N-Acetylgalactosamine-4-sulfatase (Arylsulfatase B) Regulates PD-L1 Expression in Melanoma by an HDAC3-Mediated Epigenetic Mechanism.

The effects of the enzyme N-acetylgalactosamine-4-sulfatase (Arylsulfatase B, ARSB), which removes the 4-sulfate group at the non-reducing end of chondroitin 4-sulfate, on the expression of PD-L1 were determined, and the underlying mechanism of PD-L1 expression was elucidated. Initial experiments in human melanoma cells (A375) showed that PD-L1 expression increased from 357 ± 31 to 796 ± 50 pg/mg protein (p < 10-11) when ARSB was silenced in A375 cells. In subcutaneous B16F10 murine melanomas, PD-L1 declined from 1227 ± 189 to 583 ± 110 pg/mg protein (p = 1.67 × 10-7), a decline of 52%, following treatment with exogenous, bioactive recombinant ARSB. This decline occurred in association with reduced tumor growth and prolongation of survival, as previously reported. The mechanism of regulation of PD-L1 expression by ARSB is attributed to ARSB-mediated alteration in chondroitin 4-sulfation, leading to changes in free galectin-3, c-Jun nuclear localization, HDAC3 expression, and effects of acetyl-H3 on the PD-L1 promoter. These findings indicate that changes in ARSB contribute to the expression of PD-L1 in melanoma and can thereby affect the immune checkpoint response. Exogenous ARSB acted on melanoma cells and normal melanocytes through the IGF2 receptor. The decline in PD-L1 expression by exogenous ARSB may contribute to the impact of ARSB on melanoma progression.

Increase in Chondroitin Sulfate and Decline in Arylsulfatase B May Contribute to Pathophysiology of COVID-19 Respiratory Failure.

INTRODUCTION: The potential role of accumulation of chondroitin sulfates (CSs) in the pathobiology of COVID-19 has not been examined. Accumulation may occur by increased synthesis or by decline in activity of the enzyme arylsulfatase B (ARSB; N-acetylgalactosamine-4-sulfatase) which requires oxygen for activity. METHODS: Immunostaining of lung tissue from 28 patients who died due to COVID-19 infection was performed for CS, ARSB, and carbohydrate sulfotransferase (CHST)15. Measurements of mRNA expression of CHST15 and CHST11, sulfotransferase activity, and total sulfated glycosaminoglycans (GAGs) were determined in human vascular smooth muscle cells following angiotensin (Ang) II treatment. RESULTS: CS immunostaining showed increase in intensity and distribution, and immunostaining of ARSB was diminished in COVID-19 compared to normal lung tissue. CHST15 immunostaining was prominent in vascular smooth muscle cells associated with diffuse alveolar damage due to COVID-19 or other causes. Expression of CHST15 and CHST11 which are required for synthesis of CSE and chondroitin 4-sulfate, total sulfated GAGs, and sulfotransferase activity was significantly increased following AngII exposure in vascular smooth muscle cells. Expression of Interleukin-6 (IL-6), a mediator of cytokine storm in COVID-19, was inversely associated with ARSB expression. DISCUSSION/CONCLUSION: Decline in ARSB and resulting increases in CS may contribute to the pathobiology of COVID-19, as IL-6 does. Increased expression of CHSTs following activation of Ang-converting enzyme 2 may lead to buildup of CSs.

Profound Impact of Decline in N-Acetylgalactosamine-4-Sulfatase (Arylsulfatase B) on Molecular Pathophysiology and Human Diseases.

The enzyme N-acetylgalactosamine-4-sulfatase (Arylsulfatase B; ARSB) was originally identified as a lysosomal enzyme which was deficient in Mucopolysaccharidosis VI (MPS VI; Maroteaux-Lamy Syndrome). The newly directed attention to the impact of ARSB in human pathobiology indicates a broader, more pervasive effect, encompassing roles as a tumor suppressor, transcriptional mediator, redox switch, and regulator of intracellular and extracellular-cell signaling. By controlling the degradation of chondroitin 4-sulfate and dermatan sulfate by removal or failure to remove the 4-sulfate residue at the non-reducing end of the sulfated glycosaminoglycan chain, ARSB modifies the binding or release of critical molecules into the cell milieu. These molecules, such as galectin-3 and SHP-2, in turn, influence crucial cellular processes and events which determine cell fate. Identification of ARSB at the cell membrane and in the nucleus expands perception of the potential impact of decline in ARSB activity. The regulation of availability of sulfate from chondroitin 4-sulfate and dermatan sulfate may also affect sulfate assimilation and production of vital molecules, including glutathione and cysteine. Increased attention to ARSB in mammalian cells may help to integrate and deepen our understanding of diverse biological phenomenon and to approach human diseases with new insights.

Lysosomal Proteome and Secretome Analysis Identifies Missorted Enzymes and Their Nondegraded Substrates in Mucolipidosis III Mouse Cells.

Targeting of soluble lysosomal enzymes requires mannose 6-phosphate (M6P) signals whose formation is initiated by the hexameric N-acetylglucosamine (GlcNAc)-1-phosphotransferase complex (α2ß2γ2). Upon proteolytic cleavage by site-1 protease, the α/ß-subunit precursor is catalytically activated but the functions of γ-subunits (Gnptg) in M6P modification of lysosomal enzymes are unknown. To investigate this, we analyzed the Gnptg expression in mouse tissues, primary cultured cells, and in Gnptg reporter mice in vivo, and found high amounts in the brain, eye, kidney, femur, vertebra and fibroblasts. Consecutively we performed comprehensive quantitative lysosomal proteome and M6P secretome analysis in fibroblasts of wild-type and Gnptgko mice mimicking the lysosomal storage disorder mucolipidosis III. Although the cleavage of the α/ß-precursor was not affected by Gnptg deficiency, the GlcNAc-1-phosphotransferase activity was significantly reduced. We purified lysosomes and identified 29 soluble lysosomal proteins by SILAC-based mass spectrometry exhibiting differential abundance in Gnptgko fibroblasts which was confirmed by Western blotting and enzymatic activity analysis for selected proteins. A subset of these lysosomal enzymes show also reduced M6P modifications, fail to reach lysosomes and are secreted, among them α-l-fucosidase and arylsulfatase B. Low levels of these enzymes correlate with the accumulation of non-degraded fucose-containing glycostructures and sulfated glycosaminoglycans in Gnptgko lysosomes. Incubation of Gnptgko fibroblasts with arylsulfatase B partially rescued glycosaminoglycan storage. Combinatorial treatments with other here identified missorted enzymes of this degradation pathway might further correct glycosaminoglycan accumulation and will provide a useful basis to reveal mechanisms of selective, Gnptg-dependent formation of M6P residues on lysosomal proteins.

Identification of Novel ARSB Genes Necessary for p-Benzoquinone Biosynthesis in the Larval Oral Secretion Participating in External Immune Defense in the Red Palm Weevil.

External secretions, composed of a variety of chemical components, are among the most important traits that endow insects with the ability to defend themselves against predators, parasites, or other adversities, especially pathogens. Thus, these exudates play a crucial role in external immunity. Red palm weevil larvae are prolific in this regard, producing large quantities of p-benzoquinone, which is present in their oral secretion. Benzoquinone with antimicrobial activity has been proven to be an active ingredient and key factor for external immunity in a previous study. To obtain a better understanding of the genetic and molecular basis of external immune secretions, we identify genes necessary for p-benzoquinone synthesis. Three novel ARSB genes, namely, RfARSB-0311, RfARSB-11581, and RfARSB-14322, are screened, isolated, and molecularly characterized on the basis of transcriptome data. To determine whether these genes are highly and specifically expressed in the secretory gland, we perform tissue/organ-specific expression profile analysis. The functions of these genes are further determined by examining the antimicrobial activity of the secretions and quantification of p-benzoquinone after RNAi. All the results reveal that the ARSB gene family can regulate the secretory volume of p-benzoquinone by participating in the biosynthesis of quinones, thus altering the host's external immune inhibitory efficiency.

Decline in arylsulfatase B expression increases EGFR expression by inhibiting the protein-tyrosine phosphatase SHP2 and activating JNK in prostate cells.

Epidermal growth factor receptor (EGFR) has a crucial role in cell differentiation and proliferation and cancer, and its expression appears to be up-regulated when arylsulfatase B (ARSB or GalNAc-4-sulfatase) is reduced. ARSB removes 4-sulfate groups from the nonreducing end of dermatan sulfate and chondroitin 4-sulfate (C4S), and its decreased expression has previously been reported to inhibit the activity of the ubiquitous protein-tyrosine phosphatase, nonreceptor type 11 (SHP2 or PTPN11). However, the mechanism by which decline in ARSB leads to decline in SHP2 activity is unclear. Here, we show that SHP2 binds preferentially C4S, rather than chondroitin 6-sulfate, and confirm that SHP2 activity declines when ARSB is silenced. The reduction in ARSB activity, and the resultant increase in C4S, increased the expression of EGFR (Her1/ErbB1) in human prostate stem and epithelial cells. The increased expression of EGFR occurred after 1) the decline in SHP2 activity, 2) enhanced c-Jun N-terminal kinase (JNK) activity, 3) increased nuclear DNA binding by c-Jun and c-Fos, and 4) EGFR promoter activation. In response to exogenous EGF, there was increased bromodeoxyuridine incorporation, consistent with enhanced cell proliferation. These findings indicated that ARSB and chondroitin 4-sulfation affect the activation of an important dual phosphorylation threonine-tyrosine kinase and the mRNA expression of a critical tyrosine kinase receptor in prostate cells. Restoration of ARSB activity with the associated reduction in C4S may provide a new therapeutic approach for managing malignancies in which EGFR-mediated tyrosine kinase signaling pathways are active.

Dihydrotestosterone inhibits arylsulfatase B and Dickkopf Wnt signaling pathway inhibitor (DKK)-3 leading to enhanced Wnt signaling in prostate epithelium in response to stromal Wnt3A.

BACKGROUND: In tissue microarrays, immunostaining of the enzyme arylsulfatase B (ARSB; N-acetylgalactosamine-4-sulfatase) was less in recurrent prostate cancers and in cancers with higher Gleason scores. In cultured prostate stem cells, decline in ARSB increased Wnt signaling through effects on Dickkopf Wnt Signaling Pathway Inhibitor (DKK)3. The effects of androgen exposure on ARSB and the impact of decline in ARSB on Wnt signaling in prostate tissue were unknown. METHODS: Epithelial and stromal tissues from malignant and normal human prostate were obtained by laser capture microdissection. mRNA expression of ARSB, galactose-6-sulfate-sulfatase (GALNS) and Wnt-signaling targets was determined by QPCR. Non-malignant human epithelial and stromal prostate cells were grown in tissue culture, including two-cell layer cultures. ARSB was silenced by specific siRNA, and epithelial cells were treated with stromal spent media following treatment with IWP-2, an inhibitor of Wnt secretion, and by exogenous recombinant human Wnt3A. Promoter methylation was detected using specific DKK3 and ARSB promoter primers. The effects of DHT and of ARSB overexpression on DKK expression were determined. Cell proliferation was assessed by BrdU incorporation. RESULTS: Normal stroma showed higher expression of vimentin, ARSB, and Wnt3A than epithelium. Normal epithelium had higher expression of E-cadherin, galactose 6-sulfate-sulfatase (GALNS), and DKK3 than stroma. In malignant epithelium, expression of ARSB and DKK3 declined, and expression of GALNS and Wnt signaling targets increased. In cultured prostate epithelial cells, Wnt-mediated signaling was greatest when ARSB was silenced and cells were exposed to exogenous Wnt3A. Exposure to 5α-dihydrotestosterone (DHT) increased ARSB and DKK3 promoter rmethylation, and effects of DHT on DKK3 expression were reversed when ARSB was overexpressed. CONCLUSIONS: Androgen-induced declines in ARSB and DKK3 may contribute to prostate carcinogenesis by sustained activation of Wnt signaling in prostate epithelium in response to stromal Wnt3A.

Identification of eleven different mutations including six novel, in the arylsulfatase B gene in Iranian patients with mucopolysaccharidosis type VI.

Mucopolysaccharidosis VI is a rare autosomal recessive disorder caused by the deficiency of enzyme Arylsulfatase B. The enzyme deficiency leads to the accumulation of dermatan sulfate in connective tissue which causes manifestations related to MPS VI. Up to now, three different disease causing variants are reported in Iranian patients. In this study, we scanned ARSB gene of 13 Iranian patients from 12 families in whom all parents were consanguineous and from the same ethnicity except one family that were not consanguineous but co-ethnic. We found six not previously reported disease causing variants. We extracted DNA from peripheral blood samples of patients that were previously confirmed as MPS VI by clinical, biochemical and enzymatic assays including berry-spot test and fluorimetry, followed by PCR and direct sequencing. Computational approaches were used to analyze novel variants in terms of their impact on the protein structure. 11 disease causing variants and 15 polymorphisms were found. Six disease causing variants were novel and five were previously reported of which three were in Iranian population. Four of patients, who were unrelated, two by two had the same disease causing variant and polymorphisms, which indicates a possible founder effect. Our study also implicates genotype-phenotype correlation. Computational structural modeling indicated these disease causing variants might affect structural stability and function of the protein. Data of this study confirms the existence of mutational heterogeneity in the ARSB between Iranian patients. Disease causing variants with high frequency can be used in the prenatal diagnosis and genetic counseling. Also, the existence of the same variants and polymorphisms in some of the unrelated patients indicates a possible founder effect.

Liver transplantation: New treatment for mucopolysaccharidosis type VI in rats.

BACKGROUND: Mucopolysaccharidosis (MPS) VI is a rare, autosomal recessive congenital metabolic disorder caused by deficient activity of the lysosomal metabolic enzyme, N-acetylgalactosamine 4-sulfatase. Enzyme replacement therapy (ERT) is the current treatment for MPS VI, although it involves limited compliance to the therapy and high cost. The aim of this study was to develop a new method of treatment by conducting an orthotopic liver transplantation (LTx) using an animal model of human MPS VI, and to evaluate and examine its effectiveness for treating MPS VI. METHODS: LTx was carried out from normal unaffected to affected MPS VI rats (MPR), which were then killed after LTx, and tissues from the heart, spleen, and knee joint, as well as serum, collected for biological and morphologic evaluation. RESULTS: Liver-transplanted (LTx) MPR had the same level of N-acetylgalactosamine 4-sulfatase activity in the liver and lungs as normal unaffected MPR, and the urinary secretion of mucopolysaccharides/glycosaminoglycan (GAG) in LTx MPR was significantly decreased. Furthermore, on histopathology, the spleens of LTx MPR showed elimination of vacuole cells. In the knee joints, growth plates became thinner, and on radiography the facial and cranial bones of LTx MPR were morphologically normal. CONCLUSIONS: LTx from normal to affected MPR was effective for symptoms of MPS and accumulation of GAG, suggesting that LTx could be a promising alternative approach for MPS VI.

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.

Mucopolysaccharidosis Type VI in a Great Dane Caused by a Nonsense Mutation in the ARSB Gene.

Mucopolysaccharidoses are inherited metabolic disorders that result from a deficiency of lysosomal enzymes required for the catabolism of glycosaminoglycans. Lysosomal glycosaminoglycan accumulation results in cell and organ dysfunction. This study characterized the phenotype and genotype of mucopolysaccharidosis VI in a Great Dane puppy with clinical signs of stunted growth, facial dysmorphia, skeletal deformities, corneal opacities, and increased respiratory sounds. Clinical and pathologic evaluations, urine glycosaminoglycan analyses, lysosomal enzyme assays, and ARSB sequencing were performed. The urine mucopolysaccharide spot test was strongly positive predominantly due to the accumulation of dermatan sulfate. Enzyme assays in leukocytes and tissues indicated a deficiency of arylsulfatase B (ARSB) activity. Histologic examination revealed cytoplasmic vacuoles in many tissues. Analysis of the exonic ARSB DNA sequences from the affected puppy compared to the published canine genome sequence revealed a homozygous nonsense mutation (c.295C>T) in exon 1, replacing glutamine with a premature stop codon (p.Gln99*), predicting no enzyme synthesis. A polymerase chain reaction-based restriction fragment length polymorphism test was established to assist with the clinical diagnosis and breeding of Great Danes. This genotyping test revealed that the clinically healthy parents and some other relatives of the puppy were heterozygous for the mutant allele, but all 200 clinically healthy dogs screened including 15 Great Danes were homozygous for the normal allele. This ARSB mutation is the fourth identified genetic variant causing canine mucopolysaccharidosis VI. Mucopolysaccharidosis VI is the first lysosomal storage disorder described in Great Danes but does not appear to be widespread in this breed.

Regulation of chondroitin-4-sulfotransferase (CHST11) expression by opposing effects of arylsulfatase B on BMP4 and Wnt9A.

In this report, the gene regulatory mechanism by which decline in arylsulfatase B (ARSB; N-acetylgalactosamine-4-sulfatase) reduces CHST11 (chondroitin-4-sulfotransferase; C4ST) mRNA expression in human colonic epithelial cells and in colonic epithelium of ARSB-deficient mice is presented. ARSB controls the degradation of chondroitin 4-sulfate (C4S) by removing the 4-sulfate group at the non-reducing end of the C4S chain, but has not previously been shown to affect C4S biosynthesis. The decline in CHST11 expression following ARSB reduction is attributable to effects of ARSB on bone morphogenetic protein (BMP)4, since BMP4 expression and secretion declined when ARSB was silenced. Inhibition of BMP4 by neutralizing antibody also reduced CHST11 expression. When C4S was more sulfated due to decline in ARSB, more BMP4 was sequestered by C4S in the cell membrane, and CHST11 expression declined. Exogenous recombinant BMP4, acting through a phospho-Smad3 binding site in the CHST11 promoter, increased the mRNA expression of CHST11. In contrast to the decline in BMP4 that followed decline in ARSB, Wnt9A mRNA expression was previously shown to increase when ARSB was silenced and C4S was more highly sulfated. Galectin-3 bound less to the more highly sulfated C4S, leading to increased nuclear translocation and enhanced galectin-3 interaction with Sp1 in the Wnt9A promoter. Silencing Wnt9A increased the expression of CHST11 in the colonic epithelial cells, and chromatin immunoprecipitation assay demonstrated enhancing effects of Wnt9A siRNA and exogenous BMP4 on the CHST11 promoter through the pSmad3 binding site. These findings suggest that cellular processes mediated by differential effects of Wnt9A and BMP4 can result from opposing effects on CHST11 expression.

Effect of CFTR modifiers on arylsulfatase B activity in cystic fibrosis and normal human bronchial epithelial cells.

BACKGROUND: The enzyme Arylsulfatase B (ARSB; N-acetylgalactosamine 4-sulfatase), is required for degradation of sulfated glycosaminoglycans (GAGs) which accumulate in cystic fibrosis. ARSB is reduced in cystic fibrosis cells and increases when defective CFTR is repaired by insertion of the normal gene. This study was undertaken to determine if modification of CFTR by small molecule correctors or potentiators could also increase ARSB and reduce the accumulation of chondroitin 4-sulfate (C4S). METHODS: CF bronchial epithelial cells homozygous for the F508 deletion (ACD#14071) and normal human bronchial epithelial cells (BEC) were grown and differentiated following an established protocol. Cells were treated with either VRT-532, a CFTR potentiator, or VRT-534, a CFTR corrector, or vehicle control. The impact on ARSB activity, protein and mRNA expression, C4S and total sulfated glycosaminoglycan content, Interleukin-8 and Interleukin-6 secretion, and neutrophil chemotaxis was determined by specific assays. RESULTS: The CFTR potentiator, but not the corrector, increased ARSB activity and expression to the level in the normal bronchial epithelial cells (BEC). Concomitantly, total sulfated glycosaminoglycans and C4S declined, secreted IL-8 increased, secreted IL-6 declined, and neutrophil chemotaxis to the spent media obtained from the potentiator-treated CF cells increased. CONCLUSION: The CFTR potentiator increased ARSB activity and expression and associated effects. This suggests that a critical interaction between CFTR and ARSB is related to CFTR function in regulation of a ligand-gated anion channel at the cell membrane, rather than to CFTR processing and intracellular trafficking.

Decline in arylsulfatase B and Increase in chondroitin 4-sulfotransferase combine to increase chondroitin 4-sulfate in traumatic brain injury.

In an established rat model of penetrating ballistic-like brain injury (PBBI), arylsulfatase B (ARSB; N-acetylgalactosamine 4-sulfatase) activity was significantly reduced at the ipsilateral site of injury, but unaffected at the contralateral site or in sham controls. In addition, the ARSB substrate chondroitin 4-sulfate (C4S) and total sulfated glycosaminoglycans increased. The mRNA expression of chondroitin 4-sulfotransferase 1 (C4ST1; CHST11) and the sulfotransferase activity rose at the ipsilateral site of injury (PBBI-I), indicating contributions from both increased production and reduced degradation to the accumulation of C4S. In cultured, fetal rat astrocytes, following scratch injury, the ARSB activity declined and the nuclear hypoxia inducible factor-1α increased significantly. In contrast, sulfotransferase activity and chondroitin 4-sulfotransferase expression increased following astrocyte exposure to TGF-ß1, but not following scratch. These different pathways by which C4S increased in the cell preparations were both evident in the response to injury in the PBBI-I model. Hence, findings support effects of injury because of mechanical disruption inhibiting ARSB and to chemical mediation by TGF-ß1 increasing CHST11 expression and sulfotransferase activity. The increase in C4S following traumatic brain injury is because of contributions from impaired degradation and enhanced synthesis of C4S which combine in the pathogenesis of the glial scar. This is the first report of how two mechanisms contribute to the increase in chondroitin 4-sulfate (C4S) in TBI. Following penetrating ballistic-like brain injury in a rat model and in the scratch model of injury in fetal rat astrocytes, Arylsulfatase B activity declined, leading to accumulation of C4S. TGF-ß1 exposure increased expression of chondroitin 4-sulfotransferase. Hence, the increase in C4S in TBI is attributable to both impaired degradation and enhanced synthesis, combining in the pathogenesis of the glial scar.

Arylsulfatase B modulates neurite outgrowth via astrocyte chondroitin-4-sulfate: dysregulation by ethanol.

In utero ethanol exposure causes fetal alcohol spectrum disorders, associated with reduced brain plasticity; the mechanisms of these effects are not well understood, particularly with respect to glial involvement. Astrocytes release factors that modulate neurite outgrowth. We explored the hypothesis that ethanol inhibits neurite outgrowth by increasing the levels of inhibitory chondroitin sulfate proteoglycans (CSPGs) in astrocytes. Astrocyte treatment with ethanol inhibited the activity of arylsulfatase B (ARSB), the enzyme that removes sulfate groups from chondroitin-4-sulfate (C4S) and triggers the degradation of C4S, increased total sulfated glycosaminoglycans (GAGs), C4S, and neurocan core-protein content and inhibited neurite outgrowth in neurons cocultured with ethanol-treated astrocytes in vitro, effects reversed by treatment with recombinant ARSB. Ethanol also inhibited ARSB activity and increased sulfate GAG and neurocan levels in the developing hippocampus after in vivo ethanol exposure. ARSB silencing increased the levels of sulfated GAGs, C4S, and neurocan in astrocytes and inhibited neurite outgrowth in cocultured neurons, indicating that ARSB activity directly regulates C4S and affects neurocan expression. In summary, this study reports two major findings: ARSB modulates sulfated GAG and neurocan levels in astrocytes and astrocyte-mediated neurite outgrowth in cocultured neurons; and ethanol inhibits the activity of ARSB, increases sulfated GAG, C4S, and neurocan levels, and thereby inhibits astrocyte-mediated neurite outgrowth. An unscheduled increase in CSPGs in the developing brain may lead to altered brain connectivity and to premature decrease in neuronal plasticity and therefore represents a novel mechanism by which ethanol can exert its neurodevelopmental effects.

Exogenous recombinant N-acetylgalactosamine-4-sulfatase (Arylsulfatase B; ARSB) inhibits progression of B16F10 cutaneous melanomas and modulates cell signaling.

In the syngeneic, subcutaneous B16F10 mouse model of malignant melanoma, treatment with exogenous ARSB markedly reduced tumor size and extended survival. In vivo experiments showed that local treatment with exogenous N-acetylgalactosamine-4-sulfatase (Arylsulfatase B; ARSB) led to reduced tumor growth over time (p < 0.0001) and improved the probability of survival up to 21 days (p = 0.0391). Tumor tissue from the treated mice had lower chondroitin 4-sulfate (C4S) content and lower sulfotransferase activity. The free galectin-3 declined, and the SHP2 activity increased, due to altered binding with chondroitin 4-sulfate. These changes induced effects on transcription, which were mediated by Sp1, phospho-ERK1/2, and phospho-p38 MAPK. Reduced mRNA expression of chondroitin sulfate proteoglycan 4 (CSPG4), carbohydrate sulfotransferase 15 (N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase), and matrix metalloproteinases 2 and 9 resulted. Experiments in the human melanoma cell line A375 demonstrated similar responses to exogenous ARSB as in the tumors, and inverse effects followed ARSB siRNA. ARSB, which removes the 4-sulfate group at the non-reducing end of C4S, acts as a tumor suppressor, and treatment with exogenous ARSB impacts on vital cell signaling and reduces the expression of critical genes associated with melanoma progression.

The arylsulfatase- and phospholipase-rich venom of the plutoniumid centipede Theatops posticus.

Research on centipede venoms has led to the discovery of a diverse array of novel proteins and peptides, including those with homology to previously discovered toxin families (e.g., phospholipase A2s and pM12a metalloproteases) and novel toxin families not previously detected in venoms (e.g., ß-pore forming toxins and scoloptoxins). Most of this research has focused on centipedes in the order Scolopendromorpha, particularly those in the families Scolopendridae, Cryptopidae, and Scolopocryptopidae. To generate the first high-throughput venom characterization for a centipede in the scolopendromorph family Plutoniumidae, we performed venom-gland transcriptomics and venom proteomics on two Theatops posticus. We identified a total of 64 venom toxins, 60 of which were detected in both the venom-gland transcriptome and venom proteome and four of which were only detected transcriptomically. We detected a single highly abundant arylsulfatase B (ARSB) toxin, the first ARSB toxin identified from centipede venoms. As ARSBs have been detected in other venomous species (e.g., scorpions), ARSBs in T. posticus highlights a new case of convergent evolution across venoms. Theatops posticus venom also contained a much higher abundance and diversity of phospholipase A2 toxins compared to other characterized centipede venoms. Conversely, we detected other common centipedes toxins, such as CAPs and scoloptoxins, at relatively low abundances and diversities. Our observation of a diverse set of toxins from T. posticus venom, including those from novel toxin families, emphasizes the importance of studying unexplored centipede taxonomic groups and the continued potential of centipede venoms for novel toxin discovery and unraveling the molecular mechanisms underlying trait evolution.

The remarkably enzyme-rich venom of the Big Bend Scorpion (Diplocentrus whitei).

Scorpion venoms have long been studied for their peptide discovery potential, with modern high-throughput venom-characterization techniques paving the way for the discovery of thousands of novel putative toxins. Research into these toxins has provided insight into the pathology and treatment of human diseases, even resulting in the development of one compound with Food and Drug Administration (FDA) approval. Although most of this research has focused on the toxins of scorpion species considered medically significant to humans, the venom of harmless scorpion species possess toxins that are homologous to those from medically significant species, indicating that harmless scorpion venoms may also serve as valuable sources of novel peptide variants. Furthermore, as harmless scorpions represent a vast majority of scorpion species diversity, and therefore venom toxin diversity, venoms from these species likely contain entirely new toxin classes. We sequenced the venom-gland transcriptome and venom proteome of two male Big Bend scorpions (Diplocentrus whitei), providing the first high-throughput venom characterization for a member of this genus. We identified a total of 82 toxins in the venom of D. whitei, 25 of which were identified in both the transcriptome and proteome, and 57 of which were only identified in the transcriptome. Furthermore, we identified a unique, enzyme-rich venom dominated by serine proteases and the first arylsulfatase B toxins identified in scorpions.

Increased Cerebral Serum Amyloid A2 and Parameters of Oxidation in Arylsulfatase B (N-Acetylgalactosamine-4-Sulfatase)-Null Mice.

Background: Chondroitin sulfate and chondroitin sulfate proteoglycans have been associated with Alzheimer's disease (AD), and the impact of modified chondroitin sulfates is being investigated in several animal and cell-based models of AD. Published reports have shown the role of accumulation of chondroitin 4-sulfate and decline in Arylsulfatase B (ARSB; B-acetylgalactosamine-4-sulfatase) in other pathology, including nerve injury, traumatic brain injury, and spinal cord injury. However, the impact of ARSB deficiency on AD pathobiology has not been reported, although changes in ARSB were associated with AD in two prior reports. The enzyme ARSB removes 4-sulfate groups from the non-reducing end of chondroitin 4-sulfate and dermatan sulfate and is required for their degradation. When ARSB activity declines, these sulfated glycosaminoglycans accumulate, as in the inherited disorder Mucopolysaccharidosis VI. Objective: Reports about chondroitin sulfate, chondroitin sulfate proteoglycans, and chondroitin sulfatases in AD were reviewed. Methods: Measurements of SAA2, iNOS, lipid peroxidation, chondroitin sulfate proteoglycan 4 (CSPG4), and other parameters were performed in cortex and hippocampus from ARSB-null mice and controls by QRT-PCR, ELISA, and other standard assays. Results: SAA2 mRNA expression and protein, CSPG4 mRNA, chondroitin 4-sulfate, and iNOS were increased significantly in ARSB-null mice. Measures of lipid peroxidation and redox state were significantly modified. Conclusion: Findings indicate that decline in ARSB leads to changes in expression of parameters associated with AD in the hippocampus and cortex of the ARSB-deficient mouse. Further investigation of the impact of decline in ARSB on the development of AD may provide a new approach to prevent and treat AD.

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.