interaction between the natural anti-Gal antibodies and alpha-gal epitopes on the xenografts is a prerequisite to the success of xenografts in humans. Previously, we reported that the green coffee bean alpha-galactosidase could remove all alpha-Gal epitopes from cell surface of porcine aortic valve and pericardial tissue, but it has limitations on cost effectiveness. In this study we wanted to know whether the recently produced recombinant human alpha-galactosidase A has the same effective enzymatic activity as green coffee bean alpha-galactosidase in removing alpha-Gal epitopes from the same tissues. After treating fresh porcine aortic valve and pericardial tissue with recombinant alpha-galactosidase A, each sample was stained with Griffonia simplicifolia type I isolectin B4 indirect immunoperoxidase avidin-biotin technique. We then examined whether the alpha-Gal epitopes were reduced or abolished in each consecutive concentration of recombinant alpha-galactosidase A by comparing the degree of the Griffonia simplicifolia isolectin B4 staining. As a result, the recombinant alpha-galactosidase A could remove cell surface alpha-Gals on porcine aortic valve and pericardial tissue as effectively as green coffee bean alpha-galactosidase.

Due to their ability to bind specifically to certain carbohydrate sequences, lectins are a frequently used tool in cytology, histology, and glycan analysis but also offer new options for drug targeting and drug delivery systems. For these and other potential applications, it is necessary to be certain as to the carbohydrate structures interacting with the lectin. Therefore, we used glycoproteins remodeled with glycosyltransferases and glycosidases for testing specificities of lectins from Aleuria aurantia (AAL), Erythrina cristagalli (ECL), Griffonia simplicifolia (GSL I-B(4)), Helix pomatia agglutinin (HPA), Lens culinaris (LCA), Lotus tetragonolobus (LTA), peanut (Arachis hypogaeae)(PNA), Ricinus communis (RCA I), Sambucus nigra (SNA), Vicia villosa (VVA), and wheat germ (Triticum vulgaris)(WGA) as well as reactivities of anti-carbohydrate antibodies (anti-bee venom, anti-horseradish peroxidase [anti-HRP], and anti-Lewis(x)). After enzymatic remodeling, the resulting neoglycoforms display defined carbohydrate sequences and can be used, when spotted on nitrocellulose or in enzyme-linked lectinosorbent assays, to identify the sugar moieties bound by the lectins. Transferrin with its two biantennary complex N-glycans was used as scaffold for gaining diverse N-glycosidic structures, whereas fetuin was modified using glycosidases to test the specificities of lectins toward both N- and O-glycans. In addition, alpha(1)-acid glycoprotein and Schistosoma mansoni egg extract were chosen as controls for lectin interactions with fucosylated glycans (Lewis(x) and core alpha1,3-fucose). Our data complement and expand the existing knowledge about the binding specificity of a range of commercially available lectins.

Carbohydrate mimetic peptides are designable, they can carry T-cell epitopes and circumvent tolerance. A mimic-based HIV vaccine can be a viable alternative to carbohydrate-based antigens if the diversity of epitopes found on gp120 can be recapitulated. To improve existing mimics, an attempt was made to study the structural correlates of the observed polyspecificity of carbohydrate mimetic peptides based on the Y[P/R]Y motif in more detail. A carbohydrate mimetic peptide - D002,(RGGLCYCRYRYCVCVGR), bound a number of lectins with different specificities. While this peptide reacted strongly with both Lotus and Concanavalin A (ConA) lectins, it bound to Lotus stronger than Con A. By varying the central motif RYRY, five versions were produced in MAP format and their avidity for Lotus and ConA lectins was tested by SPR. Although the kinetic parameters were similar, the version based on the sequence YPYRY had an optimal affinity for both lectins as well as improved avidity for WGA and PHA. Thus, as far as lectin specificity is concerned, YPYRY had improved multiple antigenic properties. Both RYRY and YPYRY precipitated antibodies from human IgG for intravenous use that bound to gp120 in vitro and immunoprecipitated gp120 from transfected CHO-PI cells. Thus, Y[P/R]Y motifs mimic multiple carbohydrate epitopes, many of which are found on HIV and a preimmune human IgG antibodies that bind to HIV carbohydrates cross-react to a comparable extent with both RYRY and YPYRY carbohydrate mimetic peptides.

Mucoadhesion is a topic of current interest in the design of drug delivery systems. Mucoadhesive micro-spheres exhibit a prolonged residence time at the site of application or absorption and facilitate an intimate contact with the underlying absorption surface and thus contribute to improved and/or better therapeutic performance of drugs. In recent years such mucoadhesive microspheres have been developed for oral, buccal, nasal, ocular, rectal and vaginal routes for either systemic or local effects. The objective of this article is review the principles underlying the development and evaluation of mucoadhesive microspheres and the research work carried out on these systems.

Kirkeby S, Winter HC, Goldstein IJ. Comparison of the binding properties of the mushroom Marasmius oreades lectin and Griffonia simplicifolia I-B(4) isolectin to alphagalactosyl carbohydrate antigens in the surface phase. Xenotransplantation 2004; 11: 1-8. Copyright Blackwell Munksgaard, 2004Abstract: The binding of two alpha-galactophilic lectins, Marasmius oreades agglutinin (MOA), and Griffonia simplicifolia I isolectin B(4)(GS I-B(4)) to neoglycoproteins and natural glycoproteins were compared in a surface phase assay. Neoglycoproteins carrying various alpha-galactosylated glycans and laminin from basement membrane of mouse sarcoma that contains the xenogenic Galalpha1-3Gal1-4GlcNAc epitope were immobilized in microtiter plate wells and lectin binding determined with an enzyme-linked assay. After 24 h of incubation, MOA had higher affinity for the xenogenic pentasaccharide (Galalpha1-3Gal1-4GlcNAcbeta1-3Galbeta1-4Glc) than for the Galalpha-monosaccharide. The binding properties of MOA and GS I-B(4) to the xenogenic disaccharide (Galalpha1-3Galbeta1) were comparable while the binding of MOA to the xenogenic pentasaccharide was much stronger than the binding of GS I-B(4) to the same epitope. Non-xenogenic disaccharide-coupled neoglycoproteins having galactose end groups linked alpha1-2 or alpha1-4 to Gal or linked alpha1-3 to GalNAc bound very weakly to MOA, whereas GS I-B(4) recognized all of these disaccharides with similarly high affinity. MOA also showed high affinity for laminin. The results indicate that the Marasmius oreades lectin has nearly the same affinities as does GS I-B(4) for the simple xenogenic carbohydrate antigens, but MOA has greater affinity for the pentasaccharide and is far more specific in its binding preferences than the Griffonia lectin.

Reduction of pig cell-surface alpha-galactosyl (Gal) epitope, Galalpha1, 3Galbeta1, 4GlcNAc-R, by the introduction of glycosyltransferase genes is effective in suppressing hyperacute rejection (HAR) in pig-to-human xenotransplantation. The transmission of porcine endogenous retroviruses (PERVs) has been recognized as a potential risk factor associated with xenotransplantation. In this study, effects of the introduction of glycosyltransferase genes to pig cells on the sensitivity of gammaretroviruses to human serum were investigated. Pig endothelial cells (PEC), PEC transduced with alpha1,2 fucosyltransferase (FT), alpha2,3 sialyltransferase (ST), or N-acetylglucosaminyltransferase III (GnT-III), and human embryonic kidney (HEK) 293 cells were transduced with the LacZ gene with the packaging signal of murine leukemia virus (MuLV) under the control of the long terminal repeat of MuLV by a pseudotype infection. Then, the cells were further infected with PERV subtype B (PERV-B) or feline leukemia virus subgroup B (FeLV-B). Culture supernatants of the infected cells were mixed with human serum (HS) and then inoculated to HEK293 cells. The inoculated cells were histochemically stained and lacZ-positive blue foci were counted. Glycosyltransferase activity, xenoantigenicity, and alpha-Gal epitope density in the cells were measured at the time of the infection experiments. PERV-B or FeLV-B particles from the parental PEC were efficiently neutralized by HS, while those from PEC transduced with alpha1,2FT, alpha2,3ST or GnT-III were less sensitive to HS. The transduced PEC exhibited high levels of activity of the introduced glycotransferases, and expressed fewer xenoantigens and cell-surface alpha-Gal epitopes. Our results suggest that gammaretroviruses including PERVs produced by transgenic pigs, that are generally modified to reduce the cell-surface alpha-Gal epitope to overcome the HAR in xenotransplantation, are less sensitive to HS.

OBJECTIVES: In future pig-to-man xenotransplantation it is important to master tools that identify potentially xenogenic alphagalactose (Galalpha) antigens in the doner tissue.DESIGN AND METHODS: We have measured the binding potentials of Galalpha detecting lectins and antibodies, including a naturally occurring subfraction from human serum, to Galalpha containing neoglycoproteins and mouse laminin that were immobilized on microtiter plates.RESULTS: Galalpha reactive antibodies with similar monosaccharide specificity have distinct structural preference for sugar ligands. Laminin and neoglycoproteins were treated with alpha-galactosidase and subsequently incubated with antibodies and lectins. The enzyme treatment was more deleterious on antibody binding than on lectin binding.CONCLUSION: Antibodies and lectins may bind to different galactose determinants on the glycoproteins. Two anti-Galalpha1 antibodies that both have been raised against glycans on rabbit red blood cells may recognize Galalpha-antigens with varying specificities. Binding results obtained after digestion with alpha-galactosidase indicate that some xenoreactive Galalpha groups are not directly accessible for removal by the enzyme.

Protease-activated receptors (PARs) are widely distributed in human airways, and recent evidence indicates a role for PARs in the pathophysiology of inflammatory airway disease. To further investigate the role of PARs in airway disease, we determined the expression and function of PARs in a murine model of respiratory tract viral infection. PAR-1, PAR-2, PAR-3, and PAR-4 mRNA and protein were expressed in murine airways, and confocal microscopy revealed colocalization of PAR-2 and cyclooxygenase (COX)-2 immunostaining in basal tracheal epithelial cells. Elevated levels of PAR immunostaining, which was particularly striking for PAR-1 and PAR-2, were observed in the airways of influenza A/PR-8/34 virus-infected mice compared with sham-infected mice. Furthermore, increased PAR-1 and PAR-2 expression was associated with significant changes in in vivo lung function responses. PAR-1 agonist peptide potentiated methacholine-induced increases in airway resistance in anesthetized sham-infected mice (and in indomethacin-treated, virus-infected mice), but no such potentiation was observed in virus-infected mice. PAR-2 agonist peptide transiently inhibited methacholine-induced bronchoconstriction in sham-infected mice, and this effect was prolonged in virus-infected mice. These findings suggest that during viral infection, the upregulation of PARs in the airways is coupled to increased activation of COX and enhanced generation of bronchodilatory prostanoids.

This review focuses on the recent advances in investigations of the role of cell surface carbohydrates in tumor metastasis. It also summarizes the results of extensive studies of endogenous lectins, their structure, carbohydrate specificity and biological functions with the major emphasis on the significance of lectin-cell surface carbohydrate interactions in a metastatic process. Numerous data demonstrate that malignant transformation is associated with various and complex alterations in the glycosylation process. Some of these changes might provide a selective advantage for tumor cells during their progression to more invasive and metastatic phenotype. Cell glycosylation depends on the expression and function of various glycosyltransferases and glycosidases. Recently, transfection of genes encoding various glysosyltransferases gene in sense and antisense orientation helped to bring direct evidence that changes in cell surface carbohydrates are important for the metastatic behavior of tumor cells. Cell surface carbohydrates affect tumor cell interactions with normal cells or with the extracellular matrix during metastatic spread and growth. These interactions can be mediated via tumor cell carbohydrates and their binding proteins known as endogenous lectins. The family of the discovered endogenous lectins is rapidly expanding. The number of C-type lectins has reached 50 and at least 10 galectins have been identified. The biological significance of the endogenous lectins and their possible role in tumor growth and metastasis formation has started to unravel. Some lectins recognize the 'foreign' patterns of cell surface carbohydrates expressed by microorganisms and tumor cells, and play a role in innate and adaptive immunity. It was shown that lectins affect tumor cell survival, adhesion to the endothelium or extracellular matrix, as well as tumor vascularization and other processes that are crucial for metastatic spread and growth.

The neurons labeled by isolectin B4 (IB4) in rat and mouse sensory ganglia are often regarded as non-nerve growth factor (NGF)-dependent and non-peptidergic neurons, but a considerable number of IB4-positive neurons in the dorsal root ganglion (DRG) are also shown to be immunoreactive to substance P (SP) and calcitonin gene-related peptide (CGRP), which are synthesized by NGF-dependent neurons. Therefore, we examined the relationships between the IB4-binding neurons and NGF/glial cell line-derived neurotrophic factor (GDNF)/GDNF-related proteins(GDNFs)-dependent neurons in rat DRGs by use of in situ hybridization histochemistry in serial sections. Of the DRG neurons, 42% and 22% were intensely and weakly labeled by IB4, respectively. The former neurons were small, and the latter varied in size. Of the trkA mRNA-expressing neurons, 29% and 57% were intensely and weakly labeled by IB4, respectively. On the other hand, 66% and 10% of the c-ret mRNA-expressing neurons were intensely and weakly labeled, respectively. The mRNA of somatostatin, another major neuropeptide in the sensory neurons, was exclusively expressed in the intensely IB4-labeled neurons. These findings suggest that many NGF-dependent and peptidergic sensory neurons are labeled by IB4 in rats.

The Griffonia simplicifolia-I (GS-I) isolectins have been used to probe the effect of lectin valence on their high-affinity binding to human erythrocytes. These tetrameric lectins are composed of A and B subunits and constitute a series of five isolectins (A4, A3B, A2B2, AB3, B4). The A subunit is specific for alpha-D-GalNAc end groups and binds to the blood type A determinant GalNAcalpha1, as well as to terminal alpha-D-Gal groups found on type B cells. The B subunit is specific for alpha-D-Gal end groups, and binds very specifically to type B erythrocytes. This series of isolectins is tetravalent (A4), trivalent (A3B), divalent (A2B2), and monovalent (AB3) for type A erythrocytes; thus, this system provides the opportunity to examine the effect of lectin valency on the association constants of these GS-I isolectins binding to cells. Cell binding experiments carried out using 125I-labeled GS-I isolectins and type A human erythrocytes allowed us to demonstrate that (1) the association constant of the isolectin monovalent for alpha-D-GalNAc (AB3) is virtually identical to its association constant for the haptenic sugar methyl-N-acetyl-alpha-D-galactosaminide, reported previously, and (2) the association constant of the GS-I isolectins for human type A erythrocytes increases with increasing valency of the isolectin. These results indicate that the increased affinity displayed by the GS-I isolectins for human type A erythrocytes is dependent on their multivalency, and not on an extended binding site nor on nonspecific, or noncarbohydrate, interactions of the lectin with the cell surface. These findings should be of general relevance to understanding the high-affinity interactions observed between other multivalent proteins and multivalent ligands (e.g., cell surfaces).

An alpha-D-galactosyltransferase activity has been detected in membranous fractions (42,000 x g) of Ehrlich ascites cells which transfers galactosyl groups from UDP-galactose to endogenous and exogenous acceptors. The products of the reaction contain alpha-D-galactopyranosyl groups at the nonreducing termini. A solid state assay was developed to follow alpha-D-galactosyltransferase activity in the presence of beta-D-galactosyltransferase. Examination of a variety of insolubilized exogenous acceptors indicated that the most active acceptors for the alpha-D-galactosyltransferase had the structure beta-D-Gal-(1 goes to 4)-beta-D-GlcNAc(1 goes to at their nonreducing termini. Incubation of UDP-[14C]galactose and beta-D-gal-(1 goes to 4)-D-GlcNAc (N-acetyllactosamine) or of UDP-galactose and beta-D-[14C]Gal-(1 goes to 4)-D-GlcNAc in the presence of the alpha-D-galactosyltransferase resulted in the enzymic synthesis of a 14C-labeled trisaccharide. Chemical and enzymic methods of analysis revealed the structure of the trisaccharide to be alpha-D-Gal-(1 goes to 3)-beta-D-Gal-(1 goes to 4)-D-GlcNAc. These data indicate that the alpha-D-galactosyltransferase in Ehrlich ascites cells transfers galactosyl groups to suitable acceptors to form an alpha-(1 goes to 3)-D-galactosidic linkage.

A simple and convenient method for the introduction of radiolabel onto C-6' of N-acetyllactosamine is described. 1-N-Benzyl-3-O-beta-D-galactopyranosyl-D-arabinosylamine (1) was synthesized from 3-O-beta-D-galactopyranosyl-D-arabinose as described by Lee and Lee. Compound 1 was oxidized with D-galactose oxidase, and the product reduced with KB3H4 to introduce the label at C-6'. After dilution with unlabeled material, the N-benzyl-3-O-beta-D-[6-3H]galactopyranosyl-D-arabinosylamine was converted into 2-(benzylamino)-2-deoxy-4-O-D-[6-3H]galactopyranosyl-D-glucononitrile , which was subjected to simultaneous hydrogenolysis of the benzylamino and nitrile groups. N-Acetylation of the amino group as described by Alais and Veyrières afforded the crystalline title compound in 63% yield.

A UDP-Gal:N-acetyllactosaminide alpha (1,3)-galactosyltransferase from Ehrlich ascites tumor cells has been purified over 200,000-fold to apparent electrophoretic homogeneity. The purified enzyme transfers D-galactosyl groups from UDP-Gal to beta-D-Gal-(1,4)-D-GlcNAc in alpha-linkage. The apparent Km values for donor and acceptor substrates are 12.6 microM and 1.15 mM, respectively. The trisaccharides beta-D-Gal(1,4)-beta-D-GlcNAc(1,2)- or (1,6)-D-Man exhibit a Km 5-fold lower than that of N-acetyllactosamine, and an even more pronounced effect is observed with the biantennary pentasaccharide beta-D-Gal(1,4)-beta-D-GlcNAc(1,2)-[beta-D-Gal(1, 4)-beta-D-GlcNAc-(1,6)]-D-Man (Km 0.10 mM). The transferase shows a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions with an apparent subunit molecular weight of 80,000, exhibits a pH optimum at 6.2, and requires Mn2+ ions and detergent for enzymatic activity. Specificity studies using immobilized oligosaccharides show that the minimum acceptor structure for the alpha-galactosyltransferase is N-acetyllactosamine. The narrow specificity of the alpha-galactosyltransferase is indicated by the fact that lactose, beta-D-Gal(1,3)-D-GlcNAc, and beta-D-Gal(1,4)-[alpha-L-Fuc(1,3)]-D-GlcNAc are very poor acceptors. The enzyme differs from the blood-group B-specified galactosyltransferase in that the sequence alpha-L-Fuc(1,2)-beta-D-Gal(1,4)-D-GlcNAc is not an acceptor. Oligosaccharides, glycoproteins, glycolipids, and glycosaminoglycans containing the terminal nonreducing N-acetyllactosamine unit all serve as acceptors for the enzyme.

Kinetics of binding of p-nitrophenyl 2-O-alpha-D-mannopyranosyl-alpha-D-mannopyranoside (M2) to concanaviln A (con A) were examined. The time course of formation of a M2 . con A complex is clearly biphasic, whereas the association with con A of p-nitrophenyl 2-O-methyl-alpha-D-mannopyranoside and other monosaccharides is a monophasic process. The biphasic time course of the binding of M2 to conA is most simply explained in terms of a model wherein the disaccharide can bind to con A two different ways. In the initial rapid phase of the biphasic reaction, both complexes form in amounts determined by the relative values of the rate constants for association. In the subsequent slow phase, the complexes equilibriate according to the relative values of the initial constants for formation of each complex. The enthalpy of activation for formation of the initial complexes with M2 is about 4 kcal/mol less favorable than for monosaccharides, whereas the entropy of activations about 14 e.u. more favorable for binding of the disaccharide. These differences in the activation parameters for binding M2 and monosaccharides suggest that con A interacts simultaneously with groups on both mannopyranosyl residues.

The synthesis of bioactive oligosaccharides is too tedious to scale up for commercialization. However, structurally simplified glycomimetics are commercializable, if they can be synthesized much more easily than the oligosaccharides while having a comparable bioactivity. In this study, we propose a 2-oxabutane (OB) structure as an imitation of the internal monosaccharide units in oligosaccharides. Two trimannoside and three pentamannoside OB-glycomimics were synthesized in remarkably short steps. Among them, Manα1-OB-2Man 10, a trimannoside mimic, showed eight-fold affinity toward concanavalin A (ConA) relative to methyl mannoside in latex agglutination lectin assay and equilibrium dialysis assay (EDA), while the other mimics showed three- to four-fold affinities. EDA indicated that the bindings between each mimic molecule and a ConA subsite were all in one-to-one stoichiometry and thus these mimics were monovalent ligands, excluding multivalence effect for the high affinities. The strong affinity of 10 could be explained by the occupation of two mannose binding sites of a ConA subsite by its two mannose units. Mimic 10 proved to be even a better ligand for ConA than the natural disaccharide Manα1,2Man, while been much more easy to synthesize, thereby illustrating the potential of the approach here presented.

alpha-d-Mannopyranosyl units were attached to an aromatic scaffold through disulfide linkages to obtain mono- to trivalent glycosylated ligands for lectin binding studies. Isothermal titration calorimetric (ITC) measurements indicated that binding affinities of these derivatives to Concanavalin A (Con A) were comparable to or slightly higher than that of methyl alpha-d-mannopyranoside (K(a) values in the range of 10(4)M(-1)). The stoichiometries of the lectin-ligand complexes were in agreement with the formal valencies (1-3) of the respective ligands indicating cross-linking in interactions with the di- and trivalent derivatives. Multivalency effects could not, however, be observed with the latter. These ligands were shown to bind to the carbohydrate binding site of Con A using saturation transfer difference (STD) NMR competition experiments.

A blood group B-specific lectin from the mushroom Marasmius oreades (MOA) was investigated with respect to its molecular structure and carbohydrate binding properties. SDS-PAGE mass spectrometric analysis showed it to consist of an intact (H; 33 kDa) and truncated (L; 23 kDa) subunit in addition to a small polypeptide (P; 10 kDa). Isolation in the presence of EDTA produced only the H subunits, indicating that the latter two are formed by metalloprotease cleavage of the intact H subunit. Tryptic digestion of the H, L, and P polypeptide chains followed by mass spectral analysis supports this view. The lectin strongly precipitated blood group type B substance, was nonreactive with type A substance, and reacted weakly with type H substance. Carbohydrate binding studies reveal a high affinity for Galalpha1,3Gal (but not for the isomeric alpha1,2-, alpha1,4-, and alpha1,6-disaccharides); Galalpha1,3Galbeta1,4GlcNAc; and the type B branched trisaccharide. MOA also reacts strongly with murine laminin from the Engelbreth-Holm-Swarm sarcoma and bovine thyroglobulin, both of which contain multiple Galalpha1,3Galbeta1,4GlcNAc end groups. This linear B trisaccharide is a component of porcine tissues and organs, preventing their transplantation into humans. MOA also shares carbohydrate recognition of this trisaccharide with toxin A elaborated by Clostridium difficile.

Aplysia gonad lectin (AGL), which has been shown to stimulate mitogenesis in human peripheral lymphocytes, to suppress tumor cells, and to induce neurite outgrowth and improve cell viability in cultured Aplysia neurons, exhibits a peculiar galacturonic acid/galactose specificity. The carbohydrate binding site of this lectin was characterized by enzyme-linked lectino-sorbent assay and by inhibition of AGL-glycan interactions. Examination of the lectin binding with 34 glycans revealed that it reacted strongly with the following glycoforms: most human blood group precursor (equivalent) glycoproteins (gps), two Galalpha1-->4Gal-containing gps, and two d-galacturonic acid (GalUA)-containing polysaccharides (pectins from apple and citrus fruits), but poorly with most human blood group A and H active and sialylated gps. Among the GalUA and mammalian saccharides tested for inhibition of AGL-glycan binding, GalUA mono- to trisaccharides were the most potent ones. They were 8.5 x 10(4) times more active than Gal and about 1.5 x 10(3) more active than the human blood group P(k) active disaccharide (E, Galalpha1-->4Gal). This disaccharide was 6, 28, and 120 times more efficient than Galbeta1-->3GlcNAc(I), Galbeta1-->3GalNAc(T), and Galbeta1--> 4GlcNAc (II), respectively, and 35 and 80 times more active than melibiose (Galalpha1-->6Glc) and human blood group B active disaccharide (Galalpha1-->3Gal), respectively, showing that the decreasing order of the lectin affinity toward alpha-anomers of Gal is alpha1-->4 > alpha1-->6 > alpha1-->3. From the data provided, the carbohydrate specificity of AGL can be defined as GalUAalpha1-->4 trisaccharides to mono GalUA > branched or cluster forms of E, I, and II monomeric E, I, and II, whereas GalNAc is inactive.

The association constant of Cajanus cajan lectin for methyl alpha-D-mannopyranoside was studied by equilibrium dialysis method. An attempt was also made to understand the metal ion requirements and to establish that ionizable groups are responsible for lectin-carbohydrate interaction. The N-terminal sequence up to 27 amino acid residues was found to be more than 80% homologous with other mannose-specific legume lectins of the tribe Viceae. Like concanavalin A and pea lectin it also exhibits high affinity for the sugar alpha-methyl mannose and at 37 degrees C the association constant was found to be 1.4x104 M-1. The lectin required one Ca2+ and one Mg2+ per mole and during the lectin sugar interaction two ionizable groups with pK of 3.75 and 8.3 are ionized. Whether the secondary structure is similarly affected with pH changes and presence or absence of metal ion was investigated by circular dichroism studies. Results suggested that changes in carbohydrate binding properties of the Cajanus cajan lectin due to change in pH and addition of metal ions are not accompanied by any significant change in secondary structure.

The Griffonia simplicifolia-I (GS-I) isolectins have been used to probe the effect of lectin valence on their high-affinity binding to human erythrocytes. These tetrameric lectins are composed of A and B subunits and constitute a series of five isolectins (A4, A3B, A2B2, AB3, B4). The A subunit is specific for alpha-D-GalNAc end groups and binds to the blood type A determinant GalNAcalpha1, as well as to terminal alpha-D-Gal groups found on type B cells. The B subunit is specific for alpha-D-Gal end groups, and binds very specifically to type B erythrocytes. This series of isolectins is tetravalent (A4), trivalent (A3B), divalent (A2B2), and monovalent (AB3) for type A erythrocytes; thus, this system provides the opportunity to examine the effect of lectin valency on the association constants of these GS-I isolectins binding to cells. Cell binding experiments carried out using 125I-labeled GS-I isolectins and type A human erythrocytes allowed us to demonstrate that (1) the association constant of the isolectin monovalent for alpha-D-GalNAc (AB3) is virtually identical to its association constant for the haptenic sugar methyl-N-acetyl-alpha-D-galactosaminide, reported previously, and (2) the association constant of the GS-I isolectins for human type A erythrocytes increases with increasing valency of the isolectin. These results indicate that the increased affinity displayed by the GS-I isolectins for human type A erythrocytes is dependent on their multivalency, and not on an extended binding site nor on nonspecific, or noncarbohydrate, interactions of the lectin with the cell surface. These findings should be of general relevance to understanding the high-affinity interactions observed between other multivalent proteins and multivalent ligands (e.g., cell surfaces).

The binding of carbohydrates to the hemolytic lectin CEL-III isolated from the marine invertebrate Cucumaria echinata was studied. Equilibrium dialysis data suggest that CEL-III has two carbohydrate-binding sites with equal affinity. The binding of specific carbohydrates to CEL-III induces a decrease in the fluorescence intensity at 339 nm and the shift of the fluorescence emission maximum to a wavelength shorter by 3 nm, owing to the change in the environment of tryptophan. By analyzing the change in the fluorescence intensity at 339 nm as a function of the concentration of carbohydrates, the association constants for binding of individual carbohydrates to CEL-III were calculated. The results indicate that GalNAc, lactulose, and lactose are bound by CEL-III with fairly high affinity among the carbohydrates tested. The pH-dependence profile of the association constant of lactose suggests that CEL-III binds carbohydrates with highest affinity around pH 5.0. Modification of CEL-III with N-bromosuccinimide produces an oxidized derivative, in which four tryptophan residues/mol were oxidized and had no hemolytic activity. However, two out of these four tryptophans escaped from the modification in the presence of specific saccharides and the resulting derivative retained fairly high hemolytic activity.

The carbohydrate binding properties of the Dolichos biflorus seed lectin and DB58, a vegetative tissue lectin from this plant, were compared using two types of solid phase assays. Both lectins bind to hog blood group A + H substance covalently coupled to Sepharose 4B and this binding can be inhibited with free blood group A + H substance. However, the binding of the seed lectin is inhibited by D-GalNAc whereas DB58 binding was not inhibited by any monosaccharide tested, thus suggesting that its carbohydrate combining site may be more extensive than that of the seed lectin. The activities of these two lectins also differ from one another in ability to recognize blood group A + H substance adsorbed on to plastic and in the effects of salt and urea on their carbohydrate binding activities. Neither lectin showed glycosidase activity with p-nitrophenyl alpha-D-GalNAc or p-nitrophenyl beta-D-GalNAc.

The affinity of Bandeiraea (Griffonia) simplicifolia lectin-I isolectin B4 (BSI-B4) for the isomer of human blood group B active disaccharide (B, Gal alpha 1-->3Gal), the Gal alpha 1-->4Gal galabiose ligand, was studied by quantitative precipitin (QPA) and precipitin-inhibition assays. When human blood group B, P1 and H active glycoproteins were tested by OPA. BSI-B4 reacted strongly with both the B active glycoprotein purified from human ovarian cyst fluid and a P1 active glycoprotein isolated from sheep hydatid fluid and precipitated over 86% of the lectin nitrogen added. The P1 active glycoprotein-BSI-B4 interaction was inhibited by both Gal alpha 1-->3Gal alpha 1-->methyl and Gal alpha 1-->4Gal disaccharide indicating that BSI-B4 is not only reacting with Gal alpha 1-->3Gal disaccharide, but also recognizing Gal alpha 1-->4Gal. The galabiose sequence is frequently found in the carbohydrate chains of many glycosphingolipids located at the mammalian cell membranes such as intestinal and red blood cell membranes, for E. coli ligand binding and toxin attachment.

Equilibrium dialysis studies on the binding of the Dolichos biflorus lectin with [14C]methyl alpha-D-GalNAc showed that the lectin has two combining sites/molecule and an intrinsic association constant at 3 degrees C of 4.2 X 10(3) liters mol-1. Binding studies on individual fractions (II, IV, and VII) of the lectin that differ in their chromatographic properties on concanavalin A-Sepharose gave association constants for methyl alpha-D-GalNAc of 2.2 X 10(3) liters mol-1, 3.2 X 10(3) liters mol-1, and 3.2 X 10(3) liters mol-1, respectively. Molecular exclusion chromatography of iodinated Subunits I and II of the lectin, as well as sedimentation velocity studies of the noniodinated subunits, showed that the isolated subunits form aggregates in aqueous solution. Aggregates of subunit I were capable of agglutinating blood type A erythrocytes, precipitating blood group A + H substance, and binding to blood group A + H substance in an affinity electrophoretic system. Aggregates of subunit II exhibited none of these binding properties and did not inhibit the ability of the intact lectin to agglutinate type A erythrocytes. Affinity electrophoresis of subunit I showed that it has an association constant for N-acetyl-D-galactosamine similar to that of the intact lectin. The results suggest that it is subunit I that is primarily responsible for the carbohydrate binding properties of the lectin.