Glycobiology Advance Access originally published online on December 23, 2005
Glycobiology 2006 16(4):318-325; doi:10.1093/glycob/cwj072
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A defect in exodegradative pathways provides insight into endodegradation of heparan and dermatan sulfates
2 Lysosomal Diseases Research Unit, Department of Genetic Medicine, Children, Youth and Women’s Health Service, 72 King William Road, North Adelaide, SA 5006, Australia and 3 Department of Paediatrics, University of Adelaide, Adelaide, SA 5005, Australia
1 To whom correspondence should be addressed; email: maria.fuller{at}adelaide.edu.au
Received on August 30, 2005; revised on December 7, 2005; accepted on December 19, 2005
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Abstract |
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Within cells, dermatan sulfate (DS) and heparan sulfate (HS) are degraded in two steps. The initial endohydrolysis of these polysaccharides is followed by the sequential action of lysosomal exoenzymes to reduce the resulting oligosaccharides to monosaccharides and inorganic sulfate. Mucopolysaccharidosis (MPS) type II is a lysosomal storage disorder caused by a deficiency of the exoenzyme iduronate-2-sulfatase (I2S). Consequently, partially degraded fragments of DS and HS have been shown to accumulate in the lysosomes of affected cells and are excreted in the urine. Di- to hexadecasaccharides, isolated from the urine of a MPS II patient using anion exchange and gel filtration chromatography, were identified using electrospray ionization-tandem mass spectrometry (ESI-MS/MS). These oligosaccharides were shown to have non-reducing terminal iduronate-2-sulfate residues by digestion with recombinant I2S. A pattern of growing oligosaccharide chains composed of alternating uronic acid and N-acetylhexosamine residues was identified and suggested to originate from DS. A series of oligosaccharides consisting of hexosamine/N-acetylhexosamine alternating with uronic acid residues was also identified and on the basis of the presence of unacetylated hexosamine; these oligosaccharides are proposed to derive from HS. The presence of both odd and even-length oligosaccharides suggests both endo-ß-glucuronidase and endo-N-acetylhexosaminidase activities toward both glycosaminoglycans. Furthermore, the putative HS oligosaccharide structures identified indicate that heparanase activities are directed toward regions of both low and high sulfation, while the N-acetylhexosaminidase activity acted only in regions of low sulfation in this polysaccharide.
Key words: dermatan sulfate / endohydrolase / glycosaminoglycans / heparan sulfate / mucopolysaccharidosis II
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Introduction |
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The sulfated glycosaminoglycans heparan sulfate (HS) and dermatan sulfate (DS) are present in a wide variety of cell types where they play an intricate role in the extracellular matrix (Trowbridge and Gallo, 2002
; Kramer and Yost, 2003). HS has repeating disaccharide units consisting of uronic acid (UA) alternating with 
-linked (1,4)-glucosamine (GlcN) residues. Biosynthesis of HS occurs in the Golgi. Following the synthesis of the base polymer consisting of the disaccharide repeat (glucuronic acid [GlcA] 1-4-N-acetylglucosamine [GlcNAc]), a series of enzymatic reactions occur: GlcNAc deacetylation, GlcA epimerization to iduronic acid (IdoA), and sulfate transfer to both O and N positions on IdoA and GlcN. The enzymes involved in each step show substrate structure requirements, and to some extent these requirements drive the overall final structure of HS. Consequently, the UA residue may be ß-linked (1,4)-D-GlcA, or -linked (1,4)-L-IdoA, unsulfated or with O-sulfation of the C2-hydroxyl. The amino group of GlcN may be N-sulfated, N-acetylated, or occasionally unsubstituted. The GlcN may also be sulfated on the C6-hydroxyl and sometimes on the C3-hydroxyl. The proportion of GlcA and IdoA varies considerably, not only between different species of HS but also within a particular HS chain. Likewise, the degree and type of sulfation is not stoichiometric (Lindahl et al., 1998). The modification reactions that are responsible for the structural diversity are not complete, producing a final HS molecule with a domain structure (Lyon and Gallagher, 1998). HS chains typically contain regions rich in GlcA and N-acetylated GlcN (GlcNAc) disaccharides with no sulfation (NA domains), contiguous variable-length sequences containing IdoA and GlcNS derivatives with high sulfation (NS domains), and bridging these domains are mixed sequences in which GlcNAc disaccharides and GlcNS disaccharides alternate (NA/NS domains) (Maccarana et al., 1996).
DS is composed of repeating disaccharide units consisting of UA alternating with ß-linked (1,4)-D-N-acetylgalactosamine (GalNAc) residues that may be sulfated on the C4 and/or C6 position. Some DS chains have predominantly (1,3)--linked IdoA residues with some C2 sulfation, whereas others have primarily (1,3)-ß-linked GlcA. Similar to HS, DS forms block structures of lowly sulfated GlcA-GalNAc disaccharides alternating with blocks of highly sulfated IdoA-GalNAc disaccharides (Prydz and Dalen, 2000).
The catabolism of HS and DS begins with endohydrolysis of the polysaccharide chains to oligosaccharides. Two classes of human endoenzymes have been reported that cleave at specific sites within the DS and HS polysaccharides. Hyaluronidases (endo-ß-N-acetylhexosaminidases) are a family of enzymes that degrade hyaluronan, as well as chondroitin sulfate and DS. Hyaluronidases cleave internal ß-linked (1,4)-glycosidic bonds between GalNAc and GlcA in DS (Kreil, 1995). Levels of hyaluronidase are elevated in a number of cancers, but the identity of the type of hyaluronidase expressed in most cancer tissue and cells is still unknown (Lokeshwar et al., 2001). Heparanase (endo-ß-glucuronidase) cleaves at glucuronosyl bonds within HS, resulting in smaller saccharide chains (Hulett et al., 1999; Bame, 2001). Heparanase activity has been shown to be associated with cell invasion, angiogenesis, inflammation, and tissue remodeling through a number of cell–matrix interactions (Dempsey et al., 2000; Vlodavsky and Friedmann, 2001). The action of heparanase on cell surface and extracellular matrix HS proteoglycans is thought to be a highly regulated process involving the binding and internalization of heparanse to specific cell-surface proteoglycans (syndecans), thereby limiting the extracellular accumulation and action of this endoglycosidase (Gingis-Velitski et al., 2004). Endosulfatase action on heparin has also been reported, and it is likely that similar activity will be shown to the sulfated domains of HS (Morimoto-Tomita et al., 2002).
Following partial catabolism by endoenzymes, HS and DS are degraded from their non-reducing termini by the sequential action of highly specific lysosomal exoenzymes. At least 10 lysosomal exoenzymes act to reduce these oligosaccharides to monosaccharides and inorganic sulfate to enable exit out of the lysosome. A deficiency in any one of these exoenzyme activities may result in lysosomal storage of the glycosaminoglycan (GAG) substrates with the resulting clinical manifestations of the mucopolysaccharidoses (MPS). MPS II results from a deficiency of iduronate-2-sulfatase (I2S; EC 3.1.6.13
[EC]
), which hydrolyzes the C2-sulfate ester bond of non-reducing terminal -L-IdoA residues in HS and DS (Neufeld and Meunzer, 2001). In the absence of I2S activity, the catabolism of HS and DS is blocked. Consequently, partially degraded fragments of HS and DS accumulated in the lysosomes of MPS II affected cells and are excreted in the urine. These oligosaccharide fragments are presumably produced by endohydrolase activities, followed by exohydrolase activities that terminate at the substrate for I2S. Structural characterization of these oligosaccharides can therefore provide insight into the process of endodigestion of HS and DS and detail of the substrate specificities of the endoenzymes involved. To this end, we have identified a series of oligosaccharides from MPS II patient urine that provide evidence for novel endohydrolase activities and specificities.
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Isolation of oligosaccharides in MPS II urine
A combination of anion exchange and gel filtration was used to isolate GAG-derived oligosaccharides from control and MPS II urine. From a volume of 500 mL of MPS II urine, 45 mg of UA equivalents was recovered from the DEAE column, and of this material 40% eluted in the fractionation range of the Bio-Gel P4 column (<hexadecasaccharide). In comparison, from 500 mL of urine from a control individual, only 0.2 mg of UA equivalents was recovered, and 100% of this was excluded from the Bio-Gel P4 fractionation range (>hexadecasaccharide). Urinary oligosaccharides, from the MPS II patient, eluting between 96 and 225 mL (Figure 1) were further characterized by mass spectrometry.
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Mass spectrometry of oligosaccharides
Electrospray ionization-mass spectrometry (ESI-MS) was performed in negative ion mode on each eluate fraction from the Bio-Gel P4 column. For each oligosaccharide structure, multiple sulfated species could be identified. These oligosaccharides showed characteristic multiply charged ions produced by proton abstraction and as such were identified as [M-H]– up to [M-8H]8– ions. Figure 2 shows representative spectra for oligosaccharides ranging from hepta- to decasaccharides. ESI-MS/MS was also used for further characterization of these oligosaccharides. Precursor ion scan of m/z 173, corresponding to an ionized 1-phenyl-3-methyl-5-pyrazolone (PMP) fragment, was used to support the assignments made from the ESI-MS scans. Strong signals were observed for oligosaccharide ions in higher charged states (>[M-3H]3–), whereas signals from the [M-2H]2– were weak or not detectable (data not shown). Collisionally activated dissociation-tandem mass spectrometry (CAD-MS/MS) was performed on all major oligosaccharide signals and used to confirm the assignments made from the ESI-MS spectra and to identify the residue at the reducing terminus. Oligosaccharides containing N-acetylhexosamine (HNAc) at the reducing terminus gave a product ion at m/z 256 and those with a UA at the reducing end gave a product ion of m/z 331 as has previously been described (Ramsay et al., 2003; Fuller et al., 2004). A representative product ion spectra of a tetrasaccharide (UA-HN-UA-HNAc [4S]) with a characteristic product ion m/z 256 is shown in Figure 3A, and Figure 3B shows the product ion spectra from a pentasaccharide (UA-HN-UA-HNAc-UA [4S]) with a characteristic product ion of m/z 331. Product ion analysis was performed on all major signals in the ESI-MS and in most instances enabled the identification of the reducing terminal sugar as either UA or HNAc.
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The mass spectrometric analysis of the oligosaccharides eluted from the Bio-Gel P4 column enabled the identification of 25 different oligosaccharide species ranging in size from di- to hexadecasaccharide with various numbers of sulfates. Tables I and II display a summary of the oligosaccharides present in the MPS II patient urine that were classified into series, based on their proposed structures. Two series of oligosaccharides containing UA and HNAc disaccharide repeat units were identified with 1–2 sulfates per disaccharide. The first series, with an even number of saccharides, had a UA residue on the non-reducing end and a HNAc on the reducing end (Table I, series 1). The second series consisted of an odd number of residues ranging from tri- to heptasaccharide (Table I, series 2), with UA at the reducing terminus.
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Four series of oligosaccharides containing UA, HN, and HNAc were identified with different levels of sulfation (Table II). Series 1–3 consist of an odd number of residues, with UA at the reducing terminus, and series 4 contains an even number of residues, with HNAc at the reducing terminus. The oligosaccharides in series 1 are composed of one HN with either zero, one, or two HNAc. Series 2 contains oligosaccharides with two HN and up to five HNAc. The oligosaccharide in series 3 contains three HN residues and a single HNAc. Series 4 lists oligosaccharides with an even number of residues containing one HN and up to four HNAc residues.
Enzymatic characterization of the non-reducing end
For oligosaccharides present in sufficient quantities, the non-reducing end UA was identified as iduronate-2-sulfate (Ido2S) by enzymatic cleavage with recombinant I2S and -L-iduronidase (IDUA). Figure 4 shows ESI-MS of pooled hexa- and heptasaccharide fractions isolated from MPS II urine, before and after I2S and IDUA treatment. The hexasaccharide identified by m/z ratios with the putative structure [UA-HNAc]3 with 1–4 sulfates was shown to lose 80 and 176 amu, representing S and UA respectively, following incubation with recombinant I2S and IDUA. As with the hexasaccharide, a heptasaccharide with the proposed structure UA-HN[UA-HN]/[UA-HNAc]-UA with 3–6 sulfates also showed losses of 80 and 176 amu following digestion with recombinant I2S and IDUA (Figure 4). A peak with m/z of 523.3 corresponding to IdoA hydrolyzed from the oligosaccharides was also identified in the digested sample (Figure 4B). Treatment of the oligosaccharides with only recombinant IDUA resulted in no change to the spectra, whereas treatment with I2S resulted in the disappearance of each of the oligosaccharides in the highest sulfation form (spectra not shown).
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Discussion |
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Di- to hexadecasaccharides that would appear to originate from endohydrolase activities on HS and DS, and are therefore the result of incomplete catabolism, were isolated from the urine of an MPS II patient. Mass spectrometry has enabled structural characterization of these oligosaccharides using a combination of ESI-MS and CAD-MS/MS. CAD-MS/MS was performed on all major oligosaccharide signals and used to confirm the assignments made from the ESI-MS spectra and to identify the residue at the reducing terminus. Recovery of reducing oligosaccharides following derivatization has been demonstrated to be >94% (Rozaklis et al., 2002
). In this study, we have seen no evidence of aldol reductase action on oligosaccharides to render them unreactive to PMP, and this would have been evident in the ESI-MS of the derivatized oligosaccharides (Figure 2) where underivatized oligosaccharides would also be observed. Most signals in these spectra could be attributed to PMP oligosaccharides. Enzyme treatment with recombinant I2S and IDUA identified the non-reducing residue as IdoA2S, the substrate for the enzyme deficiency in MPS II.
ESI-MS identified oligosaccharides that have a different number of residues but very similar molecular weights, which occurs as a result of the similarity between the mass of HN (161 Da) and 2 sulfates (160 Da). For example, the octasaccharide ([UA-HN]3-UA-HNAc [4S]) has a molecular weight of 2058.3, whereas the heptasaccharide ([UA-HN]2-UA-HNAc-UA [6S]) has a molecular weight of 2057.4, and when the [M-4H]4– ions are compared, the difference is m/z 513.6 compared with m/z 513.4, respectively. The mass accuracy of the instrument used in this study could not positively discriminate these structures. However, identification of the reducing terminus as either UA or HNAc and the non-reducing terminus as IdoA2S enabled this discrimination. We have previously reported characteristic product ions produced from oligosaccharides having either HNAc or UA at the reducing end following derivatization with PMP (Ramsay et al., 2003; Fuller et al., 2004).
We identified two series of oligosaccharides that are likely derived from DS (Table I) and four series of oligosaccharides likely derived from HS (Table II). Mass spectrometry cannot distinguish the structural isomers GlcNAc and GalNAc to provide discrimination of oligosaccharides derived from HS and DS, respectively. Consequently, the assignment of oligosaccharide series as derived from DS or HS was based on a number of factors including the presence of unacetylated HN residues, which are common structural features in HS but are not present in DS (Prydz and Dalen, 2000). A second factor used for this discrimination was the degree of sulfation in the extending oligosaccharide series; HS oligosaccharides extending with GlcNAc-UA disaccharides would be expected to show little change in sulfation level as the extension is into the unsulfated NA region, and this is seen in the oligosaccharides in Table II series 2 and also in Table II series 1 and series 4. In contrast, DS oligosaccharides extending with GalNAc-UA disaccharides could show an increase in sulfation from the GalNAcS and sulfated UA residues (Prydz and Dalen, 2000), and this is seen clearly in Table I series 1 and also in Table I series 2. The formation of DS-derived oligosaccharides terminating in HNAc (Table I, series 1) is consistent with the reported action of hyaluronidase to act on the GalNAc-UA linkages of DS (Kreil, 1995), and the even distribution of sulfate residues along the length of the DS oligosaccharides is consistent with the reported action of hyaluronidase to GalNAc-4-S residues (Knudson et al., 1984). Although the smaller oligosaccharides (di- to octasaccharide, Table I, series 1) had up to 2 sulfates per disaccharide, the larger (up to hexadecasaccharide) had fewer sulfates. This may result from the loss of sulfate residues in the ion source of the mass spectrometer as has been previously reported (Ramsay et al., 2003). However, these oligosaccharides may also arise in part from the structural heterogeneity present in DS. We have previously reported tri- and pentasaccharides, isolated from MPS I urine, and identified them as being derived from DS by the presence of GalNAc-4-S as the penultimate residue at the non-reducing end (Fuller et al., 2004). These oligosaccharides were suggested to derive from endohydrolase activity against the UA-GalNAc bond in DS. This concept is further supported here in MPS II, with the identification of tri-, penta-, and heptasaccharides derived from DS. However, we did not observe any significant signals from larger oligosaccharide species in this series. The limitation on the size of the oligosaccharide identified may reflect substrate specificity of the endoglucuronidase activity for terminal regions of the DS chains, although it may also be that larger species are not present in sufficient amounts to be resolved from the more abundant oligosaccharide species terminating in HNAc, seen in the complex spectra of the larger oligosaccharides.
The oligosaccharide series containing UA-HN and UA-HNAc disaccharides provides a more complex picture of endohydrolysis of HS. Two series of oligosaccharides (Table II, series 1 and 2) are likely products of heparanase activity toward HS. These series contain one or two UA-HN disaccharides with increasing numbers (up to five) of UA-HNAc disaccharides. All oligosaccharides in these series have a UA at the reducing terminus. The relative positions of the HN and HNAc residues in these oligosaccharides have not been defined, and it is likely that the structures proposed in Table II represent mixtures of structural isomers. For example, the heptasaccharide (UA-HN-[UA-HN]/[UA-HNAc]-UA) could equally have the structure UA-HN-UA-HN-UA-HNAc-UA or UA-HN-UA-HNAc-UA-HN-UA. Given the known biosynthesis of HS, it is probable that both structures are present. Interestingly, the number of sulfates associated with this series does not increase with the size of the oligosaccharide but remains relatively constant between three and seven. In light of the reported domain structure of HS with regions of high (NS) and low (NA) sulfation and mixed (NA/NS) domains (Maccarana et al., 1996), it seems probable that these two series of oligosaccharides represent either short NS domains or mixed NA/NS regions, each with an increasing extension of the NA domain. Thus, the heparanase activity appears to have specificity for both the relatively highly sulfated linkage region between the NS and NA domains (thus producing the smaller oligosaccharides) and also for the unsulfated region represented by the extending UA-GlcNAc disaccharides (NA domain). Previous studies have indicated multiple substrates for heparanase activity. For example, earlier studies (Marchetti et al., 1997; Pikas et al., 1998) indicated that human heparanase cleaved a sequence within the highly modified NS domains of HS. Heparanase from CHO cells has been proposed to have substrate specificity for the mixed NA/NS domains (Bame and Robson, 1997; Bame et al., 2000). Studies on the heparanase from a rat parathyroid cell line identified relatively undersulfated structures at the cleavage site and also proposed that the cleavage occurred at the boundary of highly sulfated and undersulfated domains (Podyma-Inoue et al., 2002). At the same time, studies on recombinant human heparanase with defined tetra- and hexasaccharide structures indicated the requirement for a highly sulfated structure containing an unsulfated GlcA at the cleavage site (Okada et al., 2002).
Although there appears to be only one candidate gene for heparanase (Toyoshima and Nakajima, 1999), it is also plausible that there are two different enzymes with specificities for either NS or NA domains in HS. Heparanase activity is also likely to be responsible for the generation of the nonasaccharide (UA-HN-[UA-HN]2/[UA-HNAc]-UA) (Table II, series 3) that represents a longer NS domain, with heparanase cleavage occurring after the first HNAc in the adjacent NA domain or a larger NA/NS domain in which the UA-HNAc disaccharide is between two UA-HNS disaccharides. The final HS-derived oligosaccharide series (Table II, series 4) represents similar structures to those seen in series 1 but with HNAc as opposed to UA at the reducing end. This suggests the action of an endohydrolase on the HNAc-UA linkage in HS. In this series of oligosaccharides, the number of sulfates does not proportionately increase with oligosaccharide length, again indicating that the extension of the UA-HNAc disaccharide is into the NA domain, and thus the endoenzyme specificity is for the unsulfated NA domain.
The absence of any oligosaccharides larger than a pentasaccharide containing only UA and HN suggests that the NA/NS domain of HS is more resistant to heparanase activity than the NA or NA/NS domains. Presumably, the larger NS regions are to be found in the high-molecular-weight structures eluting at Vo from the Bio-Gel P4 column. The structures observed appear to arise primarily from the action of heparanase in the NA and NA/NS domains of HS. The reducing terminus of each oligosaccharide is then trimmed back by the action of exoenzymes to the first IdoA2S residue where, as a result of the deficient enzyme, exodegradation is halted. A similar scheme involving an endo-N-acetylglucosaminidase activity is also proposed to account for the HS-derived oligosaccharides in Table II series 4.
The study of stored oligosaccharides from patients with defective exodigestion of GAG (MPS patients) provides a unique, global insight into the process of endohydrolysis of GAG in the endosomal/lysosomal network. In urine from an MPS II patient, we have observed a significant proportion of low-molecular-weight oligosaccharides (40% of UA in hexadecasaccharide or smaller), indicating that the action of these endoglycosidases provides a significant contribution to the total degradation of GAG in the lysosome. Characterization of the resulting oligosaccharides yields information about the endoenzymes involved and their substrate specificities. In addition to the hyaluronidase and heparanase, we also have evidence for the action of an endoglucuronidase activity on DS and an endo-N-acetylglucosaminidase activity on HS. We have also provided evidence that the majority of HS digestion occurs in the low-sulfation (NA) regions. Further detailed characterization of the stored substrates in MPS II and other MPS types will increase our understanding of these enzymes and their role in GAG turnover.
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Materials and methods |
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Materials
Recombinant human IDUA (Unger et al., 1994
) and recombinant human I2S (Bielicki et al., 1993) were each prepared from CHO-K1 expression systems. MPS II patient urine and control urine was supplied with consent.
Isolation of urinary GAG
Urine samples from an MPS II patient and an age-matched control (500 mL) were clarified by centrifugation and passed over a 30 mL column of DEAE-Sephacel previously equilibrated with 0.1 M NaCOOCH3 buffer, pH 5. The column was washed with 10 column volumes of the equilibration buffer and urinary GAG eluted in the same buffer containing 1.2 M NaCl. Fractions were assayed for UA (Blumenkrantz and Asboe-Hansen, 1973) and the GAG-containing fractions (20 mL) were pooled, lyophilized, and reconstituted in 4 mL of H2O. The pooled GAG fraction was then size-fractionated on a Bio-Gel P4 column (170 cm x 1.5 cm) in 0.5 M NH4COO. Fractions (4 mL) were collected and assayed for UA.
Derivatization of oligosaccharides
Samples from Bio-Gel P4 column fractions were lyophilized before derivatization. Samples were resuspended in 100 µl 250 mM PMP, 400 mM NH4OH, heated at 70°C for 90 min, and then acidified with a two-fold molar excess of HCOOH. Samples were made up to 500 µl with H2O and then extracted with an equal volume of CHCl3 to remove excess PMP and centrifuged at 13,000 x g for 5 min. Chloroform extraction was repeated a further two times, and the aqueous layer was lyophilized and the derivatized oligosaccharides resuspended in 500 µl of an aqueous solution of 50% (v/v) CH3CN/0.025% (v/v) HCOOH before analysis by mass spectrometry. Derivatization of samples following enzyme digestion was performed in the same way except that the aqueous phase from the first chloroform extraction was treated as follows: copolymeric solid-phase extraction cartridges (50 mg, C18 and aminopropyl) (United Chemical Technologies, Bristol, PA) were primed with methanol, (1 mL) then water (1 mL), after which the sample was applied and allowed to enter the solid phase completely. Samples were desalted with three consecutive 1 mL water washes, dried on a Supelco, Visiprep24 vacuum manifold (Sigma-Aldrich, St Louis, MO), and the remaining PMP was removed with two 1 mL chloroform washes. The columns were again dried, and derivatized oligosaccharides were eluted in an aqueous solution of 50% (v/v) CH3CN/NH4OH, pH 11.5, lyophilized, and resuspended in 50% (v/v) CH3CN/0.025% (v/v) HCOOH.
Enzymatic cleavage
Column fractions containing 10 µg of UA were lyophilized before digestion with recombinant I2S and IDUA. Enzymatic cleavage was performed at 37°C for 16 h in 50 µl of 50 mM NaCOOCH3 buffer (pH 4.5) supplemented with 0.5 mg/mL BSA and 20 µg recombinant enzyme (IDUA and/or I2S). Following digestion, the oligosaccharides were derivatized as described above and analyzed by ESI-MS in negative ion mode.
Mass spectrometry
Oligosaccharide analysis was performed by ESI-MS/MS using a PE Sciex API 3000 triple-quadrupole mass spectrometer with a turbo ionspray source and Analyst 1.1 data system. Samples were either directly infused using a Harvard Apparatus pump at 10 µl/min or injected with a Gilson 233 autosampler at 80 µl/min using a carrying solvent of 50% (v/v) CH3CN/0.025% (v/v) HCOOH in H2O. Oligosaccharides were identified on the basis of mass-to-charge (m/z) ratios by ESI-MS and further characterized using CAD-MS/MS in the negative ion mode.
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This work was supported in part by the NH & MRC (Australia), TLH Research (USA) and The Wellcome Trust (UK), grant reference number 060104Z/00/Z.
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Abbreviations |
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CAD-MS/MS, collisionally activated dissociation-tandem mass spectrometry; DS, dermatan sulfate; ESI-MS, electrospray ionization-mass spectrometry; ESI-MS/MS, electrospray ionization-tandem mass spectrometry; GAG, glycosaminoglycans; GalNAc, N-acetylgalactosamine; GlcN, glucosamine; GlcNAc, N-acetylglucosamine; HN, hexosamine; HNAc, N-acetylhexosamine; HS, heparan sulfate; I2S, iduronate-2-sulfatase; IdoA,
-L-iduronic acid; IDUA, -L-iduronidase; MPS, mucopolysaccharidosis; PMP, 1-phenyl-3-methyl-5-pyrazolone; UA, uronic acid |
References |
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