Carbohydrate Sulfotransferase Research Articles

Loss of carbohydrate sulfotransferase 6 function leads to macular corneal dystrophy phenotypes and skeletal defects in zebrafish.

The carbohydrate sulfotransferase 6 (chst6) gene is linked to macular corneal dystrophy (MCD), a rare disease that leads to bilateral blindness due to the accumulation of opaque aggregates in the corneal stroma. chst6 encodes for a keratan sulfate proteoglycan (KSPG) specific sulfotransferase. MCD patients lose sulfated KSPGs (cKS) in the cornea and the serum. The significance of serum cKS loss has not been understood. Zebrafish cornea structure is similar to that of humans and it contains high levels of sulfated cKS in the stroma. Here, zebrafish chst6 is shown to be expressed in the cornea and head structures of the embryos. An animal model of MCD is developed by generating chst6 mutant animals with CRISPR/Cas9-mediated gene editing. The dramatic decrease in cKS epitopes in the mutants was shown with ELISA and immunofluorescence. Morphological defects or alterations of jaw cartilage were detected in a minor fraction of the mutant larvae. Loss of cKS epitopes and morphological defects was fully rescued with wild-type chst6. Mutant adult zebrafish displayed all clinical manifestations of MCD, while a fraction also displayed jaw and skeleton defects. Opaque accumulations formed in the eye, which were alcian blue positive. Loss of cKS in the corneal stroma and a decrease in corneal thickness were shown. Interestingly, alteration of transforming growth factor beta-induced (BIGH3) expression which was not described in patients was also observed. This is the first report of an MCD model in a genetically tractable organism, providing a preclinical model and insight into the importance of KSPG sulfation for proper skeletal morphogenesis.

Trehalose extricates impaired mitochondrial and autophagy dysregulation in patient iPSC-derived macular corneal dystrophy disease model

BackgroundPatient-derived induced pluripotent stem cell (iPSCs) represents a powerful tool for elucidating the underlying disease mechanisms. Macular corneal dystrophy (MCD) is an intractable and progressive bilateral corneal disease affecting the corneal stroma due to mutation/s in carbohydrate sulfotransferase 6 (CHST6) gene. The underlying molecular mechanisms leading to MCD are unclear due to a lack of human contextual model and limited access to affected corneal stromal keratocytes (CSKs) from MCD patients. This has restricted the current treatment option for MCD to restorative corneal transplantation thereby lending itself to the use of iPSCs.Methodsinduced pluripotent stem cells (iPSCs) were generated from two MCD patients and a healthy participant by senai virus based reprogramming of the peripheral mononuclear blood cells (PBMCs). The iPSCs were characterized based on the expression of pluripotent markers and formation of embryoid bodies possessing tri-lineage potential. Directed differentiation of the iPSCs to corneal stromal keratocytes (CSKs) was done via intermediate induction of neural crest cells. The iCSKs were characterized by immunocytochemistry and qPCR. Proteostat staining of the iCSKs was done to validate the disease phenotype invitro. Expression of autophagy markers in the iCSKs and JC staining were visualized by immunochemistry and live-cell imaging in trehalose treated iCSKs.ResultsWe show that the MCD iPSC-derived CSKs (MCDiCSKs) exhibits impaired autophagy assessed by the profiles of autophagy-associated proteins (LAMP1, LC3II/I, p62 and Beclin-1) and mitochondrial membrane potential. Significantly higher protein aggregates in MCDiCSKs was seen compared with the control, which could be rescued upon autophagy modulation. Hence, we treated MCD-iCSKs with trehalose (autophagy inducer) and showed that it protects MCD-iCSKs from mitochondrial dysfunction and maintains autophagic degradation.ConclusionOur study highlights the possible pathological mechanisms involved in MCD. We found trehalose ameliorate the impaired mitochondrial and autophagy dysregulation in patient iPSC-derived macular corneal dystrophy disease model, which could be a potential alternative for MCD management.Graphical abstract

PARN Maintains RNA Stability to Regulate Insulin Maturation and GSIS in Pancreatic β Cells.

Diabetes, a metabolic disorder characterized by hyperglycemia, underscores the importance of normal pancreatic β-cell development and function in maintaining glucose homeostasis. Poly(A)-specific ribonuclease (PARN) serves as the principal regulator of messenger RNA (mRNA) stability, yet its specific role in pancreatic β cells remains unclear. This study utilizes mice with targeted PARN deficiency in β cells to elucidate this role. Notably, Parn conditional knockout mice present unaltered β-cell development and insulin sensitivity but reduced glucose-stimulated insulin secretion (GSIS). The observed outcomes are corroborated in NIT-1 cells. Furthermore, transcriptomic analyses reveal aberrant mRNA expression of genes crucial for insulin secretion in PARN-deficient β cells. Insights from linear amplification of complementary DNA ends and sequencing and coimmunoprecipitation experiments reveal an interaction between PARN and polypyrimidine tract-binding protein 1 (PTBP1), regulating the RNA stability of solute carrier family 30, member 8 (Slc30a8) and carbohydrate sulfotransferase 3 (Chst3). Interference with either PARN or PTBP1 disrupts this stability. These data indicate that PARN deficiency hampers GSIS and insulin maturation by destabilizing Slc30a8 and Chst3 RNAs. These findings provide compelling evidence indicating that PARN is a potential therapeutic target for enhancing insulin maturation and secretion.

Corneal microstructural changes of precise CHST6 gene mutation: a case series

BackgroundMacular corneal dystrophy (MCD) is an inherited, autosomal recessive disorder of defective keratan sulfate (KS) metabolism. It is caused by the mutations in carbohydrate sulfotransferase 6 gene (CHST6) which is essential for the sulfation of KS. Unlike the western world, MCD is the most common corneal stromal dystrophy in India, especially in south Indian population; it could be due to high frequency of consanguineous marriages.Case presentationThis study presents the clinical findings of one North Indian MCD family, including 6 patients and 3 unaffected relatives. We used slit lamp examination and in vivo confocal microscopy for assessment. Mutation screening was performed with Sanger sequencing, and corneal structure was analyzed through histochemistry and immunohistochemistry. Our comparative findings revealed that all the patients identified with the deletion of major portion of CHST6 that included the Open Reading Frame (ORF). Although all the patients showed significantly reduced central corneal thickness (CCT-250 μm), a drastic decrease in stromal keratocyte count, and depletion of Bowman’s layer compared to controls.ConclusionsThis study first time revealed that MCD patients from one family with a deletion of major portion of CHST6 that included ORF leads to severe corneal morphological changes.

CHST4 associates with high-abundance immune infiltration in hormone receptor-positive breast cancer.

Hormone receptor-positive breast cancer (HR+ BRCA) with high-risk factors such as lymph node metastasis has a relatively poor prognosis. However, the biological basis of tumor cell migration is still poorly understood, especially as some of the metastatic events occur at an early stage. Here, we identified that carbohydrate sulfotransferase 4 (CHST4), which has an important role in lymphocyte homing, was abnormally down-regulated in HR+ BRCA and associated with lymph node metastasis. By enrichment analysis and immune infiltration evaluation, we predicted the potential ability of CHST4 to enhance immune cell infiltration. Then, IHC staining further demonstrated the contribution of CHST4 to the infiltration abundance of CD8+ T cells and CD4+ T cells in HR+ BRCA. IHC staining of MECA-79 further identified the correlation between CHST4 and sulfated perpheral lymphonode vascular addressin (PNAd). Finally, we demonstrated that CHST4 was connected to increased tumor-immune cell communication by analyzing single-cell sequencing data. In summary, our study provided novel insights into the regulation of HR+ BRCA immune infiltration by CHST4.

Integrating plasma protein-centric multi-omics to identify potential therapeutic targets for pancreatic cancer

BackgroundDeciphering the role of plasma proteins in pancreatic cancer (PC) susceptibility can aid in identifying novel targets for diagnosis and treatment.MethodsWe examined the relationship between genetically determined levels of plasma proteins and PC through a systemic proteome-wide Mendelian randomization (MR) analysis utilizing cis-pQTLs from multiple centers. Rigorous sensitivity analyses, colocalization, reverse MR, replications with varying instrumental variable selections and additional datasets, as well as subsequent meta-analysis, were utilized to confirm the robustness of significant findings. The causative effect of corresponding protein-coding genes’ expression and their expression pattern in single-cell types were then investigated. Enrichment analysis, between-protein interaction and causation, knock-out mice models, and mediation analysis with established PC risk factors were applied to indicate the pathogenetic pathways. These candidate targets were ultimately prioritized upon druggability and potential side effects predicted by a phenome-wide MR.ResultsTwenty-one PC-related circulating proteins were identified in the exploratory phase with no evidence for horizontal pleiotropy or reverse causation. Of these, 11 were confirmed in a meta-analysis integrating external validations. The causality at a transcription level was repeated for neutrophil elastase, hydroxyacylglutathione hydrolase, lipase member N, protein disulfide-isomerase A5, xyloside xylosyltransferase 1. The carbohydrate sulfotransferase 11 and histo-blood group ABO system transferase exhibited high-support genetic colocalization evidence and were found to affect PC carcinogenesis partially through modulating body mass index and type 2 diabetes, respectively. Approved drugs have been established for eight candidate targets, which could potentially be repurposed for PC therapies. The phenome-wide investigation revealed 12 proteins associated with 51 non-PC traits, and interference on protein disulfide-isomerase A5 and cystatin-D would increase the risk of other malignancies.ConclusionsBy employing comprehensive methodologies, this study demonstrated a genetic predisposition linking 21 circulating proteins to PC risk. Our findings shed new light on the PC etiology and highlighted potential targets as priorities for future efforts in early diagnosis and therapeutic strategies of PC.

Robust pancreatic tumor suppression by a novel combination treatment with anti-PD-1 immune checkpoint antibody and stroma modifying RNA oligonucleotide STNM01 in mice.

e14630 Background: Tumor stromal remodeling is an obstacle for immune checkpoint blockade therapies. STNM01 is a synthetic RNA oligonucleotide that represses carbohydrate sulfotransferase 15 (CHST15), which is responsible for tumor stromal remodeling. We have previously reported that STNM01 monotherapy by intratumoral injection repressed tumor stromal remodeling while increase tumor-infiltrating T lymphocytes (TILs) in pancreatic tumor-bearing mice. STNM01 was shown to increase TILs in patients with unresectable pancreatic cancer in a Phase I/IIa trial. In the present study, we investigated whether STNM01-mediated TIL enhancement improves anti-tumor effect of anti-PD-1 immune checkpoint antibody by combination treatment in murine models of pancreatic tumor. Methods: In mouse pancreatic cancer KPC and Pan02 subcutaneous syngeneic tumor models, mice were divided into 4 groups for treatment; 1) control, 2) STNM01 monotherapy, 3) anti-PD-1 antibody monotherapy, and 4) combination therapy with STNM01 and anti-PD-1 antibody. Intratumoral injections with 0.9-1.0 mg/mL of STNM01 or control and intraperitoneal administrations of anti-PD-1 antibody or control (5mg/kg) were then performed twice a week for 2 weeks. Mice were sacrificed after 2 week-treatment from baseline, and anti-tumor effects were evaluated by immunohistochemistry for KPC and flowcytometry for Pan02 model, respectively. Results: In the KPC-implanted syngeneic mouse model, combination treatment with intratumoral STNM01 and systemic anti-PD-1 antibody synergistically and robustly suppressed tumor growth as mean % tumor growth inhibition was over 80%. Tumor-infiltrating CD4+ and CD8+ T cells significantly increased in the anti-PD-1 plus STNM01 combination therapy group compared to anti-PD-1 monotherapy group. In the Pan02-implanted syngeneic mouse model, the combination treatment with STNM01 and anti-PD-1 significantly augmented necrosis area of tumor. Flowcytometry analyses showed that tumor-infiltrating CD3+, CD4+ and CD8+ T cells significantly increased in the anti-PD-1 plus STNM01 combination therapy group compared to control group. Although FoxP3+ Treg did not change, the ratio of CD8 to FoxP3 was significantly high only in the anti-PD-1 plus STNM01 combination therapy group. Notably, Ly6C+Ly6G+ granulocytic MDSCs dramatically diminished in the anti-PD-1 plus STNM01 combination therapy group, while anti-PD-1 monotherapy had no impact on tumoral MDSCs. Conclusions: The present work represents the first report of robust synergy between systemic anti-PD-1 antibody and single stroma modifying agent through intratumoral route of administration. Combination usage of STNM01 would provide a novel and clinically feasible therapeutic option to trigger remarkable effect of immune checkpoint inhibitors to this most hard-to-treat solid tumor.

Chst9 marks a spatially and transcriptionally unique population of Oprm1-expressing neurons in the nucleus accumbens

Opioids produce addictive, analgesic, and euphoric effects via actions at mu opioid receptors (µORs). The µOR is encoded by the Oprm1 gene and is expressed in multiple brain regions that regulate reward and motivation, such as the nucleus accumbens (NAc). Oprm1 expression in NAc medium spiny neurons (MSNs) mediates opioid place preference, seeking, and consumption. However, recent single nucleus RNA sequencing (snRNA-seq) studies have revealed that multiple subpopulations of NAc neurons express Oprm1 mRNA, making it unclear which populations mediate diverse behaviors resulting from µOR activation. Using published snRNA-seq datasets from the rat NAc, we identified a novel population of MSNs that express the highest levels of Oprm1 of any NAc cell type. Here, we show that this population is selectively marked by expression of Chst9, a gene encoding a carbohydrate sulfotransferase. Notably, Chst9+ neurons exhibited more abundant expression of Oprm1 as compared to other cell types, and formed discrete cellular clusters along the medial and ventral borders of the NAc shell subregion. Moreover, CHST9 mRNA was also found to mark specific MSN populations in published human and primate snRNA-seq studies, indicating that this unique population may be conserved across species. Together, these results identify a spatially and transcriptionally distinct NAc neuron population characterized by the expression of Chst9. The abundant expression of Oprm1 in this population and the conservation of these cells across species suggests that they may play a key functional role in opioid response and identify this subpopulation as a target for further investigation.

Effect of CHST11, a novel biomarker, on the biological functionalities of clear cell renal cell carcinoma

Clear cell renal cell carcinoma (ccRCC) is a common malignant tumor, and the role of carbohydrate sulfotransferase 11 (CHST11) in this cancer remains unclear. Here, by using bioinformatics methods, we comprehensively analyzed the relationship between CHST11 and clinical significance, immune infiltration, functional enrichment, m6A methylation, and protein–protein interaction networks. We found that CHST11 expression was significantly higher in ccRCC samples than in normal tissues. Additionally, CHST11 levels correlated with the clinicopathological features of ccRCC patients and functioned as a prognostic factor for patient survival. Functional analysis revealed the involvement of CHST11 in metabolic pathways. Immune infiltration and m6A methylation analysis suggested the association of CHST11 with immune cell abundance in the tumor microenvironment and specific methylation patterns in ccRCC. The in vitro analysis of the clinical samples and ccRCC cell lines demonstrated that the overexpression of CHST11 promotes ccRCC cell proliferation, migration, and invasion, while its suppression has the opposite effect. Thus, CHST11 may play a remarkable role in the occurrence and progression of ccRCC. Functionally, CHST11 promotes the aggressiveness of ccRCC cells. These findings provide insights into the role of CHST11 in ccRCC progression.Registry and the Registration No. of the study/trial: No. 2021K034.

SARS-CoV-2 spike protein-ACE2 interaction increases carbohydrate sulfotransferases and reduces N-acetylgalactosamine-4-sulfatase by p38 MAPK

Immunostaining in lungs of patients who died with COVID-19 infection showed increased intensity and distribution of chondroitin sulfate and decline in N-acetylgalactostamine-4-sulfatase (Arylsulfatase B; ARSB). To explain these findings, human small airway epithelial cells were exposed to the SARS-CoV-2 spike protein receptor binding domain (SPRBD) and transcriptional mechanisms were investigated. Phospho-p38 MAPK and phospho-SMAD3 increased following exposure to the SPRBD, and their inhibition suppressed the promoter activation of the carbohydrate sulfotransferases CHST15 and CHST11, which contributed to chondroitin sulfate biosynthesis. Decline in ARSB was mediated by phospho-38 MAPK-induced N-terminal Rb phosphorylation and an associated increase in Rb-E2F1 binding and decline in E2F1 binding to the ARSB promoter. The increases in chondroitin sulfotransferases were inhibited when treated with phospho-p38-MAPK inhibitors, SMAD3 (SIS3) inhibitors, as well as antihistamine desloratadine and antibiotic monensin. In the mouse model of carrageenan-induced systemic inflammation, increases in phospho-p38 MAPK and expression of CHST15 and CHST11 and declines in DNA-E2F binding and ARSB expression occurred in the lung, similar to the observed effects in this SPRBD model of COVID-19 infection. Since accumulation of chondroitin sulfates is associated with fibrotic lung conditions and diffuse alveolar damage, increased attention to p38-MAPK inhibition may be beneficial in ameliorating Covid-19 infections.

CHST12: a potential prognostic biomarker related to the immunotherapy response in pancreatic adenocarcinoma.

Pancreatic adenocarcinoma (PAAD) is characterized by lower immunogenicity with a poor response rate to immune checkpoint inhibitors (ICIs) and exhibits the poorest prognosis of all solid tumors, which results in the highest tumor-related mortality among malignancies. However, the underlying mechanisms are poorly understood. In addition, diverse carbohydrate sulfotransferases (CHSTs), which are involved in the sulfation process of these structures, play an important role in the metastatic spread of tumor cells. Aberrant glycosylation is beginning to emerge as an influencing factor in tumor immunity and immunotherapy. Therefore, it might serve as a biomarker of the immunotherapeutic response in tumors. The purpose of the study was to evaluate the role of CHST12 in PAAD prognosis and its relevance to the immunotherapeutic response. A comprehensive investigation of the interactions between CHST12 expression and the immune microenvironment as well as the clinical significance of CHST12 in PAAD was conducted. Data derived from the Cancer Genome Atlas (TCGA) database were analyzed using univariate and multivariate approaches, the Tumor Immune Estimation Resource (TIMER), and Tumor Immune Dysfunction and Exclusion (TIDE) algorithms. Publicly available datasets were analyzed in this study. These data can be found on websites such as http://www.xiantao.love and https://www.proteinatlas.org. An assessment of the predictive value of CHST12 for PAAD prognosis was conducted using univariate and multivariate Cox regression analysis, Kaplan-Meier analysis, and nomograms. The TIMER algorithm calculates the proportions of six types of immune cells. The TIDE algorithm was used to indicate the characteristics of tumors that respond to ICI therapy. The mRNA and protein levels of CHST12 showed the opposite trend. CHST12 mRNA expression was significantly upregulated in PAAD. According to Cox regression analysis, CHST12 RNA expression acts as a protective factor for overall survival [hazard ratio (HR), 0.617, P < 0.04]. Functional annotation indicated that CHST12-associated differentially expressed genes (DEGs) were related to the signaling activity of receptor tyrosine kinases and the regulation of ubiquitin-protein transferase. These are usually involved in tumor development and may be related to the treatment responses of immune checkpoint inhibitors (ICIs). There was significantly higher CHST12 mRNA expression in PAAD samples than in non-malignant samples. In PAAD, elevated CHST12 mRNA expression might regulate immune cell infiltration into the tumor microenvironment (TME) and may predict clinical outcomes.

Genetic insights into carbohydrate sulfotransferase 8 and its impact on the immunotherapy efficacy of cancer

Immune checkpoint blockade (ICB) is a promising therapy for solid tumors, but its effectiveness depends on biomarkers that are not precise. Here, we utilized genome-wide association study to investigate the association between genetic variants and tumor mutation burden to interpret ICB response. We identified 16 variants (p<5×10-8) probed to 17 genes on 9 chromosomes. Subsequent analysis of one of the most significant loci in 19q13.11 suggested that the rs111308825 locus at the enhancer is causal, as its A allele impairs KLF2 binding, leading to lower carbohydrate sulfotransferase 8 (CHST8) expression. Breast cancer cells expressing CHST8 suppress Tcell activation, and Chst8 loss attenuates tumor growth in a syngeneic mouse model. Further investigation revealed that programmed death-ligand 1 (PD-L1) and its homologs could be sulfated by CHST8, resulting in M2-like macrophage enrichment in the tumor microenvironment. Finally, we confirmed that low-CHST8 tumors have better ICB response, supporting the genetic effect and clinical value of rs111308825 for ICB efficacy prediction.

A comprehensive analysis of the expression, immune infiltration, prognosis and partial experimental validation of CHST family genes in gastric cancer

Previous studies have demonstrated that carbohydrate sulfotransferase family proteins (CHSTs) play a crucial role in the extracellular matrix structural constituent and cancer progression, however, the effect of CHSTs on gastric cancer is still superficial. To investigate these, our study seeks to provide a comprehensive understanding of CHSTs' expression, immune infiltration, and prognostic implications in gastric cancer, utilizing data from the TCGA, GEO and GTEx databases. Furthermore, we conducted experimental validation to elucidate the role of CHST14 specifically in gastric cancer. Our findings suggest that most CHSTs were highly expressed in gastric cancer. Gene copy number variations further indicated prevalent CHSTs amplification in gastric cancer, pointing to its potential relevance in disease progression. Intriguingly, we noted strong positive correlations between most CHSTs and immune cell infiltration. Importantly, most members of CHSTs were related to OS and PFI with gastric cancer, with particular emphasis on CHST14 and CHST9. Multifactorial regression analysis indicates that CHST14 is an independent prognostic factor influencing the overall survival of gastric cancer patients. In further experimental validation, our results demonstrate elevated expression of CHST14 in gastric cancer, and knocking down CHST14 inhibits gastric cancer cell proliferation, invasion, migration and EMT. Additionally, CHST14 may exert its function through the regulation of the Wnt pathway. In summary, our study comprehensively analyzes the hitherto undescribed role of CHSTs in gastric cancer through the analysis of multi-omics data. Importantly, we identify CHST14 as a pivotal promoter in the malignant progression of gastric cancer, offering potential targets for gastric cancer therapy.

Abstract LB_C21: Exogenous N-acetylgalactosamine-4-sulfatase (ARSB)-mediated tumor suppression involves effects on chondroitin 4-sulfate binding with SHP-2 and galectin-3, MAPK signaling, and transcriptional events, including epigenetic regulation of PD-L1

Abstract In experiments in melanoma, prostate, colon, and mammary cells and tissues, the enzyme N-acetylgalactosamine-4-sulfatase (Arylsulfatase B: ARSB), which removes the 4-sulfate group at the non-reducing end of chondroitin 4-sulfate (C4S), acts as a tumor suppressor. Following treatment with recombinant human (rh) ARSB in previous experiments and in recent experiments in the B16F10 mouse model of malignant melanoma, specific molecular pathways have been identified that retard tumor progression. These pathways involve more or less binding with chondroitin 4-sulfate when ARSB activity is modified. Galectin-3 binding with C4S increases following exogenous ARSB, leading to lower nuclear galectin-3 and reduced expression of chondroitin sulfate proteoglycan (CSPG)4 and of carbohydrate sulfotransferase (CHST)15 in melanoma cells and tissues. Nuclear Sp1 also declines, inhibiting transcription of both CSPG4, also known as the melanoma-associated chondroitin sulfate proteoglycan (MCSP), and of CHST15, which is required for 6-sulfation of N-acetylgalactosamine 4-sulfate. These proteins are associated with more aggressive tumors, and decline in their expression is consistent with the tumor suppressive function of ARSB. Inverse to the effect on galectin-3, SHP2, the ubiquitous non-receptor tyrosine phosphatase (PTPN11), binds less to C4S following treatment with exogenous ARSB. The resulting activation of SHP2 impacts on MAPK signaling, reducing phospho-ERK1/2 and phospho(T180/Y182)-p38-MAPK. These effects lead to decline in expression of the matrix metalloproteinases MMP2 and MMP9 and thereby restrain invasiveness and metastasis. Treatment of cultured melanoma cells with siRNA for ARSB leads to opposite effects to those of exogenous ARSB, including epigenetic effects modifying Programmed Death-Ligand 1 (PD-L1) expression. Treatment with rhARSB significantly reduces PD-L1 protein and gene expression, in contrast to increases when ARSB is silenced. The effects of rhARSB on PD-L1 expression are attributable to declines in phospho-JNK and nuclear c-Jun when free galectin-3 is less and to associated increases in histone deacetylase (HDAC) 3 expression and activity. Consistent with these effects, the ARSB-silencing induced increase in PD-L1 is inhibited by peptide inhibitors of the JNK-c-Jun interaction and by HDAC3 inhibitor. ARSB silencing reduces PD-L1 promoter acetylation, as shown by chromatin immunoprecipitation with anti-acetylhistone H3 antibody. The impact of rhARSB on signaling pathways, transcriptional events, and epigenetic mechanisms demonstrates how a critical sulfation and the resulting modification of chondroitin 4-sulfate can influence vital cell processes and can suppress tumor development. Effects on subcutaneous and pulmonary melanomas demonstrate the impact of exogenous ARSB in melanoma and suggest future prospects as a therapeutic agent. Recombinant human ARSB is an approved agent for the treatment of Mucopolysaccharidosis VI and is used safely and effectively for this congenital deficiency of ARSB. Citation Format: Joanne K. Tobacman, Insug O-Sullivan, Sumit Bhattacharyya. Exogenous N-acetylgalactosamine-4-sulfatase (ARSB)-mediated tumor suppression involves effects on chondroitin 4-sulfate binding with SHP-2 and galectin-3, MAPK signaling, and transcriptional events, including epigenetic regulation of PD-L1 [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_C21.

Prenatal exposure to per- and polyfluoroalkyl substances and DNA methylation in the placenta: A prospective cohort study

Epidemiological studies regarding the relationship between per- and polyfluoroalkyl substances (PFAS) and DNA methylation were limited. We investigated the associations of maternal PFAS concentrations with placental DNA methylation and examined the mediating role of methylation changes between PFAS and infant development. We measured the concentrations of 11 PFAS in maternal plasma during early pregnancy and infant development at six months of age. We analyzed genome-wide DNA methylation in 16 placental samples using reduced representation bisulfite sequencing. Additionally, we measured DNA methylation levels using bisulfite amplicon sequencing in 345 mother-infant pairs for five candidate genes, including carbohydrate sulfotransferase 7 (CHST7), fibroblast growth factor 13 (FGF13), insulin receptor substrate 4 (IRS4), paired like homeobox 2Ap (PHOX2A), and plexin domain containing 1 (PLXDC1). We found that placental DNA methylation profiles related to PFOA mainly enriched in angiogenesis and neuronal signaling pathways. PFOA was associated with hypomethylation of IRS4 and PLXDC1, and PFNA was associated with PLXDC1 hypomethylation. There were positive associations of CHST7 methylation with PFTrDA and IRS4 methylation with PFDoA and PFTrDA. PLXDC1 hypomethylation mediated the association between PFOA and suspected developmental delay in infants. Future studies with larger sample sizes are warranted to confirm these findings.

Carbohydrate sulfotransferases: a review of emerging diagnostic and prognostic applications.

Carbohydrate sulfotransferases (CHST) catalyse the biosynthesis of proteoglycans that enable physical interactions and signalling between different neighbouring cells in physiological and pathological states. The study aim was to provide an overview of emerging diagnostic and prognostic applications of CHST. PubMed database search was conducted using the keywords "carbohydrate sulfotransferase" together with appropriate inclusion and exclusion criteria, whereby 41 publications were selected. Additionally, 40 records on CHST genetic and biochemical properties were hand-picked from UniProt, GeneCards, InterPro, and neXtProt databases. Carbohydrate sulfotransferases have been applied mainly in diagnostics of connective tissue disorders, cancer and inflammations. The lack of CHST activity was found in congenital connective tissue disorders while CHST overexpression was detected in different malignancies. Mutations of CHST3 gene cause skeletal dysplasia, chondrodysplasia, and autosomal recessive multiple joint dislocations while increased tissue expression of CHST11, CHST12 and CHST15 is an unfavourable prognostic factor in ovarian cancer, glioblastoma and pancreatic cancer, respectively. Recently, CHST11 and CHST15 overexpression in the vascular smooth muscle cells was linked to the severe lung pathology in COVID-19 patients. Promising CHST diagnostic and prognostic applications have been described but larger clinical studies and robust analytical procedures are required for the more reliable diagnostic performance estimations.

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.

Jasmonates disrupt carrageenan synthesis during carposporogenesis in the red seaweed Grateloupia imbricata

In this study, we monitored changes in cell wall sulfated galactans in the red seaweed Grateloupia imbricata after the rapid (48 h) induction of cystocarp maturation and carpospore development by the volatile hormone methyl jasmonate (MEJA). Synthesis of sulfated galactans, carrageenans, which requires sulfated UDP galactose, was followed by expression analysis of genes encoding phosphoglucomutase (PGM), galactose-1-phosphate uridyltransferase (GALT), and those responsible for sulfate assimilation (S-transporter and sulfate adenylyltransferase, SAT). In addition, the expression of carbohydrate sulfotransferase and galactose-6-sulfurylase responsible for the addition and removal of sulfate groups to the galactans backbone were evaluated. Structural changes, such as thallus softening, were analyzed using Fourier-transformed infrared spectroscopy (FTIR) spectra, and ploidy was assessed using flow cytometry. The results showed downregulation of most of the genes encoding precursors or those in charge of transformation into sulfated hexoses for cell wall synthesis. Furthermore, alterations in the FTIR spectrum of carrageenan from MEJA-induced fertile thalli, such as the disappearance and flattening of absorption bands, were observed. The promptness of these events and the ploidy of thalli and spores seem to confirm the induction of mitotic production of haploid spores by MEJA.

The role of corneal endothelium in macular corneal dystrophy development and recurrence.

Macular corneal dystrophy (MCD) is a progressive, bilateral stromal dystrophic disease that arises from mutations in carbohydrate sulfotransferase 6 (CHST6). Corneal transplantation is the ultimate therapeutic solution for MCD patients. Unfortunately, postoperative recurrence remains a significant challenge. We conducted a retrospective review of a clinical cohort comprising 102 MCD patients with 124 eyes that underwent either penetrating keratoplasty (PKP) or deep anterior lamellar keratoplasty (DALK). Our results revealed that the recurrence rate was nearly three times higher in the DALK group (39.13%, 9/23 eyes) compared with the PKP group (10.89%, 11/101 eyes), suggesting that surgical replacement of the corneal endothelium for treating MCD is advisable to prevent postoperative recurrence. Our experimental data confirmed the robust mRNA and protein expression of CHST6 in human corneal endothelium and the rodent homolog CHST5 in mouse endothelium. Selective knockdown of wild-type Chst5 in mouse corneal endothelium (ACsiChst5), but not in the corneal stroma, induced experimental MCD with similar extracellular matrix synthesis impairments and corneal thinning as observed in MCD patients. Mice carrying Chst5 point mutation also recapitulated clinical phenotypes of MCD, along with corneal endothelial abnormalities. Intracameral injection of wild-type Chst5 rescued the corneal impairments in ACsiChst5 mice and retarded the disease progression in Chst5 mutant mice. Overall, our study provides new mechanistic insights and therapeutic approaches for MCD treatment by high-lighting the role of corneal endothelium in MCD development.

DNA Variation in Spinal Pathologies: Genetics Running Down the Spine

Commentary Nearly 400 types of skeletal malformations, including those that involve the spine, have been characterized by molecular and cellular analyses1. Identification of genetic components that control the integrity of intervertebral discs—and that predispose to spinal degeneration in aging patients when mutated—represents a challenging goal. Spinal conditions that occur later in life may emerge from faulty musculoskeletal tissue repair after accidental injury and the influences of comorbidities. The collective stochastic and often undocumented events in the life of a patient can easily complicate attempts at defining genetic predispositions. To manage the statistical noise that comes from random mutations within the human population (“nature”) and the randomness of life’s events (“nurture”), it is necessary to examine massive genetic data sets with many thousands of patients. Technological advances that allow cost-effective DNA sequencing have made it feasible to compare large patient populations matched on the basis of specific codes for medical conditions. Consequently, several studies have made impressive attempts at defining genetic variations linked to a range of surgically relevant musculoskeletal complications, including degenerative rotator cuff disease2, arthroplasty3, adhesive capsulitis of the shoulder4, and end-stage knee osteoarthritis5. The current paper by Bovonratwet et al. at the Hospital for Special Surgery in New York provides the latest installment in a growing set of orthopaedic studies in this journal that investigate the relationship between genetic variations and the risk of surgery for spinal conditions. Similar to prior published efforts by Yanik et al.2, Brüggemann et al.3, and Kulm et al.4,5, the study by Bovonratwet et al. leverages the power of the UK Biobank, which has genetic data for about 400,000 patients, of whom a total of about 20,000 (∼5%) had 1 of 4 spinal conditions (i.e., lumbar spondylolisthesis, spinal stenosis, degenerative disc disease, and pseudarthrosis after spinal fusion). Results from this large data set (“training population”) were then tested using the FinnGen database (“test population”) for validation. The analysis yielded multiple different genetic variants across 7 chromosomes. On average, the authors discovered 2 distinct loci per spinal condition. These findings are consistent with the genetic complexities of spinal conditions and suggest that the diseases are polygenic, as expected. Several nucleotide variants associated with 4 common spinal conditions were located in loci encoding anonymous genes that have not been experimentally explored. Degenerative disc disease was associated with a locus containing 2 genes that affect chondrogenesis: CHST3 (for carbohydrate sulfotransferase 3) promotes sulfation of chondroitin, and SMAD3 is the inducible target of chondrogenic transforming growth factor (TGF)-β signaling. Remarkably, 1 chromosomal region significantly associated with both lumbar spondylolisthesis and spinal stenosis contained overlapping sets of genes (i.e., GFPT1 and NFU1) that encode proteins involved in cell metabolism. These genes represent attractive targets for studies on genetic causality for these 2 spinal conditions. While it is tempting to speculate about potential mechanistic implications, the genetic biomarkers for spinal conditions mapped to large regions containing several genes. Whether these genes are even expressed in cell types and tissues relevant to homeostasis and repair of spinal tissues remains to be determined. Lack of similarity among studies indicates that endogenous tissue-specific repair processes may be predominant in the genetic landscape. One major strength of this study is the control for demographics and comorbidities. Yet, even with access to nearly 400,000 people who form a fairly homogeneous study cohort (i.e., White elderly people in Britain), this population size is not nearly large enough to identify the many genes that undoubtedly contribute to spinal conditions. The observation that only a few genetic variants were replicated in the Finnish cohort may further reflect the polygenicity of disease factors and genetic differences of Finnish patients. In closing, just as our emotions can run up or down our spine depending on whether we are in a positive or negative mood, genetic variation appears to affect spinal diseases. Some DNA mutations may literally be running down our spine, by destroying either disc integrity or our capacity for repair after injury. The positive news is that recognition of genetic predispositions may enhance consideration of behavioral modifications or guide surgical decisions. The paper by Bovonratwet et al. will inspire future studies, while bringing us 1 step closer to that goal.