Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, and Division of Molecular and Vascular Medicine, BIDMC, Harvard Medical School, Boston, MA 02215
Received on November 20, 2003; revised on December 29, 2003; accepted on January 11, 2004
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Abstract |
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Key words: Glycosaminoglycans / capillary liquid chromatography / mass spectrometry / stable isotope labeling / biosynthetic mechanisms / critical groups
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Introduction |
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Two such HS structures that have been studied by us are the antithrombin III (AT III) binding site which binds to the ATIII molecule and accelerates its action, and the glycoprotein D (gD) binding site which binds to this Herpes simplex virus-1 (HSV-1) envelope protein and facilitates viral entry. The biosynthesis of these two rare motifs requires specific glucosaminyl 3-O-sulfotransferase (3-OST) enzymes, of which six isoforms have been cloned and characterized. Anticoagulant-active HS (HSact) can be produced by the action of either 3-OST-1 or 3-OST-5 (Liu et al., 1996; Xia et al., 2002
). The modifications necessary to complete formation of the gD binding site, however, can be catalyzed by any of four 3-OST isoforms (3-OST-2, 3-OST-3, 3-OST-4, and 3-OST-5) (Shukla et al., 1999
; Shukla and Spear, 2001
; Xia et al., 2002
). Because these enzymes sulfate at the C3 position of glucosamine monosaccharides within the heparan chain, the differences demonstrated by these isoforms probably reflects diversity in the regulation and generation of precursor structures. Due to this precursor specificity, the detection of a unique subset of saccharide residues can be diagnostic for the expression of certain sulfotransferases and for the elaboration of HS with critical groups that have important biological activities. However, these reactions do not convert all of the available precursor structures, resulting in substantial sequence diversity in the final chain. Consequently, these biosynthetic pathways lead to enormous structural heterogeneity in which no two chains are identical and thus render structural analysis of HS challenging.
To address these difficulties, we developed an ultra-sensitive approach that relies on the coupling of capillary high-performance liquid chromatography (HPLC) and electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS) to analyze saccharide residues derived from heparitinase digestion of HS chains. This technique allowed us to rapidly delineate the fine structural modifications elaborated on HS chains from a variety of sources as well as the spacing of critical groups within the motifs of these chains. We can now determine the expression of specific sulfotransferases and the biosynthetic pathways in which they participate within distinct tissues and individual cells.
The AT III binding site requires a minimal pentasaccharide sequence uniquely 3-O sulfated on the central glucosamine residue (Atha et al., 1984, 1987
; Kusche et al., 1988
). The specific pentasaccharide has been shown to be primarily responsible for the anticoagulant activity of heparin or heparan sulfate (Desai et al., 1998
; Petitou et al., 1987
). Commercial heparin is highly heterogeneous in nature, with different sulfation patterns along the chain and with relatively few glucuronic acids (accounting for less than 10% of the total uronic acid) (Sudo et al., 2001
). One purpose of the present study was to define the distribution of the rare glucuronic acid units within the chains of heparin in the context of mapping critical functional groups. It is known that epimerase requires N-sulfated glucosamine units at the nonreducing end of uronic acid for that residue to be epimerized (Jacobsson et al., 1984
; Kuberan et al., 2003
). Normally, GlcNAc residues precede glucuronic acids and the disaccharide unit GlcNAc-GlcA constitutes the precursor site for 3-OST-1 action (Linhardt et al., 1992; Zhang et al., 2001a
,b
). Spacing of these rare GlcNAc-glucuronic acids is unknown. The determination of spacing between these residues would help delineate the biosynthetic pathway leading to the generation of precursor sites for 3-OST-1 action.
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Results |
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The 3-OST-1 isoform is known to generate two specific disaccharides (Liu et al., 1999), one of which, GlcA-GlcNS3S6S, is readily detected as UA-GlcNS3S6S by LC/MS after enzymatic digestion (Figure 2A, inset). This disaccharide was observed as two species: one with a total labeled mass of 596 and the other 725. These values correspond to the combined mass of adduct ions formed between the disaccharide and either water or water plus the ion pairing reagent, DBA: [M-1H + H2O]1 and [M-2H + DBA + H2O]1, respectively. In addition, some smaller amount of saturated nonreducing-end terminal residues containing this 3-O-modification and having the same m/z maybe present as well. Also detected were two similar adduct ions with m/z of 594 and 723, which eluted within a peak having a slightly longer retention time (Figure 2A). These masses correspond to adduct ions formed between
UA2S-GlcNS6S and water or water along with DBA: [M-1H + H2O]1 and [M-2H + DBA + H2O]1, respectively (Figure 2A). Verification that this disaccharide residue contains 6-O-sulfate and must be
UA2S-GlcNS6S was proven by in vitro modification of CHO cell HS with recombinant 6-OST-1 and PAP34S (data not shown). This resulted in labeling of the unmodified compound in Figure 2A as well as an additional peak (predominantly m/z of 576: [M-1H]1), which appears later and coelutes with the
UA2S-GlcNS6S standard. Thus the resolution of these two trisulfated residues by LC/MS differs not only by their retention times but also by their adduct ion formation preferences (their ionic distribution). It is important to emphasize that the differences in the migration of modified disaccharides, independent of the interpretation of adduct ions, allow 3-OST-1-modified HS to be easily distinguished from unmodified HS.
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Despite the identical masses for all HS-derived trisulfated disaccharides, it was still possible to differentiate between the 3-O-labeled trisulfated disaccharide and the 6-O-containing UA2S-GlcNS6S residue. In addition to the differences in retention times between these species, they had different tendencies in forming adduct ions with DBA (Figure 2B, insets).
Overall, we found that adduct ion formation was significant for saccharides containing three or more sulfates only. As we have already demonstrated, preferential differences in adduct ion formation can be an important structural determinant that complements the other data in characterizing specific residues. The retention times and ionic distributions for each of the saccharide residues observed in mature HS are tabulated in Table II. These data allow for the easy identification of sulfotransferase product residues in labeled and unlabeled samples using LC/MS without the further need for standards. This is important, especially for 3-O-sulfate-containing residues, because model compounds of known structure are not readily available.
In vivo analysis of 3-OST isoform activity using LC/MS
To further verify the detection of specific residues by LC/MS, in vivo modified HS was analyzed based on the information derived from the in vitro studies. CHO cells expressing the ecotropic receptor for retroviral expression constructs were transduced with constructs for 3-OST-1, 3-OST-3A, or 3-OST-5, and subsequent HS purified from these cells was analyzed by LC/MS. The extracted ion current (XIC) for residues with three or more sulfates is given in Figure 3. The UA2S-GlcNS6S species was the only highly sulfated disaccharide observed in HS purified from wild type cells (Figure 3A). In contrast, 3-OST-1-transduced cells exhibited a small additional peak with retention time, mass spectrum, and ionic distribution consistent with
UA-GlcNS3S6S (m/z of 594 and 723, Figure 3B). Thus, the in vivo 3-OST-1 modification results for CHO cellderived HS were as expected from the in vitro results described. The in vivo results for 3-OST-3A were also consistent with the corresponding in vitro results (Figure 3C). Peaks for
UA2S-GlcNS3S,
UA2S-GlcNS3S6S, Tetra-A, and Tetra-B were detected as before (in vitro modification with either 3-OST-3A or 3-OST-4). Along with these species eluting at their expected positions, an additional peak was observed eluting just prior to the
UA2S-GlcNS6S + H2O peak (Figure 3C, inset). The resolved mass and ionic distribution of this additional peak are consistent with
UA2S-GlcNS3S in adduction with water alone ([M-1H + H20]1) or with DBA and water ([M-2H + H2O + DBA]1). It is less likely that this peak corresponds to terminal saturated residues due to their lower abundance.
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Purification and rechromatography of saccharide residues
For most saccharide residues, the use of LC/MS leads to adequate resolution. However, in cases when two residues are structurally very similar, separation between the two may be incomplete. We were able to enhance the detection of disaccharides that similarly elute with the use of a microfraction collector to capture residues for rechromatography in the absence of salt and other nonsaccharide contaminants that can perturb elution.
Figure 4 shows the results of a fractionation between two closely eluting trisulfated disaccharides, UA2S-GlcNS3S and
UA2S-GlcNS6S, derived from 3-OST-3A-transduced CHO cell HS. The capillary HPLC-fractionated samples were equally split between the ESI source and a robotic fraction collector, PROBOT (baiGmbH, Germany). Specific fractions containing the desired saccharides were identified by referring to the MS trace. During the first run the retention times of these two residues were not sufficiently different to give baseline separation (Figure 4A). Despite this, we were able to collect and rechromatograph fractions that contained predominantly one or the other disaccharide (Figures 4B and 4C). Another consequence of rechromatography of the sample was an increase in the intensity of the first peak (Figure 4B). This has been observed with other samples and may be attributable to the purification of the species from much of the interfering salt present in the original sample. Thus in addition to being able to resolve residues to near purity, we were able to increase the intensity for at least one of the two species. Furthermore, the microfraction collector could be employed to accumulate residues for further biological or structural analysis.
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Spatial distribution of GlcNAc-GlcA in heparin
We have shown in other studies (Zhang et al., 2001b) that the N-acetyl group is a critical structural feature that marks HS chains for the action of 3-OST-1 but not 3-OST-3A. It is not clear how HS biosynthetic pathways diverge to accommodate the needs for different precursor structures that ultimately lead to AT III or gD binding motifs. Moreover, because of the highly heterogeneous nature of the polymer, it is very difficult to determine this important spacing parameter between these rare residues. Therefore, we prepared heparin polymers with a more homogeneous sulfation pattern to aid in the determination of the spacing between GlcNAc-GlcA disaccharide units. This was accomplished by complete desulfation/re-N-sulfation of heparin polymer while preserving all other structural features as shown in Scheme 1 (Nagasawa and Inoue, 1974
). This polymer was then cleaved with recombinant heparanase to prepare oligosaccharides containing glucuronic acid at the reducing end of the cleavage site (McKenzie et al., 2003
). The resulting oligosaccharides were then subjected to LC/MS analysis to determine the size distribution of the products that correspond to the spacing between these rare disaccharide residues. The results revealed a series of oligosaccharides that range from hexasaccharides to dodecasaccharides with approximately equal amounts of each oligosaccharide. This indicates that the average spacing between glucuronic acids is approximately 8 or 10 residues (Figures 8A and 8B; Table III). The results are summarized in Table III for oligosaccharides and their molecular weights obtained from LC/MS analysis.
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Discussion |
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We determined which HS modifying enzymes are expressed and which HS structures can be synthesized in cells and tissues. We used LC/MS as an effective and simple tool for the structural analysis of HS isolated from various sources. This is an important development because antibodies against specific isoforms of biosynthetic enzymes are not available. This technique allows us to establish the presence or absence of specific sulfotransferases within cells or tissues. Furthermore, we showed that this approach also permits the delineation of biosynthetic pathways within different tissues, which has not been possible prior to this study.
We characterized the fine structure of HS purified from HEK293 cells. These cells appear to express a sulfotransferase activity similar to 3-OST isoforms that can generate the HSV-1 gD binding site. This result is consistent with the known susceptibility of HEK293 cells to HSV infection (Israel et al., 1995; Skaliter et al., 1996
). In addition to demonstrating 3-OST activity in HEK293 cells, the data in Figure 5 also show that in comparison to CHO cells, HEK293 cells express elevated levels of 6-OST activity, as indicated by the relatively large peaks for
UA-GlcNS6S and
UA2S-GlcNS6S when compared to that of
UA2S-GlcNS. This could have significant implications on the use of these cells in studying the role of HS structure in important biological systems, such as the interaction between cell factors and their receptors.
We earlier observed differences in the steady-state levels of 3-OST isoform mRNAs in various tissues. As expected from these mRNA results, significant levels of 3-O-sulfate-containing residues were detected in HS isolated from kidney, whereas no significant levels were detected in HS extracted from lung tissue. Furthermore, spleen, which had not as yet been characterized for 3-OST expression, was shown herein to express significant levels of 3-O-sulfated HS residues. In addition to differences in 3-OST expression, we were also able to detect variations in NDST, 2-OST, and 6-OST activities within these tissues. Thus we have the ability to map relative differences in HS biosynthetic enzymes, which allows us to elucidate the HS biosynthetic pathways and determine the biological role of HS sulfotransferases within both tissues and cells.
A number of studies have established that CHO cells normally express NDST-1 and NDST-2 as well as 2-OST and 6-OST-1 (Aikawa and Esko, 1999; Bai and Esko, 1996
; Zhang et al., 2001a
). Together these enzymes act in a concerted fashion to produce the HS structures normally observed in these cells. Figure 9 summarizes the complement of HS sulfotransferase activities normally found in CHO cells and the resulting modifications in HS chains (mainly at the disaccharide level). In addition, the effects on HS structure remodeling by exogenous sulfotransferases are shown. It is understood that a strong correlation between precursor site and 3-OST isoform must exist. However, the scheme outlined in Figure 9 shows only the modifications that occur on disaccharides within the nascent chain without regard to these critical biological constraints.
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Mapping of entire pathways within both individual cells and tissues is now possible and is expected to be a powerful means of defining source-specific HS modalities. Furthermore, with enzymatic dispersion of tissue samples or laser capture microdissection, it should be possible to map these structures on a cellular level and thus focus attention on specific cells within tissues.
Finally, we defined the spacing of critical biosynthetic regulatory markers along the HS chains. To this end, the positions of the relatively rare GlcNAc-GlcA units along the heparin chains were determined. This structural feature directs the action of 3-OST-1 in sulfonating glucosamine units to generate AT III binding sites. In a model system, completely desulfated and re-N-sulfated heparin with homogeneous sulfation pattern was cleaved with endoglucuronidase. This study permitted us to infer the position of the potential 3-OST-1 acceptor sites along the heparin chain. Although this was accomplished in a model system, we trust that it can now be expanded with fully sulfated HS chains to define the spacing of biosynthetic molecular markers, which leads to the placement of critical functional groups.
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Materials and methods |
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Tissue culture and retroviral transductions
CHO cells were grown in growth medium A (Ham's F-12 containing 50 U/ml penicillin, 50 mg/ml streptomycin, 2 mM glutamine, and 10% fetal calf serum by volume). A human embryonic kidney cell line (HEK293) as well as the viral packaging line (Phoenix cells, ATCC SD 3444) were grown in growth medium B (Dulbecco's modified Eagle's medium containing 10% fetal calf serum, 50 U/ml penicillin, 50 mg/ml streptomycin, and 2 mM glutamine).
For retroviral infections (transductions), a stable CHO cell line CHO[ECO] expressing the ecotropic retrovirus receptor was used as previously described.(Gu et al., 2000) The retroviral vector MVT-37 was prepared from the MSCVpac retroviral vector (Hawley et al., 1994
) by ligation of a new multiple cloning segment. The complete coding sequences for human clones of 3OST-1, 3OST-3A, 3OST-4, and 3OST-5 were subcloned into the appropriate restriction sites and the Phoenix packaging line, maintained in growth medium B, and transfected with these 3-OST constructs as previously described (Gu et al., 2000
).
Isolation of cell- and tissue-specific HS
HS was extracted from cells as described in our prior publication (Zhang et al., 2001b). Tissue samples were dissected from adult (3 months old) C57 BL6 mice (Taconic) and subsequently homogenized in phosphate buffered saline. HS from tissue samples was isolated in the same way as HS extracted from cells.
In vitro modification of HS using stable isotope
3'-Phosphoadenosine-5'-phosphosulfate (PAPS) with stable isotope 34S was prepared as previously outlined (Kuberan et al., 2003). Baculovirus-expressed 3-OST-1, 3-OST-3A, 3-OST-4, and 6OST-1 enzymes were prepared as described earlier (Kuberan et al., 2003
; Liu et al., 1999; Shworak et al., 1997
). Purification of labeled HS chains was completed as mentioned earlier.
Enzymatic degradation of HS chains
Stable isotope-labeled or unlabeled HS (10 µl) was combined with 34-µl water, 5 µl 10x digest buffer (400 mM ammonium acetate, 33 mM calcium acetate), 1 µl 0.33 mU heparitinases I, heparitinases II, and heparinase (Seikagaku, Tokyo). Digestion was allowed to proceed overnight at 37°C. Digests were lyophilized and reconstituted in 7 µl water, 1 ml ion pairing reagent DBA and then analyzed by LC/MS for heparitinase-derived residues as reported earlier (Kuberan et al., 2002).
Data analysis
The ionic masses for disaccharide residues are determined by the formula 336 + x(42) + y(80), where x = number of acetyl groups and y = number of sulfates. In addition to the raw ionic weight for each residue, the combined weight for adduct ions formed with either water or the ion pairing reagent used for reverse-phase chromatography (DBA) were also analyzed. Extracted ion current was computed as described in the documentation for the Data Explorer software provided by Applied Biosystems.
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Acknowledgements |
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Footnotes |
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1 These authors contributed equally to this article.
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Abbreviations |
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References |
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