Regulation of neutrophil function by selective targeting of glycan epitopes expressed on the integrin CD11b/CD18.

Polymorphonuclear neutrophils (PMNs) play a critical role in the innate immune response to invading pathogens. However, dysregulated mucosal trafficking of PMNs and associated epithelial tissue damage is a pathological hallmark of numerous inflammatory conditions including inflammatory bowel disease. The glycoprotein CD11b/CD18 plays a well-described role in regulating PMN transepithelial migration and PMN inflammatory functions. Previous studies have demonstrated that targeting of the N-linked glycan Lewis X on CD11b blocks PMN transepithelial migration (TEpM). Given evidence of glycosylation-dependent regulation of CD11b/CD18 function, we performed MALDI TOF Mass Spectrometry (MS) analyses on CD11b/CD18 purified from human PMNs. Unusual glycan epitopes identified on CD11b/CD18 included high Mannose oligosaccharides recognized by the Galanthus Nivalis lectin and biantennary galactosylated N-glycans recognized by the Phaseolus Vulgaris erythroagglutinin lectin. Importantly, we show that selective targeting of glycans on CD11b with such lectins results in altered intracellular signaling events that inhibit TEpM and differentially affect key PMN inflammatory functions including phagocytosis, superoxide release and apoptosis. Taken together, these data demonstrate that discrete glycan motifs expressed on CD11b/CD18 such as biantennary galactose could represent novel targets for selective manipulation of CD11b function and reduction of PMN-associated tissue damage in chronic inflammatory diseases.

Establishment of immunity against Epstein-Barr virus infection in a patient with CHARGE/complete DiGeorge syndrome after peripheral blood lymphocyte transfusion.

CHARGE syndrome is a rare congenital malformation syndrome which may share symptoms with DiGeorge syndrome. Complete DiGeorge syndrome (cDGS) is a severe form of DiGeorge syndrome, characterized by a CD3+ T-cell count of <50/mm3 due to athymia, and is fatal without immunologic intervention. We performed peripheral blood lymphocyte transfusion (PBLT) from an HLA-identical sibling without pretransplant conditioning in a CHARGE/cDGS patient with a novel CHD7 splice site mutation. Cyclosporine and short-term methotrexate were used for graft versus host disease (GVHD) prophylaxis, and neither acute nor chronic GVHD was observed. After PBLT, T-cell proliferative response to phytohemagglutinin and concanavalin A recovered, and intractable diarrhea improved. EBV infection, evidenced by a gradual increase in the viral genome copy number to a maximum of 2861 copies/µgDNA on day 42 after PBLT, resolved spontaneously. HLA A2402 restricted, EBV-specific CTLs were detected from peripheral blood on day 148, and EBV seroconversion was observed on day 181. Thus, EBV-specific immunity was successfully established by PBLT. Our results indicate that PBLT is a simple and effective therapy to reconstitute immune systems in CHARGE/DiGeorge syndrome.

Cytokines Produced by Lymphocytes in the Incompetent Great Saphenous Vein.

The pathogenesis of chronic venous disease (CVD) remains unclear, but lately inflammation is suggested to have an important role in its development. This study is aimed at assessing cytokines released by lymphocytes in patients with great saphenous vein (GSV) incompetence. In 34 patients exhibiting oscillatory flow (reflux) in GSV, blood was derived from the cubital vein and from the incompetent sapheno-femoral junction. In 12 healthy controls, blood was derived from the cubital vein. Lymphocyte culture with and without stimulation by phytohemagglutinin (PHA) was performed. Interleukins (IL) 1ß, 2, 4, 10, 12 (p70), and 17A; interleukin 1 receptor α (IL-1ra); tumor necrosis factor-α (TNF-α); interferon-gamma (IFN-γ); and RANTES were assessed in culture supernatants by the Bio-Plex assay. In both stimulated and unstimulated samples, in the examined group, IL-1ß and IFN-γ had higher concentrations and RANTES had lower concentrations when compared to those in the control group. In the examined group, IL-4 and IL-17A had higher concentrations without stimulation and TNF-α had higher concentrations with stimulation. The GSV samples had higher IL-2, IL-4, IL-12 (p70), and IFN-γ concentrations without stimulation and lower IL-2 and TNF-α concentrations with stimulation when compared to those of the upper limb in the examined group. These observations indicate that the oscillatory flow present in incompetent veins causes changes in the cytokine production by lymphocytes, promoting a proinflammatory profile. However, the relations between immunological cells, cytokines, and the endothelium require more insight.

The molecular mechanisms involved in lectin-induced human platelet aggregation.

We have compared the effect of three legume lectins, wheat germ agglutinin (WGA), Phaseolus vulgaris agglutinin (PHA) and Lens culinaris agglutinin (LCA), on the function of human platelets. We have found that WGA is more active than PHA in stimulating platelet activation/aggregation, while LCA has no effect. Studies on the mechanisms involved show that WGA and PHA induce phosphorylation/activation of PLCγ2 and increase [Ca2+]i. For the first time, it has been shown that Src/Syk pathway, the adapter protein SLP-76 and the exchange protein VAV, participate in the PLCγ2 activation by these lectins. Moreover WGA and PHA stimulate the PI3K/AKT pathway. PI3K, through its product phosphatidylinositol-3,4,5-trisphosphate activates Bruton's tyrosine kinase (BTK) and contributes to PLCγ2 activation. In conclusion, our findings suggest that PLCγ2 activation induced by WGA and PHA is regulated by Src/Syk and by PI3K/BTK pathways through their concerted action.

Effects of selective cleavage of high-mannose-type glycans of Maackia amurensis leukoagglutinin on sialic acid-binding activity.

BACKGROUND: Maackia amurensis leukoagglutinin (MAL) is a glycoprotein and sialic acid-binding lectin that is used widely in the detection and characterization of sialoglycoconjugates and human cancer cells. However, its N-linked glycan structure and role have yet to be determined. METHODS: The N-linked glycans were analyzed using high-performance liquid chromatography with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and the secondary structure was investigated using circular dichroism analysis. A hemagglutination assay was performed. Furthermore, surface plasmon resonance analysis, and fluorescence microscopy and fluorescence-activated cell-sorting analysis were conducted to assess the sialoglycoprotein-binding ability and its usefulness in the detection of human breast cancer MCF-7 cells, respectively. RESULTS: Analysis of the N-linked glycan structure of MAL confirmed the presence of eight glycans, comprising two α1,3-fucosylated paucimannosidic-type and six high-mannose-type glycans. Glycan analysis of MAL that had been treated with peptide N-glycosidase F (de-M-MAL) revealed that while the two α1,3-fucosylated paucimannosidic glycans remained attached following the treatment, the six high-mannose-type glycans had been completely cleaved from the original MAL. There were almost no secondary structural changes between MAL and de-M-MAL; however, the lectin activities exhibited by MAL, such as hemagglutination and binding to a sialoglycoprotein, were completely absent in de-M-MAL, and the ability to detect human breast cancer MCF-7 cells was 77% lower in de-M-MAL than in MAL. CONCLUSION: The high-mannose-type glycans in intact MAL are closely associated with its lectin activities. GENERAL SIGNIFICANCE: This is the first report of the N-linked glycan structure of MAL and the effect of high-mannose-type glycans on lectin activities.

Possible mechanisms of Leukoagglutinin induced apoptosis in human cells in vitro.

Leukoagglutinin is one of the phytohemagglutinin isolectins isolated from Phaseolus vulgaris. In our recent study, we showed that this lectin is able to influence the growth of human cancer cells in vitro. In addition, using the acridine orange and ethidium bromide staining, we found that leukoagglutinin can induce apoptosis. In order to understand the molecular mechanisms of induction of apoptosis, we performed computational modeling with subsequent experimental verification of theoretical data in vitro. We developed computational models of leukoagglutinin interaction with pro- (FasR and TNFR) and anti-apoptotic (IGF-1 and EGFR) receptors, and confirmed that leukoagglutinin may specifically interact with these receptors. Furthermore, we proved that leukoagglutinin can induce apoptosis in cancer (HEp-2) and non-cancer (4BL) cells, and observed that PHA-L is able to induce apoptosis through the up-regulation of Bax protein and activation of the effector caspase-3 and initiator caspase-8. However, these proteins have no effect on the Bcl-2 expression level.

Gamma delta T cells are early responders to Mycobacterium avium ssp. paratuberculosis in colostrum-replete Holstein calves.

Peripheral blood mononuclear cells (PBMC) and mesenteric node lymphocytes (MNL) were obtained from 30 calves that were assigned randomly at birth to 1 of 6 treatment groups with 5 calves per treatment in a 14-d study: (1) colostrum-deprived (CD), no vitamins; (2) colostrum-replacer (CR), no vitamins; (3) CR, vitamin A; (4) CR, vitamin D3; (5) CR, vitamin E; (6) CR, vitamins A, D3, E. Calves were injected with appropriate vitamin supplements and fed pasteurized whole milk (CD calves) or fractionated colostrum replacer (CR calves) at birth. Thereafter, all calves were fed pasteurized whole milk fortified with vitamins according to treatment group. Calves were orally inoculated with 108 cfu of Mycobacterium avium ssp. paratuberculosis (MAP) on d 1 and 3. The PBMC and MNL harvested on d 13 were analyzed by flow cytometry as fresh cells, after 3-d culture with phytohemagglutinin (PHA), and after 6-d culture with a whole-cell sonicate of MAP (MPS). Peripheral γδ T cells were a predominant lymphocyte subset in neonatal calves, with a decreased percentage noted in CD calves compared with CR calves. As well, CD25 expression was higher in γδ T cells compared with other cell subsets, regardless of treatment group. Stimulation of PBMC with PHA resulted in increased CD4+ and CD8+ subsets, whereas MNL response was dominated by expansion of B-cell subpopulations. Stimulation with PHA and MPS decreased the relative abundance of PBMC γδ T cells, but MNL γδ T cells increased upon stimulation with MPS. These results identify γδ T cells as key early responders to intracellular infection in neonatal calves and suggest that colostrum may be an important mediator of this response.

Phytohemagglutinin from Phaseolus vulgaris (PHA-E) displays a novel glycan recognition mode using a common legume lectin fold.

Phytohemagglutinin from Phaseolus vulgaris (PHA-E), a legume lectin, has an unusual specificity toward biantennary galactosylated N-glycan with bisecting N-acetylglucosamine (GlcNAc). To investigate the interaction in detail, we have solved the crystal structures of PHA-E without ligand and in complex with biantennary N-glycan derivatives. PHA-E interacts with the trisaccharide unit (Galß1-4GlcNAcß1-2Man) in a manner completely different from that of mannose/glucose-specific legume lectins. The inner mannose residue binds to a novel site on the protein, and its rotation is opposite to that occurring in the monosaccharide-binding site of other lectins around the sugar O3 axis. Saturation-transfer difference NMR using biantennary di-galactosylated and bisected glycans reveals that PHA-E interacts with both antennas almost equally. The unique carbohydrate interaction explains the glycan-binding specificity and high affinity.

Cross-platform comparison of glycan microarray formats.

Carbohydrates participate in almost every aspect of biology from protein sorting to modulating cell differentiation and cell-cell interactions. To date, the majority of data gathered on glycan expression has been obtained via analysis with either anti-glycan antibodies or lectins. A detailed understanding of the specificities of these reagents is critical to the analysis of carbohydrates in biological systems. Glycan microarrays are increasingly used to determine the binding specificity of glycan-binding proteins (GBPs). In this study, six different glycan microarray platforms with different modes of glycan presentation were compared using five well-known lectins; concanavalin A, Helix pomatia agglutinin, Maackia amurensis lectin I, Sambucus nigra agglutinin and wheat germ agglutinin. A new method (universal threshold) was developed to facilitate systematic comparisons across distinct array platforms. The strongest binders of each lectin were identified using the universal threshold across all platforms while identification of weaker binders was influenced by platform-specific factors including presentation of determinants, array composition and self-reported thresholding methods. This work compiles a rich dataset for comparative analysis of glycan array platforms and has important implications for the implementation of microarrays in the characterization of GBPs.

Trial of complete weaning from immunosuppression for liver transplant recipients: factors predictive of tolerance.

Recipients of liver transplantation (LT) may develop immunological tolerance. Factors predictive of tolerance are not clearly understood. Transplant recipients with normal liver function tests and without active viral hepatitis or autoimmune disease who presented with side effects of immunosuppression or a high risk of de novo malignancies were selected to participate in this prospective study. Twenty-four patients fulfilled the inclusion criteria and, therefore, underwent a gradual reduction of immunosuppression. Tolerance was defined as normal liver function tests after immunosuppression withdrawal. Basal clinical and immunological characteristics, including lymphocyte counts and subpopulations (T, B, natural killer, CD4(+) , CD8(+) , and regulatory T cells) and the phytohemagglutinin stimulation index (SI), were compared for tolerant and nontolerant patients. Fifteen of the 24 patients (62.5%) were tolerant at a median of 14 months (interquartile range = 8.5-22.5 months) after complete immunosuppression withdrawal. Tolerant patients had a longer median interval between transplantation and inclusion in the study (156 for tolerant patients versus 71 months for nontolerant patients, P = 0.003) and a lower median SI (7.49 for tolerant patients versus 41.73 for nontolerant patients, P = 0.01). We identified 3 groups of patients with different probabilities of tolerance: in the first group (n = 7 for an interval > 10 years and an SI < 20), 100% reached tolerance; in the second group (n = 10 for an interval > 10 years and an SI > 20 or an interval < 10 years and an SI < 20), 60% reached tolerance; and in the third group (n = 7 for an interval < 10 years and an SI > 20), 29% reached tolerance. In conclusion, a high proportion of select LT recipients can reach tolerance over the long term. Two simple basal variables-the time from transplantation and the SI-may help to identify these patients.

Biomolecule screening for efficient attachment of biofunctionalized microparticles to the zona pellucida of mammalian oocytes and embryos.

Individual tagging of oocytes and embryos through the attachment of micrometer-sized polysilicon barcodes to their zona pellucida (ZP) is a promising approach to ensure their correct identification and traceability in human assisted reproduction and in animal production programs. To provide barcodes with the capacity of binding to the ZP, they must be first biofunctionalized with a biomolecule capable of binding to the ZP of both oocytes and embryos. The aim of this work was to select, among an anti-ZP2 antibody and the two lectins wheat germ agglutinin (WGA) and phytohemagglutinin-L, the most optimal biomolecule for the eventual biofunctionalization of barcodes, using mouse oocytes and embryos and commercially available microspheres as a model. Despite the anti-ZP2 antibody showed the highest number of binding sites onto the ZP surface, as determined by field emission scanning electron microscopy, the binding of anti-ZP2-biofunctionalized microspheres to the ZP of cultured oocytes and embryos was less robust and less stable than the binding of lectin-biofunctionalized ones. WGA proved to be, among the three candidates tested, the most appropriate biomolecule to biofunctionalize microparticles with the aim to attach them to the ZP of both oocytes and embryos and to maintain them attached through oocyte activation (zona reaction) and in vitro culture up to the blastocyst stage. As saccharides recognized by WGA are highly abundant in the ZP of most mammalian species, WGA-biofuncionalized microparticles would be able to attach to the ZP of oocytes/embryos of species other than the mouse, such as humans and farm animals.

Identification of low-abundance cancer biomarker candidate TIMP1 from serum with lectin fractionation and peptide affinity enrichment by ultrahigh-resolution mass spectrometry.

As investigating a proteolytic target peptide originating from the tissue inhibitor of metalloproteinase 1 (TIMP1) known to be aberrantly glycosylated in patients with colorectal cancer (CRC), we first confirmed that TIMP1 is to be a CRC biomarker candidate in human serum. For this, we utilized matrix-assisted laser desorption/ionization (MALDI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS) showing ultrahigh-resolution and high mass accuracy. This investigation used phytohemagglutinin-L(4) (L-PHA) lectin, which shows binding affinity to the ß-1,6-N-acetylglucosamine moiety of N-linked glycan on a protein, to compare fractionated aberrant protein glycoforms from both noncancerous control and CRC serum. Each lectin-captured fraction containing aberrant glycoforms of TIMP1 was digested by trypsin, resulting in the tryptic target peptide, representative of the serum glycoprotein TIMP1. The resulting target peptide was enriched using a stable isotope standard and capture by the antipeptide antibody (SISCAPA) technique and analyzed by a 15 T MALDI FTICR mass spectrometer with high mass accuracy (Δ < 0.5 ppm to the theoretical mass value of the target peptide). Since exact measurement of multiplex isotopic peaks of the target peptide could be accomplished by virtue of high mass resolution (Rs > 400,000), robust identification of the target peptide is only achievable with 15 T FTICR MS. Also, MALDI data obtained in this study showed that the L-PHA-captured glycoforms of TIMP1 were measured in the pooled CRC serum with about 5 times higher abundance than that in the noncancerous serum, and were further proved by MRM mass analysis. These results confirm that TIMP1 in human serum is a potent CRC biomarker candidate, demonstrating that ultrahigh-resolution MS can be a powerful tool toward identifying and verifying potential protein biomarker candidates.

Role of Phytomolecules in the Treatment of Obesity: Targets, Mechanisms and Limitations.

Obesity has become a worldwide health problem. It triggers additional co-morbidities like cardiovascular diseases, cancer, depression, sleep disorders, gastrointestinal problems and many more. Excess accumulation of fat in obesity could be caused by many factors like sedentary lifestyle, consumption of high-fat diet, genetic predisposition, etc. Imbalanced energy metabolism i.e., greater energy consumption than utilisation, invariably underlies obesity. Considering the high prevalence and continuous, uncontrolled increase of this major public health issue, there is an urgent need to find appropriate therapeutic agents with minimal or no side effects. The high prevalence of obesity in recent years has led to a surge in the number of drugs available in the market that claim to control obesity. Although there is a long list of medicines and management strategies that are available, selecting the right therapeutic intervention and feasible management of obesity is a challenge. Several phytochemicals like hydroxycitric acid, flavonoids, tannins, anthocyanins, phytohaemagglutinin, thymoquinone and epigallocatechin gallate have been shown to possess promising anti-obesity properties. However, studies providing information on how various phytochemicals exert their anti-obesity effects are inadequate. This calls for more experimentation in this less explored area of research. Additionally, the complication of obesity arises when it is a result of multiple factors and associated with a number of co-morbidities. In order to handle such complexities, combinatorial therapeutic interventions become effective. In this review, we have described the medicinal chemistry of different highly effective phytochemicals which can be used in the effective treatment and management of obesity.

Characterisation of a haemagglutinin from Hokkaido red bean (Phaseolus vulgaris cv. Hokkaido red bean).

BACKGROUND: A haemagglutinin was purified from Japanese Hokkaido red beans (Phaseolus vulgaris cv. Hokkaido red bean) with a procedure that included three chromatographic media. RESULTS: Haemagglutinating activity was adsorbed on DEAE cellulose, Affi-gel blue gel and Mono S. The pure haemagglutinin was a homodimer and each subunit was around 30 kDa in molecular mass. The haemagglutinating activity of this agglutinin could not be inhibited by a variety of simple sugars at 200 mmol L(-1) concentration including alpha-L-fucose, D(+)-galactose, D(+)-glucose, D(+)-glucosamine, D(-)galactosamine, galacturonic acid, (+)-lactose, D(+)-melibose, L(-)-mannose, D(+)-mannose, D-mannosamine, D(+)-raffinose, L-rhamnose, (+)-xylose and galacturonic acid. The haemagglutinating activity was fully retained at pH 4-11 and at 0-80 degrees C, but was completely lost at extreme pH values (0-2 and 13-14) and at very high temperatures (90 degrees C and 100 degrees C). The haemagglutinin exhibited a weak mitogenic activity toward mouse splenocytes, a stronger anti-proliferative activity than Con A toward HepG2 (human hepatoma) cells and inhibited >80% of HIV-1 reverse transcriptase inhibitory activity at 3.3 micromol L(-1). It was devoid of anti-fungal activity. CONCLUSION: Hokkaido red bean haemagglutinin possesses a potent anti-proliferative effect on HepG2 cells.

Calcium redistribution contributes to the hard-to-cook phenotype and increases PHA-L lectin thermal stability in common bean low phytic acid 1 mutant seeds.

Seed phytic acid reduces mineral bioavailability by chelating minerals. Consumption of common bean seeds with the low phytic acid 1 (lpa1) mutation improved iron status in human trials but caused adverse gastrointestinal effects, presumably due to increased stability of lectin phytohemagglutinin L (PHA-L) compared to the wild type (wt). A hard-to-cook (HTC) defect observed in lpa1 seeds intensified this problem. We quantified the HTC phenotype of lpa1 common beans with three genetic backgrounds. The HTC phenotype in the lpa1 black bean line correlated with the redistribution of calcium particularly in the cell walls, providing support for the "phytase-phytate-pectin" theory of the HTC mechanism. Furthermore, the excess of free cations in the lpa1 mutation in combination with different PHA alleles affected the stability of PHA-L lectin.

AFM- and NSOM-based force spectroscopy and distribution analysis of CD69 molecules on human CD4+ T cell membrane.

Although CD69 is well known as an early T cell-activation marker, the possibility that CD69 are distributed as nano-structures on membrane for immune regulation during T cell activation has not been tested. In this study, nanoscale features of CD69 expression on activated T cells were determined using the atomic force microscopy (AFM) topographic and force-binding nanotechnology as well as near-field scanning optical microscopy (NSOM)-/fluorescence quantum dot (QD)-based nanosacle imaging. Unstimulated CD4(+) T cells showed neglectable numbers of membrane CD69 spots binding to the CD69 Ab-functinalized AFM tip, and no detectable QD-bound CD69 as examined by NSOM/QD-based imaging. In contrast, Phytohemagglutinin (PHA)-activated CD4(+) T cells expressed CD69, and displayed many force-binding spots binding to the CD69 Ab-functionalized AFM tip on about 45% of cell membrane, with mean binding-rupture forces 276 +/- 71 pN. Most CD69 molecules appeared to be expressed as 100-200 nm nanoclusters on the membrane of PHA-activated CD4(+) T cells. Meanwhile, NSOM/QD-based nanoscale imaging showed that CD69 were non-uniformly distributed as 80-200 nm nanoclusters on cell-membrane of PHA-activated CD4(+) T cells. This study represents the first demonstration of the nano-biology of CD69 expression during T cell activation.

Purification and characterization of a lectin from Phaseolus vulgaris cv. (Anasazi beans).

A lectin has been isolated from seeds of the Phaseolus vulgaris cv. "Anasazi beans" using a procedure that involved affinity chromatography on Affi-gel blue gel, fast protein liquid chromatography (FPLC)-ion exchange chromatography on Mono S, and FPLC-gel filtration on Superdex 200. The lectin was comprised of two 30-kDa subunits with substantial N-terminal sequence similarity to other Phaseolus lectins. The hemagglutinating activity of the lectin was stable within the pH range of 1-14 and the temperature range of 0-80 degrees C. The lectin potently suppressed proliferation of MCF-7 (breast cancer) cells with an IC(50) of 1.3 microM, and inhibited the activity of HIV-1 reverse transcriptase with an IC(50) of 7.6 microM. The lectin evoked a mitogenic response from murine splenocytes as evidenced by an increase in [3H-methyl]-thymidine incorporation. The lectin had no antifungal activity. It did not stimulate nitric oxide production by murine peritoneal macrophages. Chemical modification results indicated that tryptophan was crucial for the hemagglutinating activity of the lectin.

Lectin precipitation using phytohemagglutinin-L(4) coupled to avidin-agarose for serological biomarker discovery in colorectal cancer.

N-acetylglucosaminyltransferase V (GnT-V) has been reported to be upregulated in malignant cancer cells, and its targets have been sought after with regard to biomarker identification. The low capacity and high false positive rates of 2-DE gel-based lectin blots using phytohemagglutinin-L(4) (L-PHA) prompted us to develop a novel protocol for identifying GnT-V targets, in which serum proteins were subjected to immunodepletion, alkylation, and lectin precipitation using L-PHA coupled to avidin-agarose bead complexes, and tryptic digestion. Proteins captured by L-PHA conjugates were analyzed by a nano-LC-FT-ICR/LTQ MS. Here, we report 26 candidate biomarkers for colorectal cancer (CRC) that show 100% specificity and sensitivities of greater than 50%. Not only can these candidate proteins be used as analytes for validation, but the novel protocol described herein can be applied to biomarker discovery in nonCRCs.

Identification of proteins bearing beta1-6 branched N-glycans in human melanoma cell lines from different progression stages by tandem mass spectrometry analysis.

The common structural alterations in the cell-surface glycoproteins concern the highly elevated expression of tri- and tetra-antennary beta1-6-N-acetylglucosamine (beta1-6 GlcNAc) bearing N-glycans, which are recognised by Phaseolus vulgaris agglutinin (PHA-L). In this report we identified proteins bearing beta1-6 GlcNAc branched N-glycans in three human melanoma cell lines: WM35--from the primary tumour site, as well as WM239 and WM9 from different metastatic sites: the skin and the lymph node, respectively, by tandem mass spectrometry (MS/MS) on PHA-L agarose bound material, followed by immunochemical identification. Our results show that melanoma cell lines differ from each other in the number of N-glycoproteins bearing beta1-6 GlcNAc branched oligosaccharides. Among identified proteins the largest group consists of integrin subunits. In addition, L1-CAM, Mac-2 binding protein, melanoma cell adhesion molecule, intercellular adhesion molecule, melanoma associated antigen, tumour rejection antigen-1, melanoma-associated chondroitin sulfate proteoglycan 4 and lysosome-associated membrane protein (LAMP-1) were found. It was indicated that WM35 cell line showed the lowest number of proteins possessing beta1-6 GlcNAc branched N-glycans in comparison to metastatic WM9 and WM239 cell lines. Our data suggest that changes in the number of proteins being a substrate for GlcNAc-TV are better correlated with melanoma development and progression than with expression of cell adhesion molecules.

Apoptosis induction by Maackia amurensis agglutinin in childhood acute lymphoblastic leukemic cells.

Malignant transformation is known to be associated with changes in cell surface carbohydrate-architecture, which can be detected by lectins. In the present study, Maackia amurensis agglutinin (MAA), specific for NeuNAcalpha(2-->3)Gal/GalNAc showed strong binding with lymphoblasts of children having acute lymphoblastic leukemia (ALL) as compared to cells from children with non-hematological disorders ("Controls"). MAA recognized a 66 kDa sialoglycoprotein present in membrane fraction of ALL cells. Moreover, MAA induced apoptosis in ALL cells was found to be reduced significantly in presence of GM2/IgG(MAA). Thus, MAA has a potential to be used as diagnostic and therapeutic agent in case of childhood-ALL.