Sulfate Modification Research Articles

Total Synthesis of Homogeneous Variants of Hirudin P6: A Post‐Translationally Modified Anti‐Thrombotic Leech‐Derived Protein

Hirudin P6 is a leech-derived anti-thrombotic protein which possesses two post-translational modifications, O-glycosylation and tyrosine sulfation. In this study we report the ligation-based synthesis of a library of hirudin P6 proteins possessing homogeneous glycosylation and sulfation modifications. The nature of the modifications incorporated was shown to have a drastic effect on inhibition against both the fibrinogenolytic and amidolytic activities of thrombin and thus highlights a potential means for attenuating the biological activity of the protein.

Study of some biological activities of aqueous extract of ginger (Zingiber officinale)

Purpose This study was designed to evaluate the potential of two aqueous extracts of ginger rhizome under different extraction conditions (cold and hot water). Materials and methods Anticoagulant, fibrinolytic, antimicrobial, prebiotic, and antitumor activities were examined for the two aqueous extracts. The two aqueous extracts were then used for evaluation of yield, total carbohydrates, protein, and monosaccharide contents. The sulfation of crude extracts was carried out by chlorosulfonic acid as a sulfating agent in formamide. Results The results showed that sulfation modification of the two aqueous extracts increased significantly both the anticoagulation and the fibrinolytic activities, but did not affect the antimicrobial, prebiotic, or antitumor activities. However, the two native water extracts showed antibacterial activity against Escherichia coli only, but not for Staphylococcus aureus, and antifungal activity against Candida albicans. Conclusion The chemical modification of the two aqueous extracts of ginger by sulfation will increase significantly its anticoagulation and fibrinolytic activities while not affecting or improving the prebiotic and antimicrobial activities. However, the antitumor activity is high for both the native and the sulfated aqueous extracts of ginger. Thus, this result does not mean that the sulfation modification of the water extracts studied affected the antitumor activity.

Catalytic synthesis and antioxidant activity of sulfated polysaccharide from Momordica charantia L.

Sulfated derivatives of polysaccharide from Momordica charantia L. (MCPS) with different degree of sulfation (DS) were synthesized by chlorosulfonic acid method with ionic liquids as solvent. Fourier transform infrared spectra and 13C nuclear magnetic resonance spectra indicated that C-6 substitution was predominant in MCPS compared with the C-2 position. Compared with the native polysaccharide from Momordica charantia L. (MCP), MCPS exhibited more excellent antioxidant activities in vitro, which indicated that sulfated modification could enhance antioxidant activities of MCP. Furthermore, high DS and moderate molecular weight could improve the antioxidant activities of polysaccharide.

High-Field Asymmetric-Waveform Ion Mobility Spectrometry and Electron Detachment Dissociation of Isobaric Mixtures of Glycosaminoglycans

High-field asymmetric waveform ion mobility spectrometry (FAIMS) is shown to be capable of resolving isomeric and isobaric glycosaminoglycan negative ions and to have great utility for the analysis of this class of molecules when combined with Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) and tandem mass spectrometry. Electron detachment dissociation (EDD) and other ion activation methods for tandem mass spectrometry can be used to determine the sites of labile sulfate modifications and for assigning the stereochemistry of hexuronic acid residues of glycosaminoglycans (GAGs). However, mixtures with overlapping mass-to-charge values present a challenge, as their precursor species cannot be resolved by a mass analyzer prior to ion activation. FAIMS is shown to resolve two types of mass-to-charge overlaps. A mixture of chondroitin sulfate A (CSA) oligomers with 4-10 saccharides units produces ions of a single mass-to-charge by electrospray ionization, as the charge state increases in direct proportion to the degree of polymerization for these sulfated carbohydrates. FAIMS is shown to resolve the overlapping charge. A more challenging type of mass-to-charge overlap occurs for mixtures of diastereomers. FAIMS is shown to separate two sets of epimeric GAG tetramers. For the epimer pairs, the complexity of the separation is reduced when the reducing end is alkylated, suggesting that anomers are also resolved by FAIMS. The resolved components were activated by EDD and the fragment ions were analyzed by FTICR-MS. The resulting tandem mass spectra were able to distinguish the two epimers from each other.

Sulfated modification can enhance antiglycation abilities of polysaccharides from Dendrobium huoshanense

Dendrobium huoshanense is an important edible-medicinal plant with high nutritional values and health functions. A homogenous polysaccharide (DHPD1) with molecular weight of 3.2×103Da was extracted from D. huoshanense, which was mainly composed of glucose, arabinose, galactose, mannose and xylose. Chlorosulfonic acid-pyridine (CSA-Pyr) method was performed to modify the structure of DHPD1. In order to get a high degree of substitution (DS), sulfated modification conditions were optimized by response surface methodology. The maximum DS of 1.473 was obtained when the reaction condition was fixed at reaction temperature 60°C, reaction time 160min and volume ratio of Pyr to CSA 2:1. NMR spectra revealed that this sulfation occurred to C-2 and C-6 of glycosyl residues in DHPD1. After 28 days of incubation, the sulfated DHPD1 at 1.0mg/mL showed the inhibitory ability of 58.5%, which increased by 16.2% and 52.5% than that of aminoguanidine and DHPD1 at the same dosage.

Vascular biomechanical properties in mice with smooth muscle specific deletion of Ndst1.

Heparan sulfate proteoglycans act as co-receptors for many chemokines and growth factors. The sulfation pattern of the heparan sulfate chains is a critical regulatory step affecting the binding of chemokines and growth factors. N-deacetylase-N-sulfotransferase1 (Ndst1) is one of the first enzymes to catalyze sulfation. Previously published work has shown that HSPGs alter tangent moduli and stiffness of tissues and cells. We hypothesized that loss of Ndst1 in smooth muscle would lead to significant changes in heparan sulfate modification and the elastic properties of arteries. In line with this hypothesis, the axial tangent modulus was significantly decreased in aorta from mice lacking Ndst1 in smooth muscle (SM22αcre(+)Ndst1(-/-), p < 0.05, n = 5). The decrease in axial tangent modulus was associated with a significant switch in myosin and actin types and isoforms expressed in aorta and isolated aortic vascular smooth muscle cells. In contrast, no changes were found in the compliance of smaller thoracodorsal arteries of SM22αcre(+)Ndst1(-/-) mice. In summary, the major findings of this study were that targeted ablation of Ndst1 in smooth muscle cells results in altered biomechanical properties of aorta and differential expression of myosin and actin types and isoforms.

Sulphation can enhance the antioxidant activity of polysaccharides produced by Enterobacter cloacae Z0206

The protective effects of sulfated polysaccharide derivatives produced by Enterobacter cloacae Z0206 against H2O2-induced oxidative damage in RAW264.7 murine macrophages as well as the possible mechanisms governing the protective effects were studied. Sulfated polysaccharides protected RAW264.7 cells from oxidative damage and apoptosis induced by H2O2 by protecting the cellular structure; improving the activity of antioxidant enzymes, such as superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px); and inhibiting caspase-3 activation and DNA fragmentation. In addition, the sulfated polysaccharides conferred higher levels of protection from H2O2-induced oxidative damage in RAW264.7 murine macrophages compared to the native polysaccharide lacking sulfation. These results indicated that sulfated modifications might be an effective approach to enhance the antioxidant activity of polysaccharides produced by E. cloacae Z0206, and the sulfated derivatives of these polysaccharides may act as potent antioxidant agents.

Galactose 6-O-Sulfotransferases Are Not Required for the Generation of Siglec-F Ligands in Leukocytes or Lung Tissue

Eosinophil accumulation is a characteristic feature of the immune response to parasitic worms and allergens. The cell surface carbohydrate-binding receptor Siglec-F is highly expressed on eosinophils and negatively regulates their accumulation during inflammation. Although endogenous ligands for Siglec-F have yet to be biochemically defined, binding studies using glycan arrays have implicated galactose 6-O-sulfate (Gal6S) as a partial recognition determinant for this receptor. Only two sulfotransferases are known to generate Gal6S, namely keratan sulfate galactose 6-O-sulfotransferase (KSGal6ST) and chondroitin 6-O-sulfotransferase 1 (C6ST-1). Here we use mice deficient in both KSGal6ST and C6ST-1 to determine whether these sulfotransferases are required for the generation of endogenous Siglec-F ligands. First, we characterize ligand expression on leukocyte populations and find that ligands are predominantly expressed on cell types also expressing Siglec-F, namely eosinophils, neutrophils, and alveolar macrophages. We also detect Siglec-F ligand activity in bronchoalveolar lavage fluid fractions containing polymeric secreted mucins, including MUC5B. Consistent with these observations, ligands in the lung increase dramatically during infection with the parasitic nematode, Nippostrongylus brasiliensis, which is known to induce eosinophil accumulation and mucus production. Surprisingly, Gal6S is undetectable in sialylated glycans from eosinophils and BAL fluid analyzed by mass spectrometry. Furthermore, none of the ligands we describe are diminished in mice lacking KSGal6ST and C6ST-1, indicating that neither of the known galactose 6-O-sulfotransferases is required for ligand synthesis. These results establish that ligands for Siglec-F are present on several cell types that are relevant during allergic lung inflammation and argue against the widely held view that Gal6S is critical for glycan recognition by this receptor.

Sulfated modification and anti-tumor activity of laminarin

The aim of this study was to investigate the sulfated modification of laminarin and the changes in structure and antitumor activity. The chlorosulfonic acid-pyridine method was applied for sulfated modification. The molecular weights of laminarin and laminarin sulfate (LAMS) were measured by high-performance liquid chromatography (HPLC), and IR and NMR spectra were also recorded. The surface conformations of laminarin and LAMS were observed with a scanning electron microscope. The antitumor activities of the two polysaccharides were also evaluated using an MTT assay. LAMS with a sulfate content of 45.92% and a molecular weight of 16,000 was synthesized. The IR spectra of laminarin and LAMS showed the characteristic absorption peaks of a polysaccharide, and LAMS also had the characteristic absorption peaks of sulfate moieties. The NMR spectra showed that laminarin and LAMS had β-(1→3) glycosidic bonds forming the main chain, and sulfate substitution was at the hydroxyl groups of C2 and C6. Under the scanning electron microscope, there were clear differences in surface conformation between laminarin and LAMS; laminarin was cloud-like and spongy, while LAMS was block-like and flaky. The MTT results showed that laminarin and LAMS had inhibitory effects on LoVo cell growth, and the antitumor activity of LAMS was higher than that of laminarin at the same concentration. This suggests that sulfated modification was able to change the laminarin structure and markedly enhance the antitumor activity.

The SULFs, Extracellular Sulfatases for Heparan Sulfate, Promote the Migration of Corneal Epithelial Cells during Wound Repair

Corneal epithelial wound repair involves the migration of epithelial cells to cover the defect followed by the proliferation of the cells to restore thickness. Heparan sulfate proteoglycans (HSPGs) are ubiquitous extracellular molecules that bind to a plethora of growth factors, cytokines, and morphogens and thereby regulate their signaling functions. Ligand binding by HS chains depends on the pattern of four sulfation modifications, one of which is 6-O-sulfation of glucosamine (6OS). SULF1 and SULF2 are highly homologous, extracellular endosulfatases, which post-synthetically edit the sulfation status of HS by removing 6OS from intact chains. The SULFs thereby modulate multiple signaling pathways including the augmentation of Wnt/ß-catenin signaling. We found that wounding of mouse corneal epithelium stimulated SULF1 expression in superficial epithelial cells proximal to the wound edge. Sulf1−/−, but not Sulf2−/−, mice, exhibited a marked delay in healing. Furthermore, corneal epithelial cells derived from Sulf1−/− mice exhibited a reduced rate of migration in repair of a scratched monolayer compared to wild-type cells. In contrast, human primary corneal epithelial cells expressed SULF2, as did a human corneal epithelial cell line (THCE). Knockdown of SULF2 in THCE cells also slowed migration, which was restored by overexpression of either mouse SULF2 or human SULF1. The interchangeability of the two SULFs establishes their capacity for functional redundancy. Knockdown of SULF2 decreased Wnt/ß-catenin signaling in THCE cells. Extracellular antagonists of Wnt signaling reduced migration of THCE cells. However in SULF2- knockdown cells, these antagonists exerted no further effects on migration, consistent with the SULF functioning as an upstream regulator of Wnt signaling. Further understanding of the mechanistic action of the SULFs in promoting corneal repair may lead to new therapeutic approaches for the treatment of corneal injuries.

The comparison of immune-enhancing activity of sulfated polysaccharidses from Tremella and Condonpsis pilosula

Based on our previous research, four sulfated polysaccharide (sPSs) from Tremella and Condonpsis pilosula, sTPStp, sTPS70c, sCPPStp and sCPPS50c, were prepared and their effects on splenic lymphocytes proliferation in vitro and the immune response of ND vaccine in chicken were compared taking the unmodified polysaccharide (uPS) TPStp as control. The results showed that four sPSs could significantly or numerically stimulate splenic lymphocyte proliferation singly or synergistically with LPS in vitro, sTPS70c and sCPPStp demonstrated better effect; promote peripheral lymphocytes proliferation and enhance serum HI antibody titer in chickens vaccinated with ND vaccine, the actions of sPSs were stronger than that of uPS, and sTPS70c at medium dosage presented the best efficacy. These indicated that sulfation modification could improve the immune-enhancing activity of TPS and CPPS, sTPS70c possessed the strongest activity and would be expected as a component of new-type immunopotentiator.

Analisis Keragaman Genetik Temulawak (Curcuma xanthorrhiza Roxb.) Menggunakan Penanda Amplified Fragment Length Polymorphism (AFLP)

Curcuma xanthorrhiza Roxb, which is well-known as Java turmeric, has been extensively used in pharmaceutical industries in Indonesia. In spite of this commercial value, the identity of this species is commonly mistaken from other similar orange rhizomes Curcuma. Correct identity of these species is vital in pharmaceutical industries. The objective of the study was to determine genetic diversity of 32 accession Curcuma xanthorrhiza Roxb. Genomic DNA was extracted from leaf using Sodium Dodesyl Sulphate (SDS) modification. Amplified fragment length polymorphism (AFLP) was carried out according to the protocol ofAFLPTM plant mapping kit and the final polymerase chain reaction (PCR) products were separated using The Agilent 2100 Bioanalyzer. The number of fragment produced by 12 pairs primer combination of AFLP ranged from 42 to 60 with an average of 52. Data obtained was analyzed by the NTSys program. From the AFLP amplification on 32 DNA samples, it was proven that the accession of Curcuma xanthorrhiza Roxb. had a high degree of diversity. Based on analysis of AFLP and unweighted pair group with arithme average (UPGMA) it was shown that the accession of Curcuma xanthorrhiza Roxb. could be grouped into two cluster at relative ecludian distance of 0.10 (10%). Cluster I for accession from Palembang, Pacitan and Ciamis 2. Cluster II for accession from Makale, Pontianak, Kulonprogo, Mataram, Boyolali, Salatiga, Sumberejo, Bali, P. Seram, Sentolo, Purworejo, Samas Bantul, Ciamis1, Blora, Semarang, Poso, Kalsesl, Tagari, Merapi Farm, Salakaria, NTB, Menoreh, Karang Anyar, Mangunan, Medan, Toraja, dan Solok.

Sulfated Astragalus polysaccharide can regulate the inflammatory reaction induced by LPS in Caco2 cells

This study evaluates the effect of sulfated Astragalus polysaccharide (SAPS) on inflammatory reaction induced by LPS in Caco2 cells. Sulfated modification was conducted using the chlorosulfonic acid–pyridine method. Caco2 cells were cultured with 25, 50 and 100μg/mL SAPS or 100μg/mL Astragalus polysaccharide (APS) for 24h. Then, 1μg/mL LPS was added for the next 24h to trigger an inflammatory response. DMEM culture medium was used as a blank control. In present study, LPS stimulation significantly increased the mRNA expression of TNF-α, IL-1β, IL-8 and TLR4, and reduced the expression of ZO-1 and occludin. Compared with the LPS control group, APS (100μg/mL) or SAPS (100μg/mL) administration decreased the expression of TNF-α, IL-1β and IL-8. Moreover, 25μg/mL and 50μg/mL SAPS down-regulated TNF-α and IL-1β expression. APS administration (100μg/mL) up-regulated occludin expression, but did not affect ZO-1 expression. However, the expression of ZO-1 and occludin was up-regulated by lower dose SAPS administration (25μg/mL and 50μg/mL). Compared with the other groups, the expression of TLR4 was lower in the SAPS group at all concentrations of SAPS. These results suggest that SAPS was to be a more effective anti-inflammatory agent than APS in vitro.

An approach for separation and complete structural sequencing of heparin/heparan sulfate-like oligosaccharides.

As members of the glycosaminoglycan (GAG) family, heparin and heparan sulfate (HS) are responsible for mediation of a wide range of essential biological actions, most of which are mediated by specific patterns of modifications of regions of these polysaccharides. To fully understand the regulation of HS modification and the biological function of HS through its interactions with protein ligands, it is essential to know the specific HS sequences present. However, the sequencing of mixtures of HS oligosaccharides presents major challenges due to the lability of the sulfate modifications, as well as difficulties in separating isomeric HS chains. Here, we apply a sequential chemical derivatization strategy involving permethylation, desulfation, and trideuteroperacetylation to label original sulfation sites with stable and hydrophobic trideuteroacetyl groups. The derivatization chemistry differentiates between all possible heparin/HS sequences solely by glycosidic bond cleavages, without the need to generate cross-ring cleavages. This derivatization strategy combined with LC-MS/MS analysis has been used to separate and sequence five synthetic HS-like oligosaccharides of sizes up to dodecasaccharide, as well as a highly sulfated Arixtra-like heptamer. This strategy offers a unique capability for the sequencing of microgram quantities of HS oligosaccharide mixtures by LC-MS/MS.

Characterization and Properties of Recycled Polypropylene/Coconut Shell Powder Composites: Effect of Sodium Dodecyl Sulfate Modification

This research work focuses on the utilization of coconut shell powder (CSP) as filler in recycled polypropylene (rPP). Sodium Dedecyl Sulfate (SDS) was used as coupling agent in these composites. The effect of filler content and SDS on tensile properties, thermal properties, water absorption and morphology of rPP/CSP composites were investigated. In this study, modified rPP/CSP composites with SDS show significant increased tensile propertied, thermal stability, crystallinity and low water absorption compared unmodified rPP/CSP composites. Those improvements were contributed by the coupling effect of SDS.

Sulfated derivatives of 20(S)-ginsenoside Rh2 and their inhibitory effects on LPS-induced inflammatory cytokines and mediators

Ginsenoside Rh2 is one of the most important ginsenosides in ginseng with antitumor, antidiabetic, antiallergic, and anti-inflammatory effects. However, the extremely poor oral bioavailability induced by its low water solubility greatly limits the potency of Rh2 in clinical use. Therefore, in this study we sulfated 20(S)-ginsenoside Rh2 with chlorosulfonic acid and pyridine method, and got two new sulfated derivatives, Rh2-B1 and Rh2-B2, with higher water solubility. Their chemical structures were characterized by spectroscopic methods (IR, MS and NMR). Additionally, Rh2-B1 and Rh2-B2 had the greater anti-inflammatory effects than Rh2 through inhibiting inflammatory cytokines and mediators in LPS-induced mouse RAW264.7 macrophages cells. These results suggested that the sulfated modification of Rh2 improved its water solubility and the sulfated derivatives could be more potential candidates for developing as anti-inflammatory agents.

Sulfated modification can enhance antiviral activities of Achyranthes bidentata polysaccharide against porcine reproductive and respiratory syndrome virus (PRRSV) in vitro

Achyranthes bidentata polysaccharide (ABPS) was sulfated using the chlorosulfonic acid-pyridine method. The UV-spectrum of sulfated ABPS (sABPS) was characterized with two different absorbance peaks at 196.3 and 262.8nm. FT-IR spectrum of sABPS exhibited absorption bands at 1236.5cm−1 (asymmetrical SO stretching vibration) and 822.8cm−1 (symmetrical COS vibration related to a COSO3 group). Anti-PRRSV activities of sABPS were tested on MARC-145 cells by MTT method. The results showed that sABPS exhibited significant antiviral activities at lower concentrations in comparison with the non-modified ABPS treated cells, indicating that sulfated modification could enhance antiviral activities of ABPS. These findings may provide the potential value to develop a new-type drug against PRRSV infection.

Sulfated modification of the polysaccharide from Cordyceps_gunnii mycelia and its biological activities

A chemically new sulfated polysaccharide (SPS50) was prepared from the water soluble polysaccharide (PS50), isolated from Cordyceps_gunnii mycelia, by concentrated sulfuric acid method. The yield of crude SPS50 was 62.34% and its specific rotation was [α]D20=−36.75°. The structural characteristics of this chemically sulfated polysaccharide were determined based on the infrared analysis (IR), high performance liquid chromatography (HPLC) and sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE). Its biological properties including anti-oxidant and anti-tumor activities were also investigated. The results showed that the anti-oxidant capacity of SPS50 was not as good as SP50 and the anti-tumor activity of SPS50 was much better than PS50. SPS50 showed evident growth inhibition on K562 cells. The tumor inhibition ratio of SPS50 against K562 cells was 69.92%.

Immune-enhancing activity comparison of sulfated ophiopogonpolysaccharide and sulfated jujube polysaccharide

The immune-enhancing activities of four sulfated polysaccharides, sOPSt, sOPS80, sJPSt and sJPS50 picked out in our previous researches, were compared taking four corresponding unmodified polysaccharides as control. In vitro experiment, the effects of eight polysaccharides on chicken peripheral lymphocyte proliferation were determined by MTT assay. The result displayed that four sulfated polysaccharides could significantly stimulate lymphocyte proliferation, their actions were significantly or numerically stronger than those of corresponding unmodified polysaccharides, sOPS80 presented better efficacy. In vivo experiment, 300 14-day-old chickens were averagely divided into ten groups. The chickens except blank control (BC) group were vaccinated with Newcastle disease vaccine, repeated vaccination at 28 days old. At the same time of the first vaccination, the chickens in eight polysaccharides groups were injected respectively with 0.5ml (1mg) of eight polysaccharides, in vaccination control (VC) and BC group, with 0.5mL of physiological saline, once a day for three successive days. On days 7, 14, 21 and 28 after the first vaccination, the peripheral lymphocyte proliferation and serum ND antibody titer were determined. The result showed that four sulfated polysaccharides could significantly promote lymphocyte proliferation and enhance serum antibody titer. Their actions were significantly or numerically stronger than those of corresponding unmodified polysaccharides, sOPS80 possessed the best efficacy. These results indicated that sulfation modification could enhance the immune-enhancing activity of OPS and JPS, sOPS80 possessed the best efficacy and would be expected as a component drug of new-type immunopotentiator.

Sulfation modification and anticoagulant activity of the polysaccharides obtained from persimmon (Diospyros kaki L.) fruits

The optimal conditions for sulfation of polysaccharides from persimmon fruits (PFP) with chlorosulfonic acid–pyridine (CSA–Pyr) method were determined by response surface methodology. Box–Behnken design was applied to evaluate the effects of three independent variables (volume ratio of Pyr to CSA, volume ratio of PFP to SO3Pyr and reaction time) on the degree of substitution (DS), molecular weight (MW) and activated partial thromboplastin time (APTT) of sulfated polysaccharides (PFP-S). The APTT activity of PFP-S could be improved by application of various volume ratio of Pyr to CSA, volume ratio of PFP to SO3Pyr and reaction time, which was possible due to the degradation of polysaccharides to different extent and increasing of DS. The optimal conditions to obtain the strongest APTT of PFP-S were the volume ratio of CSA to Pyr of 1:8, the volume ratio of SO3Pyr to PFP of 1:3.6 and the reaction time of 3h, respectively.