Quantitation of the oligosaccharides of human serum IgG from patients with rheumatoid arthritis: a critical evaluation of different methods.

Several different chromatographic methods and a lectin-based assay have been compared for the quantitation of oligosaccharides released from immunoglobulin G (IgG). The analysis of a series of IgG samples purified from the serum of rheumatoid arthritis patients was carried out by these methods to evaluate the percentage of the glycoforms having 0, 1 or 2 galactose residues (G0, G1 and G2) in order to (a) identify the method that can be most widely used for quantitation, (b) accurately define the range of G0 values found in patients with rheumatoid arthritis, and (c) make available a series of characterised standards for distribution to clinical chemistry laboratories. The chromatographic methods involved: release of oligosaccharides by glycoamidase A after protease digestion followed by HPLC analysis of aminopyridine derivatives on reverse phase and normal phase columns; hydrazinolysis treatment with exoglycosidases (G0 mix) and Biogel P4 chromatography of 2-aminobenzamide (2-AB) derivatives; hydrazinolysis and weak anion exchange or normal phase HPLC of 2-AB derivatives; release of oligosaccharides by PNGase F and either Biogel P4 chromatography of 2-AB derivatives or HPAEC-PAD analysis of native oligosaccharides. The G0 values given by these methods compared favourably with each other and a dot blot assay of denatured IgG interaction with Ricinus communis agglutinin and Bandeiraea simplicifolialectin II. The HPLC and HPAEC methods give additional information that may be important in less routine assays.

Chemical characterisation of glycosylinositolphospholipids of Herpetomonas samuelpessoai.

The structure of two glycosylinositolphospholipids of the cell surface of the monoxenic protozoan Herpetomonas samuelpessoai have been deduced by methylation analysis, fast-atom bombardment mass spectrometry and two dimensional nuclear magnetic resonance spectroscopy. These glycolipids have features in common with the glycoinositolphospholipids of both Leishmania and Trypanosoma cruzi, resembling the former by the presence of the hybrid type core sequence Man alpha 1-->3(Man alpha 1-->6)Man alpha 1-->4GlcN alpha 1-->6 myo-inositol-1-PO4-lipid, while the 2-aminoethylphosphonate substituent on 0-6 of glucosamine and the presence of ceramide in place of glycerol lipids is more reminiscent of T. cruzi. Possible phylogenetic implications of these observations are discussed.

1H NMR analysis of novel sialylated and fucosylated lactose-based oligosaccharides having linear GlcNAc(beta 1-6) Gal and Neu5Ac(alpha 2-6) GlcNAc sequences.

Three novel oligosaccharides of human infant faeces have been fully characterised by methylation analysis and 500/600 MHz 1H NMR spectroscopy including DQF-COSY, TQF-COSY, TOCSY and ROESY experiments. The oligosaccharides were shown to be lactose-based structures two of which were substituted at C-6 of Gal with either the Le(x) trisaccharide, Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc(beta 1-, or Neu5Ac(alpha 2-6)Gal(beta 1-4)GlcNAc(beta 1-. They differ from other free oligosaccharides previously isolated from the human by having the (1-->6) linkage to Gal in the absence of a (1-->3) branch. The third oligosaccharide has Neu5Ac(alpha 2-6) linked to GlcNAc of the trisaccharide GlcNAc(beta 1-3)Gal(beta 1-4)Glc. This is a linear fragment of the disialylated tetrasaccharide sequence Neu5Ac(alpha 2-3)Gal(beta 1-3)[Neu5Ac(alpha 2-6)]GlcNAc(beta 1-found in the milk oligosaccharide disialyl LNT (the GlcNAc residue of the tetrasaccharide linked to lactose) and also of N-linked chains (GlcNAc linked to Man).

The preparation of neoglycoconjugates containing inter-saccharide phosphodiester linkages as potential anti-Leishmania vaccines.

The Leishmania express complex glycoconjugates containing phosphosaccharide repeat units at all stages of their life-cycle. One of these molecules, lipophosphoglycan (LPG) has been suggested to be a vaccine candidate. To assess the immunological properties of Leishmania phosphosaccharides, we have prepared neoglycoproteins and neoglycolipids containing synthetic Leishmania phosphosaccharide repeats. The coupling procedure uses the dec-9-enyl spacer of previously synthesised phosphosaccharides for linkage to protein and phospholipid. This alkene moiety is converted by ozonolysis to an aldehyde which is then attached to protein and phospholipid amino groups by reductive amination. The procedure produces neoglycoconjugates in good yield and without compromising the labile phosphodiester linkages within the phosphosaccharide chains.

The glycosylation pattern of humanized IgGI antibody (D1.3) expressed in CHO cells.

A humanized IgG antibody (D1.3) which retains murine complementarity determining regions specific for the antigen lysozyme has been expressed in CHO-DUKX cells. Heavy and light chain containing plasmids were co-transfected into CHO-DUKX cells and stable clones were grown in DMEM/F12 medium supplemented with 5% foetal calf serum. D1.3 antibody was purified from culture supernatants by Protein G chromatography. With the recombinant D1.3 antibody as a model, this cell culture system was shown to glycosylate the IgG Fc region in a similar manner to IgG isolated from serum. The neutral, core fucosylated biantennary oligosaccharides found are present in serum IgG and no novel carbohydrate sequences were detected. The degree of terminal agalactosylation was also similar to normal serum, in contrast to the increased levels found in rheumatoid serum. Furthermore, those oligosaccharides which lack only one terminal Gal are exclusively galactosylated on the GlcNAc(beta1,2) Man(alpha1,6) Man(beta1,4) antenna. Unambiguous identification of the exact glycosylation pattern of the antibody was carried out by a combination of specific exoglycosidase digestions, gel permeation chromatography of 2-aminobenzamide derivatives, high pH anion exchange chromatography and methylation analysis followed by gas-liquid chromatography-mass spectrometry.

Characterization of the elongating alpha-D-mannosyl phosphate transferase from three species of Leishmania using synthetic acceptor substrate analogues.

Leishmania express lipophosphoglycans and proteophosphoglycans that contain Galbeta1-4Manalpha1-P phosphosaccharide repeat structures assembled by the sequential addition of Manalpha1-P and betaGal. The synthetic acceptor substrate Galbeta1-4Manalpha1-P-decenyl and a series of analogues were used to probe Leishmania alpha-D-mannosyl phosphate transferase activity. We show that the activity detected with Galbeta1-4Manalpha1-P-decenyl is the elongating alpha-D-mannosyl phosphate transferase associated with lipophosphoglycan biosynthesis (eMPT(LPG)). Differences in the apparent K(m) values for the donor and acceptor substrates were found using L. major, L. mexicana, and L. donovani promastigote membranes, but total activity correlated with the number of lipophosphoglycan repeats. Further comparisons showed that lesion-derived L. mexicana amastigotes, that do not express lipophosphoglycan, lack eMPT(LPG) and that nondividing L. major metacyclic promastigotes contain 5-fold less eMPT(LPG) activity than dividing procyclic promastigotes. The fine specificity of promastigote eMPT(LPG) activity was determined using 24 synthetic analogues of Galbeta1-4Manalpha1-P-decenyl. The three species gave similar results: the negative charge of the phosphodiester and the C-6 hydroxyl of the alphaMan residue are essential for substrate recognition, the latter most likely acting as a hydrogen bond acceptor. The C-6' hydroxyl of the betaGal residue is required for substrate recognition as well as for catalysis. The rate of Manalpha1-P transfer declines with increasing acceptor substrate chain length. The presence of a monosaccharide substituent at the C-3 position of the terminal betaGal residue abrogates Man-P transfer, showing that chain elongation must precede side chain modification during lipophosphoglycan biosynthesis. In contrast, substitution of the penultimate phosphosaccharide repeat does not abrogate transfer but is slightly stimulatory in L. mexicana and inhibitory in L. major.