Jasna Peter-Katalinic

Heterogeneity of bovine lactotransferrin glycans. Characterization of α-D-Galp-(1→3)-β-D-Gal- and α-NeuAc-(2→6)-β-D-GalpNAc-(1→4)-β-D-GlcNAc-substituted N-linked glycans

Abstract Lactotransferrin isolated from a pool of mature bovine milk has been shown to contain N-glycosidically-linked glycans possessing N-acetylneuraminic acid, galactose, mannose, fucose, N-acetylglucosamine and N-acetylgalactosamine. The glycopeptides obtained by Pronase digestion were fractionated by concanavalin A-Sepharose affinity chromatography into three fractions: slightly retained (A), retained (B), and strongly retained (C). The structure of the glycans of the three fractions has been determined by application of methanolysis, methylation analysis, fast atom bombardment-mass spectrometry, and 1H NMR spectroscopy. Diantennary structures without GalNAc were present as partially sialylated and partially (1 → 6)-α- l -fucosylated structures in Fractions A and B. Sequences containing α- d -Galp-(1 → 3)-β- d -Gal on the α- d -Man-(1 → 6) antenna, and β- d -GalpNAc-(1 → 4)-β- d -GlcNAc and α-NeuAc-(2 → 6)-β- d -GalpNAc-(1 → 4)-β- d -GlcNAc on the α- d -Man-(1 → 3) antenna were characterized in the oligosaccharide-alditols obtained by reductive cleavage of Fraction B. A series of Man4 − 9-GlcNAc structures were identified in Fraction C after endo-N-acetyl-β- d -glucosaminidase digestion. These results show that the structures of bovine lactotransferrin glycans are more heterogeneous than those of previously characterized transferrin glycans.

Analysis of gangliosides using fast atom bombardment mass spectrometry

The native gangliosides GM3, GM1, Fuc-GM1, GD1a, GD1b, Fuc-GD1b, GT1b and GQ1b were analysed by fast atom bombardment mass spectrometry (FAB-MS) in the negative ion mode in a matrix of thioglycerol. After permethylation the same gangliosides were analysed by electron impact (EI) and FAB-MS in the positive ion mode. The negative ion mass spectra furnished information on the molecular weight, the ceramide moiety and the sequence of carbohydrate residues. The sites of attachment and the number of sialic acids present could be deduced directly from the pattern of sequence ions. After addition of sodium acetate positive ion FAB-spectra of the permethylated samples show intense pseudomolecular ions M + Na, that provide evidence on the homogeneity of the samples. In addition, the ceramide part, the oligosaccharide moiety obtained after cleavage of the glycosidic bond of the hexosamine residue, the whole carbohydrate chain and the sialic acids are represented by specific fragment ions. With EI-MS further information can be obtained on the sphingosine and fatty acid components of the ceramide residue. The data show, that the combination of soft ionization mass spectrometry with classical EI-MS gives valuable information on the structure and homogeneity of gangliosides. The method is also applicable to the structural elucidation or quantitation of more complex gangliosides or glycolipid mixtures using only micrograms of material.

Structures of acidic O-linked polylactosaminoglycans on human skim milk mucins.

O-Linked glycans were isolated from human skim milk mucins or mucin-derived high-molecular weight glycopeptides and fractionated by anion exchange chromatography into neutral and acidic alditols. Major oligosaccharides contained in the acidic fraction were purified by high performance liquid chromatography and structurally characterized by a combination of fast atom bombardment mass spectrometry, methylation analysis and 500 MHz1H-nuclear magnetic resonance spectroscopy. The structural aspects exhibited by these major species in the acidic fraction resemble those established previously for the neutral oligosaccharides from human skim milk mucins:1)the size of the alditols varies from tri- to decasaccharides2)the core structure is of the ubiquitous type 23)the backbone sequences are of the poly-N-acetyllactosamine type with a particular preponderance of linearly extended GlcNAcβ(1–3)Gal (major) or GlcNAcβ(1–6)Gal units (minor).N-Acetylneuraminic acid on monosialylated (mucin- or glycopeptide-derived) and disialylated glycans (glycopeptide derived) is linked predominantly to position C-3 of galactose.

Structural studies on oncofetal carbohydrate antigens (Ca 19-9, Ca 50, and Ca 125) carried by O-linked sialyl-oligosaccharides on human amniotic mucins☆

Mucins were extracted from human amniotic fluid in the presence of 45% vol. phenol and separated from the bulk of smaller-sized glycoproteins by exclusion on Sephacryl S400. The mucin-fraction FW, which still contained a minute proportion of mannose, strongly expressed oncofetal antigens recognized by monoclonal antibodies C 50, NS 19-9, OC 125, Leu M1, 49 H 8, and 115 C 2. The structures of the respective mucin-linked saccharides responsible for Ca 50-, Ca 19-9-, and Lea-related antigenic activities were analyzed before or after reductive beta-elimination from sialylglycoproteins, and purification of the derived alditols by gel-permeation chromatography on Bio-Gel P-4 or high performance liquid chromatography. Two ubiquitous (FW2, FW3) and three novel oligosaccharide alditols (FW5) were characterized by f.a.b.- and e.i.-m.s., combined with methylation analysis and chromium trioxide oxidation. The OC 125 epitope on mucin-carried O-glycans was destroyed during reductive cleavage of the saccharides, indicating a conformational involvement of the reducing terminal residue and its mode of conjugation to the protein. Exoglycosidase treatment of the mucin-bound antigen revealed that the epitope structure of OC 125 includes terminal beta-D-galactosyl groups, and terminal sialyl groups that are almost inaccessible to Vibrio cholerae sialidase digestion.

Carbohydrate structures of hen ovomucoid. A mass spectrometric analysis.

The apparently homogenous N‐glycosidically‐linked glycans 1, 7, 11 and 14 released by hydrazinolysis from hen ovomucoid were analysed by fast atom bombardment and electron‐impact mass spectrometry after reduction and permethylation. The spectra support the primary structures established independently [FEBS Letters (1983) 152, 145–152] using methylation analysis, partial acid hydrolysis and 500 MHz 1H NMR spectroscopy. In addition to the major constituents present in fractions 1, 7, 11 and 14, four minor components not detected by other methods could be characterized with the aid of the mass spectrometry data as: Man2GlcNAcGlcNAc‐ol, GlcNAc4Man3GlcNAc‐ol, GlcNAc6Man3GlcNAc‐ol and GalGlcNAc6Man3GlcNAc‐ol. Our results show that the physical techniques used provide valuable data on the structure of complex glycans. In addition they can be employed to ascertain the homogeneity of the compounds examined as well as to detect trace amounts of homologs that might not be noticed by other methods.

Structure of two new oligosaccharides isolated from human milk: sialylated lacto-N-fucopentaoses I and II

The structures of two minor oligosaccharides isolated from human milk have been investigated. By using f.a.b.-mass spectrometry, methylation analysis, and partial formolysis as the principal methods of structural investigation, these oligosaccharides were identified as IV2-alpha-Fuc-III6-alpha-NeuAc-LcOse4 and IV3-alpha-NeuAc-III4-alpha-Fuc-LcOse4. This latter oligosaccharide has been recently found to be the carbohydrate moiety of a ganglioside isolated from a colorectal carcinoma cell-line (see ref. 12).

Different binding capacities of influenza A and Sendai viruses to gangliosides from human granulocytes

The structures of gangliosides from human granulocytes were elucidated by fast atom bombardment mass spectrometry and by gas chromatography/mass spectrometry as their partially methylated alditol acetates. In human granulocytes besides GM3 (II3Neu5Ac-LacCer), neolacto-series gangliosides (IV3Neu5Ac-nLcOse4Cer, IV6Neu5Ac-nLcOse4Cer and VI3Neu5Ac-nLcOse6Cer) containing C24:1, and to some extent C22:0; and C16:0 fatty acid in their respective ceramide portions, were identified as major components. In this study we demonstrate that gangliosides from human granulocytes, the second most abundant cells in peripheral blood, can serve as receptors for influenza viruses A/PR/8/34 (H1N1), A/X-31 (H3N2), and a parainfluenza virus Sendai virus (HNF1, Z-strain). Viruses were found to exhibit specific adhesion to terminal Neu5Acα2-3Gal and/or Neu5Acα2-6Gal sequences as well as depending on the chain length of ganglioside carbohydrate backbones from human granulocytes, these important effector cells which represent the first line of defence in immunologically mediated reactions.

Structural studies of gangliosides from the YAC-1 mouse lymphoma cell line by immunological detection and fast atom bombardment mass spectrometry

YAC-1 cells were propagated in bioreactors in 11 and 7.51 volumes. The cells were metabolically labelled withd-[1-14C]galactose andd-[1-14C]glucosamine. The ganglioside fraction, purified by DEAE-Sepharose and silica gel column chromatography, showed on thin layer chromatography four major bands with mobilities between GM1 and GD1a. Gangliosides, obtained by further purification steps including high performance liquid chromatography on silica gel 60 columns with a gradient system of isopropanol:hexane:water, and preparative high performance TLC were characterized by (1) immunostaining of corresponding asialogangliosides obtained by mild acid hydrolysis and neuraminidase treatment and (2) fast atom bombardment mass spectrometry of native and permethylated samples and methylation analysis of GM1b ganglioside. As well as small amounts of GM2 and GM1, the major gangliosides found in the complex mixture were GM1b and GalNAc-GM1b. The structural heterogeneity of these gangliosides was cased by (a) substitution of the ceramide moiety by fatty acids of different chain length and degree of unsaturation (C16:0, C24:0, C24:1) and (b) N-substitution of the sialic acid moieties with either acetyl or glycolyl groups. Disialogangliosides were detected only in low amounts and will be the subject of further investigation. A polyclonal chicken antiserum was raised against IVNeuAc-GgOse5Cer. The antiserum was highly specific for gangliosides (IVNeuAc and IVNeuGc) and asialogangliosides with a GgOse5Cer backbone. No cross-reaction with GM1b or GgOse4Cer was observed.

MS and NMR analysis of the cross-reacting determinant glycan from Trypanosoma brucei brucei MITat 1.6 variant specific glycoprotein

The cross-reacting determinant glycan from Trypanosoma brucei brucei MITat 1.6 is known to contain galactose, mannose and non-acetylated glucosamine. The structural elucidation of this oligosaccharide has been impeded by an unusual non-glycosidic linkage to the peptide chain and a glycosidic linkage to inositol phosphate on either side of the oligosaccharide. Using two different approaches for the isolation of the glycan, namely hydrolysis to give the oligosaccharide directly or pronase digestion to yield the glycan-containing C-terminal glycophosphopeptide, the structure of this glycan was elucidated by mass spectrometry and 1H-NMR spectroscopy. There was evidence of heterogeneity in the glycan residue.

Isolation and characterization of the tetrasaccharide (bis)phosphate from the glycosyl backbone of Salmonella Minnesota and Escherichia coli re-mutant lipopolysaccharides

Abstract Intact tetrasaccharide mono- ( 1 ) and bisphosphates ( 2 ) were isolated from deep rough mutant strains of Salmonella minnesota (mutant R595) and Escherichia coli (mutant F515) and their chemical structure was unequivocally characterized by 1 H-, 13 C- and 31 P-NMR spectrometry and FAB-MS and chemical analysis as 1 and 2