Glycosaminoglycan degradation fragments in mucopolysaccharidosis I

Maria Fuller1, Peter J. Meikle and John J. Hopwood

Lysosomal Diseases Research Unit, Department of Genetic Medicine, Women's and Children's Hospital, 72 King William Road, North Adelaide, South Australia, 5006; and Department of Paediatrics, University of Adelaide, South Australia, 5005

Received on October 15, 2003; revised on December 1, 2003; accepted on December 15, 2003


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
The catabolism of glycosaminoglycans begins with endohydrolysis of polysaccharides to oligosaccharides followed by the sequential action of an array of exoenzymes to reduce these oligosaccharides to monosaccharides and inorganic sulfate. In a lysosomal storage disorder known as mucopolysaccharidosis I, caused by a deficiency of the exohydrolase {alpha}-L-iduronidase, fragments of two different glycosaminoglycans, dermatan sulfate and heparan sulfate, have been shown to accumulate. Oligosaccharides isolated from the urine of a mucopolysaccharidosis I patient using anion exchange and gel filtration chromatography were identified as di-, tri-, tetra-, penta-, and hexasaccharides using electrospray ionization–tandem mass spectrometry and shown to have nonreducing terminal {alpha}-L-iduronate residues, susceptible to digestion with {alpha}-L-iduronidase. The presence of odd and even oligosaccharides suggests both endo-ß-glucuronidase and endo-N-acetylhexosaminidase activities toward both glycosaminoglycans. Cultured skin fibroblasts from mucopolysaccharidosis I patients accumulate the same dermatan sulfate–and heparan sulfate–derived di- and trisaccharides as identified in urine, and supplementation of culture medium with recombinant {alpha}-L-iduronidase reduced their level to that of unaffected control fibroblasts. A dermatan-derived tetrasaccharide not elevated in mucopolysaccharidosis I fibroblasts transiently increased in these fibroblasts in the presence of recombinant {alpha}-L-iduronidase, indicating it is an intermediate product of catabolism. These oligosaccharides were elevated in urine samples from mucopolysaccharidosis I patients, and we suggest that these glycosaminoglycan-derived oligosaccharides may be useful biochemical markers for the identification and the clinical management of mucopolysaccharidosis I patients.

Key words: dermatan sulfate / endohydrolase / glycosaminoglycans / heparan sulfate / mucopolysaccharidosis I


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Glycosaminoglycans (GAGs) are major components of the extracellular matrix and cell surface of most cell types. GAGs exist as proteoglycans and have multiple functions that are often dependent on their sequence structure (Esko and Selleck, 2002Go; Hardingham and Fosang, 1992Go). GAGs are degraded in the lysosome by the concerted action of a number of exohydrolase activities following partial catabolism by endoenzymes (endoglycosidases, hyaluronidases, heparanases, and endosulfatases). Both endo- and exoenzyme activities toward these GAGs have highly conserved substrate structure specificities. Initially, endohydrolysis of the polysaccharide GAG chains to oligosaccharides occurs, followed by the action of up to 13 lysosomal exoenzymes to reduce these oligosaccharides to monosaccharides and inorganic sulfate to enable exit from the lysosome. A deficiency of any one of these exoenzyme activities may result in lysosomal storage of the GAG substrates for these exoenzyme activities and clinical symptoms of the mucopolysaccharidoses (MPSs).

MPS I, the most common of the 11 reported MPS disorders (Meikle et al., 1999Go), results from a deficiency in the exohydrolase {alpha}-L-iduronidase (IDUA; EC 3.2.1.76). IDUA is required for the lysosomal degradation of heparan sulfate (HS) and dermatan sulfate (DS). Failure to remove {alpha}-L-iduronic acid (IdoA) from the nonreducing end of a GAG results in the accumulation of these substrates in the lysosomes of the IDUA-deficient cells. Lysosomal storage leads to the chronic and progressive deterioration of cells, tissues, organs, and the urinary secretion of partially degraded GAGs, which may lead to the onset of a MPS I clinical phenotype within a spectrum of severity ranging from severe Hurler syndrome to relatively mild Scheie syndrome (Neufeld and Muenzer, 2001Go).

HS has repeating disaccharide units consisting of uronic acid (UA) alternating with {alpha}-linked (1,4) glucosamine residues. The UA residue may be ß-linked (1,4) D-glucuronic (GlcA) or {alpha}-linked (1,4) L-IdoA, unsulfated or with O-sulfation of the C2-hydroxyl. The amino group of the glucosamine (GlcN) residue may be N-sulfated or N-acetylated. The GlcN may also be sulfated on the C6-hydroxyl and occasionally 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 (Kjellén and Lindahl, 1991Go). HS forms block structures with GlcA-GlcNAc disaccharides alternating with blocks of highly sulfated IdoA-GlcNS disaccharides (Lindahl et al., 1998Go). DS has 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-hydroxyls. Some DS chains have predominantly (1,3) {alpha}-linked IdoA residues with some C2-hydroxyls sulfated. Other DS chains have predominantly (1,3) ß-linked GlcA. Similar to HS, DS also forms block structures of lowly sulfated GlcA-GalNAc disaccharides alternating with blocks of highly sulfated IdoA-GalNAc disaccharides (Kjellén and Lindahl, 1991Go; Prydz and Dalen, 2000Go). Consequently, there is considerable structural heterogeneity in both HS and DS.

Several classes of endohydrolases may be involved in the degradation of the HS and DS chains to oligosaccharides. There are a number of endoglycosidase activities (or hyaluronidases) that cleave internal ß-linked (1,4) glycosidic bonds between GalNAc and GlcA in DS (Kreil, 1995Go). Several endoglucuronidases, known as heparanases, degrade HS chains to oligosaccharides (Bame, 2001Go). Heparanase cleavage of HS chains occurs at specific glucuronosyl bonds defined by the nature and degree of sulfation of the GAG and the specificity of each particular heparanase (Bame and Robson, 1997Go). Another reaction by ß-hexosaminidase A may remove nonreducing end ß-linked, sulfated GlcNAc residues from keratan sulfate or nonreducing end ß-linked sulfated GalNAc from DS or chondroitin sulfate to produce sulfated N-acetylhexosamine monosaccharides in the absence of lysosomal exosulfatases (Hopwood and Elliott, 1985Go). Heparin-degrading endosulfatases have also been identified and shown to have high selectivity for glucosamine-6-sulfate (Tomita et al., 2002Go). It is predicted that such sulfatases will show corresponding activity to the sulfated domains of HS.

Small oligosaccharides have been identified in the urine of MPS patients (Byers et al., 1998Go, unpublished data). It is likely that the nonreducing terminus of the GAGs stored in MPS would be the residue that is the native substrate for the enzyme deficiency (Bach et al., 1973Go). MPS I patients would therefore be expected to lysosomally accumulate mostly HS and DS fragments with nonreducing end IdoA residues and excrete such fragments in their urine. We report that these smaller oligosaccharide fragments derived from HS and DS are likely products of endoglycosidase activities on these GAG chains. We describe the structure of di-, tri-, tetra-, penta-, and hexasaccharides isolated from the urine of MPS I patients. These low-molecular-weight GAG fragments were identified by electrospray ionization (ESI)–tandem mass spectrometry (MS/MS) and confirmed by digestion with recombinant human IDUA and two recombinant exosulfatases. The presence of these oligosaccharides in MPS I cells and urine yields information about the processes involved in the turnover of GAGs, as well as being diagnostic markers for MPS I. As an extension of these findings it should be possible to predict the fine structure of low-molecular-weight GAG fragments that would be present in other MPS disorders.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Identification and characterization of oligosaccharides in MPS I urine
GAGs were isolated from urine by a combination of anion exchange and gel filtration chromatography. The majority of UA-positive material was associated with the high-molecular-weight GAG components (>2000 Da) in the fractionated MPS I urine (Figure 1). Fractions 30 to 41 from the Bio-Gel P2 column were derivatized and oligosaccharides identified in these fractions based on mass to charge (m/z) ratios. Once identified, these oligosaccharides were further characterized by ESI-MS/MS to elucidate partial structure and for oligosaccharides with m/z 806, 509, 982, and 632 to also identify a suitable product ion for multiple-reaction monitoring (MRM). None of the oligosaccharides identified in the fractionated MPS I urine was seen by MS in the control urine. The control urine contained less than 10% of the total UA present in the urine from the MPS I patient. Fractions 30, 32, 34, 35, 37, 38, and 40 were treated with recombinant human IDUA to determine the presence of nonreducing end IdoA residues. In every instance a loss of 176 amu was observed, confirming this terminal residue as IdoA.



View larger version (11K):
[in this window]
[in a new window]
 
Fig. 1. Elution profile of MPS I and control urine from Bio-Gel P2. Urinary GAGs were isolated by a combination of anion exchange and size exclusion chromatography. Fractions of 4 ml were collected from a Bio-Gel P2 column and assayed for UA equivalents. The Vo and Vt of the column were determined at fractions 28 and 55, respectively. The open squares and solid triangles represent MPS I and control urine, respectively.

 
Following digestion with recombinant human IDUA, the fractions were subsequently treated with recombinant human N-acetylgalactosamine-4-sulfatase and recombinant caprine glucosamine-6-sulfatase. The loss of 80 amu (proposed loss of SO3) with N-acetylgalactosamine-4-sulfatase but not with glucosamine-6-sulfatase identified the residue adjacent to the IdoA as N-acetylgalactosamine-4-sulfate in the tri-, tetra-, penta-, and hexasaccharide (see Table I). The mass spectra of the enzyme digests of the tetrasaccharide (m/z 632) are shown in Figure 2. Table I indicates the oligosaccharides identified in the MPS I urine. None of these oligosaccharides were pure; a number of other oligosaccharides present in urine were also observed but at low levels similar to that in the control urine. To evaluate the use of these oligosaccharides as biochemical markers, urine samples collected from MPS I patients from a range of phenotypes (Hurler to Scheie) and age-matched unaffected individuals were analyzed by ESI-MS/MS for the MRM pairs 509/422, 630/256, 632/298, 806/295, and 982/269. There was an elevation in the oligosaccharides corresponding to the MRM pairs 509/422, 806/295, 982/269, and 632/298 in the MPS I urine samples over the controls (Figure 3).


View this table:
[in this window]
[in a new window]
 
Table I. Oligosaccharides in MPS I urine

 


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 2. Mass spectra of tetrasaccharide (m/z 632) following recombinant enzyme digests. Mass spectrum (a) shows a Q1 scan of the tetrasaccharide [M–2H]–2 in fraction 34 from the Bio-Gel P2 column, with an m/z 632.6. Spectrum (b) shows the same oligosaccharide following recombinant human IDUA digestion. The m/z 544.5 corresponds to a loss of uronic acid (176 amu). Spectrum (c) shows the oligosaccharide treated with recombinant human IDUA and then recombinant human N-acetylgalactosamine-4-sulfatase. The m/z 504.4 corresponds to a proposed loss of sulfate (80 amu) in the [M–2H]–2 ion.

 


View larger version (12K):
[in this window]
[in a new window]
 
Fig. 3. Oligosaccharides in urine from control and MPS I patients. Urine samples (1 µmole creatinine equivalent) were derivatized with 1-phenyl-3-methyl-5-pyrazolone and analyzed by MS. Center bars show the median value for each group, shaded areas show the 25th and 75th percentiles, and the top and bottom bars demarcate the limits of the range. Circles represent statistical outliers and N = number of samples in each group.

 
MPS I phenotype correction in cultured skin fibroblasts
Postconfluent cultures (2–15 weeks) of MPS I and control skin fibroblasts were harvested. Cell extracts were derivatized for MS and then analyzed for selected oligosaccharides. The effect of time in culture (aging past a state of confluence) on the accumulation of the low-molecular-weight oligosaccharides is shown in Figure 4. With the exception of the disulfated tetrasaccharide (m/z 632) and the sulfated monosaccharide (m/z 630), the tri- and disaccharides accumulated with time in culture in MPS I fibroblasts when compared with the control cell lines. The DS-derived trisaccharide (m/z 982) showed the highest response relative to the internal standard. This monosulfated trisaccharide is elevated in MPS I fibroblasts over control fibroblasts following 4 weeks in culture. This increase continues with culture time, whereas the amount of this trisaccharide in control skin fibroblasts remains negligible for at least 10 weeks in culture. The disaccharide (m/z 806) showed the next highest response in the cultured skin fibroblasts with a clear elevation in MPS I cells apparent at 6 weeks postconfluence. The HS-derived trisaccharide (m/z 509) also accumulated in MPS I fibroblasts cultured past their state of confluence.



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 4. Relative oligosaccharide levels in skin fibroblasts. A number of MPS I and control skin fibroblasts were cultured 2–15 weeks postconfluence. Cell extracts were prepared, derivatized, and analyzed by ESI-MS/MS. The circles and crosses represent the average of duplicates of the same skin fibroblast from MPS I patients and control cell lines, respectively.

 
Recombinant human IDUA was administered daily for 5 days to the culture medium of MPS I fibroblasts and at 1- and 5-days in control skin fibroblasts, both of which had been aged for 6 weeks postconfluence. The cells were then harvested, and the extracts were prepared for MS and analyzed for each MRM pair. Figure 5 shows a decrease in the amount of accumulated disaccharide (m/z 806) and the trisaccharide (m/z 982) immediately after supplementation of the culture medium with recombinant human IDUA. The trisaccharide (m/z 509) is at low levels and does not appear to alter. Interestingly, the amount of tetrasaccharide (m/z 632) increased in the presence of recombinant human IDUA. Digestion of cell extract with recombinant human IDUA showed no loss of IdoA from this tetrasaccharide. Likewise a sulfated monosaccharide (m/z 630), shown to be elevated previously in some MPS disorders (Hopwood and Elliott, 1985Go; Ramsay et al., 2003Go), also increased. This is a sulfated N-acetylhexosamine that is present in urine and fibroblasts but at comparable levels in MPS I and controls (Figures 3 and 4).



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 5. Relative oligosaccharide levels in MPS I skin fibroblasts following correction with recombinant human IDUA. MPS I and control skin fibroblasts were maintained in culture for 6 weeks postconfluence. Recombinant human IDUA was added to the culture medium of MPS I fibroblasts, which was replenished daily for up to 5 days. Cells were harvested 24 h later, derivatized for MS, and analyzed for each MRM pair. Control fibroblasts are shown in the gray boxes; recombinant human IDUA was added daily, but cells were harvested at 1 day and 5 days only. The 0 time point represents the mean of four values.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
In the absence of IDUA activity, the catabolism of HS and DS is blocked. Consequently partially degraded substrates accumulate in the lysosomes of affected cells, leading to hypertrophy of the lysosomal system, the hallmark of a lysosomal storage disorder. Fragments produced from endohydrolysis of both DS and HS will be further reduced in size from the nonreducing end by the action of exohydrolase activities to the first IdoA residue and then stop. Therefore the presence of di-, tri-, tetra-, penta-, and hexasaccharides in the urine of an MPS I patient probably represent products of endohydrolysis of HS and DS that possibly have also undergone some further digestion with exohydrolase activities that will terminate at nonreducing end IdoA residues.

The characterization of these oligosaccharides has been possible through the use of ESI-MS/MS and specific recombinant lysosomal exohydrolases. From the molecular ion mass and the fragmentation patterns, it is possible to propose the composition of the oligosaccharides as containing UA, hexosamine, N-acetylhexosamine, and sulfate, and to some extent the order/sequence of these residues. Despite MS not enabling the elucidation of stereoisomers, we have predicted N-hexosamine as GlcN or GalN by determining whether the oligosaccharides were products of endohydrolase action on HS or DS (Table I). Identification of a product ion of m/z 331 corresponding to the mass of UA and one PMP molecule (-H2O) in four oligosaccharides (m/z 509, 982, 740, and 720) indicated a UA at the reducing end (Table I). A product ion of 256 suggested an N-acetylhexosamine at the reducing end of oligosaccharides m/z 806, 434, 632, and 862 (Ramsay et al., 2003Go). All the oligosaccharides fragmented to give a product at m/z 193 (UA + H2O) indicating a UA at the nonreducing end that was confirmed as IdoA following removal with recombinant human IDUA. A small peak of tetrasaccharide is noted following IDUA digestion, which probably reflects inefficient enzyme activity at very low substrate concentrations (Figure 2). Nonetheless the possibility that there are small amounts of oligosaccharides with GlcA at the nonreducing end cannot be excluded. The tri-, tetra-, penta-, and hexasaccharide (m/z 982, 632, 720, and 862) were further characterized enzymatically and the residue adjacent to the IdoA was shown to be N-acetylgalactosamine-4-sulfate, thus indicating these four oligosaccharides are the products of endohydrolase digestion of DS. The penta- (m/z 740), tetra- (m/z 651), and trisaccharide (m/z 509) contain unacetylated hexosamine residues and therefore are likely to be derived from HS (Table I). The residue adjacent to the nonreducing IdoA of the pentasaccharide (m/z 740) was not susceptible to N-acetylgalactosamine-4-sulfatase or glucosamine-6-sulfatase, suggesting the presence of GlcN ± S. Additionally, given that the tetrasaccharide (m/z 651) has a GlcNAc at the reducing end, the residue adjacent to the nonreducing terminal IdoA was deduced as GlcN. The disaccharide (m/z 806) could be derived from either DS or HS or both.

Oligosaccharides larger than the hexasaccharide, from both DS and HS, are also likely to be present in the urine from the MPS I patient. The Bio-Gel P2 column has only fractionated oligosaccharides with a molecular weight of less than 2000 Da, so larger oligosaccharides will be in the Vo. Large amounts of UA are evident in Bio-Gel P2 fractions 25–29 (Figure 1) that must contain DS and HS polysaccharides larger than a hexasaccharide. This has been shown previously by gradient polyacrylamide gel electrophoresis (Byers et al., 1998Go). It is also noteworthy that there is a small amount of UA in the control urine, which resumably represents normal levels of UA-containing oligosaccharides. Many of these oligosaccharides were also seen in the Q1 spectra of the Bio-Gel P2 column fractions from the MPS I urine, albeit at very small levels (Figure 2).

Hyaluronidase (endo-ß-N-acetylhexosaminidase) will cleave the glycosidic bond between GalNAc and GlcA residues in DS to a minimum-sized reaction product of a tetrasaccharide (Kreil, 1995Go). Three oligosaccharides (m/z 862, 632, and 806) have GalNAc at the reducing terminus and are potentially a consequence of this hyaluronidase action. The tetrasaccharide (m/z 632) is elevated in MPS I urine over control samples (Figure 3). However, in cultured skin fibroblasts from MPS I patients a tetrasaccharide with this mass is not elevated compared with control cell lines, despite the tetrasaccharide appearing to be one of the more abundant oligosaccharides present in skin fibroblasts (Figure 4). Further investigation showed that this tetrasaccharide is resistant to digestion with recombinant human IDUA, implying that the residue at the nonreducing end may be GlcA, as opposed to IdoA, at the terminus of the tetrasaccharide identified in urine. Of interest is the transient elevation in this tetrasaccharide in MPS I fibroblasts on addition of recombinant human IDUA. This tetrasaccharide may represent the reducing terminus of larger GAG oligosaccharides subsequently degraded by the action of exohydrolases that are able to act after the addition of recombinant human IDUA and/or increased endohydrolase activity following administration of the IDUA. The accumulation of this tetrasaccharide suggests that digestion by the exohydrolase, ß-glucuronidase, is a limiting enzyme in the degradation of DS. It would appear that in skin fibroblasts, the endo-ß-N-acetylhexosaminidase activity has specificity for regions of DS containing GlcA as opposed to IdoA. A similar transient increase in the sulfated N-acetylhexosamine was also observed (Figure 5), which is presumably the result of the action of recombinant human IDUA on the disaccharide (m/z 806) as well as ß-hexosaminidase action on the exposed GalNAc-4SO4 following removal of IdoA. The disaccharide (m/z 806) identified in the MPS I urine is unlikely to be a direct product of hyaluronidase action. However, the disaccharide may derive from a hyaluronidase-produced tetrasaccharide (GlcA-GalNAc-IdoA-GalNAc) or higher oligosaccharide that has a GlcA-GalNAc disaccharide or repeating GlcA-GalNAc disaccharide at the nonreducing end with a reducing end IdoA-GalNAc disaccharide.

The trisaccharide (m/z 982) and pentasaccharide (m/z 720) derived from DS require the specific endohydrolysis of the glycosidic bond between UA and GalNAc residues within DS. This suggests the presence of an endo-ß-glucuronidase activity toward UA-GalNAc bonds in DS. Endo-ß-glucuronidase cleavage of DS has not been previously reported. Further investigation is required into the mechanisms involved in the generation of oligosaccharides with UA reducing ends.

Heparanases are also a family of endo-ß-glucuronidase activities that cleave HS into smaller oligosaccharides (Bame, 2001Go; Bame and Robson, 1997Go; Toyoshima and Nakajima, 1999Go). Despite being tissue-specific, their action is highly dependent on the structure and sulfation pattern of HS (Bai et al., 1997Go; Okada et al., 2002Go). The tri- (m/z 509) and pentasaccharide (m/z 740) are likely products of heparanase action; from their composition it is predicted that they are derived from a low sulfated region of HS, unless perhaps they are desulfated by the action of endosulfatases (Tomita et al., 2002Go). Further work is required to understand how a tetrasaccharide (m/z 651) with a reducing end GlcNAc can be produced, as there are no reports of such endo-{alpha}-N-acetylglucosaminidase activity.

Cell death and/or exocytosis may result in these low-molecular-weight compounds of the lysosome being present in body fluids at concentrations higher than that seen in unaffected individuals. Of the oligosaccharides identified in MPS I, only four could be measured in biological samples, because the hydrophobic solid phase extraction material required to desalt prior to ESI-MS/MS does not bind larger, highly sulfated oligosaccharides. Using these four oligosaccharides as surrogate measures of disease has enabled diagnosis of MPS I from urine samples (Figure 3). These urine samples were from MPS I patients with a range of phenotypes and genotypes, as well as some for whom clinical information was unavailable. Additional studies to evaluate the influence of other factors (such as age of the patient and creatinine levels) will be required to refine the relation between disease severity and the type and amount of oligosaccharide in urine. Further work with these oligosaccharides may show them to be useful biomarkers for predicting disease severity and monitoring current and future therapies for MPS I.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Materials
Recombinant human IDUA (Unger et al., 1994Go), human N-acetylgalactosamine-4-sulfatase (Anson et al., 1992Go) and caprine glucosamine-6-sulfatase (Litjens et al., 1997Go) were each prepared from Chinese hamster ovary–K1 expression systems. MPS I patient urine samples were submitted to this department for diagnosis, and control urine samples were from age-matched volunteers. All urine samples were stored at –20°C. The internal standard used was an N-acetylglucosamine-6-sulfate (d3) prepared by selective, deuterated, N-acetylation of glucosamine-6-sulfate (Ramsay et al., 2003Go).

Cell culture
Fibroblasts were cultured from skin biopsies submitted to this hospital for diagnosis (Hopwood et al., 1982Go). Skin fibroblasts from unaffected individuals and MPS I (intermediate/severe phenotype) patients were cultured in BME containing 10% (v/v) fetal calf serum. Total cell protein was determined by the method of Lowry et al. (1951)Go. Recombinant human IDUA was administered to MPS I and control fibroblasts (6 weeks postconfluence) by supplementing the culture medium with 7 mg of the recombinant enzyme/flask. The media (+recombinant human IDUA) was replenished daily for up to 5 days. MPS I fibroblasts were harvested daily and control fibroblasts were harvested at 1 and 5 days.

Isolation of urinary GAG
Urine from an MPS I 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 GAGs were eluted in the same buffer containing 1.2 M NaCl. Fractions were assayed for UA (Blumenkrantz and Asboe-Hansen, 1973Go) and the GAG-containing fractions (20 ml) were pooled, lyophilized, and reconstituted in 4 ml H2O. The pooled GAG fraction was then size-fractionated on a Bio-Gel P2 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 P2 column fractions, urine (1 µmole creatinine equivalents) and cultured skin fibroblast extracts (200 µg protein) were lyophilized prior to derivatization. Samples were resuspended in 100 µl 250 mM 1-phenyl-3-methyl-5-pyrazolone, 400 mM NH4OH containing 1 nmol of internal standard. Samples were heated at 70°C for 90 min and then acidified with a twofold 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 1-phenyl-3-methyl-5-pyrazolone and centrifuged at 13,000xg for 5 min. Solid phase extraction cartridges (25 mg C18) were primed with methanol and water, after which the sample was applied and allowed to enter the solid phase completely. Samples were desalted with three consecutive 1.0 ml water washes; dried on a Supelco, Visiprep24 vacuum manifold; and any remaining 1-phenyl-3-methyl-5-pyrazolone was removed with two CHCl3 washes. The columns were again dried thoroughly, and derivatized oligosaccharides were eluted in an aqueous solution of 50% (v/v) CH3CN/0.025% (v/v) HCOOH.

Enzymatic cleavage
Derivatized oligosaccharides in the Bio-Gel P2 column fractions (100 µl) were digested with 50 ng recombinant human IDUA in 50 mM NH4COOCH3 buffer, pH 4.0, for 24 h at 37°C. One-tenth of this digest was analyzed by ESI-MS/MS, and the remainder was digested with either 50 ng recombinant human N-acetylgalactosamine-4-sulfatase (50 mM NH4COOCH3 buffer, pH 5.6) or 50 ng recombinant caprine glucosamine-6-sulfatase (50 mM NH4COOCH3 buffer, pH 5.0) for 24 h at 37°C. These digests were also analyzed by ESI-MS/MS.

MS
Oligosaccharide analysis was performed by ESI-MS/MS using a PE Sciex API 3000 triple-quadrupole mass spectrometer with an 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 215 autosampler at 80 µl/min using a carrying solvent of 50% (v/v) CH3CN/0.025% (v/v) HCOOH in H2O. Oligosaccharides were identified based on mass to charge (m/z) ratios in Q1 scans and further characterized using product ion scans in the negative ion mode. Relative quantification of these oligosaccharides was performed using MRM in negative ion mode. The ion pairs monitored were m/z 509/422, 630/256, 632/298, 633/259 (internal standard), 806/295, and 982/269; each pair was monitored for 100 ms at unit resolution. For each measurement, consecutive scans over the injection period were averaged, and relative concentration ratios were calculated by relating the peak heights of the derivatized oligosaccharides to the peak height of the internal standard. The ion pair 630/256 represents a sulfated N-acetylhexosamine previously reported elevated in some MPS types (Ramsay et al., 2003Go).


    Acknowledgements
 
We are particularly grateful to Enzo Ranieri for sharing unpublished data describing odd and even DS oligosaccharides in MPS VI patients. We also thank Julian Adams for helpful discussions. This work was supported in part by the National Health and Medical Research Council of Australia and the Wellcome Trust (U.K.), grant reference number 060104Z/00/Z.


    Footnotes
 
1 To whom correspondence should be addressed; e-mail: maria.fuller{at}adelaide.edu.au


    Abbreviations
 
DS, dermatan sulfate; ESI, electrospry ionization; GAG, glycosaminoglycans; GlcA, D-glucuronic acid; HS, heparan sulfate; IdoA, {alpha}-L-iduronic acid; IDUA, {alpha}-L-iduronidase; MPS, mucopolysaccharidosis; MRM, multiple-reaction monitoring; MS/MS, tandem mass spectrometry; UA, uronic acid


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Anson, D.S., Taylor, J.A., Bielicki, J., Harper, G.S., Peters, C., Gibson, G.J., and Hopwood, J.J. (1992) Correction of human mucopolysaccharidoses type VI fibroblasts with recombinant N-acetylgalactosamine-4-sulfatase. Biochem. J., 284, 789–794.[ISI][Medline]

Bach, G., Eisenberg, F. Jr., Cantz, M., and Neufeld, E.F. (1973) The defect in the Hunter syndrome: deficiency of sulfoiduronate sulfatase. Proc. Natl Acad. Sci. USA, 70, 2134–2138.[Abstract]

Bai, X., Bame, K.J., Habuchi, H., Kimata, K., and Esko, J.D. (1997) Turnover of heparan sulfate depends on 2-O-sulfation of uronic acids. J. Biol. Chem., 272, 23172–23179.[Abstract/Free Full Text]

Bame, K.J. (2001) Heparanases: endoglycosidases that degrade heparan sulfate proteoglycans. Glycobiology, 11, 91R–98R.[Abstract/Free Full Text]

Bame, K.J. and Robson, K. (1997) Heparanases produce distinct populations of heparan sulfate glycosaminoglycans in Chinese hamster ovary cells. J. Biol. Chem., 272, 2245–2251.[Abstract/Free Full Text]

Blumenkrantz, N. and Asboe-Hansen, G. (1973) New method for quantitative determination of uronic acids. Anal. Biochem., 54, 484–489.[ISI][Medline]

Byers, S., Rozaklis, T., Brumfield, L.K., Ranieri, E., and Hopwood, J.J. (1998) Glycosaminoglycan accumulation and excretion in the mucopolysaccharidoses: characterization and basis of a diagnostic test for MPS. Mol. Genet. Metab., 65, 282–290.[CrossRef][ISI][Medline]

Esko, J.D. and Selleck, S.B. (2002) Order out of chaos: assembly of ligand binding sites in heparan sulfate. Ann. Rev. Biochem., 71, 435–471.[CrossRef][ISI][Medline]

Hardingham. T.E. and Fosang, A.J. (1992) Proteoglycans: many forms and many functions. FASEB J., 6, 861–870.[Abstract/Free Full Text]

Hopwood, J.J. and Elliott, H. (1985) Urinary excretion of sulfated N-acetylhexosamines in patients with various mucopolysaccharidoses. Biochem. J., 229, 579–86.[ISI][Medline]

Hopwood, J.J., Muller, V., Harrison, J.R., Carey, W.F., Elliott, H., Robertson, E.F., and Pollard, A.C. (1982) Enzymatic diagnosis of the mucopolysaccharidoses: experience of 96 cases diagnosed in a five-year period. Med. J. Aust., 1, 257–260.[ISI][Medline]

Kjellén, L. and Lindahl, U. (1991) Proteoglycans: structures and interactions. Annu. Rev. Biochem., 60, 443–475. Erratum. Annu. Rev. Biochem., (1992) 61, viii.[CrossRef][ISI][Medline]

Kreil, G. (1995) Hyaluronidases—a group of neglected enzymes. Protein Sci., 4, 1666–1669.[Abstract/Free Full Text]

Lindahl, U., Kusche-Gullberg, M., and Kjellén, L. (1998) Regulated diversity of heparan sulfate. J. Biol. Chem., 273, 24979–24982.[Free Full Text]

Litjens, T., Bielicki, J., Anson, D.S., Fridericic, K., Jones, M.Z., and Hopwood, J.J. (1997) Expression, purification and characterization of recombinant caprine N-acetylglucosamine-6-sulfatase. Biochem. J., 327, 89–94.[ISI][Medline]

Lowry, O.H., Rosebrough, N.H., Farr, A.L., and Randall, R.J. (1951) Protein measurement with the Folin-phenol reagent. J. Biol. Chem., 193, 265–275.[Free Full Text]

Meikle, P.J., Hopwood, J.J., Clague, A.E., and Carey, W.F. (1999) Prevalence of lysosomal storage disorders. J. Am. Med. Assoc., 281, 249–254.[Abstract/Free Full Text]

Neufeld, E.F. and Muenzer, J. (2001) The mucopolysaccharidoses. In Scriver, C.R., Beaudet, A.L., Sly, W.S., and Valle, D. (Eds.), The metabolic and molecular bases of inherited disease, 8th ed. McGraw-Hill, New York, pp. 3421–3452.

Okada, Y., Yamada, S., Toyoshima, M., Dong, J., Nakajima, M., and Sugahara, K. (2002) Structural recognition by recombinant human heparanase that plays critical roles in tumor metastasis. Hierachical sulfate groups with different effects and the essential target disulfated trisaccharide sequence. J. Biol. Chem., 277, 42488–42495.[Abstract/Free Full Text]

Prydz, K. and Dalen, K.T. (2000) Synthesis and sorting of proteoglycans. J. Cell Sci., 113, 193–205.[Abstract/Free Full Text]

Ramsay, S.L., Meikle, P.J., and Hopwood, J.J. (2003) Determination of monosaccharides and disaccharides in mucopolysaccharidoses patients by electrospray ionisation mass spectrometry. Mol. Genet. Metab., 78, 193–204.[CrossRef][ISI][Medline]

Tomita, M-M., Uchimura, K., Werb, Z., Hemmerich, S., and Rosen, S.D. (2002) Cloning and characterisation of two extracellular heparin-degrading endosulfatases in mice and humans. J. Biol. Chem., 277, 49175–49185.[Abstract/Free Full Text]

Toyoshima, M. and Nakajima, M. (1999) Human heparanase. Purification, characterization, cloning and expression. J. Biol. Chem., 274, 2245–2251.

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 mucopolysaccahridoses type I fibroblasts. Biochem. J., 304, 43–49.[ISI][Medline]