Martin Gohlke

RAPID ANALYSIS OF O-ACETYLATED NEURAMINIC ACIDS BY MATRIX ASSISTED LASER DESORPTION/IONIZATION TIME-OF-FLIGHT MASS SPECTROMETRY

N-Acetylneuraminic acid (a sialic acid) occurs mainly as a terminal substituent of oligosaccharides of glycoconjugates. Derivatives of neuraminic acid occur widely, substituted in the amino and hydroxy side chains, as well in the C-9 carbon skeleton. These derivatives are responsible for specific functions of sialic acids during cell-cell, cell-substrate, or cell-virus interactions. The study of O-acetylated neuraminic acids is difficult, because only small amounts are extractable from natural sources and they are generally unstable to acids and bases. We report a new method for the rapid analysis of O-acetylated neuraminic acids, using a combination of reversed phase HPLC and MALDI-TOF mass spectrometry. A mixture of neuraminic acids from bovine submaxillary gland mucins was analysed, as well as neuraminic acids variously substituted in the amino and hydroxy side chains with acetyl and glycolyl groups, respectively.

N-Glycosylation of the carcinoembryonic antigen related cell adhesion molecule, C-CAM, from rat liver: detection of oversialylated bi- and triantennary structures

Abstract Rat C-CAM is a ubiquitous, transmembrane and carcino-embryonic antigen related cell adhesion molecule. The human counterpart is known as biliary glycoprotein (BGP) or CD66a. It is involved in different cellular functions ranging from intercellular adhesion, microbial receptor activity, signaling and tumor suppression. In the present study N-glyco-sylation of C-CAM immunopurified from rat liver was analyzed in detail. The primary sequence of rat C-CAM contains 15 potential N-glycosylation sites. The N-glycans were enzymatically released from glycopeptides, fluorescently labeled with 2-aminobenzamide, and separated by two-dimensional HPLC. Oligosaccharide structures were characterized by enzymatic sequencing and MALDI-TOF-MS. Mainly bi- and triantennary complex structures were identified. The presence of type I and type II chains in the antennae of these glycans results in heterogeneous glycosylation of C-CAM. Sialylation of the sugars was found to be unusual; bi- and triantennary glycans contained three and four sialic acid residues, respectively, and this linkage seemed to be restricted to the type I chain in the antennae. Approximately 20% of the detected sugars contain these unusual numbers of sialic acids. C-CAM is the first transmembrane protein found to be over-sialylated.

The adhesive properties of recombinant soluble L-selectin are modulated by its glycosylation

The leukocyte adhesion molecule L-selectin, which mediates the initial steps of leukocyte attachment to vascular endothelium, is intensely glycosylated. Different glycoforms of L-selectin are expressed on different leukocyte subsets and differences in L-selectin glycosylation appear to be correlated with the leukocytes ability to attach to different endothelial targets. In the present study we addressed the question whether glycosylation of L-selectin influences L-selectin-ligand interactions. To obtain different glycoforms of L-selectin, recombinant proteins were expressed both in the baby hamster kidney (BHK) cell line and in the human myelogenous cell line K562, resulting in sL-sel[BHK] or sL-sel[K562], respectively. The glycosylation characteristics of the purified proteins were determined. The most striking differences in glycosylation were seen in the terminal sialylation. Each of the two proteins carried sialic acids in the alpha 2-3 position, while alpha 2-6-bound sialic acids were found exclusively on sL-sel[K562]. To investigate their adhesive properties, both recombinant sL-selectins were used in cell adhesion assays and interactions with the ligands present on various hematopoietic cell lines or activated human cardiac microvascular endothelial cells were examined. The binding capacity of sL-sel[K562] was about 1.6 fold higher compared to sL-sel[BHK] under static as well as under flow conditions. These findings indicate that the terminal sialylation pattern of L-selectin modulates its binding characteristics.

Separation of N-glycans by HPLC

Most glycoproteins carry a very heterogeneous mixture of oligosaccharides and even a single glycosylation site of a pure glycoprotein is often heterogeneously glycosylated. The structural diversity of oligosaccharides arises from linkage variants, from differences in the size and number of charges of glycans, and from differences in the monosaccharide composition of glycans. Fortunately, the biosynthetic pathway is subject to certain restrictions, so that structural diversity is limited and amenable to laboratory investigation. Different approaches have been developed to the structural characterization of oligosaccharides, including nuclear magnetic resonance (NMR), mass-spectrometry, linkage analysis by gas chromatography-mass spectrometry (GC-MS), sequence analysis using specific exoglycosidases and others, but a crucial part of these strategies is the separation of the glycan mixture into homogeneous glycan fractions. In this chapter some high-performance liquid chromatography (HPLC)-techniques are described for the isolation of oligosaccharides, in particular N-linked glycans.

Carbohydrate structures of soluble human L-selectin recombinantly expressed in baby-hamster kidney cells.

A soluble form of L‐selectin was recombinantly produced, which might be an effective therapeutic agent in inflammatory disorders, acting as an inhibitor for leucocyte endothelium adhesion. In the present study the oligosaccharide structures of soluble human L‐selectin, recombinantly expressed in baby‐hamster kidney cells, were determined. The N‐linked glycans were enzymically released and fluorescently labelled with 2‐aminobenzamide. Sialylation of the N‐glycans was analysed by anion‐exchange chromatography followed by rechromatography of the resulting fractions on amino‐phase HPLC after release of the sialic acid residues. Desialylated oligosaccharides were separated using two‐dimensional HPLC and characterized by digestion with exoglycosidases and MS. More than 30 oligosaccharide structures representing at least 95% of the overall glycosylation of this protein were determined. The results revealed that recombinant soluble human L‐selectin carries bi‐, tri‐ and tetra‐antennary sugar chains, which are fucosylated on the innermost residue of N‐acetylglucosamine. The number of sialic acid residues linked to these glycans ranges from 0 (neutral glycans) to 4 (tetrasialylated oligosaccharides). The sialic acid is found exclusively in the α2–3 linkage to galactose. In addition to the main glycans, different minor structures containing terminal N‐acetylgalactosamine, or the H (O) blood‐group determinant were also identified. O‐Glycosylation of mucin‐type sugar chains was not detected in recombinant soluble human L‐selectin.

H (0) blood group determinant is present on soluble human L‐selectin expressed in BHK‐cells

In the present study we show that the H (0) blood group determinant Fucα1‐2Galβ1‐4GlcNAcβ1‐R is present on N‐linked glycans of soluble human L‐selectin recombinantly expressed in baby hamster kidney (BHK) cells. The glycans were isolated using complementary HPLC techniques and characterized by a combination of exoglycosidase digestion and mass spectrometry. The linkage of the fucose residues was determined by incubation of the glycans with specific fucosidases. The H blood determinant Fucα1‐2Galβ1‐4GlcNAcβ1 was detected for bi‐, 2,4 branched tri‐ and tetraantennary structures. To our knowledge, the proposed oligosaccharide structures represent a new glycosylation motif for recombinant glycoproteins expressed on BHK cells.