Labelled Sialic Acids

For the study of biochemical processes several labelled sialic acids and sialic acid derivatives have been prepared. 

The doubly labelled sialic acids N-acetyl-[2-i4C,9-3H]neuraminic acid (NOhle and Schauer 1981) and N-glycolyl-[2-i4C,9-3H]neuraminic acid (NOhle et al. 1982) were prepared from sodium[2-i4C]pyruvate and either N-acetyl-[6-3H]mannosamine or N-glycolyl-[6- H]mannosamine, with the aid of the N-acetylneuraminate lyase from Clostridium perfringens. The metabolic fate of these compounds was studied after oral and intravenous application to mice and rats. 

A number of these labelled sialic acids have been converted into their CMP-analogues, and subsequently incorporated into glycoconjugates (chapter I). Sialoglycoconjugates labelled in sialic acid can also be prepared by incubations of surviving tissue slices with isotopically labelled precursors of sialic acid, as N-PH]acetyl-mannosamine. 

By using periodate oxidation/tritiated borohydride reduction (see also section VIII), sialoglycoconjugates can be labelled very easily. Van Lenten and Ashwell (1972) and Schauer et al. (1976) reported the use of this technique for the modification of sialoglycoproteins. Veh et al. (1977) have described this degradation for gangliosides see also Pfannschmidt and Schauer (1980) for additional data. 

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Our preferred method of sialic acid profiling is to use the DMB / HPLC system. The first step is release of the sialic acids from the glycoprotein by mild acid hydrolysis (typical conditions are incubation with 2 mol dm acetic acid for 2 h at 80 °C) followed by fluorescent labeling using DMB and stabilization of the tagged conjugates by reduction with sodium dithionite. Analysis of the DMB labeled sialic acids is done by HPLC with a C18 column and fluorescence detection (.7ex = 295nm,.7em= 352 nm). 

Alternatives to DMB analysis include HPLC of OPD (o-phenylenediamine) labeled sialic acids [6] as well as HPAE-PAD and hybrid variants of these procedures. 

The DMB-labeled sialic acids are applied to the HPLC equipped with a TSK-gel ODS-120T column (250 x 4.6 mm i.d., Tosoh), and a fluorescence detector (FP-2025, JASCO). The column is equilibrated using acetonitrile/methanol/water (9 7 84 by volume) at 26°C. Then 2-20 pL of the supernatants are applied to HPLC analysis for an isocratically flow at 1.0 mL/min and the DMB-labeled sialic acid is detected with a fluorescence detector at an excitation of 373 nm and an emission at 448 mn (Fig. 4). The estimate of the ratios of the quantity of internal sialic acid residues (C9(Sia)) to that of total sialic acid residues (C7(Sia) + C9(Sia)) can then be obtained. 

FIGURE 8.9 Separation of DMB-labeled sialic acids by standard HFLC. Column LudgerSep-Rl, 3 pm particle size, 4.6x150 mm. Instrument Waters Alliance 2795 with 2475 fluorescence detector. Solvent A methanol acetonitrile water (7 9 84 v/v). Flow rate 0.5 ml/ min for 30 min. Temperature 30°C. Detection Xex = 373 run, Xem 448 nm. 

FIGURE 8.10 Separation of DMB-labeled sialic acids by UPLC. Column ACQUITY... 

Detection of glycopeptides is simplified if the samples can be labelled with radioisotopes. It is possible to metabolically label complex carbohydrates with isotopic precursors and these may provide some useful structural information. A glucosamine preeursor will specifically label N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc) and sialic acid whereas N-acetyl-mannosamine will only label sialic acid (19). Radioactive mannose is incorporated as mannose and fucose. A radiolabelling ratio of 3 1,... 

In parallel experiments, samples of the culture were withdrawn and the sialic acid content of the protein was monitored by metabolically labeling sialic acid on the protein by including H-ManNAc in the culture media [30]. 

Bemacki, R. J., and Bosmann, H. B., 1973, Rat-liver-sialidase activity utilizing a tritium-labeled sialic acid derivative of glycoprotein substrates, Eur. J. Biochem. 34 425-433. 

Add 106-108cells/ml in a PBS solution (lOmM sodium phosphate, 0.15M NaCl, pFI 7.4) containing ImM sodium periodate and incubate on ice for 30 minutes in the dark. This level of periodate addition will target the oxidation only to sialic acid residues (Chapter 1, Section 4.4). If additional sites of glycosylation also are to be labeled, increase the periodate concentration to 10 mM and do the reaction at room temperature in the dark. 

Dissolve the glycoprotein(s) to be labeled in ice-cold ImM sodium periodate, lOmM sodium phosphate, 0.15 M NaCl, pH 7.4, for the exclusive oxidation of sialic acid residues. For general carbohydrate oxidation, increase the periodate concentration to 10 mM in PBS at room temperature. 

Dissolve a glycoprotein to be labeled in 50mM sodium phosphate, pH 7 (reaction buffer), at a concentration of l-10mg/ml. For sialic acid modification, place the sample in ice to cool to near 0°C. 

The following protocol describes the use of biotin-hydrazide to label glycosylated proteins at their carbohydrate residues. Control of the periodate oxidation level can result in specific labeling of sialic acid groups or general sugar residues (Chapter 1, Section 4.4). 

Figure 11.22 Azido-sialic acid-containing glycans can be labeled in vivo with biotin-PEG-phosphine using the Staudinger ligation reaction, which forms an amide bond.

The methods used for in vivo incorporation of azido-monomers and performing a labeling reaction with live cells are relatively simple. The following protocol is based on the methods of Saxon and Bertozzi (2000), which uses acetylated azidoacetylmannosamine as the azido-monomer source and a biotin-PEG-phosphine compound to biotinylate cell surface glycoproteins at the specific azide-sialic acid incorporation sites (Figure 17.19). 

Figure 17.19 An azido-sialic acid derivative that gets incorporated into glycans in cells can be labeled specifically with a biotin-phosphine tag using the Staudinger ligation process. The result is an amide bond linkage with the glycan.

Bayer, E.A., Ben-Hur, H., and Wilchek, M. (1988) Biocytin hydrazide—a selective label for sialic acids, galactose, and other sugars in glycoconjugates using avidin-biotin technology. Anal. Biochem. 170, 271-281. 

Spin-labelling of free, or cell-surface, sialic acids has been used in order to obtain information about the rate of rotational orientation of the label after attachment to macromolecules this knowledge is important in the investigation of the orientation and mobility of sialogly-coproteins in, for example, cell membranes. In a first approach, the label was introduced into the carboxyl groups by a carbodiimicle-me-diated, amidation procedure.177 This method is, however, not specific... 

Sialic acids may also be specifically stained by using peroxidase-labelled Limulus polyphemus agglutinin in combination with 3,3 -diaminobiphenyl or Aleian Blue, and the procedure was tested with various, mammalian tissues by light-microscopy.199... 

A method for selective, radioactive labelling of sialic acids, especially in cell membranes, by mild oxidation with periodate followed by reduction with borotritide, has heen described by Gahmberg and Andersson.200 This procedure can be used either for isolation and characterization of the labelled, cell-surface glycoconjugates (see, for example, Ref. 201) or for autoradiography of tissues and cells (for example, erythrocytes).142... 

The pathway of the biosynthesis of Neu5Ac demonstrates the origin of sialic acids from the cellular hexose and hexosamine pools. These sugars are, therefore, suitable components for the study of the biosynthesis of sialic acid. However, only ManNAc has been shown to be a relatively specific precursor of sialic acids, as may be seen from the distribution of radioactivity between the individual monosaccharides of glycoconjugates after incubation. Injections of radioactive ManNAc into animals, or incubation of surviving tissue slices or individual cells with this compound, give incorporation of label mainly into the sialic acids.226 227... 

Radioactive acetate is a cheaper, readily available precursor for experiments on the labelling of sialic acid in tissues or cells, and it effectively labels the N- and O-acyl groups of sialic acids.228 This method is of great value not only for the preparation of radioactive sialic acids having high specific radioactivity but also for metabolic studies of sialic acids. However, the sialic acids must be isolated before determination of the specific radioactivity, as other aeetylated hexosamines are also labelled. 

Nonphysiological compounds have also been described as influencing the overall metabolism of sialic acid. Administration of ethanol (2 g/kg) to rats significantly decreases the sialic acid content of brain tissue.246 Convulsions induced by pentylenetetrazole (6,7,8,9-tetrahy-dro-5/f-tetrazoloazepine) are accompanied by a diminution in the rate of biosynthesis of polysialogangliosicles GT, and GQn, in rat brain.227 Such ManNAc analogs as 2-acetamido-l,3,4,6-tetra-0-acetyl-2-deoxy-D-mannopyranose or the 2-(trifluoroacetamido) derivative lead to a marked lowering of the incorporation of radioactivity from labelled ManNAc into glycoconjugate sialic acids of murine, erythroleukemia (Friend) cells.247... 

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