Carbohydrate characterization of recombinant glycoproteins of pharmaceutical interest

Abstract

Carbohydrate characterization of recombinant glycoproteins entails determination of the primary structures and points of attachment of the oligosaccharide moieties. This article reviews several methods for oligosaccharide- and glycosylation-site characterization. A major recent advance in carbohydrate analysis has been the use of high-pH anion exchange (HPAE) chromatography for separation of glycoprotein-derived oligosaccharides. These separations are sensitive to molecular size, carbohydrate composition, linkage positions, and anomeric configurations. As a result, HPAE chromatography is a powerful technique for glycoprotein characterization and can serve as the basis of an "oligosaccharide map." Characterization of potential N-glycosylation sites involves determining whether each potential site is glycosylated, the extent of oligosaccharide processing at each site, and ideally, a detailed description of the distribution of oligosaccharides at each site. Several approaches to characterizing glycosylation sites are described, including peptide mapping and mass spectrometry. Treatment of a glycoprotein with endo-beta-N-acetylglucosaminidase H (endo H) followed by peptide:N-glycosidase F (PNGase F) can be used to distinguish sites that are not glycosylated from those carrying high-mannose structures and from those containing attached complex oligosaccharides. After individual glycosylation sites have been labeled by this series of reaction, the resulting peptides are characterized by automated Edman degradation. This technique is particularly valuable for characterizing peptides that contain more than one potential N-glycosylation site. An example is also given in which HPAE chromatography is used in conjunction with reversed-phase high-performance liquid chromatography tryptic mapping to obtain detailed information on the distribution of oligosaccharides at individual glycosylation sites.

Multiple post-translational modifications regulate E-cadherin transport during apoptosis

“…PNGase F hydrolyzes both types of N-glycosylation (Maley et al, 1989). Thus Endo H resistance with PNGase F sensitivity reflects export from the ER and transit through the cis-Golgi, whereas sensitivity to both glycosidases indicates retention in the ER (Spellman et al, 1990).…”

Mouse RIC-3, an Endoplasmic Reticulum Chaperone, Promotes Assembly of the α7 Acetylcholine Receptor through a Cytoplasmic Coiled-Coil Domain

“…The primary amino acid sequence of mRIC-3 contains a single consensus site (NXT/S) for N-glycosylation at N128. To determine whether this site is glycosylated, lysates of COS cells transfected with mRIC-3 were treated with Endo H or PNGase F (Spellman, 1990;Plummer and Tarentino, 1991) and then analyzed by Western blotting. Treatment with neither enzyme reduced the mobility of the protein (Fig.…”

An improved method for quantitative sugar analysis of glycoproteins

“…Oligosaccharides occupy a large part of glycoproteins and can modify the structure, dynamics and functional activities of their conjugate polypeptides. The glycosylation of a protein is cell type-specific (Kornfeld and Kornfeld, 1985) and is influenced by extra-cellular environment and the method of cell culture, especially for recombinant glycoproteins (Spellman, 1990). Thus, it is very important to characterize the structure of glycans in order to fully understand the structural basis of a glycoprotein's function.…”

“…Quantitative monosaccharide analysis of a given glycoprotein provides the molar ratio of individual sugars to protein and provides information regarding types of oligosaccharide (N-or O-glycans), the basis to design a structural elucidation strategy, and a measure for production consistency of recombinant glycoprotein therapeutics (Spellman, 1990) such as erythropoietin (EPO). Sugar composition of a glycoprotein is obtained through serial steps of cleavage of all glycosidic bonds, separation, detection and quantification of the released sugars.…”