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The binding property of Con A has been studied intensively and applied widely to glycoconjugates / glycobiology for over 80 years. However, its role and functional relationship of Con A with these mammalian structural units, glycotopes, N-glycan chains, as well as their polyvalent forms in N-glycoproteins involved in the Con A- glycan interactions have not been all defined and organzied. In this study, the recognition factors involved in these inteactions were azalyzed by our well developed method- the enzyme linked lectinosorbent (ELLSA) and inhibition assay. Based on all data obtained, it is concluded that Con A, as previous report, has a relatively broad and wide recognition ability with the Manα1→ and Glcα1→ related glycans. In addition to it reacted strongly with yeast mannan and glycogens, it also bound well with a large number of mammalian N-glycans, including the N-glycans of rat sublingual gp (RSL), human Tamm-Horsfall glycoprotein (THGP), thyroglobin and lactosferrin. The recognition specificity of Con A towards ligands, expressed by Molar Relative Potency (Molar R.P.), in a decreasing order is as follows: a1→3, a1→6 Mannopentaose (M5) and Biantennary N-linked core pentasaccharide (MDi) ≥ a1→3, a1→6 Mannotriose (M3) > Mana1→3Man (α1→3Mannobiose), Mana1→2Man (α1→2Mannobiose), Mana1→6Man (α1→6Mannobiose), Mana1→4Man (α1→4Mannobiose) > GlcNAcb1→2Man (b1→2 N-Acetyl glucosamine-mannose) > Mana1→/Glcα1→ > Man > Glc, while Gal / GalNAc were inactive. Furthermore, the Man related code system, in this study, is proposed to express by both numbers of Man and GlcNAcb1→ branches (M3 to M9 / MMono to Penta etc.) and a table of three Mana1→ and Glca1→ related biomasses of six recognition factors involved in the Con A-glycan interactions has also been demonstrated. These themes should be one of the most valuable advances since 1980s.

Human ovarian cyst glycoproteins (HOC, cyst gps) isolated from pseudomucinous type of human ovarian cyst fluids is one of the richest and pioneer sources for studying biosynthesis, structures and functional roles of blood group ABH, Lea,b,x,y, sLea and sLex active glycoproteins. After 70+ years of exploration, four top highlights are shared. (i) an updated concept of glycotopes and their internal structures in cyst gps was composited; (ii) the unknown codes of new genes in secreted cyst gps were unlocked as Lex and Ley; (iii) recognition profiles of cyst glycans and a sialic acid-rich (18%) glycan with lectins and antibodies were shown. (iv) Co-expression of Blood Group A/ A-Leb/y and B/B-Leb/y active Glycotopes in the same glycan chains were isolated and illustrated. These are the most advanced achievements since 1980.

Lectins, in combination with our established enzyme-linked lectin sorbent assay (ELLSA) and inhibition study, have been used as powerful tools in many glycoconjugate recognition studies. In this short review, we highlight the following: (i) The recognition profiles of Gal/GalNAc-specific lectins were updated and upgraded. (ii) Based on the cross-specificities of applied lectins, a new classification system was introduced. (iii) Applications of lectins for the detection and identification of N-glycan and/or Tn glycotope in glycoconjugates were intergraded. (iv) The polyvalency of the glycotopes in glycans was found to play a critical role in glycan-lectin recognition. This is an unexplored area of glycobiology and one of the most promising directions toward the coming glycoscience transformation.

Summary The small 3-O-sulfated galactose head group of sulfatides, an abundant glycosphingolipid class, poses the (sphinx-like) riddle on involvement of glycan bridging by tissue lectins (sugar code). First, synthesis of head group derivatives for functionalization of amphiphilic dendrimers is performed. Aggregation of resulting (biomimetic) vesicles, alone or in combination with lactose, demonstrates bridging by a tissue lectin (galectin-4). Physiologically, this can stabilize glycolipid-rich microdomains (rafts) and associate sulfatide-rich regions with specific glycoproteins. Further testing documents importance of heterobivalency and linker length. Structurally, sulfatide recognition by galectin-8 is shown to involve sphingosine's OH group as substitute for the 3′-hydroxyl of glucose of lactose. These discoveries underscore functionality of this small determinant on biomembranes intracellularly and on the cell surface. Moreover, they provide a role model to examine counterreceptor capacity of more complex glycans of glycosphingolipids and to start their bottom-up glycotope surface programming., Graphical abstract, Highlights • Nanoparticle programming detects sulfatide-(N)-glycan bridging by galectins-4 and -8 • Protein design (linker/domain type) is a switch for aggregation activity • Sphingosine's OH group is involved in contact building with a galectin, Supramolecular Chemistry; Biochemistry; Biophysics

The recombinant fucolectin-related protein (FRP) of unknown function, encoded by the SP2159 gene of Streptococcus pneumoniae, was expressed in E. coli. In this study, its glycan-recognition epitopes and their binding potencies were examined by enzyme-linked lectinosorbent and inhibition assays. The results indicate that FRP reacted strongly with human blood group ABH and l-Fucα1→2-active glycotopes and in their polyvalent (super) forms. When expressed by mass relative potency, the binding affinities of FRP to poly-l-Fucα1→glycotopes were about 5.0 × 105 folds higher than that of the mono-l-Fucα1→glycotope form. This unique binding property of FRP can be used as a special tool to differentiate complex forms of l-Fucα1→2 and other forms of glycotopes., Highlights • FRP encoded by the SP2159 gene of Streptococcus pneumoniae shows an unusual blood group A, B, and H binding specificity. • Ley, Leb and H determinants, containing the monomeric forms of Fucα1-2Gal-glycotope were weak ligands for FRP. • Ley, Leb and H determinants, corresponding polyvalent forms exhibit unusual strong interactions with FRP. • Lex [Galβ1-4(LFucα1-3)GlcNAc], Lea [Galβ1-3(LFucα1-4) GlcNAc], A (GalNAcα1-3Gal) and B (Galα1-3Gal) are poor inhibitors. • Lex, Lea, A and B same determinants above enhance the FRP-polyvalent LFucα1-2Gal glycotope interactions. • FRP recognizes only LFucα1→ related ligands, but do not with others, such as Man related glycans.

Hog or porcine gastric mucin resembles the human source in carrying not only blood group antigens but also the rather rare α4-GlcNAc-capped terminal epitope functionally implicated in protection against Helicobacter pylori infection. Being more readily available and reasonably well characterized, it serves as a good reagent for immunobiological studies, as well as a standard for analytical methodology developments. Current approaches in mass spectrometry (MS)-based glycomic mapping remain vastly inadequate in revealing the full complexity of glycosylation, particularly for cases such as the extremely heterogeneous O-glycosylation of mucosal mucins that can be further sulfated. We demonstrate here a novel concerted workflow that extends the conventional matrix-assisted laser desorption/ionization–mass spectrometry (MALDI-MS) mapping of permethylated glycans in positive ion mode to include a further step of sulfoglycomic analysis in negative ion mode. This was facilitated by introducing a mixed-mode solid-phase extraction step, which allows direct cleanup and simultaneous fractionation of the permethylated glycans into separate non-sulfated and sulfated pools in one single step. By distinct MALDI-MS/MS fragmentation patterns, all previously known structural features of porcine gastric mucin including the terminal epitopes and location of sulfates could be readily defined. We additionally showed that both arms of the core 2 structures could be extended via 6-O-sulfated GlcNAc to yield a series of disulfated O-glycans not previously reported, thus expanding its current glycomic coverage. However, a targeted LC-MSn analysis was required and best suited to dig even deeper into validating the occurrence of very minor structural isomers carrying the Lewis Y epitope implicated by positive antibody binding.

Owing to the weak reactivities of monomeric d Manα1 and Galβ1→3/4GlcNAcβ (Iβ/IIβ) glycotopes with Ralstonia solanacearum lectin (RSL), their recognition roles were previously ignored. In this study, the interaction intensities of RSL toward four monomeric glycotopes l Fucα1→, d Manα1→ and Iβ/IIβ within two combining sites were established by both enzyme-linked lectinosorbent and inhibition assays. It was found that high density of l Fucα1→ complex enhanced the recognition intensities at l Fucα1→ site, polyvalent d Manα1→ was essential for binding at the d Manα1→ site and polyvalent Iβ/IIβ was required at l Fucα1→ site. The peculiar recognition systems of RSL are very different from other well known microbial lectins.

Background Lactoferrin is an iron-binding protein belonging to the transferrin family. In addition to iron homeostasis, lactoferrin is also thought to have anti-microbial, anti-inflammatory, and anticancer activities. Previous studies showed that all lactoferrins are glycosylated in the human body, but the recognition roles of their carbohydrate glycotopes have not been well addressed. Methods The roles of human and bovine lactoferrins involved in lectin–N-glycan recognition processes were analyzed by enzyme-linked lectinosorbent assay with a panel of applied and microbial lectins. Results and conclusions Both native and asialo human/bovine lactoferrins reacted strongly with four Man-specific lectins — Concanavalia ensiformis agglutinin, Morniga M, Pisum sativum agglutinin, and Lens culinaris lectin. They also reacted well with PA-IIL, a L Fuc>Man-specific lectin isolated from Pseudomonas aeruginosa. Both human and bovine lactoferrins also recognized a sialic acid specific lectin-Sambucus nigra agglutinin, but not their asialo products. Both native and asialo bovine lactoferrins, but not the human ones, exhibited strong binding with a GalNAc>Gal-specific lectin-Wisteria floribunda agglutinin. Human native lactoferrins and its asialo products bound well with four Gal>GalNAc-specific type-2 ribosome inactivating protein family lectins-ricin, abrin-a, Ricinus communis agglutinin 1, and Abrus precatorius agglutinin (APA), while the bovine ones reacted only with APA. General significance This study provides essential knowledge regarding the different roles of bioactive sites of lactoferrins in lectin–N-glycan recognition processes.

Ralstonia solanacearum lectin (RSL), that might be involved in phytopathogenicity, has been defined as LFucMan specific. However, the effects of polyvalency of glycotopes and mammalian structural units on binding have not been established. In this study, recognition factors of RSL were comprehensively examined with natural multivalent glycotopes and monomeric ligands using enzyme linked lectin-sorbent and inhibition assays. Among the glycans tested, RSL reacted strongly with multivalent blood group A(h) (GalNAcalpha1-3[Fucalpha1-2]Gal) and H (Fucalpha1-2Gal) active glycotopes, followed by B(h) (Galalpha1-3[Fucalpha1-2]Gal), Le(a) (Galbeta1-3[Fucalpha1-4]GlcNAc) and Le(b) (Fucalpha1-2Galbeta1-3[Fucalpha1-4]GlcNAc) active glycotopes. But weak or negligible binding was observed for blood group precursors having Galbeta1-3/4GlcNAcbeta1- (Ibeta/IIbeta) residues or Galbeta1-3GalNAcalpha1- (Talpha), GalNAcalpha1-Ser/Thr (Tn) bearing glycoproteins. These results indicate that the density and degree of exposure of multivalent ligands of alpha1-2 linked LFuc to Gal at the non-reducing end is the most critical factor for binding. An inhibition study with monomeric ligands revealed that the combining site of RSL should be of a groove type to fit trisaccharide binding with highest complementarity to blood group H trisaccharide (H(L); Fucalpha1-2Galbeta1-4Glc). The outstandingly broad RSL saccharide-binding profile might be related to the unusually wide spectrum of plants that suffer from R. solanacearum pathogenicity and provide ideas for protective antiadhesion strategies.

Human blood group A, B, H, Ii, Le(a) and Le(b) antigens and their determinants expressed on ovarian cyst glycoproteins have been studied for over five decades. However, little is known about sialyl Le(x) and sialyl Le(a) glycotopes, which play essential roles in normal immunity, inflammation, and cancer cell metastasis. Furthermore, Le(x) and Le(y) were classified as glycotopes of unknown genes. Identification of these Lewis epitopes was hampered by the lack of specific antibodies. In this study, the occurrence of sialyl Le(x), sialyl Le(a), Le(x) and Le(y) reactivities in cyst glycoproteins was characterized by enzyme-linked immunosorbent assays. The results indicated that most human ovarian cyst glycoproteins carried Le(x) (8/25) and/or Le(y) (17/25) glycotopes. The expression (epitopes) of the new genes described in previous reports are Le(x) and Le(y) glycotopes; the reactivities of sialyl Le(x) and sialyl Le(a) glycotopes in secreted cyst glycoproteins may be affected by the conditions of purification; the relationship between Le(y) and human blood group ABH was confirmed; recognition profiles of sialyl Le(x), sialyl Le(a), Le(x) and Le(y) present in the carbohydrate chains of water-soluble cyst glycoproteins were illustrated; possible attachments of glycotopes to the internal carbohydrate complex of cyst glycoproteins have been reconstructed; proposed biosynthetic pathways for the formation of sialyl Le(a), sialyl Le(x), Le(x), Le(y), ALe(y) and BLe(y) determinant structures on Type I and Type II core structures of human ovarian cyst glycoproteins are also included in this study.

Lectins are ubiquitous proteins of nonimmune origin, present in plants, microorganisms, animals and humans which specifically bind defined monosugars or oligosaccharide structures. Great progress has been made in recent years in understanding crucial roles played by lectins in many biological processes. Elucidation of carbohydrate specificity of human and animal lectins is of great importance for better understanding of these processes. Long before the role of carbohydrate-protein interactions had been explored, many lectins, mostly of plant origin, were identified, characterized and applied as useful tools in studying glycoconjugates. This review focuses on the specificity-based lectin classification and the methods of measuring lectin-carbohydrate interactions, which are used for determination of lectin specificity or for identification and characterization of glycoconjugates with lectins of known specificity. The most frequently used quantitative methods are shortly reviewed and the methods elaborated and used in our laboratories, based on biotinylated lectins, are described. These include the microtiter plate enzyme-linked lectinosorbent assay, lectinoblotting and lectin-glycosphingolipid interaction on thin-layer plates. Some chemical modifications of lectin ligands on the microtiter plates and blots (desialylation, Smith degradation, beta-elimination), which extend the applicability of these methods, are also described.

Bacterium Klebsiella pneumoniae (KP) contains a prominent capsule. Clinical infections usually are associated with pneumonia or urinary tract infection (UTI). Emerging evidence implicates KP in severe liver abscess especially in diabetic patients. The goal of this study was to investigate the capsular polysaccharides from KP of liver abscess (hepatic-KP) and of UTI-KP. The composition of capsular polysaccharides was analyzed by capillary high-performance liquid chromatography (HPLC, Dionex system). The terminal sugars were assayed by binding ability to lectins. The results showed that the capsule of a hepatic KP (KpL1) from a diabetic patient contained fucose, while the capsule from UTI-KP (KpU1) did not. The absence of fucose was verified by the absence of detectable polymerase chain reaction (PCR) fragment for fucose synthesis genes, gmd and wcaG in KpU1. Mice infected with the KpL1 showed high fatality, whereas those infected with the KpU1 showed high survival rate. The KpL1 capsule was reactive to lectins AAA and AAL, which detect fucose, while the KpU1 capsule was reactive to lectin GNA, which detects mannose. Phagocytosis experiment in mouse peritoneal cavity indicated that the peritoneal macrophages could interact with KpU1, while rare association of KpL1 with macrophages was observed. This study revealed that different polysaccharides were displayed on the bacterial capsules of virulent KpL1 as compared with the less virulent KpU1. Interaction of KpU1 with mice peritoneal macrophages was more prominent than that of KpL1. The possession of fucose might contribute to KpL1 virulence by avoiding phagocytosis since fucose on bacteria had been implicated in immune evasion.

Carbohydrate moieties of salivary mucins play various roles in life processes, especially as a microbial trapping agent. While structural details of the salivary O-glycans from several mammalian sources are well studied, very little information is currently available for the corresponding N-glycans. The existence of N-glycans alongside O-glycans on mucin isolated from rat sublingual gland has previously been implicated by total glycosyl compositional analysis but the respective structural data are both lacking. The advent of facile glycomic mapping and sequencing methods by mass spectrometry (MS) has enabled a structural reinvestigation into many previously unsolved issues. For the first time, high energy collision induced dissociation (CID) MALDI-MS/MS as implemented on a TOF/TOF instrument was applied to permethyl derivatives of mucin type O-glycans and N-glycans, from which the linkage specific fragmentation pattern could be established. The predominant O-glycans carried on the rat sublingual mucin were defined as sialylated core 3 and 4 types whereas the N-glycans were determined to be non-bisected hybrid types similarly carrying a sialylated type II chain. The masking effect of terminal sialylation on the tight binding of rat sublingual mucin to Galbeta1-->4GlcNAc specific lectins and three oligomannose specific lectins were clearly demonstrated in this study.

Previous studies on the carbohydrate specificities of Erythrina cristagalli lectin (ECL) were mainly limited to analyzing the binding of oligo-antennary Galbeta1-->4GlcNAc (II). In this report, a wider range of recognition factors of ECL toward known mammalian ligands and glycans were examined by enzyme-linked lectinosorbent and inhibition assays, using natural polyvalent glycotopes, and a glycan array assay. From the results, it is shown that GalNAc was an active ligand, but its polyvalent structural units, in contrast to those of Gal, were poor inhibitors. Among soluble natural glycans tested for 50% molecular mass inhibition, Streptococcus pneumoniae type 14 capsular polysaccharide of polyvalent II was the most potent inhibitor; it was 2.1 x 10(4), 3.9 x 10(3) and 2.4 x 10(3) more active than Gal, tri-antennary II and monomeric II, respectively. Most type II-containing glycoproteins were also potent inhibitors, indicating that special polyvalent II and Galbeta1-related structures play critically important roles in lectin binding. Mapping all information available, it can be concluded that: [a] Galbeta1-->4GlcNAc (II) and some Galbeta1-related oligosaccharides, rather than GalNAc-related oligosaccharides, are the core structures for lectin binding; [b] their polyvalent II forms within macromolecules are a potent recognition force for ECL, while II monomer and oligo-antennary II forms play only a limited role in binding; [c] the shape of the lectin binding domains may correspond to a cavity type with Galbeta1-->4GlcNAc as the core binding site with additional one to four sugars subsites, and is most complementary to a linear trisaccharide, Galbeta1-->4GlcNAcbeta1-->6Gal. These analyses should facilitate the understanding of the binding function of ECL.

The carbohydrate binding properties of a novel member of the subfamily of galactose-specific jacalin-related lectin isolated from the bark of black mulberry ( Morus nigra ) (Morniga G) was studied in detail by enzyme-linked lectinosorbent and inhibition assays using panels of monomeric saccharides, mammalian polyvalent glycotopes and polysaccharides. Among the natural glycans tested for lectin binding, Morniga G reacted best with glycoproteins (gps) presenting a high density of tumor-associated carbohydrate antigens Tn (GalNAcα1-Ser/Thr) and T α (Galβ1-3GalNAcα1-). Their reactivities, on a nanogram basis, were up to 72.5, 3.9 × 10 3 , 6.0 × 10 3 , 8.8 × 10 3 and 2.9 × 10 4 times higher than that of Tn -containing glycopeptides (M.W. T , Tn , GalNAc and Gal, respectively. It also reacted well with many multi-antennary N -glycans with II (Galβ1-4GlcNAc) termini, ABH histo-blood group antigens and their precursors containing high densities of I / II and T / Tn glycotopes, and sialylated T / Tn . Among the mono-, di- and oligosaccharides tested, Thomsen-Friedenreich ( T ) disaccharide with aromatic aglycon [Galβ1-3GalNAcα1-benzyl ( T α 1-benzyl)] and Tn glycopeptides were the best inhibitors. Molecular modeling and docking studies indicated the occurrence of a primary GalNAcα1- and Galβ1-3GalNAc glycotope-binding site in Morniga G. Using a recently proposed system [Wu, A.M., 2003. Carbohydrate structural units in glycoproteins and polysaccharides as important ligands for Gal and GalNAc reactive lectins. J. Biomed. Sci. 10, 676–688], the binding properties of the combining sites of Morniga G can be defined as follows: (i) the monosaccharide specificity is GalNAc/Gal ≫ Man/Glc, GlcNAc and l Fuc; (ii) the mammalian glycotope specificity is T α 1-benzyl > T > Tn > GalNAcβ1-3Gal (P), while B / E (Galα1-3/4Gal), I / II (Galβ1-3/4GlcNAc), S (GalNAcβ1-4Gal), F / A (GalNAcα1-3GalNAc/Gal) and L (Galβ1-4Glc) are inactive; (iii) the most active ligand is T / Tn ; (iv) simple clustered Tn or triantennary N -glycans with II termini (Tri- II ) have limited impact; (v) high-density polyvalent glycotopes play a prominent role for enhancing Morniga G reactivity. These results provide evidence for the binding of this lectin to dense cell surface T / Tn glycoconjugates and facilitate future usage of this lectin in biotechnological and medical applications.

Carbohydrate structures between retinal neurons and retinal pigment epithelium (RPE) play an important role in maintaining the integrity of retinal adhesion to underlying RPE, and in retinal detachment pathogenesis. Since relevant knowledge is still in the primary stage, glycotopes on the adult retina of mongrel canines (dog), micropigs and Sprague-Dawley rats were examined by lectino-histochemistry, using a panel of 16 different lectins. Paraffin sections of eyes were stained with biotinylated lectins, and visualized by streptavidin-peroxidase and diaminobenzidine staining. Mapping the affinity profiles, it is concluded that: (i) all sections of the retina reacted well with Morniga M, suggesting that N-linked glycans are present in all layers of the retina; (ii) no detectable human blood group ABH active glycotopes were found among retinal layers; (iii) outer and inner segments contained glycoconjugates rich in ligands reacting with Tα (Galβ1–3GalNAcα1-Ser/Thr) and Tn (GalNAcα1-Ser/Thr) specific lectins; (iv) cone cells of retina specifically bound peanut agglutinin (PNA), which recognizes Tα residues and could be used as a specific marker for these photoreceptors; (v) the retinas of rat, dog and pig, had a similar binding profile but with different intensity; (vi) each retinal layer had its own binding characteristic. This information may provide useful background knowledge for normal retinal physiology and miscellaneous retinal diseases, including retinal detachment (RD) and age-related macular degeneration (ARMD).

Ricinus communis agglutinin (RCA1) is one of the most important applied lectins that has been widely used as a tool to study cell surfaces and to purify glycans. Although the carbohydrate specificity of RCA1 has been described, the information obtained was mainly focused on inhibition of simple Galbeta1-related oligosaccharides and simple clusters. Here, all possible recognition factors of RCA1 of glycan binding were examined by enzyme-linked lectinosorbent (ELLSA) and inhibition assays, using known mammalian Gal/GalNAc carbohydrate structural units and natural polyvalent glycans. Among the glycoproteins (gps) tested and expressed as 50% nanogram inhibition, the high-density polyvalent Galbeta1-4GlcNAc (II) glycotopes occurring in natural gps, such as Pneumococcus type 14 capsular polysaccharide which is composed of repeating poly II residues, resulted in 9.0 x 10(4), 1.5 x 10(5), 2.3 x 10(4) and 2.1 x 10(4)-fold higher affinities to RCA1 than the monomeric Gal, linear I/II and Tri-antennary-II (Tri-II). Of the ligands tested and expressed as nanomoles of 50% inhibition, Tri-II was the best, being about 2, 4, 25.6 and 33.3 times better inhibitor than Di-II, II, I (Galbeta1-3GlcNAc) and Gal, respectively. From the results of this study, it is concluded that: (a) Galbeta1-4GlcNAc and other Galbeta1-related oligosaccharides are essential for lectin binding and their polyvalent form in macromolecules should be the most important recognition factor for RCA1; (b) the combining site of RCA1 may be a groove type, recognizing Galbeta1-4GlcNAc (II) as the major binding site; (c) its combining size may be large enough to accommodate a tetrasaccharide of beta-anomeric Gal at the non-reducing end and most complementary to human blood group I Ma active trisaccharide (Galbeta1-4GlcNAcbeta1-6Gal) and lacto-N-neotetraose (Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc); (d) RCA1 has a preference for the beta-anomer of Gal oligosaccharides with a Galbeta1-4 linkage > Galbeta1-6 > or = Galbeta1-3; (e) configuration of carbon-2, -3 -4 and -6 in Gal are essential for binding; (f) hydrophobic interaction in the vicinity of the binding site useful for sugar accommodation increases affinity. These results should be helpful for understanding the functional role of RCA1 and for characterizing glycotopes of mammalian complex carbohydrates.

Cell-surface glycans are functional docking sites for tissue lectins such as the members of the galectin family. This interaction triggers a wide variety of responses; hence, there is a keen interest in defining its structural features. Toward this aim, we have used enzyme-linked lectinosorbent (ELLSA) and inhibition assays with the prototype rat galectin-5 and panels of free saccharides and glycoconjugates. Among 45 natural glycans tested for lectin binding, galectin-5 reacted best with glycoproteins (gps) presenting a high density of Galbeta1-3/4GlcNAc (I/II) and multiantennary N-glycans with II termini. Their reactivities, on a nanogram basis, were up to 4.3 x 10(2), 3.2 x 10(2), 2.5 x 10(2), and 1.7 x 10(4) times higher than monomeric Galbeta1-3/4GlcNAc (I/II), triantennary-II (Tri-II), and Gal, respectively. Galectin-5 also bound well to several blood group type B (Galalpha1-3Gal)- and A (GalNAcalpha1-3Gal)-containing gps. It reacted weakly or not at all with tumor-associated Tn (GalNAcalpha1-Ser/Thr) and sialylated gps. Among the mono-, di-, and oligosaccharides and mammalian glycoconjugates tested, blood group B-active II (Galalpha1-3Gal beta1-4GlcNAc), B-active IIbeta1-3L (Galalpha1-3Galbeta1-4GlcNAc beta1-3Galbeta1-4Glc), and Tri-II were the best. It is concluded that (1) Galbeta1-3/4GlcNAc and other Galbeta1-related oligosaccharides with alpha1-3 extensions are essential for binding, their polyvalent form in cellular glycoconjugates being a key recognition force for galectin-5; (2) the combining site of galectin-5 appears to be of a shallow-groove type sufficiently large to accommodate a substituted beta-galactoside, especially with alpha-anomeric extension at the non-reducing end (e.g., human blood group B-active II and B-active IIbeta1-3L); (3) the preference within beta-anomeric positioning is Galbeta1-4 > or = Galbeta1-3 > Galbeta1-6; and (4) hydrophobic interactions in the vicinity of the core galactose unit can enhance binding. These results are important for the systematic comparison of ligand selection in this family of adhesion/growth-regulatory effectors with potential for medical applications.

The agglutinin isolated from the seeds of Maclura pomifera (MPA) recognizes a mucin-type disaccharide sequence, Galbeta1-->3GalNAc (T) on a human erythrocyte membrane. We have utilized the enzyme-linked lectinosorbent assay (ELLSA) and inhibition assay to more systematically analyze the carbohydrate specificity of MPA with glyco-recognition factors and mammalian Gal/GalNAc structural units in lectin-glycoform interactions. From the results, it is concluded that the high densities of polyvalent GalNAcalpha1-->Ser/Thr (Tn) and Galbeta1-->3GalNAcalpha1-->Ser/Thr (T(alpha)) glycotopes in macromolecules are the most critical factors for MPA binding, being on a nanogram basis 2.0 x 10(5), 4.6 x 10(4) and 3.9 x 10(4) more active than monovalent Gal, monomeric T and Tn glycotope, respectively. Other carbohydrate structural units in mammalian glycoconjugates, such as human blood group Sd (a+) related disaccharide (GalNAcbeta1-->4Gal) and Pk/P1 active disaccharide (Galalpha1-->4Gal) were inactive. These results demonstrate that the configurations of carbon-4 and carbon-2 are essential for MPA binding and establish the importance of affinity enhancement by high-density polyvalencies of Tn/T glycotopes in MPA-glycan interactions. The overall binding profile of MPA can be defined in decreasing order as high density of polyvalent Tn/T(alpha) (M.W. > 4.0 x 10(4)) >> Tn-containing glycopeptides (M.W. monomeric T/Tn and P (GalNAcbeta1-->3Gal) > GalNAc > Gal >> Man, L: ARA: , D: Fuc and Glc (inactive). Our findings should aid in the selection of this lectin for elucidating functions of carbohydrate chains in life processes and for applications in the biomedical sciences.

The lectin extracted from the seeds of Salvia sclarea (SSL) recognizes the Tn antigen (GalNAc alpha1--Ser/Thr) expressed in certain human carcinomas. In previous studies, knowledge of the binding properties of SSL was restricted to GalNAcalpha1--related oligosaccharides and glycopeptides. Thus, the requirements of functional groups in monosaccharide and high-density polyvalent carbohydrate structural units for SSL binding and an updated affinity profile were further evaluated by enzyme-linked lectinosorbent (ELLSA) and inhibition assays. Among the glycoproteins (gps) tested for interaction, a high density of exposed Tn-containing glycoproteins such as in the armadillo salivary Tn glycoprotein and asialo ovine salivary glycoprotein reacted best with SSL. When the gps were tested for inhibition of SSL binding, which was expressed as 50% nanogram inhibition, the high density polyvalent Tn present in macromolecules was the most potent inhibitor. Among the monosaccharide and carbohydrate structural units studied, which were expressed as nanomole inhibition, GalNAc alpha1--3GalNAc beta1--3Gal alpha1--4Gal beta1--4Glc (Fp), GalNAc alpha1--3Gal beta1--4Glc (A(L)), GalNAc alpha1--3GalNAc beta1--Me (F beta), GalNAc alpha1--3GalNAc alpha1--Me (F alpha) and GalNAc alpha1--Ser/Thr (Tn) were the most active ligands, being 2.5-5.0 x 10(3) and 1.25-2.5 times more active than Gal and GalNAc, respectively. From the results, it is suggested that the combining site of SSL is a shallow groove type, recognizing the monosaccharide of GalNAc as the major binding site or Tn up to the Forssman pentasaccharide (Fp). It can be concluded that the three critical factors for SSL binding are the -NH CH(3)CO at carbon-2 in Gal, the configuration of carbon-3 in GalNAc, and the polyvalent Tn (GalNAc alpha1--Ser/Thr) present in macromolecules. These results should assist in understanding the glyco-recognition factors involved in carbohydrate-lectin interactions in biological processes. The effect of the polyvalent F alpha, F beta and GalNAc beta1--3Gal alpha1--(P alpha) glycotopes on binding should be examined. However, this is hampered by the lack of availability of suitable reagents.

Morniga M is a jacalin-related and mannose-specific lectin isolated from the bark of the mulberry (Morus nigra). In order to understand the function and application of this novel lectin, the binding property of Morniga M was studied in detail using an enzyme-linked lectinosorbent assay and lectin-glycan inhibition assay with extended glycan/ligand collection. From the results, it was found that the di-, tri-, and oligomannosyl structural units of N-glycans such as those of the bovine alpha1-acid glycoprotein (gp) and lactoferrin were the most active gps, but not the O-glycans or polysaccharides including mannan from yeast. The binding affinity of Morniga M for ligands can be ranked in decreasing order as follows: gps carrying multiple N-glycans with oligomannosyl residuesN-glycopeptide with a single trimannosyl coreTri-Man oligomer [Man alpha1--6(Man alpha1--3) Man], Penta-Man oligomer [Man alpha1--6(Man alpha1--3)Man alpha1--6(Man alpha1--3) Man]or = Man alpha1--2, 3 or 6 ManManGlcNAc, GlcL-Fuc, Gal, GalNAc (inactive), demonstrating the unique specificity of this lectin that may not only assist in our understanding of cell surface carbohydrate ligand-lectin recognition, but also provide informative guidelines for the application of this structural probe in biotechnological and clinical regimens, especially in the detection and purification of N-linked glycans.

Artocarpus lakoocha agglutinin (ALA), isolated from the seeds of A. lakoocha fruit, is a galactose-binding lectin and a potent mitogen of T and B cells. Knowledge obtained from previous studies on the affinity of ALA was limited to molecular and submolecular levels of Galbeta1-->3GalNAc (T) and its derivatives. In the present study, the carbohydrate specificity of ALA was characterized at the macromolecular level according to the mammalian Gal/GalNAc structural units and corresponding glycoconjugates by an enzyme-linked lectinosorbent (ELLSA) and inhibition assays. The results indicate that ALA binds specifically to tumor-associated carbohydrate antigens GalNAcalpha1-->Ser/Thr (Tn) and Galbeta1-->3 GalNAcalpha1-->Ser/Thr (Talpha). It barely cross-reacts with other common glycotopes on glycoproteins, including ABH blood group antigens, Galbeta1-->3/4GlcNAc (I/II) determinants, T/Tn covered by sialic acids, and N-linked plasma glycoproteins. Dense clustering structure of Tn/Talpha-containing glycoproteins tested resulted in 2.4 x 10(5)-6.7 x 10(5)-fold higher affinities to ALA than the respective GalNAc and Gal monomer. According to our results, the overall affinity of ALA for glycans can be ranked respectively: polyvalent Tn/Talpha glycotopes >> monomeric Talpha and simple clustered Tn >> monomeric Tn > GalNAc > Gal; while other glycotopes: Galalpha1-->3/4Gal (B/E), Galbeta1-->3/4GlcNAc (I/II), GalNAcalpha1-->3Gal/GalNAc (A/F), and GalNAcbeta1-->3/4Gal (P/S) were inactive. The strong specificity of ALA for Tn/Talpha cluster suggests the importance of glycotope polyvalency during carbohydrate-receptor interactions and emphasizes its value as an anti-Tn/T lectin for analysis of glycoconjugate mixtures or transformed carbohydrates.

In our recent publication, we defined core aspects of the carbohydrate specificity of domain-I of recombinant tandem-repeat-type galectin-4 from rat gastrointestinal tract (G4-N), especially its potent interaction with the linear tetrasaccharide Galbeta1-3GlcNAcbeta1-3Galbeta1-4Glc (Ibeta1-3L). The assumed role of galectin-4 as a microvillar raft stabilizer/organizer and as a malignancy-associated factor in hepatocellular and gastrointestinal carcinomas called for further refinement of its binding specificity. Thus, the effects of polyvalency of glycotopes and natural modifications of human blood group ABH/Lewis sugars at the terminal Galbeta1-core saccharides were thoroughly examined by the enzyme-linked lectinosorbent and lectin-glycan inhibition assays. The results indicate that (a) a high-density of polyvalent Galbeta1-3/4GlcNAc (I/II), Galbeta1-3GalNAc (T) and/or GalNAcalpha1-Ser/Thr (Tn) strongly favors G4-N/glycoform binding. These glycans were up to 2.3 x 10(6), 1.4 x 10(6), 8.8 x 10(5), and 1.4 x 10(5) more active than Gal, GalNAc, monomeric I/II and T, respectively; (b) while lFuc is a poor inhibitor, its presence as alpha1-2 linked to terminal Galbeta1-containing oligosaccharides, such as H active Ibeta1-3L, markedly enhances the reactivities of these ligands; (c) when blood group A (GalNAcalpha1-) or B (Galalpha1-) determinants are attached to terminal Galbeta1-3/4GlcNAc (or Glc) oligosaccharides, the reactivities are also increased; (d) with lFucalpha1-3/4 linked to sub-terminal GlcNAc, the reactivities of these haptens are reduced; and (e) short chain Le(a)/Le(x)/Le(y) and the short chains of sialyl Le(a)/Le(x) are poor inhibitors. These distinct binding features of G4-N establish the important concept of affinity enhancement by high density polyvalencies of glycotopes (vs. multi-antennary I/II) and by introduction of an ABH key sugar to Galbeta1-terminated core glycotopes. The polyvalent ligand binding properties of G4-N may help our understanding of its crucial role for cell membrane raft stability and provide salient information for the optimal design of blocking substances such as anti-tumoral glycodendrimers.

Agaricus bisporus agglutinin (ABA) isolated from edible mushroom has a potent anti-proliferative effect on malignant colon cells with considerable therapeutic potential as an anti-neoplastic agent. Since previous studies on the structural requirement for binding were limited to molecular or submolecular levels of Galβ1-3GalNAc (T; Thomsen–Friedenreich disaccharide glycotope; where Gal represents d-galactopyranose and GalNAc represents 2-acetamido-2-deoxy-d-galactopyranose) and its derivatives, the binding properties of ABA were further investigated using our collection of glycans by enzyme-linked lectinosorbent assay and lectin–glycan inhibition assay. The results indicate that polyvalent Galβ1-related glycotopes, GalNAcα1-Ser/Thr (Tn), and their cryptoforms, are the most potent factor for ABA binding. They were up to 5.5×105 and 4.7×106 times more active than monomeric T and GalNAc respectively. The affinity of ABA for ligands can be ranked as: multivalent Tα (Galβ1-3GalNAcα1-), Tn and I/II (Galβ1-3GlcNac/Galβ1-4GlcNAc, where GlcNAc represents 2-acetamido-2-deoxy-d-glucopyranose)>>>>monomeric Tα and Tn>I>>GalNAc>>>II, L (Galβ1-4Glc, where Glc represents d-glucopyranose) and Gal (inactive). These specific binding features of ABA establish the importance of affinity enhancement by high-density polyvalent (versus multiantennary I/II) glycotopes and facilitate our understanding of the lectin receptor recognition events relevant to its biological activities.

Artocarpus integrifolia agglutinin (Jacalin) from the seeds of jack fruits has attracted considerable attention for its diverse biological activities and has been recognized as a Galbeta1-->3GalNAc (T) specific lectin. In previous studies, the information of its binding was limited to the inhibition results of monosaccharides and several T related disaccharides, but its interaction with other carbohydrate structural units occurring in natural glycans has not been characterized. For this reason, the binding profile of this lectin was studied by enzyme linked lectinosorbent assay (ELLSA) with our glycan/ligand collection. Among glycoproteins (gps) tested for binding, high density of multi-Galbeta1-->3GalNAcalpha1--> (mT(alpha)) and GalNAcalpha1-->Ser/Thr (mTn) containing gps reacted most avidly with Jacalin. As inhibitors expressed as nanograms yielding 50% inhibition, these mT(alpha) and mTn containing glycans were about 7.1 x 10(3), 4.0 x 10(5), and 7.8 x 10(5) times more potent than monomeric T(alpha), GalNAc, and Gal. Of the sugars tested and expressed as nanomoles for 50% inhibition, Tn containing peptides, T(alpha), and the human P blood group active disaccharide (P(alpha), GalNAcbeta1-->3Galalpha1-->) were the best and about 283 times more active than Gal. We conclude that the most potent ligands for this lectin are mTn, mT, and possibly P(alpha) glycotopes, while GalNAcbeta1-->4Galbeta1-->, GalNAcalpha1-->3Gal, GalNAcalpha1-->3GalNAc, and Galalpha1-->3Gal determinants were poor inhibitors. Thus, the overall binding profile of Jacalin can be defined in decreasing order as high density of mTn, and mT(alpha) >>> simple Tn cluster > monomeric T(alpha) > monomeric P(alpha) > monomeric Tn > monomeric T > GalNAc > Gal > Methylalpha1-->Man z.Gt; Man and Glc (inactive). Our finding should aid in the selection of this lectin for biological applications.

Pseudomonas aeruginosa galactose- and fucose-binding lectins (PA-IL and PA-IIL) contribute to the virulence of this pathogenic bacterium, which is a major cause of morbidity and mortality in cystic fibrosis patients. The crystal structure of PA-IIL in complex with fucose reveals a tetrameric structure. Each monomer displays a nine-stranded, antiparallel b-sandwich arrangement and contains two close calcium cations that mediate the binding of fucose in a recognition mode unique among carbohydrate-protein interactions. Experimental binding studies, together with theoretical docking of fucose-containing oligosaccharides, are consistent with the assumption that antigens of the Lewis a (Le(a)) series may be the preferred ligands of this lectin. Precise knowledge of the lectin-binding site should allow a better design of new antibacterial-adhesion prophylactics.

Glycosphingolipids (GSLs) contain many carbohydrate epitopes or crypto-glycotopes for Gal and GalNAc reactive lectins. Many of them are in the nervous system and function as important receptors in various life processes. During the past two decades, 11 mammalian structural units have been used to express the binding domain of applied lectins. They are: F, GalNAc alpha1 --> 3GalNAc; A, GalNAc alpha1 --> 3Gal; T, Gal beta1 --> 3GalNAc; I, Gal beta1 --> 3GlcNAc; II, Gal beta1 --> 4GlcNAc; B, Gal alpha1 --> 3Gal; E, Gal alpha1 --> 4Gal; L, Gal beta1 --> 4Glc; P, GalNAc beta1 --> 3Gal; S, GalNAc beta1 --> 4Gal, and Tn, GalNAc alpha1 --> Ser(Thr). Although 10 of them occur in GSLs, only 3 (Lbeta, Sbeta, and Tbeta) are found in human brain, and 2 (Lbeta and IIbeta) are present in the inner structures of human blood group active GSLs. In the families of gangliosides, Lbeta and IIbeta represent 55% of the total structural units, while the other three units (Tbeta, Palpha, and Sbeta) constitute the rest. To facilitate the selection of lectins that could serve as structural probes, the carbohydrate binding specificities of Gal/GalNAc reactive lectins have been classified according to their highest affinity for the structural units and their binding properties expressed by decreasing order of reactivity. Hence, the binding relation between GSLs and Gal/GalNAc specific lectins can be established.

The binding properties of Caragana arborescens agglutinin (CAA, pea tree agglutinin) were studied by enzyme linked lectinosorbent assay (ELLSA) and by inhibition of CAA-glycan interaction. Among glycoproteins (gps) tested, CAA reacted strongly with asialo bird nest gp, asialo rat sublingual gp, human Tamm-Horsfall Sd(a(+)) urinary gp (THGP) and asialo THGP that are rich in GalNAcalpha1--, GalNAcbeta1--and/or Galbeta1--4GlcNAc residues. CAA also bound tightly with multi-valent Galbeta1--4GlcNAc (mII) containing glycoproteins (human blood group precursor gps, asialo fetuin) and asialo ovine salivary glycoprotein (Tn, GalNAcalpha1--Ser/Thr), but CAA reacted poorly or not at all with sialylated glycoproteins tested. Of the sugars tested for inhibition of binding, Forssman pentasaccharide (F(p), GalNAcalpha1--3GalNAcbeta1--3Galalpha1--4Galbeta 1--4Glc) was the best. It was about 2.3, 9.5 and 52.6 times more active than Galbeta1--4GlcNAc, GalNAc and Gal, respectively, and about 1.9 times more active than tri-antennary Galbeta1--4GlcNAc (Tri-II). These results suggest that this agglutinin is mainly specific for F(p), mII and Tn clusters. This property can be used to detect human abnormal glycotopes related to F(p) and unmasked mII/Tn clusters and to study cell growth and differentiation given the lack of toxicity of this lectin toward mouse fibroblast cells.

The binding profile of Triticum vulgaris (WGA, wheat germ) agglutinin to 23 O-glycans (GalNAc alpha1--Ser/Thr containing glycoproteins, GPs) was quantitated by the precipitin assay and its specific interactions with O-glycans were confirmed by the precipitin inhibition assay. Of the 28 glycoforms tested, six complex O-glycans (hog gastric mucins, one human blood group A active and two precursor cyst GPs) reacted strongly with WGA and completely precipitated the lectin added. All of the other human blood group A active O-glycans and human blood group precursor GPs also reacted well with the lectin and precipitated over two-thirds of the agglutinin used. They reacted 4-50 times stronger than N-glycans (asialo-fetuin and asialo-human alpha1 acid GP). The binding of WGA to O-glycans was inhibited by either p-NO2-phenyl alpha,betaGlcNAc or GalNAc. From these results, it is highly possible that cluster (multivalent) effects through the high density of weak inhibitory determinants on glycans, such as GalNAc alpha1--Ser/Thr (Tn), GalNAc at the nonreducing terminal, GlcNAc beta1--at the non-reducing end and/or as an internal residue, play important roles in precipitation, while the GlcNAc beta1--4GlcNAc disaccharide may play a minor role in the precipitation of mammalian glycan-WGA complexes.

The binding patterns of human blood group Sd(a+) and Sd(a-) Tamm-Horsfall glycoproteins (THGPs) with respect to four GalNAc specific agglutinins were studied by quantitative precipitin assay (QPA) and enzyme linked lectinosorbent assay (ELLSA). Of the native and asialo Sd(a+) and Sd(a-) THGP tested by QPA and ELLSA, only native and asialo Sd(a+) bound well with Dolichos biflorus (DBA) and Vicia villosa-B4 (VVA-B4), while Sd(a-) THGP reacted poorly with these two lectins. Neither Sd(a+) nor Sd(a-) THGPs reacted with two other GalNAc alpha-anomer specific lectins: Codium fragile subspecies tomentosoides and Artocarpus integrifolia. Furthermore, the binding of asialo Sd(a+)THGP-VVA-B4 and native Sd(a+)THGP-DBA through GalNAc beta--> was confirmed by inhibition assay. These results demonstrate that DBA and VVA-B4 are useful reagents to differentiate between Sd(a+) and Sd(a-) THGP.

The affinity of a lectin from the sponge Geodia cydonium (GCL-I) for multi-antennary Gal beta1--4GlcNAc and Gal beta1--3GalNAc ligands was studied by both the biotin/avidin-based microtiter plate lectin binding assay and the inhibition of lectin-glycoform interaction. Among the glycoforms tested for binding, GCL-I reacted strongly with three multi-antennary Gal beta1--4GlcNAc clusters containing glycoproteins (asialo human and bovine alpha1-acid gps and asialo fetuin), T (Gal beta1--3GalNAc) rich glycoprotein from porcine salivary gland, asialo bird nest gp, and human blood group A active cyst gp, while human and bovine alpha1-acid gps, fetuin, and Tn containing gps were inactive. Among the haptens tested for inhibition, tri-antennary Gal beta1--4GlcNAc (Tri-II) was about 1500, 72, and 72 times more active than GalNAc, Gal beta1--4GlcNAc (II), and Gal beta1--3GalNAc (T), respectively. Based on the present and previous results, it is proposed that tri-antennary Gal beta1--4GlcNAc and Gal beta1--3GalNAc clusters, in addition to GalNAc alpha1--3GalNAc and GalNAc alpha1--3Gal, are also important ligands for binding; and sialic acid of glycoprotein does interfere with binding.

Studies on the structures and binding properties of the glycoproteins, purified from human ovarian cyst fluids, will aid the understanding of the carbohydrate alterations occurring during the biosynthesis of blood group antigens and neoplasm formation. These glycoproteins can also serve as important biological materials to study blood group A, B, H, Le a , Le b , Le x , Le y , T and Tn determinants, precursor type I and II sequences and cold agglutinin I and i epitopes. In this study, the binding property of a cyst glycoprotein from a human blood group Le(a + ) nonsecretor individual, that contains an unusually high amount (18%) of sialic acid (HOC 350) was characterized by quantitative precipitin assay with a panel of lectins exhibiting a broad range of carbohydrate-binding specificities. Native HOC 350 reacted well only with three out of nineteen lectins tested. It precipitated about 80% of Ricinus communis (RCA 1 ), 50% of Triticum vulgaris (WGA) and 37% of Bauhinia purpurea aba (BPA) agglutinins, respectively. However, its asialo product had dramatically enhanced reactivity and reacted well with many I/II (Gal β 1 → 3 /4GcNAc), T (Gal β 1 → 3GalNAc) and Tn (GaNlAc α 1 → Ser/Thr) active lectins. It bound best to Jacalin, BPA, and abrin-a and completely precipitated all the lectins added. Asialo-HOC 350 also reacted strongly with Wistaria floribunda, Abrus precatorius agglutinin, ricin and RCA 1 and precipitated over 75% of the lectin nitrogen added, and moderately with Arachis hypogaea, Maclura pomifera , WGA, Vicia viosa -B4, Codium fragile tomentosoides and Ulex europaeus -II. But native HOC 350 and its asialo product reacted not at all or poorly with Dolichos biflorus, Helix pomatia, Lotus tetra-gonolobus, Ulex europaeus -I, Lens culinaris lectins and Con A. The lectin-glycoform interactions through bioactive sugars were confirmed by precipitin inhibition assay. Mapping the precipitation profiles of the interactions have led to the conclusion that HOC 350 contains a large number of receptors for I/II, T , and Tn active lectins. But in the untreated (or native) substance, most of these determinants are masked by sialic acids.

Unlike the human blood group Sd(a+) Tamm-Horsfall glycoprotein (THGP), the Sd(a-) one lacks terminal GalNAcbeta1--> residues at the nonreducing ends. The binding properties of this glycoprotein and its asialo product with lectins were characterized by quantitative precipitin (QPA) and precipitin inhibition assays. Among 20 lectins tested by QPA, both native and asialo Sd(a-) THGP reacted best with Abrus precatorius and Ricinus communis and completely precipitated the lectin added. They also precipitated well Wistaria floribunda (WFA), Glycine max (SBA), Bauhinia purpurea alba, abrin-a and ricin, all of which recognize the Galbeta1--> 4GlcNAcbeta1--> sequence, although at different strength. The lectin-glycan interactions were inhibited by Galbeta1--> 4GlcNAc and Galbeta1--> 4Glc. When the precipitability of Sd(a-) THGP was compared with that of the Sd(a+) phenotype, the native Sd(a-) THGP exhibited a 40% lesser affinity for WFA, SBA, WGA and mistletoe lectin-I (ML-I). Mapping the precipitation and inhibition profiles of the present study and the results of THGP Sd(a+), it is concluded that Sd(a-) THGP showed a strongly diminished affinity for GalNAcbeta1--> active lectins (SBA and WFA) than the Sd(a+) phenotype.

The affinity of Bandeiraea (Griffonia) simplicifolia lectin-I isolectin B-4 (BSI-B-4) for the isomer of human blood group B active disaccharide (B, Galα1→3Gal), the Galα1→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 QPA. 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 Galα1→3Galα1→methyl and Galα1→4Gal disaccharide indicating that BSI-B4 is not only reacting with Gal α1→3Gal disaccharide, but also recognizing Galα1→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.

The combining site of a GalNAc alpha 1---specific lectin (CFT) with Thomsen-Friedenreich (T, Gal beta 1--3-GalNAc alpha 1--Ser/Thr) activity, purified from the subspecies tomentosoides of green marine algae Codium fragile was studied by quantitative precipitin and precipitin-inhibition assays. Of 27 glycoforms tested, Tn (GalNAc alpha 1--Ser/Thr) glycoprotein from armadillo submandibular glands, and asialo porcine submandibular glycoprotein, which contains T, Tn and GalNAc alpha 1--3Gal(A) sequences, completely precipitated the lectin added, and less than 1 microgram glycoprotein was required to precipitate 50% 4.7 micrograms lectin nitrogen. However, CFT precipitated negligibly with Pneumococcus type-XIV polysaccharide and asialo human alpha 1-acid glycoprotein, that contain exclusively the human blood-type-II precursor sequence (II, Gal beta 1--4GlcNAc) at the nonreducing ends. Among the sugar inhibitors tested, the human blood A-active trisaccharide [Ah, GalNAc alpha 1--3 (LFuc alpha 1--2)Gal] was the best inhibitor; it was about twice as active as the T disaccharide. Oligosaccharides without GalNAc alpha 1--as part of their sequences were inactive, indicating that the acetamido group at C2 of galactose is essential for binding and that GalNAc is the main contributor in the T sequence for binding. From the data provided, it is clear that the combining site of CFT requires an alpha-anomer of GalNAc and recognizes Ah, internal GalNAc alpha 1--of T and Tn determinants of glycans, but not the blood group I/II (Gal beta 1--3/4GlcNAc) sequences. Consequently, CFT is a useful reagent for detecting GalNAc alpha 1---containing glycoconjugates.