Adriana E. Manzi

High-pressure liquid chromatography of sialic acids on a pellicular resin anion-exchange column with pulsed amperometric detection: A comparison with six other systems☆

A wide variety of different sialic acids have been reported in nature. Following their release and purification, detection and quantitation of these molecules is now possible by a number of techniques. We and others have previously reported high-pressure liquid chromatography separation of sialic acids with several different columns, elution methods, and detection techniques. We report here a new method for the separation of sialic acids at neutral pH on a Carbopac PA-1 anion-exchange column of pellicular resin, with pulsed amperometric detection following postcolumn addition of alkali. The major advantages of this system are the separation of a variety of sialic acids, sensitive detection (into the picomole range), and the relative ease of use for preparative purposes. Using a set of defined sialic acid standards, this method is compared and contrasted with six other HPLC methods previously described by us and by others. The advantages and disadvantages of each system are also addressed. In the final analysis, no single method is adequate to completely separate and quantitate all of the known sialic acids. However, used in appropriate combinations, these methods allow exploration of the biology of sialic acids in a manner heretofore not possible.

Carbohydrate-deficient glycoprotein syndrome: not an N-linked oligosaccharide processing defect, but an abnormality in lipid-linked oligosaccharide biosynthesis?

The carbohydrate-deficient glycoprotein syndrome (CDGS) is a developmental disease associated with an abnormally high isoelectric point of serum transferrin. Carbohydrate analyses of this glycoprotein initially suggested a defect in N-linked oligosaccharide processing, although more recent studies indicate a defect in the attachment of these sugar chains to the protein. We studied both serum glycoproteins and fibroblast-derived [2-3H]mannose-labeled oligosaccharides from CDGS patients and normal controls. While there was a decrease in the glycosylation of serum glycoproteins of affected individuals, differences were not seen in either monosaccharide composition or oligosaccharide structures. The lectin-binding profiles of glycopeptides from [2-3H]-mannose-labeled fibroblasts were likewise indistinguishable. However, the incorporation of [2-3H]mannose into both glycoproteins and the dolichol-linked oligosaccharide precursor was significantly reduced. Thus, at least in some patients, CDGS is not due to a defect in processing of N-linked oligosaccharides, but rather to defective synthesis and transfer of nascent dolichol-linked oligosaccharide precursors. This abnormality could result in both a failure to glycosylate some sites on some proteins, as well as secondary abnormalities in overall glycoprotein processing and/or function.

A Novel Anionic Modification of N-Glycans on Mammalian Endothelial Cells Is Recognized by Activated Neutrophils and Modulates Acute Inflammatory Responses

We previously reported an unusual carboxylated modification on N-glycans isolated from whole bovine lung. We have now raised IgG mAbs against the modification by immunization with biotinylated aminopyridine-derivatized glycans enriched for the anionic species and screening for Abs whose reactivities were abrogated by carboxylate neutralization of bovine lung glycopeptides. One such Ab (mAb GB3.1) was inhibited by carboxylated bovine lung glycopeptides and other multicarboxylated molecules, but not by glycopeptides in which the carboxylate groups were modified. The Ab recognized an epitope constitutively expressed on bovine, human, and other mammalian endothelial cells. Stimulated, but not resting, neutrophils bound to immobilized bovine lung glycopeptides in a carboxylate-dependent manner. The binding of activated neutrophils to immobilized bovine lung glycopeptides was inhibited both by mAb GB3.1 and by soluble glycopeptides in a carboxylate-dependent manner. The Ab also inhibited extravasation of neutrophils and monocytes in a murine model of peritoneal inflammation. This inhibition of cell trafficking correlated with the increased sequestration but reduced transmigration of leukocytes that were found to be adherent to the endothelium of the mesenteric microvasculature. Taken together, these results indicate that these novel carboxylated N-glycans are constitutively expressed on vascular endothelium and participate in acute inflammatory responses by interaction with activated neutrophils.

Overview of Glycoconjugate Analysis

Whereas DNA, RNA, and proteins are linear polymers that can usually be directly sequenced, glycans show substantially more complexity, having branching and anomeric configurations (α and β linkages). The biosynthesis of glycans, termed glycosylation, is extremely complex, is not template‐driven, varies among different cell types, and cannot be easily predicted from simple rules. This overview discusses the stereochemistry of monosaccharides and glycans and provides diagrammatic representations of monosaccharides (Fisher projections and Haworth representations) and formulas for representation of glycan chains. A glossary of terms used in glycobiology is also provided. Curr. Protoc. Protein Sci. 57:12.1.1‐12.1.10.

Direct Chemical Analysis of Glycoconjugates for Carbohydrates

This unit presents protocols for detection of different sugars bound to glycoconjugates. The solution containing the carbohydrate material is treated with a specific reagent, generating a colored reaction product that can be detected spectrophotometrically. For each assay, the absorbance of the colored solution is proportional to the amount of sugar present in the glycoconjugate.

Preparation and Analysis of Glycoconjugates

Whereas DNA, RNA, and proteins are linear polymers that can usually be directly sequenced, glycans show substantially more complexity, having branching and anomeric configurations (α and β linkages). The biosynthesis of glycans, termed glycosylation, is extremely complex, is not template-driven, varies among different cell types, and cannot be easily predicted from simple rules. This overview discusses the stereochemistry of mono- and oligosaccharides and provides diagrammatic representations of monosaccharides (Fisher projections and Haworth representations) and formulas for representation of glycan chains. A glossary of terms used in glycobiology is also provided. Curr. Protoc. Mol. Biol. 88:17.0.1-17.0.12.

Acid Hydrolysis for Release of Monosaccharides

The first step in obtaining a compositional analysis of a glycoconjugate is the release of the individual monosaccharide constituents. Mild acid hydrolysis to release fucosyl residues from glycoconjugates is described in this unit, in addition to mild acid hydrolysis to release sialic acids from glycoconjugates, along with dialysis and column chromatography procedures to purify the sialic acids. Strong acid hydrolysis to release all monosaccharides from glycoconjugates is also detailed.

Sialate 9-O-acetylesterase from rat liver

Publisher Summary A family of esterases has been discovered that appear to be specific for removal of O-acetyl esters from the 9-position of naturally occurring sialic acids. In the rat liver, the two major esterases detected with this substrate at neutral pH are a cytosolic nonglycosylated enzyme and a membrane-associated glycosylated enzyme, which are present in approximately equal amounts. The chapter discusses the biosynthetic preparation of [acetyl- 3 H]9-O-acetyl-N- acetylneuraminic acid. The assays described in this chapter are accurate and reliable even in crude extracts of tissues. With purified enzyme, the release of unlabeled free acetate from substrates can be monitored using a commercially available kit. The individual components for this acetate assay can also be purchased individually. In the case of synthetic chromogenic substrates, hydrolysis can be followed by monitoring the reaction fluorometrically. It must be emphasized that in crude tissue extracts, other “nonspecific” esterases can cleave the 4-MUOAc substrate.

Total Compositional Analysis by High‐Performance Liquid Chromatography or Gas‐Liquid Chromatography

Once the presence of carbohydrate in a glycoprotein has been confirmed, the next step is to determine the precise molar ratio of its monosaccharide constituents. The analysis involves two major phases. The release of the individual monosaccharides is achieved by methanolysis (described here), total acid hydrolysis, or enzymatic release of sialic acids. The resulting mixtures of monosaccharides are then analyzed by methods described in this unit: fractionation, characterization, and quantitation by high‐performance liquid chromatography using anion‐exchange chromatography with pulsed amperometric detection (HPAEC‐PAD) and other HPLC systems or by gas‐liquid chromatography with flame ionization detection. The identity of the individual monosaccharides is determined by comparison with known standards processed and analyzed in the same way.

HPLC methods for the fractionation and analysis of negatively charged oligosaccharides and gangliosides.

This unit describes the fractionation and analysis of anionic oligosaccharides and gangliosides using anion‐exchange high‐performance liquid chromatography (HPLC). Saccharides or gangliosides are eluted in order of the number of negative charges they possess, although the charge‐to‐mass ratio can also contribute to elution position.