Analysis of N-acetylgalactosamine-4-sulfatase protein and kinetics in mucopolysaccharidosis type VI patients.

A sensitive and specific, monoclonal antibody-based immunoquantification assay has facilitated determination of the N-acetylgalactosamine-4-sulfatase (4-sulfatase) protein content in cultured fibroblasts from normal controls and mucopolysaccharidosis type VI (MPS VI) patients. The assay enabled the quantification of 4-sulfatase protein by using a panel of seven monoclonal antibodies and has shown that fibroblasts from 16 MPS VI patients contained less than or equal to 5% of the level determined for normal controls. Fibroblasts from the most severely affected patients contained the lowest levels of 4-sulfatase protein, usually with few epitopes detected, while fibroblasts from mildly affected patients had higher levels of 4-sulfatase protein, with all seven epitopes detected. The pattern of epitope expression is proposed to reflect the conformational changes in the 4-sulfatase protein that arise from different mutations in the 4-sulfatase gene. Immunoquantification in combination with a specific and highly sensitive 4-sulfated trisaccharide-based assay of enzyme activity in these MPS VI patient fibroblasts enabled the determination of residual 4-sulfatase catalytic efficiency (kcat/Km). The capacity of fibroblasts to degrade substrate (catalytic capacity) was calculated as the product of 4-sulfatase catalytic efficiency and the content of 4-sulfatase in fibroblasts. One patient, 2357, with no clinical signs of MPS VI but with reduced 4-sulfatase activity and protein (both 5% of normal) and dermatansulfaturia, had 5% of normal catalytic capacity. The other 15 MPS VI patient fibroblasts had 0%-1.4% of the catalytic capacity of fibroblasts from normal controls and were representative of the spectrum of MPS VI clinical phenotypes, from severe to mild.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human N-acetylgalactosamine-4-sulphatase biosynthesis and maturation in normal, Maroteaux-Lamy and multiple-sulphatase-deficient fibroblasts.

The biosynthesis and maturation of N-acetylgalactosamine-4-sulphatase (4-sulphatase) was studied in normal fibroblasts and in fibroblasts from patients with either mucopolysaccharidosis type VI (MPS VI; Maroteaux-Lamy syndrome) or multiple sulphatase deficiency (MSD). Fibroblasts were incubated in culture medium containing [3H]leucine or [35S]methionine, and radiolabelled 4-sulphatase was isolated by immunoaffinity chromatography using 4-sulphatase-specific monoclonal antibodies. In normal fibroblasts a precursor of 66 kDa, detected intracellularly after 3 h and in NH4Cl-induced secretions, was processed intracellularly, within an additional 3 h, to a polypeptide of 57 kDa composed of disulphide-linked polypeptides of 43 kDa and 8 kDa. All fibroblast lines obtained from MPS VI patients, exhibiting clinical characteristics ranging from no clearly recognized symptoms to the severe classical phenotype, incorporated radioactivity into immune-purified 4-sulphatase at a rate less than 10% of that seen in normal fibroblasts. Maturation of the residual 4-sulphatase showed, variously, features which may be indicative of delayed intracellular transport, decreased intracellular stability, failure of lysosomal targetting or resistance to enzyme processing. Although some features of the residual enzyme synthesis and maturation were consistent with the patient's clinical phenotype, this was infrequent. The maturation of 4-sulphatase in fibroblasts from MSD patients was indistinguishable from that in normal fibroblasts, and the half-life of 4-sulphatase in these fibroblasts, determined after a 24 h pulse and prolonged chase, was only slightly less than that in normal fibroblasts.     

Mucopolysaccharidosis type VI: identification of three mutations in the arylsulfatase B gene of patients with the severe and mild phenotypes provides molecular evidence for genetic heterogeneity.

Mucopolysaccharidosis type VI (MPS VI; Maroteaux-Lamy disease) results from the deficient activity of the lysosomal enzyme, arylsulfatase B (ASB; N-acetylgalactosamine-4-sulfatase E.C.3.1.6.1). The enzymatic defect leads to the accumulation of the glycosaminoglycan, dermatan sulfate, primarily in connective tissue and reticuloendothelial cell lysosomes. Although MPS VI patients have normal intelligence and no neurologic abnormalities, the disease is clinically heterogeneous: severely affected individuals expire in childhood or early adolescence while those with the mild or intermediate phenotypes have a slower, milder disease course and a longer life span. The recent isolation of the full-length cDNA-encoding human ASB permitted an investigation of the molecular lesions underlying the phenotypic heterogeneity in MPS VI. The ASB cDNA-coding sequences were determined from two unrelated MPS VI patients with the severe (proband 1) and mild (proband 2) phenotypes. These patients had about 2% and 7% of normal ASB activity in cultured fibroblasts, respectively. Proband 1 was homoallelic for a T-to-C transition in nucleotide (nt) 349, which predicted a cysteine-to-arginine substitution in the ASB polypeptide at residue 117 (C117R). Proband 2 was heteroallelic, having a T-to-C transition in nt 707, which predicted a leucine-to-proline replacement at ASB residue 236 (L236P), and having a G-to-A transition in nt 1214, which predicted a cysteine-to-tyrosine substitution at ASB residue 405 (C405Y). These mutations did not occur in three other unrelated MPS VI patients or in 120 ASB alleles from normal individuals, indicating that they were not polymorphisms. The identification of these three ASB mutations documents the first evidence of molecular heterogeneity in MPS VI and provides an initial basis for genotype/phenotype correlations in this lysosomal storage disease.     

Morquio syndrome: Clinical findings in 11 patients with MPS IVA and 2 patients with MPS IVB

Summary Genetic heterogeneity of the Morquio syndrome has been known since deficiency of -galactosidase was detected in some patients in addition to the deficiency of N-acetylgalactosamine-6-sulphate sulphatase in the classical form of Morquio syndrome. The clinical findings of 11 patients with MPS IVA, the classical form, and 2 patients with MPS IV B, a variant form, are described. All of the patients with MPS IV A showed extraskeletal manifestations of their disease. The patients with MPS IV B seem to be less severely affected.     

Mucopolysaccharide accumulation in cultured skin fibroblasts derived from patients with mucolipidosis IV.

Increased concentrations of total sulfated mucopolysaccharides (MPS), threefold, and hyaluronic acid (HA), 10-fold, were found in ML IV fibroblast extracts when compared to normal controls. Such accumulations altered the distribution of MPS:HA comprised 70% of total MPS in ML IV but only 30% in control cells. Intracellular sulfated MPS was observed accumulating almost linearly in ML IV fibroblasts. "Pulse-chase" experiments indicate that both HA and the sulfated MPS remain in the ML IV cells for long periods of time; in control cells, they are rapidly removed as low molecular weight, dialyzable fragments. These data suggest that the MPS accumulation in ML IV fibroblasts, is the consequence of a catabolic block, probably involving the lysosome.     

Generation of a novel disease model mouse for mucopolysaccharidosis type VI via c. 252T>C human ARSB mutation knock-in

Mucopolysaccharidosis type VI (MPS VI) is an autosomal recessive lysosomal disorder caused by a mutation in the ARSB gene, which encodes arylsulfatase B (ARSB), and is characterized by glycosaminoglycan accumulation. Some pathogenic mutations have been identified in or near the substrate-binding pocket of ARSB, whereas many missense mutations present far from the substrate-binding pocket. Each MPS VI patient shows different severity of clinical symptoms. To understand the relationship between mutation patterns and the severity of MPS VI clinical symptoms, mutations located far from the substrate-binding pocket must be investigated using mutation knock-in mice. Here, I generated a knock-in mouse model of human ARSB Y85H mutation identified in Japanese MPS VI patients using a CRISPR-Cas9-mediated approach. The generated mouse model exhibited phenotypes similar to those of MPS VI patients, including facial features, mucopolysaccharide accumulation, and smaller body size, suggesting that this mouse will be a valuable model for understanding MPS VI pathology.     

Mucopolysaccharidoses in northern Brazil: Targeted mutation screening and urinary glycosaminoglycan excretion in patients undergoing enzyme replacement therapy

Mucopolysaccharidoses (MPS) are rare lysosomal disorders caused by the deficiency of specific lysosomal enzymes responsible for glycosaminoglycan (GAG) degradation. Enzyme Replacement Therapy (ERT) has been shown to reduce accumulation and urinary excretion of GAG, and to improve some of the patients' clinical signs. We studied biochemical and molecular characteristics of nine MPS patients (two MPS I, four MPS II and three MPS VI) undergoing ERT in northern Brazil. The responsiveness of ERT was evaluated through urinary GAG excretion measurements. Patients were screened for eight common MPS mutations, using PCR, restriction enzyme tests and direct sequencing. Two MPS I patients had the previously reported mutation p.P533R. In the MPS II patients, mutation analysis identified the mutation p.R468W, and in the MPS VI patients, polymorphisms p.V358M and p.V376M were also found. After 48 weeks of ERT, biochemical analysis showed a significantly decreased total urinary GAG excretion in patients with MPS I (p < 0.01) and MPS VI (p < 0.01). Our findings demonstrate the effect of ERT on urinary GAG excretion and suggest the adoption of a screening strategy for genotyping MPS patients living far from the main reference centers.     

Determination of the lysosomal hydrolase activity in blood collected on filter paper,an alternative to screen high risk populations

This study aimed to determine the enzymatic activity in dried blood samples collected on filter paper (DBS) for the diagnosis of the following diseases: Fabry, Pompe, Mucopolysaccharidosis type I (MPS I) and Mucopolysaccharosis type VI (MPS VI). DBS was used for high risk patientscreening, according to clinical suspicion. Plasma, leukocytes and cultured fibroblasts were used to confirm the diagnosis when necessary. Among the 529 DBS samples sent to the laboratory, 164 had abnormal results. Confirmatory materials of 73 individuals were rerouted. The frequency of diagnosis for lysosomal storage disorders was 5.9%. DBS is an alternative screening technique used in high risk populations, which should lead to earlier diagnosis for lysosomal storage disorders (LSDs), help patients get treatment sooner and improve the outcome of the disease.     

Mucopolysaccharidosis type VI (Maroteaux-Lamy syndrome): a Y210C mutation causes either altered protein handling or altered protein function of N-acetylgalactosamine 4-sulfatase at multiple points in the vacuolar network

The lysosomal hydrolase N-acetylgalactosamine 4-sulfatase (4-sulfatase) is required for the degradation of the glycosaminoglycan substrates dermatan and chondroitin sulfate. A 4-sulfatase deficiency results in the accumulation of undegraded substrate and causes the severe lysosomal storage disorder mucopolysaccharidosis type VI (MPS VI) or Maroteaux-Lamy syndrome. A wide variation in clinical severity is observed between MPS VI patients and reflects the number of different 4-sulfatase mutations that can cause the disorder. The most common 4-sulfatase mutation, Y210C, was detected in approximately 10% of MPS VI patients and has been associated with an attenuated clinical phenotype when compared to the archetypical form of MPS VI. To define the molecular defect caused by this mutation, Y210C 4-sulfatase was expressed in Chinese hamster ovary (CHO-K1) cells for protein and cell biological analysis. Biosynthetic studies revealed that Y210C 4-sulfatase was synthesized at a comparable molecular size and amount to wild-type 4-sulfatase, but there was evidence of delayed processing, traffic, and stability of the mutant protein. Thirty-three percent of the intracellular Y210C 4-sulfatase remained as a precursor form, for at least 8 h post labeling and was not processed to the mature lysosomal form. However, unlike other 4-sulfatase mutations causing MPS VI, a significant amount of Y210C 4-sulfatase escaped the endoplasmic reticulum and was either secreted from the expression cells or underwent delayed intracellular traffic. Sixty-seven percent of the intracellular Y210C 4-sulfatase was processed to the mature form (43, 8, and 7 kDa molecular mass forms) by a proteolytic processing step known to occur in endosomes-lysosomes. Treatment of Y210C CHO-K1 cells with the protein stabilizer glycerol resulted in increased amounts of Y210C 4-sulfatase in endosomes, which was eventually trafficked to the lysosome after a long, 24 h chase time. This demonstrated delayed traffic of Y210C 4-sulfatase to the lysosomal compartment. The endosomal Y210C 4-sulfatase had a low specific activity, suggesting that the mutant protein also had problems with stability. Treatment of Y210C CHO-K1 cells with the protease inhibitor ALLM resulted in an increased amount of mature Y210C 4-sulfatase localized in lysosomes, but this protein had a very low level of activity. This indicated that the mutant protein was being inactivated and degraded at an enhanced rate in the lysosomal compartment. Biochemical analysis of Y210C 4-sulfatase revealed a normal pH optimum for the mutant protein but demonstrated a reduced enzyme activity with time, also consistent with a protein stability problem. This study indicated that multiple subcellular and biochemical processes can contribute to the biogenesis of mutant protein and may in turn influence the clinical phenotype of a patient. In MPS VI patients with a Y210C allele, the composite effect of different stages of intracellular processing/handling and environment has been shown to cause a reduced level of Y210C 4-sulfatase protein and activity, resulting in an attenuated clinical phenotype.     

In vitro gene therapy of mucopolysaccharidosis type I by lentiviral vectors.

Mucopolysaccharidosis type I (MPS I) results from a deficiency in the enzyme alpha-L-iduronidase (IDUA), and is characterized by skeletal abnormalities, hepatosplenomegaly and neurological dysfunction. In this study, we used a late generation lentiviral vector to evaluate the utility of this vector system for the transfer and expression of the human IDUA cDNA in MPS I fibroblasts. We observed that the level of enzyme expression in transduced cells was 1.5-fold the level found in normal cells; the expression persisted for at least two months. In addition, transduced MPS I fibroblasts were capable of clearing intracellular radiolabeled glycosaminoglycan (GAG). Pulse-chase experiments on transduced fibroblasts showed that the recombinant enzyme was synthesized as a 76-kDa precursor form and processed to a 66-kDa mature form; it was released from transduced cells and was endocytosed into a second population of untreated MPS I fibroblasts via a mannose 6-phosphate receptor. These results suggest that the lentiviral vector may be used for the delivery and expression of the IDUA gene to cells in vivo for treatment of MPS I.     

中国人Ⅱ型MPS家系IDS基因的一种新突变的鉴定

为了研究粘多糖贮积症Ⅱ型(MPSⅡ)患者发病的分子遗传学机制, 以便为今后的产前基因诊断等创造必要的前提条件, 文章先采用尿糖胺聚糖(GAGs)定性检测法对疑似MPSⅡ的先证者进行初诊, 然后采用PCR、PCR 产物直接测序法对先证者及其家系成员进行突变检测。在检出IDS基因c.876del2新突变后, 对随机采集的120例正常对照和其他非II型MPS患者包括MPSⅠ, Ⅳ, Ⅵ三型的病人共15例的IDS基因exon 6进行序列分析, 同时采用不同物种突变点序列的保守性分析法, 以及直接测定患儿及其家庭相关成员IDS酶活性的方法对该新突变进行致病性分析。结果显示: 先证者尿检呈强阳性(GAGs +++); 其IDS基因exon 6编码区内存在c.876-877 del TC新缺失突变, 为半合子突变, 而其母、其姐为杂合突变; 正常对照和其他非II型MPS患者的IDS基因exon 6的检测结果均未发现该突变; 不同物种氨基酸序列的同源性比对显示: c.876-877 del TC突变所在的位置即p.292-293的苯丙氨酸(F)谷氨酰胺(Q)高度保守; 酶活性测定的结果显示: 先证者的IDS酶活性仅为2.3 nmol/4 h/mL, 大大低于正常值, 而其父的为641.9 nmol/4 h/mL, 其母的血浆酶活性为95.8 nmol/ 4h/mL, 其姐的为103.2 nmol/4 h/mL。说明所发现的c.876-877 del TC缺失移码突变是一种新的病理性突变, 是该MPSⅡ患儿发病的根本内因。     

Preferential hydroxylation of type IV collagen by lysyl hydroxylase from Ehlers-Danlos syndrome type VI fibroblasts

Normal and Ehlers-Danlos syndrome type VI human skin and cornea fibroblasts were assayed for lysyl hydroxylase activity using two different collagen types as substrates. The enzyme from normal fibroblasts hydroxylated type I collagen more readily than type IV collagen. In the diseased cells the enzyme activity was significantly reduced, and the residual activity was preferentially directed towards type IV collagen. This suggests the existence of isoenzymes of lysyl hydroxylase or an alteration in the Ehlers-Danlos syndrome type VI that affects the binding of type I collagen more than that of type IV collagen.     

Cytochemical and electron microscopic research on the peripheral blood leukocytes and skin fibroblasts in mucopolysaccharidoses

Cytochemical studies of the peripheral blood leucocytes in patients suffering from mucopolysaccharidoses (MPS) have revealed metachromatic granules in the cell cytoplasm. Electron microscopy of these cells has shown multiple cytoplasmic vacuoles. It is supposed that metachromatic granules in blood leucocytes of patients with MPS observed in the photo-optical studies correspond to the vacuoles found under electron microscope. The obtained data have shown that peripheral blood leucocytes and skin fibroblasts have the common ultrastructure in MPS patients. The data of electron histochemical studies testify to that the vacuoles of skin fibroblasts are filled with glycosaminoglycans.     

Incidence of the mucopolysaccharidoses in Northern Ireland

An epidemiological study of the mucopolysaccharidoses (MPS) in Northern Ireland using multiple ascertainment sources was carried out and the incidence rate for the period 1958–1985 was estimated. An incidence of approximately 1 in 76 000 live births was obtained for MPS 1H (Hurler phenotype); 1 in 280 000 for MPS 1 H/S (Hurler/Scheie phenotype); 1 in 140 000 live births (1 in 72 000 male live births) for MPS II (Hunter syndrome); 1 in 280 000 for MPS III (Sanfilippo syndrome) and 1 in 76 000 for MPS IV A (Morquio syndrome type A). No cases of MPS IS (Scheie phenotype), MPS IV B (Morquio syndrome type B) or MPS VI (Maroteaux–Lamy syndrome) were ascertained during the study period. Three cases of non-immune hydrops fetalis born to consanguineous parents were thought to be due to β-glucuronidase deficiency (MPS VII) on the basis of placental histology and enzyme studies on both parents but no living cases of MPS VII were ascertained. The overall incidence for all types of mucopolysaccharidosis was approximately 1 in 25 000 live births. A comparison is made with incidence estimates obtained from other published studies. Received: 25 May 1997 / Accepted: 22 August 1997     

Feline mucopolysaccharidosis VI: purification and characterization of the resident arylsulfatase B activity

Hepatic arylsulfatase B (ASB) from normal and mucopolysaccharidosis VI (MPS VI) cats was purified over 2,800- and 1,800-fold, respectively, and their physical and kinetic properties were characterized. In contrast to the normal feline enzyme, the partially purified MPS VI residual activity had a 100-fold greater Km value and was markedly less stable to thermal, cryo-, and pH-inactivation. In addition, the MPS VI enzyme had a more negative charge as determined by its migration on polyacrylamide gel electrophoresis and its elution profile on cation exchange chromatography. Finally, the MPS VI activity had approximately half the apparent molecular weight of the normal feline enzyme, which was a homodimer, suggesting that the genetic mutation in feline MPS VI altered the subunit association as well as the kinetic and stability properties of the mutant protein.     

Pattern of collagen synthesis and chemotactic response of fibroblasts derived from mucopolysaccharidosis patients

For comparative studies on the migratory potential we screened fibroblast strains derived from mucopolysaccharidosis (MPS) patients regarding their differential response to chemotactic stimuli and analysed their production of extracellular matrix components. Indirect immunofluorescence staining of MPS-fibroblasts showed the same distribution of type I and type III collagen and of fibronectin as in controls. Biochemical quantification of type I and type III collagen demonstrated an unaltered ratio of these collagen types, although the total amount of newly synthesized collagens was slightly reduced in fibroblasts from MPS patients. Whereas the synthesis of major extracellular matrix components was close to normal, the response of the MPS cells to chemotactic stimuli was greatly affected. Chemotactic migration was improved when fibroblasts were pretreated with medium conditioned by normal fibroblasts, although they never reached normal levels.     

Design and synthesis of substrates for newborn screening of Maroteaux–Lamy and Morquio A syndromes

In continued efforts to develop enzymatic assays for lysosomal storage diseases appropriate for newborn screening laboratories we have synthesized novel and specific enzyme substrates for Maroteaux–Lamy (MPS VI) and Morquio A (MPS IVA) diseases. The sulfated monosaccharide derivatives were found to be converted to product by the respective enzyme in blood from healthy patients but not by blood from patients with the relevant lysosomal storage disease. The latter result shows that the designed substrates are highly selective for the respective enzymes.     

Immunoquantification and enzyme kinetics of alpha-L-iduronidase in cultured fibroblasts from normal controls and mucopolysaccharidosis type I patients.

alpha-L-Iduronidase activity is deficient in mucopolysaccharidosis type I (MPS I; Hurler syndrome, Scheie syndrome) patients and results in the disruption of the sequential degradation of the glycosaminoglycans dermatan sulfate and heparan sulfate. A monoclonal antibody-based immunoquantification assay has been developed for alpha-L-iduronidase, which enables the detection of at least 16 pg alpha-L-iduronidase protein. Cultured human skin fibroblasts from 12 normal controls contained 17-54 ng alpha-L-iduronidase protein/mg extracted cell protein. Fibroblasts from 23 MPS I patients were assayed for alpha-L-iduronidase protein content. Fibroblast extracts from one MPS I patient contained at least six times the level of alpha-L-iduronidase protein for normal controls--but contained no associated enzyme activity--and is proposed to represent a mutation affecting the active site of the enzyme. Fibroblast extracts from 11 MPS I patients contained 0.05-2.03 ng alpha-L-iduronidase protein/mg extracted cell protein, whereas immunodetectable protein could not be detected in the other 11 patients. Four fibroblast extracts with no immunodetectable alpha-L-iduronidase protein had residual alpha-L-iduronidase activity, suggesting that the mutant alpha-L-iduronidase in cultured cells from these MPS I patients has been modified to mask or remove the epitopes detected by two monoclonal antibodies used in the quantification assay. Both the absence of immunoreactivity in a mild MPS I patient and high protein level in a severe MPS I patient present limitations to the use of immunoquantification analysis as a sole measure of patient phenotype. Enzyme kinetic analysis of alpha-L-iduronidase from MPS I fibroblasts revealed a number of patients with either abnormal substrate binding or catalytic activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hurler syndrome: a patient with abnormally high levels of alpha-L-iduronidase protein.

Mucopolysaccharidosis type I (MPS I: McKusick 25280) is a clinically heterogenous lysosomal storage disorder which is caused by a variable deficiency in alpha-L-iduronidase activity (alpha-L-iduronide iduronohydrolase, EC 3.2.1.76). Cultured fibroblasts from an MPS I patient (cell line 2827) with a severe clinical phenotype (Hurler syndrome) have been characterized using immunochemical and biochemical techniques. Using a specific immunoquantification assay, we have demonstrated that cell line 2827 had an alpha-L-iduronidase protein content (189 ng/mg of extracted cell protein) at least six times greater than the mean level found in normal control fibroblasts (30 ng/mg of extracted cell protein). This was the only MPS I cell line, from a group of 23 MPS I patients, that contained greater than 7% of the mean level of alpha-L-iduronidase protein detected in normal controls. Cell line 2827 had very low alpha-L-iduronidase activity toward the fluorogenic substrate 4-methylumbelliferyl-alpha-L-iduronide, and a radiolabeled disaccharide substrate derived from heparin. Maturation studies of alpha-L-iduronidase in cell line 2827 showed apparently normal levels of alpha-L-iduronidase synthesis with delayed processing to the mature form. Subcellular fractionation experiments demonstrated alpha-L-iduronidase protein in lysosomal-enriched fractions isolated from cell line 2827, suggesting a normal cell distribution and supporting the proposed delayed processing. It is proposed that the MPS I patient described has an alpha-L-iduronidase gene mutation which affects both the active site and post-translational processing of the enzyme. This mutation must be structurally conservative because it does not result in instability either during maturation or in the lysosome.