ABSTRACT
Alpha2,3-sialylation of the lactosamine type N-glycans with trans-sialidase from Trypanosoma cruzi is reported. Trans-sialidase (160 kDa, pI 5.35-5.65) and its catalytic fragment (70 kDa, pI 6.0-6.3) were isolated from T. cruzi cells and immobilized on ConA-Sepharose. The resulting preparation retained activity for several months and was repeatedly used for obtaining mono-, di-, tri-, and tetrasialylated 7-amino-4-metylcoumarine-labeled oligosaccharides with various numbers of antennas and for alpha2,3-sialylation of glycans within glycoproteins and neoglycoconjugates.
Subject(s)
Glycoproteins/chemistry , N-Acetylneuraminic Acid/chemistry , Neuraminidase/chemistry , Sepharose/analogs & derivatives , Trypanosoma cruzi/enzymology , Animals , Chromatography, Gel , Enzymes, Immobilized , Neuraminidase/isolation & purification , Polysaccharides/chemistry , Sepharose/chemistryABSTRACT
Surface-enhanced Raman scattering (SERS) spectroscopy was used to study the structure of carbohydrate chains in glycosylated forms of alpha 1-acid glycoprotein (AGP) and in pseudoglycoproteins obtained by transferring the carbohydrate chains of AGP to a polyacrylamide carrier. It was found that AGP-D glycoform and pseudoglycoproteins containing three or more glycans per molecule, which possess high immunomodulating activity, have a specific spatial organization of carbohydrate chains. This organization is maintained by the interaction of neighboring glycans with each other and does not depend on the nature of the carrier (whether it is polypeptide or polyacrylamide).
Subject(s)
Adjuvants, Immunologic/chemistry , Carbohydrates/chemistry , Orosomucoid/chemistry , Acrylic Resins/chemistry , Carbohydrate Sequence , Humans , Molecular Sequence Data , Peptides/chemistry , Protein Conformation , Spectrum Analysis, Raman/methods , Structure-Activity RelationshipABSTRACT
The interaction of human peripheral blood leukocytes with alpha 1-acid glycoprotein (AGP), its glycoforms as well as neoglyco-conjugates representing carbohydrate chains of AGP or its fragments was studied by flow cytometry. It was shown that the main target cells for AGP as well as for conjugates of its carbohydrate chains with polyacrylamide (PAA) are monocytes and polymorphonuclear leukocytes but not lymphocytes. The interaction of AGP with monocytes and granulocytes are mediated by its carbohydrate chains: the binding of AGP with cells was inhibited by AGP, AGP oligosaccharides as well as conjugates of oligosaccharides and its fragments with PAA. The data obtained show the existence of monocyte (and granulocyte) receptors which interact with complex type sialooligosaccharides of AGP.
Subject(s)
Carbohydrates/chemistry , Glycoconjugates/chemistry , Leukocytes/metabolism , Orosomucoid/metabolism , Carbohydrate Sequence , Humans , Molecular Sequence Data , Orosomucoid/chemistry , Protein Binding , Reference ValuesABSTRACT
The immunochemically pure alpha 1-acid glycoprotein (aAGP) from ascitic fluid of patients with stomach cancer was separated by chromatography on Con A-Sepharose into Con A-nonbound (aAGP-1, 43, 5 kDa, 70%) and Con A-bound (aAGP-2, 41.5 kDa, 24% and aAGP-3, 40.0 kDa, 5%) forms, differing in the monosaccharide composition. Comparative study of structures of their N-carbohydrate chains with the aid of the HPLC of fluorescence-labelled oligosaccharides showed that the molecular forms differ by the ratio of the di-, tri-, and tetraantennary carbohydrate N-chains of a complex type. The molecular forms of aAGP differ from nAGP by amounts of Le(x)-fragments and agalacto-oligosaccharides.
Subject(s)
Ascites/metabolism , Carbohydrates/chemistry , Orosomucoid/isolation & purification , Stomach Neoplasms/metabolism , Carbohydrate Conformation , Chromatography, High Pressure Liquid , Chromatography, Ion Exchange , Fluorescent Dyes , Humans , Orosomucoid/chemistry , Stomach Neoplasms/pathologyABSTRACT
Translocation of carbohydrate glycoprotein N-chains onto soluble polyacrylamide was proposed as a method for studying the biological role of carbohydrate chains. N-linked carbohydrate chains of alpha 1-acid glycoprotein (AGP) were aminated at the reducing GlcNAc moiety and covalently attached to polyacrylamide (PAA). Thus "pseudo-AGP" was obtained where peptide core was replaced with PAA. The synthetic model mimics AGP by M(r) and carbohydrate content as well as the ratio of tetra-, tri- and diantennary and mono-, di-, tri- and tetrasialo chains. It was shown that the conjugate inhibits proliferation of lymphocytes like the parent AGP. Therefore, the property of AGP to inhibit the lymphocyte proliferation is attributed to its carbohydrate chains, whereas peptide core serves as carrier providing polyvalent interaction of multiple carbohydrate chains with cell.
Subject(s)
Acrylic Resins/chemistry , Adjuvants, Immunologic/chemistry , Orosomucoid/chemistry , Adjuvants, Immunologic/pharmacology , Carbohydrate Sequence , Cell Division/drug effects , Humans , Lymphocytes/cytology , Lymphocytes/drug effects , Molecular Sequence Data , Orosomucoid/pharmacologyABSTRACT
alpha 1-Acid glycoprotein isolated from healthy individuals blood was separated on Con A-Sepharose into three fractions: non-bound (AGP-1, 84%, 43.5 kDa), Con A-bound (AGP-2, 14%, 41.3 kDa), and Con A-tightly bound (AGP-3, 2%, 39.6 kDa). Amino acid compositions of these fractions were similar but carbohydrate ones differed. HPLC analysis of 7-amino-4-methylcoumarin derivatives of the oligosaccharides in combination with their sequential exoglycosidase digestion showed that AGP-1, AGP-2, and AGP-3 have the same set of oligosaccharides and differ only by their proposition. A minor quantity of agalacto-oligosaccharides (with a terminal GlcNAc residue) was identified.
Subject(s)
Carbohydrates/chemistry , Orosomucoid/metabolism , Amino Acids/analysis , Fluorescent Dyes , Galactose/chemistry , Humans , Monosaccharides/analysisABSTRACT
4-(N-Methylcoumarin-7-yl) glycamines were employed in studying asparagine-linked carbohydrate chains of acid desialylated fetuin. The procedure was optimised for the reductive amination of oligosaccharides with 7-amino-4-methylcoumarin in the presence of Na(CN)BH3 to lead to oligosaccharide glycamines (AMC-OS). AMC-OS were obtained from dextran oligosaccharides and from oligosaccharides released by hydrazinolysis of asparagine-linked sugar chains of asialofetuin. Reverse-phase HPLC and exclusion HPLC with fluorimetric quantitation of AMC-OS is described. TSK Gel 2000 SW column was calibrated using dextran AMC-OS to give linear relationship ln Ni = k(ti/tr)+b, where ti/tr is retention time of the AMC-OS relatively to the reference AMC-trisaccharide, and Ni is calibration unit value, characterizing molecular size of AMC-OS. Three AMC-OS, Gal3GlcNAc3Man3GlcNAc2-AMC (I) and (II), and Gal2GlcNAc3Man3GlcNAc2AMC (III), were obtained from asialofetuin in a molar ration of 1:1.8:0.1. Acid treatment of AMK-OS (II) in desialylation conditions also gave AMC-OS (III), thus suggesting a partial degalactosylation of the glycoprotein sugar chains during the desialylation. Consequent digestion of AMC-OS (II) and (III) with Jack bean beta-galactosidase and beta-N-acetylhexosaminidase led to the same AMC-OS, Man3GlcNAc2AMC. The final digestion product of AMC-OS (I) was GalGlcNAcMan3GlcNAc2AMC, suggesting a structural difference in one of the antennas of the minor sugar chain of asialofetuin. The monosaccharide quantitation and exoglycosidase sequencing were carried out at a 100 pmole level.
Subject(s)
Asparagine/analysis , Coumarins , Fluorescent Dyes , Glycoproteins/analysis , Chromatography, High Pressure Liquid , Hydrolysis , Oligosaccharides/analysisABSTRACT
Antigenic preparations from human tumour (TMG) and normal mammary gland (NMG) tissues were isolated, using water extraction followed by precipitation of proteins with perchloric acid, which were further separated into fractions A, B and C by gel filtration on Sepharose 6B. Glycoprotein fractions B and C were further separated by IEC on DEAE cellulose to give fractions B1-BIV and C1-CIV. A set of glycoproteins (Mr = 13 000-122 000; pI approximately 3-7.5) were detected in TMC and NMG, the glycoproteins specific for TMG or NMG as well as common for both TMG and NMG being found. The major glycoproteins (Mr = 46 000), i. e. GP-1 from TMG not found in NMG and GP-2 and GP-3 from NMG not found in TMG were isolated and characterized both biochemically and serologically.
Subject(s)
Breast Neoplasms/analysis , Breast/analysis , Glycoproteins/analysis , Neoplasm Proteins/analysis , Antigens, Neoplasm/analysis , Chromatography, Gel , Glycoproteins/blood , Glycoproteins/isolation & purification , Humans , Isoelectric PointABSTRACT
beta-Galactosidase from Alternaria tenius was purified to homogeneity from the cultural fluid using acetone precipitation, ion-exchange chromatography on DEAE-cellulose, adsorption on hydroxylapatite and affinity chromatography on N-(beta-D-galactopyranosyl-thiocarbamoyl)-beta-aminocaproyl-AN-Sepharose 4B. The enzyme homogeneity was demonstrated by ultracentrifugation and polyacrylamide gel electrophoresis with SDS or without it. The specific activity of the homogeneous enzyme is 160 u. per mg of protein; mol. weight as determined by various methods is 142 000-176 000, pI = 4.6, temperature optimum is 60-65 degrees, pH optima for o-nitrophenyl-beta-D-galactopyranoside (o-NPG) and lactose are 3.8--4.4 and 3.6--4.8, respectively. The Km values for o-NPG and lactose are 0.21 . 10(-3) and 6.57 . 10-3 M, respectively. The enzyme is a glycoprotein and contains up to 30% of carbohydrates. EDTA and pCMB have no effect on the beta-galactosidase activity. Galactose acts as a competitive inhibitor, while glucose has no inhibiting effect on the enzyme activity.
