CBP70, a glycosylated nuclear lectin

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

Structural studies on IgG oligosaccharides of patients with primary Sjögren's syndrome

Sjögren's syndrome (SS) is an autoimmune disease, and some patients have been found to have SS complicated with rheumatoid arthritis (RA), in which IgG is known to carry abnormal N-linked oligosaccharides. In order to investigate the relationship between SS and RA, the structures of N-linked oligosaccharides of IgG from 12 primary SS patients without RA, 9 RA patients, and 8 healthy individuals were analyzed using reversed-phase high-performance liquid chromatography, in combination with sequential exoglycosidase treatment and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. All of the IgG samples obtained from primary SS patients, RA patients, and healthy individuals contained the same series of biantennary complex-type oligosaccharides, but the ratio of each oligosaccharide differed among these 3 groups. The incidence of galactose-lacking N-linked oligosaccharides obtained from the IgG of RA patients was significantly higher than that from healthy individuals, but that from the serum IgG of primary SS patients varied among individuals. The patients with primary SS were classified into two groups based on the galactosylation levels of IgG oligosaccharides; one group exhibits galactosylation levels as low as those of RA patients and another exhibits levels similar to those of healthy individuals. Measurement of levels of rheumatoid factor (RF) revealed that primary SS patients with a high incidence of RF belonged to the low galactosylation group, as did RA patients. These results suggest that appearance of IgG carrying abnormal N-linked oligosaccharides in primary SS may be related to future complication with RA.     

Carbohydrate binding specificity of immobilized Psathyrella velutina lectin.

The carbohydrate binding specificity of Psathyrella velutina lectin (PVL) was thoroughly investigated by analyzing the behavior of various complex-type oligosaccharides and human milk oligosaccharides on a PVL-Affi-Gel 10 column. Basically, the lectin interacts with the nonreducing terminal beta-N-acetylglucosamine residue, but does not show any affinity for the nonreducing terminal N-acetylgalactosamine or N-acetylneuraminic acid residue. Substitution of the terminal N-acetylglucosamine residues of oligosaccharides by galactose completely abolishes their affinity to the column. GlcNAc beta 1----3Gal beta 1----4sorbitol binds to the column, but GlcNAc beta 1----6Gal beta 1----4sorbitol is only retarded in the column. The behavior of degalactosylated N-linked oligosaccharides is quite interesting. Although all degalactosylated monoantennary sugar chain isomers are retarded in the column, those with the GlcNAc beta 1----2Man group interact more strongly with the column than those with the GlcNAc beta 1----4Man group or the GlcNAc beta 1----6Man group. The degalactosylated bi- and triantennary sugar chains bind to the column, but the tetraantennary ones are only retarded in the column. These results indicated that the binding affinity is not simply determined by the number of terminal N-acetylglucosamine residues. Addition of the bisecting N-acetylglucosamine residue reduces the affinity of oligosaccharides to the column, but addition of an alpha-fucosyl residue at the C-6 position of the proximal N-acetylglucosamine residue does not affect the behavior of oligosaccharides in the column. These results indicated that the binding specificity of PVL is quite different from those of other N-acetylglucosamine-binding lectins from higher plants, which interact preferentially with the GlcNAc beta 1----4 residue.     

G2/M cell cycle arrest by an N-acetyl-D-glucosamine specific lectin from Psathyrella asperospora

A new N-acetyl-D-glucosamine (GlcNAc) specific lectin was identified and purified from the fruiting body of the Australian indigenous mushroom Psathyrella asperospora. The functional lectin, named PAL, showed hemagglutination activity against neuraminidase treated rabbit and human blood types A, B and O, and exhibited high binding specificity towards GlcNAc, as well as mucin and fetuin, but not against asialofetuin. PAL purified to homogeneity by a combination of ammonium sulfate precipitation, chitin affinity chromatography and size exclusion chromatography, was monomeric with a molecular mass of 41.8 kDa, was stable at temperatures up to 55 °C and between pH 6–10, and did not require divalent cations for optimal activity. De novo sequencing of PAL using LC-MS/MS, identified 10 tryptic peptides that revealed substantial sequence similarity to the GlcNAc recognizing lectins from Psathyrella velutina (PVL) and Agrocybe aegerita (AAL-II) in both the carbohydrate binding and calcium binding sites. Significantly, PAL was also found to exert a potent anti-proliferative effect on HT29 cells (IC₅₀ 0.48 μM) that was approximately 3-fold greater than that observed on VERO cells; a difference found to be due to the differential expression of cell surface GlcNAc on HT29 and VERO cells. Further characterization of this activity using propidium iodine staining revealed that PAL induced cell cycle arrest at G₂/M phase in a manner dependent on its ability to bind GlcNAc.     

Single molecular dynamic interactions between glycophorin A and lectin as probed by atomic force microscopy

Glycophorin A (GpA) is one of the most abundant transmembrane proteins in human erythrocytes and its interaction with lectins has been studied as model systems for erythrocyte related biological processes. We performed a force measurement study using the force mode of atomic force microscopy (AFM) to investigate the single molecular level biophysical mechanisms involved in GpA-lectin interactions. GpA was mounted on a mica surface or natively presented on the erythrocyte membrane and probed with an AFM tip coated with the monomeric but multivalent Psathyrella velutina lectin (PVL) through covalent crosslinkers. A dynamic force spectroscopy study revealed similar interaction properties in both cases, with the unbinding force centering around 60 pN with a weak loading rate dependence. Hence we identified the presence of one energy barrier in the unbinding process. Force profile analysis showed that more than 70% of GpAs are free of cytoskeletal associations in agreement with previous reports.     

Purification and properties of an N-acetylglucosamine-specific lectin from Psathyrella velutina mushroom

A lectin in the fruiting bodies of Psathyrella velutina was purified by affinity chromatography on a chitin column and subsequent ion-exchange chromatography. P. velutina lectin (PVL) tends to aggregate irreversibly in buffered saline, but the addition of glycerol (10%, v/v) to lectin solutions was found to prevent aggregate formation. PVL is assumed to occur as a monomer of a polypeptide of Mr = 40,000 as determined by gel filtration and by gel electrophoresis in the presence of sodium dodecyl sulfate. PVL is specific for N-acetylglucosamine (GlcNAc). It was determined by equilibrium dialysis to have four binding sites/polypeptide molecule showing an average intrinsic association constant of K0 = 6.4 x 10(3) M-1 toward this sugar. The binding specificity of the lectin was studied by hemagglutination inhibition assays and by avidin-biotin-mediated enzyme immunoassays using various GlcNAc-containing saccharides. The results indicate that methyl N-acetyl beta-glucosaminide was a slightly better inhibitor than the corresponding alpha-anomer. PVL binds well to oligosaccharides bearing nonreducing terminal beta-GlcNAc linked 1----6 or 1----3 but poorly to those having a 1----4 linkage, such as N-acetylated chito-oligosaccharides. It also binds to the subterminal GlcNAc moiety when it is substituted at the C-6 position but does not interact with the moiety when substituted either at C-3 or C-4. Thus, these results show that PVL is quite different in its binding specificity from other GlcNAc-binding lectins of higher plants since they bind preferentially to beta-GlcNAc in 1----4 linkage and they have a high affinity for chitin oligosaccharides.     

A Recombinant Fungal Lectin for Labeling Truncated Glycans on Human Cancer Cells

Cell surface glycoconjugates present alterations of their structures in chronic diseases and distinct oligosaccharide epitopes have been associated with cancer. Among them, truncated glycans present terminal non-reducing β-N-acetylglucosamine (GlcNAc) residues that are rare on healthy tissues. Lectins from unconventional sources such as fungi or algi provide novel markers that bind specifically to such epitopes, but their availability may be challenging. A GlcNAc-binding lectin from the fruiting body of the fungus Psathyrella velutina (PVL) has been produced in good yield in bacterial culture. A strong specificity for terminal GlcNAc residues was evidenced by glycan array. Affinity values obtained by microcalorimetry and surface plasmon resonance demonstrated a micromolar affinity for GlcNAcβ1-3Gal epitopes and for biantennary N-glycans with GlcNAcβ1-2Man capped branches. Crystal structure of PVL complexed with GlcNAcβ1-3Gal established the structural basis of the specificity. Labeling of several types of cancer cells and use of inhibitors of glycan metabolism indicated that rPVL binds to terminal GlcNAc but also to sialic acid (Neu5Ac). Analysis of glycosyltransferase expression confirmed the higher amount of GlcNAc present on cancer cells. rPVL binding is specific to cancer tissue and weak or no labeling is observed for healthy ones, except for stomach glands that present unique αGlcNAc-presenting mucins. In lung, breast and colon carcinomas, a clear delineation could be observed between cancer regions and surrounding healthy tissues. PVL is therefore a useful tool for labeling agalacto-glycans in cancer or other diseases.     

Galactosylation of IgG from rheumatoid arthritis (RA) patients – changes during therapy

It is well documented that serum IgG from rheumatoid arthritis (RA) patients exhibits decreased galactosylation of its conservative N-glycans (Asn-297) in CH2 domains of the heavy chains; it has been shown that this agalactosylation is proportional to disease severity. In the present investigation we analyzed galactosylation of IgG derived from the patients using a modified ELISA-plate test, biosensor BIAcore and total sugar analysis (GC-MS). For ELISA and BIAcore the binding of IgG preparations, purified from the patients’ sera, to two lectins: Ricinus communis (RCA-I) and Griffonia simplicifolia (GSL-II) was applied. Based on ELISA-plate test an agalactosylation factor (AF, a relative ratio of GSL-II/RCA-I binding) was calculated, which was proportional to actual disease severity. Repeated testing of several patients before and after treatment with methotrexate (MTX) alone or in combination with Remicade (a chimeric antibody anti-TNF-α) supplied results indicating an increase of IgG galactosylation during the treatment. This introductory observation suggests that IgG galactosylation may be an additional indicator of the RA patients’ improvement.     

Fine sugar specificity of the mistletoe (Viscum album) lectin I

The behaviour ofN-acetyllactosamine-type oligosaccharides and glycopeptides on a column of mistletoe lectin I (MLI) immobilized on Sepharose 4B was examined. The immobilized lectin does not show any affinity for asialo-N-glycosylpeptides and related oligosaccharides, which possess one to four unmaskedN-acetyllactosamine sequences. However, substitution of at least one of theN-acetyllactosamine sequences by sialic acid residues, either at O-3 or O-6 of galactose, slightly enhances the affinity of the lectin. Such sialylatedN-glycosylpeptides or oligosaccharides are eluted from the lectin column by the starting buffer as retarded fractions. Surprisingly, the affinity of the immobilized MLI is higher for P1 antigen-containing glycopeptide isolated from turtle-dove ovomucoid and for glycopeptides from bovine thyroglobulin containing terminal non-reducing Gal1–3Gal sequences. These structures are strongly bound on the lectin column and their elution is obtained with 0.15M galactose in the starting buffer.In memory of Hartmut Franz.     

Fine sugar specificity of theButea frondosa seed lectin

Various monosaccharides and oligosaccharides were used to define the specificity of theButea frondosa lectin using the hapten inhibition technique of human erythrocyte agglutination. AlthoughB. frondosa lectin exhibited higher affinity forN-acetylgalactosamine, lactose andN-acetyllactosamine appeared to be relatively good inhibitors of haemagglutination. The behaviour ofN-acetyllactosamine-type oligosaccharides and glycopeptides on a column ofB. frondosa lectin immobilized on Sepharose 4B showed that the sugar-binding specificity of the lectin is directed towards unmaskedN-acetyllactosamine sequences. Substitution of theseN-acetyllactosamine sequences by sialic acid residues completely abolished the affinity of the lectin for the saccharides. The presence of one or several Fuc(1-3)GlcNAc groups completely inhibited the interaction between the glycopeptides and the lectin. Substitution of the core -mannose residue by an additional bisecting (1-4)GlcNAc residue decreases the affinity of the lectin for these structures as compared with the unsubstituted ones.     

耐盐绒毛白蜡特异性SCAR分子标记的鉴定

该研究以耐盐型和盐敏感型绒毛白蜡及其F1代为材料,采用混合品系分析法进行RAPD分析。结果显示:在随机选取的150个10碱基随机引物中,仅有引物S20在耐盐基因池和盐敏感基因池间扩增出特异而可重复的592bp的多态性片段,命名为S20-592。获得的RAPD标记S20-592经克隆、测序、重新设计一对特异性引物转化成更稳定的SCAR标记。通过F1代个体验证,耐盐型个体均能扩增出此差异条带而盐敏感型个体中不能扩增出此差异条带,证明该SCAR标记的特异引物可用于耐盐绒毛白蜡物种的快速分子鉴定。     

S-Nitrosylation of mannose binding lectin regulates its functional activities and the formation of autoantibody in rheumatoid arthritis

The possibility of post-translational modifications of mannose binding lectin (MBL) leading to functional impairment of the MBL pathway and the presence of anti-MBL autoantibodies were reported earlier in rheumatoid arthritis (RA). MBL was observed to be S-nitrosylated (S-nitrosated) in vitro. HepG2 cells were stimulated with 10% synovial fluid from RA patients to produce increased levels of MBL and nitric oxide. Under these experimental conditions MBL was observed to be S-nitrosated using biotin switch assay. The plasma of RA patients was also found to contain higher levels of S-nitrosylated MBL (SNO-MBL) in comparison to the healthy controls. Functional activities of SNO-MBL were compared with normal MBL. Mannan binding and C4 deposition ability of MBL was found to decrease after S-nitrosylation. It was also observed that S-nitrosylation of MBL leads to a decrease in the bacterial phagocytosis and apoptotic cell binding as measured by fluorescence microscopy and FACS analysis. These results indicate that the carbohydrate binding ability of MBL was affected by S-nitrosylation (S-nitrosation). High levels of anti-MBL autoantibodies were detected against SNO-MBL in plasma of RA patients in comparison to normal MBL suggesting a role of SNO-MBL in generation of autoantibodies in RA patients.     

Isolation and partial characterization of a lectin from ground elder (Aegopodium podagraria) rhizomes

A lectin has been isolated from rhizomes of ground elder (Aegopodium podagraria) using a combination of affinity chromatography on erythrocyte membrane proteins immobilized on cross-linked agarose and hydroxyapatite, and ion-exchange chromatography. The molecular structure of the lectin was determined by gelfiltration, sucrose density-gradient centrifugation and gel electrophoresis under denaturing conditions. It has an unusually high M_r (about 480000) and is most probably an octamer composed of two distinct types of subunits with slightly different M_r (about 60000). Hapten inhibition assays indicated that the Aegopodium lectin is preferentially inhibited by N-acetylgalactosamine. Nevertheless, it does not agglutinate preferentially blood-group-A erythrocytes. The ground-elder lectin is a typical non-seed lectin, which occurs virtually exclusively in the underground rhizomes. In this organ it is an abundant protein as it represents up to 5% of the total protein content. The lectin content of the rhizome tissue varies strongly according to its particular location along the organ. In addition, the lectin content changes dramatically as a function of the seasons. The ground-elder lectin differs from all other plant lectins by its unusually high molecular weight. In addition, it is the first lectin to be isolated from a species of the family Apiaceae.Abbreviations APA Aegopodium podagraria agglutinin - PBS phosphate-buffered saline - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Detection of disease-specific augmentation of abnormal immunoglobulin G in sera of patients with rheumatoid arthritis

Galactose-free immunoglobulin G (IgG), which is known to be higher in the sera of patients with rheumatoid arthritis, was prepared from IgG of healthy volunteers using enzymes. Its reactivity to lectins was analyzed. The galactose-free IgG showed no reactivity to Ricinus communis agglutinin 120 but displayed greater reactivity to concanavalin A and Lens culinaris lectin than did intact human IgG. Then, IgG in serum samples was bound to protein A immobilized on a nitrocellulose membrane, and its reactivity to biotinylated concanavalin A was measured with streptavidin-conjugated horseradish peroxidase. When the reactivity to concanavalin A of IgG in sera from healthy individuals and patients with rheumatoid arthritis (RA), osteoarthritis, systemic lupus erythematosus, or hepatic disease was compared, higher levels were shown in patients with RA, notably in 60% of the seronegative patients and 80% of the early phase patients. Therefore, it was suggested that augmentation of the abnormal IgG in sera was highly specific to patients with RA and that this novel serum test could be very useful for an accurate diagnosis of this disease.     

The isolation of a rat alveolar macrophage lectin

A lectin in rat alveolar macrophage membranes with a high affinity for binding ligands containing L-fucose and N-acetyl-D-glucosamine has been isolated by affinity chromatography on Fuc-BSA-Sepharose (where Fuc is fucosyl and BSA is bovine serum albumin). The lectin was extracted from rat lung homogenates with Triton X-100, absorbed from the extract onto Fuc-BSA-Sepharose in the presence of Ca2+ and eluted by removal of Ca2+. After a second adsorption to and elution from Fuc-BSA-Sepharose, three protein species were detected electrophoretically in fractions that bind Fuc-BSA. One, which was the mannose/N-acetylglucosamine lectin (Mr = 32,000) found earlier in hepatocytes, was removed by adsorption on anti-lectin IgG-Sepharose. Another (Mr = 46,000) was removed by adsorption to Fuc-BSA-Sepharose and elution with galactose. The remaining lectin (Mr = 180,000) bound fucose and N-acetylglucosamine but not galactose. Binding was maximal between pH 6.5 and 9.0 and dependent on Ca2+. Immunocytological analysis with rabbit anti-lectin IgG and fluorescein-labeled goat anti-rabbit IgG revealed the lectin to be in rat alveolar macrophages and nonparenchymal cells of liver. Thus, the lectin appears to be present in macrophages and is likely involved in receptor-mediated endocytosis. It is distinctly different structurally from the hepatocyte lectin with a similar ligand-binding specificity.     

Biophysical characterization and structural determination of the potent cytotoxic Psathyrella asperospora lectin

A lectin with strong cytotoxic effect on human colon cancer HT29 and monkey kidney VERO cells was recently identified from the Australian indigenous mushroom Psathyrella asperospora and named PAL. We herein present its biochemical and structural analysis using a multidisciplinary approach. Glycan arrays revealed binding preference towards N‐acetylglucosamine (GlcNAc) and, to a lesser extent, towards sialic acid (Neu5Ac). Submicromolar and millimolar affinity was measured by surface plasmon resonance for GlcNAc and NeuAc, respectively. The structure of PAL was resolved by X‐ray crystallography, elucidating both the protein's amino acid sequence as well as the molecular basis rationalizing its binding specificity. Proteins 2017; 85:969–975. © 2016 Wiley Periodicals, Inc.     

Investigation into the Concanavalin A reactivity, fucosylation and oligosaccharide microheterogeneity of α1-acid glycoprotein expressed in the sera of patients with rheumatoid arthritis

α₁-Acid glycoprotein (AGP) exists as an heterogeneous population of glycosylated variants (glycoforms) in plasma. The concentration of AGP increases some 2–5 fold in certain pathophysiological states exemplified by the chronic inflammatory disease, rheumatoid arthritis (RA). Moreover, the expressed glycosylation pattern alters in such conditions, indicating functional significance that is likely to be related to the oligosaccharide heterogeneity. We have investigated the heterogeneity of AGP glycosylation using the technique of high pH anion-exchange chromatography (HPAEC). AGP was isolated from the blood of RA sufferers, partially separated by Concanavalin A (Con A) affinity chromatography into bound and non-bound fractions and was enzymatically deglycosylated. Chromatography on the pellicular HPAE resin at pH 13 separated the released oligosaccharides and allowed a comparison of profiles in terms of branching and fucosylation. Results demonstrate an abnormal RA AGP glycosylation, with a tendency towards tri- and tetra-antennary oligosaccharides and enhanced fucosylation, in addition to the possible existence of penta-sialylated RA AGP glycoforms.     

A lectin from <Emphasis Type="Italic">Sesbania aculeata</Emphasis> (<Emphasis Type="Italic">Dhaincha</Emphasis>) roots and its possible function

A lectin was isolated from the roots of Sesbania aculeata. This is a glucose specific lectin having 39 kDa subunit molecular weight. The expression of this lectin was found to be developmentally regulated and observed to be the highest in the second week. The lectin was purified by affinity chromatography using Sephadex G-50 and found to have 28% homology with Arabidopsis thaliana lectin-like protein (accession No. CAA62665). The lectin binds with lipopolysaccharide isolated from different rhizobial strains indicating the plants interaction with multiple rhizobial species. Published in Russian in Biokhimiya, 2009, Vol. 74, No. 3, pp. 404–411.     

Purification and Characterization of a Novel β-<Emphasis Type="SmallCaps">d</Emphasis>-Galactosides-Specific Lectin from <Emphasis Type="Italic">Clitoria ternatea</Emphasis>

A lectin present in seeds of Clitoria ternatea agglutinated trypsin-treated human B erythrocytes. The sugar specificity assay indicated that lectin belongs to Gal/Gal NAc-specific group. Hence the lectin, designated C. ternatea agglutinin (CTA), was purified by the combination of acetic acid precipitation, salt fractionation and affinity chromatography. HPLC gel filtration, SDS-polyacrylamide gel electrophoresis and mass spectrometry indicated that the native lectin is composed of two identical subunits of molecular weight 34.7 kDa associated by non covalent bonds. The N-terminal sequence of CTA shared homology with Glycine max and Pisum sativum. Complete sequence was also found to be homologous to S-64 protein of Glycine max, suggesting that CTA probably exhibits both hemagglutination and probably sugar uptake activity. The carbohydrate binding specificity of the lectin was investigated by quantitative turbidity measurements, and percent inhibition assays. Based on these assays, we conclude that CTA binds β-d-galactosides, and also may has an extended specificity towards non-reducing terminal Neu5Acα2,6Gal.