Journal of applied glycoscience最新文献

Glycogen is a highly branched storage polysaccharide found mainly in the liver and the muscles. Glycogen is also present in the skin, but its functional role is poorly understood. Recently, it has been reported that glycogen plays an important role in intracellular signal transduction. In the epidermis of the skin, keratinocytes are the predominant cells that produce ceramide. Ceramides are lipids composed of sphingosine, and prevent water loss, as well as protecting the skin against environmental stressors. In this study, we investigated the effects of glycogen on ceramide production in cultured keratinocytes. Thin-layer chromatography revealed that incubation of keratinocytes with 2 % glycogen enhanced the cellular amount of ceramide NS (ceramide 2) by 3.4-fold compared to the control. We also found that glycogen regulated the mRNA expression levels of signaling molecules of the sphingomyelin-ceramide pathway by quantitative real-time PCR. The activity of sphingomyelinase was also significantly enhanced by 2.5-fold in cultures with 1 % glycogen compared to the control. Moreover, glycogen increased the ATP production by 1.5-fold compared to the control, while glucose did not affect the production. Western blotting showed that phosphorylation of Akt, a cellular signaling molecule, was inhibited in the presence of glycogen in cultured keratinocytes. This study shows that glycogen upregulates the ceramide production pathway from sphingomyelin in epidermal keratinocytes, and provides new insights into the role of glycogen in cellular signal transduction.

Human urinary trypsin inhibitor (UTI) is a proteoglycan composed of one core protein covalently linked to one glycosaminoglycan, which is a low sulfated chondroitin 4-sulfate. It is used as an anti-inflammatory medicine based on the protease inhibitory activity of the core protein. However, functions of the chondroitin sulfate have not been clarified. Recently, we succeeded in remodeling the UTI chondroitin sulfate to hyaluronan to create hyaluronan hybrid UTI, without changing the core protein. Here, we investigated the effect of the remodeled chondroitin sulfate on the activities of serine proteases. Native UTI showed stronger protease inhibitory activity than hyaluronan hybrid UTI or hydrolyzed glycosaminoglycan UTI. Chondroitin 4-sulfate chains with a small peptide derived from the native UTI did not show any protease inhibitory activity. These results suggest that the chondroitin sulfate chain linked covalently to core protein enhances protease inhibitor activity of UTI although the chondroitin sulfate chain itself does not.

Starch biosynthetic enzymes form multi-protein complexes consisting of starch synthase (SS) I, SSIIa, and starch branching enzyme (BE) IIb, which synthesize amylopectin clusters. This study analyzed the starch properties in two double mutant rice lines lacking SSIIa and BEIIb, one of which expressed an inactive BEIIb protein. The ss2a be2b lines showed similar or greater seed weight than the be2b lines, and plant growth was not affected. The ss2a line showed increased short amylopectin chains resulting in a lower gelatinization temperature. Starch granule morphology and A-type crystallinity were similar between the ss2a line and the wild type, except for a mild chalky seed phenotype in the ss2a line. However, the starch phenotype of the ss2a be2b lines, which was similar to that of be2b but not ss2a, was characterized by increased long amylopectin chains, abnormal starch granules, and B-type crystallinity. The similarity in phenotype between the ss2a be2b and be2b lines may be attributed to the inability of the be2b mutants to generate short amylopectin branches, which serve as primers for SSIIa. Therefore, the presence or absence of SSIIa hardly affected the amylopectin structure under the be2b background. The amylose content was significantly higher in the ss2a be2b lines than in the be2b lines. Starch crystallinity was greater in ss2a be2b lines than in be2b lines, despite the fact that starch crystallinity is generally negatively correlated with amylose content. This suggests that the formation of a double helix between long amylopectin chains and amylose affects starch crystallinity in the ss2a be2b mutants.

Cellobiohydrolase I from Trichoderma reesei ( Tr Cel7A) is one of the best-studied cellulases, exhibiting high activity towards crystalline cellulose. Tryptophan residues at subsites -7 and -4 (Trp40 and Trp38 respectively) are located at the entrance and middle of the tunnel-like active site of Tr Cel7A, and are conserved among the GH family 7 cellobiohydrolases. Trp40 of Tr Cel7A is important for the recruitment of cellulose chain ends on the substrate surface, but the role of Trp38 is less clear. Comparison of the effects of W38A and W40A mutations on the binding energies of sugar units at the two subsites indicated that the contribution of Trp38 to the binding was greater than that of Trp40. In addition, the smooth gradient of binding energy was broken in W38A mutant. To clarify the importance of Trp38, the activities of Tr Cel7A WT and W38A towards crystalline cellulose and amorphous cellulose were compared. W38A was more active than WT towards amorphous cellulose, whereas its activity towards crystalline cellulose was only one-tenth of that of WT. To quantify the effect of mutation at subsite -4, we measured kinetic parameters of Tr Cel7A WT, W40A and W38A towards cello-oligosaccharides. All combinations of enzymes and substrates showed substrate inhibition, and comparison of the inhibition constants showed that the Trp38 residue increases the velocity of substrate intake ( k _on for forming productive complex) from the minus side of the subsites. These results indicate a key role of Trp38 residue in processively loading the reducing-end of cellulose chain into the catalytic tunnel.

Most functional biopharmaceuticals such as antibodies are glycoproteins carrying N-linked oligosaccharides (N-glycans). In animal cells, these glycans are generally expressed as heterogeneous glycoforms that are difficult to separate into a pure form. The structure of these glycans directly affects several biological aspects of the glycoproteins, especially binding affinity. Therefore, the preparation of glycoproteins with well-defined and homogeneous glycoforms is necessary for functional studies and improved efficacy, particularly for biopharmaceuticals. This review describes the recent remarkable progress in the development and production of biopharmaceutical glycan-modified antibodies, through the use of glycan remodeling using microbial endoglycosidases and sophisticated glycoengineering techniques utilizing microbial enzymatic reaction mechanisms.

The genus Pestalotiopsis are endophytic fungi that have recently been identified as cellulolytic system producers. We herein cloned a gene coding for a xylanase belonging to glycoside hydrolase (GH) family 10 (PesXyn10A) from Pestalotiopsis sp. AN-7, which was isolated from the soil of a mangrove forest. This protein was heterologously expressed by Pichia pastoris as a host, and its enzymatic properties were characterized. PesXyn10A was produced as a glycosylated protein and coincident to theoretical molecular weight (35.3 kDa) after deglycosylation by peptide-NfF-glycosidase F. Purified recombinant PesXyn10A exhibited maximal activity at pH 6.0 and 50 °C, and activity was maintained at 90 % at pH 5.0 and temperatures lower than 30 °C for 24 h. The substrate specificity of PesXyn10A was limited and it hydrolyzed glucuronoxylan and arabinoxylan, but not β-glucan. The final hydrolysis products from birchwood xylan were xylose, xylobiose, and 1,2³-α-D-(4-O-methyl-glucuronyl)-1,4-β-D-xylotriose. The addition of metallic salts (NaCl, KCl, MgCl₂, and CaCl₂) activated PesXyn10A for xylan degradation, and maximal activation by these divalent cations was approximately 160 % at a concentration of 5 mM. The thermostability of PesXyn10A significantly increased in the presence of 50 mM NaCl or 5 mM MgCl₂. The present results suggest that the presence of metallic salts at a low concentration, similar to brackish water, exerts positive effects on the enzyme activity and thermal stability of PesXyn10A.

Utilizing transglycosylation reaction catalyzed by β- N -acetylhexosaminidase of Stenotrophomonas maltophilia , β-D-fructofuranosyl-(2↔1)-α- N , N ´diacetylchitobioside (GlcNAc ₂ -Fru) was synthesized from N -acetylsucrosamine and N , N ´-diacetylchitobiose (GlcNAc ₂ ), and β-D-fructofuranosyl-(2↔1)-α- N , N ´, N ´´-triacetylchitotrioside (GlcNAc ₃ -Fru) was synthesized from GlcNAc ₂ -Fru and GlcNAc ₂ . Through purification by charcoal column chromatography, pure GlcNAc ₂ -Fru and GlcNAc ₃ -Fru were obtained in molar yields of 33.0 % and 11.7 % from GlcNAc ₂ , respectively. The structures of these oligosaccharides were confirmed by comparing instrumental analysis data of fragments obtained by enzymatic hydrolysis and acid hydrolysis of them with known data of these fragments.

Hexokinases catalyze glucose phosphorylation at the first step in glycolysis in eukaryotes. In the budding yeast Saccharomyces cerevisiae , three enzymes for glucose phosphorylation have long been known: Hxk1, Hxk2, and Glk1. In this study, we focus on Emi2, a previously uncharacterized hexokinase-like protein of S. cerevisiae . Our data show that the recombinant Emi2 protein (rEmi2), expressed in Escherichia coli , possesses glucose-phosphorylating activity in the presence of ATP and Mg ²⁺ . It was also found that rEmi2 phosphorylates not only glucose but also fructose, mannose and glucosamine in vitro . In addition, we examined changes in the level of endogenous Emi2 protein in S. cerevisiae in the presence or absence of glucose and a non-fermentable carbon source. We found that the expression of Emi2 protein is tightly suppressed during proliferation in high glucose, while it is strongly upregulated in response to glucose limitation and the presence of a non-fermentable carbon source. Our data suggest that the expression of the endogenous Emi2 protein in S. cerevisiae is regulated under the control of Hxk2 in response to glucose availability in the environment.

Glycopolymers have attracted increased attention as functional polymeric materials, and simple methods for synthesizing glycopolymers remain needed. This paper reports the aqueous one-pot and chemoenzymatic synthesis of four types of glycopolymers via two reactions: the β-galactosidase-catalyzed glycomonomer synthesis using 4,6-dimetoxy triazinyl β-D-galactopyranoside and hydroxy group-containing (meth)acrylamide and (meth)acrylate derivatives as the activated glycosyl donor substrate and as the glycomonomer precursors, respectively, followed by radical copolymerization of the resulting glycomonomer and excess glycomonomer precursor without isolating the glycomonomers. The resulting glycopolymers bearing galactose moieties exhibited specific and strong interactions with the lectin peanut agglutinin as glycoclusters.

The browning, gelatinization of starch, water sorption, glass transition, and caking properties of freeze-dried maca ( Lepidium meyenii Walpers) powders were investigated and compared with a commercial maca powder. The freeze-dried maca powders had lower optical density (browning) and higher enthalpy change for starch gelatinization than the commercial maca. This resulted from a difference in thermal history. The equilibrium water contents of the freeze-dried maca powders were higher than those of commercial maca at each water activity ( a _w ) because of differences in amorphous part. The glass transition temperature ( T _g ) was evaluated by differential scanning calorimetry. There was a negligible difference in the anhydrous T _g (79.5-80.2 ºC) among the samples. The T _g -depression of freeze-dried maca powders induced by water sorption was more gradual than that of the commercial maca due to a difference in water insoluble material content. From the results, critical water activity ( a _wc ) was determined as the a _w at which T _g becomes 25 ºC. There was negligible caking below a _w = 0.328. At higher a _w , the degree of caking remarkably increased with a large variation depending on the samples. The degree of caking could be described uniformly as a function of a _w / a _wc . From these results, we propose an empirical approach to predict the caking of maca powders.