iduronate and Mucopolysaccharidosis-I

Mucopolysaccharidoses (MPSs) are complex lysosomal storage disorders that result in the accumulation of glycosaminoglycans (GAGs) in urine, blood, and tissues. Lysosomal enzymes responsible for GAG degradation are defective in MPSs. GAGs including chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS), and keratan sulfate (KS) are disease-specific biomarkers for MPSs. This article describes a stable isotope dilution-tandem mass spectrometric method for quantifying CS, DS, and HS in urine samples. The GAGs are methanolyzed to uronic or iduronic acid-N-acetylhexosamine or iduronic acid-N-sulfo-glucosamine dimers and mixed with internal standards derived from deuteriomethanolysis of GAG standards. Specific dimers derived from HS, DS, and CS are separated by ultra-performance liquid chromatography (UPLC) and analyzed by electrospray ionization tandem mass spectrometry (MS/MS) using selected reaction monitoring for each targeted GAG product and its corresponding internal standard. This UPLC-MS/MS GAG assay is useful for identifying patients with MPS types I, II, III, VI, and VII. © 2023 Wiley Periodicals LLC. Basic Protocol: Urinary GAG analysis by ESI-MS/MS Support Protocol 1: Prepare calibration samples Support Protocol 2: Preparation of stable isotope-labeled internal standards Support Protocol 3: Preparation of quality controls for GAG analysis in urine Support Protocol 4: Optimization of the methanolysis time Support Protocol 5: Measurement of the concentration of methanolic HCl.

Topics: Chondroitin Sulfates; Chromatography, Liquid; Dermatan Sulfate; Glycosaminoglycans; Heparitin Sulfate; Humans; Iduronic Acid; Isotopes; Mucopolysaccharidoses; Mucopolysaccharidosis I; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry

Mucopolysaccharidosis type I (MPS-I) is a rare lysosomal storage disorder caused by deficiency of the enzyme alpha-L-iduronidase, which removes iduronic acid in both chondroitin/dermatan sulfate (CS/DS) and heparan sulfate (HS) and thereby contributes to the catabolism of glycosaminoglycans (GAGs). To ameliorate this genetic defect, the patients are currently treated by enzyme replacement and bone marrow transplantation, which have a number of drawbacks. This study was designed to develop an alternative treatment by inhibition of iduronic acid formation. By screening the Prestwick drug library, we identified ebselen as a potent inhibitor of enzymes that produce iduronic acid in CS/DS and HS. Ebselen efficiently inhibited iduronic acid formation during CS/DS synthesis in cultured fibroblasts. Treatment of MPS-I fibroblasts with ebselen not only reduced accumulation of CS/DS but also promoted GAG degradation. In early Xenopus embryos, this drug phenocopied the effect of downregulation of DS-epimerase 1, the main enzyme responsible for iduronic production in CS/DS, suggesting that ebselen inhibits iduronic acid production in vivo. However, ebselen failed to ameliorate the CS/DS and GAG burden in MPS-I mice. Nevertheless, the results propose a potential of iduronic acid substrate reduction therapy for MPS-I patients.

Topics: Dose-Response Relationship, Drug; Fibroblasts; Glycosaminoglycans; HEK293 Cells; Humans; Iduronic Acid; Isoindoles; Molecular Structure; Mucopolysaccharidosis I; Organoselenium Compounds; Structure-Activity Relationship

Preparation of substituent-diverse, triazole-iduronic acid hybrid molecules by click reaction of an azido iduronic acid derivative with randomly chosen alkynes is described. Library members were screened for their ability to inhibit α-l-iduronidase, and hit molecules and analogues were then investigated for their ability to stabilize rh-α-IDUA in a thermal denaturation study. This work resulted in the discovery of the first small molecules that can be used to stabilize exogenous rh-α-IDUA protein in vitro.

Topics: Click Chemistry; Drug Discovery; Enzyme Stability; Humans; Iduronic Acid; Iduronidase; Molecular Structure; Mucopolysaccharidosis I; Small Molecule Libraries; Triazoles

Mucopolysaccharide (MPS) diseases are characterized by accumulation of glycosaminoglycans (GAGs) due to deficiencies in lysosomal enzymes responsible for GAG breakdown. Using a murine model of MPSI Hurler (MPSIH), we have quantified the heparan sulfate (HS) accumulation resulting from α-l-iduronidase (Idua) deficiency. HS levels were significantly increased in liver and brain tissue from 12-week-old Idua(-/-) mice by 87- and 20-fold, respectively. In addition, HS chains were shown to contain significantly increased N-, 2-O-, and 6-O-sulfation. Disaccharide compositional analyses also uncovered an HS disaccharide uniquely enriched in MPSIH, representing the terminal iduronic acid residue capping the non-reducing end of the HS chain, where no further degradation can occur in the absence of Idua. Critically, we identified that excess HS, some of which is colocalized to the Golgi secretory pathway, acts as a positive regulator of HS-sulfation, increasing the N-sulfotransferase activity of HS-modifying N-deacetylase/N-sulfotransferase enzymes. This mechanism may have severe implications during disease progression but, now identified, could help direct improved therapeutic strategies.

Topics: Animals; Disease Models, Animal; Golgi Apparatus; Heparitin Sulfate; Humans; Iduronic Acid; Iduronidase; Mice; Mice, Knockout; Mucopolysaccharidosis I; Sulfotransferases

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-beta-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.

Topics: Animals; Cells, Cultured; Cricetinae; Dermatan Sulfate; Fibroblasts; Glucuronidase; Glycosaminoglycans; Heparitin Sulfate; Humans; Iduronic Acid; Iduronidase; Mucopolysaccharidosis I; Skin; Spectrometry, Mass, Electrospray Ionization

Using 4-methylumbelliferyl-alpha-L-iduronide as a substrate, alpha-L-iduronidase activity was measured in leukocytes and in lymphoblastoid cells obtained from patients with alpha-L-iduronidase deficiency and from obligate heterozygotes for this disease. There was complete discrimination between alpha-L-iduronide in leukocytes and in lymphoblastoid cells from the patients and controls. However, overlap was observed between values of the activity in the obligate heterozygotes and those in the controls. 4-Methylumbelliferyl-alpha-L-iduronidase activity because of greater sensitivity, easier assay procedure and shorter incubation period.

Topics: Female; Fluorometry; Glycoside Hydrolases; Humans; Hymecromone; Iduronic Acid; Iduronidase; Male; Mucopolysaccharidoses; Mucopolysaccharidosis I; Umbelliferones

4-Methylumbelliferyl-alpha-l-iduronide provided a more sensitive method than phenyl-alpha-l-iduronide for the estimation of alpha-l-iduronidase in cultured cells and could be used to diagnose Hurler's disease. The 4-methylumbelliferyl derivative was no more useful than the phenyl derivative for the detection of heterozygotes. All ten lysosomal enzymes tested could be used as reference enzymes when cell extracts were prepared by freeze/thawing in formate buffer pH 3.5 containing 150 mmol/l sodium chloride.

Topics: Amniotic Fluid; Clinical Enzyme Tests; Female; Fibroblasts; Genetic Carrier Screening; Glycoside Hydrolases; Humans; Hydrolases; Hymecromone; Iduronic Acid; Iduronidase; Kinetics; Mucopolysaccharidosis I; Pregnancy; Skin; Substrate Specificity; Umbelliferones; Uronic Acids

1. Homogenates of cultured skin fibroblasts derived from patients with alpha-L-iduronidase-deficiency disorders (Hurler and Scheie syndromes) were capable of hydrolysing iduronosyl anhydro-[1-3H]mannitol 6-sulphate although at considerably reduced rates compared with normal controls. 2. The Vmax. values of alpha-L-iduronidase from patients with Hurler or Scheie syndromes and from normal controls were 11, 12 and 833 pmol min-1 mg-1 of protein respectively; the corresponding apparent Km values were 656, 50 and 53 mumol/l respectively. The alpha-L-iduronidases from normal and Scheie fibroblast homogenates were shown to exhibit pH optima at 3.6 and 4.1 and were competitively inhibited by both chloride and sulphate ions: Hurler alpha-L-iduronidase activity exhibited the pH optimum at 3.8 and was also inhibited by chloride and to a lesser extent by sulphate ions. 3. The thermal stability of Hurler, Scheie and normal alpha-L-iduronidase activities at 55 degrees C gave half-lives of approximately 1.0, 2.5 and 1.0 h respectively. 4. These biochemical findings clearly demonstrate enzyme differences for these two clinically distinct phenotypes and provide biochemical evidence that the Hurler and Scheie syndromes result from different allelic mutations.

Topics: Child; Chlorides; Fibroblasts; Glucaric Acid; Glucuronidase; Humans; Hydrogen-Ion Concentration; Hydrolysis; Iduronic Acid; Iduronidase; Kinetics; Lactones; Mannitol; Mucopolysaccharidoses; Mucopolysaccharidosis I; Proteins; Sulfates

A fluorogenic substrate for alpha-L-iduronidase, 4-methylumbelliferyl alpha-L-iduronide, has been newly synthesized and the enzyme activity has been measured in urine samples obtained from normal persons and patients suffering from mucopolysaccharidosis. Urine samples derived from a patient with Scheie syndrome showed greatly reduced activity compared with a normal adult at a similar age. This patient exhibited a high level of urinary excretion of dermatan sulfate and heparan sulfate, which could be interpreted in terms of her low alpha-L-iduronidase activity. The use of the fluorogenic substrate has some advantages over existing methods because of the high sensitivity and the relative ease of handling, and it should be useful not only for diagnosis but also for following the purification process of the enzyme.

Topics: Adult; Child, Preschool; Dermatan Sulfate; Female; Fluorometry; Glycoside Hydrolases; Heparitin Sulfate; Humans; Hymecromone; Iduronic Acid; Iduronidase; Male; Middle Aged; Mucopolysaccharidoses; Mucopolysaccharidosis I