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
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Abstract |
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Key words: dermatan sulfate / endohydrolase / glycosaminoglycans / heparan sulfate / mucopolysaccharidosis I
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Introduction |
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MPS I, the most common of the 11 reported MPS disorders (Meikle et al., 1999), results from a deficiency in the exohydrolase
-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
-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, 2001
).
HS has repeating disaccharide units consisting of uronic acid (UA) alternating with -linked (1,4) glucosamine residues. The UA residue may be ß-linked (1,4) D-glucuronic (GlcA) or
-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, 1991
). HS forms block structures with GlcA-GlcNAc disaccharides alternating with blocks of highly sulfated IdoA-GlcNS disaccharides (Lindahl et al., 1998
). 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)
-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, 1991
; Prydz and Dalen, 2000
). 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, 1995). Several endoglucuronidases, known as heparanases, degrade HS chains to oligosaccharides (Bame, 2001
). 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, 1997
). 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, 1985
). Heparin-degrading endosulfatases have also been identified and shown to have high selectivity for glucosamine-6-sulfate (Tomita et al., 2002
). 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., 1998, 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., 1973
). 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.
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Results |
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Discussion |
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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., 2003). 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 2529 (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., 1998). 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, 1995). 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, 2001; Bame and Robson, 1997
; Toyoshima and Nakajima, 1999
). Despite being tissue-specific, their action is highly dependent on the structure and sulfation pattern of HS (Bai et al., 1997
; Okada et al., 2002
). 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., 2002
). 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-
-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.
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Materials and methods |
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Cell culture
Fibroblasts were cultured from skin biopsies submitted to this hospital for diagnosis (Hopwood et al., 1982). 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)
. 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, 1973) 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., 2003).
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Acknowledgements |
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Footnotes |
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Abbreviations |
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References |
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