RESUMEN
3'-Phosphoadenosine-5'-phosphosulfate (PAPS) is the bioactive form of sulfate and is involved in all biological sulfation reactions. The enzymatic transformation method for PAPS is promising, but the low efficiency and high cost of enzyme purification and storage restrict its practical applications. Here, we reported PAPS biosynthesis with a protein crystalline inclusion (PCI)-based enzyme immobilization system. First, the in vivo crystalline inclusion protein CipA was identified as an efficient auto-assembly tag for immobilizing the bifunctional PAPS synthase (ASAK). After characterizing the pyrophosphokinase activity of a polyphosphate exonuclease PaPPX from Pseudomonas aeruginosa, and optimizing the linker fragment, auto-assembled enzymes ASAK-PT-CipA and PaPPX-PT-CipA were constructed. Then, the auto-assembled enzymes ASAK-PT-CipA and PaPPX-PT-CipA with high stability were co-expressed and immobilized for constructing a transformation system. The highest transformation rate of PAPS from ATP and sulfate reached 90%, and the immobilized enzyme can be reused 10 times. The present work provided a convenient, efficient, and easy to be enlarged auto-immobilization system for PAPS biosynthesis from ATP and sulfate. The immobilization system also represented a new approach to reduce the production cost of PAPS by facilitating the purification, storage, and reuse of related enzymes, and it would boost the studies on biotechnological production of glycosaminoglycans and sulfur-containing natural compounds.
Asunto(s)
Enzimas Inmovilizadas , Sulfato Adenililtransferasa , Sulfato Adenililtransferasa/genética , Sulfato Adenililtransferasa/química , Sulfato Adenililtransferasa/metabolismo , Sulfatos/metabolismo , Fosfoadenosina Fosfosulfato/metabolismo , Adenosina Trifosfato/metabolismoRESUMEN
Hyaluronan (HA) is a glycosaminoglycan polymer involved in cell phenotype change, inflammation modulation, and tumor metastasis progression. HA oligosaccharides have a higher solubility and drug-forming ability than polysaccharides. HA tetrasaccharide was reported as the smallest fragment required for inhibiting triple-negative breast cancer, but the anti-tumor activity of HA tetrasaccharide (HA4) and its sulfated derivatives in lung cancer is still unknown. In this study, HA4 was prepared via HA degradation by chondroitinase ABC (CSABC), while its sulfated derivatives were prepared by sulfur pyridine trioxide complex in N, N-dimethylformamide (DMF). Then, the anti-tumor activity was detected via MTT assay and xenograft tumor experiments, while the expression level change of apoptosis genes was analyzed by qRT-PCR. Electrospray mass spectrometry (ESI-MS) analysis showed several HA4 sulfated derivatives, GlcA2GlcNAc2 (SO3H)n contains 0-6 sulfation groups, which mainly contain 3-6, 2-3, and 0-1 sulfation groups were classified as HA4S1, HA4S2, and HA4S3, respectively. After the addition of 1.82 mg/mL HA4, HA4S1, HA4S2, and HA4S3, the cell viability of A549 cells was reduced to 81.2 %, 62.1 %, 50.3 %, and 65.9 %, respectively. Thus, HA4S2 was chosen for further measurement, the qRT-PCR results showed it significantly up-regulated the expression of genes in the apoptosis pathway. Moreover, HA4S2 exhibited stronger antitumor activity than HA4 in vivo and the tumor inhibition rate reached 36.90 %. In summary, this study indicated that the CSABC enzyme could effectively degrade HA into oligosaccharides, and sulfation modification was an effective method to enhance the antitumor activity of HA tetrasaccharides.
Asunto(s)
Adenocarcinoma del Pulmón , Ácido Hialurónico , Células A549 , Adenocarcinoma del Pulmón/tratamiento farmacológico , Condroitina ABC Liasa , Dimetilformamida , Humanos , Ácido Hialurónico/química , Ácido Hialurónico/metabolismo , Ácido Hialurónico/farmacología , Oligosacáridos/química , Polímeros , Piridinas , Sulfatos , Azufre , Óxidos de AzufreRESUMEN
OBJECTIVE: The formation of kidney stone is closely related to cell injury and crystal adhesion. METHOD: The sulfur trioxide-pyridine method was used to sulfate raw Undaria pinnatifida polysaccharide (UPP) with a molecular weight (Mw) of 8.33 kDa. Four polysaccharides with the sulfate group (-OSO3-) contents of 1.59% (UPP0), 6.03% (UPP1), 20.83% (UPP2), and 36.39% (UPP3) were obtained. The antioxidant activity of the four UPPs, the difference in oxidative damage inflicted by nano-CaOx monohydrate (nano-COM) on human proximal tubular epithelial (HK-2) cells before and after protection by UPPs, and the inhibitory effect on nano-COM adhesion were explored. RESULTS: Structural characterization showed that sulfation was successful. As the -OSO3- content in the UPPs was increased, the antioxidant activity and capability of the UPPs to regulate the growth of calcium oxalate (CaOx) crystals gradually increased. The damage caused by nano-COM crystals to HK-2 cells under protection by UPPs was weakened. This effect enhanced cell viability, enabled the maintenance of good cell morphology, reduced reactive oxygen species (ROS) levels, and inhibited the decrease in mitochondrial membrane potential, as well as decreased the eversion of phosphatidylserine (PS) and the expression of the adhesion proteins osteopontin (OPN), heat shock protein (HSP 90), and Annexin A1 (ANXA1). The adhesion of nano-COM to HK-2 cells was inhibited under the protection by UPPs. CONCLUSION: UPP3 with the highest content of -OSO3- presented the best antioxidant activity and crystal regulation ability, while UPP2 with the second highest -OSO3- content showed optimal cell protection ability and crystal adhesion inhibition ability. The biological activity of UPPs was regulated by Mw and -OSO3- content. UPP2 with moderate -OSO3- content may become a potential drug for preventing CaOx stones.
Asunto(s)
Cálculos Renales , Undaria , Antioxidantes/farmacología , Oxalato de Calcio/química , Células Epiteliales , Humanos , Cálculos Renales/tratamiento farmacológico , Polisacáridos/farmacología , Sulfatos/farmacología , Undaria/metabolismoRESUMEN
Chondroitin sulfate (CS) extracted from animal tissues has been widely used as nutraceutical and pharmaceutical products for osteoarthritis treatment. Here we developed an efficient sulfation-modification system for large scale preparation of CSA in vitro. First, the expression level of C4ST was improved by 30 times with fusion of the chaperone SUMO. Then, glycerol as a protein stabilizer was found to improve rat AST IV stability during the regeneration of cofactor PAPS. Then peptide linkers or protein scaffolds were employed to assemble AST IV and C4ST into artificial complexes to bring the enzymes and PAPS spatially closer and enhance the catalytic efficiency of chondroitin sulfation. Eventually, the system was scaled up to 1 L system and 15 g chondroitin was converted to CSA in 24 h, with a 98 % conversion. The present study made a step further towards the industrial production of CSA with different sulfation degrees.
Asunto(s)
Arilsulfotransferasa/metabolismo , Sulfatos de Condroitina/biosíntesis , Ingeniería Metabólica/métodos , Sulfotransferasas/metabolismo , Adenosina Difosfato/metabolismo , Animales , Escherichia coli/enzimología , Escherichia coli/genética , Cinética , Organismos Modificados Genéticamente/metabolismo , Plásmidos/genética , Ratas , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomycetales/genética , Saccharomycetales/metabolismo , Solubilidad , Biología Sintética/métodosRESUMEN
The aim of this study was to investigate the effect of sulfated modification on the in vitro antioxidant and hypoglycemic activities of Sargassum pallidum polysaccharides (SPP). Three sulfated derivatives (S-SPP1-4, S-SPP1-6 and S-SPP1-8) were prepared using chlorosulfonic acid-pyridine method under different reaction conditions. Physicochemical characterization indicated that sulfated modification had successfully occurred. The degrees of substitution (DS) of S-SPP1-4, S-SPP1-6 and S-SPP1-8 were determined to be ranging from 0.85 to 1.19. Sulfated modification resulted in some changes in chemical component, molecular weight and monosaccharide composition of SPP. Among these sulfated polysaccharides, S-SPP1-4 exhibited the best DPPH and ABTS radical scavenging activities, while S-SPP1-8 exhibited the best α-glucosidase inhibitory activity and promoting-effect on glucose consumption in insulin resistant (IR)-HepG2 cells. Furthermore, all the sulfated derivatives exhibited better hypoglycemic ability than native SPP. These results suggest that appropriately sulfated modification could enhance the antioxidant and hypoglycemic activities of S. pallidum polysaccharides, which might be used as an alternative derivative of natural antioxidant and hypoglycemic agent.
Asunto(s)
Polisacáridos/química , Polisacáridos/farmacología , Sargassum/química , Sulfatos/química , Antioxidantes/química , Antioxidantes/farmacología , Glucosa/metabolismo , Inhibidores de Glicósido Hidrolasas/química , Inhibidores de Glicósido Hidrolasas/farmacología , Células Hep G2 , Humanos , Hipoglucemiantes/química , Hipoglucemiantes/farmacología , Peso Molecular , Monosacáridos/análisis , Relación Estructura-ActividadRESUMEN
Heparan sulfate is a component of the extracellular matrix (ECM) that modulates individual development and cell growth through its interaction with growth factors. Structurally, heparan sulfate consists of repeating linear sulfated poly-anionic disaccharide structures. The K5 polysaccharide has the same structure as heparosan, and is the capsular polysaccharide of Escherichia coli K5 strain which serves as a precursor in heparin and heparan sulfate biosynthesis. Here, we prepared sulfated K5 polysaccharides that are structurally similar to heparan sulfate and investigated their biocompatibility and bioactivity in stem cell chondrogenic differentiation. Briefly, sulfation groups were added to -NH- and/or -OH of a precursor heparosan and the modified heparosan was qualitatively analyzed by FT-IR, (1)H NMR, and (13)C NMR techniques. Cell viability was not significantly affected by the sulfated K5 capsular polysaccharide. Relative mRNA expression of the chondrogenic differentiation marker COL2A1 was significantly upregulated in cells treated with the N,O-sulfated K5 polysaccharide confirming that the sulfated K5 capsular polysaccharide is able to stimulate chondrogenic differentiation. The main sulfation pattern for chondrogenic activity is N,6-O sulfation and the activity was not proportional to the sulfation level. This type of mimic was prepared in nearly a gram scale, supporting further structural study and 3 dimension stem cell culture. Together, the results of this study show that sulfated K5 capsular polysaccharides are able to stimulate chondrogenic differentiation without affecting cell viability.
Asunto(s)
Materiales Biocompatibles/química , Materiales Biocompatibles/farmacología , Diferenciación Celular/efectos de los fármacos , Condrogénesis/efectos de los fármacos , Heparitina Sulfato/química , Heparitina Sulfato/farmacología , Células Madre Mesenquimatosas/citología , Animales , Materiales Biocompatibles/síntesis química , Células de la Médula Ósea/citología , Condrocitos/citología , Colágeno Tipo II/genética , Regulación de la Expresión Génica/efectos de los fármacos , Heparitina Sulfato/síntesis química , Células Madre Mesenquimatosas/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Sulfatos/químicaRESUMEN
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.