Synthetic glycopeptides reveal specific binding pattern and conformational change at O-mannosylated position of α-dystroglycan by POMGnT1 catalyzed GlcNAc modification

Structural and functional effects of core M1 type glycan modification catalyzed by protein O-linked mannose β1,2-N-acetylglucosaminyltransferase 1 (POMGnT1) were investigated using a core M1 glycoform focused library of an α-dystroglycan fragment, ³⁷²TRGAIIQTPTLGPIQPTRV³⁹⁰. Evanescent-field fluorescence-assisted microarray system illuminated the specific binding pattern of plant lectins that can discriminate the glycan structure of core M1 glycan of the library. The comparative NMR analysis of synthetic glycopeptide having different length of the O-mannosylated glycans revealed a conformational change of the peptide backbone along with core M1 disaccharide formation. No long-range NOE signals of glycan-amino acid nor inter amino acid indicate the conformational change is induced by steric hindrance of core M1, the sole 1,2-O-modified form among protein binding sugar residue found in mammals.     

Purification of human urinary prokallikrein. Identification of the site of activation by the metalloproteinase thermolysin.

Human urinary active kallikrein and prokallikrein were separated on DEAE-cellulose and octyl-Sepharose columns and both purified to homogeneity by affinity chromatography, gel filtration and hydrophobic h.p.l.c. Prokallikrein was monitored during purification by trypsin activation followed by determination of both amidase and kininogenase activity. After trypsin activation, purified prokallikrein had a specific kininogenase activity of 39.4 micrograms of bradykinin equivalent/min per mg and amidase activity of 16.5 mumol/min per mg with D-Val-Leu-Arg-7-amino-4-trifluoromethylcoumarin. Purified active kallikrein had a specific activity of 47 micrograms of bradykinin/min per mg. The molecular mass of prokallikrein was 48 kDa on electrophoresis and 53 kDa on gel filtration whereas active kallikrein gave values of 46 kDa and 53 kDa respectively. Antisera to active and prokallikrein were obtained. In double immunodiffusion and immunoelectrophoresis, antiserum to active kallikrein reacted with active and pro-kallikrein. Antiserum to prokallikrein contained antibodies to determinants not found in active kallikrein, presumably due to the presence of the activation peptide in the proenzyme. Human prokallikrein can be activated by thermolysin, trypsin and human plasma kallikrein. Activation of 50% of the prokallikrein (1.35 microM) was achieved in 30 min with 25 nM-thermolysin, 78 nM-trypsin or 180 nM-human plasma kallikrein. Thus thermolysin was the most effective activator. Thermolysin activated prokallikrein by releasing active kallikrein with N-terminal Ile1-Val2. Thus human tissue (glandular) prokallikrein can be activated by two types of enzymes: serine proteinases, which cleave at the C-terminus of basic amino acids, and by a metalloproteinase that cleaves at the N-terminus of hydrophobic amino acids.     

Relationships between functional subclasses and information contained in active‐site and ligand‐binding residues in diverse superfamilies

To investigate the relationships between functional subclasses and sequence and structural information contained in the active‐site and ligand‐binding residues (LBRs), we performed a detailed analysis of seven diverse enzyme superfamilies: aldolase class I, TIM‐barrel glycosidases, α/β‐hydrolases, P‐loop containing nucleotide triphosphate hydrolases, collagenase, Zn peptidases, and glutamine phosphoribosylpyrophosphate, subunit 1, domain 1. These homologous superfamilies, as defined in CATH, were selected from the enzyme catalytic‐mechanism database. We defined active‐site and LBRs based solely on the literature information and complex structures in the Protein Data Bank. From a structure‐based multiple sequence alignment for each CATH homologous superfamily, we extracted subsequences consisting of the aligned positions that were used as an active‐site or a ligand‐binding site by at least one sequence. Using both the subsequences and full‐length alignments, we performed cluster analysis with three sequence distance measures. We showed that the cluster analysis using the subsequences was able to detect functional subclasses more accurately than the clustering using the full‐length alignments. The subsequences determined by only the literature information and complex structures, thus, had sufficient information to detect the functional subclasses. Detailed examination of the clustering results provided new insights into the mechanism of functional diversification for these superfamilies. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Chemical composition and immunobiological activities of sodium dodecyl sulphate extracts from the cell envelopes of Actinobacillus actinomycetemcomitans, Bacteroides gingivalis and Fusobacterium nucleatum

The chemical composition and immunobiological activities in vivo and in vitro of sodium dodecyl sulphate extracts (SDS-SE) derived from periodontopathic bacteria (three strains of Actinobacillus actinomycetemcomitans, two strains of Bacteroides gingivalis, and one strain of Fusobacterium nucleatum) were investigated. The main components of SDS-SE were protein and lipid, with negligible amounts of peptidoglycan and lipopolysaccharide. Immunopotentiating activity was detected in both delayed-type hypersensitivity and antibody formation against the elicitation of a protein antigen with the SDS-SE preparations of A. actinomycetemcomitans ATCC 29524 and B. gingivalis 381 and 1021. On the other hand the SDS-SE of A. actinomycetemcomitans ATCC 29522 enhanced only the induction of a delayed-type hypersensitivity response. All the SDS-SE preparations had mitogenic activity to splenocytes from BALB/c nu/nu, C3H/HeN and C3H/HeJ mice. Migration-stimulating activity for human peripheral blood monocytes was detected especially in the SDS-SE preparations of A. actinomycetemcomitans ATCC 29524 and Y4. All of the SDS-SE samples inhibited [3H]thymidine uptake in human gingival fibroblasts and caused degradation of the cells. The results suggest that the cell membrane components extractable with sodium dodecyl sulphate from periodontopathic bacteria are involved in the pathogenesis of periodontal disease.     

An ability of isolated strains to efficiently cooperate in ethanolic fermentation of agricultural plant refuse under initially aerobic thermophilic conditions: oxygen deletion process appended to consolidated bioprocessing (CBP)

For bioconversion of bean curd refuse, a processing by-product of bean curd, ethanol-producing anaerobic thermophiles (strains kpu03 and kpu04) were newly isolated. Both of them degraded hemicellulose, but not cellulose at all. Phylogenetically, strains kpu03 and kpu04 belong to the Clostridium and Thermoanaerobacterium genus, respectively. Aerobic thermophiles degrading cellulose were also isolated newly. Among them, strain kpuB3 particularly enhanced ethanol production by anaerobic strain kpu04 in the aerobic bean curd refuse medium. Strain kpuB3 belongs to the Geobacillus genus phylogenetically. The co-culture also significantly reduced CH(3)SH production, leading to the prevention of offensive odor. These results demonstrate that cellulolytic aerobe cooperated with hemicellulolytic anaerobe in ethanolic fermentation by not only synergistic effect but also deletion of oxygen from the vessels, providing a new model of oxygen deletion process appended to consolidated bioprocessing (CBP).     

Redescription of Papilloma luteum and description of a new Praon species (Hymenoptera: Braconidae; Aphidiinae) parasitic on Kurisakia querciphila (Homoptera: Aphididae: Thelaxinae) in Japan,with notes on synonymy and life cycle of P. luteum

Two aphidiine species were found to be associated with a thelaxine aphid, Kurisakia querciphila on Quercus serrata and Q. acutissima in Japan. One of them is identified as Papilloma luteum and redescribed, and the other is described as a new species, Praon kurisakiae. Sinoaphidius zhejiangensis is synonymized with P. luteum. Papilloma luteum aestivated within a unique shaped and black “diapausing mummy”. Aestivating adults of P. luteum emerging from “diapausing mummies” in the autumn were darker and smaller than non‐aestivating ones. The aphidiine parasitoids of Thelaxinae are reviewed; the systematic position of Papilloma and life cycles of K. querciphila and P. luteum are discussed.     

A Single Amino Acid in the M1 Protein Responsible for the Different Pathogenic Potentials of H5N1 Highly Pathogenic Avian Influenza Virus Strains

Two highly pathogenic avian influenza virus strains, A/duck/Hokkaido/WZ83/2010 (H5N1) (WZ83) and A/duck/Hokkaido/WZ101/2010 (H5N1) (WZ101), which were isolated from wild ducks in Japan, were found to be genetically similar, with only two amino acid differences in their M1 and PB1 proteins at positions 43 and 317, respectively. We found that both WZ83 and WZ101 caused lethal infection in chickens but WZ101 killed them more rapidly than WZ83. Interestingly, ducks experimentally infected with WZ83 showed no or only mild clinical symptoms, whereas WZ101 was highly lethal. We then generated reassortants between these viruses and found that exchange of the M gene segment completely switched the pathogenic phenotype in both chickens and ducks, indicating that the difference in the pathogenicity for these avian species between WZ83 and WZ101 was determined by only a single amino acid in the M1 protein. It was also found that WZ101 showed higher pathogenicity than WZ83 in mice and that WZ83, whose M gene was replaced with that of WZ101, showed higher pathogenicity than wild-type WZ83, although this reassortant virus was not fully pathogenic compared to wild-type WZ101. These results suggest that the amino acid at position 43 of the M1 protein is one of the factors contributing to the pathogenicity of H5N1 highly pathogenic avian influenza viruses in both avian and mammalian hosts.