Glycoqueuing: Isomer-Specific Quantification for Sialylation-Focused Glycomics.

Changes of α-2,3-/α-2,6-linked sialic acids (SAs) in sialylglycans have been found to be closely related with some diseases. However, accurate quantification of sialylglycans at the isomeric level remains challenging due to their instability, structural complexity, and low mass spectrometry (MS) detection sensitivity. Herein, we propose an analytical strategy named "glycoqueuing", which allows sequential chromatographic elution and high-sensitivity MS quantification of various sialylglycan isomers based on isotopic labeling followed by analysis via online reversed-phase high performance liquid chromatography coupling with MS (RP-HPLC-MS). The new method was validated by detailed structural identification and quantification of fetal bovine serum (FBS) N-linked sialylglycan isomers, during which many branching isomers were successfully differentiated, and 28 sialylglycan compositions with Neu5Gc residues were analyzed. The method was successfully applied to isomer-specific, quantitative comparison of sialylated N-glycans between bovine and rabbit immunoglobulin G (IgG) and the search for serum sialylated N-glycan biomarker candidates of hepatocellular carcinoma, during which a 55% increase of α-2,6-sialylated fucosylated N-glycans was revealed, demonstrating the great applicability and potential clinical usage of the method.

Experimental approaches to interfere with the polysialylation of the neural cell adhesion molecule in vitro and in vivo.

Sialic acid (Sia) is expressed as terminal sugar in many glycoconjugates and plays an important role during development and regeneration. Addition of homopolymers of Sia (polysialic acid; polySia/PSA) is a unique and highly regulated post-translational modification of the neural cell adhesion molecule (NCAM). The presence of polySia affects NCAM-dependent cell adhesion and plays an important role during brain development, neural regeneration, and plastic processes including learning and memory. PolySia-NCAM is expressed on several neuroendocrine tumors of high malignancy and correlates with poor prognosis. Two closely related enzymes, the polysialyltransferases ST8SiaII and ST8SiaIV, catalyze the biosynthesis of polySia. This review summarizes recent knowledge on Sia biosynthesis and the correlation between Sia biosynthesis and polysialylation of NCAM and report on approaches to modify the degree of polySia on NCAM in vitro and in vivo. First, we describe the inhibition of polysialylation of NCAM in ST8SiaII-expressing cells using synthetic Sia precursors. Second, we demonstrate that the key enzyme of the Sia biosynthesis (UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase) regulates and limits the synthesis of polySia by controlling the cellular Sia concentration.

Victor Ginsburg's influence on my research of the role of sialic acids in biological recognition.

Sialic acids are monosaccharides with relatively strong acidity which belong to the most important molecules of higher animals and also occur in some microorganisms. They are bound to complex carbohydrates and occupy prominent positions, especially in cell membranes. Their structural diversity is high and, correspondingly, the mechanisms for their biosynthesis complex. Sialic acids are involved in a great number of cell functions. Due to their cell surface location these acidic molecules shield macromolecules and cells from enzymatic and immunological attacks and thus contribute to innate immunity. In contrast to this masking role, enabling, for example, blood cells and serum glycoproteins a longer life-time, sialic acids also represent recognition sites for various physiological receptors, such as the selectins and siglecs, as well as for toxins and microorganisms and thus allow their colonization. The recognition function of sialic acids can again be masked by O-acetylation, which modifies the interaction with receptors. Many viruses use sialic acids for the infection of cells. As sialic acids play also a decisive role in tumor biology, they prove to be rather versatile molecules that modulate biological and pathological cellular events in a sensitive way. Thus, they are most prominent representatives of mediators of molecular and cellular recognition.

Sialidases.

Sialic acids are a family of nine-carbon acidic sugars found at the nonreducing terminus of many glycoconjugates. Sialidases can remove these sugar units selectively from cell surfaces, membranes, or purified glycoconjugates. In this unit, sialidase digestion of purified glycoproteins is described as is treatment of intact cells. The physical properties of the four most useful sialidases are discussed along with their relative activities against sialic acids with different modifications and in different linkages.

Polysialic acid and the regulation of cell interactions.

Polysialic acid, a unique glycosylation of the neural cell adhesion molecule, is highly regulated in its expression. Its function is manifested in the modulation of cell interactions, probably through its unusual physical properties. Recent advances have clarified the enzymatic mechanism of polysialic acid biosynthesis, expanded its role in cell migration and axon guidance, and suggested that it promotes plasticity in the adult nervous system.

Surface properties of crosslinked and crosslinked-permeabilized erythrocytes as studied by partitioning in aqueous polymer two-phase systems.

Non-charge- and charge-sensitive dextran/polyethyleneglycol two-phase systems have been used to study the surface properties of red blood cells stabilized by crosslinking with dimethyl suberimidate and permeabilized with digitonin. While crosslinked red blood cells show similar hydrophobic- and charge-related surface properties as control cells, the digitonin treatment changes their surface properties. The changes in hydrophobicity are related to the cell total lipid and cholesterol content while the changes in the charge are related to the sialic acid released by neuraminidase.

Electrophoresis of bovine erythrocyte sialic acids: existence of additional band in trypanotolerant Ndama cattle.

Mild acid-hydrolysis of erythrocyte surface sialic acids of the trypanotolerant Ndama and the trypanosusceptible White/Fulani Zebu breeds of cattle was performed. The cleaved sialic acids from the two breeds of cattle were simultaneously subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS-PAGE), along with commercial standard N-acetylneuraminic acid (MW 309.28), blank gel and plasma proteins. The cleaved sialic acids migrated in the globulin fractions, as shown by the plasma protein electrophoresis. While the Ndama and the Zebu erythrocytes had one migrating band each of which coincided with the standard N-acetylneuraminic acid, the Ndama had another trailing band of sialic acid, of an estimated molecular weight of 30 kDa, which may account for the higher erythrocyte sialic acid concentrations of the Ndama. This additional band was absent in the Zebu. All these bands were readily reproducible.

Identification of new sialic acids derived from glycoprotein of bovine submandibular gland.

Improvements in the isolation procedure and the analytical equipment enabled the detection of seven novel sialic acids in bovine submandibular gland glycoprotein: N-acetyl-8-O-acetylneuraminic acid, N-acetyl-8,9-di-O-acetylneuraminic acid, N-acetyl-7,8,9-tri-O-acetylneuraminic acid, N-glycoloyl-7-O-acetylneuraminic acid, N-glycoloyl-7,9-di-O-acetylneuraminic acid, N-glycoloyl-8,9-di-O-acetylneuraminic acid, and N-glycoloyl-7,8,9-tri-O-acetylneuraminic acid. There are also indications for the presence of N-glycoloyl-8-O-acetylneuraminic acid. In addition, the sialic acids already known to occur in this tissue, namely N-acetylneuraminic acid, N-acetyl-7-O-acetylneuraminic acid, N-acetyl-9-O-acetylneuraminic acid, N-acetyl-7,9-di-O-acetylneuraminic acid, N-glycoloylneuraminic acid, and N-glycoloyl-9-O-acetylneuraminic acid could be identified. Sialic acids were released from the mucin by mild acid hydrolysis and prefractionated on a Dowex 2X8 anion-exchange column (formate form) by elution with a 0-0.6 M gradient of formic acid. The four pools of sialic acids obtained in this way were each further fractionated by column chromatography on cellulose with n-butanol/n-propanol/water (1/2/1, v/v/v) as eluent. By this procedure, N-acetylneuraminic acid, N-acetyl-7-O-acetylneuraminic acid, N-acetyl-9-O-acetylneuraminic acid, N-glycoloylneuraminic acid, and N-glycoloyl-9-O-acetylneuraminic acid were obtained in pure form. The other sialic acids could be enriched sufficiently in different fractions for structural identification. Analyses of sialic acids were carried out by one-dimensional and two-dimensional thin-layer chromatography, gas-liquid chromatography, and gas-liquid chromatography-mass spectrometry.