BECKMAN. A p p l i c a t i o n I n f o r m a t i o n. Resolution of Glycoforms of Ribonuclease B by P/ACE Capillary Electrophoresis. P r o t e i n...

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1 A-1745B A p p l i c a t i o n I n f o r m a t i o n P r o t e i n Resolution of Glycoforms of Ribonuclease B by P/ACE Capillary Electrophoresis Pauline Rudd and Eva Coghill Glycobiology Institute, Dept. of Biochemistry University of Oxford Oxford, UK Introduction Glycoproteins generally consist of populations of glycosylated variants (glycoforms) of a single protein. Molecules comprising a unique amino acid sequence may be diversified by a range of oligosaccharides covalently bound to the protein either through the nitrogen of an asparagine side chain (N-linked) or the oxygen in the side chain of a serine or threonine residue (O-linked). Each potential glycosylation site is normally able to accommodate many alternative oligosaccharide structures, allowing the generation of discrete populations of a structurally modified protein. The relative proportions of such glycoforms are found to be reproducible, not random, and depend on the environment in which the protein is glycosylated. Factors which control glycosylation include physiological state as well as the type of organism, tissue, and cell in which the glycoprotein is made. (1,2) Manufacturing processes also influence glycosylation and these, as well as procedures for isolating glycoproteins, may result in the inadvertent selection of particular glycoform populations. Such considerations are important not only because glycosylation modifies structures but because it may also modulate the function of a protein. (3) The limitations of some of the current techniques used for the analysis of glycoforms are discussed below: 1. Analysis of all oligosaccharide structures covalently bound to a protein with a single glycosylation site. Sugars, released by hydrazinolysis or with endoglycosidase enzymes, are analyzed by exoglycosidase sequencing. The number and relative proportions of the released oligosaccharides is equal to the number and proportion of the individual glycoforms. In practice, however, hydrazinolysis is a difficult technique requiring a high degree of skill. Endoglycosidases do not release all sugars in equimolar proportions; some are sterically protected from the enzyme and may not be released at all, while others are released preferentially. (4) 2. Analysis of the oligosaccharide structures associated with a single site within a protein with more than one glycosylation site. Proteolytic enzymes are used to prepare a set of glycopeptides, each of which contains one of the glycosylation sites. Analysis of the sugars released from each glycopeptide enables the number and relative proportions of the glycoforms at each site to be defined. However, site analysis is usu- BECKMAN

2 ally very time consuming and difficult, especially if there are multiple glycosylation sites. Moreover, although the glycosylation may be fully defined at individual sites, the information about the glycosylation of specific sets of glycoforms is lost. It cannot be assumed that the intact molecule can accommodate all the permutations and combinations of sugars present at each of the sites. 3. Sequential lectin affinity chromatography. This technique enables individual sets of glycoforms to be separated before analysis. However, a glycoprotein may have multiple sites each associated with a range of oligosaccharides, making the results of sequential lectin affinity chromatography difficult to interpret. In this bulletin, capillary electrophoresis (CE) was used to resolve glycoforms and to follow the time course of the enzymatic digestion of a glycoprotein. Ribonuclease B (RNase B) was selected as a model glycoprotein. (5) It is demonstrated that CE avoids some of the above problems associated with the current methodologies which involve analysis of released sugars. Experimental Capillary Electrophoresis of RNAse B A P/ACE System 2100 with Gold software (version 7.11) was used for the CE experiments. The capillary cartridge contained a 72-cm (65 cm to detector) 50-µm i.d. uncoated capillary. The temperature of the capillary cartridge was maintained at 30 C. The run buffer was 20 mm sodium phosphate, 50 mm SDS, 5 mm sodium tetraborate, ph 7.2. The run voltage conditions were 1 kv for 1 min, 20 kv for 19 min. Detection was at 200 nm. Samples were injected by pressure for 1.5 s. The relative proportions of the glycoform populations in the intact glycoprotein were obtained by integration of the peak data at time zero. Release and Radioactive Labeling of Asparaginelinked Oligosaccharides from RNAse B. Bovine pancreatic RNAse B (Sigma Chemicals, St. Louis, MO) was cryogenically dried over activated charcoal and subjected to hydrazinolysis, re-n-acetylation and reduction with sodium borohydride according to standard procedures. (6) Size Exclusion Chromatography (SEC) 2 nmoles of radiolabeled sugar ( CPM) were dissolved in 180 ml water. Dextran hydrolysate (20 µl; 20 mg/ml) was added and the mixture applied to a cm Bio-Gel P-4 gel size exclusion chromatography (SEC) column (> 400 mesh). The eluant was monitored with radioactivity and refractive index detection systems. Exoglycosidase Digestion of Oligomannose Structures of RNAse B Ribonuclease B was digested with Jack Bean α-mannosidase (10 mm sodium citrate ph 4.5/ 0.2 mm zinc acetate; enzyme:substrate ratio 5 Units:1 mg). The course of the reaction, which was carried out in the CE autosampler vials at approximately 30 C, was followed by directly injecting the digestion mixture into the capillary at programmed time intervals. Results and Discussion Glycoform Analysis RNAse is a glycoprotein (molecular mass 15.5 kd) containing a single N-glycosylation site at Asn 34. As is the case with other glycoproteins, it consists of a population of glycosylated variants in which a single amino acid sequence is diversified by conjugated oligosaccharides. For a protein containing a single glycosylation site, the number and relative proportions of the different glycoforms is equal to the number and relative proportions of the different oligosaccharides associated with the site. This allows such glycoform populations to be characterized from an analysis of their oligosaccharide structures. Figure 1 shows the CE electropherogram of oligomannose glycoforms of RNAse B. Analysis of the sugars released by hydrazine from RNAse B by SEC revealed that this glycoprotein consists of a mixture of five discrete populations, each characterized by one of the oligomannose structures from the series Man 9 to Man 5 (Figure 2). Comparison of Figures 1 and 2 shows that the relative proportions of these glycoforms correlates with the percentages of the five populations observed when the intact glycoprotein is resolved by CE. Table 1 compares the percentage of each glycoform as determined from the peak areas of the CE and SEC data. Excellent correlation between these data sets demonstrates that CE offers a direct method of analyzing the glycoforms in their correct molar proportions at the protein level. 2

3 Man5 Table 1. The Relative Proportions of the Glycoforms of Ribonuclease B Absorbance Man8 Man6 Man7 SEC % CE % Man Man Man Man Man Proportions were determined by Bio-Gel P-4 SEC and CE of the glycoprotein. The percentages were obtained by integration of the peak area data in Figures 1 and 2. Man Time (min) Figure 1. CE profile showing the resolution of the naturally occurring oligomannose glycoforms of bovine pancreatic RNAse B Furthermore, it appears that, in this instance, CE is a sensitive technique for the quantification of minor glycoform populations. Although the glycoform containing Man 9 oligosaccharides comprises only 4% of the total population, it was able to be quantified accurately by integration of the CE peak data. glucose units Radioactivity Man9 Man8 Man7 Man6 Man Time (min) Figure 2. Size exclusion chromatogram of the N-linked oligomannose oligosaccharides released by hydrazinolysis from RNAse B and radiolabeled by reduction with tritiated sodium borohydride. The figure shows radioactivity (vertical axis) plotted against retention time. Numerical superscripts refer to the elution volume of glucose oligomers (dextran hydrolysate) in glucose units, as detected simultaneously by the refractive index monitor (data not shown). Peaks 1-5 indicate the elution time of the oligomannose series Man 5 (1) to Man 9 (5). 3

4 Monitoring of Enzymatic Digestion The enzymatic digestion of RNAse B by J. Bean α mannosidase was followed by CE (Figure 3). The enzyme reduced all the populations of RNAse B to a single population of the Man 1 glycoform in 95 min (See Figure 4 for the structures of Man 9 and Man 1 glycoforms of RNAse B). CE is a simple and fast way to follow enzymatic digestions of oligosaccharides still attached to a glycoprotein. In this case the sample was digested in the CE autosampler vials and injections were made directly into the capillary from the reaction mixture. This technique eliminates the need to take aliquots manually throughout the time course and to stop the reaction with reagents which may subsequently interfere with the CE analysis. Such studies may allow further insight into the mechanism of enzyme digestions and into the role of the protein in oligosaccharide biosynthesis. Conclusion The above-described work is a novel application of CE which has opened up new possibilities in the field of glycobiology. The glycoform populations have been separated, without destruction, in their correct molar proportions, directly at the protein level in a single step. In the case of a protein with a single glycosylation site, routine analysis of the glycoform populations by CE at the protein level is possible once the peaks have been assigned by analyzing the released sugars (e.g., by mass spectrometry, anion exchange chromatography, or size exclusion chromatography). When a protein has multiple glycosylation sites, the population of sugars at each site may be similarly analyzed following the isolation of each site by proteolytic cleavage. Complete resolution and identification of every glycoform of a glycoprotein may not always be possible or necessary even if it contains only one glycosylation site. For example, when glycoproteins are manufactured in vitro, the nature of the glycosylation may differ both from the naturally occurring substance and from previous preparations. If a complete analysis of glycosylation is not possible or appropriate, a sensitive fingerprinting of the glycosylation by CE may be sufficient to indicate whether or not significant glycosylation changes have occurred. This novel application of CE to the analysis of the glycosylated variants of ribonuclease B may, therefore, represent a useful advance in the technology available for the study of variations in glycoform populations. Absorbance RNAse Man5 T RNAse Man4 T 35 min RNAse Man2 T 55 min T 95 min RNAse Man Time (min) Figure 3. Capillary electrophoresis profiles of RNAse B showing part of the time course for the digestion of the glycoprotein with Jack Bean α-mannosidase which converts the population of naturally-occurring glycoforms to a single population of RNAse Man

5 Manα(1 2)Manα(1 6) RNAse Man 9 Manα(1 6) Manα(1 2)Manα(1 3) Manβ(1 4)GlcNacβ(1 4)GlcNacβ(1 N)Asn Manα(1 2)Manα(1 2)Manα(1 3) Jack Bean α-mannosidase RNAse Man 1 Manβ(1 4)GlcNacβ(1 4)GlcNacβ(1 N)Asn Figure 4. The oligomannose structure associated with the Man 9 glycoform of RNAse B, and the Man 1 glycoform which results from the digestion of RNAse B with J. Bean α-mannosidase References 1. Parekh, R. B., Dwek, R. A., Sutton, B. J., Fernandes, D. L., Leung, A., Stanworth, D., Rademacher, T.W., Mizuochi, T., Taniguchi, T., Matsuta, K., Takeuchi, F., Nagano, Y., Miyamolo, T., Kobata, A. Association of rheumatoid arthritis and primary osteoarthritis with changes in the glycosylation pattern of total serum lgg. Nature 316, (1985) 2. Parekh, R. B., Dwek, R. A., Thomas, J. R., Opdenakker, G., Rademacher, T. W., Wittwer, A. J., Howard, S. C., Nelson, R., Siegel, N. R., Jennings, M. G., Harakas, N. K., Feder, J. Cell-type-specific and site-specific N-glycosylation of type 1 and type 2 human tissue plasminogen activator. Biochemistry 28, (1989) 3. Parekh, R. B., Dwek, R. A., Rudd, P. M., Thomas, J. R., Rademacher, T. W., Warren, T., Wun, T.-C., Hebert, B., Reitz, B., Palmier, M., Ramabhadran, T., Tiemeier, D. C. N- glycosylation and in vitro enzymatic activity of human recombinant tissue plasminogen activator expressed in chinese hamster ovary cells and a murine cell line. Biochemistry 28, (1989) 4. Ashford, D. A., Dwek, R. A., Welply, J. K., Amatayakul, S., Homans, S. W., Lis, H., Taylor, G. N., Sharon, N., Rademacher, T. W. The β1 2 D-Xylose and α1-3-l-fucose substituted N linked oligosaccharides from Erythrina cristagalli lectin. Isolation, characterization and comparison with other legume lectins. Eur. J. Biochem. 166, (1987) 5. Amatayakul-Chantler, S., Ferguson, M. A. J., Dwek, R. A., Rademacher, T. W., Parekh, R. B., Crandall, I. E., Newell, P. C. Cell surface oligosaccharides on Dictyostelium during development. J. Cell Science 99, (1991) 6. Rudd, P. M., Scragg, I. G., Coghill, E. C., Dwek, R. A. Separation and analysis of the glycoform populations of ribonuclease B using capillary electrophoresis. Glycoconjugate Journal 9, (1992) All trademarks are the property of their respective owners. 5

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