RESUMEN
Solution nuclear magnetic resonance (NMR) analysis of polysaccharides can provide valuable information not only on their primary structures but also on their conformation, dynamics, and interactions under physiological conditions. One of the main problems is that non-anomeric 1H signals typically overlap, and this often hinders detailed NMR analysis. Isotope enrichment, such as with 13C and 15N, will add a new dimension to the NMR spectra of polysaccharides, and spectral analysis can be performed with enhanced sensitivity using isolated peaks. For this purpose, here we have prepared uniformly 13C- and/or 15N-labeled chondroitin polysaccharides -4)-ß-D-glucuronopyranosyl-(1-3)-2-acetamido-2-deoxy-ß-D-galactopyranosyl-(1- with molecular weights in the range from 310 to 460 k by bacterial fermentation. The enrichment ratios for 13C and 15N were 98.9 and 99.8%, respectively, based on the mass spectrometric analysis of the constituent chondroitin disaccharides. 1H and 13C NMR signals were assigned mainly based on HSQC and 13C-detection experiments including INADEQUATE, HETCOR, and HETCOR-TOCSY. The carbonyl carbon signal of the N-acetyl-ß-D-galactosamine residue was unambiguously distinguished from the C6 carbon of the ß-D-glucuronic acid residue by the observation of 13C peak splitting due to 1JCN coupling in 13C- and 15N-labeled chondroitin. The T2* and T1 were measured and indicate that both rigid and mobile sites are present in the long sequence of chondroitin. The conformation, dynamics, and interactions of chondroitin and its derivatives will be further analyzed based on the results obtained in this study.
Asunto(s)
Isótopos de Carbono , Espectroscopía de Resonancia Magnética , Peso Molecular , Isótopos de Nitrógeno , Espectroscopía de Resonancia Magnética/métodos , Condroitín/químicaRESUMEN
The structures and amounts of glycosaminoglycan (GAG) produced by cells have attracted much interest because GAG biosynthesis activity can change in cellular processes such as disease and differentiation. ß-Xylosides, also called saccharide primers, have been used as artificial acceptors not only to generate GAG oligosaccharides in cells and tissues but also to investigate their biosynthetic pathways. Various analytical methods have been applied to confirm the structure and amounts of GAG oligosaccharides elongated using saccharide primers, yet sample preparation processes such as solid-phase extraction in analysis can cause experimental error and disrupt accurate comparative quantification of glycosylated products. In this study, we developed a new quantification method using a deuterium-labeled saccharide primer. The "heavy" and "light" primers were chemically synthesized, and priming abilities were confirmed by liquid chromatography-tandem mass spectrometry. Relative peak areas of light/heavy products showed good linearity and were well correlated with the theoretical amounts of glycosylated products. Then, as a validation study, we carried out a biosynthesis inhibition assay using known GAG biosynthesis inhibitors. According to the relative quantification using saccharide primers, differences in the mode-of-action among the four GAG biosynthesis inhibitors were dependent on the GAG biosynthetic pathway. Our results indicate that the method will likely forge a new path for comparative glycosaminoglycomics using cultured cells and tissues.
Asunto(s)
Glicosaminoglicanos/análisis , Glicósidos/química , Marcaje Isotópico , Oligosacáridos/química , Azaserina/farmacología , Brefeldino A/farmacología , Línea Celular , Genisteína/farmacología , Glicosaminoglicanos/antagonistas & inhibidores , Glicosaminoglicanos/biosíntesis , Glicosilación , Humanos , Estructura Molecular , Rodaminas/farmacologíaRESUMEN
Chondroitin sulfate (CS) is a family of glycosaminoglycans and have a wide range of applications in dietary supplements and pharmaceutical drugs. In this study, we evaluated the effects of several types of CS, differing in their sulfated positions, on the human colonic microbiota and their metabolites. CS (CSA, CSC, and CSE) and non-sulfated chondroitin (CH) were added into an in vitro human colonic microbiota model with fecal samples from 10 healthy individuals. CS addition showed a tendency to increase the relative abundance of Bacteroides, Eubacterium, and Faecalibacterium, and CSC and CSE addition significantly increased the total number of eubacteria in the culture of the Kobe University Human Intestinal Microbiota Model. CSE addition also resulted in a significant increase in short-chain fatty acid (SCFA) levels. Furthermore, addition with CSC and CSE increased the levels of a wide range of metabolites including lysine, ornithine, and Ile-Pro-Pro, which could have beneficial effects on the host. However, significant increases in the total number of eubacteria, relative abundance of Bacteroides, and SCFA levels were also observed after addition with CH, and the trends in the effects of CH addition on metabolite concentrations were identical to those of CSC and CSE addition. These results provide novel insight into the contribution of the colonic microbiota to the beneficial effects of dietary CS.
Asunto(s)
Sulfatos de Condroitina , Microbiota , Humanos , Fermentación , Sulfatos , Glicosaminoglicanos , Bacteroides , Eubacterium , Óxidos de AzufreRESUMEN
Preoperative nutritional status is an important prognostic factor in gastric cancer patients. This study will evaluate whether preoperative oral dysfunction is associated with prognostic nutrition index (PNI). This case-control study analyzed 95 patients who underwent oral function management. We assessed the following parameters: body mass index, stage of gastric cancer, C-reactive protein, total lymphocyte count, albumin, and prognostic nutritional index. The patients were divided into groups with prognostic nutritional indexes <45 and >45. Logistic regression analysis was used to assess the association between the measurements of oral function and the prognostic nutritional index. Univariate analysis of factors associated with decreased oral function and prognostic nutritional index showed significant differences between the two groups in C-reactive protein, neutrophils, and tongue pressure (p<0.01). However, oral hygiene, oral dryness, occlusal force, tongue-lip motor function, masticatory function, and swallowing function were not significantly different. Multivariate analysis showed that C-reactive protein (odds ratio: 0.12, 95% confidence interval: 0.30-0.45, p<0.01) and tongue pressure (odds ratio: 3.62, 95% confidence interval: 1.04-12.60, p<0.05) were independent risk factors for oral hypofunction. Oral function decreased in perioperative patients with gastric cancer, and decreased tongue pressure is associated with a decreased prognostic nutritional index.
Asunto(s)
Evaluación Nutricional , Neoplasias Gástricas , Humanos , Estudios Retrospectivos , Estudios de Casos y Controles , Pronóstico , Neoplasias Gástricas/complicaciones , Neoplasias Gástricas/cirugía , Proteína C-Reactiva , Presión , Lengua , Estado NutricionalRESUMEN
ß-Xylosides have been used as an artificial initiator of glycosaminoglycan (GAG) biosynthesis to investigate its mechanism and to obtain these oligosaccharides. In GAG biosynthesis, phosphorylation on the xylose residue is a crucial step. However, little attention has been paid to phosphorylated oligosaccharides obtained from ß-xylosides. In a previous study, we demonstrated that a novel ß-xyloside, N-lauryl-O-ß-xyloyranosyl-serinamide (Xyl-Ser-C12), had excellent GAG-type oligosaccharide priming ability, whereas phosphorylated oligosaccharides were not found in the primed oligosaccharides. This study examines the potential of Xyl-Ser-C12 and three of its derivatives for use as a probe to investigate the GAG biosynthesis mechanism. Glycosylated products were obtained by incubation of the ß-xylosides in normal human dermal fibroblast cells and compared by liquid chromatography-electrospray ionization-mass spectrometry. By the optimized method to detect phosphorylated products, Xyl-Ser-C12 was demonstrated to prime not only GAG-type oligosaccharides but also a variety of xylose-phosphorylated products. Among the synthesized ß-xylosides, those consisting of xylosyl-serine primed large amounts of phosphorylated and GAG-type oligosaccharides, whereas the others primed sialyloligosaccharides mainly. The majority of the phosphorylated products were considered to be GAG intermediates, which are less observed in nature. To our best knowledge, this is the first report showing that the amino acid residues around the Xyl attachment position strongly affect the phosphorylation efficiency and GAG chain-priming ability of ß-xylosides. This study leads to the possibility of the use of ß-xyloside as a probe to observe the Xyl phosphorylation process during GAG biosynthesis and investigate comparative glycosaminoglycomics between different cells.
RESUMEN
This study examines the non-covalent interactions between glycosaminoglycan (GAG) oligosaccharides using nanoelectrospray ionization mass spectrometry (nanoESI-MS). It is the first time that interactions between oligosaccharides have been observed using MS. The importance of interactions between GAGs has recently attracted much interest because they are related to biological functions. For instance, hyaluronic acid (HA) is known to associate with chondroitin sulfates (CSs), although the details of the interaction remain unclear. In general, non-covalent interactions between glycans are too weak to detect by general means. In this work, we applied nanoESI-MS with high sensitivity, which is widely used to observe non-covalent interactions, to investigate the interaction between HA and CSs. HA and CS oligosaccharides are used to discuss the results in a simplified manner. Our approach is aimed at interpreting the behavior of GAG polysaccharides from the information obtained using the oligosaccharides. HA and CS tetrasaccharides were demonstrated to associate to form heterodimer ions that were easily detected using nanoESI-MS. We also determined the stoichiometry of the interaction and calculated the K(d) values of the interactions between HA and CS tetrasaccharides. How these structures affect the strength and stability of the non-covalent complexes is discussed. Further study of the interactions between HA and CS oligosaccharides will clarify the biological meaning of the coexistence of HA and CS in body fluids and tissues.