Discovery of a class of glycosaminoglycan lyases with ultrabroad substrate spectrum and their substrate structure preferences.

Glycosaminoglycan (GAG) lyases are often strictly substrate specific, and it is especially difficult to simultaneously degrade GAGs with different types of glycosidic bonds. Herein, we found a new class of GAG lyases (GAGases) from different bacteria. These GAGases belong to polysaccharide lyase 35 family and share quite low homology with the identified GAG lyases. The most surprising thing is that GAGases can not only degrade three types of GAGs: HA, CS and HS, but even one of them can also degrade alginate. Further investigation of structural preferences revealed that GAGases selectively act on GAG domains composed of non/6-O-/N-sulfated hexosamines and d-glucoronic acids, as well as on alginate domains composed of d-mannuronic acids. Additionally, GAG lyases were once speculated to have evolved from alginate lyases, but no transitional enzymes have been found. The discovery of GAGases not only broadens the category of GAG lyases, provides new enzymatic tools for the structural and functional studies of GAGs with specific structures, but also provides candidates for the evolution of GAG lyases.

Enzymatic comparison of two homologous enzymes reveals N-terminal domain of chondroitinase ABC I regulates substrate selection and product generation.

Chondroitinase ABC-type I (CSase ABC I), which can digest both chondroitin sulfate (CS) and dermatan sulfate (DS) in an endolytic manner, is an essential tool in structural and functional studies of CS/DS. Although a few CSase ABC I have been identified from bacteria, the substrate-degrading pattern and regulatory mechanisms of them have rarely been investigated. Herein, two CSase ABC I, IM3796 and IM1634, were identified from the intestinal metagenome of CS-fed mice. They show high sequence homology (query coverage: 88.00%, percent identity: 90.10%) except for an extra peptide (Met1-His109) at the N-terminus in IM1634, but their enzymatic properties are very different. IM3796 prefers to degrade 6-O-sulfated GalNAc residue-enriched CS into tetra- and disaccharides. In contrast, IM1634 exhibits nearly a thousand times more activity than IM3796 and can completely digest CS/DS with various sulfation patterns to produce disaccharides, unlike most CSase ABC I. Structure modeling showed that IM3796 did not contain an N-terminal domain composed of two ß-sheets, which is found in IM1634 and other CSase ABC I. Furthermore, deletion of the N-terminal domain (Met1-His109) from IM1634 caused the enzymatic properties of the variant IM1634-T109 to be similar to those of IM3796, and conversely, grafting this domain to IM3796 increased the similarity of the variant IM3796-A109 to IM1634. In conclusion, the comparative study of the new CSase ABC I provides two unique tools for CS/DS-related studies and applications and, more importantly, reveals the critical role of the N-terminal domain in regulating the substrate binding and degradation of these enzymes.

Characterizing neuroinflammation and identifying prenatal diagnostic markers for neural tube defects through integrated multi-omics analysis.

BACKGROUND: Neural Tube Defects (NTDs) are congenital malformations of the central nervous system resulting from the incomplete closure of the neural tube during early embryonic development. Neuroinflammation refers to the inflammatory response in the nervous system, typically resulting from damage to neural tissue. Immune-related processes have been identified in NTDs, however, the detailed relationship and underlying mechanisms between neuroinflammation and NTDs remain largely unclear. In this study, we utilized integrated multi-omics analysis to explore the role of neuroinflammation in NTDs and identify potential prenatal diagnostic markers using a murine model. METHODS: Nine public datasets from Gene Expression Omnibus (GEO) and ArrayExpress were mined using integrated multi-omics analysis to characterize the molecular landscape associated with neuroinflammation in NTDs. Special attention was given to the involvement of macrophages in neuroinflammation within amniotic fluid, as well as the dynamics of macrophage polarization and their interactions with neural cells at single-cell resolution. We also used qPCR assay to validate the key TFs and candidate prenatal diagnostic genes identified through the integrated analysis in a retinoic acid-induced NTDs mouse model. RESULTS: Our analysis indicated that neuroinflammation is a critical pathological feature of NTDs, regulated both transcriptionally and epigenetically within central nervous system tissues. Key alterations in gene expression and pathways highlighted the crucial role of STATs molecules in the JAK-STAT signaling pathway in regulating NTDs-associated neuroinflammation. Furthermore, single-cell resolution analysis revealed significant polarization of macrophages and their interaction with neural cells in amniotic fluid, underscoring their central role in mediating neuroinflammation associated with NTDs. Finally, we identified a set of six potential prenatal diagnostic genes, including FABP7, CRMP1, SCG3, SLC16A10, RNASE6 and RNASE1, which were subsequently validated in a murine NTDs model, indicating their promise as prospective markers for prenatal diagnosis of NTDs. CONCLUSIONS: Our study emphasizes the pivotal role of neuroinflammation in the progression of NTDs and underlines the potential of specific inflammatory and neural markers as novel prenatal diagnostic tools. These findings provide important clues for further understanding the underlying mechanisms between neuroinflammation and NTDs, and offer valuable insights for the future development of prenatal diagnostics.

A mutated glycosaminoglycan-binding domain functions as a novel probe to selectively target heparin-like epitopes on tumor cells.

The high heterogeneity and mutation rate of cancer cells often lead to the failure of targeted therapy, and therefore, new targets for multitarget therapy of tumors are urgently needed. Aberrantly expressed glycosaminoglycans (GAGs) have been shown to be involved in tumorigenesis and are promising new targets. Recently, the GAG-binding domain rVAR2 of the Plasmodium falciparum VAR2CSA protein was identified as a probe targeting cancer-associated chondroitin sulfate A-like epitopes. In this study, we found that rVAR2 could also bind to heparin (Hep) and chondroitin sulfate E. Therefore, we used rVAR2 as a model to establish a method based on random mutagenesis of the GAG-binding protein and phage display to identify and optimize probes targeting tumor GAGs. We identified a new probe, VAR2HP, which selectively recognized Hep by interacting with unique epitopes consisting of a decasaccharide structure that contains at least three HexA2S(1-4)GlcNS6S disaccharides. Moreover, we found that these Hep-like epitopes were overexpressed in various cancer cells. Most importantly, our in vivo experiments showed that VAR2HP had good biocompatibility and preferentially localizes to tumors, which indicates that VAR2HP has great application potential in tumor diagnosis and targeted therapy. In conclusion, this study provides a strategy for the discovery of novel tumor-associated GAG epitopes and their specific probes.

Hypomethylation of GDNF family receptor alpha 1 promotes epithelial-mesenchymal transition and predicts metastasis of colorectal cancer.

Tumor metastasis is the major cause of poor prognosis and mortality in colorectal cancer (CRC). However, early diagnosis of highly metastatic CRC is currently difficult. In the present study, we screened for a novel biomarker, GDNF family receptor alpha 1 (GFRA1) based on the expression and methylation data in CRC patients from The Cancer Genome Altlas (TCGA), followed by further analysis of the correlation between the GFRA1 expression, methylation, and prognosis of patients. Our results show DNA hypomethylation-mediated upregulation of GFRA1 in invasive CRC, and it was found to be correlated with poor prognosis of CRC patients. Furthermore, GFRA1 methylation-modified sequences were found to have potential as methylation diagnostic markers of highly metastatic CRC. The targeted demethylation of GFRA1 by dCas9-TET1CD and gRNA promoted CRC metastasis in vivo and in vitro. Mechanistically, demethylation of GFRA1 induces epithelial-mesenchymal transition (EMT) by promoting AKT phosphorylation and increasing c-Jun expression in CRC cells. Collectively, our findings indicate that GFRA1 hypomethylation can promote CRC invasion via inducing EMT, and thus, GFRA1 methylation can be used as a biomarker for the early diagnosis of highly metastasis CRC.

Host and gut microbial tryptophan metabolism and type 2 diabetes: an integrative analysis of host genetics, diet, gut microbiome and circulating metabolites in cohort studies.

OBJECTIVE: Tryptophan can be catabolised to various metabolites through host kynurenine and microbial indole pathways. We aimed to examine relationships of host and microbial tryptophan metabolites with incident type 2 diabetes (T2D), host genetics, diet and gut microbiota. METHOD: We analysed associations between circulating levels of 11 tryptophan metabolites and incident T2D in 9180 participants of diverse racial/ethnic backgrounds from five cohorts. We examined host genome-wide variants, dietary intake and gut microbiome associated with these metabolites. RESULTS: Tryptophan, four kynurenine-pathway metabolites (kynurenine, kynurenate, xanthurenate and quinolinate) and indolelactate were positively associated with T2D risk, while indolepropionate was inversely associated with T2D risk. We identified multiple host genetic variants, dietary factors, gut bacteria and their potential interplay associated with these T2D-relaetd metabolites. Intakes of fibre-rich foods, but not protein/tryptophan-rich foods, were the dietary factors most strongly associated with tryptophan metabolites. The fibre-indolepropionate association was partially explained by indolepropionate-associated gut bacteria, mostly fibre-using Firmicutes. We identified a novel association between a host functional LCT variant (determining lactase persistence) and serum indolepropionate, which might be related to a host gene-diet interaction on gut Bifidobacterium, a probiotic bacterium significantly associated with indolepropionate independent of other fibre-related bacteria. Higher milk intake was associated with higher levels of gut Bifidobacterium and serum indolepropionate only among genetically lactase non-persistent individuals. CONCLUSION: Higher milk intake among lactase non-persistent individuals, and higher fibre intake were associated with a favourable profile of circulating tryptophan metabolites for T2D, potentially through the host-microbial cross-talk shifting tryptophan metabolism toward gut microbial indolepropionate production.

Investigation of action pattern of a novel chondroitin sulfate/dermatan sulfate 4-O-endosulfatase.

Recently, a novel CS/DS 4-O-endosulfatase was identified from a marine bacterium and its catalytic mechanism was investigated further (Wang, W., et. al (2015) J. Biol. Chem.290, 7823-7832; Wang, S., et. al (2019) Front. Microbiol.10, 1309). In the study herein, we provide new insight about the structural characteristics of the substrate which determine the activity of this enzyme. The substrate specificities of the 4-O-endosulfatase were probed by using libraries of structure-defined CS/DS oligosaccharides issued from synthetic and enzymatic sources. We found that this 4-O-endosulfatase effectively remove the 4-O-sulfate of disaccharide sequences GlcUAß1-3GalNAc(4S) or GlcUAß1-3GalNAc(4S,6S) in all tested hexasaccharides. The sulfated GalNac residue is resistant to the enzyme when adjacent uronic residues are sulfated as shown by the lack of enzymatic desulfation of GlcUAß1-3GalNAc(4S) connected to a disaccharide GlcUA(2S)ß1-3GalNAc(6S) in an octasaccharide. The 3-O-sulfation of GlcUA was also shown to hinder the action of this enzyme. The 4-O-endosulfatase exhibited an oriented action from the reducing to the non-reducing whatever the saturation or not of the non-reducing end. Finally, the activity of the 4-O-endosulfatase decreases with the increase in substrate size. With the deeper understanding of this novel 4-O-endosulfatase, such chondroitin sulfate (CS)/dermatan sulfate (DS) sulfatase is a useful tool for exploring the structure-function relationship of CS/DS.

Circ_0001247 functions as a miR-1270 sponge to accelerate cervical cancer progression by up-regulating ZEB2 expression level.

BACKGROUND: There is increasing evidence that circular RNA (circRNA) disorders have an impact on the progression of various malignancies. The expression characteristics, function and underlying mechanism of circ_0001247 in cervical cancer (CC) have not been confirmed. METHODS: GSE147483 datasets of circRNAs expression in CC cell line and normal cervical cell line were retrieved from GEO database, and the circRNA with significant difference was selected; circ_0001247, miR-1270, and Zinc finger E-box binding homeobox 2 (ZEB2) expressions in CC tissues and cell lines were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) assay; cell counting kit-8 (CCK-8) assay and BrdU assay were applied to monitor the proliferative ability of CC cells; Transwell assay was conducted to examine the migration and invasion of CC cells, and flow cytometry was used to evaluate the apoptosis; Western blot assay was adopted to detect ZEB2 protein expressions; dual-luciferase report gene assay was used to verify the targeting relationship between circ_0001247 and miR-1270, and miR-1270 and the 3'UTR of ZEB2. RESULTS: Analysis of GSE147483 suggested that circ_0001247 could probably be an oncogenic circRNA in CC. Compared with that in adjacent tissues and normal cervical epithelial cells, circ_0001247 expression in CC tissues and cell lines was significantly increased; knocking down circ_0001247 expression could inhibit the proliferation and metastasis of CC cells, and promote apoptosis, while circ_0001247 overexpression worked oppositely; circ_0001247 sponged miR-1270 in CC cells; miR-1270 diminished the promoting effect of circ_0001247 by inactivating the ZEB2. CONCLUSION: Circ_0001247 promotes progression of CC by sponging miR-1270 to upregulate ZEB2 expression level.

Temporal DNA methylation pattern and targeted therapy in colitis-associated cancer.

DNA methylation plays a crucial role in the pathogenesis of various diseases, including colorectal cancer (CRC). However, the global and temporal DNA methylation pattern during initiation and progression of colitis-associated cancer (CAC) are still unknown, including the potential therapeutic strategy of targeting methylation for CAC. In the present study, the global DNA methylation pattern was determined at different time points during CAC using DNA methylation sequencing, followed by the Starburst plot integrating alterations and potential functional prediction analysis. After demonstrating the regulatory role of DNA methyltransferases (DNMTs) on the expression of hub-genes in CRC cells, DNMT inhibitors were administered to treat CAC mice. Our results indicated that 811 genes were hypermethylated at different time points during initiation and progression of CAC. Genes that were downregulated and hypermethylated during CAC, including hub-genes BAD and inositol polyphosphate phosphatase-like 1 (INPPL1), were involved in MAPK signaling pathways, kit receptor signaling pathways, apoptosis and EGF/EGFR signaling pathways. Upregulated DNMTs (DNMT1, DNMT3A and DNMT3B) mediated downregulation and hypermethylation of BAD and INPPL1 in CAC and CRC cells. Low doses of DNMT inhibitors (decitabine (DAC) and azacitidine (AZA)) exerted efficient antitumor effects in CAC, accompanied with upregulation of BAD and INPPL1 expression, and apoptosis induction. In summary, the present study demonstrates the temporal DNA methylation pattern during CAC and provides a novel therapeutic strategy for treating this disease.

A chondroitin sulfate and hyaluronic acid lyase with poor activity to glucuronyl 4,6-O-disulfated N-acetylgalactosamine (E-type)-containing structures.

GlcUAß1-3GalNAc(4S,6S) (E unit)-rich domains have been shown to play key roles in various biological functions of chondroitin sulfate (CS). However, an enzyme that can specifically isolate such domains through the selective digestion of other domains in polysaccharides has not yet been reported. Here, we identified a glycosaminoglycan lyase from a marine bacterium Vibrio sp. FC509. This enzyme efficiently degraded hyaluronic acid (HA) and CS variants, but not E unit-rich CS-E, into unsaturated disaccharides; therefore, we designated this enzyme a CS-E-resisted HA/CS lyase (HCLase Er). We isolated a series of resistant oligosaccharides from the final product of a low-sulfated CS-E exhaustively digested by HCLase Er and found that the E units were dramatically accumulate in these resistant oligosaccharides. By determining the structures of several resistant tetrasaccharides, we observed that all of them possessed a Δ4,5HexUAα1-3GalNAc(4S,6S) at their non-reducing ends, indicating that the disulfation of GalNAc abrogates HCLase Er activity on the ß1-4 linkage between the E unit and the following disaccharide. Δ4,5HexUAα1-3GalNAc(4S,6S)ß1-4GlcUAß1-3GalNAc(4S,6S) was most strongly resistant to HCLase Er. To our knowledge, this study is the first reporting a glycosaminoglycan lyase specifically inhibited by both 4-O- and 6-O-sulfation of GalNAc. Site-directed and truncation mutagenesis experiments indicated that HCLase Er may use a general acid-base catalysis mechanism and that an extra domain (Gly739-Gln796) is critical for its activity. This enzyme will be a useful tool for structural analyses and for preparing bioactive oligosaccharides of HA and CS variants, particularly from E unit-rich CS chains.

Synthesis and Enhanced Photocatalytic Activity of Porous SrTiO3/TiO2 Composites.

Porous photocatalysts have attracted significant attention for their large specific surface area, numerous surface catalytic active sites, and high photocatalytic activity. In this study, porous SrTiO3/TiO2 composites were successfully fabricated through a hydrothermal approach utilizing porous TiO2 as a substrate. The as-synthesized SrTiO3/TiO2 composites were then characterized by X-ray diffraction, scanning electron microscopy (SEM), transmission electron microscopy, Brunauer-Emmett-Teller (BET), and ultraviolet-visible spectroscopy (UV-Vis) analysis. The results of SEM and BET indicate that such composites have a porous structure and large surface area. Compared to unadulterated TiO2, SrTiO3 /TiO2 composites exhibit higher photocatalytic performance for the photodegradation of rhodamine B under UV-Vis irradiation. Additionally, it was found that when the content of SrTiO3 reaches 20%, it achieves the maximum photodegradation efficiency of 98.6% under UV-Vis irradiation over 60 min. These results demonstrate that SrTiO3/TiO2 composites are a promising material in terms of environmental cleanliness.

Sequencing of chondroitin sulfate oligosaccharides using a novel exolyase from a marine bacterium that degrades hyaluronan and chondroitin sulfate/dermatan sulfate.

Glycosaminoglycans (GAGs) are a family of chemically heterogeneous polysaccharides that play important roles in physiological and pathological processes. Owing to the structural complexity of GAGs, their sophisticated chemical structures and biological functions have not been extensively studied. Lyases that cleave GAGs are important tools for structural analysis. Although various GAG lyases have been identified, exolytic lyases with unique enzymatic property are urgently needed for GAG sequencing. In the present study, a putative exolytic GAG lyase from a marine bacterium was recombinantly expressed and characterized in detail. Since it showed exolytic lyase activity toward hyaluronan (HA), chondroitin sulfate (CS), and dermatan sulfate (DS), it was designated as HCDLase. This novel exolyase exhibited the highest activity in Tris-HCl buffer (pH 7.0) at 30°C. Especially, it showed a specific activity that released 2-aminobenzamide (2-AB)-labeled disaccharides from the reducing end of 2-AB-labeled CS oligosaccharides, which suggest that HCDLase is not only a novel exolytic lyase that can split disaccharide residues from the reducing termini of sugar chains but also a useful tool for the sequencing of CS chains. Notably, HCDLase could not digest 2-AB-labeled oligosaccharides from HA, DS, or unsulfated chondroitin, which indicated that sulfates and bond types affect the catalytic activity of HCDLase. Finally, this enzyme combined with CSase ABC was successfully applied for the sequencing of several CS hexa- and octasaccharides with complex structures. The identification of HCDLase provides a useful tool for CS-related research and applications.

Hyaluronidase and Chondroitinase.

Glycosaminoglycans (GAGs) are important constituents of the extracellular matrix that make significant contributions to biological processes and have been implicated in a wide variety of diseases. GAG-degrading enzymes with different activities have been found in various animals and microorganisms, and they play an irreplaceable role in the structure and function studies of GAGs. As two kind of important GAG-degrading enzymes, hyaluronidase (HAase) and chondroitinase (CSase) have been widely studied and increasing evidence has shown that, in most cases, their substrate specificities overlap and thus the "HAase" or "CSase" terms may be improper or even misnomers. Different from previous reviews, this article combines HAase and CSase together to discuss the traditional classification, substrate specificity, degradation pattern, new resources and naming of these enzymes.

Cloning and characterization of a novel chondroitin sulfate/dermatan sulfate 4-O-endosulfatase from a marine bacterium.

Sulfatases are potentially useful tools for structure-function studies of glycosaminoglycans (GAGs). To date, various GAG exosulfatases have been identified in eukaryotes and prokaryotes. However, endosulfatases that act on GAGs have rarely been reported. Recently, a novel HA and CS lyase (HCLase) was identified for the first time from a marine bacterium (Han, W., Wang, W., Zhao, M., Sugahara, K., and Li, F. (2014) J. Biol. Chem. 289, 27886-27898). In this study, a putative sulfatase gene, closely linked to the hclase gene in the genome, was recombinantly expressed and characterized in detail. The recombinant protein showed a specific N-acetylgalactosamine-4-O-sulfatase activity that removes 4-O-sulfate from both disaccharides and polysaccharides of chondroitin sulfate (CS)/dermatan sulfate (DS), suggesting that this sulfatase represents a novel endosulfatase. The novel endosulfatase exhibited maximal reaction rate in a phosphate buffer (pH 8.0) at 30 °C and effectively removed 17-65% of 4-O-sulfates from various CS and DS and thus significantly inhibited the interactions of CS and DS with a positively supercharged fluorescent protein. Moreover, this endosulfatase significantly promoted the digestion of CS by HCLase, suggesting that it enhances the digestion of CS/DS by the bacterium. Therefore, this endosulfatase is a potential tool for use in CS/DS-related studies and applications.

A novel eliminase from a marine bacterium that degrades hyaluronan and chondroitin sulfate.

Lyases cleave glycosaminoglycans (GAGs) in an eliminative mechanism and are important tools for the structural analysis and oligosaccharide preparation of GAGs. Various GAG lyases have been identified from terrestrial but not marine organisms even though marine animals are rich in GAGs with unique structures and functions. Herein we isolated a novel GAG lyase for the first time from the marine bacterium Vibrio sp. FC509 and then recombinantly expressed and characterized it. It showed strong lyase activity toward hyaluronan (HA) and chondroitin sulfate (CS) and was designated as HA and CS lyase (HCLase). It exhibited the highest activities to both substrates at pH 8.0 and 0.5 m NaCl at 30 °C. Its activity toward HA was less sensitive to pH than its CS lyase activity. As with most other marine enzymes, HCLase is a halophilic enzyme and very stable at temperatures from 0 to 40 °C for up to 24 h, but its activity is independent of divalent metal ions. The specific activity of HCLase against HA and CS reached a markedly high level of hundreds of thousands units/mg of protein under optimum conditions. The HCLase-resistant tetrasaccharide Δ(4,5)HexUAα1-3GalNAc(6-O-sulfate)ß1-4GlcUA(2-O-sulfate)ß1-3GalNAc(6-O-sulfate) was isolated from CS-D, the structure of which indicated that HCLase could not cleave the galactosaminidic linkage bound to 2-O-sulfated d-glucuronic acid (GlcUA) in CS chains. Site-directed mutagenesis indicated that HCLase may work via a catalytic mechanism in which Tyr-His acts as the Brønsted base and acid. Thus, the identification of HCLase provides a useful tool for HA- and CS-related research and applications.

Supercharged fluorescent protein as a versatile probe for the detection of glycosaminoglycans in vitro and in vivo.

Glycosaminoglycans (GAGs) are linear acidic heteropolysaccharides that are ubiquitously expressed in animal tissues and participate in various life processes. To date, the detection and visualization of GAGs in complex biological samples and living organisms remain a challenge because of the lack of powerful biocompatible probes. In this study, a superpositively charged green fluorescent protein (ScGFP) was shown great potential in GAG detection for the first time. First, on the basis of the phenomenon of GAGs dose-dependently inhibiting the fluorescence quenching of ScGFP by graphene oxide, a simple and highly sensitive signal-on homogeneous platform was established for detecting and quantifying GAGs, even in complex samples such as heparin in citrated plasma and oversulfated chondroitin sulfate in heparin. Furthermore, ScGFP with excellent stability and biocompatibility could be easily used as a highly sensitive and selective probe to visualize different types of GAGs in vitro and in vivo through combination with specific GAG-degrading enzymes. This study introduces a versatile probe for GAG detection, which is easy to prepare and which shows a high practical value in basic research and medical applications.

Astragaloside IV protects against lung injury and pulmonary fibrosis in COPD by targeting GTP-GDP domain of RAS and downregulating the RAS/RAF/FoxO signaling pathway.

BACKGROUND: Pulmonary fibrosis is a chronic progressive interstitial lung disease characterized by the replacement of lung parenchyma with fibrous scar tissue, usually as the final stage of lung injury like COPD. Astragaloside IV (AST), a bioactive compound found in the Astragalus membranaceus (Fisch.) used in traditional Chinese medicine, has been shown to improve pulmonary function and exhibit anti-pulmonary fibrosis effects. However, the exact molecular mechanisms through which it combats pulmonary fibrosis, especially in COPD, remain unclear. PURPOSE: This study aimed to identify the potential therapeutic target and molecular mechanisms for AST in improving lung injury especially treating COPD type pulmonary fibrosis both in vivo and in vitro. METHODS: Multi lung injury models were established in mice using lipopolysaccharide (LPS), cigarette smoke (CS), or LPS plus CS to simulate the processes of pulmonary fibrosis in COPD. The effect of AST on lung function protection was evaluated, and proteomic and metabolomic analysis were applied to identify the signaling pathway affected by AST and to find potential targets of AST. The interaction between AST and wild-type and mutant RAS proteins was studied. The RAS/RAF/FoxO signaling pathway was stimulated in BEAS-2B cells and in mice lung tissues by LPS plus CS to investigate the anti-pulmonary fibrosis mechanism of AST analyzed by western blotting. The regulatory effects of AST on the RAS/RAF/FoxO pathway dependent on RAS were further confirmed using RAS siRNA. RESULTS: RAS was predicted and identified as the target protein of AST in anti-pulmonary fibrosis in COPD and improving lung function. The administration of AST was observed to impede the conversion of fibroblasts into myofibroblasts, reduce the manifestation of inflammatory factors and extracellular matrix, and hinder the activation of epithelial mesenchymal transition (EMT). Furthermore, AST significantly suppressed the RAS/RAF/FoxO signaling pathway in both in vitro and in vivo settings. CONCLUSION: AST exhibited lung function protection and anti-pulmonary fibrosis effect by inhibiting the GTP-GDP domain of RAS, which downregulated the RAS/RAF/FoxO signaling pathway. This study revealed AST as a natural candidate molecule for the protection of pulmonary fibrosis in COPD.

A novel 4-O-endosulfatase with high potential for the structure-function studies of chondroitin sulfate/dermatan sulfate.

The sulfation patterns of chondroitin sulfate (CS)/dermatan sulfate (DS), which encode unique biological information, play critical roles in the various biological functions of CS/DS chains. CS/DS sulfatases, which can specifically hydrolyze sulfate groups, could potentially be essential tools for deciphering and changing the biological information encoded by these sulfation patterns. However, endosulfatase with high activity to efficiently hydrolyze the sulfate groups inside CS/DS polysaccharides have rarely been identified, which hinders the practical applications of CS/DS sulfatases. Herein, a novel CS/DS 4-O-endosulfatase (endoBI4SF) with a strong ability to completely remove 4-O-sulfated groups inside various CS/DS polysaccharides was identified and successfully used to investigate the biological roles of 4-O-sulfated CS/DS in vitro and in vivo. This study provides a much-needed tool to tailor the sulfation patterns and explore the related functions of 4-O-sulfated CS/DS chains in vitro and in vivo.

Data on cloning, expression and biochemical characteristics of a chondroitin sulfate/dermatan sulfate 4-O-endosulfatase.

The data shown in this article are related to the published paper entitled "A novel 4-O-endosulfatase with high potential for the structure-function studies of chondroitin sulfate/dermatan sulfate" in Carbohydrate Polymers. In this article, the phylogenetic analysis, cloning, expression, purification, specificity and biochemical characteristics of the identified chondroitin sulfate/dermatan sulfate 4-O-endosulfatase (endoBI4SF) are described in detail. The recombinant endoBI4SF with a molecular mass of 59.13 kDa can can specifically hydrolyze the 4-O- but not 2-O- and 6-O-sulfate groups in the oligo-/polysaccharides of chondroitin sulfate/dermatan sulfate and show the maximum reaction rate in 50 mM Tris-HCl buffer (pH 7.0) at 50°C, which can be a very useful tool for the structural and functional studies of chondroitin sulfate/dermatan sulfate.