Purification and partial characterization of a novel lectin from elder (Sambucus nigra L.) fruit

A previously unknown haemagglutinin, named Sambucus nigra agglutinin-III (SNA-III), has been purified from the fruit of the elder (Sambucus nigra). Whereas elder bark agglutinin I (SNA-I) is highly specific for terminal alpha 2,6-linked sialic acid residues, SNA-III displays a high affinity for oligosaccharides containing exposed N-acetylgalactosamine and galactose residues. Different N-terminal sequences and the amino acid composition distinguish the fruit lectin from elder bark agglutinin II (SNA-II), which shows a similar carbohydrate specificity. The 40-fold higher affinity of SNA-III for asialofetuin than for human asialo-alpha 1-acid glycoprotein and human asialotransferrin respectively suggests a preference for O-linked glycans. SNA-III occurs mainly as a monomeric glycoprotein, but tends to form di- and oligo-meric aggregates. This aggregation seems to mediate the multivalent interaction, leading to agglutination. SDS/PAGE revealed two major polypeptides with apparent molecular masses of 32 and 33 kDa respectively. This heterogeneity is probably a result of proteolysis in the C-terminal region. Binding to concanavalin A and susceptibility to peptide: N-glycosidase F indicated the presence of N-glycosidically linked oligosaccharides.

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Carbohydrate characterization of purified thyroxine-binding globulin by lectin blot and isoelectric focusing

Jugoslovenska medicinska biohemija ◽

10.2298/jmh0304319p ◽

2003 ◽

Vol 22 (4) ◽

pp. 319-323 ◽

Cited By ~ 1

Author(s):

Ivana Petrovic ◽

Svetlana Savin-Zegarac ◽

Ivona Baricevic ◽

Dubravka Cvejic

Keyword(s):

Sialic Acid ◽

Isoelectric Focusing ◽

Blot Analysis ◽

Wheat Germ ◽

Sambucus Nigra ◽

Thyroxine Binding Globulin ◽

Sambucus Nigra Agglutinin ◽

Lectin Blot ◽

Wheat Germ Lectin

The structure of carbohydrate moiety of purified thyroxine-binding globulin (TBG) was examined by lectin blot and isoelectric focusing (IEF). In lectin blot, TBG reacted positively with the following lectins: Sambucus nigra agglutinin (SNA I), Ricinus commuais agglutinin (RCA I), wheat germ lectin (WGA), phytoheamagglutinin (PHA) and pea lectin (PSA). The obtained results indicate that purified TBG contains N-linked oligosaccharide chains consisting of mannose, galactose, N-acetylglucosamine and sialic acid. Isoelectric focusing of TBG at pl 4.2-4.6 revealed three bands, which confirmed that isolated TBG had retained its structure with?out desialylation. Lectin blot analysis and IEF can be considered to be useful tools in the study of TBG glycosylation.

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Effects of Sialic Acid Substitutions on Recognition by Sambucus nigra Agglutinin and Maackia amurensis Hemagglutinin

Analytical Biochemistry ◽

10.1006/abio.2001.5539 ◽

2002 ◽

Vol 303 (1) ◽

pp. 98-104 ◽

Cited By ~ 40

Author(s):

Els C.M. Brinkman-Van der Linden ◽

Justin L. Sonnenburg ◽

Ajit Varki

Keyword(s):

Sialic Acid ◽

Sambucus Nigra ◽

Maackia Amurensis ◽

Sambucus Nigra Agglutinin

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α2,6-Linked sialic acid acts as a receptor for Feline calicivirus

Journal of General Virology ◽

10.1099/vir.0.82158-0 ◽

2007 ◽

Vol 88 (1) ◽

pp. 177-186 ◽

Cited By ~ 74

Author(s):

Amanda D. Stuart ◽

T. David K. Brown

Keyword(s):

Sialic Acid ◽

Adhesion Molecule ◽

Sodium Periodate ◽

Metabolic Inhibitors ◽

Feline Calicivirus ◽

Sambucus Nigra ◽

Virus Binding ◽

Maackia Amurensis ◽

The Family

Feline calicivirus (FCV) is a major causative agent of respiratory disease in cats. It is also one of the few cultivatable members of the family Caliciviridae. It has recently been reported that FCV binding is in part due to interaction with junction adhesion molecule-A. This report describes the characterization of additional receptor components for FCV. Chemical treatment of cells with sodium periodate showed that FCV recognized carbohydrate moieties on the surface of permissive cells. Enzymic treatment with Vibrio cholerae neuraminidase demonstrated that sialic acid was a major determinant of virus binding. Further characterization using linkage-specific lectins from Maackia amurensis and Sambucus nigra revealed that FCV recognized sialic acid with an α2,6 linkage. Using various proteases and metabolic inhibitors, it was shown that α2,6-linked sialic acid recognized by FCV is present on an N-linked glycoprotein.

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Isolation and characterization of a glycoprotein from human colostrum

Biochemical Journal ◽

10.1042/bj1350875 ◽

1973 ◽

Vol 135 (4) ◽

pp. 875-880 ◽

Cited By ~ 9

Author(s):

James H. Nichols ◽

Anatoly Bezkorovainy

Keyword(s):

Molecular Weight ◽

Sialic Acid ◽

Blood Group ◽

Group Activity ◽

Acid Glycoprotein ◽

Isolation And Characterization ◽

Human Colostrum ◽

No Absorption

A glycoprotein was isolated from the M-1 acid glycoprotein fraction of human colostrum. It had a molecular weight of 31200 and contained 27% galactose, 21.7% hexosamine, 8.0% fucose and 10.8% sialic acid by weight. The glycoprotein had no absorption maxima in the 240–300nm region, and was virtually free of ABH(O) and M and N blood-group activity. Alkaline borohydride cleavage of the glycoprotein resulted predominantly in the destruction of threonine and galactosamine.

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Isolation and characterization of high affinity and highly stable anti-Chikungunya virus antibodies using ALTHEA Gold Libraries™

10.21203/rs.3.rs-34990/v1 ◽

2020 ◽

Author(s):

Martha Pedraza-Escalona ◽

Omar Guzmán-Bringas ◽

Ivan Arrieta-Oliva ◽

Keyla Gómez-Castellano ◽

Juana Salinas-Trujano ◽

...

Keyword(s):

Chikungunya Virus ◽

Dynamic Range ◽

Sandwich Elisa ◽

Diagnostic Methods ◽

Diagnostic Tools ◽

Chikv Infection ◽

High Affinity ◽

Isolation And Characterization ◽

Sds Page

Abstract Background More than three million infections were attributed to Chikungunya virus (CHIKV) in the 2014–2016 outbreak in Mexico, Central and South America, with over 500 deaths directly or indirectly related to this viral disease. CHIKV outbreaks are recurrent and no vaccine nor approved therapeutics exist to prevent or treat CHIKV infection. Reliable and robust diagnostic methods are thus critical to control future CHIKV outbreaks. Direct CHIKV detection in serum samples via highly specific and high affinity anti-CHIKV antibodies has shown to be an early and effective clinical diagnosis. Methods Chikungunya virions isolated from serum of a patient in Veracruz, México, were purified and characterized via electron microscopy, SDS-PAGE and binding to diverse well-characterized anti-CHIKV monoclonal antibodies. UV-inactivated CHIKV particles were used as selector in a solid-phase panning coupled with ELISA-based screening and Next-Generation Sequencing to discover specific and high affinity anti-CHIKV antibodies from ALTHEA Gold Libraries™. Results The CHIKV isolate showed the typical morphology of the virus. Protein bands in the SDS-PAGE were consistent with the size of its capsid proteins. UV-inactivated CHIKV particles bound tightly the control antibodies. The lead antibodies here obtained showed high expression yield, monomeric content over 95% after a single-step Protein A purification, and importantly, a thermal stability above 75oC. Most of the antibodies recognized linear epitopes on E2, including the highest affinity antibody called C7. A sandwich ELISA implemented with C7 and a potent neutralizing antibody isolated elsewhere, also specific for E2 but recognizing a discontinuous epitope, showed a dynamic range of 0.2–40.0 µγ/mL of UV-inactivated CHIKV purified preparation. The number of CHIKV particles estimated based on the concentration of E2 in the extract suggested that the assay could detect clinically meaningful amounts of CHIKV in serum. Conclusions The newly discovered antibodies offer valuable tools for characterization of CHIKV isolates and development of robust diagnostic tools for CHIKV infection surveillance. Application of ALTHEA Gold Libraries™ in combination with viral particles other than CHIKV could expedite the discovery and development of antibodies for detection and control of emergent and quickly spreading viral outbreaks such as SARS-CoV-2 (COVID-19).

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Partial characterization of the heteropolysaccharide associated with the 7S domain of type IV collagen from placenta

Biochemistry and Cell Biology ◽

10.1139/o87-064 ◽

1987 ◽

Vol 65 (5) ◽

pp. 501-506 ◽

Cited By ~ 6

Author(s):

James R. A. Leushner

Keyword(s):

Sialic Acid ◽

Amino Acid ◽

Chemical Analysis ◽

Amino Acid Analysis ◽

Molecular Sieve ◽

Type Iv Collagen ◽

Relative Mass ◽

Type Iv ◽

Structure Formula

A major heteropolysaccharide fraction was isolated from the 7S domain of human placental type IV collagen. Analyses revealed that it was an asparagine-linked oligosaccharide. Characterization using molecular sieve chromatography, exoglycosidase and endoglycosidase digestion, and chemical analysis suggested a bianternnary complex with the following structure:[Formula: see text]A microheterogeneity was noted with respect to the addition of the fucose and sialic acid residues. Analysis of component polypeptides of the 7S fraction following endoglycosidase treatment suggested that the most obvious site of heteropolysaccharide attachment was in a polypeptide of relative mass 40 000. Amino acid analysis of this isolated polypeptide indicated that it was rich in collagenous sequences and also contained half-cystine residues.

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Purification and characterization of three extracellular (1→3)-β-d-glucan glucohydrolases from the filamentous fungus Acremonium persicinum

Biochemical Journal ◽

10.1042/bj3080733 ◽

1995 ◽

Vol 308 (3) ◽

pp. 733-741 ◽

Cited By ~ 25

Author(s):

S M Pitson ◽

R J Seviour ◽

B M McDougall ◽

J R Woodward ◽

B A Stone

Keyword(s):

Filamentous Fungus ◽

Gel Filtration ◽

High Specificity ◽

Major Product ◽

Ph Optimum ◽

Sds Page ◽

Molecular Masses ◽

Monomeric Proteins ◽

Ph Range

Three (1-->3)-beta-D-glucanases (GNs) were isolated from the culture filtrates of the filamentous fungus Acremonium persicinum and purified by (NH4)2SO4 precipitation followed by anion-exchange and gel-filtration chromatography. Homogeneity of the purified proteins was confirmed by SDS/PAGE, isoelectric focusing and N-terminal amino acid sequencing. All three GNs (GN I, II and III) are non-glycosylated, monomeric proteins with apparent molecular masses, estimated by SDS/PAGE, of 81, 85 and 89 kDa respectively. pI values for the three enzymes are 5.3, 5.1, and 4.4 respectively. The pH optimum for GN I is 6.5, and 5.0 for GN II and III. All three purified enzymes displayed stability over the pH range 4.5-10.0. Optimum activities for GN I, II and III were recorded at 65, 55 and 60 degrees C respectively, with both GN II and III having short-term stability up to 50 degrees C and GN I up to 55 degrees C. The purified GNs have high specificity for (1-->3)-beta-linkages and hydrolysed a range of (1-->3)-beta- and (1-->3)(1-->6)-beta-D-glucans, with laminarin from Laminaria digitata being the most rapidly hydrolysed substrate of those tested. K(m) values for GN I, II, and III against L. digitata laminarin were 0.1, 0.23 and 0.22 mg/ml respectively. D-Glucono-1,5-lactone does not inhibit any of the three GNs, some metals ions are mild inhibitors, and N-bromosuccinimide and KMnO4 are strong inhibitors. All three GNs acted in an exo-hydrolytic manner, determined by the release of alpha-glucose as the initial and major product of hydrolysis of (1-->3)-beta-D-glucans, and confirmed by viscometric analysis and the inability to cleave periodate-oxidized laminarin, and may be classified as (1-->3)-beta-D-glucan glucohydrolases (EC 3.2.1.58).

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Characterization of MAX.3 antigen, a glycoprotein expressed on mature macrophages, dendritic cells and blood platelets: identity with CD84

Biochemical Journal ◽

10.1042/bj3460729 ◽

2000 ◽

Vol 346 (3) ◽

pp. 729-736 ◽

Cited By ~ 8

Author(s):

Stefan W. KRAUSE ◽

Michael REHLI ◽

Sven HEINZ ◽

Reinhard EBNER ◽

Reinhard ANDREESEN

Keyword(s):

Dendritic Cells ◽

Blood Platelets ◽

Cell Types ◽

Variable Expression ◽

Surface Molecules ◽

Sds Page ◽

Molecular Masses ◽

Antigen A

MAX.3 is a monoclonal antibody that preferentially reacts with mature macrophages (MAC), monocyte-derived dendritic cells, megakaryocytes and platelets. In this study, we describe the characterization, purification and identification of the MAX.3 antigen. Immunoprecipitation and SDS/PAGE revealed different molecular masses of MAX.3 antigen in MAC (60-90 kDa) and platelets (58-64 kDa), whereas a similar size (45 kDa) was observed in both cell types after digestion with N-glycosidase F. Lectin affinity and sequential treatment with different glycosidases suggests complex type glycosylation of MAX.3 antigen in MAC and hybrid type glycosylation in platelets. Amino acid sequencing led to the identification of a corresponding cDNA clone and showed its identity to the sequence of the CD84 antigen, a member of the CD2 family of cell surface molecules. MAX.3/CD84 was further studied by immunohistochemistry and a variable expression was found on tissue MAC, confirming this antigen to be mainly a marker for MAC in situ.

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Biochemical characterization of tektins from sperm flagellar doublet microtubules

The Journal of Cell Biology ◽

10.1083/jcb.104.4.1069 ◽

1987 ◽

Vol 104 (4) ◽

pp. 1069-1075 ◽

Cited By ~ 31

Author(s):

RW Linck ◽

RE Stephens

Keyword(s):

Amino Acid ◽

Biochemical Characterization ◽

Peptide Mapping ◽

Amino Acid Content ◽

Gradient System ◽

Intermediate Filament Proteins ◽

Sds Page ◽

Helical Content ◽

Protein Components

Tektins, protein components of stable protofilaments from sea urchin sperm flagellar outer doublet microtubules (Linck, R. W., and G. L. Langevin, 1982, J. Cell Sci., 58:1-22), are separable by preparative SDS PAGE into 47-, 51-, and 55-kD equimolar components. High resolution two-dimensional tryptic peptide mapping reveals 63-67% coincidence among peptides of the 51-kD tektin chain and its 47- and 55-kD counterparts, greater than 70% coincidence between the 47- and 55-kD tektins, but little obvious similarity to either alpha- or beta-tubulin. With reverse-phase HPLC on a C18 column, using 6 M guanidine-HCl solubilization and a 0.1% trifluoroacetic acid/CH3CN gradient system (Stephens, R. E., 1984, J. Cell Biol. 90:37a [Abstr.]), the relatively less hydrophobic 51-kD tektin elutes at greater than 45% CH3CN, immediately followed by the 55-kD chain. The 47-kD tektin is substantially more hydrophobic, eluting between the two tubulins. The amino acid compositions of the tektins are very similar to each other but totally distinct from tubulin chains, being characterized by a greater than 50% higher arginine plus lysine content (in good agreement with the number of tryptic peptides) and about half the content of glycine, histidine, proline, and tyrosine. The proline content correlates well with the fact that tektin filaments have twice as much alpha-helical content as tubulin. Total hydrophobic amino acid content correlates with HPLC elution times for the tektins but not tubulins. The average amino acid composition of the tektins indicates that they resemble intermediate filament proteins, as originally postulated from structural, solubility, and electrophoretic properties. Tektins have higher cysteine and tryptophan contents than desmin and vimentin, which characteristically have only one residue of each, more closely resembling certain keratins in these amino acids.

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