Heparin is a very important anticoagulant drug. Currently, heparin is mainly extracted from porcine mucosa. However, animal-derived heparin shows low anticoagulant activity due to the low proportion of the anticoagulant active unit, the GlcNS6S-GlcA-GlcNS6S3S-Ido2S-GlcNS6S pentasaccharide. In this study we proposed an enzymatic strategy to sulfate the animal-sourced heparin to increase the proportion of anticoagulant pentasaccharide and the anticoagulant activity. First, three sulfotransferases HS2ST, HS6ST, and HS3ST were expressed tentatively in Escherichia coli and Pichia pastoris. After measuring the sulfotransferase activity, we confirmed P. pastoris GS115 is the better host for sulfotransferases production. Then, the maltose binding protein (MBP) and thioredoxin (TrxA) were fused separately to the N-terminal of sulfotransferases to increase enzyme solubility. As a result, the yields of HS2ST and HS6ST were increased to (839±14) U/L and (792±23) U/L, respectively. Subsequent sulfation of the animal-sourced heparin with the recombinant HS2ST, HS6ST and HS3ST increased the anticoagulant activity from (76±2) IU/mg to (189±17) IU/mg.
Animals ; Escherichia coli ; Heparin ; chemistry ; Oligosaccharides ; chemistry ; Pichia ; Sulfotransferases ; biosynthesis ; Swine

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is an idiopathic chronic, progressive interstitial lung disease with a diagnosed median survival of 3-5 years. IPF is associated with an increased risk of lung cancer. Therefore, exploring the shared pathogenic genes and molecular pathways between IPF and lung adenocarcinoma (LUAD) holds significant importance for the development of novel therapeutic approaches and personalized precision treatment strategies for IPF combined with lung cancer. METHODS: Bioinformatics analysis was conducted using publicly available gene expression datasets of IPF and LUAD from the Gene Expression Omnibus (GEO) database. Weighted gene co-expression network analysis was employed to identify common genes involved in the progression of both diseases, followed by functional enrichment analysis. Subsequently, additional datasets were used to pinpoint the core shared genes between the two diseases. The relationship between core shared genes and prognosis, as well as their expression patterns, clinical relevance, genetic characteristics, and immune-related functions in LUAD, were analyzed using The Cancer Genome Atlas (TCGA) database and single-cell RNA sequencing datasets. Finally, potential therapeutic drugs related to the identified genes were screened through drug databases. RESULTS: A total of 529 shared genes between IPF and LUAD were identified. Among them, SULF1 emerged as a core shared gene associated with poor prognosis. It exhibited significantly elevated expression levels in LUAD tissues, concomitant with high mutation rates, genomic heterogeneity, and an immunosuppressive microenvironment. Subsequent single-cell RNA-seq analysis revealed that the high expression of SULF1 primarily originated from tumor-associated fibroblasts. This study further demonstrated an association between SULF1 expression and tumor drug sensitivity, and it identified potential small-molecule drugs targeting SULF1 highly expressed fibroblasts. CONCLUSIONS This study identified a set of shared molecular pathways and core genes between IPF and LUAD. Notably, SULF1 may serve as a potential immune-related biomarker and therapeutic target for both diseases.
Humans ; Lung Neoplasms/genetics* ; Adenocarcinoma of Lung/genetics* ; Idiopathic Pulmonary Fibrosis/genetics* ; Adenocarcinoma ; Cancer-Associated Fibroblasts ; Prognosis ; Tumor Microenvironment ; Sulfotransferases

OBJECTIVETo investigate the effect of HS3ST3B1 on hepatitis B virus (HBV) replication.

METHODSHepG2 cells were classified into 7 groups according to the plasmids transfected: (1) Blank group, no plasmid transfected; 2. Positive control, transfected with pCH9-HBV which permits HBV replication; (3) Negative control, transfected with pCH9-HBV + pcDNA3.1 + pTZU6+1; (4) Treatment A, transfected with pCH9-HBV + pCDNA3.1-HS3ST3B1 + pTZU6+1; (5) Interference A, transfected with pCH9-HBV + pCDNA3.1-HS3ST3B1 + psh1126 (a plasmid to interfere HS3ST3B1 expression); (6) Treatment B, transfected with pCH9-HBV + pTZU6+1; (7) Interference B, transfected with pCH9-HBV + psh1126. The levels of HBV DNA were detected in the above groups by Southern blotting. HBV total RNA of Negative control, Treatment A and Interference A were quantified by Real-time PCR to determine the influence of HS3ST3B1 over-expression on the HBV RNA transcription. The activity of the four HBV promoters [core promoter (cp), x promoter(xp), surface antigen promoter1(sp1), surface antigen promoter2 (sp2)] were assayed by Dual-Luciferase Reporter Assay System. The data was analyzed using one way ANOVA, with P < 0.05 indicating statistically meaningful difference.

RESULTSouthern blot data revealed the level of HBV DNA in Treatment A and Interference A accounted for 10% +/- 2% and 31% +/- 4% of that in control. Compared with control, a statistical difference existed between Treatment A and Control, with F value equalling to 20.8 and P value equalling to 0.034 respectively. A statistical difference also existed between Interfere A and Treatment A, with F value equalling to 24.9 and P value equalling to 0.021 respectively. The level of HBV DNA in Experiment B was raised by 130% +/- 11% as compared to that in Interference B, and the levels of HBV DNA showed a dose-dependent decrease when H7 cells were transfected with 0.5, 1.0, 1.5 microg pCDNA3.1-HS3ST3B1 respectively. Statistical differences existed between control and H7 transfected with different dose of pCDNA3.1-HS3ST3B1, with F values equalling to 22.7, 20.3, 26.5 and P values equalling to 0.029, 0.041 and 0.015 respectively. Real-time PCR revealed that the HBV total RNA in Treatment A accounted for 17.0% +/- 2.7% of that in control and there was a statistical difference between Treatment A and control, with F value equalling to 25.6 and P value equalling to 0.018. In addition, HBV DNA in Interference A was restored to 74.0% +/- 3.9% of that in control, and there was also a statistical difference between Treatment A and Interference A, with F value equalling to 21.3 and P value equalling to 0.032. However, the down regulation of HBV total RNA had nothing to do with HBV promoters activity.

CONCLUSIONHS3ST3B1 can inhibit HBV replication and reduce the level of HBV total RNA, but the downregulation of HBV total RNA may not be the result of direct interaction of HS3ST3B1 and HBV promoters.

DNA Replication ; DNA, Viral ; biosynthesis ; Hep G2 Cells ; Hepatitis B virus ; genetics ; physiology ; Humans ; Plasmids ; Sulfotransferases ; genetics ; Transfection ; Virus Replication

The paper summarizes the interactions between luteolin (glucosides) and drug-metabolizing enzyme from the literature of recent years and our research work. The metabolism of luteolin is chiefly mediated by phase II metabolic enzyme. Its glucosides are firstly hydrolyzed into aglycone in intestinal tract, and then absorbed and metabolized. Luteolin has the effect on the induction of CYP3A, and on the inhibition of CYPIA, 1B and 2E. Also, luteolin is an effective inhibitor of CYP2B6, CYP2C9 and CYP2D6. Luteolin can induce and inhibit UGTs and SULTs. It can also inhibit multi ABC transport proteins. Understanding the interactions between luteolin (glucosides) and drug-metabolizing enzyme has an important significance in guiding clinical use of the drug.
ATP-Binding Cassette Transporters ; metabolism ; Animals ; Aryl Hydrocarbon Hydroxylases ; metabolism ; Drug Interactions ; Enzyme Induction ; Glucuronosyltransferase ; metabolism ; Humans ; Luteolin ; metabolism ; Microsomes, Liver ; metabolism ; Sulfotransferases ; metabolism

Under the catalysis of a variety of metabolic enzymes in vivo, such as UDP-glucuronyl transferases, cytochrome P450, carboxylesterase, sulfotransferase, butyrylcholinesterase, catechol-O-methyl transferase and 6-morphine dehydrogenase, the drugs perform glucuronidation, hydrolysis, oxidation, sulfonation and other reactions, then translate into active or inactive metabolites, which are excreted through urination, bile or the other pathways at last. Different drugs own their different metabolic pathways. This paper introduces the studies about the metabolism of drugs in human and animal in recent years, such as morphine-like drugs, amphetamine, ketamine, cannabis and cocaine, and reviews the research progress about the sites of metabolism, metabolic enzymes, metabolites and physiological activity of those drugs metabolic in vivo.
Alcohol Oxidoreductases/metabolism* ; Animals ; Carboxylesterase/metabolism* ; Catechol O-Methyltransferase/metabolism* ; Cholinesterases/metabolism* ; Cytochrome P-450 Enzyme System/metabolism* ; Glucuronosyltransferase/metabolism* ; Humans ; Illicit Drugs/metabolism* ; Oxidation-Reduction ; Sulfotransferases/metabolism*

OBJECTIVETo investigate the expression of estrogen sulfotransferase (EST) in the mammary gland of hypertrophic breast and its significance.

METHODSEST expression in the mammary gland was detected by EnVision two step method of immunohistochemistry in 15 cases with normal breasts and 32 cases with hypertrophic breasts, including 19 gland-associated cases and 13 fat-associated cases.

RESULTSThe positive expression rate of EST in mammary gland was 34.4% (11/32) in hypertrophic group and 93.3% (14/15) in normal group, showing a significant difference between the two groups (P < 0.01). The positive expression rate of EST was 10.5% (2/19) in gland-associated group and 69.2% (9/ 13) in fat-associated group, showing a significant difference between the two groups (P < 0.01).

CONCLUSIONSDecrease or deletion of EST in the mammary gland may be related to the development of hypertrophic breast, especially gland-associated hypertrophic breast.

Adult ; Breast ; abnormalities ; enzymology ; Case-Control Studies ; Female ; Humans ; Hypertrophy ; enzymology ; Mammary Glands, Human ; enzymology ; Middle Aged ; Sulfotransferases ; metabolism ; Young Adult

OBJECTIVETo explore the relationship between SULF2 and WRN promoter methylation and chemosensitivity to irinotecan, and also the clinicopathological features in patients with gastric cancer.

METHODSThe chemosensitivity to irinotecan was tested by MTT assay. The methylation of SULF2 and WRN promoter in the fresh gastric cancer tissues was detected by methylation specific PCR. The differences of chemosensitivity and clinicopathological features of the methylation group were compared with that of the non-methylation group. The tumor growth in nude mice bearing human gastric cancer xenografts treated with CPT-11was also observed.

RESULTSThe methylation rates of SULF2 and WRN were 28.4% (29/102) and 23.5% (24/102), respectively. There were no significant association between promoter methylation and clinicopathological features of patients including age, gender, histologic type, lymphatic invasion, and TNM Stage. In all the 102 cases, there were 30 cases of irrinotecan-sensitive group, and 72 cases of the irrinotecan-resistant group. The SULF2 methylation rate was 46.7% (14/30)in the sensitive group, and 20.8% (15/72) in the resistant group (P = 0.008),The WRN methylation rate was 33.3% (10/30) in the sensitive group, and 19.4% (14/72) in the resistant group (P = 0.214). Gastric cancer tissues were more sensitive to irrinotecan when both the genes were methylated. The nude mice bearing human gastric cancer xenografts with SULF2 methylation were more sensitive to irrinotecan.

CONCLUSIONSThe detection of SULF2 and WRN promoter methylation may provide evidence for screening and targeting the most sensitive gastric cancer subpopulation suitable for personalized irrinotecan chemotherapy.

Antineoplastic Agents, Phytogenic ; pharmacology ; Camptothecin ; analogs & derivatives ; pharmacology ; DNA Methylation ; Exodeoxyribonucleases ; metabolism ; Humans ; Methylation ; Promoter Regions, Genetic ; RecQ Helicases ; metabolism ; Stomach Neoplasms ; metabolism ; Sulfotransferases ; metabolism ; Werner Syndrome Helicase

To report a novel mutation within the CHST6 gene, as well as describe light and electron microscopic features of a case of macular corneal dystrophy. A 59-year old woman with macular corneal dystrophy in both eyes who had decreased visual acuity underwent penetrating keratoplasty. Further studies including light and electron microscopy, as well as DNA analysis were performed. Light microscopy of the cornea revealed glycosaminoglycan deposits in the keratocytes and endothelial cells, as well as extracellularly within the stroma. All samples stained positively with alcian blue, colloidal iron, and periodic acid-Schiff. Electron microscopy showed keratocytes distended by membrane-bound intracytoplasmic vacuoles containing electron-dense fibrillogranular material. These vacuoles were present in the endothelial cells and between stromal lamellae. Some of the vacuoles contained dense osmophilic whorls. A novel homozygous mutation (c.613 C>T [p.Arg205Trp]) was identified within the whole coding region of CHST6. A novel CHST6 mutation was detected in a Korean macular corneal dystrophy patient.
Corneal Dystrophies, Hereditary/diagnosis/*genetics/metabolism ; Corneal Keratocytes/ultrastructure ; DNA/*genetics ; DNA Mutational Analysis ; Female ; Humans ; Microscopy, Electron ; Middle Aged ; *Mutation, Missense ; Pedigree ; Polymerase Chain Reaction ; Republic of Korea ; Sulfotransferases/*genetics/metabolism

OBJECTIVETo investigate the role of sulfated tyrosine in regulating the activity of tyrosylprotein sulfotransferases (TPST) 1 and TPST2.

METHODSConstructs of TPST 1 and TPST2 were amplified by polymerase chain reaction (PCR), then fused into immunoglobulin G1 Fc region. All the variants in which sulfated tyrosines were mutated to phenylalanine were made by the PCR-based Quick Change method and confirmed by sequencing the entire reading frame. Small hairpin RNA (shRNA) constructs-targeting nucleotides 259-275 of TPST1 and nucleotides 73-94 of TPST2 were generated and subcloned into pBluescript. Human embryonic kidney (HEK) 293T cells were transfected with these plasmids. One day later, cells were split: one part was labeled with 35S-cysteine and methionine or 35S-Na2SO3 overnight, the second part was used for 125I labeled binding experiment, and the third part was retained for binding and flow cytometry.

RESULTSTyrosines at position 326 of TPST1 and position 325 of TPST2 were sulfated posttranslationally. Tyrosine sulfation of TPSTs was effectively inhibited by sulfation inhibitors, including specific shRNAs and non-specific NaCIO3. shRNAs reduced the sulfation of C3a receptor and C5a receptor, and partially blocked the binding of these two receptors to their respective ligands.

CONCLUSIONSThe activities of TPSTs were regulated by tyrosine sulfation. Inhibition of sulfotyrosine decreases the binding ability of C3a receptor and C5a receptor to their respective ligands.

Cell Line ; Complement C3a ; metabolism ; Complement C5a ; metabolism ; Humans ; Protein Binding ; Protein Processing, Post-Translational ; Receptor, Anaphylatoxin C5a ; metabolism ; Receptors, Complement ; metabolism ; Sulfotransferases ; genetics ; metabolism ; Transfection ; Tyrosine ; analogs & derivatives ; metabolism

In this study,liquiritigenin sulfonation was characterized using recombinant human sulfotransferases( SULTs). The chemical structure of liquiritigenin sulfate was determined by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry( UPLC-Q-TOF-MS/MS). Then model fitting and parameter estimation were performed using the Graphpad Prism V5 software. Various SULT enzymes( SULT1 A1,1 A2,1 A3,1 B1,1 C2,1 C4,1 E1 and 2 A1) were able to catalyze the formation of liquiritigenin-7-O-sulfate. Sulfonation of liquiritigenin-7-hydroxy( 7-OH) by these eight SULT enzymes consistently displayed the classical Michaelis-Menten profile. According to the intrinsic clearance( CLint) value,the sulfonation rates of liquiritigenin-7-OH by expressed SULT enzymes followed the following rank order: SULT1 C4 > SULT1 A3 > SULT1 E1 > SULT1 A1 > SULT1 A2 > SULT1 B1 >SULT1 C2>SULT2 A1. Further,liquiritigenin-7-O-sulfonation was significantly correlated with the SULT1 A3 protein levels( P<0. 05).Then,human embryonic kidney( HEK) 293 cells over expressing SULT1 A3( named as HEK-SULT1 A3 cells) were conducted. As a result,liquiritigenin-7-O-sulfate( L-7-S) was rapidly generated upon incubation of the cells with liquiritigenin. Consistent with SULT1 A3,sulfonation of liquiritigenin-7-OH in HEK-SULT1 A3 cells also followed the Michaelis-Menten kinetics. The derived Vmaxvalues was( 0. 315±0. 009) μmol·min-1·g-1,Kmwas( 7. 04±0. 680) μmol·L^-1,and CLintwas( 0. 045±0. 005) L·min^-1·g^-1. Moreover,the sulfonation characters of liquiritigenin( 7-OH) in SULT1 A3 were strongly correlated with that in HEK-SULT1 A3 cells( P<0. 001).The results indicated that HEK-SULT1 A3 cells have shown the catalytic function of SULT1 A3 enzymes. In conclusion,liquiritigenin was subjected to efficient sulfonation,and SULT1 A3 enzyme plays an important role in the sulfonation of liquiritigenin-7-OH. Significant sulfonation should be the main reason for the low bioavailability of liquiritigenin. In addition,HEK-SULT1 A3 cells were conducted and successfully used to evaluate liquiritigenin sulfonation,which will provide an appropriate tool to accurately depict the sulfonation disposition of liquiritigenin in vivo.
Arylsulfotransferase ; Flavanones/metabolism* ; Humans ; Tandem Mass Spectrometry