The articular and the craniocervical abnormalities are of confusing age of onset in patients with Maroteaux-Lamy disease (MPS VI).

BACKGROUND: Maroteaux-Lamy disease (MPS Type VI) is an autosomal recessive lysosomal storage disorder. Skeletal abnormalities are vast. Early recognition may facilitate timely diagnosis and intervention, leading to improved patient outcomes. The most challenging is when patients manifest a constellation of craniocervical and articular deformities with variable age of onset. METHODS: We collected 15 patients with MPS VI (aged from 6 years-58 years). From within our practice in Pediatric Orthopedics, we present patients with MPS type VI who were found to manifest a diverse and confusing clinical presentation of hip deformities and cervical cord compression. Stem cell transplants were proposed as treatment tool and enzyme replacement therapy has been instituted in some patients. RESULTS: The spectrum of the clinical involvement in our group of patients was supported firstly via the clinical phenotype followed by assessment of the biochemical defect, which has been detected through the deficiency of N-acetylgalactosamine-4-sulfatase (arylsulphatase B) leading to increased excretion of dermatan sulphate. Secondly, through the molecular genetic results, which showed homozygous or compound heterozygous mutation in the ARSB gene on chromosome 5q14. Hip replacements and decompression operations have been performed to restore function and to alleviate pain in the former and life saving procedure in the latter. CONCLUSIONS: The efforts in searching for the etiological diagnosis in patients with skeletal dysplasia/MPSs has not been rewarding as many had anticipated. This emerged from several facts such as improper clinical documentation, missing diagnostic pointers in radiographic interpretations, limited knowledge in skeletal dysplasia and its variants, and the reliance on underpowered studies. Physicians and radiologists are required to appreciate and assess the diverse phenotypic and the radiographic variability of MPS VI. The importance of considering MPS in the differential diagnosis of other forms skeletal dysplasia is mandatory. Finally, we stress that the value of early diagnosis is to overcome dreadful complications.

A Possible Role for Arylsulfatase G in Dermatan Sulfate Metabolism.

Perturbations of glycosaminoglycan metabolism lead to mucopolysaccharidoses (MPS)-lysosomal storage diseases. One type of MPS (type VI) is associated with a deficiency of arylsulfatase B (ARSB), for which we previously established a cellular model using pulmonary artery endothelial cells with a silenced ARSB gene. Here, we explored the effects of silencing the ARSB gene on the growth of human pulmonary artery smooth muscle cells in the presence of different concentrations of dermatan sulfate (DS). The viability of pulmonary artery smooth muscle cells with a silenced ARSB gene was stimulated by the dermatan sulfate. In contrast, the growth of pulmonary artery endothelial cells was not affected. As shown by microarray analysis, the expression of the arylsulfatase G (ARSG) in pulmonary artery smooth muscle cells increased after silencing the arylsulfatase B gene, but the expression of genes encoding other enzymes involved in the degradation of dermatan sulfate did not. The active site of arylsulfatase G closely resembles that of arylsulfatase B, as shown by molecular modeling. Together, these results lead us to propose that arylsulfatase G can take part in DS degradation; therefore, it can affect the functioning of the cells with a silenced arylsulfatase B gene.

Translation arrest as a protein quality control system for aberrant translation of the 3'-UTR in mammalian cells.

Read-through or mutations of a stop codon resulting in translation of the 3'-UTR produce potentially toxic C-terminally extended proteins. However, quality control mechanisms for such proteins are poorly understood in mammalian cells. Here, a comprehensive analysis of the 3'-UTRs of genes associated with hereditary diseases identified novel arrest-inducing sequences in the 3'-UTRs of 23 genes that can repress the levels of their protein products. In silico analysis revealed that the hydrophobicity of the polypeptides encoded in the 3'-UTRs is correlated with arrest efficiency. These results provide new insight into quality control mechanisms mediated by 3'-UTRs to prevent the production of C-terminally extended cytotoxic proteins.

Mucopolysaccharidosis type VI (MPS VI) and molecular analysis: Review and classification of published variants in the ARSB gene.

Maroteaux-Lamy syndrome (MPS VI) is an autosomal recessive lysosomal storage disorder caused by pathogenic ARSB gene variants, commonly diagnosed through clinical findings and deficiency of the arylsulfatase B (ASB) enzyme. Detection of ARSB pathogenic variants can independently confirm diagnosis and render genetic counseling possible. In this review, we collect and summarize 908 alleles (201 distinct variants, including 3 polymorphisms previously considered as disease-causing variants) from 478 individuals diagnosed with MPS VI, identified from literature and public databases. Each variant is further analyzed for clinical classification according to American College of Medical Genetics and Genomics (ACMG) guidelines. Results highlight the heterogeneity of ARSB alleles, with most unique variants (59.5%) identified as missense and 31.7% of unique alleles appearing once. Only 18% of distinct variants were previously recorded in public databases with supporting evidence and clinical significance. ACMG recommends publishing clinical and biochemical data that accurately characterize pathogenicity of new variants in association with reporting specific alleles. Variants analyzed were sent to ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/), and MPS VI locus-specific database (http://mps6-database.org) where they will be available. High clinical suspicion coupled with diagnostic testing for deficient ASB activity and timely submission and classification of ARSB variants with biochemical and clinical data in public databases is essential for timely diagnosis of MPS VI.

Deletion of exon 4 in the N-acetylgalactosamine-4-sulfatase gene in a Taiwanese patient with mucopolysaccharidosis type VI.

Mucopolysaccharidosis type VI (MPS VI) is a rare autosomal recessive disorder caused by a deficiency of N-acetylgalactosamine-4-sulfatase (ARSB), one of the enzymes required for the degradation of dermatan sulfate (DS). Accumulation of DS in connective tissue causes growth failure, resulting in short stature. Here, we observed a 5-year-old girl who was the only one affected member of her family and who presented with an exaggerated, convex curvature of the back at the age of one year. Abnormal excretion of DS in the urine and extremely low leukocyte ARSB activity were noted. The patient was suspected to have MPS VI. Direct DNA sequencing indicated that there was no mutation in the coding region of ARSB. However, RT-PCR analysis of RNA prepared from blood samples indicated the deletion of the entire exon 4. Further analysis of the genomic DNA by quantitative PCR confirmed a homozygous deletion of exon 4, an unusual intragenic deletion in ARSB. The deletion led to a truncated protein that lacks most of the catalytic domain. The patient received recombinant human ARSB as enzyme replacement therapy (ERT) at an early stage (2 years), and responded positively in terms of skeletal development and other developmental milestones. The early identification of type VI MPS patients and subsequent treatment with ERT may be beneficial for the clinical outcome of MPS VI patients. In addition, detailed gene analysis may enhance the ability to provide genetic counseling to families of patients affected by MPS VI.

Exploiting 4-sulphate N-acetyl galactosamine decorated gelatin nanoparticles for effective targeting to professional phagocytes in vitro and in vivo.

The present study was focused on the development of surface modified gelatin nanoparticles (SGNPs) using novel ligand 4-sulfated N-acetyl galactosamine (4-SO(4)GalNAc) for specific targeting to macrophages. The gelatin has been modified with the potential targeting moiety 4-SO(4)GalNAc, which was further used for the preparation of modified nanoparticles. The nanoparticles have been prepared by two step desolvation method. The SGNPs and unmodified gelatin nanoparticles (GNPs) were loaded with doxorubicin (DxR) and its targeting potential was compared. Developed DxR-loaded SGNPs (DxR-SGNPs) were found to have negative zeta potential (-19.8 ± 0.22 mV) whereas DxR-loaded GNPs (DxR-GNPs) have the positive zeta potential of around +12.2 ± 0.36 mV. The mean particle size of DxR-SGNPs and DxR-GNPs was found to be 283 ± 7 and 134 ± 5 nm, respectively. Flow cytometric data confirmed the enhanced uptake of DxR-SGNPs in J774A.1 and PBMC when compared with DxR-GNPs. Intracellular localization studies indicate that the fluorescence intensity of DxR-SGNPs was significantly higher when compared to DxR-GNPs. DxR-SGNPs rendered significantly higher localization of DxR in liver and spleen as compared to DxR-GNPs after i.v. administration. The study stipulates that 4-SO(4)GalNAc assures for targeting resident macrophages.

Database of the clinical phenotypes, genotypes and mutant arylsulfatase B structures in mucopolysaccharidosis type VI.

Mucopolysaccharidosis type VI (MPS VI) is a genetic disorder caused by a deficiency of arylsulfatase B (ARSB). In our previous study, we investigated the structural changes in ARSB caused by amino acid substitutions associated with MPS VI, and revealed that such structural changes in ARSB were correlated with the clinical phenotypes. To the best of our knowledge, there is no database containing the structures of mutant ARSBs. Here, we built a database of clinical phenotypes, genotypes and structures of mutant ARSBs (http://mps6-database.org). This database can be accessed via the Internet, and is user friendly being equipped with powerful computational tools. This database will be useful for a better understanding of MPS VI.

Segregation analysis in a family at risk for the Maroteaux-Lamy syndrome conclusively reveals c.1151G>A (p.S384N) as to be a polymorphism.

Maroteaux-Lamy syndrome is an autosomal-recessive disorder due to the deficit of the lysosomal enzyme, arylsulfatase B (ARSB). Among the numerous genomic lesions reported till now, the sequence variant, c.1151G>A (p.S384N), has been associated with a severe phenotype in more than 10% of the patients. We now report the first in vivo demonstration of the polymorphic nature of p.S384N, revealed during the segregation analysis in a family at risk for Maroteaux-Lamy syndrome. The proband, compound heterozygous for c.[944G>A]+[245T>G] (p.[R315Q]+[L82R]), did not carry the p.S384N change, which was instead present in two healthy members of the family, in trans with the causative mutations, p.R315Q and p.L82R, respectively. The hypothesis that p.S384N was a polymorphism was further addressed by reverse dot-blot analysis of 400 control alleles, estimating an allele frequency of 4.5%. To predict the consequences of p.R315Q, p.L82R and p.S384N, we also modeled and compared the three amino-acid changes in the three-dimensional ARSB structure. The in silico analysis predicted a local protein misfolding in the presence of p.R315Q and p.L82R. On the contrary, no evident problem was predicted in the case of p.S384N, occurring on the protein surface, far from the active site. Overall, these findings strongly support the hypothesis that the non-synonymous change p.S384N is a polymorphism. Moreover, our results emphasize the need for caution in drawing conclusions from a novel variant allele before screening at least 50 healthy control subjects.

Structural and clinical implications of amino acid substitutions in N-acetylgalactosamine-4-sulfatase: insight into mucopolysaccharidosis type VI.

To elucidate the basis of mucopolysaccharidosis type VI (MPS VI) from the point of view of enzyme structure, we built structural models of mutant N-acetylgalactosamine-4-sulfatase (4S) resulting from 34 missense mutations (17 severe and 17 attenuated), and analyzed the influence of each amino acid replacement on the structure by calculating the number of atoms affected. Then, we calculated the average of solvent-accessible surface area value of the residues for which a substitution was identified in the severe MPS VI group and compared it with that in the attenuated MPS VI group. In the severe MPS VI group, the number of atoms influenced by a mutation was generally larger than that in the attenuated MPS VI group in both the main chain and the side chain, and residues associated with the mutations found in the severe MPS VI group tended to be less solvent-accessible than those in the attenuated MPS VI group. Furthermore, we analyzed the structural changes in 4S caused by six amino acid substitutions, for which the expressed proteins have been characterized, by means of color imaging. The results revealed that R95Q, G144R, H393P, and C521Y cause large structural changes, and that they are associated with the severe phenotype. On the other hand, G137V and Y210C are thought to cause small structural changes in a limited region resulting in the attenuated phenotype. Structural study is useful for elucidating the basis of MPS VI and predicting the influence of amino acid substitutions on clinical outcome, although there are a couple of exceptional cases.

Human sulfatases: a structural perspective to catalysis.

The sulfatase family of enzymes catalyzes hydrolysis of sulfate ester bonds of a wide variety of substrates. Seventeen genes have been identified in this class of sulfatases, many of which are associated with genetic disorders leading to reduction or loss of function of the corresponding enzymes. Amino acid sequence homology suggests that the enzymes have similar overall folds, mechanisms of action, and bivalent metal ion-binding sites. A catalytic cysteine residue, strictly conserved in prokaryotic and eukaryotic sulfatases, is post-translationally modified into a formylglycine. Hydroxylation of the formylglycine residue by a water molecule forming the activated hydroxylformylglycine (a formylglycine hydrate or a gem-diol) is a necessary step for the enzyme's sulfatase activity. Crystal structures of three human sulfatases, arylsulfatases A and B(ARSA and ARSB), and estrone/dehydroepiandrosterone sulfatase or steroid sulfatase (STS), also known as arylsulfatase C, have been determined. While ARSA and ARSB are water-soluble enzymes, STS has a hydrophobic domain and is an integral membrane protein of the endoplasmic reticulum. In this article, we compare and contrast sulfatase structures and revisit the proposed catalytic mechanism in light of available structural and functional data. Examination of the STS active site reveals substrate-specific interactions previously identified as the estrogen-recognition motif. Because of the proximity of the catalytic cleft of STS to the membrane surface, the lipid bilayer has a critical role in the constitution of the active site, unlike other sulfatases.

Recognition of arylsulfatase A and B by the UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-phosphotransferase.

The critical step for sorting of lysosomal enzymes is the recognition by a Golgi-located phosphotransferase. The topogenic structure common to all lysosomal enzymes essential for this recognition is still not well defined, except that lysine residues seem to play a critical role. Here we have substituted surface-located lysine residues of lysosomal arylsulfatases A and B. In lysosomal arylsulfatase A only substitution of lysine residue 457 caused a reduction of phosphorylation to 33% and increased secretion of the mutant enzyme. In contrast to critical lysines in various other lysosomal enzymes, lysine 457 is not located in an unstructured loop region but in a helix. It is not strictly conserved among six homologous lysosomal sulfatases. Based on three-dimensional structure comparison, lysines 497 and 507 in arylsulfatase B are in a similar position as lysine 457 of arylsulfatase A. Also, the position of oligosaccharide side chains phosphorylated in arylsulfatase A is similar in arylsulfatase B. Despite the high degree of structural homology between these two sulfatases substitution of lysines 497 and 507 in arylsulfatase B has no effect on the sorting and phosphorylation of this sulfatase. Thus, highly homologous lysosomal arylsulfatases A and B did not develop a single conserved phosphotransferase recognition signal, demonstrating the high variability of this signal even in evolutionary closely related enzymes.

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.

Mucopolysaccharidosis type VI: Structural and clinical implications of mutations in N-acetylgalactosamine-4-sulfatase.

Mucopolysaccharidosis type VI (MPS-VI) is an autosomal recessive lysosomal storage disorder caused by the deficiency of N-acetylgalactosamine-4-sulfatase (4S; or ARSB). Mutations in the 4S gene are responsible for 4S deficiency, which leads to the intralysosomal storage of partially degraded glycosaminoglycans, dermatan sulfate, and chondroitin 4-sulfate. To date, a total of 45 clinically relevant mutations have been identified in the human 4S gene. Missense mutations are the largest group, with 31 identified mutations. Nonsense mutations and small insertions or deletions comprise the remainder, with seven mutations each. Six polymorphisms have also been reported: two amino acid substitutions and four silent transitions. Mapping of the missense mutations onto the 4S structure shows that they are distributed throughout the three subunits of the mature 4S polypeptide. Mutations have been identified in active site residues, in residues adjacent to the active site, in potential substrate binding residues, in residues exposed on the surface, and in residues buried within the protein core. Missense mutations have also been identified in disulfide crosslinks. Molecular modeling of MPS-VI mutations onto the 4S structure suggests that the majority cause 4S deficiency via destabilization and the consequent reduction of 4S protein concentration. The vast majority of MPS-VI mutant alleles are either unique to a patient or are present in a small number of patients. So far, no common mutations have been described. Therefore, screening of the general population for MPS-VI alleles will be difficult.

Enzyme replacement therapy in a feline model of MPS VI: modification of enzyme structure and dose frequency.

Enzyme replacement therapy (ERT) in the MPS VI cat is effective at reducing or eliminating pathology in most connective tissues. One exception is that cartilage and chondrocytes remained distended with extensive lysosomal vacuolation after long-term, high-dose ERT. In this study, we demonstrate that recombinant human N-acetylgalactosamine-4-sulphatase (4S) is taken up by chondrocytes via a mannose-6-phosphate-dependent mechanism and is effective at removing MPS storage. In vitro, the penetration of 4S into articular cartilage is low (partitioning coefficient = 0.06) and i.v. administered enzyme does not distribute significantly into articular cartilage in vivo. To alter the tissue distribution of 4S, the enzyme was coupled to ethylene diamine or poly-L-lysine, increasing its overall charge and diffusion into cartilage, and the dosing frequency of unmodified 4S was increased. Modification resulted in active 4S that maintained its ability to correct MPS storage and increased the partitioning coefficient of 4S into cartilage by 77% and 50% for ethylene diamine and poly-L-lysine, respectively. However, in vivo ERT studies demonstrated that response to therapy was not significantly improved by either the enzyme modifications or change to the dosing regimen, when compared with ERT with unmodified enzyme. Distribution experiments indicated the majority of enzyme is taken up by the liver irrespective of modification. To optimize therapy and improve the amount of enzyme reaching cartilage and other tissues demonstrating poor uptake, it may be necessary to bypass the liver or prolong plasma half-life so that proportionately more enzyme is delivered to other tissues.

Changes in glycosylation of rat liver arylsulfatase B in relation to age.

The aim of this study was to determine how glycosylation of the rat liver arylsulfatase B was influenced by the age of the animal. The enzyme was purified from a liver lysosomal fraction obtained from male Wistar rats aged 18 days of gestation, 1 week, and 1, 1.5, 3 and 18 months by an affinity chromatography. Examination of the carbohydrate structures was performed after electrophoresis and blotting, followed by a very sensitive detection system with a set of six highly specific digoxygenin-labelled lectins. After densitometric measurement of the intensity of a digoxigenin-labelled lectin binding to arylsulfatase B, it could be stated that, at least, changes in sialylation are related to the growth and development of rats. Sialylation increases while fucosylation slightly decreases with age of the animal.

Maroteaux-lamy syndrome: five novel mutations and their structural localization.

Maroteaux-Lamy syndrome (mucopolysaccharidosis type VI, MPS VI) is an autosomal recessive disorder due to the deficiency of the lysosomal enzyme N-acetylgalactosamine-4-sulfatase (arylsulfatase B, ASB). Mutation analysis in Maroteaux-Lamy syndrome resulted in the identification of approximately 40 molecular defects underlying a great genetic heterogeneity. Here we report five novel mutations in Italian subjects: S65F, P116H, R315Q, Q503X, P531R; each defect was confirmed by restriction enzyme or amplification refractory mutation system (ARMS) analysis. We also performed a three-dimensional (3-D) structure analysis of the alterations identified by us, and of an additional 22 point mutations reported by other groups, in an attempt to draw helpful information about their possible effects on protein conformation.

Recombinant caprine 3H-[N-acetylglucosamine-6-sulfatase] and human 3H-[N-acetylgalactosamine-4-sulfatase]: plasma clearance, tissue distribution, and cellular uptake in the rat.

The use of recombinant lysosomal enzymes for enzyme replacement therapy (ERT) is likely to be a necessary component of effective treatment regimens for lysosomal storage diseases (LSDs). The mechanism and rate of uptake into target cells, rate of disappearance of the enzyme from plasma, and its tissue distribution are important factors to assess the need for possible modifications to the enzyme, particularly for LSDs that affect the central nervous system (CNS). Two recombinant lysosomal enzymes, caprine N-acetylglucosamine-6-sulfatase (rc6S) and human N-acetylgalactosamine-4-sulfatase (rh4S), deficient in MPS IIID and MPS VI, respectively, were radiolabeled and purified. The major portion (>77%) of each recombinant enzyme contained the mannose-6-phosphate (M6P) recognition marker as demonstrated by their ability to bind to a M6P receptor affinity column. The uptake of 3H-rc6S and 3H-rh4S into cultured rat brain cells was also inhibited by the addition of 5 mM M6P to the culture medium. After iv administration of 0.4-0.5 mg/kg of 3H-rc6S and 1 mg/kg of 3H-rh4S to the rat, both enzymes were rapidly lost from the circulation in a biphasic fashion (t1/2 for 3H-rc6S = 1.25+/-0.15 min and 37.17+/-23.29 min; t1/2 for 3H-rh4S = 0.41 and 5.3 min). At this dose, about 6% of 3H-rc6S, but only 0.49% of 3H-rh4S, remained in the plasma 4 h after administration, whereas approx 30% of 3H-rc6S and more than 50% of 3H-rh4S was found in the liver. At doses of 1.6-2.0 mg/kg of 3H-rc6S and 1 mg/kg 3H-rh4S, but not at the lower dose of 3H-rc6S, trace levels of both 3H-rc6S and 3H-rh4S were detected in the brain. The low level of enzyme recovered from the brain suggests that modification of rc6S will be necessary to achieve sufficient enzyme uptake into the CNS for effective therapy of MPS IIID.