Heparin sequencing

Sally E. Stringer12,4, Balbant S. Kandola4, David A. Pye3,4 and John T. Gallagher5

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
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Heparin is a highly sulfated glycosaminoglycan widely used as an anticoagulant. Modifications in its relatively uniform structure appear to be key to its recognition and modulation of serine proteases, growth factors, chemokines, and extracellular proteins, as has been most clearly demonstrated in the antithrombin binding site. We sequenced the major oligosaccharides released from mastocytoma heparin by partial nitrous acid using a highly sensitive technique tailored for sequencing of metabolically radiolabeled heparin. It utilizes partial nitrous acid cleavage to allow simultaneous sequencing of the internal components of the oligosaccharide under investigation by specific lysosomal exoenzymes. Sequencing revealed that although the majority of the heparin disaccharides are N-, 2-O-, and 6-O-sulfated, the less sulfated disaccharides (lacking 2-O- or 6-O-sulfates) seem to be spaced out along the chain. The technique may be particularly useful for characterizing heparin from novel sources, such as the glial progenitor cells and Ascidia, as well as for sequencing protein binding sites.

Key words: exoenzymes / glycosaminoglycan / heparin / proteoglycan / sequencing


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Heparin is a highly sulfated polysaccharide that has been used for many years as an anticoagulant (for review see Conrad, 1998Go). Its physiological role is still unclear, because blood coagulation is mostly achieved by endothelial cell-derived heparan sulfate (HS) proteoglycans (Kojima et al., 1992Go). Originally heparin was thought to be produced exclusively by connective tissue mast cells as the polysaccharide component of the intracellular proteoglycan serglycin, but it has recently been discovered somewhat suprisingly in glial progenitor cells as a component of a cell surface proteoglycan (Stringer et al., 1999Go). This finding inspires the thought that other yet undiscovered niches of heparin expression may occur and could be critical for the complex process of mammalian development. Heparin has also recently been discovered in the invertebrate Ascidia, with almost identical disaccharide composition to the glial heparin (Cavalcante et al., 2000Go). A heparin-like glycosaminoglycan (denoted as mactin) has previously been found in some species of mollusc (Dietrich et al., 1985Go).

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., 1992Go; Conrad, 1998Go). 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., 1980Go). 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, 1994Go). 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., 1999Go). 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., 1999Go; Vives et al., 1999Go; Juhasz and Biemann, 1994Go; Venkataraman et al., 1999Go). 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)Go 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)Go. 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
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Optimization of partial nitrous acid conditions
Partial nitrous acid treatment, which cleaves at a proportion of the {alpha}-D-N-sulfoglucosamine (GlcNS) residues, converting these to anhydromannose, was used to generate a range of different sized oligosaccharides from mastocytoma heparin. The length of time of deamination, dilution of the reagents, and temperature were optimized to isolate the greatest number of oligosaccharides in the hexasaccharide to decasaccharide size. During this process we also confirmed that both mild and extensive nitrous acid degradation conditions released the same main species of oligosaccharides. Figure 1A depicts 35S-heparin partially degraded by a short treatment with nitrous acid (30 min at room temperature in 19 mM HCl, 1 mM NaNO2) and Figure 1B a milder but longer treatment (half reaction stopped at 90 min and half at 120 min, 9.5 mM HCl, 500 µM NaNO2 on ice), which have been separated on a Biogel P10 column. In Figure 1A the hexa-, octa-, and decasaccharides were 4.4%, 6.4%, and 5.6% of the total material and in Figure 1B 12.6%, 7.9%, and 7.2%, respectively (discounting the residual 5% chondroitin sulfate seen in the void peak). The latter milder conditions were deemed favorable, allowing more time control on the extend of the digest, and the deamination time was optimized for each new heparin preparation from the mastocytoma cells.



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Fig. 1. Size separation of heparin oligosaccharides. Mouse mastocytoma cell heparin was degraded by partial nitrous acid treatment. A depicts a short scission (30 min at room temperature in 19 mM HCl, 1 mM NaNO2) and B a milder but longer scission (half reaction stopped at 90 and half at 120 min, 9.5 mM HCl, 500 µM NaNO2 on ice). The products of the reaction have been separated on a Biogel P10 size exclusion column (1x1200 mm) at a flow rate of 5 ml/h in 0.25 ammonium bicarbonate. Elution profiles were monitored by scintillation counting of 35S. In C the octasaccharide peak from A has been isolated and rerun on the same column.

 
Size fractionation of heparin oligosaccharides
The central points from each oligosaccharide peak eluted on the P10 were pooled to isolate single sized pools. Figure 1C shows that the octasaccharide peak from the preparation in Figure 1A reruns as a single peak on the same P10 column. Further confirmation of the size purity of the pooled species was obtained from the lack of overlap in the position of the different-sized species on the polyacrylamide gels (Figure 2, insets).



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Fig. 2. Isolation of individual heparin oligosaccharide species from sized pools by SAX-HPLC and PAGE. Sized dp6 (A), dp8 (B), and dp10 (C) oligosaccharide populations were applied to a ProPac PA-1 column eluted with a linear gradient of 0–2 M NaCl, pH 3.5, at a flow rate of 1 ml/min, and 0.5-ml fractions collected. Elution profiles were monitored by scintillation counting of 35S. Central fractions of peaks from each SAX-HPLC run were pooled and further analyzed by western blotting (insets).

 
Separation of heparin oligosaccharides accordingto charge-density
Sized oligosaccharide pools were separated according to charge-density by strong anion exchange (SAX)-HPLC. The HPLC profiles for the hexa-, octa-, and decasaccharides isolated in Figure 1B are shown in Figure 2. These profiles were reproducible between heparin preparations, with 6b, 8a, and 10a consistently being the dominant species, except for the material from the short deamination shown in Figure 1A, where noticeably octasaccharide 8b was most prevalent (data not shown). The separated oligosaccharide species were stringently pooled, often only as a single HPLC fraction and rerun on the HPLC to confirm they were a single peak. However, when subsequently separated on a polyacrylamide gel a number of these peaks were found to contain two or more oligosaccharide species (Figure 2 inset). Those which were single species, including the predominant peaks 6b, 8a, and 10a, were selected for sequencing.

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 {alpha}-L-iduronate 2-O-sulfate (IdoA 2S)–GlcNS 6-O-sulfate (6S) (Montgomery et al., 1992Go). 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 s—far 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.



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Fig. 3. Theoretical oligosaccharides released from hexa-, octa-, and decasaccharides by partial nitrous acid treatment. Each sphere represents a disaccharide, with the different shading patterns depicting different disaccharide types. A demonstrates the partial nitrous acid scission of a decasaccharide (dp10), in which all the disaccharides are different. This sequence yields two octasaccharides (dp8), three hexasaccharides (dp6), four tetrasaccharides (dp4), and five disaccharides (dp2). B shows the partial nitrous acid scission of a decasaccharide in which only one disaccharide is different from the rest (as seen in this study). In this case two octasaccharide species, two hexasaccharides, three tetrasaccharides, and two disaccharides are released. The products in A and B would all exist simultaneously. The inset dashed-line and dotted-line triangles show the maximum number of different products from scission of an octa- and a hexasaccharide, respectively.

 
Sequencing of the major hexa-, octa-, and decasaccharide heparin oligosaccharides by SAX-HPLC
Partial nitrous acid profiles for the major species, 6b, 8a, and 10a, were very simple (Figure 4A–C). The presence of single species of dp2 s, dp4 s, dp6 s, and dp8 s in these profiles immediately indicated that 10a (as well as 8a and 6b) must have a uniform structure, with all the disaccharides being identical and all N-sulfated. In addition the elution positions suggested that 6b and 8a were breakdown products of 10a and 6b of 8a. The elution position of the single dp2 species in each profile (Figure 4A–C, ISMS) was compared to disaccharide standards and shown to be IdoA(2S)-GlcNS(6S), (IdoA(2S)–anhydromannose [aMan] (6S) after nitrous acid treatment), the major constituent of mast cell heparin. This was confirmed by disaccharide analysis (Figure 4D, E). So 6b, 8a, and 10a must be [IdoA(2S)-GlcNS(6S]n-IdoA(2S)-aMan(6S), where n=2, 3, and 4, respectively.



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Fig. 4. Partial nitrous acid scission profiles of hexasaccharide 6b, octasaccharide 8a, and decasaccharide 10a. Fragment profiles of partial nitrous acid scission of (A) hexasaccharide 6b, (B) octasaccharide 8a, and (C) decasaccharide 10a resolved on a ProPac PA-1 column with a linear 0–2 M NaCl, pH 3.0, gradient over 120 min. (D) Disaccharide standards (E) representative profile of disaccharides generated by complete low-pH nitrous acid deamination of 6b, 8a, or 10a on a ProPac PA-1 column with a stepped gradient of 0–120 mM NaCl, pH 3.0, in 40 min and 120–400 mM NaCl, pH 3.0, in 55 min at a flow rate of 1 ml/min. GM, glucuronate-anhydromannose; IM, iduronate-anhydromannose; MS, anhydromannose-6-sulfate; GSM, glucuronate-2-O-sulfate-anhydromannose; ISM, iduronate-2-O-sulfate-anhydromannose; GMS/IMS, glucuronate-anhydromannose-6-O-sulfate/iduronate-anhydromannose-6-O-sulfate; ISMS, iduronate-2-O-sulfate-anhydromannose-6-O-sulfate; S, 35SO4.

 
Sequence analysis of 6b, 8a, and 10a is therefore very straightforward to interpret and succinctly demonstrates the main aspects of this technique. Figure 5 (up to the dashed line in each profile) depicts the sequencing of 8a. The profiles from sequencing of 10a were exactly the same as 8a, apart from the additional decasaccharide peak, so this has been added to the right of the dashed line in each profile. The peaks to the left of the dotted line are representative of sequencing of 6b. Treatment of the deamination products in Figure 5A with iduronidase resulted in no changes in their SAX-HPLC position (Figure 5B), the enzyme being inhibited by the presence of the 2S group on each of the nonreducing terminal iduronates (see Figure 5F for the oligosaccharide sequences). {alpha}-L-Iduronate-2-sulfatase (2-sulfatase) treatment, on the other hand, resulted in a shift to the left of all the component species, as removal of the 2S reduces the charge-density (Figure 5C, F). Note that the disaccharide is now in the appropriate position for IdoA-aMan(6S) (IMS) by comparison to disaccharide standards, although this cannot be distinguished from other monosulfated disaccharides on this HPLC gradient. A small shift to the right of all the oligosaccharides is seen with additional removal of the iduronates (i.e., as well as the 2Ss) (compare Figure 5D to C) with the monosaccharide aMan(6S) (MS), now appearing at the other side of the sulfur peak. Then a final shift to the left of the peaks occurs when the 6S is also removed (Figure 5E). The {alpha}-D-glucosamine-6-sulfatase (6-sulfatase) did not, however, appear to work as efficiently on the aMan(6S).



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Fig. 5. Sequence analysis of hexasaccharide 6b, octasaccharide 8a, and decasaccharide 10a. (A) Composite plot of the fragment profiles generated bypartial nitrous acid scission of 8a and 10a resolved on a ProPac PA-1 column with a linear 0–2 M NaCl, pH 3.0, gradient over 120 min. (The 10a decasaccharide peak was added to the 8a profile, see Figure 4B and C). Consequently the entire profile represents 10a, to the left of the dashed line 8aand to the left of the dotted line 6a after partial nitrous acid treatment. Aliquots of these fragments were subsequently digested with iduronidase (B),2-sulfatase (C), iduronidase and 2-sulfatase (D), and iduronidase, 2-sulfatase, and 6-sulfatase (E). Fractions of 0.5 ml were analyzed for 35S byscintillation counting. F shows the sequence of all the fragments at each stage of the analysis. MS, anhydromannose-6-sulfate; IMS, iduronate-anhydromannose-6-O-sulfate; ISMS, iduronate-2-O-sulfate-anhydromannose-6-O-sulfate; S, 35SO4.

 
Sequencing of the major hexa-, octa-, and decasaccharide heparin oligosaccharides by PAGE
Analysis of the 8a and 10a sequencing reactions by PAGE and electrotransfer to nylon membrane was also carried out, and this confirmed the results of the SAX-HPLC analysis (Figure 6A, B). In both 8a and 10a there were no shifts of the peaks with iduronidase treatment in the HPLC profiles (compare 5A to B) and similarly no movement of the 8a bands in Figure 6A. There was a shift downward of all the 8a and 10a bands after treatment with 2-sulfatase (Figure 6A and B, 2S) and a further significant downward shift when the IdoA is also removed by iduronidase plus 2-sulfatase (Ido + 2S). Additional removal of the 6S (Ido + 2S + 6S) results in another small drop of band positions. Unfortunately it is difficult to detect the smaller oligosaccharides, dp2 s and dp3 s (dp4 after removal of IdoA), with this technique, probably because they bind very weakly to the membrane.



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Fig. 6. Sequence analysis of octasaccharide 8a and decasaccharide 10a by PAGE. (A) Partial nitrous acid fragments, and A and B, exoenzyme-treated partial nitrous acid fragments, of octasaccharide 8a (A) and decasaccharide 10a (B) have been resolved on a 16x12x0.75 cm PAGE gel consisting of 30% acrylamide/5% crosslinker at a constant current of 25 mA for 3–4 h, then blotted. The bands were detected by autoradiography. The positions of the original decasaccharide, octasaccharide, hexasaccharide, tetrasaccharide, and disaccharide bands are indicated. The treatments were as follows, pNA, partial nitrous acid; Ido, iduronidase; 2-S, 2-sulfatase, Ido + 2-S, iduronidase plus 2-sulfatase; Ido + 2-S + 6-S, iduronidase, 2-sulfatase and 6-sulfatase. ISMS, iduronate-2-O-sulfate-anhydromannose-6-O-sulfate. The order of the lanes in A was rearranged from that in the original gel for consistency with B.

 
Sequencing of octasaccharide 8c
Octasaccharide 8c, like 8a, was also an oligosaccharide species that was homogeneous by PAGE analysis as well as by SAX-HPLC (Figure 2B). The slightly lower position of this band on the PAGE gel compared to 8a (Figure 2B inset) was equivalent to the loss of a single sulfate group, as seen with the sequencing gel of dp10a (Figure 6B). The partial nitrous acid profile of 8c revealed that it contained a hexasaccharide eluting at the same position as 6e as part of its structure (Figure 7A). Interestingly, 6e was found by PAGE to be two oligosaccharide species (Figure 2A inset), and the partial nitrous acid profile of 6e (data not shown) contained all three dp4 s seen in the partial nitrous acid profile of 8c (Figure 7A), this would not be possible from a single hexasaccharide species. However extension of the HPLC gradient in the region of the hexasaccharides (see Figure 7 legend) did not result in separation of these species in 6e. It was noticeable that there was only a low level of dp6b present in Figure 5A, which may have resulted from increased susceptibility of this hexasaccharide to partial nitrous acid or decreased susceptibility to it being released from 8c.



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Fig. 7. Sequence analysis of octasaccharide 8c. (A) Fragment profile generated by partial nitrous acid scission of 8b resolved on a ProPac PA-1 columnwith a linear 0–2 M NaCl gradient over 120 min. Aliquots of these fragments were subsequently digested with iduronidase (B), iduronidase and2-sulfatase (C), and iduronidase, 2-sulfatase, and 6-sulfatase (D). Fractions of 0.5 ml were analyzed for 35S by scintillation counting. (E) Representative profile of disaccharides generated by complete low-pH nitrous acid deamination of 8c on a ProPac PA-1 column with a stepped gradient of 0–120 mMNaCl, pH 3.0, in 40 min and 120–400 mM NaCl, pH 3.0, in 55 min, at a flow rate of 1 ml/min. F shows the sequence of all the fragments at each stageof the analysis. MS, anhydromannose-6-sulfate; GSM, glucuronate-2-O-sulfate-anhydromannose; GMS/IMS, glucuronate-anhydromannose-6-O-sulfate/iduronate-anhydromannose-6-O-sulfate; ISMS, iduronate-2-O-sulfate-anhydromannose-6-O-sulfate; S, 35SO4.

 
The pair of dp4 s eluting at around 100 in the 8C partial nitrous acid profile (Figure 7A) have been together labeled as dp4b; this is because sometimes they coeluted as a single peak, and they moved together throughout the sequencing treatment. Disaccharide analysis of dp4b revealed that it contains approximately equal levels of IdoA(2S)-aMan(6S) and of ß-D-glucuronate (GlcA)/IdoA-aMan(6S) (Figure 7E, compared to standards in Figure 4D) when the difference in radioactivity of the singly and doubly sulfated species is accounted for. From this we deduce that one of the dp4bs most likely contains the GlcA epimer and the other the IdoA epimer of the latter disaccharide. Consequently this enables us to identify the dp2 s in the partial nitrous acid profile of 8c as IdoA(2S)-aMan(6S), GlcA-aMan(6S), and IdoA-aMan(6S), the latter two initially eluting in the same position.

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 {Delta}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).



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Fig. 8. Sequence analysis of hexasaccharide 6d. (A) Fragment profile generated by partial nitrous acid scission of 6d resolved on a ProPac PA-1 column with a linear 0–2 M NaCl gradient over 120 min. Aliquots of these fragments were subsequently digested with iduronidase (B), iduronidase and 2-sulfatase (C), and iduronidase, 2-sulfatase, and 6-sulfatase (D). Fractions of 0.5 ml were analyzed for 35S by scintillation counting. E shows the sequence of allthe fragments at each stage of the analysis. MS, anhydromannose-6-sulfate; GSM, glucuronate-2-O-sulfate-anhydromannose; ISMS,iduronate-2-O-sulfate-anhydromannose-6-O(/3-O?)-sulfate; S, 35SO4.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
The present study demonstrates the successful implementation of a technique tailored for sequencing minute quantities of metabolically radiolabeled heparin or highly sulfated HS oligosaccharides without the requirement for end-labeling. This methodology has similarities to the HS step-sequencing method described previously by Merry et al. (1999)Go because it utilizes the partial nitrous acid cleavage to allow simultaneous sequencing of the internal components of the oligosaccharide under investigation by specific lysosomal exoenzymes. However it is novel in the use of partial nitrous acid for preparation of the oligoaccharides to sequence. In contrast to enzymatic cleavage by heparinase III, partial nitrous acid scission preserves the susceptibility of the nonreducing end of the oligosaccharides to all the exoenzymes. Another advantage of this technique for sequencing metabolically labeled heparin is the low cost of the SO4 radiolabel.

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)Go, 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., 1992Go). 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.


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Table I. Sequence comparisons for heparin oligosaccharides analyzed so far

 
The sequence of oligosaccharide 6d, IdoA(2S)-GlcNS(6S)-IdoA(2S)-GlcNS(3S?)-GlcA(2S)-aMan, was particularly interesting because of the possible presence of the 3S in the central disaccharide. Availability of 3-O-sulfatase in the near future will improve sequence analysis of rare oligosaccharides like this.

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 structure–function relationships of heparins from biological sources, such as cell culture.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Materials
Mastocytoma cells were a kind gift from Dr. J. Esko (Montgomery et al., 1992Go). Cell culture medium and donor calf serum were from Invitrogen Life Technologies (Paisley, Scotland). Na235SO4 (1050–1600 Ci/mmol) was from NEN Life Sciences (Stevenage, UK). DEAE sephacel, Sephadex G50, and PD10 columns were from Amersham Pharmacia Biotech (St. Albans, UK), Biogel P10 (fine grade) was from Biorad (Hemel Hampstead, UK), and ProPac PA1 analytical columns from Dionex (Camberley, Surrey, UK). Chondroitinase ABC (EC 4.2.2.4) was from Seikagaku Kogyo (Tokyo). BioTrace HP positively charged membrane (0.45 µm pore size) was from Pall-Gelman. Human 2-sulfatase (Bielicki et al., 1993Go), human {alpha}-L-iduronidase (Unger et al., 1994Go), and caprine {alpha}-D-N-acetylglucosamine-6-sulfatase (Litjens et al., 1997Go) were all purified recombinant enzymes kindly provided by John Hopwood (Adelaide) and are now available from Oxford Glycoscience (Abingdon, UK). Anhydromannose disaccharide standards were from Chirozyme.

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 90–120 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)Go. 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)Go. 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 0–120 mM NaCl, pH 3.0, in 40 min and 120–400 mM NaCl, pH 3.0, in 55 min, at a flow rate of 1 ml/min. Fractions of 0.25 ml were collected from 23–110 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., 1999Go).


    Acknowledgements
 
We would like to thank Chris Bishop for preparing mastocytoma heparin and John Hopwood (Adelaide) for the provision of exoenzymes. This work was funded by Cancer Research UK.


    Footnotes
 
2 Present address: Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455, USA Back

3 Present address: Department of Chemistry and Biological Sciences, University of Huddersfield, UK Back

1 To whom correspondence should be addressed; e-mail: sallyelizabethstringer{at}yahoo.co.uk Back


    Abbreviations
 
2-sulfatase, {alpha}-L-iduronate-2-sulfatase; 2S, 2-O-sulfate; 3S, 3-O-sulfate; 6-sulfatase, {alpha}-D-glucosamine-6-sulfatase; 6S, 6-O-sulfate; aMan, anhydromannose; dp, degree of polymerization; GlcA, {alpha}-D-glucuronate; GlcNS, {alpha}-D-N-sulfoglucosamine; HPLC, high-performance liquid chromatography; HS, heparan sulfate; IdoA, {alpha}-L-iduronate; UA, {Delta}4,5-unsaturated hexuronate; PAGE, polyacrylamide gel electrophoresis; SAX, strong anion exchange.


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
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