Toshihiko Toida

The proteoglycan bikunin has a defined sequence

Proteoglycans are complex glycoconjugates that regulate critical biological pathways in all higher organisms. Bikunin, the simplest proteoglycan having a single glycosaminoglycan chain, is a serine protease inhibitor used to treat acute pancreatitis. Unlike the template driven synthesis of nucleic acids and proteins, Golgi synthesized glycosaminoglycans are not believed to have predictable or deterministic sequence. Bikunin peptidoglycosaminoglycans were prepared and fractionated to obtain a collection of size similar and charge similar chains. Fourier transform mass spectral analysis identified a small number of parent molecular-ions corresponding to mono-compositional peptidoglycosaminoglycans. Fragmentation using collision induced dissociation surprisingly afforded a single sequence for each mono-compositional parent-ion, unequivocally demonstrating the presence of a defined sequence. The common biosynthetic pathway for all proteoglycans suggests that even more structurally complex proteoglycans, such as heparan sulfate, may have defined sequences, requiring a readjustment of our understanding of information storage in complex glycans.

Structural analysis of bikunin glycosaminoglycan.

The structure of an intact glycosaminoglycan (GAG) chain of the bikunin proteoglycan (PG) was analyzed using a combined top-down and bottom-up sequencing strategy. PGs are proteins with one or more linear, high-molecular weight, sulfated GAG polysaccharides O-linked to serine or threonine residues. GAGs are often responsible for the biological functions of PGs, and subtle variations in the GAG structure have pronounced physiological effects. Bikunin is a serine protease inhibitor found in human amniotic fluid, plasma, and urine. Bikunin is posttranslationally modified with a chondroitin sulfate (CS) chain, O-linked to a serine residue of the core protein. Recent studies have shown that the CS chain of bikunin plays an important role in the physiological and pathological functions of this PG. While no PG or GAG has yet been sequenced, bikunin, the least complex PG, offers a compelling target. Electrospray ionization Fourier transform-ion cyclotron resonance mass spectrometry (ESI FTICR-MS) permitted the identification of several major components in the GAG mixture having molecular masses in a range of 5505-7102 Da. This is the first report of a mass spectrum of an intact GAG component of a PG. FTICR-MS analysis of a size-uniform fraction of bikunin GAG mixture obtained by preparative polyacrylamide gel electrophoresis, allowed the determination of chain length and number of sulfo groups in the intact GAGs.

Binding of spermine and ifenprodil to a purified, soluble regulatory domain of the N-methyl-d-aspartate receptor

The binding of spermine and ifenprodil to the amino terminal regulatory (R) domain of the N‐methyl‐D‐aspartate receptor was studied using purified regulatory domains of the NR1, NR2A and NR2B subunits, termed NR1‐R, NR2A‐R and NR2B‐R. The R domains were over‐expressed in Escherichia coli and purified to near homogeneity. The Kd values for binding of [14C]spermine to NR1‐R, NR2A‐R and NR2B‐R were 19, 140, and 33u2003μM, respectively. [3H]Ifenprodil bound to NR1‐R (Kd, 0.18u2003μM) and NR2B‐R (Kd, 0.21u2003μM), but not to NR2A‐R at the concentrations tested (0.1–0.8u2003μM). These Kd values were confirmed by circular dichroism measurements. The Kd values reflected their effective concentrations at intact NR1/NR2A and NR1/NR2B receptors. The results suggest that effects of spermine and ifenprodil on NMDA receptors occur through binding to the regulatory domains of the NR1, NR2A and NR2B subunits. The binding capacity of spermine or ifenprodil to a mixture of NR1‐R and NR2A‐R or NR1‐R and NR2B‐R was additive with that of each individual R domain. Binding of spermine to NR1‐R and NR2B‐R was not inhibited by ifenprodil and vice versa, indicating that the binding sites for spermine and ifenprodil on NR1‐R and NR2B‐R are distinct.

Brain infarction correlates more closely with acrolein than with reactive oxygen species.

Although it is thought that the major factor responsible for cell damage is reactive oxygen species (ROS), our recent studies have shown that acrolein is more toxic than ROS. Thus, the relative importance of acrolein and ROS in cell damage during brain infarction was compared using photochemically induced thrombosis model mice. The levels of acrolein-conjugated albumin, and of 4-hydroxynonenal (HNE)-conjugated albumin and 8-OHdG were evaluated as indicators of damage produced by acrolein and ROS, respectively. The increase in acrolein-conjugated albumin was much greater than the increase in HNE-conjugated albumin or 8-OHdG, suggesting that acrolein is more strongly involved in cell damage than ROS during brain infarction. It was also shown that infarction led more readily to RNA damage than to DNA or phospholipid damage. As a consequence, polyamines were released from RNA, and acrolein was produced from polyamines, especially from spermine by spermine oxidase. Production of acrolein from spermine by spermine oxidase was clarified using spermine synthase-deficient Gy mice and transglutaminase 2-knockout mice, in which spermine content is negligible or spermidine/spermine N(1)-acetyltransferase activity is elevated.

Correlation between images of silent brain infarction, carotid atherosclerosis and white matter hyperintensity, and plasma levels of acrolein, IL-6 and CRP

OBJECTIVEnWe found previously that the measurement of plasma levels of protein-conjugated acrolein (PC-Acro) together with IL-6 and CRP can be used to identify silent brain infarction (SBI) with high sensitivity and specificity. The aim of this study was to clarify how three biochemical markers are correlated to SBI, carotid atherosclerosis (CA) and white matter hyperintensity (WMH).nnnMETHODSnThe levels of PC-Acro, IL-6 and CRP in plasma were measured by ELISA. SBI and WMH were evaluated by MRI, and CA was evaluated by duplex carotid ultrasonography.nnnRESULTSnA total of 790 apparently healthy volunteers were classified into 260 control, 214 SBI, 263 CA and 245 WMH subjects, which included 187 subjects with two or three pathologies. When the combined measurements of PC-Acro, IL-6 and CRP were evaluated together with age, using a receiver operating characteristic curve and artificial neural networks, the relative risk value (RRV), an indicator of tissue damage, was in the order SBI with CA (0.90)>SBI (0.80)>CA (0.76)>WMH with CA (0.65)>WMH (0.46)>control (0.14). RRV was also correlated with severity in each group of SBI, CA and WMH.nnnCONCLUSIONnThe RRV supports the idea that the degree of risk to develop a stroke is in the order SBI>CA>WMH.

Characterization and antitumor effect of a novel polysaccharide from Grifola frondosa

A novel polysaccharide, MZF, with a molecular mass of 23 kDa was isolated from Grifola frondosa . Results from methylation and (1)H NMR led to the conclusion that MZF is a heteropolysaccharide consisting of -->6)-alpha-D-Galp-(1--> (36.2%), -->3)-alpha-L-Fucp-(1--> (14.5%), -->6)-alpha-D-Manp-(1--> (9.4%), -->3)-beta-D-Glcp-(1--> (10.1%), alpha-D-Manp-(1--> (23.2%), and -->3,6)-beta-D-Glcp-(1--> (6.5%). Although MZF did not affect the proliferation of colon-26 cells in vitro, it significantly inhibited tumor growth in BALB/cA mice inoculated with colon-26 cancer cells. Moreover, MZF significantly induced the proliferation of splenocytes and peritoneal macrophages. The mRNA expression of IL-12p40, IL-2 and IFN-gamma were increased significantly in MZF-treated spleen. Furthermore, MZF augmented the percentage of IFN-gamma-producing cells in both splenic CD4(+) and CD8(+) T cells and tumor infiltrating CD4(+) and CD8(+) T cells and enhanced the cytotoxic activity of NK cells and CTLs. These results indicate that MZF is a novel effective immunomodulator that has antitumor activity associated with induced cell-mediated immunity.

CS lyases: structure, activity, and applications in analysis and the treatment of diseases.

Publisher Summary Modern analytical methods, including nuclear magnetic resonance (NMR) and mass spectrometry (MS), are widely used for the determination of chondroitin sulfate (CS) structure. While modern spectroscopic techniques provide limited information on the structure of the intact CS polysaccharide, it is often useful to utilize chondroitin lyases to prepare oligosaccharides for more detailed structural determination. The structure, activity, and specificity of these enzymes are discussed in this chapter. These lyases can be combined with separation methods, such as chromatography and electrophoresis, for the preparation of CS oligosaccharides for biological evaluations, as well as for disaccharide analysis, oligosaccharide mapping, and polysaccharide sequencing. These enzymes have also been shown to have direct therapeutic value. This chapter examines the various applications of this important class of enzymes. CS is the predominant glycosaminoglycan (GAG) present in aggrecan, the major proteoglycan (PG) of cartilage. Because of the sulfo and carboxyl moieties of their GAG components, PGs concentrate large amounts of negative charge in the extracellular matrix. This has direct osmotic effects on these tissues in which the GAGs are under hydrated because of constraints imposed by the collagen fiber network, giving cartilage its shock‐absorbing function.

Photolytic depolymerization of alginate.

A photochemical reaction has been developed for the partial de-polymerization of sodium alginate, a polysaccharide utilized in medicine, pharmacy, basic sciences and foods. An aqueous solution of sodium alginate was photochemically depolymerized to approximately 40% of its average molecular weight using ultraviolet light in the presence of titanium dioxide catalyst at pH 7 over a period of 3h. The products were separated giving four fractions all having an average molecular weight that was smaller than that of the starting material. Characterization of the guluronate (G) and mannuronate (M) contents, and determination of the M/G ratio of photochemically depolymerized alginate, were accomplished using (1)H NMR spectroscopy. The resulting M/G ratio was compared to that obtained for alginate fractions produced by acid hydrolysis. The M and G content, of each alginate fraction, was also assigned with regards to their occurrence in G-rich, M-rich or M/G heteropolymeric domains. This new depolymerization method might also be applicable in the preparation of alginate oligosaccharides for use in the food and pharmaceutical industries.

Intense correlation between protein-conjugated acrolein and primary Sjögren's syndrome

BACKGROUNDnWe recently found that an increased plasma concentration of protein-conjugated acrolein is a good biomarker for stroke. Therefore we determine whether the concentration of protein-conjugated acrolein is increased in saliva from patients with primary Sjögrens syndrome.nnnMETHODSnStimulated whole-mixed saliva was collected from 10 patients and 13 control subjects. The concentration of protein-conjugated acrolein in saliva and plasma was measured by either Western blotting or enzyme-linked immunosorbent assay.nnnRESULTSnThe concentration of protein-conjugated acrolein, especially albumin-conjugated acrolein, was greatly increased in saliva from patients with primary Sjögrens syndrome (p<0.001). The concentration of protein-conjugated acrolein was inversely correlated with the flow rate of saliva.nnnCONCLUSIONnThe results indicate that the concentration of protein-conjugated acrolein, a marker of cell or tissue damage, in saliva is well correlated with seriousness of primary Sjögrens syndrome.

Structural characterization of glycosaminoglycans from zebrafish in different ages

The zebrafish (Danio rerio) is a popular model organism for the study of developmental biology, disease mechanisms, and drug discovery. Glycosaminoglycans (GAGs), located on animal cell membranes and in the extracellular matrix, are important molecules in cellular communication during development, in normal physiology and pathophysiology. Vertebrates commonly contain a variety of GAGs including chondroitin/dermatan sulfates, heparin/heparan sulfate, hyaluronan and keratan sulfate. Zebrafish might represent an excellent experimental organism to study the biological roles of GAGs. A recent study showing the absence of heparan sulfate in adult zebrafish, suggested a more detailed evaluation of the GAGs present in this important model organism needed to be undertaken. This report aimed at examining the structural alterations of different GAGs at the molecular level at different developmental stages. GAGs were isolated and purified from zebrafish in different stages in development ranging from 0.5xa0days to adult. The content and disaccharide composition of chondroitin sulfate and heparan sulfate were determined using chemical assays, liquid chromotography and mass spectrometry. The presence of HS in adult fish was also confirmed using 1H-NMR.