Induction of Anti-influenza Immunity by Modified Green Fluorescent Protein (GFP) Carrying Hemagglutinin-derived Epitope Structure

The development of vaccination methods that can overcome the emergence of new types of influenza strains caused by escape mutations is desirable to avoid future pandemics. Here, a novel type of immunogen was designed that targeted the conformation of a highly conserved region of influenza A virus hemagglutinin (HA) composed of two separate sequences that associate to form an anti-parallel β-sheet structure. Our previous study identified this β-sheet region as the structural core in the epitope of a characteristic antibody (B-1) that strongly neutralizes a wide variety of strains within the H3N2 serotype, and therefore this β-sheet region was considered a good target to induce broadly reactive immunity against the influenza A virus. To design the immunogen, residues derived from the B-1 epitope were introduced directly onto a part of enhanced green fluorescent protein (EGFP), whose surface is mostly composed of β-sheets. Through site-directed mutagenesis, several modified EGFPs with an epitope-mimicking structure embedded in their surface were prepared. Two EGFP variants, differing from wild-type (parental) EGFP by only five and nine residues, induced mice to produce antibodies that specifically bind to H3-type HA and neutralize H3N2 virus. Moreover, three of five mice immunized with each of these EGFP variants followed by a booster with equivalent mCherry variants acquired anti-viral immunity against challenge with H3N2 virus at a lethal dosage. In contrast to conventional methods, such as split HA vaccine, preparation of this type of immunogen requires less time and is therefore expected to be quickly responsive to newly emerged influenza viral strains.     

EXTL2, a Member of the EXT Family of Tumor Suppressors,Controls Glycosaminoglycan Biosynthesis in a Xylose Kinase-dependent Manner

Mutant alleles of EXT1 or EXT2, two members of the EXT gene family, are causative agents in hereditary multiple exostoses, and their gene products function together as a polymerase in the biosynthesis of heparan sulfate. EXTL2, one of three EXT-like genes in the human genome that are homologous to EXT1 and EXT2, encodes a transferase that adds not only GlcNAc but also N-acetylgalactosamine to the glycosaminoglycan (GAG)-protein linkage region via an α1,4-linkage. However, both the role of EXTL2 in the biosynthesis of GAGs and the biological significance of EXTL2 remain unclear. Here we show that EXTL2 transfers a GlcNAc residue to the tetrasaccharide linkage region that is phosphorylated by a xylose kinase 1 (FAM20B) and thereby terminates chain elongation. We isolated an oligosaccharide from the mouse liver, which was not detected in EXTL2 knock-out mice. Based on structural analysis by a combination of glycosidase digestion and 500-MHz ¹H NMR spectroscopy, the oligosaccharide was found to be GlcNAcα1-4GlcUAβ1–3Galβ1–3Galβ1–4Xyl(2-O-phosphate), which was considered to be a biosynthetic intermediate of an immature GAG chain. Indeed, EXTL2 specifically transferred a GlcNAc residue to a phosphorylated linkage tetrasaccharide, GlcUAβ1–3Galβ1–3Galβ1–4Xyl(2-O-phosphate). Remarkably, the phosphorylated linkage pentasaccharide generated by EXTL2 was not used as an acceptor for heparan sulfate or chondroitin sulfate polymerases. Moreover, production of GAGs was significantly higher in EXTL2 knock-out mice than in wild-type mice. These results indicate that EXTL2 functions to suppress GAG biosynthesis that is enhanced by a xylose kinase and that the EXTL2-dependent mechanism that regulates GAG biosynthesis might be a “quality control system” for proteoglycans.     

An Exopolygalacturonase from a Strain of Acrocylindrium

An exopolygalacturonase was partially purified from mycelial extracts of a strain of Acrocylindrium. The enzyme was most active at pH 4.5 and showed a higher affinity for polygalac turo nic acid than for oligogalacturonic acids. The enzyme was found to hydrolyze glycosidic linkages at the non-reducing end of polygalacturonic acid molecules, giving only monogalacturonic acid as the reaction product.     

Studies on Respiratory Enzymes in Rice Kernel

Rice embryo peroxidase 556 was purified to the extent as indicated by the absorbance ratio, RZ greater than 4.0. The enzyme was found to be major basic component among isoenzymes of rice embryo. The preparation was homogeneous as examined by sedimentation analysis, and the sedimentation coefficient, s°_20,w, was 3.76 S. The prosthetic group of the enzyme was identified as protohematin and its content was 1.36%. The minimum molecular weight was calculated to be 46,700. From the typical spectra of ligand-enzyme compounds, peroxidase 556 was found to react with carbon monoxide, cyanide, fluoride, and azide. However, at neutral pH, neither fluoride nor azide reacted with the enzyme. The high affinity of the enzyme to ammonia was one of the most remarkable characteristics of the enzyme. The hydrogen peroxide compounds I and II have been observed in the enzymic reaction, and therefore rice embryo peroxidase 556 is also concluded to follow the common reaction mechanism of plant peroxidases. Overall results show the close resemblance of rice embryo peroxidase 556 with wheat germ peroxidase 556 and hemoprotein 550.     

Polylysine Produced by Streptomyces

The xylitol dehydrogenase gene (xdh) of Bacillus pallidus was cloned and overexpressed in Escherichia coli using pQE60 vector, for the first time. The open reading frame of 759 bp encoded a 253 amino acid protein with a calculated molecular mass of 27,333 Da. The recombinant xylitol dehydrogenase (XDH) was purified to homogeneity by three-step column chromatography, producing a single SDS–PAGE band of 28 kDa apparent molecular mass. The enzyme exhibited maximal activity at 55 °C in glycine-NaOH buffer pH 11.0, with 66% of initial enzyme activity retained after incubation at 40 °C for 1 h. In further application of the recombinant bacterium to L-xylulose production from xylitol (initial concentration 5%) using a resting cell reaction, 35% L-xylulose was produced within 24 h. This result indicates that this recombinant XDH is applicable in the large-scale production of L-xylulose.     

Isoenzymes of Japanese-radish Peroxidase

Japanese-radish root contained eighteen isoenzymes of peroxidase distinguishable on polyacrylamide gel electropherograms. The isoenzymes were found to be quite similar to those of horseradish peroxidase, although their quantities were different between two plants. The acidic components were the major isoenzyme in Japanese-radish peroxidase, while the neutral ones were the major one in horseradish. The chromatographic purification of the isoenzymes was performed on CM- and DEAE-Sephadex columns to characterize the components. The components in the preparations purified by the previously reported procedures of Morita et al. were also identified.