Different Degrees Of Sulfation Research Articles

Effect of glycosaminoglycans with different degrees of sulfation on chondrogenesis.

This study aims to investigate the effects and mechanisms of chondroitin sulfate (CS), dermatan sulfate (DS), and heparin (HEP) on chondrogenesis of murine chondrogenic cell line (ATDC5) cells and the maintenance of murine articular cartilage in vitro. ATDC5 and articular cartilage tissue explant were cultured in the medium containing different sulfated glycosaminoglycans. Cell proliferation, differentiation, cartilage formation, and mechanism were observed using cell proliferation assay, Alcian blue staining, real-time quantitative polymerase chain reaction (RT-qPCR), and Western blot, respectively. Results showed that HEP and DS primarily activated the bone morphogenetic protein (BMP) signal pathway, while CS primarily activated the protein kinase B (AKT) signal pathway, further promoted ATDC5 cell proliferation and matrix production, and increased Sox9, Col2a1, and Aggrecan expression. This study investigated the differences and mechanisms of different sulfated glycosaminoglycans in chondrogenesis and cartilage homeostasis maintenance. HEP promotes cartilage formation and maintains the normal state of cartilage tissue in vitro, while CS plays a more effective role in the regeneration of damaged cartilage tissue.

Preparation of fully bio-based multilayers composed of heparin-like carboxymethylcellulose sodium and chitosan to functionalize poly (l-lactic acid) film for cardiovascular implant applications

In this study, heparin-like polysaccharides were successfully produced by sulfation of carboxymethylcellulose sodium, then a fully biobased bilayer composed of sulfated carboxymethylcellulose sodium (SCMC) and chitosan (CS) was composited on the surface of Poly (L-lactic acid) (PLA) through layer-by-layer (LBL) assembly for the potential blood-contact application such as bioresorbable vascular scaffold. The preliminary structure and bioactivity of SCMC with different degree of sulfation were investigated, and the SCMC with best performance was selected. The surface chemical compositions, morphologies and wettability of SCMC/CS multilayer-modified PLA films were researched by X-ray photoelectron spectrometer, scanning electron microscopy and water contact angle meter. A series of anticoagulation tests of SCMC/CS multilayer-modified PLA films were performed. In term of (SCMC/CS)15 multilayer-modified PLA film, the protein adsorption and plate adhesion decreased by 44.6 % and 71.5 %, respectively, the activated partial thromboplastin time prolonged by 11.9 times and thrombin time exceed 300 s, the contact activation and hemolysis rate significantly reduced compared with unmodified PLA film. Besides, this modified PLA films performed good cytocompatibility to L929 fibroblast cells, excellent anti-inflammatory and antibacterial abilities. In conclusion, the multifunctional SCMC/CS multilayer-modified PLA films with hemocompatibility, cytocompatibility, anti-inflammatory and antibacterial properties may have promising potential in future clinical applications.

The effects of sulfated hyaluronan in breast, lung and colorectal carcinoma and monocytes/macrophages cells: Its role in angiogenesis and tumor progression.

Hyaluronan (HA) is a component of the extracellular matrix (ECM) it is the main non-sulfated glycosaminoglycan able to modulate cell behavior in the healthy and tumor context. Sulfated hyaluronan (sHA) is a biomaterial derived from chemical modifications of HA, since this molecule is not naturally sulfated. The HA sulfation modifies several properties of the native molecule, acquiring antitumor properties in different cancers. In this study, we evaluated the action of sHA of ~30-60 kDa with different degrees of sulfation (0.7 sHA1 and 2.5 sHA3) on tumor cells of a breast, lung, and colorectal cancer model and its action on other cells of the tumor microenvironment, such as endothelial and monocytes/macrophage cells. Our data showed that in breast and lung tumor cells, sHA3 is able to modulate cell viability, cytotoxicity, and proliferation, but no effects were observed on colorectal cancer cells. In 3D cultures of breast and lung cancer cells, sHA3 diminished the size of the tumorsphere and modulated total HA levels. In these tumor models, treatment of monocytes/macrophages with sHA3 showed a downregulation of the expression of angiogenic factors. We also observed a decrease in endothelial cell migration and modulation of the hyaluronan-binding protein TSG-6. In the breast in vivo xenograft model, monocytes/macrophages preincubated with sHA1 or sHA3 decreased tumor vasculature, TSG-6 and HA levels. Besides, in silico analysis showed an association of TSG-6, HAS2, and IL-8 with biological processes implicated in the progression of the tumor. Taken together, our data indicate that sHA in a breast and lung tumor context is able to induce an antiangiogenic action on tumor cells as well as in monocytes/macrophages (Mo/MØ) by modulation of endothelial migration, angiogenic factors, and vessel formation.

Assessment of Anti-microbial and Anti-oxidant Activities of Modified Guar Gum

Apart from drug delivery potential of polysaccharides or their chemical derivatives, some chemically modified polysaccharides particularly sulfated polysaccharides showed significant biological property. In this study, guar gum was sulfated by reacting with various ratios of CSA (chlorosulfonic acid) and pyridine to obtain the product with different degree of sulfation (DS). The sulfated guar gum derivatives were examined by FTIR, NMR and degree of sulfation analysed and were further evaluated for their potential antibacterial activity by agar diffusion method. Results obtained from the study indicated that derivatives having higher degree of sulfation exhibited moderate activity against the selected bacterial strains at 200 µg∙ml − 1 . However, the sulfated guar gum did not show any significant antifungal activity at any concentration studied. The sulfated guar gum demonstrated dose-dependent DPPH and H 2 O 2 scavenging activity with maximum activity noted at 2 mg∙ml − 1 concentration. The sulfation of partially oxidized guar gum caused significant improvement of antioxidant activity to that observed for non-oxidized sulfated guar gum derivative. This study revealed that the extent of sulfation and molecular weight had significant impact on antimicrobial and antioxidant activities of guar gum.

Ultra-high-performance liquid chromatography charge transfer dissociation mass spectrometry (UHPLC-CTD-MS) as a tool for analyzing the structural heterogeneity in carrageenan oligosaccharides.

Ultra-high-performance liquid chromatography (UHPLC) with charge transfer dissociation mass spectrometry (CTD-MS) is presented for the analysis of a mixture of complex sulfated oligosaccharides. The mixture contained kappa (κ), iota (ι), and lambda (λ) carrageenans that contain anhydro bridges, different degrees of sulfation ranging from one to three per dimer, different positioning of the sulfate groups along the backbone, and varying degrees of polymerization (DP) between 4 and 12. Optimization studies using standard mixtures of carrageenans helped establish the optimal conditions for online UHPLC-CTD-MS/MS analysis. Optimization included (1) UHPLC conditions; (2) ion source conditions, such as the capillary voltage, drying gas and nebulizing gas temperature, and flow rate; and (3) CTD-MS conditions, including data-dependent CTD-MS. The UHPLC-CTD results were contrasted with UHPLC-CID results of the same mixture on the same instrument. Whereas CID tends to produce B/Y and C/Z ions with many neutral losses, CTD produced more abundant A/X ions and less abundant neutral losses, which enabled more confident structural detail. The results demonstrate that He-CTD is compatible with the timescale of UHPLC and provides more structural information about carrageenans compared to state-of-the-art methods like UHPLC-CID analysis.

Biomimetic sulfated glycosaminoglycans maintain differentiation markers of breast epithelial cells and preferentially inhibit proliferation of cancer cells

Glycosaminoglycans (GAG) are key elements involved in various physiological and pathological processes including cancer. Several GAG-based drugs have been developed showing significant results and potential use as cancer therapeutics. We previously reported that alginate sulfate (AlgSulf), a GAG-mimetic, reduces the proliferation of lung adenocarcinoma cells. In this study, we evaluated the preferential effect of AlgSulf on tumorigenic and nontumorigenic mammary epithelial cells in 2D, 3D, and coculture conditions. AlgSulf were synthesized with different degrees of sulfation (DSs) varying from 0 to 2.7 and used at 100 µg/mL on HMT-3522 S1 (S1) nontumorigenic mammary epithelial cells and their tumorigenic counterparts HMT-3522 T4–2 (T4–2) cells. The anti-tumor properties of AlgSulf were assessed using trypan blue and bromodeoxyuridine proliferation (BrdU) assays, immunofluorescence staining and transwell invasion assay. Binding of insulin and epidermal growth factor (EGF) to sulfated substrates was measured using QCM-D and ELISA. In 2D, the cell growth rate of cells treated with AlgSulf was consistently lower compared to untreated controls (p<0.001) and surpassed the effect of the native GAG heparin (positive control). In 3D, AlgSulf preferentially hindered the growth rate and the invasion potential of tumorigenic T4–2 nodules while maintaining the formation of differentiated polarized nontumorigenic S1 acini. The preferential growth inhibition of tumorigenic cells by AlgSulf was confirmed in a coculture system (p<0.001). In the ELISA assay, a trend of EGF binding was detected for sulfated polysaccharides while QCM-D analysis showed negligible binding of insulin and EGF to sulfated substrates. The preferential effect mediated by the mimetic sulfated GAGs on cancer cells may in part be growth factor dependent. Our findings suggest a potential anticancer therapeutic role of AlgSulf for the development of anticancer drugs.

Regulation of Laminaria Polysaccharides with Different Degrees of Sulfation during the Growth of Calcium Oxalate Crystals and their Protective Effects on Renal Epithelial Cells.

The original Laminaria polysaccharide (LP0) was sulfated using the sulfur trioxide-pyridine method, and four sulfated Laminaria polysaccharides (SLPs) were obtained, namely, SLP1, SLP2, SLP3, and SLP4. The sulfated (–OSO3–) contents were 8.58%, 15.1%, 22.8%, and 31.3%, respectively. The structures of the polysaccharides were characterized using a Fourier transform infrared (FT-IR) spectrometer and nuclear magnetic resonance (NMR) techniques. SLPs showed better antioxidant activity than LP0, increased the concentration of soluble Ca2+ in the solution, reduced the amount of CaOx precipitation and degree of CaOx crystal aggregation, induced COD crystal formation, and protected HK-2 cells from damage caused by nanometer calcium oxalate crystals. These effects can inhibit the formation of CaOx kidney stones. The biological activity of the polysaccharides increased with the content of –OSO3−, that is, the biological activities of the polysaccharides had the following order: LP0 < SLP1 < SLP2 < SLP3 < SLP4. These results reveal that SLPs with high –OSO3− contents are potential drugs for effectively inhibiting the formation of CaOx stones.

Biomimetic Sulfated GAGs Maintain Differentiation Markers of Breast Epithelial Cells and Preferentially Inhibit Proliferation of Cancer Cells

Glycosaminoglycans (GAG) are key elements involved in various physiological and pathological processes including cancer. Several GAG-based drugs have been developed showing significant results and potential use as cancer therapeutics. We previously reported that alginate sulfate (AlgSulf), a GAG-mimetic, reduces the proliferation of lung adenocarcinoma cells. In this study, we evaluated the preferential effect of AlgSulf on tumorigenic and nontumorigenic mammary epithelial cells in 2D, 3D, and coculture conditions. AlgSulf were synthesized with different degrees of sulfation (DSs) varying from 0 to 2.7 and used at 100 µg/mL on HMT-3522 S1 (S1) nontumorigenic mammary epithelial cells and their tumorigenic counterparts HMT-3522 T4-2 (T4-2) cells. The anti-tumor properties of AlgSulf were assessed using trypan blue assay, immunofluorescence staining and transwell invasion assay. Binding of insulin and epidermal growth factor (EGF) to sulfated substrates was measured using QCM-D and ELISA. In 2D, the cell growth rate of cells treated with AlgSulf was consistently lower compared to untreated controls (p<0.001) and surpassed the effect of the native GAG heparin (positive control). In 3D, AlgSulf preferentially hindered the growth rate and the invasion potential of tumorigenic T4-2 nodules while maintaining the formation of differentiated polarized nontumorigenic S1 acini. The preferential growth inhibition of tumorigenic cells by AlgSulf was confirmed in a coculture system (p<0.001). In QCM-D and ELISA binding studies, negligible binding of insulin and EGF to sulfated substrates was reported. The preferential effect mediated by the mimetic sulfated GAGs on cancer cells seems to be independent of growth factors binding. Our findings suggest a potential anticancer therapeutic role of AlgSulf for the development of anticancer drugs.

Synthesis and Properties of Interpolymer Complexes Based on Chitosan and Sulfated Arabinogalactan

Interpolymer complexes based on chitosan and arabinogalactan with a different degree of sulfation (7.7–40.8%) stable for 30 days are synthesized. The resulting complexes are characterized by scanning electron microscopy, IR spectroscopy, dynamic light scattering, and ζ-potential electrophoretic measurements. The main mechanism of complex formation is considered in terms of the electrostatic bonding of polyions. New complexes show promise as biosorbents and drug delivery systems.

Effect of Sulfation Route and Subsequent Oxidation on Derivatization Degree and Biocompatibility of Cellulose Sulfates.

Sulfated cellulose (CS) represents an interesting biopolymer due to bioactivity comparable to heparin. However, use of CS for making surface coatings or hydrogels requires the presence of reactive groups for covalent reactions. Here, an approach is presented to oxidize cellulose sulfates for subsequent cross-linking reactions with amino groups to form imine bonds. Cellulose is sulfated by direct sulfation or acetosulfation, followed by a Malaprade oxidation. The CS obtained is characterized by elemental analysis and 13 C-NMR spectroscopy. The resulting oxidized cellulose sulfates (oxCS) have different degrees of sulfation ranging from 0.79 to 1.13 and oxidation degrees from 0.18 to 0.34, but also different mass average molecular mass (MW ). Toxicity studies are carried out with mouse 3T3 fibroblasts exposed to aqueous solutions of oxCS. The results show that all oxCS are non-toxic at lower concentrations (0.5 mg mL-1 ), but with both increasing degree of oxidation and concentrations, toxic effects are observed particularly for acetosulfated and lesser for direct sulfated oxCS, which is related to a decrease in the MW of the products. It is concluded that oxCS obtained by direct sulfation with MW above 70 kDa may represent a biocompatible material for the applications suggested above.

Fabrication of Nanofibrous PVA/Alginate-Sulfate Substrates for Growth Factor Delivery.

Providing affinity sites on alginate (ALG) matrix enables specific binding of growth factors to the polymer backbone and allows their release in a controlled fashion. In this study, we used a blend of alginate sulfate (ALG-S) and polyvinyl alcohol (PVA) to fabricate electrospun scaffolds capable of delivering a heparin-like growth factor, transforming growth factor-beta1 (TGF-β1). The alginate was sulfated with different degrees of sulfation (DS, from 0.8, 3.4 to 12.4%) by a simple process. The success of sulfation was determined by Fourier-transform infrared spectroscopy (FTIR), nuclear magnetic resonance spectroscopy (NMR), elemental analysis, ultraviolet (UV) spectroscopy and staining with dimethylmethylene blue. The physical-mechanical properties of nanofibrous mats were characterized by scanning electron microscopy (SEM), FTIR, energy-dispersive X-ray spectroscopy (EDX), tensile strength and mass loss analysis. Additionally, the release kinetics of transforming growth factor-β1 (TGF-β1) from PVA/ALG-S and PVA/ALG scaffolds were compared. The results showed that the binding and entrapment of TGF-β1 to the nanofibrous scaffolds are improved by the addition of sulfate group to alginate. In conclusion, our results support that nanofibrous scaffold based on PVA/ALG-S can deliver growth factors in tissue engineering application. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 403-413, 2019.

Synthesis, physicochemical characterization and biological evaluation of chitosan sulfate as heparan sulfate mimics

Despite the relevant biological functions of heparan sulfate (HS) glycosaminoglycans, their limited availability and the chemical heterogeneity from natural sources hamper their use for biomedical applications. Chitosan sulfates (ChS) exhibit structural similarity to HSs and may mimic their biological functions. We prepared a variety of ChS with different degree of sulfation to evaluate their ability to mimic HS in protein binding and to promote neural cell division and differentiation. The structure of the products was characterized using various spectroscopic and analytical methods. The study of their interaction with different growth factors showed that ChS bound to the proteins similarly or even better than heparin. In cell cultures, a transition effect on cell number was observed as a function of ChS concentration. Differences in promoting the expression of the differentiation markers were also found depending on the degree of sulfation and modification in the chitosan.

Sulfated Laminaria japonica polysaccharides inhibit macrophage foam cell formation

In this work, a purified Laminaria japonica polysaccharide (LJP61A) was chemically modified to obtain three sulfated polysaccharides (SLJP1, SLJP2 and SLJP3) with different degrees of sulfation using the method of chlorosulfonic acid/pyridine. The effects and underlying mechanism of SLJP1, SLJP2 and SLJP3 on the suppression of macrophage foam cell formation were further investigated using the model of oxidized low-density lipoprotein (ox-LDL)-induced foam cell formation. Results exhibited that the macrophage foam cell formation induced by ox-LDL could be significantly alleviated by these sulfated polysaccharides in a dose-dependent manner. Meanwhile, the enhancement of PPAR-γ mRNA expression in ox-LDL induced macrophages was remarkably inhibited by these sulfated polysaccharides. Moreover, the cellular inflammation induced by ox-LDL could also be remarkably mitigated by these sulfated polysaccharides. These results indicated that the sulfated L. japonica polysaccharides could inhibit the conversion of macrophage into foam cell via obstructing PPAR-γ activation and alleviating cellular inflammation.

Sulfated modification and anticoagulant activity of pumpkin (Cucurbita pepo, Lady Godiva) polysaccharide

Sulfated modification of pumpkin polysaccharide using CAS with pyridines as catalysts under different conditions was conducted to obtain different degrees of sulfation on a laboratory scale. Anticoagulant activities of pumpkin polysaccharide and its sulfated derivatives were also investigated employing various established in vitro systems. Results showed that addition of high ratio of CAS/pyridine under constant conditions could increase the degree of substitution. Sulfate substitution was further confirmed by the FT-IR and 13C NMR analysis. The df values between 2.11–2.73 indicated the relatively expanded conformation of the sulfated derivatives. The sulfated polysaccharides showed higher anticoagulant activities through activated partial thrombosis time (aPTT), thrombin time (TT), prothrombin time (PT) and anti-Xa activity assay, which revealed that better anticoagulant activities could be obtained when DS remained higher and Mw maintained in a moderate range.

How members of the human gut microbiota overcome the sulfation problem posed by glycosaminoglycans

The human microbiota, which plays an important role in health and disease, uses complex carbohydrates as a major source of nutrients. Utilization hierarchy indicates that the host glycosaminoglycans heparin (Hep) and heparan sulfate (HS) are high-priority carbohydrates for Bacteroides thetaiotaomicron, a prominent member of the human microbiota. The sulfation patterns of these glycosaminoglycans are highly variable, which presents a significant enzymatic challenge to the polysaccharide lyases and sulfatases that mediate degradation. It is possible that the bacterium recruits lyases with highly plastic specificities and expresses a repertoire of enzymes that target substructures of the glycosaminoglycans with variable sulfation or that the glycans are desulfated before cleavage by the lyases. To distinguish between these mechanisms, the components of the B. thetaiotaomicron Hep/HS degrading apparatus were analyzed. The data showed that the bacterium expressed a single-surface endo-acting lyase that cleaved HS, reflecting its higher molecular weight compared with Hep. Both Hep and HS oligosaccharides imported into the periplasm were degraded by a repertoire of lyases, with each enzyme displaying specificity for substructures within these glycosaminoglycans that display a different degree of sulfation. Furthermore, the crystal structures of a key surface glycan binding protein, which is able to bind both Hep and HS, and periplasmic sulfatases reveal the major specificity determinants for these proteins. The locus described here is highly conserved within the human gut Bacteroides, indicating that the model developed is of generic relevance to this important microbial community.

Sulfated Alginate as a Mimic of Sulfated Glycosaminoglycans: Binding of Growth Factors and Effect on Stem Cell Behavior.

Sulfated glycosaminoglycans (GAGs) are principal elements of the extracellular matrix, where they are involved in a plethora of signaling pathways mainly via interactions with diverse proteins such as growth factors and cytokines. However, the mechanisms that drive these interactions are not yet clear, mostly because of the difficulty to access large quantities of homogeneously sulfated natural GAGs. In this work, GAG mimics are synthesized from readily available alginate with different degrees of sulfation (DS, from 0.8 to 2.6) by simple process. The effect of the DS is determined on the binding of basic fibroblast growth factor (FGF-2). The enzyme-linked immunosorbent assay demonstrates that the binding of FGF-2 is significantly greater for alginates with high DS as compared to unmodified and low sulfated analogs. These results are further applied to engineer FGF-2-loaded substrates for stem cell culturing using the layer-by-layer approach. All films support the attachment and growth of adipose derived stem cells (ADSCs). Noteworthy, highly sulfated alginates maintain the stemness of the ADSCs that exhibit remarkably long filopodia. These results can be exploited in the engineering of novel substrates that induce targeted cell behavior via controlled protein delivery and for tissue engineering constructs applicable in various regenerative approaches.

Preparation and sulfation of an α-glucan from Actinidia chinensis roots and their potential activities

In this study, one homopolysaccharide (ACPA1) with a molecular weight (Mw) of 5.5×103g/mol was prepared from the roots of Actinidia chinensis. It was characterized as an α-d-glucan consisting of predominant 4-linked α-d-Glcp residues branched at O-6. Three sulfated derivatives of ACPA1 (SA1, SA2 and SA3) with different degrees of sulfation (DS) were obtained by SO3-pyridine procedure. Moisture-preserving tests demonstrated that the sulfated derivatives with the highest DS values, SA1 and SA2, exhibited better moisture-preserving abilities than ACPA1 and SA3. All sulfated derivatives exerted stimulatory effects on phagocytosis activity and nitric oxide production by RAW 264.7 macrophages while ACPA1 did not. These findings suggested that the sulfated derivatives of ACPA1 might be used as moisture-preserving or immunopotentiating reagents.

In vitro anticancer activity of the laminarans from Far Eastern brown seaweeds and their sulfated derivatives

Brown seaweeds have drawn worldwide attention due to their involvement in many industrial applications. They produce neutral low molecular weight polysaccharides (laminarans) with beneficial biological activities. However, the anticancer activity and molecular mechanism of the laminarans and their sulfated derivatives have not been investigated in detail. Herein, the laminarans from brown seaweeds Saccharina cichorioides, Saccharina japonica, and Fucus evanescens were isolated. The laminarans from S. cichorioides and S. japonica were confirmed to contain a main chain of β-(1→3)-D-glucopyranose with single branches at C6. The laminaran from F. evanescens consisted of not only β-(1→3)-linked D- glucopyranose but also includes single β-(1→6)-linked D-glucose residues. The branches at C6 are presented as glucose or as gentiobiose. The sulfated laminarans with different degrees of sulfation were obtained by the chlorosulfonic acid-pyridine assay. In modified polysaccharides, the positions of sulfates are directly predetermined by the structure of native laminarans. The laminarans and their sulfated derivatives inhibited proliferation, colony formation, and migration of human colorectal adenocarcinoma, melanoma, and breast adenocarcinoma cells in different manners. The sulfated laminaran from F. evanescens possessed the highest anticancer activity in vitro and effectively prevented migration of breast adenocarcinoma cells by inhibiting of the Matrix Metalloproteinases 2 and 9 activity.

Time-dependent localization of high- and low-sulfated keratan sulfates in the song nuclei of developing zebra finches.

Keratan sulfate proteoglycans (KSPGs) and chondroitin sulfate proteoglycans (CSPGs) consist of a protein core with covalently attached glycosaminoglycan side chain. Although CSPGs are known to regulate the end of the critical period, the role of KSPGs in brain development remains unclear. Young male zebra finches memorise song templates during development. The brain regions that are responsible for song learning, known as song nuclei, are recognized as a suitable model for the study of brain development. To understand the potential role of KSPGs, here we examined the localization of KSs with different degrees of sulfation in the brain of developing male zebra finches. Exclusively in the song nuclei, an increase in expression of 5-D-4-positive (5-D-4(+)) high-sulfated KS started after hatching, and reached a plateau at the end of the sensory period, during which the young bird listens to and memorises the song of an adult tutor. By contrast, weak and ubiquitous expression of BCD-4(+) low-sulfated KS remained unchanged until the end of the sensory period, and first increased in the song nuclei at the end of the sensorimotor period, during which the young bird produces plastic songs. Immunoblot analysis showed that phosphacan was a common core protein of 5-D-4(+) KS and BCD-4(+) KS. Finally, we confirmed that the sulfotransferase responsible for the synthesis of high-sulfated KS was exclusively localised in the song nuclei. Our observations suggest that time-dependent localization of KSPGs with different sulfation patterns in the song nuclei may underlie song learning in developing male zebra finches.

Inhibitory effects of sulfated lentinan with different degree of sulfation against tobacco mosaic virus (TMV) in tobacco seedlings

The inhibitory effects of sulfated lentinan with different degrees of sulfation against tobacco mosaic virus (TMV) and the underlying mechanism were investigated. The results indicated that plants treated with increasing concentrations of sulfated lentinan, with increasing numbers of treatments and with increasing time after treatment had a decrease in the number of necrotic lesions, indicating a long-term protection against TMV that mimics vaccination. In addition, the levels of TMV-capsid protein (CP) transcripts decreased in distant leaves, indicating that sulfated lentinan induces systemic protection against TMV. The activities of the defense enzymes phenylalanine ammonia lyase (PAL) and lipoxygenase (LOX) and the amounts of several phenylpropanoid compounds (PPCs) were measured in control and treated plants without infection. A progressive increase in PAL activity was observed with increasing time after treatment, together with the accumulation of free and conjugated PPCs. In contrast, LOX activity remained unchanged. Interestingly, the increase in PAL activity showed a linear correlation with the decrease in necrotic lesions and the decrease in TMV-CP transcript level. Thus, sulfated lentinan induced systemic and long-term protection against TMV in tobacco plants that is determined, at least in part, by a sustained activation of PAL and the accumulation of PPCs with potential antiviral activity.