ProfileThoseSugars is a new service launched by BCCM/LMBP in cooperation with Prof. N. Callewaert, Director of the Unit for Molecular Glycobiology of the host laboratory (Department for Molecular Biomedical Research (DMBR), VIB – Department of Molecular Biology, Ghent University). The service provides the analysis of protein-linked glycans, for which Prof. N. Callewaert and Prof. R. Contreras adapted the technique from the use of polyacrylamide gel-based sequencers to multicapillary DNA sequencers, which represent the state of the art today. The protocol – recently published in Nature Protocols (Laroy et al., 2006) – satisfies the glyco-analytical needs of many projects and can form the basis of ‘glycomics’ studies, in which robustness, high throughput, high sensitivity and reliable quantification are of paramount importance.

1. Introduction

Glycosylation is the enzymatic addition of sugars or oligosaccharides to macromolecules such as proteins (glycoproteins) and lipids (glycolipids). In most cases, glycosylation occurs in the secretory pathway, i.e. in the endoplasmic reticulum and the Golgi apparatus. Consequently, more than 90% of all secreted and membrane bound proteins are glycosylated. Together with other post- and cotranslational modifications, of which phosphorylation is probably the most studied, glycosylation helps in diversifying gene products. Depending on the protein structure both during and after folding, specific sites are modified with carbohydrates. Although all cells in an organism have the same genomic information, the physiological state of a specific cell and its environment will determine what part of the glycosylation machinery is active. Consequently, a particular glycoprotein should not be considered a single entity but rather as a group of different glycoforms. Different glycoforms of the same protein may have different functional, kinetic or physical properties.
The so-called paradox of glycosylation states that no specific function can be associated with a specific carbohydrate structure. The exact role of a carbohydrate depends on its interactions with the protein backbone and with other proteins, carbohydrates or other types of molecules. Thus a specific carbohydrate structure can serve many functions. Obviously, some general functions can be assigned to specific structures, but they usually do not tell the whole story. Terminal sialic acid molecules on glycoproteins are known to protect them from clearance from the blood by liver receptors. However, as part of a larger terminal carbohydrate structure, it may influence many other functions or properties of the specific protein in a specific environment. This illustrates that, for a full comprehension of complex life, glycomics may be as essential as proteomics and genomics. As the condition of the cell largely controls which part of the glycosylation machinery is active at any time, changes in these conditions will alter the glycosylation. One can also look at it in another way: the types of glycoconjugates produced by a cell, tissue or organism reflect their current physiological state. In pathology, this knowledge can be used in two ways. First, these changes can indicate a certain disease as do changes in the protein concentration. Second, the altered glycosylation can be studied as a function of the pathology. In the former, the focus is diagnosis whereas, in the latter, the primary concern is therapy.

Figure 2: Sample preparation

2. Glycosylation in diagnosis

Many diagnostic methods currently used in the clinic rely on glycosylation changes. For example, some of the current applications are based on the occurrence of different isoforms of a protein. When a charged sugar is involved, the protein isoforms have different isoelectric points, which facilitate their separation and give them predictive value. For example, specific changes in isoforms of human transferrin are directly related to alcohol abuse. Therefore, carbohydrate deficient transferrin (CDT) is currently used to monitor alcoholic patients under treatment. The same thing is true for the detection of abuse of exogenous erythropoietin (EPO) in sports. In this case, the test is based on differences in EPO isoforms between human endogenous and recombinantly expressed EPO.
In a recent study on liver disease, the assumption was made that different active glycosylation machineries are active in healthy and diseased hepatocytes (Callewaert et al., 2004). As the majority of glycoproteins in the serum are synthesized by these cells, the total N-glycome of these proteins may reflect the condition of the liver. By means of DSA-FACE, total serum N-glycoprofiles were obtained. Clear changes in the N-glycan profiles were observed in cirrhosis. Based on these profiles, the GlycoCirrhoTest was defined, which allows differentiation of cirrhosis from non-cirrhotic chronic hepatitis. A more detailed study of the glycan profiles revealed delicate changes in the different fibrosis stadia. Obviously, this is of considerable clinical importance, as a biopsy is still the only practical starting point for grading fibrosis.

3. Glycosylation and therapy

N-glycosylation is one of the most common post-translational modifications encountered on biopharmaceuticals. While the modification is often critical to achieve efficient folding and secretion of the glycoprotein drug, its notorious heterogeneity can hinder efficient downstream processing and can significantly complicate the structural characterization of the molecule. Moreover, the type of N-glycosylation can have a tremendous impact on the pharmacokinetics of a drug, as receptors with affinity for certain carbohydrate types are present on the liver endothelium and on hepatocytes, which cause rapîd clearance of glycoproteins modified with their cognate carbohydrate ligands. A well-known example concerns the N-glycosylation of erythropoietin (EPO), where large multi-antennary N-glycans are necessary to avoid renal clearance of this small protein and achieve the long residence time in the circulation, which is necessary for therapeutic efficacy. Taking this concept one step further, second-generation EPO (AraNESP, Amgen) has been engineered to contain an extra N-glycosylation site, which makes the glycoprotein molecule even larger, thus effectively increasing the circulation time from days to weeks.

4. Tools for N-glycan structural profiling

4.1 Limitations and needs

One of the major current obstacles for the use of carbohydrates in diagnosis has been the lack of appropriate analytic techniques. Carbohydrate recognizing proteins, NMR, Xray crystallography and mass spectrometry (MS) all have their limitations. Because the amount of biological sample available is frequently small, and because of the characteristics of oligosaccharides, the perfect technique for glycodiagnosis should combine high sensitivity, high resolving power (including the isomeric and isobaric structures), high throughput, and low cost (material and personnel)

Moreover, in biopharmaceutical engineering campaigns, the availability of robust, highresolution and high-sensitivity N-glycan structural profiling tools is of paramount importance in monitoring production process variations for their influence on product N-glycosylation and in ensuring batch-tobatch reproducibility. High throughput at relatively low cost and an analytics platform that can be operated by non-specialist technicians are certainly desirable features.

4.2 DSA-FACE as the solution

Carbohydrates can be separated electrophoretically on polyacrylamide gels. To allow detection and in some cases, to add charge, the oligosaccharides are fluorescently labeled. Classical FACE (fluorophore assisted carbohydrate electrophoresis) allows separation and detection of oligosaccharides albeit with relatively low sensitivity and poor resolving power. A major breakthrough came when slab gel DNA-sequencing equipment was successfully used for the profiling and analysis of oligosaccharides. When an appropriate dye is used, usually APTS (8-aminopyrene- 1,3,6-trisulfonic acid), DSA-FACE (DNA-Sequencer Aided FACE) usually allows separation with high resolution and sensitivity (Callewaert et al., 2001).
In the DMBR Unit, Molecular Glycobiology, the workhorse analytical technique is not MALDI-TOF mass spectrometry, as in many other laboratories in this field, but N-glycan profiling on the Applied Biosystems gel-based or capillary array-based DNAsequencers, which are very familiar to any laboratory that has ever performed DNA-sequencing or genetic analysis. This method has enabled N-glycan profiling to become teins and the DNA-sequencing, the latter being executed by technicians on over 10,000 samples per year on a single 4-capillary instrument.
Samples of glycoproteins are worked up through an optimized multi-step 96-well plate-based protocol, and the released Nglycans are labeled with the fluorophore 8-aminopyrene-1,3,6-trisulfonic acid (originally developed for carbohydrate labeling at Beckman), followed by the removal of the excess label. The protocol has recently been described in detail (Laroy et al., 2006). The workflow for sample preparation is provided in Figure 2. Along similar lines, Prof. N. Callewaert and his team have been using N-glycan profiling on DNA-sequencers in an analysis service for academic scientists and the biopharmaceutical industry. The high sensitivity of the integrated analytical protocols is a very significant advantage of the method, with 1-5 micrograms of protein being sufficient to obtain results of exoglycosidase array sequencing. This enables to implement N-glycan profiling at a much earlier stage of cell-line development than has hitherto been possible. Expertise was gained for the analysis of MAb N-glycosylation at an early stage of clone selection (24-well plate culture scale) for the analysis of MAbs and total glycoprotein extracts produced in plants (proteins extracted from parts of single Arabidopsis plants are sufficient), for the analysis of N-glycans derived from Westernblotted protein bands, for recombinant Factor IX secreted by muscular tissue implants, for EPO produced in the novel expression system Leishmania tarentolae, for N-glycosylation analysis of antigens derived from parasitic trematodes, and many more other applications.
If structural information on the observed N-glycans is desired, exoglycosidase array sequencing, can be performed either on the total mixture if the major compounds are of interest, or on HPLC-purified minor compounds from the mixture. The integration of a normal phase ion-pairing HPLC option in our standard workflow allows for such complete analysis of complex mixtures. An example is given in Figure 3.

Figure 3: Exoglycosidase sequencing example. The N-glycans from total serum protein of a liver cirrhosis patient were analyzed upon digestion with mixtures of exoglycosidases. From careful analysis of peak abundances and mobility shifts between the different panels, the structure of the main components can easily be derived. For similar analysis of minor compounds in the mixture, prior HPLC separation of the mixture has been implemented (see Fig. 2).

Contacts

Through the ProfileThoseSugars service BCCM/LMBP offers customized contract analytical services (specialized in miniaturized comparative analysis of larger series of samples) built on DSA-FACE technology and performed by the developers of this technology. For more information, see the website: http://bccm.belspo.be/about/lmbp.php
Please contact Kristien Neyts at Kristien. Neyts@dmbr.UGent.be. In concert with Prof. N. Callewaert and his team, she will be happy to discuss your glycoanalysis requirements and advise you on the most appropriate strategies and the options suitable for your specific applications.

Literature

• Callewaert, N. et al. Noninvasive diagnosis of liver cirrhosis using DNA sequencer-based total serum protein glycomics. Nature Medicine 10, 429-434, 2004.
  
  
• Callewaert N, Geysens S, Molemans F, Contreras R. Ultrasensitive profiling and sequencing of N-linked oligosaccharides using standard DNA-sequencing equipment. Glycobiology 11, 275-81, 2001.
  
  
• Laroy W, Contreras R, Callewaert N. Glycome mapping on DNA sequencing equipment. Nature Protocols 1, 397-405, 2006.