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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.

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.

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.

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.

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.