Serum-based Comprehensive N-Glycans Profiling Analysis in Different Gastric Disease Stages by Porous Graphitic Carbon Liquid Chromatography-Mass Spectrometry Associated With Potential Marker Discovery.

BACKGROUND/AIM: N-glycans are potential serum biomarkers due to their aberrant structure and abundance alteration during disease progression. Few studies have been associated with relative quantitative N-glycans profiling during different gastric disease stages. In this study, we conducted an investigation on the profiling of N-glycans in patients with gastric disease, as well as in healthy controls. MATERIALS AND METHODS: In this study, the porous graphitization carbon chromatography-high resolution Fourier transform mass spectrometry (PGC-FTMS) method was applied to assess comprehensive N-glycans profiling in patients at different stages of gastric disease, including gastritis, atrophic gastritis, gastric ulcer, gastric polyps, and gastric cancer. RESULTS: A total of 45 N-glycans (relative abundance >0.1%) were detected, and 9 N-glycans were found to be potential biomarkers for gastric disease detection. Along with the progression of gastric disease, the abundance of sialylated N-glycans increased, while that of core-fucosylated N-glycans decreased. Multivariate statistical analysis demonstrated that N-glycans profiling between gastritis and healthy controls had significant differences. The characteristic N-glycans distinguished gastric cancer from healthy controls, which had strong clinical diagnostic value. CONCLUSION: The relative quantitative profile of N-glycans in different gastric disease stages was revealed and serum N-glycans are proposed for distinguishing gastric disease stages in clinical application.

Targeting lectin-like oxidized low-density lipoprotein receptor-1 triggers autophagic program in esophageal cancer.

Autophagy is a highly conserved catabolic process to maintain cellular homeostasis. However, dysfunctional autophagy contributes to a context-dependent role in cancer. Here, we clarified the exact role of autophagy modulated by the scavenger receptor lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) in esophageal cancer (EC). A comprehensive analysis in various cancers displayed that LOX-1 was upregulated the most in EC tissues and associated with poor prognosis of patients. Deletion of LOX-1 ex vivo and in vivo suppresses EC development by inducing autophagic cell death. Receptor for activated C kinase 1 (RACK1) was identified as a signal adapter of LOX-1, which incented RAS/MEK/ERK pathway and TFEB nuclear export signal and safeguarded tumorigenesis. A sulfated polysaccharide fucoidan extracted from brown seaweed was found to bind with LOX-1 and mediate its proteasomal degradation but not the lysosome pathway, leading to autophagy-related cell death in EC. These results reveal a central contribution of LOX-1 to EC development and provide genetic ablation or bioactive polysaccharide as an effective intervention for EC therapy.

Highly sialylated mucin-type glycopeptide from porcine intestinal mucosa after heparin extraction: O-glycan profiling and immunological activity evaluation.

Mucins are the major proteins that distributed on the intestinal mucosa layer and protect the intestine from pathogens infection. The composition of intestinal mucin O-glycans can affect the health of the gastrointestinal tract in pigs. Porcine intestinal mucosa is widely used as the main raw material of heparin extraction. The heparin extraction residues rich in mucins were usually wasted. The structure of mucin derived O-glycans in porcine intestinal mucosa are currently unknown. In this study, we isolated the mucins from the heparin extraction residues and profiled the O-glycans. After heparin extraction, mucin was digested with trypsin, and separated by strong anion exchange chromatography. The mucin derived O-glycans were release by alkaline ß elimination, and analyzed by ultra high performance liquid chromatography-porous graphitized carbon-Fourier transform mass spectrometry (UPLC-PGC-FTMS/MS). Thirty five kinds of O-glycans were identified, most of which were Core 3-derived glycans. In particular, the O-glycans containing sialic acid Neu5Ac accounted for 71.93% of the total O-glycans, which were different from that of other species, including mouse intestine, fish intestine, and porcine colon. The high content sialylated mucin may explain its effect in biological processes. Furthermore, the immunological activity results indicated that the porcine intestinal mucin could promote phagocytosis and proliferation without any cytotoxic effects, which may aid in the development of immunomodulators.

Two different fucosylated chondroitin sulfates: Structural elucidation, stimulating hematopoiesis and immune-enhancing effects.

Two fucosylated chondroitin sulfates FCShp and FCSht were isolated from the sea cucumber Holothuria polii and Holothuria tubulosa, respectively. The NMR spectroscopy and HILIC-FTMS methods were applied for their detailed structural characterization. Chemical analysis indicated that the two FCSs all contained a chondroitin sulfate backbone chondroitin sulfate-like core and fucosyl branches of α-L-Fuc2,4S, α-L-Fuc4S or α-L-Fuc3,4S linked to O-3 of glucuronic acid residues. The main branches of FCShp and FCSht were monofucose, and the small amounts of di-, tri- and tetrafucose with α-1,3-linkage type were also detected. Finally, we investigated the immunomodulatory function of FCShp and FCSht in cyclophosphamide (CTX)-induced immunosuppressed mouse models. The results showed that FCShp and FCSht had beneficial effects on hematopoietic function recovery in CTX-induced bone marrow suppression mice. Notably, the α-L-Fuc2,4S was more important to the activity than α-L-Fuc3,4S. These results provided basis for developing the drugs to reduce side effects of chemotherapy.

Anti-diabetic activities of agaropectin-derived oligosaccharides from Gloiopeltis furcata via regulation of mitochondrial function.

The aim of the present study was to investigate whether agaropectin-derived oligosaccharides from Gloiopeltis furcata (SAOs) exert an anti-diabetic effect in sodium palmitate (PA)-induced insulin resistant HepG2 cells. We found that SAOs were co-localized with mitochondria and regulated mitochondrial function. SAOs reduced respiratory chain activities, which led to reduced respiratory oxygen consumption and increased the cellular ADP/ATP ratio in a certain degree of dose-dependent manner. Thus, SAOs alleviated the oxidative stress state in PA-treated cells and, moreover, concurrently regulated the ROS-JNK-IRS-1 pathway. As a result, SAOs enhanced insulin sensitivity and glucose metabolism by activating the IRS-1-AKT-GSK-3ß-GS pathway. Additionally, SAOs activated AMPK through both PKA-LKB1 and mitochondrial-regulated energy metabolism pathways. Therefore, SAOs decreased accumulation of lipids and improved lipid metabolism via regulating HMGCR, ACC and SREBP-1 proteins in HepG2 cells. Taken together, we conclude that SAOs could significantly ameliorate diabetic states in vitro via regulating mitochondria and their downstream signaling pathways.

Fucoidan from sea cucumber Holothuria polii: Structural elucidation and stimulation of hematopoietic activity.

The structural elucidation of polysaccharides is essential for understanding their structure-bioactivity relationship and related drug development. In this study, fucoidan (Fuchp) was extracted and purified from sea cucumber Holothuria polii. Its sulfate content was 39.5 ± 1.4%, and the "weight-average" molecular mass was 103.1 ± 2.8 kDa. The primary structure of Fuchp was clarified using a combination of acid degradation, tandem mass spectrometry, and nuclear magnetic resonance spectroscopy analysis. As a result, Fuchp was found to be composed of a tetrafucose repeating unit [→3-α-l-Fucp-1 â†’ 3-α-l-Fucp2(OSO3-)-1 â†’ 3-α-l-Fucp2(OSO3-)-1 â†’ 3-α-l-Fucp2,4(OSO3-)-1→]. The stimulating hematopoiesis was further evaluated in a mouse model induced by cyclophosphamide. Based on these findings, intraperitoneally administered Fuchp may accelerate the recovery of white blood cells and neutrophils, in which its activity exceeded that of recombinant human granulocyte colony-stimulating factor (rhG-CSF). Meanwhile, in the background of cyclophosphamide-induced immunosuppression, treatment with Fuchp reduces platelet aggregation caused by CTX, so it might have the effect of reducing the risk of thrombosis. Therefore, Fuchp can be exploited as potentially promising stimulator of hematopoiesis in the future.

Anti-Metabolic Syndrome Effects of Fucoidan from Fucus vesiculosus via Reactive Oxygen Species-Mediated Regulation of JNK, Akt, and AMPK Signaling.

Recent studies have reported that dietary fiber improved metabolic syndrome (MetS). However, the effects of fucoidans on MetS were still not clear. In this study, we evaluated the activity of fucoidan from Fucus vesiculosus (FvF) on attenuating MetS and first elucidated the underlying mechanism. In vitro, FvF treatment remarkably lowered the level of reactive oxygen species (ROS) compared with the sodium palmitate (PA)-induced insulin resistance (IR) group. The phosphorylation level of c-Jun N-terminal kinase (JNK) was significantly decreased, while phosphorylation of protein kinase B (pAkt) level increased, compared with that of the HepG2 cells treated with PA. Thus, FvF increased glucose consumption and relieved IR via ROS-mediated JNK and Akt signaling pathways. In addition, these changes were accompanied by the activation of adenosine 5'-monophosphate-ativated protein kinase (AMPK) and its downstream targets (e.g., HMG-CoA reductase (HMGCR), acetyl-CoA carboxylase (ACC), and sterol-regulatory element-binding protein-1c (SREBP-1C)), which improved lipid metabolism in IR HepG2 cells. In vivo, FvF improved hyperglycemia and decreased serum insulin level in mice with MetS. Furthermore, we evaluated the inhibition of glucose transport by in vitro (Caco-2 monolayer model), semi-in vivo (everted gut sac model) and oral glucose tolerance test (OGTT), which indicated that FvF could significantly reduce the absorption of glucose into the blood stream, thus it could improve blood-glucose levels and IR in mice with MetS. Moreover, FvF decreased serum triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) levels and liver lipid accumulation, while increased the serum high density lipoprotein cholesterol (HDL-C) level in mice with MetS. Therefore, FvF could be considered as a potential candidate for the treatment of MetS by alleviating IR, inhibiting glucose transportation, and regulating lipid metabolism.

Gangliosides profiling in serum of breast cancer patient: GM3 as a potential diagnostic biomarker.

Gangliosides altered during the pathological conditions and particularly in cancers. Here, we aimed to profile the gangliosides in breast cancer serum and propose potential biomarkers. LC-FTMS method was first used to identify all the ganglioside species in serum, then LC-MS/MS-MRM method was employed to quantitate the levels of gangliosides in serum from healthy volunteers and patients with benign breast tumor or breast cancer. 49 ganglioside species were determined, including GM1, GM2, GM3, GD1, GD3 and GT1 species. Compared to healthy volunteers, the levels of GM1, GM2, GM3, GD1 and GD3 displayed a rising trend in breast cancer patients. In particular, as the major glycosphingolipid component, GM3 showed excellent diagnostic accuracy in cancer serum (AUC > 0.9). PCA profile of the GM3 species showed clear distinction between normal and cancer serum. What's more, ROC curve proved great diagnostic accuracy of GM3 between cancer and benign serum. In addition, GM3 was discovered as a diagnostic marker to differentiate luminal B subtype from other subtypes. Furthermore, a positive correlation between GM3 and Ki-67 status of patients was identified. In conclusion, our results introduced the alteration patterns of serum gangliosides in breast cancer and suggested serum GM3 as a potential diagnostic biomarker in breast cancer diagnosis and luminal B subtype distinction.

Comprehensive N-Glycome Profiling of Cells and Tissues for Breast Cancer Diagnosis.

Aberrant protein glycosylation is observed in the progression of many types of diseases, including different cancers. In this study, we assess differential N-glycan patterns of human breast cancer cells and tissues by PGC-ESI-MS/MS. Compared with mammary epithelial cells, high-mannose glycans were significantly elevated in breast cancer cells. However, the alteration of N-glycans in tissues was more obvious than that in cells. Sixty-three kinds of different N-glycans were stably identified, and 38 types of them exhibited significant differences between para-carcinoma and breast cancer tissues. High-mannose glycans and core-fucosylated glycans were increased in the breast cancer tissues, while bisected glycans and sialylated glycans were decreased. Moreover, a total of 27 types of N-glycans displayed evident differences between benign breast tumor and breast cancer tissues, and most of them including bisected and sialylated glycans exhibited decreased relative abundances in cancer tissues. Overall, three high-mannose N-glycans (F0H6N2S0, F0H7N2S0, F0H8N2S0) exhibited significant diagnostic accuracy in both breast cancer cells and tissues, suggesting their potential role in biomarkers. Furthermore, a negative correlation between sialylated glycans and age of patients was identified. In conclusion, our results may be beneficial to understand the role that N-glycan plays on the progression of breast cancer and propose potential diagnostic biomarkers.

MicroRNA 125a-5p Inhibits Cell Proliferation and Induces Apoptosis in Hepatitis B Virus-Related Hepatocellular Carcinoma by Downregulation of ErbB3.

MicroRNAs, a class of endogenous noncoding RNAs, regulate gene expression at the posttranscriptional level and thus take part in multiple biological processes. An increasing number of miRNAs have been found to be dysregulated in hepatocellular carcinoma (HCC) and are involved in liver tumorigenesis. In this study, miR-125a-5p was found to be obviously downregulated much more in hepatitis B virus (HBV)-related HCC. To investigate the effects of miR-125a-5p, miR-125a-5p was overexpressed in HepG2.2.15 and HepG3X cells. The findings have indicated that overexpression of miR-125a-5p dramatically inhibited cell proliferation and induced cell apoptosis. Furthermore, overexpression of miR-125a-5p could significantly decrease the secretion of HBsAg and HBeAg. In concordance to this, the expression of ErbB3 was upregulated in human HBV-related HCC tissue, HepG2.2.15 cells, and HepG3X cells. miR-125a-5p directly targeted ErbB3 and reduced both mRNA and protein levels of ErbB3, which promoted cell proliferation and suppressed cell apoptosis in HCC cells. Our results provide new insights into the function of miR-125a-5p in HBV-related HCC. It is beneficial to gain insight into the mechanism of HBV infection and pathophysiology of HBV-related HCC.

Marine polysaccharides attenuate metabolic syndrome by fermentation products and altering gut microbiota: An overview.

Marine polysaccharides (MPs), including plant, animal, and microbial-derived polysaccharides, can alleviate metabolic syndrome (MetS) by different regulation mechanisms. MPs and their derivatives can attenuate MetS by vary cellular signal pathways, such as peroxisome proliferator-activated receptor, 5' adenosine monophosphate-activated protein kinase, and CCAAT/enhancer binding protein-α. Also, most of MPs cannot be degraded by human innate enzymes, but they can be degraded and fermented by human gut microbiota. The final metabolic products of these polysaccharides are usually short-chain fatty acids (SCFAs), which can change the gut microbiota ecology by altering the existing percentage of special microorganisms. In addition, the SCFAs and changed gut microbiota can regulate enteroendocrine hormone secretion, blood glucose, lipid metabolism levels, and other MetS symptoms. Here, we summarize the up-to-date findings on the effects of MPs, particularly marine microbial-derived polysaccharides, and their metabolites on attenuating MetS.

Structure and immunomodulatory activity of a sulfated agarose with pyruvate and xylose substitutes from Polysiphonia senticulosa Harvey.

A charged and size uniform polysaccharide PS2 was prepared from red seaweed Polysiphonia senticulosa Harvey. Based on nuclear magnetic resonance (NMR), fourier transform infrared (FTIR) spectroscopy, and electrospray ionization collision-induced-dissociation mass spectrometry (ESI-CID-MSn) analysis, PS2 was determined to be a novel 6-O-sulfated agarose which was mainly composed of a repeating unit of alternating (1→3)-linked 6-sulfated-ß-d-galactose and (1→4)-linked 3,6-anhydro-α-l-galactose in association with small amount of pyruvate substituted on galactose residues and xylose (Xyl) branched at C2 position of 3,6-anhydro-galactose. The immunomodulatory activity of PS2 was evaluated on RAW264.7 cells, and the results showed PS2 could increase nitric oxide (NO) production and enhanced the phagocytic activity of macrophages. Moreover, PS2 could significantly promote proliferation of T lymphocyte and potentiated the nature kill cell (NK) activity. These findings suggest that PS2 could be potentially developed as a novel valuable immunomodulator.

Carrageenan-induced colitis is associated with decreased population of anti-inflammatory bacterium, Akkermansia muciniphila, in the gut microbiota of C57BL/6J mice.

Carrageenan as a food additive has been used for years. However, controversy exists regarding to the safety of carrageenan and accumulating evidence indicates that it could induce colitis in experimental models. Here, to provide more information on this issue and solve the debate, we studied and compared in detail the toxic effects of different isomers of carrageenan (κ-, ι-, and λ-) on the colon of C57BL/6J mice. Interestingly, all isomers of carrageenan were found to induce colitis with a comparable activity. Given that carrageenan is unabsorbed after oral administration, and also in light of the fact that gut microbiota plays a pivotal role in the pathogenesis of colitis, we further investigated the effect of carrageenan on gut microbiota using high-throughput sequencing. Intriguingly, carrageenan-induced colitis was observed to be robustly correlated with changes in the composition of gut microbiota. Specifically, all carrageenans significantly decreased the abundance of a potent anti-inflammatory bacterium, Akkermansia muciniphila, in the gut, which is highly relevant for understanding the toxic effect of carrageenan. Altogether, our results corroborate previous studies demonstrating harmful gastrointestinal effect of carrageenan and, from a gut microbiota perspective, shed new light into the mechanism by which carrageenan induces colitis in experimental animals.

Glycosaminoglycans and glycolipids as potential biomarkers in lung cancer.

In this report, we used liquid chromatography-mass spectrometry and Western blotting to analyze the content and structure of glycosaminoglycans, glycolipids and selected proteins to compare differences between patient-matched normal and cancerous lung tissues obtained from lung cancer patients. The cancer tissue samples contained over twice as much chondroitin sulfate (CS)/dermatan sulfate (DS) as did the normal tissue samples, while the amount of heparan sulfate (HS) and hyaluronan (HA) in normal and cancer tissues were not significantly different. In HS, several minor disaccharide components, including NS6S, NS2S and 2S were significantly lower in cancer tissues, while the levels of major disaccharides, TriS, NS and 0S disaccharides were not significantly different in normal and cancer tissues. In regards to CS/DS, the level of 4S disaccharide (the major component of CS-type A and DS) decreased and the level of 6S disaccharide (the major component of CS- type C) increased in cancer tissues. We also compared the content and structure of GAGs in lung tissues from smoking and non-smoking patients. Analysis of the glycolipids showed all lipids present in these lung tissues, with the exception of sphingomyelin were higher in cancer tissues than in normal tissues. Western analysis showed that syndecan 1 and 2 proteoglycans displayed much higher expression in cancer tissue/biopsy samples. This investigation begins to provide an understanding of patho-physiological roles on glycosaminoglycans and glycolipids and might be useful in identifying potential biomarkers in lung cancer.

In Vivo Anti-Cancer Mechanism of Low-Molecular-Weight Fucosylated Chondroitin Sulfate (LFCS) from Sea Cucumber Cucumaria frondosa.

The low-molecular-weight fucosylated chondroitin sulfate (LFCS) was prepared from native fucosylated chondroitin sulfate (FCS), which was extracted and isolated from sea cucumber Cucumaria frondosa, and the anti-cancer mechanism of LFCS on mouse Lewis lung carcinoma (LLC) was investigated. The results showed that LFCS remarkably inhibited LLC growth and metastasis in a dose-dependent manner. LFCS induced cell cycle arrest by increasing p53/p21 expression and apoptosis through activation of caspase-3 activity in LLC cells. Meanwhile, LFCS suppressed the expression of vascular endothelial growth factor (VEGF), increased the expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) and downregulated the matrix metalloproteinases (MMPs) level. Furthermore, LFCS significantly suppressed the activation of ERK1/2/p38 MAPK/NF-κB pathway, which played a prime role in expression of MMPs. All of these data indicate LFCS may be used as anti-cancer drug candidates and deserve further study.

Fibroblast growth factor-based signaling through synthetic heparan sulfate blocks copolymers studied using high cell density three-dimensional cell printing.

Four well-defined heparan sulfate (HS) block copolymers containing S-domains (high sulfo group content) placed adjacent to N-domains (low sulfo group content) were chemoenzymatically synthesized and characterized. The domain lengths in these HS block co-polymers were ~40 saccharide units. Microtiter 96-well and three-dimensional cell-based microarray assays utilizing murine immortalized bone marrow (BaF3) cells were developed to evaluate the activity of these HS block co-polymers. Each recombinant BaF3 cell line expresses only a single type of fibroblast growth factor receptor (FGFR) but produces neither HS nor fibroblast growth factors (FGFs). In the presence of different FGFs, BaF3 cell proliferation showed clear differences for the four HS block co-polymers examined. These data were used to examine the two proposed signaling models, the symmetric FGF2-HS2-FGFR2 ternary complex model and the asymmetric FGF2-HS1-FGFR2 ternary complex model. In the symmetric FGF2-HS2-FGFR2 model, two acidic HS chains bind in a basic canyon located on the top face of the FGF2-FGFR2 protein complex. In this model the S-domains at the non-reducing ends of the two HS proteoglycan chains are proposed to interact with the FGF2-FGFR2 protein complex. In contrast, in the asymmetric FGF2-HS1-FGFR2 model, a single HS chain interacts with the FGF2-FGFR2 protein complex through a single S-domain that can be located at any position within an HS chain. Our data comparing a series of synthetically prepared HS block copolymers support a preference for the symmetric FGF2-HS2-FGFR2 ternary complex model.

Carbohydrate-containing molecules as potential biomarkers in colon cancer.

Glycans play a critical role in physiological and pathological processes through interaction with a variety of ligands. Altered expression and dysregulation of these molecules can cause aberrant cellular function such as malignancy. Glycomics provide information of the structure and function of glycans, glycolipids, and glycoproteins such as proteoglycans, and may help to predict cancer development and progression as biomarkers. In this report, we compared the expression of proteoglycans, the content and structure of glycosaminoglycans and glycolipids between patient-matched normal and cancer tissues obtained from colon cancer patients. Tumor-related proteoglycans, glypican-3, and syndecan-1 showed downregulation in cancer tissues compared to normal tissues. In cancer tissue, the total amount of chondroitin sulfate (CS)/dermatan sulfate and heparan sulfate were lower and, interestingly, the level of disaccharide units of both 4S6S (CS-E) and 6S (CS-C) were higher compared to normal tissue. Also, overall lipids including glycolipids, a major glycomics target, were analyzed by hydrophilic interaction liquid chromatography mass spectrometry. Increase of lyso-phosphatidylcholine (phospholipid), sphingomyelin (sphigolipid), and four types of glycolipids (glucosylceramide, lactosylceramide, monosialic acid ganglioside, and globoside 4) in cancer tissue showed the possibility as potential biomarkers in colon cancer. While requiring the need for careful interpretation, this type of broad investigation gives us a better understanding of pathophysiological roles on glycosaminoglycans and glycolipids and might be a powerful tool for colon cancer diagnosis.

Effects of polysaccharides from abalone (Haliotis discus hannai Ino) on HepG2 cell proliferation.

Three polysaccharides, AAP, AVAP I, and AVAP II, were isolated from abalone Haliotis discus hannai Ino. The polysaccharides' compositions were analysed, and their effects on HepG2 cell proliferation were assessed. AVAP I had a greater growth-stimulatory effect than AAP or AVAP II. The oligosaccharide of AVAP I (Oli-AVAP I) exhibited the same growth effects, but rhamnose, the primary monosaccharide of AVAP I and Oli-AVAP I, did not exhibit this activity. Moreover, AVAP I dramatically reduced the mRNA levels of CDK6 and Cyclin E1 but significantly increased Cyclin B1, CDK1 and Cyclin F. Interestingly, AVAP I remained able to induce cell proliferation in a low serum concentration medium. AVAP I could therefore promote HepG2 cell proliferation by regulating gene expression and accelerating the cell cycle process. AVAP I may be useful as a serum supplement for stimulating the proliferation of mammalian cells. Our results offer a comprehensive method for utilising the abalone viscera, which is usually discarded as waste.

Bioengineering murine mastocytoma cells to produce anticoagulant heparin.

Heparin (HP), an important anticoagulant polysaccharide, is produced in a complex biosynthetic pathway in connective tissue-type mast cells. Both the structure and size of HP are critical factors determining the anticoagulation activity. A murine mastocytoma (MST) cell line was used as a model system to gain insight into this pathway. As reported, MST cells produce a highly sulfated HP-like polysaccharide that lacks anticoagulant activity (Montgomery RI, Lidholt K, Flay NW, Liang J, Vertel B, Lindahl U, Esko JD. 1992. Stable heparin-producing cell lines derived from the Furth murine mastocytoma. Proc Natl Acad Sci USA 89:11327-11331). Here, we show that transfection of MST cells with a retroviral vector containing heparan sulfate 3-O-sulfotransferase-1 (Hs3st1) restores anticoagulant activity. The MST lines express N-acetylglucosamine N-deacetylase/N-sulfotransferase-1, uronosyl 2-O-sulfotransferase and glucosaminyl 6-O-sulfotransferase-1, which are sufficient to make the highly sulfated HP. Overexpression of Hs3st1 in MST-10H cells resulted in a change in the composition of heparan sulfate (HS)/HP and CS/dermatan sulfate (DS) glycosaminoglycans. The cell-associated HS/HP closely resembles HP with 3-O-sulfo group-containing glucosamine residues and shows anticoagulant activity. This study contributes toward a better understanding of the HP biosynthetic pathway with the goal of providing tools to better control the biosynthesis of HP chains with different structures and activities.

Immobilized enzymes to convert N-sulfo, N-acetyl heparosan to a critical intermediate in the production of bioengineered heparin.

Heparin is a critically important anticoagulant drug that is prepared from pig intestine. In 2007-2008, there was a crisis in the heparin market when the raw material was adulterated with the toxic polysaccharide, oversulfated chondroitin sulfate, which was associated with 100 deaths in the U.S. alone. As the result of this crisis, our laboratory and others have been actively pursuing alternative sources for this critical drug, including synthetic heparins and bioengineered heparin. In assessing the bioengineering processing costs it has become clear that the use of both enzyme-catalyzed cofactor recycling and enzyme immobilization will be needed for commercialization. In the current study, we examine the use of immobilization of C5-epimerase and 2-O-sulfotransferase involved in the first enzymatic step in the bioengineered heparin process, as well as arylsulfotransferase-IV involved in cofactor recycling in all three enzymatic steps. We report the successful immobilization of all three enzymes and their use in converting N-sulfo, N-acetyl heparosan into N-sulfo, N-acetyl 2-O-sulfo heparin.