Sult2b1 deficiency exacerbates ischemic stroke by promoting pro-inflammatory macrophage polarization in mice.

Rationale: Stroke is a leading causes of human death worldwide. Ischemic damage induces the sterile neuroinflammation, which directly determines the recovery of patients. Lipids, a major component of the brain, significantly altered after stroke. Cholesterol sulfate, a naturally occurring analog of cholesterol, can directly regulate immune cell activation, indicating the possible involvement of cholesterol metabolites in neuroinflammation. Sulfotransferase family 2b member 1 (Sult2b1) is the key enzyme that catalyzes the synthesis of cholesterol sulfate. This study aimed to investigate the function of Sult2b1 and cholesterol sulfate in the neuroinflammation after ischemic stroke. Methods and Results: Sult2b1-/- and wild-type mice were subjected to transient middle cerebral artery occlusion. Our data showed that Sult2b1-/- mice had larger infarction and worse neurological scores. To determine whether immune cells were involved in the worsening stroke outcome in Sult2b1-/- mice, bone marrow transplantation, immune cell depletion, and adoptive monocyte transfer were performed. Combined with CyTOF and immunofluorescence techniques, we demonstrated that after stroke, the peripheral monocyte-derived macrophages were the dominant cell type promoting the pro-inflammatory status in Sult2b1-/-mice. Using primary bone marrow-derived macrophages, we showed that cholesterol sulfate could attenuate the pro-inflammatory polarization of macrophages under both normal and oxygen-glucose deprivation conditions by regulating the levels of nicotinamide adenine dinucleotide phosphate (NADPH), reactive oxygen species (ROS), and activating the AMP-activated protein kinase (AMPK) - cAMP responsive element-binding protein (CREB) signaling pathway. Conclusions:Sult2b1-/- promoted the polarization of macrophages into pro-inflammatory status. This trend could be attenuated by adding cholesterol sulfate, which promotes the polarization of macrophages into anti-inflammatory status by metabolic regulation. In this study, we established an inflammation-metabolism axis during the macrophage polarization after ischemic stroke.

Adult-onset CNS myelin sulfatide deficiency is sufficient to cause Alzheimer's disease-like neuroinflammation and cognitive impairment.

BACKGROUND: Human genetic association studies point to immune response and lipid metabolism, in addition to amyloid-beta (Aß) and tau, as major pathways in Alzheimer's disease (AD) etiology. Accumulating evidence suggests that chronic neuroinflammation, mainly mediated by microglia and astrocytes, plays a causative role in neurodegeneration in AD. Our group and others have reported early and dramatic losses of brain sulfatide in AD cases and animal models that are mediated by ApoE in an isoform-dependent manner and accelerated by Aß accumulation. To date, it remains unclear if changes in specific brain lipids are sufficient to drive AD-related pathology. METHODS: To study the consequences of CNS sulfatide deficiency and gain insights into the underlying mechanisms, we developed a novel mouse model of adult-onset myelin sulfatide deficiency, i.e., tamoxifen-inducible myelinating glia-specific cerebroside sulfotransferase (CST) conditional knockout mice (CSTfl/fl/Plp1-CreERT), took advantage of constitutive CST knockout mice (CST-/-), and generated CST/ApoE double knockout mice (CST-/-/ApoE-/-), and assessed these mice using a broad range of methodologies including lipidomics, RNA profiling, behavioral testing, PLX3397-mediated microglia depletion, mass spectrometry (MS) imaging, immunofluorescence, electron microscopy, and Western blot. RESULTS: We found that mild central nervous system (CNS) sulfatide losses within myelinating cells are sufficient to activate disease-associated microglia and astrocytes, and to increase the expression of AD risk genes (e.g., Apoe, Trem2, Cd33, and Mmp12), as well as previously established causal regulators of the immune/microglia network in late-onset AD (e.g., Tyrobp, Dock, and Fcerg1), leading to chronic AD-like neuroinflammation and mild cognitive impairment. Notably, neuroinflammation and mild cognitive impairment showed gender differences, being more pronounced in females than males. Subsequent mechanistic studies demonstrated that although CNS sulfatide losses led to ApoE upregulation, genetically-induced myelin sulfatide deficiency led to neuroinflammation independently of ApoE. These results, together with our previous studies (sulfatide deficiency in the context of AD is mediated by ApoE and accelerated by Aß accumulation) placed both Aß and ApoE upstream of sulfatide deficiency-induced neuroinflammation, and suggested a positive feedback loop where sulfatide losses may be amplified by increased ApoE expression. We also demonstrated that CNS sulfatide deficiency-induced astrogliosis and ApoE upregulation are not secondary to microgliosis, and that astrogliosis and microgliosis seem to be driven by activation of STAT3 and PU.1/Spi1 transcription factors, respectively. CONCLUSION: Our results strongly suggest that sulfatide deficiency is an important contributor and driver of neuroinflammation and mild cognitive impairment in AD pathology.

Knockdown of carbohydrate sulfotransferase 12 decreases the proliferation and mobility of glioblastoma cells via the WNT/ß-catenin pathway.

Glioblastoma (GBM) is a common malignant tumor of the brain. Members of the carbohydrate sulfotransferase (CHST) family are deregulated in various cancer types. However, limited data are available on the role of the members of the CHST family in the development of GBM. The present study aimed to identify the role of significant members of the CHST family in GBM and explore the effects and molecular mechanisms of these significant members on GBM cell proliferation and mobility. In the current study, we demonstrated that CHST12 is the only member of CHST family that is upregulated in GBM tissues and associated with a lower survival rate according to the data obtained from The Cancer Genome Atlas. Similarly, the expression of CHST12 increased in GBM tissues than in adjacent tissues and had an important diagnostic value in distinguishing tumor tissues from adjacent tissues. The high expression of CHST12 indicated a lower overall survival rate, was negatively associated with the Karnofsky Performance Scale score, was positively associated with the KI67 expression rate, and was an independent risk factor for GBM. Knockdown of CHST12 significantly decreased GBM cell proliferation and mobility and inhibited the Wnt/ß-catenin pathway. Restoration of ß-catenin expression in GBM cells reversed the inhibitory effects of CHST12 knockdown on GBM cell proliferation and mobility. In conclusion, the present study demonstrated that CHST12 may be a novel biomarker for GBM; it regulates GBM cell proliferation and mobility via the WNT/ß-catenin pathway.

Regulation of fractone heparan sulfate composition in young and aged subventricular zone neurogenic niches.

Fractones, specialized extracellular matrix structures found in the subventricular zone (SVZ) neurogenic niche, can capture growth factors, such as basic fibroblast growth factor, from the extracellular milieu through a heparin-binding mechanism for neural stem cell (NSC) presentation, which promotes neurogenesis. During aging, a decline in neurogenesis correlates with a change in the composition of heparan sulfate (HS) within fractones. In this study, we used antibodies that recognize specific short oligosaccharides with varying sulfation to evaluate the HS composition in fractones in young and aged brains. To further understand the conditions that regulate 6-O sulfation levels and its impact on neurogenesis, we used endosulfatase Sulf1 and Sulf2 double knockout (DKO) mice. Fractones in the SVZ of Sulf1/2 DKO mice showed immunoreactivity for the HS epitope, suggesting higher 6-O sulfation. While neurogenesis declined in the aged SVZ of both wild-type and Sulf1/2 DKO mice, we observed a larger number of neuroblasts in the young and aged SVZ of Sulf1/2 DKO mice. Together, these results show that the removal of 6-O-sulfation in fractones HS by endosulfatases inhibits neurogenesis in the SVZ. Our findings advance the current understanding regarding the extracellular environment that is best suited for NSCs to thrive, which is critical for the design of future stem cell therapies.

Systematic investigation of the skin in Chst14-/- mice: A model for skin fragility in musculocontractural Ehlers-Danlos syndrome caused by CHST14 variants (mcEDS-CHST14).

Loss-of-function variants in CHST14 cause a dermatan 4-O-sulfotransferase deficiency named musculocontractural Ehlers-Danlos syndrome-CHST14 (mcEDS-CHST14), resulting in complete depletion of the dermatan sulfate moiety of decorin glycosaminoglycan (GAG) chains, which is replaced by chondroitin sulfate. Recently, we uncovered structural alteration of GAG chains in the skin of patients with mcEDS-CHST14. Here, we conducted the first systematic investigation of Chst14 gene-deleted homozygote (Chst14-/-) mice. We used skin samples of wild-type (Chst14+/+) and Chst14-/- mice. Mechanical fragility of the skin was measured with a tensile test. Pathology was observed using light microscopy, decorin immunohistochemistry and electron microscopy (EM) including cupromeronic blue (CB) staining. Quantification of chondroitin sulfate and dermatan sulfate was performed using enzymatic digestion followed by anion-exchange HPLC. In Chst14-/- mice, skin tensile strength was significantly decreased compared with that in Chst14+/+ mice. EM showed that collagen fibrils were oriented in various directions to form disorganized collagen fibers in the reticular layer. Through EM-based CB staining, rod-shaped linear GAG chains were found to be attached at one end to collagen fibrils and protruded outside of the fibrils, in contrast to them being round and wrapping the collagen fibrils in Chst14+/+ mice. A very low level of dermatan sulfate disaccharides was detected in the skin of Chst14-/- mice by anion-exchange chromatography. Chst14-/- mice, exhibiting similar abnormalities in the GAG structure of decorin and collagen networks in the skin, could be a reasonable model for skin fragility of patients with mcEDS-CHST14, shedding light on the role of dermatan sulfate in maintaining skin strength.

Reduced cellular binding affinity has profoundly different impacts on the spread of distinct poxviruses.

Poxviruses are large enveloped viruses that replicate exclusively in the cytoplasm. Like all viruses, their replication cycle begins with virion adsorption to the cell surface. Unlike most other viral families, however, no unique poxviral receptor has ever been identified. In the absence of a unique receptor, poxviruses are instead thought to adhere to the cell surface primarily through electrostatic interactions between the positively charged viral envelope proteins and the negatively charged sulfate groups on cellular glycosaminoglycans (GAGs). While these negatively charged GAGs are an integral part of all eukaryotic membranes, their specific expression and sulfation patterns differ between cell types. Critically, while poxviral binding has been extensively studied using virally centered genetic strategies, the impact of cell-intrinsic changes to GAG charge has never been examined. Here we show that loss of heparin sulfation, accomplished by deleting the enzyme N-Deacetylase and N-Sulfotransferase-1 (NDST1) which is essential for GAG sulfation, significantly reduces the binding affinity of both vaccinia and myxoma viruses to the cell surface. Strikingly, however, while this lowered binding affinity inhibits the subsequent spread of myxoma virus, it actually enhances the overall spread of vaccinia by generating more diffuse regions of infection. These data indicate that cell-intrinsic GAG sulfation plays a major role in poxviral infection, however, this role varies significantly between different members of the poxviridae.

Hydroxysteroid sulfotransferase 2B1 affects gastric epithelial function and carcinogenesis induced by a carcinogenic agent.

BACKGROUND: A healthy gastric mucosal epithelium exhibits tumor-suppressive properties. Gastric epithelial cell dysfunction contributes to gastric cancer development. Oxysterols provided from food or cholesterol oxidation in the gastric epithelium may be further sulfated by hydroxysteroid sulfotransferase 2B1 (SULT2B1), which is highly abundant in the gastric epithelium. However, the effects of SULT2B1 on gastric epithelial function and gastric carcinogenesis are unclear. METHODS: A mouse gastric tumor model was established using carcinogenic agent 3-methylcholanthrene (3-MCA). A SULT2B1 deletion (SULT2B1-/-) human gastric epithelial line GES-1 was constructed by CRISPR/CAS9 genome editing system. RESULTS: The gastric tumor incidence was higher in the SULT2B1-/- mice than in the wild-type (WT) mice. In gastric epithelial cells, adenovirus-mediated SULT2B1b overexpression reduced the levels of oxysterols, such as 24(R/S),25-epoxycholesterol (24(R/S),25-EC) and 27-hydroxycholesterol (27HC). This condition also increased PI3K/AKT signaling to promote gastric epithelial cell proliferation, epithelization, and epithelial development. However, SULT2B1 deletion or SULT2B1 knockdown suppressed PI3K/AKT signaling, epithelial cell epithelization, and wound healing and induced gastric epithelial cell malignant transition upon 3-MCA induction. CONCLUSIONS: The abundant SULT2B1 expression in normal gastric epithelium might maintain epithelial function via the PI3K/AKT signaling pathway and suppress gastric carcinogenesis induced by a carcinogenic agent.

Loss of endothelial sulfatase-1 after experimental sepsis attenuates subsequent pulmonary inflammatory responses.

Sepsis patients are at increased risk for hospital-acquired pulmonary infections, potentially due to postseptic immunosuppression known as the compensatory anti-inflammatory response syndrome (CARS). CARS has been attributed to leukocyte dysfunction, with an unclear role for endothelial cells. The pulmonary circulation is lined by an endothelial glycocalyx, a heparan sulfate-rich layer essential to pulmonary homeostasis. Heparan sulfate degradation occurs early in sepsis, leading to lung injury. Endothelial synthesis of new heparan sulfates subsequently allows for glycocalyx reconstitution and endothelial recovery. We hypothesized that remodeling of the reconstituted endothelial glycocalyx, mediated by alterations in the endothelial machinery responsible for heparan sulfate synthesis, contributes to CARS. Seventy-two hours after experimental sepsis, coincident with glycocalyx reconstitution, mice demonstrated impaired neutrophil and protein influx in response to intratracheal lipopolysaccharide (LPS). The postseptic reconstituted glycocalyx was structurally remodeled, with enrichment of heparan sulfate disaccharides sulfated at the 6-O position of glucosamine. Increased 6-O-sulfation coincided with loss of endothelial sulfatase-1 (Sulf-1), an enzyme that specifically removes 6-O-sulfates from heparan sulfate. Intravenous administration of Sulf-1 to postseptic mice restored the pulmonary response to LPS, suggesting that loss of Sulf-1 was necessary for postseptic suppression of pulmonary inflammation. Endothelial-specific knockout mice demonstrated that loss of Sulf-1 was not sufficient to induce immunosuppression in non-septic mice. Knockdown of Sulf-1 in human pulmonary microvascular endothelial cells resulted in downregulation of the adhesion molecule ICAM-1. Taken together, our study indicates that loss of endothelial Sulf-1 is necessary for postseptic suppression of pulmonary inflammation, representing a novel endothelial contributor to CARS.

Analysis of A4gnt Knockout Mice Reveals an Essential Role for Gastric Sulfomucins in Preventing Gastritis Cystica Profunda.

Gastric adenocarcinoma cells secrete sulfomucins, but their role in gastric tumorigenesis remains unclear. To address that question, we generated A4gnt/Chst4 double-knockout (DKO) mice by crossing A4gnt knockout (KO) mice, which spontaneously develop gastric adenocarcinoma, with Chst4 KO mice, which are deficient in the sulfotransferase GlcNAc6ST-2. A4gnt/Chst4 DKO mice lack gastric sulfomucins but developed gastric adenocarcinoma. Unexpectedly, severe gastric erosion occurred in A4gnt/Chst4 DKO mice at as early as 3 weeks of age, and with aging these lesions were accompanied by gastritis cystica profunda (GCP). Cxcl1, Cxcl5, Ccl2, and Cxcr2 transcripts in gastric mucosa of 5-week-old A4gnt/Chst4 DKO mice exhibiting both hyperplasia and severe erosion were significantly upregulated relative to age-matched A4gnt KO mice, which showed hyperplasia alone. However, upregulation of these genes disappeared in 50-week-old A4gnt/Chst4 DKO mice exhibiting high-grade dysplasia/adenocarcinoma and GCP. Moreover, Cxcl1 and Cxcr2 were downregulated in A4gnt/Chst4 DKO mice relative to age-matched A4gnt KO mice exhibiting adenocarcinoma alone. These combined results indicate that the presence of sulfomucins prevents severe gastric erosion followed by GCP in A4gnt KO mice by transiently regulating a set of inflammation-related genes, Cxcl1, Cxcl5, Ccl2, and Cxcr2 at 5 weeks of age, although sulfomucins were not directly associated with gastric cancer development.

Heparan Sulfate Sulfation by Hs2st Restricts Astroglial Precursor Somal Translocation in Developing Mouse Forebrain by a Non-Cell-Autonomous Mechanism.

Heparan sulfate (HS) is a cell surface and extracellular matrix carbohydrate extensively modified by differential sulfation. HS interacts physically with canonical fibroblast growth factor (FGF) proteins that signal through the extracellular signal regulated kinase (ERK)/mitogen activated protein kinase (MAPK) pathway. At the embryonic mouse telencephalic midline, FGF/ERK signaling drives astroglial precursor somal translocation from the ventricular zone of the corticoseptal boundary (CSB) to the induseum griseum (IG), producing a focus of Slit2-expressing astroglial guidepost cells essential for interhemispheric corpus callosum (CC) axon navigation. Here, we investigated the cell and molecular function of a specific form of HS sulfation, 2-O HS sulfation catalyzed by the enzyme Hs2st, in midline astroglial development and in regulating FGF protein levels and interaction with HS. Hs2st-/- embryos of either sex exhibit a grossly enlarged IG due to precocious astroglial translocation and conditional Hs2st mutagenesis and ex vivo culture experiments show that Hs2st is not required cell autonomously by CC axons or by the IG astroglial cell lineage, but rather acts non-cell autonomously to suppress the transmission of translocation signals to astroglial precursors. Rescue of the Hs2st-/- astroglial translocation phenotype by pharmacologically inhibiting FGF signaling shows that the normal role of Hs2st is to suppress FGF-mediated astroglial translocation. We demonstrate a selective action of Hs2st on FGF protein by showing that Hs2st (but not Hs6st1) normally suppresses the levels of Fgf17 protein in the CSB region in vivo and use a biochemical assay to show that Hs2st (but not Hs6st1) facilitates a physical interaction between the Fgf17 protein and HS.SIGNIFICANCE STATEMENT We report a novel non-cell-autonomous mechanism regulating cell signaling in developing brain. Using the developing mouse telencephalic midline as an exemplar, we show that the specific sulfation modification of the cell surface and extracellular carbohydrate heparan sulfate (HS) performed by Hs2st suppresses the supply of translocation signals to astroglial precursors by a non-cell-autonomous mechanism. We further show that Hs2st modification selectively facilitates a physical interaction between Fgf17 and HS and suppresses Fgf17 protein levels in vivo, strongly suggesting that Hs2st acts selectively on Fgf17 signaling. HS interacts with many signaling proteins potentially encoding numerous selective interactions important in development and disease, so this class of mechanism may apply more broadly to other biological systems.

Spinal manifestations in 12 patients with musculocontractural Ehlers-Danlos syndrome caused by CHST14/D4ST1 deficiency (mcEDS-CHST14).

Musculocontractural Ehlers-Danlos syndrome caused by mutations in CHST14 (mcEDS-CHST14) is a recently delineated disorder, characterized by craniofacial, skeletal, visceral, and ocular malformations; and progressive cutaneous, skeletal, vascular, and visceral fragility-related manifestations. Spinal lesions, though one of the most serious complications, have not been investigated systematically. In this study, we report detailed and comprehensive information about spinal lesions of 12 patients with a mean age at the first visit of 13.4 years. Eight patients (66.7%) had scoliosis with a Cobb angle ≥10°, including one with severe scoliosis with a Cobb angle ≥45°. Five patients (41.7%) had kyphosis at the thoracolumbar junction with a kyphotic angle ≥20°. Three patients (25%) developed severe thoracolumbar kyphosis with a kyphotic angle ≥50° accompanied by thoracic lordosis with a wedge-like vertebral deformity and anterior vertebral osteophyte at the thoracolumbar junction, and two of them underwent surgical correction: complicated by fistula formation in one and performed safely and effectively through two-staged operation in the other. Six patients (50.0%) had cervical kyphosis, all of whom except one had kyphosis ≥20° at the thoracolumbar level. Two patients (16.7%) had atlantoaxial subluxation, and 10 patients (83.3%) had cervical vertebral malformations. Patients with mcEDS-CHST14 are susceptible to develop scoliosis, thoracolumbar kyphosis, and cervical kyphosis; and are recommended to have regular surveillance including total spine radiology. The present findings also suggest the critical role of dermatan sulfate in the development and maintenance of the spine.

HS6ST1 Insufficiency Causes Self-Limited Delayed Puberty in Contrast With Other GnRH Deficiency Genes.

Context: Self-limited delayed puberty (DP) segregates in an autosomal-dominant pattern, but the genetic basis is largely unknown. Although DP is sometimes seen in relatives of patients with hypogonadotropic hypogonadism (HH), mutations in genes known to cause HH that segregate with the trait of familial self-limited DP have not yet been identified. Objective: To assess the contribution of mutations in genes known to cause HH to the phenotype of self-limited DP. Design, Patients, and Setting: We performed whole-exome sequencing in 67 probands and 93 relatives from a large cohort of familial self-limited DP, validated the pathogenicity of the identified gene variant in vitro, and examined the tissue expression and functional requirement of the mouse homolog in vivo. Results: A potentially pathogenic gene variant segregating with DP was identified in 1 of 28 known HH genes examined. This pathogenic variant occurred in HS6ST1 in one pedigree and segregated with the trait in the six affected members with heterozygous transmission (P = 3.01 × 10-5). Biochemical analysis showed that this mutation reduced sulfotransferase activity in vitro. Hs6st1 mRNA was expressed in peripubertal wild-type mouse hypothalamus. GnRH neuron counts were similar in Hs6st1+/- and Hs6st1+/+ mice, but vaginal opening was delayed in Hs6st1+/- mice despite normal postnatal growth. Conclusions: We have linked a deleterious mutation in HS6ST1 to familial self-limited DP and show that heterozygous Hs6st1 loss causes DP in mice. In this study, the observed overlap in potentially pathogenic mutations contributing to the phenotypes of self-limited DP and HH was limited to this one gene.

Hemoglobin adducts of furfuryl alcohol in genetically modified mouse models: Role of endogenous sulfotransferases 1a1 and 1d1 and transgenic human sulfotransferases 1A1/1A2.

Furfuryl alcohol (FFA) is a heat-induced food contaminant. Conversion by sulfotransferases (SULT) yields 2-sulfoxymethylfuran, which is prone to react with DNA and proteins. In order to monitor the internal FFA exposure we developed a technique for the mass spectrometric quantification of the adduct N-((furan-2-yl)methyl)-valine (FFA-Val) after cleavage from the N-termini of hemoglobin. In the current study the method was applied to investigate the influence of different SULT forms on the adduct formation in wild-type mice and three genetically modified mouse models treated with FFA. Two lines were devoid of endogenous Sult1a1 or Sult1d1, while another mouse line carried a transgene of human SULT1A1/1A2 in the Sult1a1/1d1 double knockout background. The Sult1d1 knockout did not influence adduct formation, whereas the lack of Sult1a1 reduced mean FFA-Val levels by 80% and 58% in male and female mice, respectively, in comparison to FFA-treated wild-type mice. The levels of FFA-Val in the humanized mice were elevated by factors of 2.7 (males) and 2.2 (females) as compared to the wild-type, indicating that SULT1A1/1A2 play a central role for FFA bioactivation also in humans. The excellent correlation between adduct levels in hepatic DNA and hemoglobin (r2 = 0.97) indicated that 2-sulfoxymethylfuran of hepatic origin is sufficiently stable to enter circulation and pass the cellular membrane of erythrocytes. This is a prerequisite for the application of FFA-Val as a biomarker of internal FFA exposure.

Airway glycomic and allergic inflammatory consequences resulting from keratan sulfate galactose 6-O-sulfotransferase (CHST1) deficiency.

Siglec-F is a pro-apoptotic receptor on mouse eosinophils that recognizes 6'-sulfated sialyl Lewis X and 6'-sulfated sialyl N-acetyl-lactosamine as well as multivalent sialyl N-acetyl-lactosamine structures on glycan arrays. We hypothesized that attenuation of the carbohydrate sulfotransferase 1 (CHST1) gene encoding keratan sulfate galactose 6-O-sulfotransferase, an enzyme likely required for 6'-sulfation of some of these putative Siglec-F glycan ligands, would result in decreased Siglec-F lung ligand levels and enhanced allergic eosinophilic airway inflammation. Tissue analysis detected CHST1 expression predominantly not only in parenchymal cells but not in airway epithelium, the latter being a location where Siglec-F ligands are located. Western blotting of lung extracts with Siglec-F-Fc fusion proteins detected ≈500 kDa and ≈200 kDa candidate Siglec-F ligands that were not appreciably altered in CHST1-/- lungs compared with normal mouse lungs. Characterization of the O-linked glycans of lung tissue and bronchoalveolar lavage fluid detected altered sialylation but minimal change in sulfation. Eosinophilic airway inflammation was induced in wild-type (WT) and CHST1-/- mice via sensitization to ovalbumin (OVA) and repeated airway challenge. After OVA sensitization and challenge, Siglec-F ligands on airway cells, and numbers of eosinophils and neutrophils accumulating in the airways, both increased to a similar degree in WT and CHST1-/- mouse lungs, while macrophages and lymphocytes increased significantly more in CHST1-/- mouse airway compared with normal mouse lungs. Therefore, keratan sulfate galactose 6-O-sulfotransferase does not contribute to the synthesis of glycan ligands for Siglec-F in the airways, although its absence results in exaggerated accumulation of airway macrophages and lymphocytes.

Biological functions of sulfoglycolipids and the EMARS method for identification of co-clustered molecules in the membrane microdomains.

Two major sulfoglycolipids, sulfatide (SO3-3Gal-ceramide) and seminolipid (SO3-3Gal-alkylacylglycerol) exist in mammals. Sulfatide is abundant in the myelin sheath and seminolipid is unique to the spermatogenic cells. The carbohydrate moiety of sulfatide and seminolipid is identical and synthesized by common enzymes: ceramide galactosyltransferase (CGT) and cerebroside sulfotransferase (CST). We have purified CST homogenously, cloned the CST gene and generated CST-knockout mice. CST-null mice completely lack sulfoglycolipids all over the body. Analysis of CST-null mice has revealed that sulfatide is an essential component for the axo-glial junction at the paranode region and regulates terminal differentiation of oligodendrocytes, and that seminolipid is responsible for the formation of a functional lactate transporter assembly to take up the critical energy source for spermatocytes. We have developed a new analytical method termed EMARS to identify co-clustered molecules in the membrane microdomains in order to elucidate the functional molecules that collaborate with sulfoglycolipids.

A 590 kb deletion caused by non-allelic homologous recombination between two LINE-1 elements in a patient with mesomelia-synostosis syndrome.

Mesomelia-synostoses syndrome (MSS) is a rare, autosomal-dominant, syndromal osteochondrodysplasia characterized by mesomelic limb shortening, acral synostoses, and multiple congenital malformations due to a non-recurrent deletion at 8q13 that always encompasses two coding-genes, SULF1 and SLCO5A1. To date, five unrelated patients have been reported worldwide, and MMS was previously proposed to not be a genomic disorder associated with deletions recurring from non-allelic homologous recombination (NAHR) in at least two analyzed cases. We conducted targeted gene panel sequencing and subsequent array-based copy number analysis in an 11-year-old undiagnosed Japanese female patient with multiple congenital anomalies that included mesomelic limb shortening and detected a novel 590 Kb deletion at 8q13 encompassing the same gene set as reported previously, resulting in the diagnosis of MSS. Breakpoint sequences of the deleted region in our case demonstrated the first LINE-1s (L1s)-mediated unequal NAHR event utilizing two distant L1 elements as homology substrates in this disease, which may represent a novel causative mechanism of the 8q13 deletion, expanding the range of mechanisms involved in the chromosomal rearrangements responsible for MSS.

Defect in dermatan sulfate in urine of patients with Ehlers-Danlos syndrome caused by a CHST14/D4ST1 deficiency.

PURPOSE: Dermatan sulfate (DS) plays a number of roles in a wide range of biological activities such as cell signaling and tissue morphogenesis through interactions with various extracellular matrix proteins including collagen. Mutations in the carbohydrate sulfotransferase 14 gene (CHST14) encoding CHST14/dermatan 4-O-sulfotransferase-1 (D4ST1), which is responsible for the biosynthesis of DS, cause a recently delineated form of Ehlers-Danlos syndrome (EDS, musculocontractural type 1), an autosomal recessive connective tissue disorder characterized by congenital malformations (specific craniofacial features, and congenital multiple contractures) and progressive fragility-related complications (skin hyperextensibility, bruisability, and fragility with atrophic scars; recurrent dislocations; progressive talipes or spinal deformities; and large subcutaneous hematomas). In an attempt to develop a diagnostic screening method for this type of EDS, the amount of DS in the urine of patients was analyzed. METHODS: Urinary DS was quantified by an anion-exchange chromatography after treatment with DS-specific degrading enzyme. RESULTS: DS was not detected in the urine of patients with homo- or compound heterozygous mutations in CHST14. These results suggest that the quantification of DS in urine is applicable to an initial diagnosis of DS-defective EDS. CONCLUSIONS: This is the first study to perform a urinary disaccharide compositional analysis of chondroitin sulfate (CS)/DS chains in patients with EDS caused by a CHST14/D4ST1 deficiency, and demonstrated the absence of DS chains. This result suggests systemic DS depletion in this disorder, and also proposes the usefulness of a urinary disaccharide compositional analysis of CS/DS chains as a non-invasive screening method for this disorder.

Pivotal Role of Carbohydrate Sulfotransferase 15 in Fibrosis and Mucosal Healing in Mouse Colitis.

Induction of mucosal healing (MH) is an important treatment goal in inflammatory bowel disease (IBD). Although the molecular mechanisms underlying MH in IBD is not fully explored, local fibrosis would contribute to interfere mucosal repair. Carbohydrate sulfotransferase 15 (CHST15), which catalyzes sulfation of chondroitin sulfate to produce rare E-disaccharide units, is a novel mediator to create local fibrosis. Here we have used siRNA-based approach of silencing CHST15 in dextran sulfate sodium (DSS) induced colitis in mice, human colon fibroblasts and cancer cell lines. In a DSS-induced acute colitis model, CHST15 siRNA reduced CHST15 mRNA in the colon, serum IL-6, disease activity index (DAI) and accumulation of F4/80+ macrophages and ER-TR7+ fibroblasts, while increased Ki-67+ epithelial cells. In DSS-induced chronic colitis models, CHST15 siRNA reduced CHST15 mRNA in the colon, DAI, alpha-smooth muscle actin+ fibroblasts and collagen deposition, while enhanced MH as evidenced by reduced histological and endoscopic scores. We also found that endoscopic submucosal injection achieved effective pancolonic delivery of CHST15 siRNA in mice. In human CCD-18 Co cells, CHST15 siRNA inhibited the expression of CHST15 mRNA and selectively reduced E-units, a specific product biosynthesized by CHST15, in the culture supernatant. CHST15 siRNA significantly suppressed vimentin in both TGF-ß-stimulated CCD18-Co cells and HCT116 cells while up-regulated BMP7 and E-cadherin in HCT116 cells. The present study demonstrated that blockade CHST15 represses colonic fibrosis and enhances MH partly though reversing EMT pathway, illustrating a novel therapeutic opportunity to refractory and fibrotic lesions in IBD.