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Purpose Attachment of Helicobacter pylori to the mucous epithelial cells and the mucous layer is said to be a crucial step for infection development. Sugar antigens of gastric mucins (MUC5AC, MUC1) can act as receptors for bacterial adhesins. The aim of the study was to investigate if Lotus tetragonolobus and Maackia amurensis lectins influence the level of MUC1, MUC5AC, Lewis b, H type 1, sialyl Lewis x, phospho-IκBα and interleukin 8 in Helicobacter pylori infected gastric cancer cells. Materials and methods The study was performed with one clinical H. pylori strain and CRL-1739 gastric cancer cells. To assess the levels of mentioned factors immunosorbent ELISA assays were used. Results Coculture of cells with bacteria had no clear effect on almost all examined structures. After coculture with H. pylori and lectins, a decrease of the level of both mucins, Lewis b and H type 1 antigens was observed. Lectins addition had no effect on sialyl Lewis x. Maackia amurensis caused slight increase of phospho-IκBα while interleukin 8 level was decreased. Conclusions Lotus tetragonolobus and Maackia amurensis lectins can mediate in binding of Helicobacter pylori to gastric epithelium.

Background Liposomes, used to improve the therapeutic index of new and established drugs, have advanced with the insertion of active targeting. The lectin from Lotus tetragonolobus (LTL), which binds glycans containing alpha-1,2-linked fucose, reveals surface regionalized glycoepitopes in highly proliferative cells not detectable in normally growing cells. In contrast, other lectins localize the corresponding glycoepitopes all over the cell surface. LTL also proved able to penetrate the cells by an unconventional uptake mechanism. Methods We used confocal laser microscopy to detect and localize LTL-positive glycoepitopes and lectin uptake in two cancer cell lines. We then constructed doxorubicin-loaded liposomes functionalized with LTL. Intracellular delivery of the drug was determined in vitro and in vivo by confocal and electron microscopy. Results We confirmed the specific localization of Lotus binding sites and the lectin uptake mechanism in the two cell lines and determined that LTL-functionalized liposomes loaded with doxorubicin greatly increased intracellular delivery of the drug, compared to unmodified doxorubicin-loaded liposomes. The LTL-Dox-L mechanism of entry and drug delivery was different to that of Dox-L and other liposomal preparations. LTL-Dox-L entered the cells one by one in tiny tubules that never fused with lysosomes. LTL-Dox-L injected in mice with melanoma specifically delivered loaded Dox to the cytoplasm of tumor cells. Conclusions Liposome functionalization with LTL promises to broaden the therapeutic potential of liposomal doxorubicin treatment, decreasing non-specific toxicity. General significance Doxorubicin-LTL functionalized liposomes promise to be useful in the development of new cancer chemotherapy protocols.

Glycoproteins of human milk are multifunctional molecules, and their fucosylated variants are potentially active molecules in immunological events ensuring breastfed infants optimal development and protection against infection diseases. The expression of fucosylated glycotopes may correspond to milk maturation stages. The relative amounts of fucosylated glycotopes of human skim milk glycoproteins over the course of lactation from the 2(nd) day to the 47(th) day were analyzed in colostrums, transitional and mature milk samples of 43 healthy mothers by lectin-blotting using α1-2-, α1-6-, and α1-3-fucose specific biotinylated Ulex europaeus (UEA), Lens culinaris (LCA), and Lotus tetragonolobus (LTA) lectins, respectively. The reactivities of UEA and LCA with the milk glycoproteins showed the highest expression of α1-2- and α1-6-fucosylated glycotopes on colostrum glycoproteins. The level of UEA-reactive glycoproteins from the beginning of lactation to the 14(th) day was high and relatively stable in contrast to LCA-reactive glycoproteins, the level of which significantly decreased from 2-3 to 7-8 days then remained almost unchanged until the 12(th)-14(th) days. Next, during the progression of lactation the reactivities with both lectins declined significantly. Eighty percent of α1-2- and/or α1-6-fucosylated glycoproteins showed a high negative correlation with milk maturation. In contrast, most of the analyzed milk glycoproteins were not recognized or weakly recognized by LTA and remained at a low unchanged level over lactation. Only a 30-kDa milk glycoprotein was evidently LTA-reactive, showing a negative correlation with milk maturation. The gradual decline of high expression of α1-2- and α1-6-, but not α1-3-, fucoses on human milk glycoproteins of healthy mothers over lactation was associated with milk maturation.

Glycoconjugates play roles in many physiological and pathological processes. Previous works have shown important functions mediated by glycans in spermatogenesis, and the carbohydrate composition of testis has been studied by several approaches, including lectin-histochemical methods. However, the testis of Xenopus laevis, an animal model extensively employed in biochemical, cell and developmental research, has not yet been analysed. The aim of this work was to carry out a histochemical study of the fucose (Fuc)-containing glycoconjugates of Xenopus testis by means of lectins, combined with deglycosylation pretreatments. Four Fuc-binding lectins were used: orange peel (Aleuria aurantia) lectin (AAL), gorse seed (Ulex europaeus) agglutinin-I (UEA-I), fresh water eel (Anguilla anguilla) agglutinin (AAA), and asparagus pea (Lotus tetragonolobus) agglutinin (LTA), each recognizing different forms of fucosylated glycans. Labelling with UEA-I, which preferably binds Fucalpha(1,2) containing oligosaccharides, did not show any appreciable staining. LTA, specific for Fucalpha(1,3), and AAA, which binds Fucalpha(1,2), labelled spermatocytes and spermatids, but no labelling was seen when the histochemical procedure was carried out after either beta-elimination (which removes O-linked oligosaccharides) or incubation with PNGase F (which removes N-linked oligosaccharides), suggesting that fucosylated glycans are of both N- and O-linked types. AAL, which has its highest affinity to Fucalpha(1,6), but also recognizes Fucalpha(1,2) and Fucalpha(1,3), labelled the whole testis, and the staining remained when the histochemical method was performed after either beta-elimination or incubation with PNGase F. Labelling with AAL could be explained by the fact that this lectin could be binding to diverse fucosylated glycans in N- and O-glycans, and even in glycolipids. The importance of these glycans is discussed.

Present study aimed at exploring the potential of α-l-fucose specific, LTA (Lotus tetragonolobus from Winged or Asparagus pea) as a homing device for nanocarriers to target the M cell for elicitation of strong immune response. LTA grafted poly(lactic-co-glycolic acids) (PLGA) nanoparticles encapsulating hepatitis B surface antigen (HBsAg) was developed and characterized for shape, size, polydispersity index, zeta potential, and antigen loading efficiency. The peyer's patch uptake was studied by using confocal laser scanning microscopy technique using dual staining technique. The immune stimulating potential was assessed by measuring anti-HBsAg titer in serum of balb/c mice. Induction of the mucosal immunity was assessed by estimating secretory immunoglobulin A level in the salivary, intestinal, and vaginal secretion and cytokine (interleukin-2 and interferon-γ) levels in the spleen homogenates. Furthermore, IgG1 and IgG2a isotype were determined to confirm the T(H)1/T(H)2 mixed immune response. The LTA anchored PLGA nanoparticles have demonstrated approximately four-fold increase in the degree of interaction with the bovine submaxillary mucin (BSM). The results demonstrated that LTA anchored PLGA nanoparticles elicited strong mucosal and systemic response and hence could be a promising carrier adjuvant for the M cell targeted oral mucosal immunization against Hepatitis B.

Lotus tetragonolobus lectin (LTA) is a fucose-specific legume lectin. Although several studies report a diverse combination of biological activities for LTA, little is known about the mechanisms involved in l -fucosyl oligosaccharide recognition. The crystal structure of LTA at 2.0 A resolution reveals a different legume lectin tetramer. Its structure consists of a homotetramer composed of two back-to-back GS4-like dimers arranged in a new mode, resulting in a novel tetramer. The LTA N -linked carbohydrate at Asn4 and the unusual LTA dimer–dimer interaction are related to its particular mode of tetramerization. In addition, we used small angle X-ray scattering to investigate the quaternary structure of LTA in solution and to compare it to the crystalline structure. Although the crystal structure of LTA has revealed a conserved metal-binding site, its l -fucose-binding site presents some punctual differences. Our investigation of the new tetramer of LTA and its fucose-binding site is essential for further studies related to cross-linking between LTA and complex divalent l -fucosyl carbohydrates.

Thirty kidney tumours of various histological type were histochemically investigated under the light microscope by means of the ABC-method. We used four biotinylated lectins which are known to bind also to normal renal tubular epithelial cells of different nephron segments. The nuclear grade, the histological growth pattern, the cell type and the histogenesis of the tumours were studied. The lectin of Lotus tetragonolobus bound to nine out of ten renal cell carcinomas with low nuclear grade, but in contrast no binding was seen in eight poorly differentiated ones. Four carcinomas mimicking collecting duct epithelium were also negative after treatment with this lectin, but showed strong staining after treatment with peanut agglutinin, Dolichos biflorus agglutinin and soybean agglutinin. Five oncocytomas showed a high affinity only for Dolichos biflorus agglutinin and only one case was positive with Lotus tetragonolobus agglutinin; three tubulo-papillary adenomas of the renal cortex without any oncocytes were negative with all of the lectins used. The value of lectin histochemistry in tumour pathology and its significance in routine pathological examination of kidney tumours are discussed.

The putative expression of distinct terminally fucosylated glycoconjugates among murine lymphocyte subpopulations was sought using a Biomphalaria alexandrina -derived lectin (BaSI), of proven specificity to a fucosyllactose determinant, and the fucose-binding lectin from Lotus tetragonolobus seeds. Direct labeling of isolated lymphocyte subsets in suspension as well as immuno-histochemical and two-dimensional Western blotting assays demonstrated the exclusive expression of the BaSI-reactive ligand among multiple isoforms of two major 95 and 92 kDa and a minor 82 kDa acidic glycoproteins, selectively localized to the splenic marginal zone B lymphocytes of adult mice. The expression of the L. tetragonolobus lectin-reactive ligand was, on the other hand, restricted primarily to a single homogeneous 50 kDa acidic glycoprotein associated with a subset of the mature (PNA − ) medullary thymocytes of adult mice as well as a minority of the immature (PNA + ) thymocytes within the deep cortical region in newly born mice. The significance of these findings is discussed in relation to mechanisms that govern lymphocyte development and homing.

A new, powerful method is presented for screening the binding in real time and taking place under dynamic conditions of oligosaccharides to lectins. The approach combines an SPR biosensor and HPLC profiling with fluorescence detection, and is applicable to complex mixtures of oligosaccharides in terms of ligand-fishing. Labeling the oligosaccharides with 2-aminobenzamide ensures a detection level in the fmol range. In an explorative study the binding of RNase B-derived oligomannose-type N-glycans to biosensor-immobilized concanavalin A (Con A) was examined, and an affinity ranking could be established for Man(5)GlcNAc(2) to Man(9)GlcNAc(2), as monitored by HPLC. In subsequent experiments and using well-defined labeled as well as nonlabeled oligosaccharides, it was found that the fluorescent tag does not interfere with the binding and that the optimum epitope for the interaction with Con A comprises the tetramannoside unit Manalpha2Manalpha6(Manalpha3)Man[D(3)B(A)4'], rather than the generally accepted trimannoside Manalpha6 (Manalpha3)Man [B(A)4' or 4(4')3]. In a similar experimental setup, the interaction of various fucosylated human milk oligosaccharides with the fucose-binding lectin from Lotus tetragonolobus purpureaus was studied, and it appeared that oligosaccharides containing blood group H could selectively be retained and eluted from the lectin-coated surface. Finally, using the same lectin and a mixture of O-glycans derived from bovine submaxillary gland mucin, minor constituents but containing fucose could selectively be picked from the analyte solution as demonstrated by HPLC profiling.

The interaction of three lectins (wheat germ, Ulex europaeus I, and Lotus tetragonolobus agglutinins: WGA, UEA-I and LTA) with either N-acetyl- d -glucosamine or l -fucose neoglycolipids incorporated into phospholipid monolayers and liposome bilayers was studied at the air/water interface and in bulk solution. The results show that for both systems studied, synthesized neoglycolipids were capable of binding their specific lectin and that, in general, the binding of lectins increased with the increase in the molar fraction of the saccharide derivative incorporated in either the monolayers or bilayers. However, whereas for UEA-I, molecular recognition was enhanced by a strong hydrophobic interaction, for WGA and LTA successful recognition was predominantly related to the distance between neighboring sugar groups. The observed lengthy adsorption times of these lectins onto their specific ligands were attributed to interfacial conformational changes occurring in the proteins upon their adsorption at the interfaces.

The most common cause of infant mortality is diarrhea; the most common cause of bacterial diarrhea is Campylobacter jejuni, which is also the primary cause of motor neuron paralysis. The first step in campylobacter pathogenesis is adherence to intestinal mucosa. We found that such binding was inhibited in vitro by human milk and, with high avidity, by alpha1,2-fucosylated carbohydrate moieties containing the H(O) blood group epitope (Fuc alpha 1,2Gal beta 1,4GlcNAc em leader ). In studies on the mechanism of adherence, campylobacter, which normally does not bind to Chinese hamster ovary cells, bound avidly when the cells were transfected with a human alpha1,2-fucosyltransferase gene that caused overexpression of H-2 antigen; binding was specifically inhibited by H-2 ligands (lectins Ulex europaeus and Lotus tetragonolobus and H-2 monoclonal antibody), H-2 mimetics, and human milk oligosaccharides. Human milk oligosaccharides inhibited campylobacter colonization of mice in vivo and human intestinal mucosa ex vivo. Campylobacter colonization of nursing mouse pups was inhibited if their dams had been transfected with a human alpha1,2-fucosyltransferase gene that caused expression of H(O) antigen in milk. We conclude that campylobacter binding to intestinal H-2 antigen is essential for infection. Milk fucosyloligosaccharides and specific fucosyl alpha1,2-linked molecules inhibit this binding and may represent a novel class of antimicrobial agents.

The interaction of sialyl Lewis(x), Lewis(x), and alpha-L-Fuc-(1-->3)-beta-D-GlcNAc with isolectin A from Lotus tetragonolobus (LTL-A), and with Aleuria aurantia agglutinin (AAA) was studied using NMR experiments and surface plasmon resonance. Both lectins are specific for fucose residues. From NMR experiments it was concluded that alpha-L-Fuc-(1-->3)-beta-D-GlcNAc and Lewis(x) bound to both lectins, whereas sialyl Lewis(x) only bound to AAA. Increased line broadening of 1H NMR signals of the carbohydrate ligands upon binding to AAA and LTL-A suggested that AAA bound to the ligands more tightly. Further comparison of line widths showed that for both lectins binding strengths decreased from alpha-L-Fuc-(1-->3)-beta-D-GlcNAc to Lewis(x) and were lowest for sialyl Lewis(x). Surface plasmon resonance measurements were then employed to yield accurate dissociation constants. TrNOESY, QUIET-trNOESY, and trROESY experiments delivered bioactive conformations of the carbohydrate ligands, and STD NMR experiments allowed a precise epitope mapping of the carbohydrates bound to the lectins. The bioactive conformation of Lewis(x) bound to LTL-A, or AAA revealed an unusual orientation of the fucose residue, with negative values for both dihedral angles, phi and psi, at the alpha(1-->3)-glycosidic linkage. A similar distortion of the fucose orientation was also observed for sialyl Lewis(x) bound to AAA. From STD NMR experiments it followed that only the L-fucose residues are in intimate contact with the protein. Presumably steric interactions are responsible for locking the sialic acid residue of sialyl Lewis(x) in one out of many orientations that are present in aqueous solution. The sialic acid residue of sialyl Lewis(x) bound to AAA adopts an orientation similar to that in the corresponding sialyl Lewis(x)/E-selectin complex.

Our autoradiographic 3H-fucose incorporation study of the brains of 20-day-old rat fetuses showed that the synthesis of fucosylated glycans is significantly higher in the ventricular germinative zone of the forebrain hemisphere than in the more superficial layers, including the cortical plate. Intense incorporation of 3H-fucose also occurred in the choroid plexus, both its epithelial and stromal component, in the primordial ependymal lining of the lateral ventricles, meninges and capillaries of the forebrain parenchyma. In the lateral ventricles, densely labeled microprecipitates of the cerebrospinal fluid (CSF) were occasionally observed. The histiotopic differences in 3H-fucose labeling were absent, or were much less expressed, in the autoradiograms prepared from unfixed cryostat sections containing mainly unincorporated isotope. This indicates that the blood-mediated supply of 3H-fucose to the studied brain compartments was essentially equal and our incorporation data reflect actual differences in the rate of fucosylation within the forebrain hemispheres. The cytochemical lectin-binding assay, carried out with Ulex europaeus and Lotus tetragonolobus agglutinins, showed that regions with a higher rate of 3H-fucose incorporation were also richer in fucose-bearing glycoconjugates. The study revealed that the periventricular regions and the CSF of fetal rat forebrain form a fucosylated glycan-enriched complex, which represents a new chemoarchitectonic feature that may be of importance for maintaining the germinative properties of the ventricular neuroepithelium and the growth of the hemispheric ventricles.

Purpose. To investigate in vivo the fate and the behavior of lectin-particle conjugates after oral administration.

This study was conducted to examine the lymph node metastasis-related carbohydrate epitopes of cancer cells in primary lesions of gastric cancer with submucosal invasion (sm gastric cancer). A total of 118 formalin-fixed and paraffin-embedded surgical specimens were studied. Carbohydrate epitopes were de-tected histochemically using 17 kinds of biotin-labeled lectins and three kinds of monoclonal antibodies (mAbs) against Thomsen-Friedenreich antigen (HB-T1), Tn (HB-Tn1), and sialyl Tn antigens (HB-STn1). The node-positive group showed significantly lower reactivities with Ricinus communis I (RCA-I), Lotus tetragonolobus (Lotus), Triticum vulgaris (WGA), and Concanavalia ensiformis (ConA), and higher reac-tivities with HB-STn1. When sm gastric cancer was classified into differentiated and undifferentiated types, lower reactivities with RCA-I and ConA were observed in the differentiated type cancers, whereas lower re-activities with Lotus, WGA, and higher reactivity with HB-STn1 were observed in the undifferentiated type cancers. Nodal status was able to be predicted with high precision by a combination of these carbohydrates and histological subtypes. Since neuraminidase treatment rarely changed the reactivity of cancer cells with these lectins, we conclude that the loss of certain kinds of carbohydrates that are restricted by histological subtypes is related to lymph node metastasis in sm gastric cancer.

In previous studies we found that sperm binding activity in the vitelline coat of the freshwater bivalve Unio elongatulus is located on the O-linked oligosaccharide chains of gp273, one of the two major components of the extracellular coat, and that fucose plays a key role in this interaction. In this paper we report the partial characterization of a large glycopeptide (about 140 kDa) obtained by cyanogen bromide fragmentation of gp273, that maintains sperm binding activity. Lectin blotting revealed that the glycopeptide reacted with lectins from Arachis hypogaea (PNA) and Lotus tetragonolobus (LTA) but not Canavalia ensiformis (ConA). No other PNA-positive fragments could be detected in the electrophoretic pattern of fragmented gp273 but several ConA-positive fragments of lower molecular weight were present indicating that all the O-linked chains are clustered together in this fragment. Two-dimensional gel electrophoresis of the fragment revealed it to be acidic in nature in contrast with the neutral character of the whole gp273 molecule. Competition binding assay showed that this fragment is a strong inhibitor of the interaction, whereas no effect was detected using the ConA-positive peptides. This confirms that the sperm receptor activity of gp273 is related to its O-linked chains. The immunodominance of this fragment is also discussed.

Human parotid salivas were collected from patients with secondary Sjogren's syndrome and controls without disease or with drug-induced xerostomia. Parotid glycoproteins were separated by gradient sodium dodecyl sulphate gel electrophoresis (SDS-PAGE), electroblotted onto nitrocellulose membrane and probed with biotinylated lectins of characterised sugar specificities. The binding patterns of lectins from Maclura pomifera (MPA) and Arachis hypogaea (PNA) indicated that many parotid glycoproteins have sialylated O-linked glycans and that sialylation is not affected by disease. Binding by lectins from Ricinus communis (RCA-1), Limax flavus (LFA), Lotus tetragonolobus (LTA) and Ulex europaeus (UEA-1) appeared unaltered in secondary Sjogren's syndrome, suggesting no obvious change in N-glycosylation of parotid glycoproteins. Variations in binding patterns of most lectins was attributable to subject-to-subject variations in recognised polymorphic proteins. Dolichos biflorus agglutinin (DBA) consistently showed increased binding to a 75 kDa (Mr) protein in salivas from patients with secondary Sjogren's syndrome. The binding protein was identified as lactoferrin but found not to contain N-acetylgalactosamine, the sugar to which DBA binds. Binding of DBA to lactoferrin was dependent upon its saturation with iron, modified SDS-PAGE under nonreducing conditions resolved iron-free and iron-saturated lactoferrins and demonstrated increased levels of the iron-saturated form in secondary Sjogren's syndrome. Lectin binding studies of purified lactoferrins from saliva, milk, and polymorphonuclear neutrophils suggested that raised levels of lactoferrin in saliva originate from salivary cells and not from inflammatory cells. These results suggest that DBA binding provides greater specificity as an indicator of salivary gland disease than measurement of lactoferrin levels alone.

It has been postulated that carbohydrates are involved in a variety of cell-cell interactions including blastocyst implantation. In primates, there are only limited investigations on the ultrastructural localisation of the cyclic changes in uterine epithelial surface carbohydrates. Our aim was to investigate such changes during the pre-ovulatory and pre-implantation stages of the reproductive cycle in the marmoset monkey. After fixation of endometrial tissues, avidin-ferritin lectin cytochemistry was employed for apical surface glycan detection at the ultrastructural level. Five lectins were used including Canavalia ensiformis (Con A), Lotus tetragonolobus (LTA), Glycine max (SBA), Phytolacca americana (PWM) and Triticum vulgaris (WGA). Morphometry was used to quantitate changes in the intensity of lectin staining by determining the total number of ferritin particles per unit length of membrane. Surface and intra-cytoplasmic vesicles, stained by the lectins, were also examined. Quantitative ferritin assessment showed that 1 day before presumed implantation (days 11 to 12 after ovulation in the marmoset monkey) there was a significant increase in Con A, LTA and SBA staining on the apical uterine epithelial plasma membrane compared to the pre-ovulatory phase and earlier stages of pregnancy (days 4–8 after ovulation). A significant increase in PWM was also detected from early pregnancy to pre-implantation stages. All lectins except WGA produced reproducible staining within reproductive cycle groups. The greatest variation and intensity of epithelial surface staining was observed with WGA and the weakest with LTA. The patchy staining with LTA compared with thick coverage by WGA indicated the complexity of the carbohydrate arrangement in the glycocalyx of the uterine surface plasma membrane. Reduction of WGA reactivity after neuraminidase treatment suggested that the lectin binding might be related to the presence of heavily sialylated apical uterine membrane glycoconjugates. This is the first high-resolution study in primates to report quantitative cyclic changes in fucosyl, galactosyl, glucosyl, and mannosyl sugar residues of the apical uterine epithelial glycocalyx. The findings support the concept that uterine epithelial glycocalyx surface carbohydrates play a role in preparing a receptive uterine surface. Anat Rec 255:241–251, 1999. © 1999 Wiley-Liss, Inc.

Plant and animal lectins with various carbohydrate specificities were used to type 35 Irish clinical isolates of Helicobacter pylori and the type strain NCTC 11637 in a microtiter plate assay. Initially, a panel of eight lectins with the indicated primary specificities were used: Anguilla anguilla (AAA), Lotus tetragonolobus (Lotus A), and Ulex europaeus I (UEA I), specific for α- l -fucose; Solanum tuberosum (STA) and Triticum vulgaris (WGA), specific for β- N -acetylglucosamine; Glycine max (SBA), specific for β- N -acetylgalactosamine; Erythrina cristagali (ECA), specific for β-galactose and β- N -acetylgalactosamine; and Lens culinaris (LCA), specific for α-mannose and α-glucose. Three of the lectins (SBA, STA, and LCA) were not useful in aiding in strain discrimination. An optimized panel of five lectins (AAA, ECA, Lotus A, UEA I, and WGA) grouped all 36 strains tested into eight lectin reaction patterns. For optimal typing, pretreatment by washing bacteria with a low-pH buffer to allow protein release, followed by proteolytic degradation to eliminate autoagglutination, was used. Lectin types of treated samples were stable and reproducible. No strain proved to be untypeable by this system. Electrophoretic and immunoblotting analyses of lipopolysaccharides (LPSs) indicated that the lectins interact primarily, but not solely, with the O side chain of H. pylori LPS.

A new method, the brush border lectin agglutination assay (BBLAA), has been developed to measure the capacity of functional lectins to agglutinate the intestinal brush border membrane. Lectins were incubated with purified chick small intestinal brush border vesicles, agglutinated membrane was isolated by low speed centrifugation and then assayed using alkaline phosphatase as a marker. The brush border agglutination activity of purified plant lectins with various carbohydrate binding specificity was compared. Tomato ( Lycopersicon esculentum ) lectin, and wheat germ ( Triticum vulgaris ) agglutinin bind N -acetyl-d-glucosamine and were found to have the highest activity. An exponential sigmoid equation fit the data obtained with wheat germ agglutinin, which is consistent with a cooperative mechanism of brush border agglutination. The N -acetyl-d-galactosamine specific lectin from soybean ( Glycine max ), and the l-fucose specific lectin from asparagus pea ( Lotus tetragonolobus ) had intermediate activity. Of the mixed carbohydrate binding isolectins from red kidney bean ( Phaseolus vulgaris ), Pha-E had intermediate activity, and Pha-L no detectable activity. Jack bean ( Canavalia ensiformus ) lectin, concanavalin A, (Con A) binds glucose/mannose residues and had marginal activity. Succinylation of con A eliminated brush border agglutination activity. The BBLAA has distinct advantages when compared to other lectin assay systems in that it measures functional agglutination activity directed toward the target membrane on the intestinal epithelium, does not require antibody or enzyme conjugation, and can be used to measure total functional lectins in extracts from diets or meals.

The investigation of N- and O-linked glycoproteins using capillary-zone electrophoresis interfaced with nanoelectrospray mass spectrometry is described. The combination of high-resolution separation with high-sensitivity mass spectrometric detection provides analysis of glycoprotein digests at sample loadings of high femtomoles to low picomoles. Stepped-orifice voltage scanning is used to identify glycopeptides in complex proteolytic digests. Further structural information is obtained using capillary zone electrophoresis (CZE)–MS–MS to elucidate the composition of both N- and O-linked glycopeptide oligosaccharides. Collisional activation in the orifice/skimmer region is used to generate first-generation fragment ions which undergo subsequent dissociation in the r.f.-only collision cell of the triple quadrupole mass spectrometer. These experiments provided informative peptide backbone fragment ions usually not available from fragment ion spectra of multiply protonated glycopeptide ions. These methods were applied to the characterization of α-amylase inhibitor 1, a lectin from Lotus tetragonolobus , two N-linked glycoproteins, and to κ-casein, a glycoprotein comprising O-linked sialylated glycans.

Some years ago, a lectin designated CBP70 that recognized glucose (Glc) but had a stronger affinity for N-acetylglucosamine (GlcNAc), was first isolated from HL60 cell nuclei. Recently, a cytoplasmic form of this lectin was described, and one 82 kDa nuclear ligand was characterized for the nuclear CBP70. In the present study, the use of Pronase digestion and the trifluoromethanesulphonic acid (TFMS) procedure strongly suggest that the nuclear and the cytoplasmic CBP70 have a same 23 kDa polypeptide backbone and, consequently, could be the same protein. In order to know the protein better and to obtain the best recombinant possible in the future, the post-translational modification of the nuclear and cytoplasmic CBP70 was analyzed in terms of glycosylation. Severals lines of evidence indicate that both forms of CBP70 are N- and O-glycosylated. Surprisingly, this glycosylation pattern differs between the two forms, as revealed by β-elimination, hydrazinolysis, peptide-N-glycosydase F (PNGase F), and TFMS reactions. The two preparations were analyzed by affinity chromatography on immobilized lectins [Ricinus communis-I agglutinin (RCA-I), Arachis hypogaea agglutinin (PNA), Galanthus nivalis agglutinin (GNA), and wheat germ agglutinin (WGA)] and by lectin-blotting analysis [Sambucus nigra agglutinin (SNA), Maackia amurensis agglutinin (MAA), Lotus tetragonolobus (Lotus), succinylated-WGA, and Psathyrella velutina agglutinin (PVA)]. Both forms of CBP70 have the following sugar moities: terminal βGal residues, Galβ1–3 GalNAc, Man α1–3 Man, sialic acid α2–6 linked to Gal or GalNAc; and sialic acid α2–3 linked to Gal. However, only nuclear CBP70 have terminal GlcNAc and α-L-fucose residues. All these data are consistent with the fact that different glycosylation pattern found for each form of CBP70 might act as a complementary signal for cellular targeting. J. Cell. Biochem. 66:370–385, 1997. © 1997 Wiley-Liss, Inc.

The interactions of sugar containing N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers with Tetragonolobus purpureas (Lotus tetragonolobus) lectin immobilized on 4% beaded agarose have been studied. HPMA copolymers containing side-chains terminating in N-acylated amino sugars, namely, fucosylamine, galactosamine, glucosamine and mannosamine were synthesized and their biorecognition evaluated. As expected, only fucosylamine containing HPMA copolymers showed specific binding to the lectin. The dissociation constants (K d ) for HPMA copolymers were determined by frontal affinity chromatography. The binding constant was dependent on the amount of fucosylamine residues per macromolecule. An increase in fucosylamine content from 9,9 to 33,2 mol-% resulted in a decrease of K d by one order of magnitude. Incorporation of hydrophobic side-chains such as glycylglycyl-5-[4-(2-aminoethylcarbamoyl)phenylazo]salicylic acid, 1'-(β-hydroxyethyl)-3',3'-dimethyl-6-nitrospiro(2H-1-benzopyran-2,2'-indoline) (SP), or 1'-(β-glycylglycyloxyethyl)-3',3'-dimethyl-6-nitrospiro(2H-1-benzopyran-2,2'-indoline) (GG-SP) into the copolymers increased the non-specific binding to the lectin. Photoinduced isomerization of bound spiropyrans to merocyanines was accompanied by changes in the conformation and biorecognition of HPMA copolymers. The results obtained demonstrated the impact of the structure and solution conformation of sugar containing HPMA copolymers on their biorecognition.

Secretory cell glycoconjugates of the external nasal glands of ruin lizard and seps were investigated by prelectin staining methods (periodic acid-Schiff, alcian blue, high iron diamine) and lectin histochemistry in combination with neuraminidase digestion. Five plant lectins (Canavalia ensiformis, Triticum vulgare, Glycine max, Lotus tetragonolobus and Arachys hypogaea) conjugated with horseradish peroxidase were tested. Two different secretory cell types could be distinguished in the glandular tubules in both species examined. Lectin histochemistry revealed a microheterogeneity of mucins produced by the different secretory cell types of the two lizards regarding to the distribution pattern of glycosidic residues in glycoproteins.

The important roles played by glycoconjugates in the recognition of gametes and in fertilization are well documented. In the present study, the nature and distribution of carbohydrate residues of the plasma membrane of spermatozoa of Drosophila melanogaster were characterized by use of FITC-conjugated lectins as probes. The plasma membrane bound agglutinins from Concanavalia ensiformis (Con A) and Pisum sativum (PSA), native and succinylated agglutinins from wheat germ (WGA and s-WGA), the A4 isoform of agglutinin-I from Griffonia simplicifolia (GSA-I A4), and, to a lesser extent, the lectins from Dolichus biflorus (DBA), Lotus tetragonolobus (LTA), and Glycine maximus (SBA). Each lectin gave a specific pattern of binding. The extent of binding of Con A, WGA, s-WGA, and GSA-I A4 over the acrosomal region was greater than over nonacrosomal regions, indicating the concentration of α-mannose/α-glucose, β-N-acetylglucosamine, and α-N-acetylgalactosamine residues in this area of the plasma membrane. The surface of the sperm failed to react with lectins from Ricinus communis (RCA-I), Limulus polyphemus (LPA), and Limax flavus (LFA) and with the B4 isoform of agglutinin-I from Griffonia simplicifolia-I (GSA-I B4). The plasma membrane over the nucleus did not react with any of the lectins tested. Quantitative analysis of binding of Con A, s-WGA, and GSA-I A4 to spermatozoa showed that only Con A bound consistently to the sperm surface, showing high affinity for the acrosomal area of the plasma membrane. The other lectins tested bound only to limited and variably sized fractions of the total population of sperm. Therefore, only residues of α-mannose/α-glucose are a constitutive component of the plasma membrane, and they are characteristic of the acrosomal area. Con A can be used as a marker of the acrosome portion of sperm from Drosophila for visualization and assessment of acrosome status; labelling with FITC-conjugated Con A provides a simple and reliable method for visualization of the acrosome of Drosophila sperm that is otherwise detectable only by ultrastructural examination. © 1995 Wiley-Liss, Inc.

Proteolytic enzymes could be very important in spread of cancer, but the role of the body's natural inhibitors of these enzymes in this process is unknown. One such inhibitor is the serum glycoprotein, alpha-1-proteinase inhibitor (API). In previous studies we showed that the fucose-specific lectin, lotus tetragonolobus, extracted high amounts of API in cancer when patients were unresponsive to treatment. The aim of this study was to determine whether the carbohydrate structure of API is altered in cancer. API was isolated from the sera of healthy women and women with breast or ovarian cancer. By means of high-performance anion-exchange chromatography, cancer API was shown to contain more fucose and less N-acetylglucosamine than healthy API. Further investigation of the purified specimens using a lectin-binding assay suggested that the cancer API was less branched and contained more alpha 2-6 and less alpha 2-3 sialic acid. Observations from both methods were consistent with an increase in bi-antennary chains terminating in alpha 2-6 sialic acid and possibly more alpha 1-6 fucose in the core of the unit. These distinctive changes could have important consequences for the function of API in cancer and may help to develop more precise markers for monitoring pathological progression in this disease.

Lectins of different binding specificity were used to analyze the oligosaccharide chains of the 220- and 180-kD proteins of the Unio elongatulus egg vitelline coat (vc). The lectins ConA and RCA1 reacted with both glycoproteins, and four other lectins reacted with one or other vc components. The lectin from Galanthus nivalis, which recognizes terminal mannose residues of N-linked high mannose type oligosaccharide chains, bound specifically to the 180-kD protein. Binding sites for this lectin were found throughout the vc of the differentiating oocyte and the mature egg. Lectins specific for the O-linked oligosaccharide chains, such as AIA and PNA, reacted only with the 220-kD protein species. Binding sites for these lectins were found only in the crater region. The presence of fucosyl residues on the glycan chains was investigated with lectins from Lotus tetragonolobus and Aleuria aurantia. The latter was positive on both glycoproteins, whereas LTA was only positive to the 220-kD species. The binding sites of both these lectins were in the same areas as those of PNA and AIA. These results suggest that while the 180-kD protein is part of the entire vc structure, the 220-kD protein is prevalently accumulated in the crater region. Since this is where sperm recognition and interaction take place, it has been suggested the 220-kD protein acts as a ligand molecule in the sperm-egg interaction. © 1995 Wiley-Liss, Inc.

Summary An assay using lectin derivatized agarose beads to rapidly and inexpensively identifycell surface lectin receptors was recently described by Latham et al. (1995). In this earlier study, the assay was tested on large, early stage sea urchin embryo cells. In this study this assay was used to examine lectin receptors on small, later stage sea urchin embryo cells that are more typical of cells that most investigators deal with, to ascertain if cell size is a determining factor in the assay's validity. The results indicated that the assay is a valid method to identify lectin receptors on small as well as large cells. Twenty-three hour Strongylocentrotus purpuratus embryo cells strongly bound Triticum vulgaris , concanavalin A, Artocarpus integrifolia and Vicia villosa using both the agarose bead and fluorescence assays, while three other lectins, Ulex europaeus I , Lotus tetragonolobus and Lens culinaris did not strongly bind to the cells using these two assays. As in earlier studies agglutinability results did not correlate well with results using the two other assays. In all cases where lectin bead binding, fluorescent lectin binding or lectin-mediated agglutination occurred, specific sugars reduced the observed binding. The second part of this study examined the putative role of concanavalin A receptors in a specific cellular interaction: sperm-egg binding. Concanavalin A inhibited fertilization of dejellied sea urchin eggs when their vitelline layers were intact and to a lesser extent when their vitelline layers were removed. This effect was counteracted by alpha methyl glucose. The major differences between these studies and previous work is that here concanavalin A was washed out after incubation with eggs, making it more likely that results reflect binding to cell surface lectin receptors rather than toxicity. In addition, performing the experiments on eggs with or without vitelline layers provided information on the location of concanavalin A receptors that may function in sperm-egg interaction.

Background Besides α-Gal expression, the differences of glycosylation and antigenicity between adult pig islets (APIs) and neonatal porcine islet-like cell clusters (NPCCs) are altogether unclear. In this study, lectin microarray analyses of NPCCs were performed and the results compared with the corresponding values for wild-type APIs and NPCCs from α-Gal transferase knockout (GalT-KO) pig. Methods NPCCs were isolated from 1–3-d-old neonatal wild-type pigs and cultured for 1 d, 5 d, and 9 d, using a previously described technique. Alternatively, the isoration of APIs were isolated based on the method for human islets. Results In a comparison between NPCCs and APIs, all of the NPCCs showed higher signals for Sambucus nigra, Sambucus sieboldiana, and Trichosanthes japonica I and the binding of α2,6 sialc acid, whereas the APIs showed stronger signals for Lotus tetragonolobus, Aleuria aurantia, Narcissus pseudonarcissus, and Galanthus nivalis, suggesting that APIs contain high levels of high-mannose forms. Among the NPCCs, NPCC (day1) appeared to be richer than the others in Lotus tetragonolobus, Narcissus pseudonarcissus, Galanthus nivalis, and Urtica dioica, implying the presence of high-mannose forms. However, as a whole, the signals for many lectins for NPCCs were very similar. The NPCCs from a GalT-KO pig indicated not only the downregulation of α-Gal expression but α-GalNAc as well, and α2-6 sialic acid was upregulated. Conclusions The results reported herein contain useful information for the future production of immunomodified pigs with less antigenicity than GalT-KO pigs toward clinical applications of NPCCs.

There is increasing evidence for changes in fucosylation in cancer. Previously, we showed that the fucose-specific lectin,Lotus tetragonolobus, extracts an abnormal form of haptoglobin (Hp) from cancer sera. This study investigates the monosaccharide content of Hp obtained from women with ovarian and breast cancer at different stages of their disease. In both cancers, Hp fucose was low when the disease was benign or in remission and much higher when the disease was progressive. This occurred whether the data was expressed per mole of protein or per three mannose residues. Changes in other monosaccharides were minor compared with fucose. There were small increases in theN-acetylglucosamine and galactose content (per three mannoses) in ovarian cancer, suggesting that some glycan chains have increased branching. The latter was independent of disease activity which may be due to some indirect cause such as cytotoxic therapy or an inflammatory response. When ovarian cancer patients were in remission, the number of glycosylation sites on Hp was reduced. Hp isolated from patients with early, but not advanced breast cancer also appeared to have increased glycan branching. The increased fucosylated Hp may interfere with fucose-mediated adhesion reactions of cancer cells.

Summary Mucous cells and enteroendocrine cells of the pyloric region of the ruin lizard ( Podarcis sicula campestris De Betta) have been examined by lectin histochemical and immunohistochemical methods. Binding to five plant lectins ( Canavalia ensiformis , Con A; Triticum vulgare , wheat germ, WGL; Lotus tetragonolobus , winged pea, WPL; Glycine max , soybean, SBL; Arachis hypogaea , peanut, PNL) was performed to characterize glycoconjugates in the secretory products of superficial and glandular mucous cells. Lectin histochemistry revealed the presence of N-acetyl-D-galactosamine, D-galactose and N-acetyl-D-glucosamine in the pyloric superficial cells. Mucous glandular cells mainly contained neutral glycoproteins with terminal residues of galactose, N-acetyl-D-glucosamine and N-acetyl-D-galactosamine. These cells did not react with Con A after periodate oxidation-sodium borohydride reduction (Paradoxical Con A staining). In the pyloric glands three different types of endocrine cells were identified immunohistochemically: gastrin-, serotonin- and somatostatin-immunoreactive cells; VIP-, bombesin- or cholecystokinin-immunoreactive cells have not been found in the pyloric mucosa.

Horseradish peroxidase-conjugated lectins were used on tissue sections to localize the main secretory glycoproteins in cat submandibular glands and on Western blots to evaluate their movement into saliva with selective nerve stimulation. Central acinar cells bound lectins from Arachis hypogaea (PNA) specific for the terminal disaccharide Gal beta 1, 3GalNac, Griffonia simplifolia (GSA I-B4) specific for terminal alpha Gal, and Lotus tetragonolobus (LTA) specific for fucose. Lectins from Limax flavus (LFA) specific for sialic acid and Dolichos biflorus (DBA) specific for terminal alpha GalNac reacted preferentially with demilunar cells, whereas apical granules in striated ducts were recognized principally by LTA. Parasympathetic stimulation promoted the release of lectin-reactive glycoconjugates from both central and demilunar cells. In contrast, sympathetic stimulation caused almost complete release of LTA-reactive granules in striated ducts and only moderate secretion from demilunar cells. Lectin blots of stimulated saliva discriminated many of the constituent bands, providing information about their glycosylation. Several bands were common to both parasympathetic and sympathetic saliva, and many bands gave wider ranges of lectin binding than anticipated from the histochemistry. The major component in parasympathetic saliva was a glycoconjugate of less than 12 KD which reacted with every lectin tested. Lectin blots of sympathetic saliva showed a prominent diffuse LTA-reactive band around 33 KD, which was attributed to tissue kallikrein. The identity and cellular origin of most bands in stimulated submandibular saliva are still unclear but the technique shows considerable promise for improving the recognition and characterization of individual glycoconjugates.

Cancer sera have high levels of an abnormal form of haptoglobin (Hp) that can be extracted from blood using the fucose-specific lectin, Lotus tetragonolobus. In order to investigate the carbohydrate abnormality that is responsible for this effect, the monosaccharide composition of Hp has been measured in Hp preparations isolated from blood samples of healthy women and women with ovarian cancer. The fucose content (g/100 g protein) of Hp was considerably elevated (7-fold) in ovarian cancer; whereas the concentrations of the other sugars were not significantly increased. There were significant increases in the galactose and N-acetylglucosamine contents of the ‘cancer’ Hp if the levels were expressed relative to the mannose content. This latter finding suggests that there are more oligosaccharide branches on the ‘cancer’ Hp. The results indicate that Hp is extracted by lotus lectin from ovarian cancer sera because of the increased fucose levels and provide further support for a disturbance in fucose metabolism in cancer.

The lectin purified from Erythrina corallodendron seeds which binds N-acetyllactosamine greater than N-acetyl-D-galactosamine greater than alpha and beta galactosides greater than D-galactose was examined for its ABO(H) blood group specificity. It has been shown that this lectin causes the strongest hemagglutination of O(H) and weakest of Oh(Bombay) red blood cells, and interacts with the H antigen in association with the I antigen. The reactions of Erythrina corallodendron and Erythrina indica lectins (which are similar in sugar specificity) with erythrocytes of different ABO(H) and Ii blood groups (the I bloods were all from adults and the i from either cord or adult bloods) revealed the following order of activity: O(H)I greater than A2 I greater than O(H)i adult greater than A2BI greater than BI greater than O(H)i cord greater than A1I greater than A1i adult greater than Bi cord greater than A1BI greater than Ai cord greater than ABi cord greater than OhI. The Erythrina indica lectin showed a lower differentiation between the agglutination of O(H) and Oh erythrocytes. Both Erythrina lectins exhibited H/HI blood group preference but were not inhibited by the saliva from ABO(H) "secretors". Thus they may be classified with the Cytisus sessilifolius, Lotus tetragonolobus and Laburnum alpinum lectins which are inhibited by lactose but not by H blood group substances in secretions.

The lectin histochemistry of ovarian mucinous cystadenoma was studied using a panel of lectins comprising Triticum vulgaris, Lotus tetragonolobus, Arachis hypogaea, Griffonia simplicifolia I, concanavalin A, and Dolichus biflorus. All 23 cases examined in this study stained extensively with T. vulgaris. Of all the lectins, D. biflorus was the least reactive. Concanavalin A stained mainly the perinuclear zone, whereas the other lectins frequently stained the cytoplasm, glycocalyx, and extracellular luminal mucin. This pattern of lectin reactivity resembles endocervical more than intestinal epithelium and suggests that ovarian mucinous cystadenomas are of mullerian nature. The lack of difference in lectin reactivity between cystadenomas with and without goblet/Paneth cells supports a common histogenetic origin of both groups.

The complete amino acid sequences of the Ulex europeus anti-H(O) lectins I and II were determined by using a protein sequencer. After digestion with endoproteinases Lys-C and Asp-N of the lectins reduced with 2-mercaptoethanol and modified with iodoacetamide, the resulting peptides were purified by reversed-phase high-performance liquid chromatography and subjected to sequence analysis. The complete primary structures of these two Ulex lectins I and II were compared with those of nine lectins already determined, including that of Lotus tetragonolobus anti-H(O) lectin which we have determined. Extensive homologies were found among them.

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.

It was previously reported that sera from ovarian cancer patients contained abnormal forms of alpha-1-proteinase inhibitor (API) that predicted unresponsiveness to chemotherapy. These molecules were detected by extracting the sera with the fucose-specific lectin, lotus tetragonolobus, and analysing the extracts by electrophoresis. In a new study of ovarian cancer, we report that API cannot be extracted by lotus from fresh sera, unless the specimens are subjected to repeated cycles of freezing and thawing. Furthermore, increases in lotus-extractable API in the pretreated sera are also associated with a poor response to chemotherapy. This would suggest that lotus is extracting the same molecules in both studies. It seems likely that the discrepancy between the two studies is due to the length of time the specimens were stored prior to analysis. We present evidence suggesting that the API extracted by lotus is present in serum as soluble complexes, the detailed composition of which is unclear. The change in the properties of API in cancer may be very important with respect to tumour spread.

Peroxidase conjugated lectins were used to analyse the glycoproteins of small intestinal mucins in normal infants and those with cystic fibrosis to ascertain whether there are any detectable histochemical differences in saccharide composition. A significant decrease in Lotus tetragonolobus (LTG) binding fucose was shown in normal small intestinal mucin starting around 36 weeks' gestation with total absence of staining at term and beyond. In contrast, the age matched patients with cystic fibrosis showed persistent and intense LTG binding of fucose. These results provide the first clear histochemical evidence that cystic fibrosis mucin is abnormal and confirm the findings of previous biochemical studies.

The milk fat globule membrane antibodies HMFG1, HMFG2, NCRC 11 and four of the Mam 6 series, and the lectins peanut agglutinin, wheat germ agglutinin, Concanavalin A, Lotus tetragonolobus and Ulex europaeus I have been applied to 115 stage I and II breast carcinomas (median follow up = 36 months) to assess their value as prognostic markers. Of the milk fat globule membrane antibodies only NCRC 11 staining showed a relationship to development of recurrent disease and overall survival, but this did not act as an independent indicator over and above that provided by histological grade. None of the lectins gave prognostic information, including those whose binding related to node status or grade. It is concluded that for short-term prognosis none of the markers can given independent prognostic information over and above that provided by histological evaluation.

We have recently observed that certain asparagine-linked oligosaccharides are multivalent and capable of binding and precipitating with the D-mannose-specific lectin concanavalin A [cf. Bhattacharyya, L., & Brewer, C. F. (1989) Eur. J. Biochem. 178, 721-726] and with a variety of D-galactose-specific lectins [Bhattacharyya, L., Haraldsson, M., & Brewer, C. F. (1988) Biochemistry 27, 1034-1041]. In the present study, we have examined the binding and precipitating activities of a variety of mono- and biantennary L-fucosyl oligosaccharides with three L-fucose-specific isolectins from Lotus tetragonolobus, LTL-A, LTL-B, and LTL-C. The results show that certain difucosyl biantennary oligosaccharides are capable of cross-linking and precipitating with tetrameric isolectins, LTL-A and LTL-C, but not with dimeric isolectin, LTL-B. Quantitative precipitation analyses show that biantennary oligosaccharides containing the Lewis(x) antigen (or type 2 chain of Lewis(a)), Gal beta (1-4)[Fuc alpha (1-3)]GlcNAc, at the nonreducing terminus of each arm are bivalent ligands. However, a biantennary oligosaccharide containing a Lewis(x) determinant on one arm and a type 2 chain of blood group H(O) determinant, Fuc alpha (1-2)Gal beta (1-4)GlcNAc, on the other arm and a monoantennary oligosaccharide containing two fucose residues (analogue of the Lewis(y) antigen) bind but do not precipitate with the isolectins, indicating that the positions and linkage of fucose residues are critical for cross-linking.(ABSTRACT TRUNCATED AT 250 WORDS)

The complete amino acid sequence of the Lotus tetragonolobus lectin was determined by a protein sequencer after digestion with endoproteinases of Lys-C and Asp-N, and compared with those of other leguminous plant lectins.

We studied the differentiation of the polarized site of sperm entrance in the egg of a freshwater bivalve, Unio elongatulus. As previously shown, in this egg model the property of sperm recognition and binding is restricted to a region characterized by a wrinkled surface which surrounds a truncated cone or crater region, at the vegetal pole of the egg. The crater is formed during oogenesis at a site opposite the oocyte attachment to the ovarian wall. It first appears as a small bleb, and later detaches, leaving in the oocyte an open narrow process filled with an orderly array of microtubules. Here the vitelline coat differentiates from the rest of the oocyte. The role of the microtubules in the formation of the region and the differential distribution of the vitelline coat components is discussed. The synthesis during development of fucosyl containing glycoproteins and their presence in an electrophoretic pattern of isolated vitelline coats were also studied using Lotus tetragonolobus (LTA) conjugated with fluorescein and peroxidase.

Lotus tetragonolobus Linnaeus, Species Plantarum 2: 773. 1753. "Habitat in Siciliae collibus." RCN: 5686. Lectotype (Domínguez & Galiano in Lagascalia 8: 209. 1979): Herb. Linn. No. 931.2 (LINN). Current name: Lotus tetragonolobus L. (Fabaceae: Faboideae)., Published as part of Jarvis, Charlie, 2007, Chapter 7: Linnaean Plant Names and their Types (part L), pp. 610-650 in Order out of Chaos. Linnaean Plant Types and their Types, London :Linnaean Society of London in association with the Natural History Museum on page 643, DOI: 10.5281/zenodo.291971

The seed lectin from Lotus tetragonolobus (LTA) has been crystallized. The best crystals grew over several days and were obtained using the vapour-diffusion method at a constant temperature of 293 K. A complete structural data set was collected at 2.00 angstroms resolution using a synchrotron-radiation source. LTA crystals were found to be monoclinic, belonging to space group P2(1), with unit-cell parameters a = 68.89, b = 65.83, c = 102.53 angstroms, alpha = gamma = 90, beta = 92 degrees. Molecular replacement yielded a solution with a correlation coefficient and R factor of 34.4 and 51.6%, respectively. Preliminary analysis of the molecular-replacement solution indicates a new quaternary association in the LTA structure. Crystallographic refinement is under way.

The gastric glands of Triturus carnifex (Amphibia, Caudata) have been examined by histochemical and immunohistochemical methods with particular regard to hydrochloric acid and pepsinogen secretion. Fundic glands consist of mucous neck cells, endocrine cells and oxynticopeptic cells producing both pepsinogen and hydrochloric acid. The neck cells showed an unexpected distribution pattern which was only observed in the oral fundus, and produced neutral mucins with glycosidic residues of GalNAc and Gal beta1,3GalNAc, and in this respect they differ from the neck cells of anuran amphibians. The secretion of pepsinogen and hydrochloric acid as demonstrated by immunolabelling with anti-H,K-ATPase and with anti-pepsinogen, respectively, seems not to vary significantly along the longitudinal axis of the stomach. The mechanism of gastric acid secretion seems to be mediated by an ATPase, having similar features to the mammalian gastric H,K-ATPase, and is localised in the luminal membrane and in the subapical cytoplasm of the oxynticopeptic cells. Unusually, the same cytoplasmic areas revealed binding specificity for the winged pea lectin (WPA) from Lotus tetragonolobus, even after beta elimination, indicating the presence of fucosyl residues in N-linked oligosaccharidic chains in glycoproteins of beta-H,K-ATPase subunits.

A total of 309 Helicobacter pylori isolates from 18 different countries were analyzed with a previously developed lectin typing system. The system was developed by using a proteolytic pretreatment to enhance the carbohydrate fraction of the sample. Four lectins from Ulex europaeus , Lotus tetragonolobus , Erythrina cristigali , and Triticum vulgaris were used to type the strains. The lectins were chosen for their specificities for sugars commonly encountered in the lipopolysaccharide of H. pylori . The isolates were received from their parent institutions as pellets of biomass and were typed at one of three centers (in Ireland, Sweden, and Estonia). All 16 possible lectin reaction patterns were observed in the study, with the isolates with the predominant pattern exhibiting reactions with all the lectins in the panel. For European patients suffering from gastritis, an association was noted between lectin reaction pattern MH4 and atrophic chronic gastritis; isolates with lectin reaction pattern MH4 were isolated from patients with atrophic chronic gastritis, whereas isolates with this pattern were not isolated from patients with chronic gastritis ( P = 0.0006). In addition, statistically significant relationships were noted between the lectin reaction pattern and the associated pathology of isolates from the Swedish population. Isolates with patterns MH13 and MH16, which had low lectin reactivities, correlated with nonulcer disease ( P = 0.0025 and P = 0.0002, respectively), and all four isolates from adenocarcinoma patients were characterized as possessing reaction pattern MH16. In contrast, isolates with lectin reaction patterns MH1 and MH10, which had high lectin reactivities, were associated with ulcer disease ( P = 0.046 and P = 0.0022, respectively).

Expression of N-cadherin an adhesion molecule of the cadherin family, in tumor cells is associated with their increased invasive potential. Many studies suggested the role of N-linked oligosaccharides as important factors that contribute to metastasis by influencing tumor cell invasion and adhesion. N-cadherin is a heavily glycosylated protein. We have analysed the carbohydrate profile of this protein synthesized in human melanoma cell lines: WM35 from the primary tumor site and WM239, WM9, and A375 from different metastatic sites. N-cadherin was immunoprecipitated with anti-human N-cadherin polyclonal antibodies. Characterisation of its carbohydrate moieties was carried out by SDS/PAGE electrophoresis and blotting, followed by immunochemical identification of the N-cadherin polypeptides and analysis of their glycans using highly specific digoxigenin or biotin labelled lectins. The positive reaction of N-cadherin from the WM35 cell line with Galanthus nivalis agglutinin (GNA), Datura stramonium agglutinin (DSA) and Sambucus nigra agglutinin (SNA) indicated the presence of high-mannose type glycans and biantennary complex type oligosaccharides with alpha2-6 linked sialic acid. N-cadherin from WM239, WM9, and A375 cell lines gave a positive reaction with Phaseolus vulgaris leukoagglutinin (L-PHA) and lotus Tetragonolobus purpureas agglutinin (LTA). This indicated the presence of tri- or tetra-antennary complex type glycans with alpha-fucose. In addition, N-cadherin from WM9 (lymphomodus metastatic site) and A375 (solid tumor metastatic site) contained complex type chains with alpha2-3 sialic acid (positive reaction with Maackia amurensis agglutinin--MAA). The results demonstrated that N-glycans of N-cadherin are altered in metastatic melanomas in a way characteristic for invasive tumor cells.