Preparation and characterization of truncated human lipopolysaccharide-binding protein in Escherichia coli

Lipopolysaccharide (LPS) is a component of the outer membrane of Gram-negative bacteria, and is the causative agent of endotoxin shock. LPS induces signal transduction in immune cells when it is recognized by the cell surface complex of toll-like receptor 4 (TLR4) and MD-2. The complex recognizes the lipid A structure in LPS, which is buried in the membrane of the outer envelope. To present the Lipid A structure to the TLR4/MD-2, processing of LPS by LPS-binding protein (LBP) and CD14 is required. In previous studies, we expressed recombinant proteins of human MD-2 and CD14 as fusion proteins with thioredoxin in Escherichia coli, and demonstrated their specific binding abilities to LPS. In this study, we prepared a recombinant fusion protein containing 212 amino terminal residues of human LBP (HLB212) by using the same expression system. The recombinant protein expressed in E. coli was purified as a complex form with host LPS. The binding was not affected by high concentrations of salt, but was prevented by low concentrations of various detergents. Both rough-type LPS lacking the O antigen and smooth-type LPS with the antigen bound to HLBP212. Therefore, oligosaccharide repeats appeared to be unnecessary for the binding. A nonpathogenic penta-acylated LPS also bound to HLBP212, but the binding was weaker than that of the wild type. The hydrophobic interaction between the LBP and acyl chains of lipid A appears to be important for the binding. The recombinant proteins of LPS-binding molecules would be useful for analyzing the defense mechanism against infections.     

Genetic evidence supporting the existence of two diverged groups in the goby Gymnogobius castaneus

The genetic population structure of the Japanese freshwater goby Gymnogobius castaneus was investigated on the basis of analysis of gene products of 19 allozyme loci. Two diverged groups were detected, one being endemic to the Kanto region and the other extensively distributed in eastern Japan. These two groups were distinguishable from each other by a complete allelic substitution in one locus, G3PDH*. In the Kanto region, both groups were distributed in the same river basin, being distinguishable by a complete allelic substitution in four loci, G3PDH*, GPI-2*, PGDH*, and PGM*. These results suggest that these two groups showed reproductive isolation.     

Synthesis and biological evaluation of 9-(5',5'-difluoro-5'-phosphonopentyl)guanine derivatives for PNP-inhibitors

9-(5',5'-Difluoro-5'-phosphonopentyl)guanine (DFPP-G) and its hypoxanthine analogue (DFPP-H) were modified by introducing a methyl group to all possible positions of the linker connecting a purine and difluoromethylenephosphonic acid moiety to evaluate the effects of the methyl group on inhibition against purine nucleoside phosphorylase. The methyl group on the linker affected the inhibition in a positional-dependent manner. Inhibitory potency of alpha-methyl and beta-methyl-substituted analogues of DFPP-H increased by about 600- to 1000-fold upon converting to cyclopropane nucleotide analogue (+/-)-4.     

Roles of Protein-disulfide Isomerase-mediated Disulfide Bond Formation of Yeast Mnl1p in Endoplasmic Reticulum-associated Degradation

The endoplasmic reticulum (ER) has a strict protein quality control system. Misfolded proteins generated in the ER are degraded by the ER-associated degradation (ERAD). Yeast Mnl1p consists of an N-terminal mannosidase homology domain and a less conserved C-terminal domain and facilitates the ERAD of glycoproteins. We found that Mnl1p is an ER luminal protein with a cleavable signal sequence and stably interacts with a protein-disulfide isomerase (PDI). Analyses of a series of Mnl1p mutants revealed that interactions between the C-terminal domain of Mnl1p and PDI, which include an intermolecular disulfide bond, are essential for subsequent introduction of a disulfide bond into the mannosidase homology domain of Mnl1p by PDI. This disulfide bond is essential for the ERAD activity of Mnl1p and in turn stabilizes the prolonged association of PDI with Mnl1p. Close interdependence between Mnl1p and PDI suggests that these two proteins form a functional unit in the ERAD pathway.The endoplasmic reticulum (ER)² is the first organelle in the secretory pathway of eukaryotic cells and provides an optimum environment for maturation of newly synthesized secretory and membrane proteins. Protein folding/assembly in the ER is aided by molecular chaperones and folding enzymes. Molecular chaperones in the ER assist folding of newly synthesized proteins and prevent them from premature misfolding and/or aggregate formation (1, 2). Protein folding in the ER is often associated with formation of disulfide bonds, which contribute to stabilization of native, functional states of proteins. Disulfide bond formation could be a rate-limiting step of protein folding both in vitro and in vivo (3, 4), and the ER has a set of folding enzymes including protein-disulfide isomerase (PDI) and its homologs that catalyze disulfide bond formation (5, 6).In parallel, protein folding/assembly in the ER relies on the inherent failsafe mechanism, i.e. the ER quality control system, to ensure that only correctly folded and/or assembled proteins can exit the ER. Misfolded or aberrant proteins are retained in the ER for refolding by ER-resident chaperones, whereas terminally misfolded proteins are degraded by the mechanism known as ER-associated degradation (ERAD). The ERAD consists of recognition and processing of aberrant substrate proteins, retrotranslocation across the ER membrane, and subsequent proteasome-dependent degradation in the cytosol. More than 20 different components have been identified to be involved in this process in yeast and mammals (7).The majority of proteins synthesized in the ER are glycoproteins, in which N-linked glycans are not only important for folding but also crucial for their ERAD if they fail in folding. Specifically, trimming of one or more mannose residues of Man₉GlcNAc₂ oligosaccharide and recognition of the modified mannose moiety represent a key step for selection of terminally misfolded proteins for disposal (8). A mannosidase I-like protein, Mnl1p/Htm1p (yeast), and EDEM (mammals, ER degradation enhancing α-mannosidase-like protein) were identified as candidates for lectins that recognize ERAD substrates with modified mannose moieties (9–11). Both Mnl1p and EDEM contain an N-terminal mannosidase homology domain (MHD), which lacks cysteine residues conserved among α1,2-mannosidase family members and is proposed to function in recognition of mannose-trimmed carbohydrate chains (supplemental Fig. S1). However, whether Mnl1p or EDEM indeed functions as an ERAD-substrate-binding lectin or has a mannosidase activity is still in debate (11–15), and Yos9p was suggested to take the role of ERAD-substrate binding lectin (14, 16–18). Mnl1p, but not EDEM, has a large C-terminal extension, which does not show any homology to known functional domains and is conserved only among fungal Mnl1p homologs (supplemental Fig. S1).After recognition of the modified mannose signal for degradation, aberrant proteins are maintained or converted to be retrotranslocation competent by ER chaperones including BiP (19). PDI was also indicated to be involved in these steps in the ERAD by, for example, its possible chaperone-like functions (20–23). The yeast PDI, Pdi1p, contains four thioredoxin-like domains, two of which have a CGHC motif as active sites, followed by a C-terminal extension containing the ER retention signal. During its catalytic cycle, PDI transiently forms a mixed disulfide intermediate with its substrate through an intermolecular disulfide bond between the cysteine residues of the active site of PDI and the substrate molecule.Here we report identification of PDI as an Mnl1p-interacting protein. Stable interactions between the C-terminal domain of Mnl1p and PDI involve intermolecular disulfide bonds. Stably interacting PDI is required for formation of the functionally essential intramolecular disulfide bond in the MHD of Mnl1p, which in turn stabilizes and prolongs the Mnl1p-PDI interactions. Possible roles for those stable interactions between Mnl1p and PDI in the ERAD will be discussed.     

Bisulfite sequencing and dinucleotide content analysis of 15 imprinted mouse differentially methylated regions (DMRs): paternally methylated DMRs contain less CpGs than maternally methylated DMRs

Imprinted genes in mammals show monoallelic expression dependent on parental origin and are often associated with differentially methylated regions (DMRs). There are two classes of DMR: primary DMRs acquire gamete-specific methylation in either spermatogenesis or oogenesis and maintain the allelic methylation differences throughout development; secondary DMRs establish differential methylation patterns after fertilization. Targeted disruption of some primary DMRs showed that they dictate the allelic expression of nearby imprinted genes and the establishment of the allelic methylation of secondary DMRs. However, how primary DMRs are recognized by the imprinting machinery is unknown. As a step toward elucidating the sequence features of the primary DMRs, we have determined the extents and boundaries of 15 primary mouse DMRs (including 12 maternally methylated and three paternally methylated DMRs) in 12.5-dpc embryos by bisulfite sequencing. We found that the average size of the DMRs was 3.2 kb and that their average G+C content was 54%. Dinucleotide content analysis of the DMR sequences revealed that, although they are generally CpG rich, the paternally methylated DMRs contain less CpGs than the maternally methylated DMRs. Our findings provide a basis for the further characterization of DMRs.     

Minor but key chlorophylls in Photosystem II

A ‘metal-free’ chlorophyll (Chl) a, pheophytin (Phe) a, functions as the primary electron acceptor in PS II. On the basis of Phe a/PS II = 2, Phe a content is postulated as an index for estimation of the stoichiometry of pigments and photosystems. We found Phe a in a Chl d-dominant cyanobacterium Acaryochloris marina, whereas Phe d was absent. The minimum Chl a:Phe a ratio was 2:2, indicating that the primary electron donor is Chl a, accessory is Chl d, and the primary electron acceptor is Phe a in PS II of A. marina. Chl d was artificially formed by the treatment of Chl a with papain in aqueous organic solvents. Further, we will raise a key question on the mechanisms of water oxidation in PS II.