Japonica Agglutinin Research Articles

Disaccharide-tag for highly sensitive identification of O-GlcNAc-modified proteins in mammalian cells.

O-GlcNAcylation is the only sugar modification for proteins present in the cytoplasm and nucleus and is thought to be involved in the regulation of protein function and localization. Currently, several methods are known for detecting O-GlcNAcylated proteins using monoclonal antibodies or wheat germ agglutinin, but these methods have some limitations in their sensitivity and quantitative comparison. We developed a new disaccharide-tag method to overcome these problems. This is a method in which a soluble GalNAc transferase is expressed intracellularly, extended to a disaccharide of GalNAc-GlcNAc, and detected using a Wisteria japonica agglutinin specific to this disaccharide. We verified the method using human c-Rel protein and also highly sensitively compared the difference in O-GlcNAc modification of intracellular proteins associated with differentiation from embryonic stem cell (ESC) to epiblast-like cells (EpiLC). As one example of such a modification, a novel O-GlcNAc modification was found in the transcription factor Sox2 at residue Ser263, and the modification site could be identified by nano liquid chromatography-mass spectrometry.

Effectiveness of Selenium on Chondrocyte Glycoprotein Glycosylation Which Play Important Roles in the Pathogenesis of an Endemic Osteoarthritis, Kashin-Beck Disease.

In this study, we aimed to explore the effectiveness of selenium on the chondrocyte glycoprotein glycosylation which plays important roles in the pathogenesis of Kashin-Beck disease (KBD). Cartilage samples were collected from KBD patients after total knee replacement surgery. Chondrocytes were cultured with sodium selenium. The group of chondrocytes which were cultured without adding sodium selenium was considered as control group. Lectin microarray was used to screen the differences in lectin levels between KBD and KBD with selenium groups. Stronger signals for Bandeiraea simplicifolia (BS-I), Hippeastrum hybrid lectin (HHL), Pisum sativum agglutinin (PSA), Psophocarpus tetragonolobus lectin I (PTL-I), Psophocarpus tetragonolobus lectin II (PTL-II), Sophora japonica agglutinin (SJA), Lotus tetragonolobus lectin (LTL), and Triticum vulgaris (WGA) were observed in the KBD group. Meanwhile, Aleuria aurantia lectin (AAL), Lens culinaris agglutinin (LCA), Lycopersicon esculentum (tomato) lectin (LEL), Peanut agglutinin (PNA), and Sambucus nigra lectin (SNA) signals were lower in the KBD group. Selenium may have the function of influence the expression levels of carbohydrate chains Galα1,3-Gal, high mannose, and GlcNAc.

Roles of glycoprotein glycosylation in the pathogenesis of an endemic osteoarthritis, Kashin–Beck disease, and effectiveness evaluation of sodium hyaluronate treatment

Background/aim We aimed to explore the roles of glycoprotein glycosylation in the pathogenesis of Kashin–Beck disease (KBD), and evaluated the effectiveness of sodium hyaluronate treatment. Materials and methods Blood and saliva were collected from KBD patients before and after the injection of sodium hyaluronate. Normal healthy subjects were included as controls. Saliva and serum lectin microarrays and saliva and serum microarray verifications were used to screen and confirm the differences in lectin levels among the three groups. Results In saliva lectin microarray, bindings to Sophora japonica agglutinin (SJA), Griffonia (Bandeiraea) simplicifolia lectin I (GSL-I), Euonymus europaeus lectin (EEL), Maackia amurensis lectin II (MAL-II), Sambucus nigra lectin (SNA), Hippeastrum hybrid lectin (HHL), and Aleuria aurantia lectin (AAL) were higher in the untreated KBD patients than in the control group. Increased levels of HHL, MAL-II, and GSL-I in the untreated KBD patients discriminated them in particular from the treated ones. Jacalin was lower in the untreated KBD patients compared to the treated KBD and control groups. In serum lectin microarray, HHL and peanut agglutinin (PNA) were increased in the untreated KBD group in comparison to the control one. AAL, Phaseolus vulgaris agglutinin (E+L) (PHA-E+L), and Psophocarpus tetragonolobus lectin I (PTL-I) were lower in the untreated KBD patients compared to the treated KBD and control groups. Hyaluronate treatment appeared to normalize SNA, AAL, and MAL-II levels in saliva, and HHL, PNA, AAL, PTL-I, and PHA-E+L levels in serum. Saliva reversed microarray verification confirmed significant differences between the groups in SNA and Jacalin, in particular for GSL-I levels, while serum reversed microarray verification indicated that HHL, PNA, and AAL levels returned to normal levels after the hyaluronate treatment. Lectin blot confirmed significant differences in HHL, AAL, and Jacalin in saliva, and HHL, PNA, PHA-E+L, and AAL in serum. Conclusion HHL in saliva and serum may be a valuable diagnostic biomarker of KBD, and it may be used as follow-up for the hyaluronate treatment.

Lectin histochemical studies on the olfactory gland and two types of gland in vomeronasal organ of the brown bear

Olfaction is mediated by the vomeronasal and main olfactory systems, and the peripheral vomeronasal organ (VNO) processes species-specific chemicals that are associated with various behaviors in mammals. Sensory epithelial surfaces of the olfactory mucosa and VNO are covered by mucosal fluid that contains secretory products derived from associated glands, and glycoconjugates in the mucosal fluid are involved in odorant reception. The VNO of brown bears contains two types of glands; submucosal vomeronasal glands (VNG) and multicellular intraepithelial glands (MIG). The present study determined the labelling profiles of 21 lectins in the olfactory glands (OG), VNG and MIG of young male brown bears. The OG reacted with 12 lectins, and the VNG and MIG were positive for seven and eight lectins, respectively. Six lectins bound only to the OG, while four reacted with both or either of the VNG and MIG, but not the OG. The differences of lectin labelling pattern between the OG and glands in the VNO suggest that glycans in covering mucosal fluids differ between the olfactory mucosa and VNO. In addition, Bandeiraea simplicifolia lectin-I, Sophora japonica agglutinin and Jacalin reacted with the MIG but not the VNG, whereas Datura stramonium lectin and concanavalin A bound to the VNG, but not the MIG. These findings indicate that the properties of secretory substances differ between the two types of glands in the bear VNO, and that the various secretions from these two types of glands may function in the lumen of VNO together.

Clinical significance of GalNAcylated glycans in cholangiocarcinoma: Values for diagnosis and prognosis

Cancer cells exhibited the aberrant cancer-associated glycans that are potential biomarkers for diagnosis and monitoring of the cancer. In this study, Sophora japonica agglutinin (SJA) was used to detect SJA-specific N-acetylgalactosamine-associated glycans (SNAG) in liver tissues and sera from cholangiocarcinoma (CCA) patients. Whether SNAG could be the diagnostic and prognostic markers for CCA was evaluated. SJA-histochemistry revealed that SNAG was undetec2 in normal bile ducts but was highly expressed in hyperplastic/dysplastic bile ducts and CCA. SNAG was negative in hepatocytes and hepatoma tissues indicating SNAG as a differential marker of CCA and hepatoma. SJA-histochemistry of CCA hamster tissues revealed the involvement of SNAG in the early pathogenesis of bile duct epithelia and CCA development. A SJA-based ELISA was successfully developed to determine SNAG in serum. Serum-SNAG from CCA patients was significantly higher than those of non-CCA control groups with the diagnostic values of 59.5% sensitivity and 73.6% specificity, comparable to those of serum CA19-9. High levels of serum SNAG (≥69AU/ml) indicated poor survival of CCA patients. Taken together, SNAG was first demonstrated here to be a glycobiomarker for diagnosis and prognosis of CCA. Association of SNAG with pathogenesis of bile ducts and CCA development were suggested. (198).

Structure and Mucopolysaccaride Type of Major Salivary Glands of the Sunda Porcupines ( Hystrixjavanica )

Sunda porcupines are one of the rodent species endemic to Indonesia. There is lack information related to the anatomical structure of their major salivary glands. The study aims to identify the anatomical structures and types of mucopolysaccharides produce d by the major salivary glands of Sunda porcupine. Four tissue samplesof major salivary glands of Sunda porcupine were processed for paraffin method and analyzed by Hematoxylin - Eosin, Alcian Blue - Periodic Acid Schiff, and lectin histochemistry for saphora japonica agglutinin and wheat germ agglutinin . The parotid gland found in the preauricular region and along the posterior surface of the mandible, while the submandibular and sublingual glands were located on the floor of the mouth posterior to each mandib ular canine. The parotid gland was divided into two lobules, each composed by different types of acini in a separate lobulation. HE staining showed that parotid gland looks unique because in the anterior lobe, the acini are dominated by serous cell - type, w hile in posterior lobe are composed by mixed of serous and mucous cell - types. The acini of submandibular gland consist of serous cells - type, but sublingual gland acini covered by mucous cell - type. All of three major salivary glands have complete duct syste m comprising intercalated, striated and excretory ducts. The acini of parotid, submandibular, and sublingual glands contain acid and neutral, neutral, and acid mucopolysaccharides, respectively. Lectin staining using SJA and WGA indicates that acini in sal ivary glands of sunda porcupine contain sugar residue of N - acetylgalactosamine and N - acetylglucosamine which is a derivative of galactose and glucose by the order of intensity from weak to strong in the parotid, sublingual and submandibular glands.The pres ent results provide for the first time data on the anatomical structure and mucopolysaccharides type produced by major salivary glands of Sunda porcupines. Sunda porcupine, major salivary gland, anatomical structure, mucopolysaccharides.

Lectin Histochemical Studies on the Vomeronasal Organ of the Sheep

The vomeronasal organ of sheep was examined using lectin histochemistry in order to compare the types and amounts of the glycoconjugates among various components of the vomeronasal sensory and non-sensory epithelia. In the vomeronasal sensory epithelium, Dolichos biflorus agglutinin (DBA) stained particular cells, located at the same level as the vomeronasal receptor cells, while the distribution, shape and number of the stained cells did not correspond to those of the vomeronasal receptor cells. Datura stramonium lectin (DSL), Concanavalin A (Con A), Phaseolus vulgaris agglutinin-E (PHA-E) and Phaseolus vulgaris agglutinin-L (PHA-L) labeled the basal cells of both vomeronasal sensory and non-sensory epithelia. While, Wheat germ agglutinin (WGA), Succinylated-wheat germ agglutinin (s-WGA), Lycopersicon esculentum lectin (LEL), Solanum tuberosum lectin (STL) and Ricinus communis agglutinin-I (RCA-120) labeled the basal cells of the sensory epithelium, and Bandeiraea simplicifolia lectin-I (BSL-I) stained the basal cells of the non-sensory epithelium, respectively. Seventeen lectins labeled the free border of both vomeronasal sensory and non-sensory epithelia, while Sophora japonica agglutinin (SJA), Jacalin and Pisum sativum agglutinin (PSA) labeled neither free border of the sensory nor that of non-sensory epithelia. The expression pattern of glycoconjugate was similar, but not identical, in the free border between the sensory and non-sensory epithelia. These results indicate that there are dissimilar features in the type and amount of glycoconjugates between the vomeronasal sensory and non-sensory epithelia, and at the same time, among the various cell types either in the vomeronasal sensory or non-sensory epithelium.

Lectin histochemical studies on the olfactory epithelium and vomeronasal organ in the Japanese striped snake, Elaphe quadrivirgata

The olfactory epithelium and the vomeronasal organ of the Japanese striped snake were examined by lectin histochemistry. Of the 21 lectins used in the study, all lectins except succinylated-wheat germ agglutinin (s-WGA) showed similar binding patterns in the vomeronasal receptor cells and the olfactory receptor cells with varying intensities. The binding patterns of s-WGA varied among individuals in the vomeronasal and olfactory receptor cells, respectively. Four lectins, Bandeiraea simplicifolia lectin-II (BSL-II), Dolichos biflorus agglutinin (DBA), Sophora japonica agglutinin (SJA), and Erythrina cristagalli lectin (ECL) stained secretory granules and the organelles in the olfactory supporting cells and did not stain them in the vomeronasal supporting cells. These results suggest that the glycoconjugate moieties are similar in the vomeronasal and olfactory receptor cells of the Japanese striped snake.

Characterization of glycans in the developmental stages of Myxobolus cerebralis (Myxozoa), the causative agent of whirling disease

Glycans and sugar-binding molecules (lectins) form an interactive recognition system, which may enable parasitic organisms to adhere to host cells and migrate into target tissues. The aim of the present study was to analyse surface-associated glycans in the developmental stages of Myxobolus cerebralis (Hofer), the causative agent of whirling disease. A panel of biotin-labelled plant lectins was used to detect a broad spectrum of glycan motifs with high specificity. Binding sites were detected histochemically in the tissue sections of infected rainbow trout, Oncorhynchus mykiss (Walbaum), and infected Tubifex tubifex (Müller), and were characterized by light, fluorescence and transmission electron microscopy. With mannose-specific lectins [Lens culinaris agglutinin, Pisum sativum agglutinin, Canavalia ensiformis agglutinin (LCA, PSA, CanA)] mannose-containing glycans were detected in all the developmental stages and host tissues. No binding sites for galactose-specific lectins were present in M. cerebralis spores but reactivity with host tissues occurred. Diversity in glycans was detected by N-acetyl-D-galactosamine-specific lectins in sporoplasm cells of M. cerebralis and triactinomyxon spores. In the group of lectins with monosaccharide-specificity for N-acetyl-D-glucosamine (GlcNAc), the reactivity of Datura stramonium agglutinin (DSA), Lycopersicon esculentum agglutinin (LEA) and Solanum tuberosum agglutinin (STA) was restricted to polar capsules whereas Griffonia simplicifolia agglutinin II (GSA II) also bound to sporoplasm cells of stages in the fish host but not in those present in infected T. tubifex. Moreover, Triticum vulgaris (wheat germ) agglutinin (WGA) and succinylated WGA indicated the presence of N-acetyl-D-glucosamine polymers in polar capsules. No specificity for spores was observed concerning 'bisected'N-glycans and no reactivity in parasitic stages was observed with the fucose-binding lectin Ulex europaeus agglutinin (UEA) I, Sambucus nigra agglutinin (SNA) (specific for alpha2,6-sialylated glycans) and Maackia amurensis agglutinin (MAAI) (specific for alpha2,3-sialylated glycans). Arachis hypogaea (peanut) agglutinin (PNA), Erythrina cristagalli agglutinin (ECA), GSA I, Sophora japonica agglutinin (SJA), Dolichos biflorus agglutinin (DBA) and GSA II detected reactive sites solely confined to the developmental stages of M. cerebralis and were not reactive in the fish host. These parasite-specific glycans may play a role in the adhesion process of the parasite to fish epidermis prior to infection, but may provide protection to the host by activating the complement system, or stimulating an adaptive immune response as putative antigens.

Lectin blot studies on proteins of Myxobolus cerebralis, the causative agent of whirling disease

It is known that Myxobolus cerebralis antigens, both surficial and secreted, are key modulators for, or targets of, host immune system compounds. We undertook SDS-PAGE glycoprotein characterisation of M. cerebralis developmental stages isolated from infected rainbow trout and Western blot analyses using selected biotin-labelled plant lectins (GSA-I, PHA-E, SJA, GSA-II) and anti-triactinomyxon polyclonal antibodies. Glycoproteins were isolated with lectin-affinity chromatography, and prominent bands were characterised by matrix-assisted laser desorption/ionisation-mass spectrometry (MALDI/MS). We identified glycoproteins of M. cerebralis myxospores that contained carbohydrate motifs reactive with Phaseolus vulgaris erythroagglutinin (proteins 20 to 209 kDa, PHA-E), Sophora japonica agglutinin (proteins 7 to 70 kDa, SJA), Griffonia simplicifolia Agglutinin I (proteins 10 to 209 kDa, GSA-I) and G. simplicifolia Agglutinin II (proteins 5 to 40 kDa, GSA-II). Mcgp33, a glycoprotein isolated by lectin-affinity chromatography, was reactive with SJA (about 33 kDa). Antiserum produced against M. cerebralis triactinomyxons was found to have differences in the antigenicity of isolated glycoproteins from both M. cerebralis myxospores and actinospores. We also demonstrated modified antigen expression, especially involving the glycoprotein Mcgp33, in different developmental stages of M. cerebralis.

A Sialic Acid-Specific Lectin from the Mushroom Paecilomyces Japonica that Exhibits Hemagglutination Activity and Cytotoxicity

The mushroom Paecilomyces japonica, grown on the silkworm larvae, has been used in Asia as a nutraceutical, tea, and Chinese medicine. In the present study, a sialic acid-specific lectin has been purified from the mushroom P. japonica using affinity chromatography on a fetuin-agarose column. Electrophoretical analyses indicated that this lectin, designated P. japonica agglutinin (PJA), is an acidic protein with a molecular mass of 16 kDa, and has no intermolecular disulfide bonds. PJA induced hemagglutination activity in human ABO, mouse, rat, and rabbit erythrocytes. This activity was inhibited by sialic acid and sialoglycoproteins, but not by any other carbohydrates. PJA was stable at pH 4.0-8.0, and at temperatures below 55 degrees C. The activity of PJA was independent of EDTA and divalent cations. In addition, PJA exerts cytotoxic effects on the following cancer cell lines: human stomach cancer SNU-1, human pancreas cancer AsPc-1, and human breast cancer MDA-MB-231.

Solubility-insolubility interconversion of sophoragrin, a mannose/glucose-specific lectin in Sophora japonica (Japanese pagoda tree) bark, regulated by the sugar-specific interaction.

Sophoragrin, a mannose/glucose-specific lectin in Sophora japonica (Japanese pagoda tree) bark, was the first lectin found to show self-aggregation that is dependent on the sugar concentration accompanying the interconversion between solubility and insolubility [Ueno, Ogawa, Matsumoto and Seno (1991) J. Biol. Chem. 266, 3146-3153]. The interconversion is regulated by the concentrations of Ca(2+) and specific sugars: mannose, glucose or sucrose. The specific glycotopes for sophoragrin were found in the sophoragrin subunit and an endogenous galactose-specific lectin, B-SJA-I (bark S. japonica agglutinin I), and the lectin subunit that binds to the glycotope was identified by photoaffinity glycan probes. Remarkably, the insoluble polymer of sophoragrin is dissociated by interaction with B-SJA-I into various soluble complexes. Based on these results, self-aggregation of sophoragrin was shown to be a unique homopolymerization due to the sugar-specific interaction. An immunostaining study indicated that sophoragrin localizes mainly in vacuoles of parenchymal cells coincidently with B-SJA-I. These results indicate that sophoragrin can sequester endogenous glycoprotein ligands via sugar-specific interactions, thus providing new insights into the occurrence and significance of the intravacuolar interaction shown by a legume lectin.

Derivation of nephrogenic adenomas from renal tubular cells in kidney-transplant recipients.

Nephrogenic adenomas are benign, tumor-like lesions within the urothelial mucosa of the urinary tract that are not uncommon in renal-transplant recipients. We investigated the origin of nephrogenic adenomas in renal-transplant recipients. Tissue sections were analyzed by fluorescence in situ hybridization with the use of probes for the X and Y chromosomes, by immunohistochemical methods with the use of antibodies to renal tubular antigens, and by lectin histochemical methods. Forty-six nephrogenic adenomas from 29 patients were analyzed. All nephrogenic adenomas in 14 female recipients of transplants from male donors and 10 male recipients of transplants from female donors showed the same sex-chromosome status as the donor kidney, but not the same sex-chromosome status as the recipient's surrounding bladder tissue. The nephrogenic adenomas from all 6 female recipients of transplants from female donors showed female chromosomes, and those from the 16 male recipients of transplants from male donors showed male chromosomes. The presence of aquaporin 1, PAX2, and lectin-binding capacity for peanut agglutinin, Lotus tetragonolobus agglutinin, and Sophora japonica agglutinin in nephrogenic adenomas indicated an origin from renal tubular cells. Nephrogenic adenomas in renal-transplant recipients are derived from tubular cells of the renal transplants and are not metaplastic proliferations of the recipient's bladder urothelium.

Lectin staining for urine cytologic monitoring after kidney transplantation.

Urine cytology, although considered a valuable diagnostic tool in the monitoring of kidney graft function, is hampered by difficulty in differentiating the nucleated non-squamous cells in urine using conventional techniques. We have now developed a method for the simple identification of urinary cell types by lectin staining. Acetone-fixed cytopreparations of urinary sediments were incubated with the lectin combination Sophora Japonica agglutinin (SJA; rhodamine-labelled) and Erythrina cristagalli agglutinin (ECA; fluorescein isothiocyanate (FITC)-labelled) for 15 min, followed by staining of the nuclei with 4',6-diamidino-2-phenylindole (DAPI). The courses of 38 patients were serially monitored after kidney transplantation during the period in hospital. Nucleated urinary cell types could be easily identified from one specimen by their characteristic lectin-binding pattern using triple-immunofluorescence microscopy (FITC/rhodamine/ultra violet), permitting a differentiation between proximal (SJA+/ECA+) and distal tubules (SJA-/ECA+), collecting ducts (SJA+/ECA-) and lymphocytes (SJA-/ECA-). Stable graft function was characterized by low numbers of lymphocytes, tubular cells and urothelia. During rejection episodes, but not graft dysfunction unrelated to rejection, urinary excretion of lymphocytes as well as of distal tubular cells (from 1.0 to 6.0 and from 1.4 to 4.0 per 10 high-power fields, respectively) increased significantly up to 3 days prior to clinical diagnosis. Lectin staining facilitates unambiguous differentiation of the urinary cell types, in particular the various tubular epithelial cells, which are otherwise difficult to identify. This technique provides a rapid and easily applicable tool to evaluate the significance of the respective cell types in the monitoring of kidney graft function.

Analyte-receptor binding and dissociation kinetics for biosensor applications: a fractal analysis

A fractal analysis of confirmative nature only is presented for analyte-receptor binding and dissociation kinetics for biosensor applications. Data taken from the literature may be modeled, in the case of binding using a single-fractal analysis or a dual-fractal analysis. The dual-fractal analysis represents a change in the binding mechanism as the reaction progresses on the surface. Relationships are presented for the binding and dissociation rate coefficients as a function of their corresponding fractal dimension, D f or the degree of heterogeneity that exists on the surface. When analyte-receptor binding or dissociation is involved, an increase in the heterogeneity on the surface (increase in D f) leads to an increase in the binding and in the dissociation rate coefficient. It is suggested that an increase in the degree of heterogeneity on the surface leads to an increase in the turbulence on the surface owing to the irregularities on the surface. This turbulence promotes mixing, minimizes diffusional limitations, and leads subsequently to an increase in the binding and in the dissociation rate coefficient (Martin S.J., Granstaff, V.E., Frye, G.C., Anal. Chem., 65, (1991) 2910). The binding and the dissociation rate coefficient are rather sensitive to the degree of heterogeneity, D f,bind and D f,diss, respectively, that exists on the biosensor surface. For example, the order of dependence on D f,bind is 19.2 for the binding rate coefficient, k bind for the binding of 0.03–1.0 μM SH-2L d in solution to 2C TCR immobilized on a surface plasmon resonance (SPR) biosensor (Corr, M., Salnetz, A.E., Boyd, L.F., Jelonek, M.T., Khilko, S., Al-Ramadi, B.K., Kim, Y.S., Maher, S.E., Bothwell, A.L.M., Margulies, D.H., Science, 265, (1994) 946). The order of dependence on D f,diss is −6.22 for the dissociation rate coefficient, k diss for the dissociation of 250–1000 nM Sophora japonica agglutinin (SJA)-lactose complex from the SPR surface. In general, the technique is applicable to other reactions occurring on different types of surfaces, such as cell-surface reactions.

Increase of glycocalyx and altered lectin agglutination profiles of Pasteurella haemolytica A1 after incubation in bovine subcutaneous tissue chambers in vivo or in ruminant serum in vitro.

Pasteurella haemolytica serotype A1 (bovine strain OK) was incubated for 2 and 6 h in bovine subcutaneous tissue chambers in vivo, and ovine strain 82-25 and bovine strain L011 were incubated in vitro for 2 h in heat-inactivated ovine or bovine serum from which gamma globulin had been depleted by protein G affinity chromatography to assess changes in morphology and lectin agglutination profiles (strains 82-25 and L101 only). Cells, removed from chambers after 2 h, were covered with an extensive, dense glycocalyx extending approximately 0.5 microm from the cell surface. In many cells, the glycocalyx was separated from the cell surface by a clear, electron-transparent area. Cells, removed at 6 h, were covered with a sparse glycocalyx of fine fibers 0.2 to 0.3 microm from the cell surface. Strains 82-25 and L101, incubated for 2 h in heat-inactivated ovine or bovine serum or in heat-inactivated ovine or bovine serum depleted of gamma globulin by protein G affinity chromatography, were also covered with a glycocalyx. The glycocalyx did not bind protein A-colloidal gold and therefore did not contain aggregates of accumulated antibody. Strains 82-25 and L101 were incubated individually for 2 h in 10 mM sodium phosphate buffer (pH 7.2) containing 0.14 M NaCl, 0.5 mM CaCl2, and 0.15 mM MgCl2 or with this buffer and either 25% heat-inactivated, gamma globulin-depleted ovine serum or 25% heat-inactivated, gamma globulin-depleted bovine serum. Agglutination profiles were then determined with 17 lectins in 10 mM HEPES-buffered saline (pH 8.4) with 0.1 mM CaCl2 and 0.08% sodium azide. Profiles did not vary with 10 of 17 lectins. However, profiles did vary with peanut agglutinin, Phaseolus vulgaris leucoagglutinin, Sophora japonica agglutinin, Maackia amurensis lectin II, Narcissus pseudonarcissus (daffodil) lectin, Griffonia simplicifolia lectin I, and Pisum sativum agglutinin. Altered profiles indicate a change in the bacterial cell surface, possibly by adsorption or alteration of surface carbohydrate moieties by serum constituents.

Analysis of lectin binding properties on human Burkitt's lymphoma cell lines that show high spontaneous metastasis to distant organs in SCID mice: the binding sites for soybean agglutinin lectin masked by sialylation are closely associated with metastatic lymphoma cells.

Alterations in cell surface carbohydrates on human lymphoma cell lines with different spontaneous metastatic potential in the severe combined immunodeficiency (SCID) mouse model were analyzed. A difference in cell surface carbohydrates between high- (HBL-2, HBL-7 and HBL-8) and no- or low-(HBL-4, HBL-6, Daudi and Raji) spontaneous metastatic human lymphoma cell lines were analyzed on a FACScan using fluorescein-isothiocyanate (FITC)-conjugated lectins. The most consistent difference in lectin binding properties was found with soybean agglutinin (SBA) lectin. High-metastatic lymphoma cells (HBL-7 and HBL-8 cells) in vitro were found to bind the SBA lectin, but the cells in vivo (in primary tumors and metastatic tumors of SCID mice) showed considerably reduced SBA lectin binding. In addition, HBL-2 cells that almost did not bind SBA lectin in vitro and in vivo showed high spontaneous metastasis. Neuraminidase treatment revealed that SBA lectin binding sites were masked by sialic acid. On the other hand, no- or low-metastatic lymphoma cells in vitro and in vivo were found to bind SBA lectin. HBL-8 cell clones without SBA lectin binding showed high spontaneous metastasis to distant organs in SCID mice but HBL-8 cell clones with SBA lectin binding showed very low spontaneous metastasis. Sophora Japonica agglutinin (SJA) lectin is able to recognize the carbohydrates in common with SBA lectin, but it does not appear to be associated with metastatic capacity. These results suggest that the sialylation of particular carbohydrate residues on human lymphoma cells that are recognized by SBA lectin may be associated with the spontaneously metastatic capacity of human lymphoma cell lines in our SCID mouse model.

6'-sulfated sialyl Lewis x is a major capping group of GlyCAM-1.

The binding of L-selectin to the HEV-derived ligand GlyCAM-1 bears a strict requirement for oligosaccharide sulfation. In the companion study [Hemmerich, S., Bertozzi, C.R., Leffler, H., & Rosen, S. D. (1994) Biochemistry 33, 4820-4829], we identified the major sulfated mono- and disaccharides of GlyCAM-1 as Gal-6-SO4, GlcNAc-6-SO4, (SO4-6)Gal beta 1-->4GlcNAc, and Gal beta 1-->4(SO4-6)GlcNAc. Sialic acid and fucose are also critical to the recognition determinants on GlyCAM-1. However, the hydrolysis conditions employed in the previous study resulted in cleavage of these moieties, precluding their positional assignment. Here, we employ lectins of defined specificity in conjunction with specific exoglycosidases to identify a major GlyCAM-1 capping structure that includes all three critical elements. The complementary reactivity of Maackia amurensis agglutinin with fully sialylated, undersulfated GlyCAM-1 and Sambucus nigra agglutinin/Trichosanthes japonica agglutinin with desialylated but normally sulfated GlyCAM-1 indicates the presence of terminal 6'-sulfated sialyllactosamine. alpha (1-->3/4)Fucosidase removes fucose almost quantitatively from asialo-GlyCAM-1 while substantially enhancing its binding to Lycopersican esculentum agglutinin (specific for beta 1-->4-linked GlcNAc), indicating the presence of Fuc in an alpha 1-->3 linkage to GlcNAc. The strict requirement for desialylation to achieve defucosylation indicates a proximal location of Fuc with respect to terminal sialic acid. The nature of the capping group was further defined by studying the effects of sulfation, sialylation, and fucosylation on the ability of exo-beta(1-->4)galactosidase to release [3H]Gal from GlyCAM-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in glycoconjugate expression during early chick embryo development: A lectin‐binding study

A selection of lectins was used to investigate developmentally regulated changes in the distribution of cell surface oligosaccharides during the gastrulation and neurulation stages of early chick embryo development. Lectins from three specificity classes were used: glucose/mannose specificity (concanavalin A [Con A], Lens culinaris agglutinin [LCA], Pisum sativum agglutinin [PSA]); N-acetylglucosamine specificity (Lycopersicon esculentum agglutinin [LEA], wheat germ agglutinin [WGA], succinylated WGA [sWGA]); N-acetylgalactosamine/galactose specificity (Dolichos biflorus agglutinin [DBA], soybean agglutinin [SBA], Sophora japonica agglutinin [SJA], Bandeiraea (Griffonia) simplicifolia lectin I [BSL I], peanut agglutinin [PNA], Artocarpus integrifolia lectin [Jacalin], Ricinus communis agglutinin-1 [RCA-1], Erythrina cristagalli lectin [ECL]). At gastrulation stages, patterns of lectin binding could be distinguished in the epiblast, mesoderm, and endoderm cell layers. The primitive streak failed to bind any of the lectins, but LEA and WGA bound to the epiblast in regions lateral to the streak, indicating the loss of some glucosamine residues medially in preparation for the ingression movements of gastrulation. Several lectins showed marked binding to the mesoderm cells after their passage through the primitive streak; these were LCA, PSA, WGA, sWGA, BSL, and most particularly PNA. Therefore, the epithelial-mesenchymal transformation from epiblast to mesoderm at the primitive streak is accompanied by cell surface oligosaccharide changes in the epiblast and mesoderm that involve all classes of lectins including the PNA-binding sequence Gal beta 1-3GalNAc. Ultrastructurally, PNA was shown to bind extracellularly to matrix fibrils. Jacalin, having the same sugar specificity as PNA, but binding to serine/threonine linked chains rather than asparagine linked chains showed no binding to the mesoderm.(ABSTRACT TRUNCATED AT 250 WORDS)

Difference in the ability of blood group-specific lectins and monoclonal antibodies to recognize the ABH antigens in human tissues

Twelve different kinds of blood group-specific lectins have been used along with monoclonal anti-A, -B and -H antibodies for detecting the corresponding antigens in selected human tissues. Although most of the lectins recognized the antigens in the tissue sections examined, they displayed marked differences in their recognition patterns in certain tissues. Helix asparsa agglutinin (HAA), Helix pomatia agglutinin (HPA) and monoclonal anti-A antibody recognized A antigens in the mucous cells of salivary glands from blood group A or AB nonsecretor as well as secretor individuals, whereas Dolichos biflorus agglutinin (DBA), Griffonia simplicifolia agglutinin-I (GSA-I), Sophora japonica agglutinin (SJA) and Vicia villosa agglutinin (VVA) did not bind to them from nonsecretors. A antigens in endothelial cells, lateral membrane of pancreatic acinar cells and small mucouslike cells of submandibular glands from some individuals were likewise recognized by HAA and HPA but not by other blood group A-specific lections. In contrast, both HAA and HPA did not recognize the A antigens in mucous cells of Brunner's glands while other A-specific lectins and monoclonal anti-A antibody reacted specifically with the antigens. Such a difference was not observed with lectins specific for blood group B. However, the B antigens in Brunner's glands were recognized by these lectins but not with monoclonal anti-B antibody. The difference in labelling ability was also noted among the blood group H-specific lectins and monoclonal anti-H antibody in endothelial cells of blood vessels. Ulex europaeus agglutinin-I reacted with these cells irrespective of ABO and the secretor status of the individuals, while Anguilla anguilla agglutinin and monoclonal anti-H antibody reacted only with those cells from blood group O individuals. No reaction was observed with Lotus tetragonolobus agglutinin in these tissue sites. These results suggest a great diversity of blood group antigens in different human tissues.