Efficient transfer of sialo-oligosaccharide onto proteins by combined use of a glycosynthase-like mutant of Mucor hiemalis endoglycosidase and synthetic sialo-complex-type sugar oxazoline

Background

An efficient method for synthesizing homogenous glycoproteins is essential for elucidating the structural and functional roles of glycans of glycoproteins. We have focused on the transglycosylation activity of endo-β-N-acetylglucosaminidase from Mucor hiemalis (Endo-M) as a tool for glycoconjugate syntheses, since it can transfer en bloc the oligosaccharide of not only high-mannose type but also complex-type N-glycan onto various acceptors having an N-acetylglucosamine residue. However, there are two major bottlenecks for its practical application: the low yield of the transglycosylation product and the difficulty to obtain the activated sugar oxazoline substrate, especially the sialo-complex type one.

Methods

We carried out the transglycosylation using a glycosynthase-like N175Q mutant of Endo-M, which was found to possess enhanced transglycosylation activity with sugar oxazoline as a donor substrate, in combination with an easy preparation of the sialo-complex-type sugar oxazoline from natural sialoglycopeptide in egg yolk.

Results

Endo-M-N175Q showed efficient transglycosylation toward sialo-complex-type sugar oxazoline onto bioactive peptides and bovine ribonuclease B, and each sialylated compound was obtained in significantly high yield.

Conclusions

Highly efficient and simple chemo-enzymatic syntheses of various sialylated compounds were enabled, by a combination of a simple synthesis of sialo-complex-type sugar oxazoline and the Endo-M-N175Q catalyzed transglycosylation.

General significance

Our method would be very useful for a practical synthesis of biologically important glycopeptides and glycoproteins.     

Detection of Sapovirus in oysters

SaV sequences which are either genetically identical or similar were detected from oysters, feces from gastroenteritis patients, and domestic wastewater samples in geographically close areas. This is the first report of the detection of SaV in oysters which meet the legal requirements for raw consumption in Japan.     

Fucoxanthin regulates adipocytokine mRNA expression in white adipose tissue of diabetic/obese KK-A mice

Fucoxanthin, a marine carotenoid found in edible brown seaweeds, attenuates white adipose tissue (WAT) weight gain and hyperglycemia in diabetic/obese KK-A^y mice, although it does not affect these parameters in lean C57BL/6J mice. In perigonadal and mesenteric WATs of KK-A^y mice fed fucoxanthin, mRNA expression levels of monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-α (TNF-α), which are considered to induce insulin resistance, were markedly reduced compared to control mice. In contrast to KK-A^y mice, fucoxanthin did not alter MCP-1 and TNF-α mRNA expression levels in the WAT of lean C57BL/6J mice. Interleukin-6 (IL-6) and plasminogen activator inhibitor-1 mRNA expression levels in WAT were also decreased by fucoxanthin in KK-A^y mice. In differentiating 3T3-F442A adipocytes, fucoxanthinol, which is a fucoxanthin metabolite found in WAT, attenuated TNF-α-induced MCP-1 and IL-6 mRNA overexpression and protein secretion into the culture medium. In addition, fucoxanthinol decreased TNF-α, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) mRNA expression in RAW264.7 macrophage-like cells stimulated by palmitic acid. These findings indicate that fucoxanthin regulates mRNA expression of inflammatory adipocytokines involved in insulin resistance, iNOS, and COX-2 in WAT and has specific effects on diabetic/obese KK-A^y mice, but not on lean C57BL/6J mice.     

A novel ryanodine receptor expressed in pancreatic islets by alternative splicing from type 2 ryanodine receptor gene

Cyclic ADP-ribose (cADPR), a potent Ca²⁺ mobilizing intracellular messenger synthesized by CD38, regulates the opening of ryanodine receptors (RyRs). Increases in intracellular Ca²⁺ concentrations in pancreatic islets, resulting from Ca²⁺ mobilization from RyRs as well as Ca²⁺ influx from extracellular sources, are important in insulin secretion by glucose. In the present study, by screening a rat islet cDNA library, we isolated a novel RyR cDNA (the islet-type RyR), which is generated from the RyR2 gene by alternative splicing of exons 4 and 75. When the expression vectors for the islet-type and the authentic RyRs were transfected into HEK293 cells, the islet-type RyR2 as well as the authentic one showed high affinity [³H]ryanodine binding. Intracellular Ca²⁺ release in the islet-type RyR2-transfected cells was enhanced in the presence of cADPR but not in the authentic RyR2-transfected cells. The islet-type RyR2 mRNA was expressed in a variety of tissues such as in pancreatic islets, cerebrum, and cerebellum, whereas the authentic RyR2 mRNA was predominantly expressed in heart and aorta. These results suggest that the islet-type RyR2 may be an intracellular target for cADPR signaling.     

<Emphasis Type="Italic">Dioscorea opposita</Emphasis> Thunb. α-mannosidase belongs to the glycosyl hydrolase family 38

α-Mannosidase (EC 3.2.1.24) was purified from ‘Iseimo’, a native variety of Dioscorea opposita Thunb. Before purification, we investigated the composition of a viscous polysaccharide that interferes with column chromatography procedures. The polysaccharide consisted mainly of mannose, and also contained uronic acid. We used the cationic detergent cetylpyridinium chloride (CPC) to remove the polysaccharide. CPC treatment decreased viscosity without affecting α-mannosidase activity. We purified α-mannosidase 2,650-fold. The optimal pH of the enzyme was 6.0 and the optimum temperature was 65°C. The K _m value for p-nitrophenyl-α-d-mannopyranoside was 0.35 ± 0.03 mM. Activity was inhibited by swainsonine but not kifunensine. The enzyme cleaved α-1,2 linkages preferentially to α-1,6 and α-1,3 linkages. The M _r of purified α-mannosidase was estimated to be 250–260 kDa by gel filtration and native-PAGE. Four polypeptides (86, 83, 80, and 28 kDa) were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is unclear whether the polypeptides are encoded by one gene or multiple genes. However, N-terminal sequence analysis suggested that post-translational cleavage and/or glycosylation resulted in the three large fragments, if these amino acids were encoded by the same gene. Homology searches and characterization of the enzyme’s properties indicated that Iseimo α-mannosidase belongs to the glycoside hydrolase family 38 proteins, and to the Class II mannosidase group.     

Production of biologically active IgG hinge-tag soluble epidermal growth factor receptors (ErbB)

The extracellular domains (ECD) of epidermal growth factor receptors, ErbB1, 2, 3 and 4, were designed as soluble dimeric forms. Each ECD was fused to a short hinge region derived from IgG, such that the stable dimer could be formed with disulfide bridges. This hinge-tagged design minimized the molecular weight to approximately 50% of the conventional Fc-fusion design without an Fc domain of IgG. The refolded dimers could be easily analyzed and characterized by SDS-PAGE. Hinge-tagged soluble ErbBs demonstrated significant affinity for betacellulin and heregulin. The IgG hinge-tag should be a simple method to design soluble dimers that would be useful for high throughput screening of ligands, antagonists or derivatives.     

Metabolic profiling and cytological analysis of proanthocyanidins in immature seeds of Arabidopsis thaliana flavonoid accumulation mutants

Arabidopsis TRANSPARENT TESTA19 (TT19) encodes a glutathione‐S‐transferase (GST)‐like protein that is involved in the accumulation of proanthocyanidins (PAs) in the seed coat. PA accumulation sites in tt19 immature seeds were observed as small vacuolar‐like structures, whereas those in tt12, a mutant of the tonoplast‐bound transporter of PAs, and tt12 tt19 were observed at peripheral regions of small vacuoles. We found that tt19 immature seeds had small spherical structures showing unique thick morphology by differential interference contrast microscopy. The distribution pattern of the thick structures overlapped the location of PA accumulation sites, and the thick structures were outlined with GFP‐TT12 proteins in tt19. PA analysis showed higher (eightfold) levels of solvent‐insoluble PAs in tt19 immature seeds compared with the wild type. Metabolic profiling of the solvent‐soluble fraction by LC‐MS demonstrated that PA derivatives such as epicatechins and epicatechin oligomers, although highly accumulated in the wild type, were absent in tt19. We also revealed that tt12 specifically accumulated glycosylated epicatechins, the putative transport substrates for TT12. tt12 tt19 showed a similar metabolic profile to tt19. Given the cytosolic localization of functional GFP‐TT19 proteins, our results suggest that TT19, which acts prior to TT12, functions in the cytosol to maintain the regular accumulation of PA precursors, such as epicatechin and glycosylated epicatechin, in the vacuole. The PA pathway in the Arabidopsis seed coat is discussed in relation to the subcellular localization of PA metabolites.     

Latency of the Lipid A Deacylase PagL Is Involved in Producing a Robust Permeation Barrier in the Outer Membrane of Salmonella enterica

Lipid A deacylase PagL, which detoxifies endotoxin, is latent in Salmonella enterica. This study determined the biological significance of this latency. PagL latency was beneficial for bacteria in producing a robust permeation barrier through lipid A modifications under host-mimetic conditions that induced the modification enzymes, including PagL.The outer layer of the outer membrane in enteric Gram-negative bacteria is exclusively occupied by lipopolysaccharide (LPS), which contains lipid A as the membrane anchor, while the inner layer contains phospholipids. This asymmetric lipid bilayer serves as a permeation barrier to a large number of noxious compounds. The strength of this barrier is due to the strong lateral interactions between LPS molecules and the low fluidity of the saturated fatty acid portion of lipid A in the outer membrane (reviewed in reference 20). Large hydrophilic compounds are excluded by narrow porin channels, and lipophilic compounds cross the asymmetric bilayer very slowly.The prototype lipid A structure synthesized in Salmonella enterica serovar Typhimurium (S. Typhimurium) is shown in Fig. ​Fig.11 A. In S. Typhimurium, lipid A is further modified by enzymes that are induced upon activation of the two-component regulatory system PhoP-PhoQ (Fig. ​(Fig.1B)1B) (9). PhoP-PhoQ is essential for Salmonella virulence (3, 6, 18), and PhoP-PhoQ-regulated lipid A modifications are involved in many aspects of virulence. PhoQ is a sensor histidine kinase that responds to environmental conditions, including those within mammalian tissues. The host environment is experimentally mimicked by magnesium limitation and/or mild acid pH in the culture medium (3, 4, 6, 18, 21). In response to specific environmental signals, PhoQ phosphorylates PhoP, leading to the activation of pagL and pagP, which encode outer membrane lipid A 3-O-deacylase and outer membrane lipid A palmitoyltransferase, respectively (2, 22). Lipid A 3-O-deacylation by PagL and palmitoylation by PagP reduce the ability of lipid A to activate host Toll-like receptor 4, indicating that PhoP-PhoQ-dependent lipid A modifications help pathogens evade innate immune recognition (12). The regulation of lpxO, which encodes lipid A hydroxylase, is also mediated, at least in part, by PhoP-PhoQ (5, 9). Activation of PhoP-PhoQ leads to the activation of a second two-component regulatory system, PmrA-PmrB (8, 10). PmrA-PmrB promotes the attachment of aminoarabinose and phosphoethanolamine to phosphate groups on lipid A, which are involved in bacterial resistance to cationic antimicrobial peptides (7, 15). Furthermore, PhoP-PhoQ activation produces a more robust permeation barrier in the outer membrane, and lipid A modifications are involved in the generation of this enhanced barrier (19). Mg²⁺ ions decreased membrane permeability strongly in a phoP-null strain but only modestly in a PhoP-constitutive strain (19), implying a biological relevance of lipid A modifications by magnesium limitation.Open in a separate windowFIG. 1.Structures of the prototype lipid A (A) and modified lipid A (B) of S. Typhimurium.Previous studies did not detect PagL-dependent lipid A deacylation when S. Typhimurium was grown under PhoP-PhoQ-activating conditions that induce PagL expression (11, 13, 22). In contrast, PagL-dependent lipid A deacylation was observed in pmrA-null and pmrE-null strains, both of which lacked aminoarabinose modification of lipid A (11, 13). These findings cannot be simply ascribed to the substrate specificity of PagL, since many lipid A species that are not modified with aminoarabinose exist in S. Typhimurium grown under PhoP-PhoQ-activating conditions (13). Therefore, it is thought that PagL is latent under these conditions and that aminoarabinose modification of lipid A is involved in the regulation of latency (13). PagL latency is consistent with an emerging paradigm of outer membrane enzyme regulation (1). It should be noted that PagL-dependent lipid A deacylation, which is beneficial for invading bacteria by allowing them to avoid Toll-like receptor 4 responses, would occur under some specific conditions such as those which activate PhoP-PhoQ without induction of lipid A aminoarabinose modification. Furthermore, we have identified several amino acid residues in the extracellular loops of PagL that are essential for latency but not for deacylase activity (17). The amino acid residues essential for latency were also necessary for PagL to associate with LPS (16). However, the biological significance of latency remains unknown.The influx rate of a lipophilic agent, ethidium bromide, is increased by a pmrA-null mutation in an S. Typhimurium strain with a PhoP-constitutive phenotype (19). The rate-limiting step of this influx is crossing of the asymmetric bilayer in the outer membrane. Therefore, these observations suggest that pmrA-dependent lipid A modifications, such as aminoarabinose and phosphoethanolamine attachment, help generate a more robust permeation barrier through PhoP-PhoQ activation. On the other hand, lipid A is deacylated by PagL in a pmrA strain under PhoP-PhoQ-activating conditions (13). These observations led us to examine whether PagL-dependent lipid A deacylation increases the membrane permeability of the pmrA mutant strain.     

Transglycosylation reaction catalyzed by a class V chitinase from cycad, Cycas revoluta: A study involving site-directed mutagenesis,HPLC, and real-time ESI-MS

Class V chitinase from cycad, Cycas revoluta, (CrChi-A) is the first plant chitinase that has been found to possess transglycosylation activity. To identify the structural determinants that bring about transglycosylation activity, we mutated two aromatic residues, Phe166 and Trp197, which are likely located in the acceptor binding site, and the mutated enzymes (F166A, W197A) were characterized. When the time-courses of the enzymatic reaction toward chitin oligosaccharides were monitored by HPLC, the specific activity was decreased to about 5–10% of that of the wild type and the amounts of transglycosylation products were significantly reduced by the individual mutations. From comparison between the reaction time-courses obtained by HPLC and real-time ESI-MS, we found that the transglycosylation reaction takes place under the conditions used for HPLC but not under the ESI-MS conditions. The higher substrate concentration (5 mM) used for the HPLC determination is likely to bring about chitinase-catalyzed transglycosylation. Kinetic analysis of the time-courses obtained by HPLC indicated that the sugar residue affinity of + 1 subsite was strongly reduced in both mutated enzymes, as compared with that of the wild type. The IC₅₀ value for the inhibitor allosamidin determined by real-time ESI-MS was not significantly affected by the individual mutations, indicating that the state of the allosamidin binding site (from − 3 to − 1 subsites) was not changed in the mutated enzymes. We concluded that the aromatic side chains of Phe166 and Trp197 in CrChi-A participate in the transglycosylation acceptor binding, thus controlling the transglycosylation activity of the enzyme.