4 Drug Development Group, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester M20 4BX, UK
5 Medical Oncology Department, University of Manchester, Manchester M20 4BX, UK
Received on May 24, 2002; revised on August 14, 2002; accepted on August 21, 2002
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: exoenzymes / glycosaminoglycan / heparin / proteoglycan / sequencing
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Heparin consists of alternating uronic acid, either D-glucuronic or L-iduronic, and D-glucosamine; the glucosamine can be N-acetylated or N-sulfated (Linhardt et al., 1992; Conrad, 1998
). The disaccharides can be further O-sulfated at C6 and/or C3 of the D-glucosamine and at C2 of the uronic acid residue. This highly acidic structure has been found to bind to more than 100 proteins, with the basic amino acids arginine and lysine being critical constituents of many protein binding sites. These proteins range from growth factors, cytokines, serine proteases, and proteins involved in lipid metabolism to components of the extracellular matrix. It is thought that many of them recognize specific sulfation patterns within the polysaccharide chain, the prototypic sequence being the antithrombin-binding pentasaccharide, where a rare 3-O-sulfate group is one of the critical components (Lindahl et al., 1980
). In another example, less sulfated areas of heparin were thought to be essential within the binding sites for a number of chemokines (Witt and Lander, 1994
). Heparin is able to affect the activity of a large number of these ligands in vitro, but it is likely that in many cases the physiological effector will be the chemically related, more ubiquitously expressed HS. In contrast to heparin, for which the majority of glucosamines are N-sulfated, only about 50% are N-sulfated in HS (Stringer et al., 1999
). The N-sulfated disaccharides in HS occur in blocks, which are also rich in O-sulfation, and these sulfated domains have been found to be important for the activation of growth factors such as basic fibroblast growth factor (Walker et al., 1999). However, due to the lower cost of heparin compared to HS and its often greater potency in binding proteins of therapeutic interest, such as a number of angiogenic growth factors, heparin is of great interest to the research community.
Over the past few years there has been a great deal of research aimed at developing techniques for sequencing of HS and heparin, to enable characterization of specific protein binding sites and comparison of polysaccharides from different tissue sources. These include approaches where the oligosaccharides are end-labeled by fluorescence or radioisotopes allowing their subsequent detection by high-performance liquid chromatography (HPLC) and polyacrylamide gel electrophoresis (PAGE), and the use of mass spectrometry (Turnbull et al., 1999; Vives et al., 1999
; Juhasz and Biemann, 1994
; Venkataraman et al., 1999
). Most of these methodologies have been developed with nonmetabolically labeled sources of HS and heparin in mind, which are available commercially. One technique by Merry et al. (1999)
has been developed for sequencing of the small amounts of HS that can be metabolically 3H-labeled and isolated from cultured cells. This article describes a complementary sequencing technique specifically designed for the trace quantities of heparin that can be obtained from tissue culture. In the present study, due to the highly sulfated heparin structure we are able to use the much less expensive 35SO4 for metabolic radiolabeling. Partial nitrous acid scission was used for initial oligosaccharide isolation as opposed to heparinase III used by Merry et al. (1999)
. Heparinase III produces oligosaccharides that are unsaturated at the nonreducing end; this prevents treatment of this terminal disaccharide with most of the sequencing enzymes and makes it difficult to identify, but it has the advantage of providing a reference point to the nonreducing end. In contrast, the oligosaccharides produced by partial nitrous acid treatment have the advantage that the nonreducing end is fully susceptible to exoenzyme scission. The technique is ideal for sequencing heparin isolated directly from native sources, such as the mastocytoma cells, glial progenitor cells, or Ascidia, although it could also be used for highly sulfated regions of HS. It is not suited to sequencing of low sulfated regions of HS, such as the regions between the sulfated domains where the level of incorporation of the 35SO4 will be minimal and the partial nitrous acid will not cleave the chain.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Predicted outcome of partial nitrous acid cleavage ofindividual heparin oligosaccharide species
Individual purified oligosaccharides from Figure 2 were partially depolymerized and the scission products separated by SAX-HPLC. Theoretically partial nitrous acid depolymerization of a dp6 (degree of polymerization is 6) could release two species of dp4 s and three dp2 s, if all the disaccharides were different and assuming all internal glucosamines are N-sulfated (Figure 3A). A dp10 could yield two dp8 s, three dp6 s, four dp4 s, and five dp2 s (Figure 3A). Heparin is relatively uniform in structure, and sequencing methods for oligosaccharides may have to identify the location of only one or perhaps two disaccharides that differ from the predominant -L-iduronate 2-O-sulfate (IdoA 2S)GlcNS 6-O-sulfate (6S) (Montgomery et al., 1992
). If, for example, only one internal disaccharide was disulfated (as oppose to trisulfated) in a dp10 fragment, then partial nitrous acid treatment would yield two dp8 s, two dp6 s, three dp4 s, and two dp2 sfar fewer species than when all the disaccharides are different (Figure 3B). Consequently, the partial nitrous acid profile of oligosaccharides, especially combined with disaccharide analysis, immediately gives clues to the sequence.
|
|
|
|
|
Treatment of the components of 8c (Figure 7A) with iduronidase resulted in no shifts of the peaks, apart from IdoA-aMan(6S) (fraction 37) to aMan(6S) (fraction 31) (Figure 7B). In contrast, combined 2-sulfatase and iduronidase treatment resulted in movement of the 8, 6 s, 4 s, as well as the IdoA-aMan(6S) (Figure 7C), demonstrating that there were IdoA(2S) groups at disaccharide positions 1, 2, and 3 of the octasaccharide (counting from the nonreducing end, Figure 7F). Additional treatment with 6-sulfatase again shifted the dp8, dp6 s, and dp4 s, showing disaccharides 1, 2, and 3 also had 6S groups. Consequently the remaining fourth position disaccharide must be the GlcA/IdoA-aMan(6S). PAGE confirmed the partial nitrous acid components of 8C and the appropriate movement of the dp8 and dp6 bands with enzyme treatments (data not shown), although again the smaller oligosaccharides were difficult to detect.
Sequencing of hexasaccharide 6d
Figure 8A shows the partial nitrous acid depolymerization profile of dp6d, which had a predominant species by PAGE (upper band, Figure 2A). It released two dp4 s and two dp2 s (Figure 8). None of the peaks shift on treatment with iduronidase (Figure 8B), but the 6, 4 s, and IdoA(2S)-aMan(6S/ 3-O-sulfate [3S]?) all shifted with combined 2-sulfatase and iduronidase treatment (Figure 8C), indicating there were IdoA(2S) residues at disaccharide positions 1 and 2 (Figure 8E). Because the disaccharide at fraction 32 did not move with combined 2-sulfatase and iduronidase treatment, it is probably GlcA-aMan(6S) or GlcA(2S)-aMan; disaccharide analysis revealed that it was GlcA(2S)-aMan (data not shown). We already know from the iduronidase and iduronidase/2-sulfatase treatments that positions 1 and 2 are IdoA(2S), so the GlcA-aMan(6S) must be in the third position (Figure 8E). The dp6 and dp4a' shifted further with 6-sulfatase treatment, demonstrating the disaccharide at position 1 was 6-sulfated and not the disaccharide at position 2 (Figure 8E). This would so far give us the sequence IdoA(2S)-GlcNS(6S)-IdoA(2S)-GlcNS-GlcA(2S)-aMan. However, there is no IdoA(2S)-aMan disaccharide released and SAX-HPLC elution positions of dp4a', which from its sequencing must be the nonreducing terminal dp4, suggest that it is initially a pentasulfated dp4 like the IdoA(2S)-GlcNS(6S)-IdoA(2S)-aMan(6S) released from 6b, 8a, 10a, 8c, and 6e. As dp4a' in 6d does not have a reducing terminal 6S we surmise that it may have a 3S to achieve the same charge-density. Nitrous acid deamination/disaccharide anal-ysis of dp6d only showed two peaks, the Glc(2S)-aMan and a peak eluting in the same place as IdoA(2S)-aMan(6S), but an unidentified peak that may be 4,5-unsaturated hexuronate (UA) (2S)GlcNS(3S) was easily separated from the IdoA(2S)-GlcNS(6S) when heparinase I, II, and III depolymerisation/disaccharide analysis was carried out, this eluted shortly after the UA(2S)-GlcNS(6S) standard (data not shown).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our initial isolation of heparin oligosaccharides for sequencing was by size-exclusion gel chromatography and SAX-HPLC. However, the purity of the major oligosaccharides released from heparin for which the sequences are shown was confirmed by PAGE analysis. Analogous to Vives et al. (2001), we found that a number of reproducible single peaks on the SAX-HPLC could contain up to four main oligosaccharide species distinguishable by PAGE. The sequencing described was carried out on oligosaccharides that were largely homogeneous by SAX-HPLC and PAGE analysis.
The major analysis of the oligosaccharide sequencing was carried out by SAX-HPLC. Because all of the components released from the oligosaccharides by the partial nitrous acid were susceptible to the exoenzymes, the profiles were relatively straightforward to interpret. Structures were confirmed by analysis of disaccharide composition of sequenced fragments. We also investigated the use PAGE/electroblotted gels for analysis of the sequencing steps. This procedure was largely satisfactory, but despite prolonged exposure of the blots the disaccharide was barely visible, making SAX-HPLC the preferred methodology. The advantage of PAGE was that there was a large shift with removal of iduronidase, which was sometimes more difficult to discern by change in elution position on SAX-HPLC. The blots may have advantages though in a situation where components of an oligosaccharide are more easily separated by PAGE than by SAX-HPLC.
Oligosaccharides were sequenced that had been released from preparations of mastocytoma heparin degraded to a variety of extents to enable an overall picture of mastocytoma heparin structure to be obtained. This demonstrated that the majority of oligosaccharides contained only disaccharides that were N-, 2-O-, and 6-O-sulfated (i.e., dp6b, 8a, and 10a) in agreement with this being the most common disaccharide in mastocytoma heparin (Montgomery et al., 1992). Several oligosaccharides released by partial nitrous acid had a single O-sulfate group "missing" in one of the disaccharide (e.g., dp6e, dp8c). There also appears from PAGE analysis of 8d and 10c (lower bands) to be rare oligosaccharides present with two sulfate groups "missing" and disaccharide analysis (data not shown) indicated that in the case of 8d a single disaccharide lacked O-sulfation. This low level of variation reduces the sequencing problem to identifying the position of a single variant disaccharide (Table I). Our findings suggest that the disaccharides within heparin that are not trisulfated are spaced out across the molecule, as opposed to being clustered like the N-acetylated domains of HS. Such detailed analysis of the overall structure of the heparin chain has not been carried out before.
|
The data presented in this article give further insights into the structure of heparin than previous studies. The technique described is easily carried out with standard laboratory equipment and will enable many groups to investigate structurefunction relationships of heparins from biological sources, such as cell culture.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell culture and heparin isolation
Mastocytoma cells were cultured at 37°C (5% CO2 in air) in Dulbecco's modified Eagle medium F12 Nut mix with 5% fetal calf serum, 2 mM glutamine, penicillin (100 U/ml), and streptomycin (100 µg/ml). Radiolabeling of the cells was carried out in the culture medium without the antibiotics with the addition of Na235SO4 (20 µCi/ml) for 72 h. The cell suspension was then treated with pronase enzyme (100 µg/ml) at 37°C for around 16 h before addition of 1% triton for a further 3 h at room temperature. The suspension was then loaded onto a DEAE sephacel column (1x10 cm) equilibrated in 0.4 M NaCl, 20 mM phosphate buffer, pH 7.3. The column was washed in the same buffer at 10 ml/h until the phenol red from the culture medium was removed. Bound material was eluted with 1.5 M NaCl, 20 mM phosphate buffer, pH 7.3. Radioactive fractions were pooled and desalted on a Sephadex G50 column (1x30 cm) run in 0.1 M ammonium bicarbonate at 10 ml/h. The radiolabeled glycosaminoglycans were freeze-dried, then treated with alkali borohydride to remove any attached peptides, desalted again, and the chondroitinase ABC was treated.
Preparation of oligosaccharides
For each preparation of heparin a small amount (10,000 cpm) was tested to find the optimum time or combination of times (around 90120 min) for its partial breakdown into a range of intermediates by dilute nitrous acid (9.5 mM HCl, 500 µM NaNO2) on ice, before treatment of the whole sample. The reaction was stopped by addition of ice-cold sodium acetate pH 5 to 0.12 M. The resultant oligosaccharides were separated by Biogel P10 column chromatography (1x1.2 m) run at 5 ml/h in 0.25 M ammonium bicarbonate. Peak positions of di-, tetra-, hexa-, octa-, and decasaccharides were determined by comparison to the prior elution positions of oligosaccharides on the same column, and these were pooled and freeze-dried two to three times. Each sized pool was then injected via a 2-ml loop onto a ProPac PA-1 SAX-HPLC column (4.6x 250 mm) and separated with a linear gradient from 0 to 2 M NaCl pH 3.0 over 120 min at a flow rate of 1 ml/min. Samples (0.5 ml) were collected, aliquots analyzed for radioactivity by liquid scintillation counting, and the separated oligosaccharide species pooled. Each oligosaccharide pool was desalted on a PD10 column in distilled water and freeze-dried. Each pool was rerun on the SAX-HPLC to confirm that it was a single peak.
Blotting
Each oligosaccharide pool was also analyzed by blotting to determine the level of purity. Each species (5000 cpm) was loaded onto polyacrylamide gels, which were prepared and run as in Vives et al. (2001). The gel was then blotted onto BioTrace HP positively charged membrane (0.45 µm pore size), sandwiched between six pieces of filter paper (everything having been presoaked for at least 10 min in 10 mM Tris, 0.5mM ethylenediamine tetra-acetic acid, 5 mM sodium acetate, pH 7.8), on a semi-dry blotter for 2 h at 200 mA. The membrane was dried and exposed against film at -80°C overnight, then for longer, up to 2 weeks, if necessary.
Sequencing
Each oligosaccharide species (100,000 cpm) was treated with dilute nitrous acid (19 mM HCl, 1 mM NaNO2) in 100 µl on ice. Aliquots of 25 µl were stopped with 37.5 µl of ice-cold 0.2 M sodium acetate pH 5 at 15, 30, 60, and 120 min. The four samples were then pooled together and 1/10th of the mixture separated by SAX-HPLC, as in the preparation of oligosaccharides. The remaining sample was desalted on a PD10 column and lyophilized, prior to treatment with the exoenzymes. Digests were set up in a total volume of 25 µl of 40 mM sodium acetate, pH 4.5. 2-Sulfatase and iduronidase were used either singly or in combination. 6-Sulfatase was only used in combination with the other two enzymes. The reactions were incubated for 24 h at 37°C. Samples were then made up to 2 ml with distilled water/HCL, pH 3.0, and analyzed by SAX-HPLC as previous.
Disaccharide analysis
Samples of 5000 cpm of each oligosaccharide species were completely depolymerized by treatment with low pH nitrous acid, as described by Shively and Conrad (1976). P10 Biogel chromatography was utilized to confirm that the heparin was completely degraded to disaccharides. The disaccharides were then injected via a 2-ml loop onto a ProPac PA-1 SAX-HPLC column (4.6x250 mm), which was washed for 2 min in distilled water/HCL, pH 3.0, then separated via a stepped gradient of 0120 mM NaCl, pH 3.0, in 40 min and 120400 mM NaCl, pH 3.0, in 55 min, at a flow rate of 1 ml/min. Fractions of 0.25 ml were collected from 23110 and 1 ml over the rest of the profile. Aliquots were analyzed for radioactivity by liquid scintillation counting. An identical run was carried out with anhydromannose disaccharide standards to enable identification of the samples' disaccharide components. To aid identification of some oligosaccharides, heparinase enzyme I, II, and III depolymerization and disaccharide analysis was also carried out (Stringer et al., 1999
).
![]() |
Acknowledgements |
---|
![]() |
Footnotes |
---|
3 Present address: Department of Chemistry and Biological Sciences, University of Huddersfield, UK
1 To whom correspondence should be addressed; e-mail: sallyelizabethstringer{at}yahoo.co.uk
![]() |
Abbreviations |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cavalcante, M.C., Allodi, S., Valente, A.P., Straus, A.H., Takahashi, H.K., Mourao, P.A., and Pavao, M.S. (2000) Occurrence of heparin in the invertebrate styela plicata (Tunicata) is restricted to cell layers facing the outside environment. An ancient role in defense? J. Biol. Chem., 275, 3618936186.
Conrad, H.E. (1998) Heparin-binding proteins. Academic Press, San Diego, CA.
Dietrich, C.P., de Paiva, J.F., Moraes, C.T., Takahashi, H.K., Porcionatto, M.A., and Nader, H.B. (1985) Isolation and characterization of a heparin with high anticoagulant activity from Anomalocardia brasiliana. Biochim. Biophys. Acta, 843, 17.[ISI][Medline]
Juhasz, P. and Biemann, K. (1994) Mass spectrometric molecular-weight determination of highly acidic compounds of biological significance via their complexes with basic polypeptides. Proc. Natl Acad. Sci. USA, 91, 43334337.[Abstract]
Kojima, T., Leone, C.W., Marchildon, G.A., Marcum, J.A., and Rosenberg, R.D. (1992) Isolation and characterization of heparan sulfate proteoglycans produced by cloned rat microvascular endothelial cells. J. Biol. Chem., 267, 48594569.
Lindahl, U., Backstrom, G., Thunberg, L., and Leder, I.G. (1980) Evidence for a 3-O-sulfated D-glucosamine residue in the antithrombin-binding sequence of heparin. Proc. Natl Acad. Sci. USA, 77, 65516555.[Abstract]
Linhardt, R.J., Ampofo, S.A., Fareed, J., Hoppensteadt, D., Mulliken, J.B., and Folkman, J. (1992) Isolation and characterization of human heparin. Biochemistry, 31, 1244112445.[ISI][Medline]
Litjens, T., Bielicki, J., Anson, D.S., Friderici, K., Jones, M.Z., and Hopwood, J.J. (1997) Expression, purification and characterization of recombinant caprine N-acetylglucosamine-6-sulphatase. Biochem. J., 327, 8994.[ISI][Medline]
Merry, C.L., Lyon, M., Deakin, J.A., Hopwood, J.J., and Gallagher, J.T. (1999) Highly sensitive sequencing of the sulfated domains of heparan sulfate. J. Biol. Chem., 274, 1845518462.
Montgomery, R.I., Lidholt, K., Flay, N.W., Liang, J., Vertel, B., Lindahl, U., and Esko, J.D. (1992) Stable heparin-producing cell lines derived from the Furth murine mastocytoma. Proc. Natl Acad. Sci. USA, 89, 1132711331.[Abstract]
Shively, J.E. and Conrad, H.E. (1976) Formation of anhydrosugars in the chemical depolymerization of heparin. Biochemistry, 15, 39323942.[ISI][Medline]
Stringer, S.E., Mayer-Proschel, M., Kalyani, A., Rao, M., and Gallagher, J.T. (1999) Heparin is a unique marker of progenitors in the glial cell lineage. J. Biol. Chem., 274, 2545525460.
Turnbull, J.E., Hopwood, J.J., and Gallagher, J.T. (1999) A strategy for rapid sequencing of heparan sulfate and heparin saccharides. Proc. Natl Acad. Sci. USA, 96, 26982703.
Unger, E.G., Durrant, J., Anson, D.S., and Hopwood, J.J. (1994) Recombinant alpha-L-iduronidase: characterization of the purified enzyme and correction of mucopolysaccharidosis type I fibroblasts. Biochem. J., 304, 4349.[ISI][Medline]
Venkataraman, G., Shriver, Z., Raman, R., and Sasisekharan, R. (1999) Sequencing complex polysaccharides. Science, 286, 537542.
Vives, R.R., Pye, D.A., Salmivirta, M., Hopwood, J.J., Lindahl, U., and Gallagher, J.T. (1999) Sequence analysis of heparan sulphate and heparin oligosaccharides. Biochem. J., 339, 767773.[CrossRef][ISI][Medline]
Vives, R.R., Goodger, S., and Pye, D.A. (2001) Combined strong anion-exchange HPLC and PAGE approach for the purification of heparan sulphate oligosaccharides. Biochem. J., 354, 141147.[CrossRef][ISI][Medline]
Walker, A., Turnbull, J.E., and Gallagher, J.T. (1994) Specific heparan sulfate saccharides mediate the activity of basic fibroblast growth factor. J. Biol. Chem., 269, 931935.
Witt, D.P. and Lander, A.D. (1994) Differential binding of chemokines to glycosaminoglycan subpopulations. Curr. Biol., 4, 394400.[ISI][Medline]