Lectin Affinity High-performance Liquid Chromatography Research Articles

Salivary Lactoferrin Is Recognized by the Human Herpesvirus-8

Human herpesvirus-8 (HHV-8) is commonly detected in all epidemiologic forms of Kaposi's sarcoma. Despite the broad cellular tropism of HHV-8, studies on mucosal shedding of HHV-8 have shown that infectious particles are restricted to saliva isolated from the oropharynx. We used biotinylated purified HHV-8 particles in a direct binding assay to whole clarified human salivary samples isolated from HHV-8-infected and uninfected individuals. We found that the major binding activity was carried out by a protein of 78-kDa size, which was further characterized as human lactoferrin (hLf) using 2-D electrophoresis and MALDI-ToF analysis. Preliminary comparison of HHV-8 binding activity of 76 salivary samples from HHV-8-infected and uninfected individuals showed that 7.8% of the uninfected population exhibited a form of Lf not recognized by HHV-8. Deglycosylation of hLf by PNGase F did not reduce HHV-8 binding activity, whereas endoproteinase cleavage of native hLf generated a non-glycosylated 8-kDa peptide recognized by HHV-8 particles and was located at the position Ala606-Tyr679 in the native hLf amino acid sequence, corresponding to the C-terminal region of the glycoprotein. This work identify the lactoferrin in saliva as a ligand for HHV-8 and suggests that this glycoprotein could be used as a carrier for the viral particles.

Comparative Study of Glycosyltransferase Activities in Caco-2 Cells before and after Enterocytic Differentiation Using Lectin-Affinity High-Performance Liquid Chromatography

Human colonic adenocarcinoma Caco-2 cells differentiate into enterocytes by induction with sodium butyrate after confluence. Our previous studies have shown that there are high levels of H type 1 blood group antigen and core 2 structure present in O-glycans of the glycoproteins from these differentiated cells and these O-glycans appear to be indispensable for the process of differentiation of the cells (J. Amano and M. Oshima, 1999, J. Biol. Chem. 274, 21209–21216). Here, we have determined the glycosyltransferase activities using lectin-affinity HPLC because the method enabled easy separation and identification of mixtures of isomeric oligosaccharide structures due to the high resolution and reproducibility. The activities of β3-galactosyltransferase, α2-fucosyltransferase, which are responsible for H type 1 antigen biosynthesis, and core 2 β6-N-acetylglucosaminyltransferase in differentiated Caco-2 cells were higher than those in undifferentiated cells. These results demonstrate that an increase in specific glycosyltransferase activities brought on a change of the O-glycan structures during differentiation.

1206 The role of neurotrophin for the neurite extension and cell migration of cerebellar granule cells in vitro

We have developed a lectin affinity high-performance liquid chromatography technique for analysis of oligosaccharides using columns of silica-bound lectins. Purified leukoagglutinating phytohemagglutinin (L-PHA), concanavalin A (Con A), Datura stramonium agglutinin (DSA), and Vicia villosa agglutinin (VVA) were covalently coupled to periodate-oxidized diol-silica by reductive amination. Homogeneous oligosaccharides of known structure, purified following release from Asn with N-glycanase and reduction with NaBH4, were tested for their ability to interact with the silica-bound lectins. The characteristic elution position obtained for each oligosaccharide was reproducible and correlated with specific structural features. The oligosaccharide specificities displayed by silica-bound L-PHA, Con A, and DSA were virtually identical to those established utilizing lectin-agarose conjugates. Analysis of oligosaccharides by lectin affinity HPLC allowed further definition of the specificity of VVA for N-glycanase-released, reduced oligosaccharides. Lectin affinity HPLC is rapid and convenient, providing an important structure-specific dimension to oligosaccharide analysis. This technique is particularly useful when utilized in conjunction with anion-exchange and ion-suppression amine adsorption HPLC methods, which fractionate on the basis of charge and size, respectively. In addition to their utility for oligosaccharide characterization, these affinity columns demonstrate the high degree of oligosaccharide specificity displayed by plant and animal lectins.

14] High-performance liquid chromatography of oligosaccharides

Publisher Summary This chapter proposes separation methods primarily for oligosaccharides that are radiolabeled by metabolic incorporation of 3H- or 14C-labeled sugar, metabolic incorporation of 35SO4 or 32PO4, or chemical incorporation of 3H at the reducing terminus by reduction with NaB3H4. The most common strategy is to first separate the radiolabeled oligosaccharides into different groups on the basis of their anionic character, using anion-exchange high-performance liquid chromatography (HPLC). Oligosaccharides differing in the number and/or character of their anionic groups are then further fractionated on the basis of size, using ion suppression-amine adsorption (ISAA) HPLC. The effects of chemical modifications and enzymatic digestions on individual species are subsequently monitored by anion-exchange HPLC and ISAA–HPLC along with other methods such as lectin affinity HPLC. Four methods for separation of anionic oligosaccharides are illustrated in the chapter. Fractionation of oligosaccharides bearing zero to four anionic moieties, using MicroPak AX-10 or AX-5 columns and a phosphate buffer (pH 4.0), is effective for oligosaccharides bearing terminal sialic acid. Oligosaccharides-bearing sulfate or phosphate can be fractionated under the same conditions; however, the concentration of the initial eluant is reduced to 2.5 mM in order to retain monosulfated species.

Characterization of oligosaccharides by lectin affinity high-performance liquid chromatography

Alterations of the oligosaccharide structures of glycoproteins are associated with differentiation, malignant transformation, and expression of the same protein in different cell types. The potential biological importance of oligosaccharides has resulted in a growing need for detailed structural information. When glycoproteins are available in limited quantities and/or bear highly heterogeneous oligosaccharides, characterization of their oligosaccharides is difficult. We have developed an efficient approach for obtaining detailed information about oligosaccharides by determining structural ‘fingerprints’ using lectin affinity high-performance liquid chromatography.

Rapid assay of β‐galactosidase and sialyltransferase by lectin affinity high performance liquid chromatography with fluorescence detection

Fluorescein isothiocyanate (FITC)-labelled asialotransferrin and pyridyl aminated oligosaccharides were prepared from asialotransferrin and human milk using affinity chromatography and high performance liquid chromatography (HPLC), respectively. These substances were incubated with galactosidase or sialyltransferase and then examined by lectin affinity HPLC. The elution patterns changed according to the period of incubation and amount of enzyme. This analytical method using lectin affinity HPLC with fluorescence labelled glycoprotein or oligosaccharides as the substrates has great value for detecting these enzyme under the same chromatographic conditions. In addition, differences were noted in the activity of beta-galactosidase toward oligosaccharides having the Gal beta(1----3)GlcNAc or Gal beta(1----4)GlcNAc structure at reducing termini.

The asparagine-linked oligosaccharides on bovine fetuin. Structural analysis of N-glycanase-released oligosaccharides by 500-megahertz 1H NMR spectroscopy.

The structures of the entire population of sialylated asparagine-linked oligosaccharides present on bovine fetuin were elucidated. Asparagine-linked oligosaccharides were released from fetuin with N-glycanase, radiolabeled by reduction with NaB[3H]4, and fractionated by anion-exchange high performance liquid chromatography (HPLC), ion-suppression amine adsorption HPLC, and concanavalin A affinity chromatography. The 3H-labeled oligosaccharide fractions obtained were analyzed by 500-MHz 1H nuclear magnetic resonance spectroscopy, revealing the presence of 23 distinct oligosaccharide structures. These oligosaccharides differed in extent of sialylation (3% mono-, 35% di-, 54% tri-, and 8% tetrasialylated), number of peripheral branches (17% di- and 83% tribranched), linkage (alpha 2,3 versus alpha 2,6) and location of sialic acid moieties, and linkage (beta 1,4 versus beta 1,3) of galactose residues. This represents the first time that the asparagine-linked oligosaccharides of fetuin have been successfully fractionated and characterized as sialylated species. The sialylated oligosaccharides derived from fetuin were also used to further define the specificities of the lectins leukoagglutinating phytohemagglutinin and Ricinus communis agglutinin I. The behavior of these oligosaccharides during lectin affinity HPLC further establishes the structural features which predominate in the interaction of oligosaccharides with leukoagglutinating phytohemagglutinin and R. communis agglutinin I.

Lectin affinity high-performance liquid chromatography: Interactions of N-glycanase-released oligosaccharides with leukoagglutinating phytohemagglutinin, concanavalin A, Datura stramonium agglutinin, and Vicia villosa agglutinin

We have developed a lectin affinity high-performance liquid chromatography technique for analysis of oligosaccharides using columns of silica-bound lectins. Purified leukoagglutinating phytohemagglutinin (L-PHA), concanavalin A (Con A), Datura stramonium agglutinin (DSA), and Vicia villosa agglutinin (VVA) were covalently coupled to periodate-oxidized diol-silica by reductive amination. Homogeneous oligosaccharides of known structure, purified following release from Asn with N-glycanase and reduction with NaBH 4, were tested for their ability to interact with the silica-bound lectins. The characteristic elution position obtained for each oligosaccharide was reproducible and correlated with specific structural features. The oligosaccharide specificities displayed by silica-bound L-PHA, Con A, and DSA were virtually identical to those established utilizing lectin-agarose conjugates. Analysis of oligosaccharides by lectin affinity HPLC allowed further definition of the specificity of VVA for N-glycanase-released, reduced oligosaccharides. Lectin affinity HPLC is rapid and convenient, providing an important structure-specific dimension to oligosaccharide analysis. This technique is particularly useful when utilized in conjunction with anion-exchange and ion-suppression amine adsorption HPLC methods, which fractionate on the basis of charge and size, respectively. In addition to their utility for oligosaccharide characterization, these affinity columns demonstrate the high degree of oligosaccharide specificity displayed by plant and animal lectins.

Lectin affinity high-performance liquid chromatography. Interactions of N-glycanase-released oligosaccharides with Ricinus communis agglutinin I and Ricinus communis agglutinin II.

The structural determinants required for interaction of oligosaccharides with Ricinus communis agglutinin I (RCAI) and Ricinus communis agglutinin II (RCAII) have been studied by lectin affinity high-performance liquid chromatography (HPLC). Homogeneous oligosaccharides of known structure, purified following release from Asn with N-glycanase and reduction with NaBH4, were tested for their ability to interact with columns of silica-bound RCAI and RCAII. The characteristic elution position obtained for each oligosaccharide was reproducible and correlated with specific structural features. RCAI binds oligosaccharides bearing terminal beta 1,4-linked Gal but not those containing terminal beta 1,4-linked GalNAc. In contrast, RCAII binds structures with either terminal beta 1,4-linked Gal or beta 1,4-linked GalNAc. Both lectins display a greater affinity for structures with terminal beta 1,4-rather than beta 1,3-linked Gal, although RCAII interacts more strongly than RCAI with oligosaccharides containing terminal beta 1,3-linked Gal. Whereas terminal alpha 2,6-linked sialic acid partially inhibits oligosaccharide-RCAI interaction, terminal alpha 2,3-linked sialic acid abolishes interaction with the lectin. In contrast, alpha 2,3- and alpha 2,6-linked sialic acid equally inhibit but do not abolish oligosaccharide interaction with RCAII. RCAI and RCAII discriminate between N-acetyllactosamine-type branches arising from different core Man residues of dibranched complex-type oligosaccharides; RCAI has a preference for the branch attached to the alpha 1,3-linked core Man and RCAII has a preference for the branch attached to the alpha 1,6-linked core Man. RCAII but not RCAI interacts with certain di- and tribranched oligosaccharides devoid of either Gal or GalNAc but bearing terminal GlcNAc, indicating an important role for GlcNAc in RCAII interaction. These findings suggest that N-acetyllactosamine is the primary feature required for oligosaccharide recognition by both RCAI and RCAII but that lectin interaction is strongly modulated by other structural features. Thus, the oligosaccharide specificities of RCAI and RCAII are distinct, depending on many different structural features including terminal sugar moieties, peripheral branching pattern, and sugar linkages.

Assay of glycosidase by lectin affinity high-performance liquid chromatography

Human transferrin was incubated with sialidase and β-galactosidase and then examined by lectin affinity high-performance liquid chromatography (HPLC)

Lectin affinity high-performance liquid chromatography columns for the resolution of nucleotide sugars

Lectin affinity high-performance liquid chromatography columns for the resolution of nucleotide sugars

Rapid resolution of nucleotide sugars by lectin affinity high-performance liquid chromatography

Rapid resolution of nucleotide sugars by lectin affinity high-performance liquid chromatography