Arylsulfatase B Research Articles

The miR-31b targets arylsulfatase B to regulate the ovarian development of Bactrocera dorsalis.

Reproduction is the basis of insect population growth and evolution, and encompasses ovarian development, reproductive behavior, and fecundity. Bactrocera dorsalis is a globally significant agricultural pest that is subject to quarantine, with mated females that can lay over 3000 eggs. The post-transcriptional regulation of ovarian development remains unclear. Here, miR-31b is shown to be involved in regulating Bactrocera dorsalis ovarian development. CRISPR/Cas9 was used to generate miR-31b loss-of-function mutations in Bactrocera dorsalis. The removal of miR-31b resulted in severely impaired ovarian development in adults, with phenotypes that included dramatically reduced egg production and hatching rates. The relationship between miR-31b and its target gene arylsulfatase B (ARSB) was subsequently identified using the methods of bioinformatics, transcriptomic sequencing, quantitative polymerase chain reaction (qPCR), RNA pull-down and dual-luciferase reporter assay. Finally, miR-31b was confirmed to bind the target gene arylsulfatase B to affect metabolism and thereby further hindered ovarian development of Bactrocera dorsalis. Overall, these results provide new insights into molecular mechanisms at the post-transcriptional level in regulating ovarian development and insect reproduction, consequently providing potential targets to control arthropod pests through the reproductive strategy. © 2024 Society of Chemical Industry.

Synergistic Impact of ARSB, TP53, and Maspin Gene Expressions on Survival Outcomes in Colorectal Cancer: A Comprehensive Clinicopathological Analysis

(1) Background: Colorectal cancer (CRC) remains a significant cause of morbidity and mortality worldwide, with its prognosis influenced by genetic and clinicopathological factors. This study investigates the associations between the gene expressions of Arylsulfatase B (ARSB), TP53, and Maspin, alongside traditional clinicopathological features, and their impact on CRC survival outcomes. (2) Methods: 70 consecutive CRC cases were analyzed for ARSB, TP53, and Maspin gene expression using RT-qPCR, and their protein levels were assessed through immunohistochemistry. Clinicopathological parameters—age, gender, tumor localization, macroscopic and microscopic aspects, lymph node ratio, pT stage, and tumor budding—were evaluated for their prognostic significance. Kaplan–Meier survival analysis with Cox proportional hazards regression was used to determine their impact on overall survival. (3) Results: No significant survival differences were observed based on age, gender, tumor localization, and macroscopic aspect. The microscopic aspect and pT stage showed significant associations with survival, with poorer outcomes in G3 and pT3/pT4 stages, respectively. Immunohistochemical positivity for ARSB and Maspin indicated a longer survival, while TP53 protein expression alone did not significantly impact the prognosis. Dual high gene expression (ARSB + TP53, TP53 + Maspin) and triple high gene expression (ARSB + TP53 + Maspin) were significantly associated with better survival outcomes. (4) Conclusions: The combined gene expression profile of ARSB, TP53, and Maspin presents a novel prognostic marker in CRC, offering insights into the molecular dynamics of cancer cells and potential therapeutic targets. These findings emphasize the importance of integrating molecular markers with traditional clinicopathological factors for a more accurate prognostication and personalized treatment approach in CRC.

Skeletal phenotype amelioration in Mucopolysaccharidosis VI requires intervention at the earliest stages of postnatal development.

Mucopolysaccharidosis VI (MPS VI) is a rare lysosomal disease arising from impaired function of the enzyme Arylsulfatase B (ARSB). This causes aberrant accumulation of dermatan sulfate, a glycosaminoglycan (GAG) abundant in cartilage. While clinical severity varies along with age at first symptom manifestation, MPS VI usually presents early and strongly affects the skeleton. Current enzyme replacement therapy (ERT) does not provide effective treatment for the skeletal manifestations of MPS VI. This lack of efficacy may be due to inability of ERT to reach affected cells, or irreversibility of disease. To address the question of reversibility of skeletal phenotypes, we generated a conditional by inversion (COIN) mouse model of MPS VI, ArsbCOIN/COIN, wherein Arsb is initially null and can be restored to wild type using Cre. We restored Arsb at different times during postnatal development, using a tamoxifen-dependent global Cre driver. By restoring Arsb at postnatal days 7, 21, and 56-70 (P7, P21, and P56-P70), we determined that skeletal phenotypes can be fully rescued if Arsb restoration occurs at P7, while only achieving partial rescue at P21, and no significant rescue at P56-70. This work has highlighted the importance of early intervention in MPS VI patients to maximize therapeutic impact.

Case study of two Iraqi patients with Mucopolysaccharidosis (Hurler syndrome "type I" and Maroteaux-Lamy syndrome "type VI") treated with Hematopoietic Stem Cell Transplantation (HSCT)

Mucopolysaccharidosis I (MPS I) or Hurler and Mucopolysaccharidosis VI (MPS VI) or Maroteaux-Lamy syndrome are infrequent genetic disorder inherited as an autosomal recessive disease attributed to genetic
 
 variants genetic variant causing α-L iduronidase (IDUA) and arylsulfatase B (ARSB)enzyme deficiency, respectively. Here, two cases of children suffering from MPS disorder were described, the first case was MPS I while the second case was MPS VI and both cases were treated with allogenic Hematopoietic Stem Cell Transplantation approach in order to limit skeletal deterioration and retard neurocognitive alterations and hence, improve the quality of life of affected children. Following Transplantations outcomes reveal a full engraftment of donor cells as well as improvement of recipient enzymatic activity, enzyme replacement therapy post-transplantation will augment transplantation clinical outcomes. Transplantation will be more successful if the disease diagnosed early before the severe irreversible symptoms ensue.

Modeling cartilage pathology in mucopolysaccharidosis VI using iPSCs reveals early dysregulation of chondrogenic and metabolic gene expression.

Mucopolysaccharidosis type VI (MPS VI) is a metabolic disorder caused by disease-associated variants in the Arylsulfatase B (ARSB) gene, resulting in ARSB enzyme deficiency, lysosomal glycosaminoglycan accumulation, and cartilage and bone pathology. The molecular response to MPS VI that results in cartilage pathology in human patients is largely unknown. Here, we generated a disease model to study the early stages of cartilage pathology in MPS VI. We generated iPSCs from four patients and isogenic controls by inserting the ARSB cDNA in the AAVS1 safe harbor locus using CRISPR/Cas9. Using an optimized chondrogenic differentiation protocol, we found Periodic acid-Schiff positive inclusions in hiPSC-derived chondrogenic cells with MPS VI. Genome-wide mRNA expression analysis showed that hiPSC-derived chondrogenic cells with MPS VI downregulated expression of genes involved in TGF-β/BMP signalling, and upregulated expression of inhibitors of the Wnt/β-catenin signalling pathway. Expression of genes involved in apoptosis and growth was upregulated, while expression of genes involved in glycosaminoglycan metabolism was dysregulated in hiPSC-derived chondrogenic cells with MPS VI. These results suggest that human ARSB deficiency in MPS VI causes changes in the transcriptional program underlying the early stages of chondrogenic differentiation and metabolism.

Optimisation of culture conditions for a producer clone coexpressing arylsulfatase B and a formylglycine-generating enzyme in order to increase the yield of arylsulfatase B

Maroteaux—Lamy syndrome (mucopolysaccharidosis type VI) is an orphan genetic disease caused by mutations in the arylsulfatase B gene (ARSB), which encodes the lysosomal enzyme arylsulfatase B (ASB). The relevance of the study lies in the need of a Russian recombinant ASB product for patients with the disease in the Russian Federation. Previously, the authors have developed producer lines coexpressing the target ASB enzyme with an auxiliary formylglycine-generating enzyme (FGE), based on Chinese hamster ovary (CHO) cells. Further development of the recombinant ASB preparation places priority on increasing the enzyme yield. The aim of this study was to increase the productivity of producer clones by optimising the culture process and adding calcium chloride and copper sulfate to the culture medium. Materials and methods: a suspension-adapted CHO cell line was used. Monoclonal cell lines were developed using Cell Metric and ClonePix FL systems. The concentration of ASB in the culture liquid was determined using the enzyme-linked immunosorbent assay (ELISA). The authors analysed batch culture and/or fed-batch culture in media supplemented with various concentrations of copper sulfate and calcium chloride. Results: the combined addition of copper sulfate and calcium chloride at concentrations of 300 μM during batch culture of producer clones coexpressing ASB and FGE increases viability and specific productivity of the cells up to 4.58±1.62 pg/ (cell×day). The cultivation of the lead producer clone coexpressing ASB and FGE under fed-batch conditions for 12 days and the addition of copper sulfate to the growth medium at the concentration of 300 μM allow for increasing the yield of the active lysosomal enzyme, arylsulfatase B, to 420 mg/L. Conclusions: the cultivation of producer clones coexpressing ASB and FGE under fed-batch conditions with copper sulfate added to the medium significantly improves cell line growth properties and the ASB yield. This approach to the selection of culture conditions for producer cell lines can be applied to other enzymes of the sulfatase family.

Early enzyme replacement therapy prevents dental and craniofacial abnormalities in a mouse model of mucopolysaccharidosis type VI.

Mucopolysaccharidosis VI (MPS VI) is a hereditary lysosomal storage disease caused by the absence of the enzyme arylsulfatase B (ARSB). Craniofacial defects are common in MPS VI patients and manifest as abnormalities of the facial bones, teeth, and temporomandibular joints. Although enzyme replacement therapy (ERT) is the treatment of choice for MPS VI, the effects on the craniofacial and dental structures are still poorly understood. In this study, we used an Arsb-deficient mouse model (Arsb m/m ) that mimics MPS VI to investigate the effects of ERT on dental and craniofacial structures and compared these results with clinical and radiological observations from three MPS VI patients. Using micro-computed tomography, we found that the craniofacial phenotype of the Arsb m/m mice was characterized by bone exostoses at the insertion points of the masseter muscles and an overall increased volume of the jaw bone. An early start of ERT (at 4 weeks of age for 20 weeks) resulted in a moderate improvement of these jaw anomalies, while a late start of ERT (at 12 weeks of age for 12 weeks) showed no effect on the craniofacial skeleton. While teeth typically developed in Arsb m/m mice, we observed a pronounced loss of tooth-bearing alveolar bone. This alveolar bone loss, which has not been described before in MPS VI, was also observed in one of the MPS VI patients. Interestingly, only an early start of ERT led to a complete normalization of the alveolar bone in Arsb m/m mice. The temporomandibular joints in Arsb m/m mice were deformed and had a porous articular surface. Histological analysis revealed a loss of physiological cartilage layering, which was also reflected in an altered proteoglycan content in the cartilage of Arsb m/m mice. These abnormalities could only be partially corrected by an early start of ERT. In conclusion, our results show that an early start of ERT in Arsb m/m mice achieves the best therapeutic effects for tooth, bone, and temporomandibular joint development. As the MPS VI mouse model in this study resembles the clinical findings in MPS VI patients, our results suggest enzyme replacement therapy should be started as early as possible.

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.

Liver-Directed Adeno-Associated Virus–Mediated Gene Therapy for Mucopolysaccharidosis Type VI

BackgroundMucopolysaccharidosis type VI (MPS VI) is an inherited multisystem lysosomal disorder due to arylsulfatase B (ARSB) deficiency that leads to widespread accumulation of glycosaminoglycans (GAG), which are excreted in increased amounts in urine. MPS VI is characterized by progressive dysostosis multiplex, connective tissue and cardiac involvement, and hepatosplenomegaly. Enzyme replacement therapy (ERT) is available but requires life-long and costly intravenous infusions; moreover, it has limited efficacy on diseased skeleton and cardiac valves, compromised pulmonary function, and corneal opacities.MethodsWe enrolled nine patients with MPS VI 4 years of age or older in a phase 1/2 open-label gene therapy study. After ERT was interrupted, patients each received a single intravenous infusion of an adeno-associated viral vector serotype 8 expressing ARSB. Participants were sequentially enrolled in one of three dose cohorts: low (three patients), intermediate (two patients), or high (four patients). The primary outcome was safety; biochemical and clinical end points were secondary outcomes.ResultsThe infusions occurred without severe adverse events attributable to the vector, meeting the prespecified end point. Participants in the low and intermediate dose cohorts displayed stable serum ARSB of approximately 20% of the mean healthy value but returned to ERT by 14 months after gene therapy because of increased urinary GAG. Participants in the high-dose cohort had sustained serum ARSB of 30% to 100% of the mean healthy value and a modest urinary GAG increase that did not reach a concentration at which ERT reintroduction was needed. In the high-dose group, there was no clinical deterioration for up to 2 years after gene therapy.ConclusionsLiver-directed gene therapy for participants with MPS VI did not have a dose-limiting side-effect and adverse event profile; high-dose treatment resulted in ARSB expression over at least 24 months with preliminary evidence of disease stabilization. (Funded by the Telethon Foundation ETS, the European Commission Seventh Framework Programme, and the Isaac Foundation; ClinicalTrials.gov number, NCT03173521; EudraCT number, 2016-002328-10.)

Chondroitin sulfate proteoglycan 4,6 sulfation regulates sympathetic nerve regeneration after myocardial infarction.

Sympathetic denervation of the heart following ischemia/reperfusion induced myocardial infarction (MI) is sustained by chondroitin sulfate proteoglycans (CSPGs) in the cardiac scar. Denervation predicts risk of sudden cardiac death in humans. Blocking CSPG signaling restores sympathetic axon outgrowth into the cardiac scar, decreasing arrhythmia susceptibility. Axon growth inhibition by CSPGs can depend on the sulfation status of the glycosaminoglycan (CS-GAG) side chains. Tandem sulfation of CS-GAGs at the 4th (4S) and 6th (6S) positions of n-acetyl-galactosamine inhibits outgrowth in several types of central neurons, but we don't know if sulfation is similarly critical during peripheral nerve regeneration. We asked if CSPG sulfation prevented sympathetic axon outgrowth after MI. Reducing 4S with the 4-sulfatase enzyme Arylsulfatase-B (ARSB) enhanced outgrowth of dissociated rat sympathetic neurons over CSPGs. Likewise, reducing 4S with ARSB restored axon outgrowth from mouse sympathetic ganglia co-cultured with cardiac scar tissue. We quantified enzymes responsible for adding and removing sulfation, and found that CHST15 (4S dependent 6-sulfotransferase) was upregulated, and ARSB was downregulated after MI. This suggests a mechanism for production and maintenance of sulfated CSPGs in the cardiac scar. We decreased 4S,6S CS-GAGs in vivo by transient siRNA knockdown of Chst15 after MI, and found that reducing 4S,6S restored tyrosine hydroxylase (TH) positive sympathetic nerve fibers in the cardiac scar. Reinnervation reduced isoproterenol induced arrhythmias. Our results suggest that modulating CSPG-sulfation after MI may be a therapeutic target to promote sympathetic nerve regeneration in the cardiac scar and reduce post-MI cardiac arrhythmias.

Increasing productivity of arylsulfatase B-producing cell line by coexpression of formylglycine-generating enzyme

Mucopolysaccharidosis type VI (Maroteaux–Lamy syndrome) is an orphan genetic disease caused by deficiency of the lysosomal enzyme arylsulfatase B (ASB). The need to develop a highly productive cell line for the production of recombinant ASB, is behind the concept and relevance of this study. The most promising approach seems to be the development of CHO producer cell lines coexpressing the target ASB enzyme and an auxiliary formylglycine-generating enzyme (FGE). At the same time, it is important from a practical perspective to have the possibility of cultivating producer cell lines as suspensions free of serum or other components of animal origin. The aim of the study was to develop highly productive cell lines for the production of recombinant ASB by coexpression of the auxiliary FGE. Materials and methods: a suspension CHO cell line was used in the study. CHO cells were transfected by electroporation using the MaxCyte STX system. Monoclonal cell lines were obtained with the help of the Cell Metric system. Enzyme-linked immunosorbent assay was used for determination of ASB concentration in the culture fluid. Culture fluid samples were analysed using polyacrylamide gel electrophoresis and Western blotting. The mRNA level was measured by real-time polymerase chain reaction. Results: producer cell lines coexpressing the target ASB enzyme and auxiliary FGE were obtained. An increase in the yield of the active target ASB enzyme from 2 to 100 mg/L was achieved by selecting the optimal ratio of plasmids during transfection. The highest yield of the target ASB enzyme was achieved at the 90:10 ratio (%) of plasmids encoding the ASB and FGE genes, respectively. Conclusions: the authors developed highly productive cell lines for the production of recombinant ASB, which coexpress the target and auxiliary enzymes. The coexpression of ASB and FGE improves the growth and production characteristics of the cell line, probably due to the modification of the ASB active site. The obtained results will help resolve the problem of low enzyme yield, which is typical of this class of medicines.

O-Sulfation disposition of curcumin and quercetin in SULT1A3 overexpressing HEK293 cells: the role of arylsulfatase B in cellular O-sulfation regulated by transporters.

Extensive phase II metabolic reactions (i.e., glucuronidation and sulfation) have resulted in low bioavailability and decreased biological effects of curcumin and quercetin. Compared to glucuronidation, information on the sulfation disposition of curcumin and quercetin is limited. In this study, we identified that BCRP and MRP4 played a critical role in the cellular excretion of curcumin-O-sulfate (C-O-S) and quercetin-O-sulfate (Q-O-S) by integrating chemical inhibition with transporter knock-down experiments. Inhibited excretion of sulfate (C-O-S and Q-O-S) caused significant reductions in cellular O-sulfation of curcumin (a maximal 74.4% reduction) and quercetin (a maximal 76.9% reduction), revealing a strong interplay of sulfation with efflux transport. It was further identified that arylsulfatase B (ARSB) played a crucial role in the regulation of cellular O-sulfation by transporters. ARSB overexpression significantly enhanced the reduction effect of MK-571 on the cellular O-sulfation (fmet) of the model compound (38.8% reduction for curcumin and 44.2% reduction for quercetin). On the contrary, ARSB knockdown could reverse the effect of MK-571 on the O-sulfation disposition of the model compound (29.7% increase for curcumin and 47.3% increase for quercetin). Taken together, ARSB has been proven to be involved in cellular O-sulfation, accounting for transporter-dependent O-sulfation of curcumin and quercetin. A better understanding of the interplay beneath metabolism and transport will contribute to the exact prediction of in vivo drug disposition and drug-drug interactions.

Deep intronic variant in the ARSB gene as the genetic cause for Maroteaux-Lamy syndrome (MPS VI).

Maroteaux-Lamy syndrome (MPS-VI) is a rare autosomal-recessive disorder with a wide spectrum of clinical manifestations, ranging from an attenuated to a rapidly progressive disease. It is caused by variants in ARSB, which encodes the lysosomal arylsulfatase B (ARSB) enzyme, part of the degradation process of glycosaminoglycans in lysosomes. Over 220 variants have been reported so far, with a majority of missense variants. We hereby report two siblings of Bedouin origin with a diagnosis of MPS-VI. Western blots in patient fibroblasts revealed total absence of ARSB protein production. Complete sequencing of the coding region of ARSB did not identify a candidate disease-associated variant. However, deep sequencing of the noncoding region of ARSB by whole genome sequencing (WGS) revealed a c.1142+581A to G variant. The variant is located within intron 5 and fully segregated with the disease in the family. Determination of the genetic cause for these patients enabled targeted treatment by enzyme replacement therapy, along with appropriate genetic counseling and prenatal diagnosis for the family. These results highlight the advantage of WGS as a powerful tool, for improving the diagnostic rate of rare disease-causing variants, and emphasize the importance of studying deep intronic sequence variation as a cause of monogenic disorders.

Compound heterozygous missense mutations in a Chinese mucopolysaccharidosis type VI patient: a case report

BackgroundMucopolysaccharidosis type VI (MPS VI) is a rare autosomal recessive inherited disease caused by mutations in the arylsulfatase B (ARSB) gene. MPS VI is a multisystemic disease resulting from a deficiency in arylsulfatase B causing an accumulation of glycosaminoglycans in the tissues and organs of the body.In this report, we present the case of a 16-year-old Chinese male who presented with vision loss caused by corneal opacity. MPS VI was confirmed by genetic diagnosis.Case presentationA 16-year-old Chinese male presented with a one-year history of binocular vision loss. The best-corrected visual acuity was 0.25 in the right eye and 0.5 in the left eye. Although slit-lamp examination revealed corneal opacification in both eyes, the ocular examinations of his parents were normal. At the same time, the patient presented with kyphotic deformity, short stature, joint and skeletal malformation, thick lips, long fingers, and coarse facial features. Genetic assessments revealed that ARSB was the causative gene. Compound heterozygous missense mutations were found in the ARSB gene, namely c.1325G > A (p. Thr442Met) (M1) and c.1197G > C (p. Phe399Leu) (M2). Genetic diagnosis confirmed that the patient had MPS VI.ConclusionsThis paper reports a case of MPS VI confirmed by genetic diagnosis.MPS VI is a multisystem metabolic disease, with corneal opacity as a concomitant ocular symptom. As it is difficult for ophthalmologists to definitively diagnose MPS VI, genetic testing is useful for disease confirmation.

A Novel Rat Model with Long Range Optic Nerve Injury to Study Retinal Ganglion Cells Endogenous Regeneration

In adult mammals, axon regeneration is limited within the lesion site after injury to the optic nerve. Changes in the microenvironment of lesion sites play an important role in retinal ganglion cells (RGCs) axon regeneration along with other intrinsic factors. In this study, the effect of the lesion site on the microenvironment and axon growth was evaluated using a refined optic nerve crush (ONC) injury model, in which the injury range was extended compared to classical injury. The number of regenerated axons labeled anterogradely with cholera toxin B fragment (CTB) was significantly increased in the long-range crush injury (LI) group compared to the ONC group at distances of 500, 1000 and 1500 µm from the initial site of the injury. These data confirmed that RGC axons can regenerate inside the lesion site. Immunofluorescence and proteomic analysis showed that the microenvironment at the lesion site was highly heterogeneous. The levels of myelin-associated inhibitors, chondroitin-sulfate proteoglycans (CSPGs) and other axon growth inhibitors decreased inside the lesion site compared to the posterior segment of the optic nerve lesion site. The expression of multiple lysosome-related enzymes, metabolic inhibitors including cholesterol esterase, cathepsin B, D, Z and arylsulfatase B (ARSB) were significantly increased inside the lesion site for the LI group compared to the normal optic nerves. Our results suggest that the model of long range optic nerve injury is more useful towards understanding the lesion microenvironment and the endogenous regeneration of RGCs. Also, we showed that myelin and neurocan (a CSPG) are differently expressed in the optic nerve between the interior and posterior lesion sites and may explain why axons cannot reach the brain through the lesion site.

Abstract B21: Arylsulfatase B (ARSB) is reduced in melanoma metastases and decline in ARSB increases programmed death-ligand (PDL) 1

Abstract Arylsulfatase B (ARSB; N-acetylgalactosamine-4-sulfatase) is the enzyme that removes 4-sulfate groups from chondroitin 4-sulfate (C4S) and dermatan sulfate. In previous work, we have shown decline in ARSB with increasing aggressiveness of melanoma cell lines. C4S increased as ARSB declined in the cultured melanoma cell lines. Decline in ARSB was associated with increased expression of chondroitin sulfate proteoglycan (CSPG) 4 and matrix metalloproteinase (MMP) 2. The increase in CSPG4 was attributed to reduced binding of galectin-3 with C4S, and the increase of MMP2 was attributed to inhibition of SHP2, the tyrosine-protein phosphatase nonreceptor type 11 (PTPN11), due to increased binding with C4S. The invasiveness of the cultured melanoma cells increased when ARSB was silenced by siRNA. To further address the role of ARSB in melanoma progression, metastatic melanoma tissue was obtained from the biorepository of the University of Illinois at Chicago. ARSB activity and expression were reduced in the metastatic tissues and ARSB promoter methylation was increased. ARSB promoter methylation was also increased in cultured metastatic melanoma cells compared to normal melanocytes and to radial growth phase melanoma cells. In the metastatic tissues from skin and liver, SHP2 activity declined, and mRNA expression of MMP2 and CSPG4 increased. When ARSB was silenced by siRNA in the cultured normal melanocytes, Programmed Death-Ligand (PD-L) 1 increased from 78 ± 7 pg/mg protein to 128 ± 11 pg/mg protein by ELISA (p<0.001). In a metastatic melanoma cell line, PD-L1 increased from baseline value of 407 ± 34 pg/mg to 462 ± 26 pg/mg when ARSB was silenced. These findings indicate that decline in ARSB contributes to the progression of metastatic melanoma and to the immune checkpoint responsiveness of the melanoma cells. Further studies are required to determine how ARSB leads to increased PD-L1 expression and how treatment with recombinant ARSB can retard the progression of melanoma. Citation Format: Joanne K. Tobacman, Sumit Bhattacharyya, Leo Feferman. Arylsulfatase B (ARSB) is reduced in melanoma metastases and decline in ARSB increases programmed death-ligand (PDL) 1 [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr B21.

A Generic Assay to Detect Aberrant ARSB Splicing and mRNA Degradation for the Molecular Diagnosis of MPS VI

Identification and characterization of disease-associated variants in monogenic disorders is an important aspect of diagnosis, genetic counseling, prediction of disease severity, and development of therapy. However, the effects of disease-associated variants on pre-mRNA splicing and mRNA degradation are difficult to predict and often missed. Here we present a generic assay for unbiased identification and quantification of arylsulfatase B (ARSB) mRNA for molecular diagnosis of patients with mucopolysaccharidosis VI (MPS VI). We found that healthy control individuals have inefficient ARSB splicing because of natural skipping of exon 5 and inclusion of two pseudoexons in introns 5 and 6. Analyses of 12 MPS VI patients with 10 different genotypes resulted in identification of a 151-bp intron inclusion caused by the c.1142+2T>C variant and detection of low ARSB expression from alleles with the c.629A>G variant. A special case showed skipping of exon 4 and low ARSB expression. Although no disease-associated DNA variant could be identified in this patient, the molecular diagnosis could be made based on RNA. These results highlight the relevance of RNA-based analyses to establish a molecular diagnosis of MPS VI. We speculate that inefficient natural splicing of ARSB may be a target for therapy based on promotion of canonical splicing.

Abstract 4699: Decline in Arylsulfatase B (ARSB) increases PD-L1 expression in melanoma, hepatic, prostate, and mononuclear cells

Abstract Arylsulfatase B (ARSB; N-acetylgalactosamine-4-sulfatase) is the enzyme that removes 4-sulfate groups from chondroitin 4-sulfate (C4S) and dermatan sulfate. In previous work, we have shown decline in ARSB with increasing aggressiveness of melanoma cell lines and in association with more aggressive prostate cancers. As ARSB decreases, chondroitin 4-sulfate increases, leading to increased binding of SHP2 (PTP11; non-receptor tyrosine phosphatase) and reduced binding of galectin-3. Subsequent effects include increase in phospho-ERK1/2 and transcriptional effects, including hypermethylation of the DKK3 promoter and reduced expression of DKK (Dickkopf WNT pathway signaling inhibitor) 3, leading to activation of Wnt signaling. Other transcriptional events following silencing of ARSB include: increased expression of CSPG4 and MMP-2 in melanoma cells; increased expression of versican in prostate epithelial cells; increased expression of Wnt9A in colonic epithelial cells; and increased MITF-mediated expression of GPNMB in hepatic cells. Invasiveness of cultured melanoma cells increased when ARSB was silenced by siRNA. To further address the role of ARSB, metastatic melanoma tissue was obtained from the biorepository of the University of Illinois at Chicago. Expression of CSPG4 and MMP2 was increased in the metastatic tissues and SHP2 activity was reduced. When ARSB was silenced by siRNA in cultured normal melanocytes (PCS 200-013, ATCC), Programmed Death-Ligand (PD-L)1 increased from 78 ± 7 pg/mg protein to 128 ± 11 pg/mg protein (ELISA assay; p<0.001). In metastatic melanoma cells (1205 Lu), PD-L1 increased from baseline value of 407 ± 34 pg/mg to 462 ± 26 pg/mg when ARSB was silenced. ARSB activity and expression were reduced in the skin metastases, attributable to increased ARSB promoter methylation. In the skin metastases, PD-L1 increased from 103 ± 2.4 pg/mg protein to 457 ± 49 pg/ml protein. In malignant HepG2 cells, when ARSB was silenced, PD-L1 mRNA expression increased to 1.6 times baseline. In human prostate cell lines, including metastatic PC-3 cells, normal prostate epithelial cells and normal prostate stromal cells, PD-L1 mRNA expression increased to 1.8 times, 2.2 times, and 1.6 times the baseline level when ARSB was silenced. In mononuclear cells of prediabetes participants in a dietary study of carrageenan withdrawal, PD-L1 expression declined by 62% in participants on the no-carrageenan diet over a 12-week period, as ARSB activity increased by 34%. These findings indicate that changes in ARSB contribute to the expression of PD-L1 in human cells and can thereby affect the immune checkpoint response. Further studies are required to determine how decline in ARSB and the resulting increase in N-acetylgalactosamine-4-sulfation lead to increased PD-L1 expression and how treatment with recombinant ARSB might affect the immune responses. Citation Format: Joanne Kramer Tobacman, Sumit Bhattacharyya, Leo Feferman. Decline in Arylsulfatase B (ARSB) increases PD-L1 expression in melanoma, hepatic, prostate, and mononuclear cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4699.

Abstract 4698: Increased CHST15 expression follows declines in Arylsulfatase B (ARSB) and DKK3 and disinhibition of non-canonical WNT signaling

Abstract CHST15 (carbohydrate sulfotransferase 15; chondroitin 4-sulfate-6-sulfotransferase), is the sulfotransferase enzyme which adds 6-sulfate to chondroitin 4-sulfate to make chondroitin 4,6-sulfate, known as CSE. Increased CHST15 expression has been identified in malignant pancreas, renal, colon, ovary, and prostate tissues. We now report a pathway by which expression of CHST15 is increased following decline in the enzyme ARSB (arylsulfatase B; N-acetylgalactosamine-4-sulfatase), which is required for removal of 4-sulfate groups from chondroitin 4-sulfate (C4S). Previous work has shown that decline in ARSB and the resulting increase in C4S leads to decline in activity of SHP2, a ubiquitous non-receptor tyrosine phosphatase, due to enhanced binding of SHP2 with C4S when ARSB is reduced. Decline in SHP2 leads to enhanced ERK1/2 phosphorylation and subsequent downstream effects, including hypermethylation of the DKK (Dickkopf WNT signaling pathway inhibitor)3 promoter and suppressed DKK3 expression. Since DKK3 acts to inhibit Wnt signaling, Wnt signaling is enhanced in prostate epithelium when ARSB is reduced. Experiments were performed to identify the pathway by which CHST15 expression is up-regulated when ARSB is reduced and to assess the role of Wnt. The impact of ARSB silencing on CHST15 expression was determined in normal and malignant human prostate cells, in normal and malignant prostate tissue obtained by laser-capture microdissection, in HepG2 cells, and in prostate tissue of the ARSB-null mouse. Effects of selective inhibitors of Wnt and MAPK signaling pathways on CHST15 expression were evaluated. CHST15 expression was increased in malignant prostate epithelial tissue, in ARSB-null mouse prostate tissue, and following ARSB silencing in human prostate epithelial cells. The activation of Wnt signaling which followed declines in ARSB and DKK3 was required for increased CHST15 expression. Inhibition of Wnt3A blocked the increase in CHST15 expression in prostate epithelial cells co-cultured with prostate stromal cells, whereas DKK3 antibody increased CHST15. Inhibition of Rac-1 GTPase and of phospho-p38 MAPK signaling blocked the observed increase in CHST15 expression, whereas inhibition of JNK, Sp1, or of Rho kinase had no effect. These results indicate that the increase in epithelial CHST15 occurred in the malignant prostate cells and tissue due to effects of non-canonical Wnt signaling leading to enhanced phospho-p38 MAPK. The resultant increase in CHST15 when ARSB activity is reduced may contribute to determination of epithelial vs. stromal characteristics. Citation Format: Joanne Kramer Tobacman, Sumit Bhattacharyya, Leo Feferman. Increased CHST15 expression follows declines in Arylsulfatase B (ARSB) and DKK3 and disinhibition of non-canonical WNT signaling [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4698.

Increased CHST15 follows decline in arylsulfatase B (ARSB) and disinhibition of non-canonical WNT signaling: potential impact on epithelial and mesenchymal identity.

Expression of CHST15 (carbohydrate sulfotransferase 15; chondroitin 4-sulfate-6-sulfotransferase; BRAG), the sulfotransferase enzyme that adds 6-sulfate to chondroitin 4-sulfate (C4S) to make chondroitin 4,6-disulfate (chondroitin sulfate E, CSE), was increased in malignant prostate epithelium obtained by laser capture microdissection and following arylsulfatase B (ARSB; N-acetylgalactosamine-4-sulfatase) silencing in human prostate epithelial cells. Experiments in normal and malignant human prostate epithelial and stromal cells and tissues, in HepG2 cells, and in the ARSB-null mouse were performed to determine the pathway by which CHST15 expression is up-regulated when ARSB expression is reduced. Effects of Wnt-containing prostate stromal cell spent media and selective inhibitors of WNT, JNK, p38, SHP2, β-catenin, Rho, and Rac-1 signaling pathways were determined. Activation of WNT signaling followed declines in ARSB and Dickkopf WNT Signaling Pathway Inhibitor (DKK)3 and was required for increased CHST15 expression. The increase in expression of CHST15 followed activation of non-canonical WNT signaling and involved Wnt3A, Rac-1 GTPase, phospho-p38 MAPK, and nuclear DNA-bound GATA-3. Inhibition of JNK, Sp1, β-catenin nuclear translocation, or Rho kinase had no effect. Consistent with higher expression of CHST15 in prostate epithelium, disaccharide analysis showed higher levels of CSE and chondroitin 6-sulfate (C6S) disaccharides in prostate epithelial cells. In contrast, chondroitin 4-sulfate (C4S) disaccharides were greater in prostate stromal cells. CSE may contribute to increased C4S in malignant epithelium when GALNS (N-aceytylgalactosamine-6-sulfate sulfatase) is increased and ARSB is reduced. These effects increase chondroitin 4-sulfates and reduce chondroitin 6-sulfates, consistent with enhanced stromal characteristics and epithelial-mesenchymal transition.