Maackia Amurensis Agglutinin Research Articles

Modifications of carbohydrate residues in the sheep oviductal ampulla after superovulation

Epithelium of oviductal ampulla was studied in normal and in superovulated sheep using morphologic analysis and lectin glycohistochemistry. The lining epithelium consisted of two types of cells, ciliated and nonciliated cells. Unlike superovulated samples, the nonciliated cells from control ewes showed apical protrusions indicating an apocrine secretory activity. The ciliated cells showed lectin-binding sites mainly at the level of the cilia which bound all the used lectins except Peanut agglutinin, suggesting the lack of glycans terminating with Galβ1,3GalNAc. In superovulated specimens, the ciliated cells with high mannosylated glycans Concanavalin A (Con A) and GlcNAc and GalNac termini Griffonia simplicifolia agglutinin II (GSA II) and Dolicurus biflorus agglutinin (DBA) decreased. The luminal surface of nonciliated cells showed all investigated sugar residues in controls, whereas it was lacking in high mannosylated (Con A) and terminal GalNAcα1,3(LFucα1,2)Galβ1,3/4GlcNAcβ1 sequence (DBA) in superovulated ewes. Apical protrusions from control ampullae nonciliated cells showed glycans containing mannose, GlcNac, GalNAc, galactose, and α2,3-linked sialic acid (Con A, KOH-sialidase- Wheat germ agglutnin [WGA], GSA II, SBA, Griffonia simplicifolia agglutinin-isolectin B4 [GSA I-B4], Maackia amurensis agglutinin II [MAL II]). The supranuclear cytoplasm of nonciliated cells expressed terminal GlcNAc (GSA II) in all specimens, also O-linked glycans (mucin-type glycans) with GalNAc and sialic acid termini (Helix pomatia agglutinin [HPA] and MAL II) in control animals, and also N-linked glycans with fucose, galactose, lactosamine, and α2,3-linked sialic acid termini (Ulex europaeus agglutinin I [UEA I], GSA I-B4, Ricinus communis agglutinin120 [RCA120], and Sambucus nigra agglutinin [SNA] ) in superovulated ewes. These results report for the first time that the superovulation treatment affects the secretory activity and the glycan pattern of the epithelium lining the sheep oviductal ampulla.

Salivary glyco-sialylation changes monitors oral carcinogenesis.

Alterations in cell membrane glycosylation play important role in oral carcinogenesis. The present study evaluated salivary sialylation changes i.e. total sialic acid (TSA), sialidase activity, linkage specific (α2-3 and α2-6) sialoproteins and sialyl transferase (ST) activity in controls, patients with oral precancerous conditions (OPC) and oral cancer. Subjects enrolled included 100 controls, 50 patients with OPC, 100 oral cancer patients, and 30 post treatment follow-ups. TSA was estimated by spectrophotometric method, sialidase activity by spectrofluorometric assay and linkage specific biotinylated lectins (α2-3: sambucus nigra agglutinin and α2-6: maackia amurensis agglutinin) were used to detect α-2,3 and α-2,6 STs and sialoproteins by ELISA and dot blot respectively. An increasing trend of salivary TSA/TP ratio, sialidase activity, α2-3 sialoproteins, α-2,3 and α-2,6 ST activities was observed from controls to patients with OPC to oral cancer patients and levels were significantly elevated in oral cancer patients as compared to the controls. Sialidase activity exhibited significant association with metastasis and infiltration. Sialidase activity, TSA/TP ratio, α-2,3 and α-2,6 ST activities were found to be higher in patients with metastasis as compared to patients without metastasis. A progressive increase in TSA/TP ratio, sialidase activity, α2-3 and α2-6 sialoproteins was observed from controls to early to advanced stage of the disease. Sialidase activity, α2-3 and α2-6 sialoproteins and ST activities were found to be decreased in complete responders; while levels were elevated in non-responders. The results documented utility of salivary sialylation endpoints, a non invasive tool in monitoring of oral carcinogenesis.

Purification and identification of α 2–3 linked sialoglycoprotein and α 2–6 linked sialoglycoprotein in edible bird’s nest

The distribution of α 2–3 and α 2–6 linked sialoglycoproteins in edible bird’s nest (EBN) was analyzed by Maackia amurensis agglutinin (MAA) and Sambucus nigra agglutinin (SNA) lectin blotting. Q Sepharose Fast Flow, Superdex 75 and Sephadex G-25 columns were combined to enrich sialoglycoproteins from EBN extraction. For purification and identification of α 2–3 linked sialoglycoprotein and α 2–6 linked sialoglycoprotein, MAA-Sepharose-4B, SNA-Sepharose-4B lectin affinity chromatography and matrix-assisted laser desorption–ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) tandem mass spectrometry (MS) were employed successively. The contents of protein, carbohydrate and sialic acid of EBN extraction were 65.43, 20.97 and 10.47 %, respectively; EBN extraction contained high-abundance glycoproteins with the molecular weights of 128 kDa (19.1 %), 106 kDa (18.5 %) and 43 kDa (29.4 %); the 43 kDa glycoproteins contained terminal α 2–3 and α 2–6 sialic acid linkage were successively enriched by three chromatography columns; α 2–3 linked sialoglycoprotein and α 2–6 linked sialoglycoprotein were further purified by affinity chromatography eluted with 0.3 and 0.5 M lactose, respectively. In addition, MALDI-TOF/TOF MS analysis showed that α 2–3 linked sialoglycoprotein was an acidic mammalian chitinase-like protein, and α 2–6 linked sialoglycoprotein was an acidic mammalian chitinase. This method is proved to be a simple and effective approach to purify sialoglycoproteins from high-abundance glycoproteins in EBN.

Association of IgA secretory component sialylation with leucocytospermia of infertile men - a pilot study

The aim of our pilot study was to check whether the differences in IgA secretory component (SC) sialylation are associated with leucocytospermia. In normozoospermic and leucocytospermic seminal plasmas, 78-kDa and 63-kDa SC immunoreactive bands were observed. The SC sialylation was analysed by lectin blotting, using sialo-specific lectins MAA (Maackia amurensis agglutinin) and SNA (Sambucus nigra agglutinin). Specific reactivity of 63-kDa SC with MAA and SNA was higher than 78-kDa SC in both analysed seminal groups. The analysis of seminal SC sialylation might be a valuable diagnosis tools for the evaluation of fertility problems related with leucocytospermia.

Galactose 6-O-Sulfotransferases Are Not Required for the Generation of Siglec-F Ligands in Leukocytes or Lung Tissue

Eosinophil accumulation is a characteristic feature of the immune response to parasitic worms and allergens. The cell surface carbohydrate-binding receptor Siglec-F is highly expressed on eosinophils and negatively regulates their accumulation during inflammation. Although endogenous ligands for Siglec-F have yet to be biochemically defined, binding studies using glycan arrays have implicated galactose 6-O-sulfate (Gal6S) as a partial recognition determinant for this receptor. Only two sulfotransferases are known to generate Gal6S, namely keratan sulfate galactose 6-O-sulfotransferase (KSGal6ST) and chondroitin 6-O-sulfotransferase 1 (C6ST-1). Here we use mice deficient in both KSGal6ST and C6ST-1 to determine whether these sulfotransferases are required for the generation of endogenous Siglec-F ligands. First, we characterize ligand expression on leukocyte populations and find that ligands are predominantly expressed on cell types also expressing Siglec-F, namely eosinophils, neutrophils, and alveolar macrophages. We also detect Siglec-F ligand activity in bronchoalveolar lavage fluid fractions containing polymeric secreted mucins, including MUC5B. Consistent with these observations, ligands in the lung increase dramatically during infection with the parasitic nematode, Nippostrongylus brasiliensis, which is known to induce eosinophil accumulation and mucus production. Surprisingly, Gal6S is undetectable in sialylated glycans from eosinophils and BAL fluid analyzed by mass spectrometry. Furthermore, none of the ligands we describe are diminished in mice lacking KSGal6ST and C6ST-1, indicating that neither of the known galactose 6-O-sulfotransferases is required for ligand synthesis. These results establish that ligands for Siglec-F are present on several cell types that are relevant during allergic lung inflammation and argue against the widely held view that Gal6S is critical for glycan recognition by this receptor.

Preferential Lectin Binding of Cancer Cells upon Sialic Acid Treatment Under Nutrient Deprivation

The terminal monosaccharide of glycoconjugates on a eukaryotic cell surface is typically a sialic acid (Neu5Ac). Increased sialylation usually indicates progression and poor prognosis of most carcinomas. Here, we utilize two human mammary epithelial cell lines, HB4A (breast normal cells) and T47D (breast cancer cells), as a model system to demonstrate differential surface glycans when treated with sialic acid under nutrient deprivation. Under a starved condition, sialic acid treatment of both cells resulted in increased activities of α2→3/6 sialyltransferases as demonstrated by solid phase assay using lectin binding. However, a very strong Maackia amurensis agglutinin I (MAL-I) staining on the membrane of sialic acid-treated T47D cells was observed, indicating an increase of Neu5Acα2→3Gal on the cell surface. To our knowledge, this is a first report showing the utility of lectins, particularly MAL-I, as a means to discriminate between normal and cancer cells after sialic acid treatment under nutrient deprivation. This method is sensitive and allows selective detection of glycan sialylation on a cancer cell surface.

Potential importance of Maackia amurensis agglutinin in non-small cell lung cancer

Maackia amurensis agglutinin is a NeuNAcα (2-3) Galβ (1-4) GlcNAc/Glc-specific lectin, which was shown to have diagnostic potential in cancers of different origin. In a previous report, we demonstrated that GM3 specific IgG from bronchoalveolar lavage fluid (BALF) of non-small cell lung cancer (NSCLC) patients interacted with ∼66kDa membrane glycoprotein band of NSCLC cell lines, which was also recognised by this lectin. This observation prompted us to assess the potential of Maackia amurensis agglutinin in NSCLC. Accordingly, we examined the reactivity of this lectin with NSCLC cell lines as well as the tissue biopsies and cells obtained from fine needle aspirations of NSCLC patients. Maackia amurensis agglutinin showed strong reactivity, specifically with cells and biopsy samples of NSCLC origin. Furthermore, this lectin was found to induce apoptosis in NSCLC cells. The mechanism of this lectin-induced apoptosis involved downregulation of Bcl-XL, upregulation of Bax, release of cytochrome c and activation of procaspase-3. Collectively our results have suggested that Maackia amurensis agglutinin may have the potential to serve as a unique probe for detection of NSCLC and also as a specific apoptosis-inducing agent in NSCLC cells.

Detection of Glycomic Alterations Induced by Overexpression of P-Glycoprotein on the Surfaces of L1210 Cells Using Sialic Acid Binding Lectins

P-glycoprotein (P-gp) overexpression is the most frequently observed cause of multidrug resistance in neoplastic cells. In our experiments, P-gp was expressed in L1210 mice leukemia cells (S cells) by selection with vincristine (R cells) or transfection with the gene encoding human P-gp (T cells). Remodeling of cell surface sugars is associated with P-gp expression in L1210 cells as a secondary cellular response. In this study, we monitored the alteration of cell surface saccharides by Sambucus nigra agglutinin (SNA), wheat germ agglutinin (WGA) and Maackia amurensis agglutinin (MAA). Sialic acid is predominantly linked to the surface of S, R and T cells via α-2,6 branched sugars that tightly bind SNA. The presence of sialic acid linked to the cell surface via α-2,3 branched sugars was negligible, and the binding of MAA (recognizing this branch) was much less pronounced than SNA. WGA induced greater cell death than SNA, which was bound to the cell surface and agglutinated all three L1210 cell-variants more effectively than WGA. Thus, the ability of lectins to induce cell death did not correlate with their binding efficiency and agglutination potency. Compared to S cells, P-gp positive R and T cells contain a higher amount of N-acetyl-glucosamine on their cell surface, which is associated with improved WGA binding. Both P-gp positive variants of L1210 cells are strongly resistant to vincristine as P-gp prototypical drug. This resistance could not be altered by liberalization of terminal sialyl residues from the cell surface by sialidase.

Glycosylation-Related Gene Expression Is Dysregulated in Multiple Myeloma and Overexpression of the Sialyltransferase ST3GAL6 Is Associated with Inferior Survival.

AbstractAbstract 2931 BackgroundAlterations in glycosylation are associated with malignant transformation, with growing evidence implicating the dysregulation of glycosylation genes (glyco-genes) in multiple myeloma (MM). P selectin glycoprotein ligand-1 (PSGL1), recently reported to be important in MM cell adhesion and trafficking is over expressed by MM cells. Importantly, the generation of functional selectin ligands requires post-translational modification of scaffold proteins by glycosyltransferases and sialyltransferases generating selectin ligands such as Sialyl Lewis X (sLex). This background led us to undertake a comprehensive evaluation of the role glyco-genes in MM disease progression. MethodsAnalyzing publically available microarray transcriptomic datasets (Mayo Clinic GSE6477, University of Arkansas (UAMS) GSE24080, GSE2658) we sought to identify dysregulated glyco-genes in MM. The most significantly dysregulated genes were validated using real time quantitative PCR (RT_PCR) in the MM cell lines RPMI8226 and MMIR and in primary MM patient CD138 positive cells. The prognostic significance of any dysregulated genes was analyzed using Kaplan Meier survival estimates. Membrane protein extracts from MM cell lines were applied directly to lectin microarrays following fluorescent labeling to generate cell surface glycan profiles. Protein expression was assessed by immunohistochemistry (IHC) on primary MM bone marrow sections from MM patients and healthy controls. Lectin based flow cytometry was used to assess for the binding of sialic acid lectins to MM cell line RPMI8226. ResultsGene expression microarray dataset analysis confirmed the over-expression of genes related to the process of glycosylation in MM. Seventy-three genes were differentially expressed (> 2 fold change in MM from combined datasets). Nineteen genes were dysregulated in smoldering MM, 12 of which were common to MM. Thirteen were differentially regulated in MGUS, 6 of which were common to both MM and MGUS. One of these genes, the sialyltransferase ST3GAL6 (ST3 beta-galactosidase, alpha-2,3-sialyltransferase 6), which plays a critical role in generation of functional selectin ligands such as sLex, was upregulated in MM (fold change = 2.67) and smoldering MM (fold change = 2.22) but was absent in MGUS. Patients from GSE24080 (n=509) were stratified into two groups based on their expression intensities for ST3GAL6. Patients with higher normalized intensity values corresponding to probeset ID 210942_s_at (ST3GAL6) were found to have a lower median overall survival compared to their lower expressing counterparts. The difference in overall survival observed was 5.13 months (p <0.001 CI 1.3, 8.9). The association with reduced survival was independently verified in the MRC Myeloma IX microarray dataset (n=260). In this dataset using the median expression of ST3GAL6 as a cutoff there was a statistically significant reduction on overall survival with higher expression of ST3GAL6 (median OS 35.7 vs. 48 months, log rank test p=0.04)RT-PCR analysis validated the over expression of glycogenes, including ST3GAL6 in MM cell lines and primary samples compared to healthy controls. We observed a trend for higher fold changes for ST3GAL6 in samples from patients with relapsed/refractory disease compared to those with responsive disease. IHC for ST3GAL6 on primary bone marrow sections from MM patients (n=57), demonstrated specific Golgi staining compared to controls. Consistent with the over expression of ST3GAL6 lectin microarray analysis of membrane protein extracts from MM cell lines RPMI8226 and MMIR showed binding to sialic acid-specific lectins Maackia amurensis agglutinin (MAA), which is specific for a2–3 linked sialic acids, and Sambucus nigra(SNA) which is specific for a2–6 linked sialic acids. This pattern was confirmed using biotinylated lectin based flow cytometry, which demonstrated a shift in allophycocyanin (APC) median fluorescence intensity for these lectins on RPMI8226 cells. ConclusionsGlyco-genes, including the sialyltransferase ST3GAL6, are differentially regulated in all stages of MM with potential effects on MM biology and survival. Upregulation of ST3GAL6 may play an important role in MM cell trafficking and in this analysis is associated with inferior survival. Studies are ongoing to address the roles of ST3GAL6 over expression and altered sialylation in MM cell adhesion and trafficking. Disclosures:Morgan:Novartis: Consultancy; Celgene: Consultancy; J&J: Consultancy.

Cell Surface Glycosylation and Glycosaminoglycan Composition Profiles in Immature and Mature Intervertebral Disks

Introduction Back pain is a predominant cause of disability, and the major cause of this disability is intervertebral disk (IVD) degeneration.1 Proteoglycan (PG) content of the IVD is decreased during degeneration, and glycosaminoglycan (GAG) composition is altered.2 In this study, the cell surface glycosylation and GAG composition profiles of immature and mature ovine IVD tissue were profiled in an effort to understand potential signals involved in cell-extracellular matrix cross-talk during maturation and degeneration. Materials and Methods IVD tissue from L3-L4 and L4-L5 lumbar segments were harvested from 3- and 11-month-old animals. One portion of the tissue was fixed for histochemical profiling with a panel of fluorescently-labeled lectins ( Maackia amurensis agglutinin, Sambucus nigra agglutinin I, Ulex europaeus agglutinin I, Concanavalin A (jack bean lectin), Wisteria floribunda agglutinin). The remaining portion was digested with proteinase K and the sulfated GAG (s-GAG) content was quantified by dimethylmethylene blue (DMMB) assay. The digested tissue was treated with chondroitinase ABC (ChABC) and analyzed by HPLC. Results The glycosylation expression level differed between annulus fibrosus (AF) and nucleus pulposus (NP) tissue as indicated by quantitative analysis using lectin histochemistry. All lectins stained cells and extracellular matrix (ECM) of the IVD independently of the tissue type. The cells demonstrated greater lectin binding compared to the ECM and the intensity of staining differed according to the tissue-type. With maturity, the expression profiles of cell surface glycosylation changed, notably for the n a-(2?6)-linked sialic acid (SNA-I) and terminal GalNAc (WFA staining) expression with a 10-fold and 2-fold increase of expression at 11 months old, respectively. DMMB assay showed that NP tissue contained threefold more s-GAGs than AF and cartilage in 3-month-old samples. In the 11-month-old IVD samples, no difference was observed between AF and NP, but overall content of s-GAG was higher than cartilage. HPLC analysis of the ChABC-digested IVD tissue revealed that the quantity and ratio of s-GAG components differed from NP to AF tissue. Moreover, a change in sulfation pattern was observed with the maturity of the disk ( Fig. 1 ). Indeed, an inversion of the occurrence of Ddi-4S and Ddi-6S disaccharides was observed. A significant increase of Ddi-4S disaccharides with a significant decrease of Ddi-6S disaccharides was noted in the 11-month-old IVD AF tissue compared to 3 month old. A similar trend, albeit not significant, was observed for the NP tissue. Cartilage tissue did not present a significant difference in percentage of sulfated disaccharides with maturity. [Figure: see text] Conclusion GAG sulfation pattern was shown to change with maturation. Sulfation is critical for maintaining water content and specific patterns of sulfation are influential in cellular signaling and differentiation events. Furthermore, high changes in glycosylation profiles at a cellular and extracellular level were observed. A better understanding of these phenomena (sulfation and glycosylation profiles changes) will allow us to design therapeutic strategies for IVD regeneration. Acknowledgements AO Foundation (S-09-7P), Science Foundation Ireland, Research Frontiers Programme (07/RFP/ENMF482), Science Foundation Ireland, 07/SRC/B1163. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared Fritz JM, et al., Physical therapy for ccute low back pain: Associations with subsequent healthcare costs. Spine 2008;33(16):1800–1805 O'Halloran D, et al. Tissue-engineering approach to regenerating the intervertebral disk. Tissue Engineering 2007;13(8):1927–1954

Cell Surface Glycosylation and Glycosaminoglycan Composition Profiles in Immature and Mature Intervertebral Disks

IntroductionBack pain is a predominant cause of disability, and the major cause of this disability is intervertebral disk (IVD) degeneration.1 Proteoglycan (PG) content of the IVD is decreased during degeneration, and glycosaminoglycan (GAG) composition is altered.2 In this study, the cell surface glycosylation and GAG composition profiles of immature and mature ovine IVD tissue were profiled in an effort to understand potential signals involved in cell-extracellular matrix cross-talk during maturation and degeneration.Materials and MethodsIVD tissue from L3-L4 and L4-L5 lumbar segments were harvested from 3- and 11-month-old animals. One portion of the tissue was fixed for histochemical profiling with a panel of fluorescently-labeled lectins (Maackia amurensis agglutinin, Sambucus nigra agglutinin I, Ulex europaeus agglutinin I, Concanavalin A (jack bean lectin), Wisteria floribunda agglutinin). The remaining portion was digested with proteinase K and the sulfated GAG (s-GAG) content was quantified by dimethylmethylene blue (DMMB) assay. The digested tissue was treated with chondroitinase ABC (ChABC) and analyzed by HPLC.ResultsThe glycosylation expression level differed between annulus fibrosus (AF) and nucleus pulposus (NP) tissue as indicated by quantitative analysis using lectin histochemistry. All lectins stained cells and extracellular matrix (ECM) of the IVD independently of the tissue type. The cells demonstrated greater lectin binding compared to the ECM and the intensity of staining differed according to the tissue-type. With maturity, the expression profiles of cell surface glycosylation changed, notably for the n a-(2?6)-linked sialic acid (SNA-I) and terminal GalNAc (WFA staining) expression with a 10-fold and 2-fold increase of expression at 11 months old, respectively.DMMB assay showed that NP tissue contained threefold more s-GAGs than AF and cartilage in 3-month-old samples. In the 11-month-old IVD samples, no difference was observed between AF and NP, but overall content of s-GAG was higher than cartilage. HPLC analysis of the ChABC-digested IVD tissue revealed that the quantity and ratio of s-GAG components differed from NP to AF tissue. Moreover, a change in sulfation pattern was observed with the maturity of the disk (Fig. 1). Indeed, an inversion of the occurrence of Ddi-4S and Ddi-6S disaccharides was observed. A significant increase of Ddi-4S disaccharides with a significant decrease of Ddi-6S disaccharides was noted in the 11-month-old IVD AF tissue compared to 3 month old. A similar trend, albeit not significant, was observed for the NP tissue. Cartilage tissue did not present a significant difference in percentage of sulfated disaccharides with maturity.DownloadOpen in new tabDownload in PowerPointFigure 1 Percentage of nonsulfated and sulfated chondroitin sulfate in ovine nucleus pulposus (A), annulus fibrosus (B), and cartilage (C) at 3- and 11-month-old. Data were represented relative to the total sGAG and as mean ± standard error of the mean. *, ** represent significant differences at p < 0.05 and p < 0.01, respectively.ConclusionGAG sulfation pattern was shown to change with maturation. Sulfation is critical for maintaining water content and specific patterns of sulfation are influential in cellular signaling and differentiation events. Furthermore, high changes in glycosylation profiles at a cellular and extracellular level were observed. A better understanding of these phenomena (sulfation and glycosylation profiles changes) will allow us to design therapeutic strategies for IVD regeneration.Acknowledgements AO Foundation (S-09-7P), Science Foundation Ireland, Research Frontiers Programme (07/RFP/ENMF482), Science Foundation Ireland, 07/SRC/B1163.I confirm having declared any potential conflict of interest for all authors listed on this abstractYesDisclosure of InterestNone declaredFritz JM, et al., Physical therapy for ccute low back pain: Associations with subsequent healthcare costs. Spine 2008;33(16):1800–1805O'Halloran D, et al. Tissue-engineering approach to regenerating the intervertebral disk. Tissue Engineering 2007;13(8):1927–1954

Associated expression of α2,3sialylated type 2 chain structures with lymph node metastasis in distal colorectal cancer

One of the typical modifications on the surface of cancer cells is sialylation of terminal carbohydrates. The expression of several types of sialylation of glycoconjugates was investigated in colorectal cancer. The cancer tissue specimens obtained from 65 colorectal cancer patients were stained with sialic acid-binding lectins from Maackia amurensis (MAM), Sambucus sieboldiana (SSA), Maackia amurensis agglutinin (MAA) and monoclonal antibodies, and compared with their clinicopathological features. Cancer tissue specimens from 44.6% of patients had positive staining with MAM, which recognized α2,3sialylated type 2 chain (NeuAcα2,3Galβ1,4GlcNAcβR) structures, but normal colorectal mucosa showed only weak staining with MAM was observed. More lymph node metastases and lymphatic invasion were seen in patients with positive staining with MAM (P < 0.01), while not with other lectins or antibodies that recognized sialylated glycoconjugates or sialyl Lewis-related antigens. The five-year survival rate of patients with MAM-positive staining was significantly lower than that with MAM-negative staining when including T0-1 cases, but there was no difference in cases with T2-4. There was no difference in the patients' survival rates when the tissues were stained with MAA, SSA or PNA lectins. α2,3Sialylated type 2 chain structures were predominantly expressed in colorectal tissues associated with the malignant transformation, in particular, with lymphatic spread of distal colorectal adenocarcinomas.

NEU1 Sialidase Expressed in Human Airway Epithelia Regulates Epidermal Growth Factor Receptor (EGFR) and MUC1 Protein Signaling

Epithelial cells (ECs) lining the airways provide a protective barrier between the external environment and the internal host milieu. These same airway epithelia express receptors that respond to danger signals and initiate repair programs. Because the sialylation state of a receptor can influence its function and is dictated in part by sialidase activity, we asked whether airway epithelia express catalytically active sialidase(s). Human primary small airway and A549 ECs expressed NEU1 sialidase at the mRNA and protein levels, and NEU1 accounted for >70% of EC sialidase activity. Blotting with Maackia amurensis and peanut agglutinin lectins established epidermal growth factor receptor (EGFR) and MUC1 as in vivo substrates for NEU1. NEU1 associated with EGFR and MUC1, and NEU1-EGFR association was regulated by EGF stimulation. NEU1 overexpression diminished EGF-stimulated EGFR Tyr-1068 autophosphorylation by up to 44% but enhanced MUC1-dependent Pseudomonas aeruginosa adhesion by 1.6-1.7-fold and flagellin-stimulated ERK1/2 activation by 1.7-1.9-fold. In contrast, NEU1 depletion increased EGFR activation (1.5-fold) and diminished MUC1-mediated bacterial adhesion (38-56%) and signaling (73%). These data indicate for the first time that human airway epithelia express catalytically active NEU1 sialidase that regulates EGFR- and MUC1-dependent signaling and bacterial adhesion. NEU1 catalytic activity may offer an additional level of regulation over the airway epithelial response to ligands, pathogens, and injurious stimuli.

Sialylation of the Fas Death Receptor by ST6Gal-I Provides Protection against Fas-mediated Apoptosis in Colon Carcinoma Cells

The glycosyltransferase, ST6Gal-I, adds sialic acid in an α2-6 linkage to the N-glycans of membrane and secreted glycoproteins. Up-regulation of ST6Gal-I occurs in many cancers, including colon carcinoma, and correlates with metastasis and poor prognosis. However, mechanisms by which ST6Gal-I facilitates tumor progression remain poorly understood due to limited knowledge of enzyme substrates. Herein we identify the death receptor, Fas (CD95), as an ST6Gal-I substrate, and show that α2-6 sialylation of Fas confers protection against Fas-mediated apoptosis. Intriguingly, differences in ST6Gal-I activity do not affect the function of DR4 or DR5 death receptors upon treatment with TRAIL, implicating a selective effect of ST6Gal-I on the Fas receptor. Using ST6Gal-I knockdown and forced overexpression colon carcinoma cell models, we find that α2-6 sialylation of Fas prevents apoptosis stimulated by FasL as well as the Fas-activating antibody, CH11, as evidenced by decreased activation of caspases 8 and 3. We also show that α2-6 sialylation of Fas does not alter the binding of CH11, but rather inhibits the capacity of Fas to induce apoptosis by blocking the association of FADD with Fas cytoplasmic tails, an event that initiates death-inducing signaling complex formation. Furthermore, α2-6 sialylation of Fas inhibits Fas internalization, which is required for apoptotic signaling. Although dysregulated Fas activity is a well known mechanism through which tumors evade apoptosis, the current study is the first to link Fas insensitivity to the actions of a specific sialyltransferase. This finding establishes a new paradigm by which death receptor function is impaired for the self-protection of tumors against apoptosis.

Alteration of the sialylation pattern and memory deficits by injection of Aβ(25–35) into the hippocampus of rats

Sialic acid in glycoconjugates participates in important cellular functions associated with normal development, growth, and communication. Therefore we evaluated the sialylation pattern and memory deficits caused by the injection of Aβ(25–35) into the hippocampus (Hp) of rats. The eight-arm maze spatial-learning and memory test indicated that the injection of Aβ(25–35) into subfield CA1 of the Hp impaired both learning and memory. The sialylation pattern was examined using sialic acid-specific lectins. Our results showed that Maackia amurensis agglutinin (MAA, specific for Neu5Acα2,3Gal) showed reactivity in the CA1 and dentate gyrus (DG) subfields of the Hp mainly in the group injected with vehicle, whereas Macrobrachium rosenbergii lectin (MRL, specific for Neu5,9,7Ac) and Sambucus nigra agglutinin (SNA, specific for Neu5Acα2,6Gal-GalNAc) had increased reactivity in the CA1 and DG subfields of the Hp in the Aβ(25–35)-injected group. The staining pattern of the antibody specific for polysialic acid (a linear homopolymer of α-2,8-linked sialic acid) increased in the CA1 and DG subfields of the Hp of the Aβ(25–35) group compared to the control group. Our results suggest that injection of Aβ(25–35) causes impairment in spatial memory and alters the sialylation pattern in response to compensatory reorganization and-or sprouting of dendrites and axons of the surviving neurons.

Sialic acid profiles in the respiratory tracts of selected species of raptors: evidence for potential binding sites for human and avian influenza A viruses

Context The ability of influenza A viruses to recognise and bind to cell surface receptors such as sialic acid linked to galactose by an α2,3 linkage (SAα2,3-gal) and sialic acid linked to galactose by an α2,6 linkage (SAα2,6-gal) is a major determinant of influenza A virus infection. Although the epidemiological surveys of influenza A virus infection in raptors suggest that some raptor species are susceptible to influenza A viruses under natural conditions, the sialic acid profiles in the respiratory and intestinal tracts of raptors are unknown. Aims To examine the sialic acid receptor profiles in the respiratory tracts of the selected raptor species and assess the potential susceptibility of raptors to avian and human influenza viruses and the role of raptors in the epidemiology and evolution of influenza A viruses. Methods The lectin immunohistochemistry staining method was used to examine the sialic acid profiles in the respiratory tracts of eight different species of raptors. Key results A strong staining with Maackia amurensis agglutinin (MAA), specific for sialic acid linked to galactose by an α2,3 linkage (SAα2,3-gal), was observed in the epithelial cells of the respiratory tract of Accipiter nisus and Falco tinnunculus. However, a positive staining for both MAA and Sambucus nigra agglutinin (SNA), specific for sialic acid linked to galactose by an α2,6 linkage (SAα2,6-gal), was detected in the epithelial cells of the upper respiratory tract of Accipiter gularis, Buteo buteo, Otus sunia, Bubo bubo and Asio otus, and in the epithelial cells of the alveoli of Buteo buteo, Falco peregrinus, Otus sunia and Bubo bubo. Conclusions Both avian and human influenza A virus receptors are expressed in six species of raptors examined. There are some variations in the type and distribution of sialic acid receptor expression among different raptor species. No correlation between phylogeny of birds and their sialic acid receptor distributions was observed. Implications Since SAα2,3-gal and SAα2,6-gal are often considered as the primary receptors for avian influenza A viruses and human influenza A viruses, respectively, our data suggest that raptors could be a potential host for avian and human influenza A viruses.

Alterations of glycan branching and differential expression of sialic acid on alpha fetoprotein among hepatitis patients

The level of serum glycoproteins and their glycosylation pattern change in liver diseases including hepatocellular carcinoma (HCC). Some of them, especially alpha fetoprotein (AFP), serve as useful biomarkers for HCC. The present investigation showed high level of AFP in hepatitis B cirrhosis (HBV-LC) and hepatitis C cirrhosis (HCV-LC) patients. However, increase of AFP level was not significantly high in chronic hepatitis B (HBV-CH) as determined by ELISA using monoclonal anti-human AFP (mAb-AFP). The differential expression of sialic acid linkage was observed in HBV-CH and HCV-LC by ELISA; the former bound strongly with Sambucus nigra agglutinin (SNA), which has exclusive binding specificity for NeuAcα2-6-, whereas HCV-LC reacted preferably with Maackia amurensis agglutinin (MAA) which recognizes NeuAcα2-3-. There was significantly high glycan branching in HBV-LC and HCV-LC in comparison to controls as illustrated by concanavalin A. This was further confirmed by Phaseolus vulgaris erythroagglutinin (E-PHA) and Datura stramonium agglutinin (DSA). Enhanced fucosylation of AFP was observed in HBV-LC, HCV-LC and HCC patients by ELISA using fucose binding Aleuria aurantia lectin; however, maximum binding was found in HCC. Fucosylation with α1-6 linkage was further confirmed by Lens culinaris agglutinin (LCA). From the above results it is concluded that the changes in concentration of AFP, differential expression of sialic acid, increase of glycan branching and fucosylation have a prognostic value of hepatitis and it could be possible that lectin-based assay with AFP can aid in diagnosis of hepatitis diseases besides clinical examination and routine laboratory investigation.

Nanomonitor: A Miniature Electronic Biosensor for Glycan Biomarker Detection

The goal of our research is to develop an ultrasensitive diagnostic platform called 'NanoMonitor' to enable rapid label-free analysis of a highly promising class of biomarkers called glycans (oligosaccharide chains attached to proteins) with high sensitivity and selectivity. The glycosylation of fetuin - a serum protein - and extracts from a human pancreatic cancer line was analyzed to demonstrate the capabilities of the NanoMonitor. The NanoMonitor device consists of a silicon chip with an array of gold electrodes forming multiple sensor sites and works on the principle of electrochemical impedance spectroscopy. Each sensor site is overlaid with a nanoporous alumina membrane that forms a high density of nanowells on top of each electrode. Lectins (proteins that bind to and recognize specific glycan structures) are conjugated to the surface of the electrode. When specific glycans from a test sample bind to lectins at the base of each nanowell, a perturbation of electrical double-layer occurs, which results in a change in the impedance. Using the lectins Sambucs nigra agglutinin (SNA) and Maackia amurensis agglutinin (MAA), subtle variations to the glycan chains of fetuin were investigated. Protein extracts from BXPC-3, a cultured human pancreatic cancer cell line were also analyzed for binding to SNA and MAA lectins. The performance of the NanoMonitor was compared to a conventional laboratory technique: lectin-based enzyme linked immunosorbent assay (ELISA). The NanoMonitor was used to identify glycoform variants of fetuin and global differences in glycosylation of protein extracts from cultured human pancreatic cancerous versus normal cells. While results from NanoMonitor correlate very well with results from lectin-based ELISA, the NanoMonitor is rapid, completely label free, requires just 10 microl of sample, is approximately five orders of magnitude more sensitive and highly selective over a broad dynamic range of glycoprotein concentrations. Based on its performance metrics, the NanoMonitor has excellent potential for development as a point-of-care handheld electronic biosensor device for routine detection of glycan biomarkers from clinical samples.

Molecular characterization of pig α2,3-Gal-β1,3-GalNAc-α2,6-sialyltransferase (pST6GalNAc IV) gene specific for Neu5Acα2-3Galβ1-3GalNAc trisaccharide structure

Sialic acids of glycoconjugates play crucial roles in various biological processes, such as cell-cell communication and cell-substrate interaction. A sisalyltransferase, ST6GalNAc IV (Neu5Ac-alpha2,3-Gal-beta1,3-GalNAc-alpha2,6-sialyltransferase), catalyzes the formation of alpha2-6-linkages onto GalNAc residues of O-glycosidically linked Ser/Thr of proteins. In this study, we cloned the pig ST6GalNAc IV (pST6GalNAc IV) and investigated its functional characterization. pST6GalNAc IV cDNA has been isolated from pig liver tissues and it contains an entire open reading frame (ORF, 906 bp) coding for 302 amino acid residues. Entire ORF of pST6GalNAc IV containing sialylmotif 'L'-(Large), 'S'-(Small) and '-VS' (Very small) has a high degree of sequence similarity with Homo sapiens (90%), Pan troglodytes (91%) and Mus musculus (87%). Expression of pST6GalNAc IV mRNA in various pig tissues was identified by reverse transcription polymerase chain reaction (RT-PCR) analysis. pST6GalNAc IV mRNA was highly expressed in tongue, muscle and heart, whereas it was not expressed in pancreas. For functional characterization of pST6GalNAc IV gene in pig kidney PK15 cells, we have also established pST6GalNAc IV-transfected PK15 cells, which are stably expressing the pST6GalNAc IV gene. The glycosylation pattern of pST6GalNAc IV-transfected PK15 cells was detected by flow cytometry and immunofluorescence analysis with Maackia amurensis agglutinin (MAA), Maackia amurensis hemagglutinin (MAL II), Sambucus nigra agglutinin (SNA) and peanut agglutinin (PNA) lectins. The specific carbohydrate structures of Neu5Acalpha2-3Galbeta1-3(Neu5Acalpha2-6)GalNAc tetrasaccharide or Neu5Acalpha2-6GalNAc disaccharide recognized by MAL-II and SNA were revealed to be newly synthesized by pST6GalNAc IV. From the results, it was suggested that the pig pST6GalNAc IV gene is capable of synthesizing Neu5Acalpha2-3Galbeta1-3(Neu5Acalpha2-6)GalNAc tetrasaccharide structures on O-glycoproteins.

Terminal monosaccharide screening of synovial immunoglobulins G and A for the early detection of rheumatoid arthritis

The expressions of some terminal glycotopes of synovial immunoglobulins G, A, and M were analysed in relation to rheumatoid arthritis (RA) progression defined according to early and advanced radiological changes in patients' hands. The relative amounts of terminal monosaccharides were determined by lectin-immunoblotting of immunoglobulin preparations using appropriate lectins able to recognize alpha2,6-linked (Sambucus nigra agglutinin) and alpha2,3-linked (Maackia amurensis agglutinin) sialic acid, galactose (Ricinus communis agglutinin I), N-acetylglucosamine (Griffonia simplicifolia agglutinin II) as well as alpha1,6-linked (Aleuria aurantia lectin), alpha1,3-linked (Lotus tetragonolobus agglutinin), and alpha1,2-linked (Ulex europaeus agglutinin) fucose. The results indicate differences between early and advanced RA stages in the terminal sugar exposition of synovial IgG and IgA, but not IgM. The galactose-deficient glycotope with exposed N-acetylglucosamine of the synovial 33.1-kDa IgG fragment appeared exclusively in the early stage of RA. In contrast, this glycotope of intact synovial IgG and IgA was present in both groups, although with higher proportions in advanced RA. The proportions of the sialyl and fucosyl determinants of intact synovial A and G immunoglobulins were clearly lower in the early RA group than in the advanced. The analysis of terminal oligosaccharide exposition in IgG, IgG fragments, and IgA present in the synovial fluid of RA patients might be applicable as a stage-specific marker in the diagnosis and therapy of RA patients.