Novel carbohydrate-binding activity of bovine liver beta-glucuronidase toward lactose/N-acetyllactosamine sequences

Beta-glucuronidase is a lysosomal enzyme that plays an essential role in normal turnover of glycosaminoglycans and remodeling of the extracellular matrix components in both physiological and inflammatory states. The regulation mechanisms of enzyme activity and protein targeting of beta-glucuronidase have implications for the development of a variety of therapeutics. In this study, the effectiveness of various carbohydrate-immobilized adsorbents for the isolation of bovine liver beta-glucuronidase (BLG) from other glycosidases was tested. Beta-glucuronidase and contaminating glycosidases in commercial BLG preparations bound to and were coeluted from adsorbents immobilized with the substrate or an inhibitor of beta-glucuronidase, whereas beta-glucuronidase was found to bind exclusively with lactamyl-Sepharose among the adsorbents tested and to be effectively separated from other enzymes. Binding and elution studies demonstrated that the interaction of beta-glucuronidase with lactamyl-Sepharose is pH dependent and carbohydrate specific. BLG was purified to homogeneity by lactamyl affinity chromatography and subsequent anion-exchange high-performance liquid chromatography (HPLC). Lactose was found to activate beta-glucuronidase noncompetitively, indicating that the lactose-binding site is different from the substrate-binding site. Binding studies with biotinyl glycoproteins, lipids, and synthetic sugar probes revealed that beta-glucuronidase binds to N-acetyllactosamine/lactose-containing glycoconjugates at neutral pH. The results indicated the presence of N-acetyllactosamine/lactose-binding activity in BLG and provided an effective purification method utilizing the novel carbohydrate binding activity. The biological significance of the carbohydrate-specific interaction of beta-glucuronidase, which is different from the substrate recognition, is discussed.     

Critical Role of Nucleostemin in Pre-rRNA Processing

Nucleostemin is a nucleolar protein widely expressed in proliferating cells. Nucleostemin is involved in the regulation of cell proliferation, and both depletion and overexpression of nucleostemin induce cell cycle arrest through the p53 signaling pathway. Although the presence of p53-independent functions of nucleostemin has been previously suggested, the identities of these additional functions remained to be investigated. Here, we show that nucleostemin has a novel role as an integrated component of ribosome biogenesis, particularly pre-rRNA processing. Nucleostemin forms a large protein complex (>700 kDa) that co-fractionates with the pre-60 S ribosomal subunit in a sucrose gradient. This complex contains proteins related to pre-rRNA processing, such as Pes1, DDX21, and EBP2, in addition to several ribosomal proteins. We show that the nucleolar retention of DDX21 and EBP2 is dependent on the presence of nucleostemin in the nucleolus. Furthermore, the knockdown of nucleostemin delays the processing of 32 S pre-rRNA into 28 S rRNA. This is accompanied by a substantial decrease of protein synthesis as well as the levels of rRNAs and some mRNAs. In addition, overexpressed nucleostemin significantly promotes the processing of 32 S pre-rRNA. Collectively, these biochemical and functional studies demonstrate a novel role of nucleostemin in ribosome biogenesis. This is a key aspect of the role of nucleostemin in regulating cell proliferation.Nucleostemin (NS)² is a nucleolar protein preferentially expressed in actively proliferating cells. The structure of NS is characterized by two GTP-binding domains, which are involved in the regulation of its dynamic shuttling between the nucleolus and nucleoplasm (1). NS was originally identified as a nucleolar protein prominently expressed in rat neural stem cells and down-regulated during differentiation of these cells in vitro (2). The same authors also found that NS is widely expressed in neural precursor cells in early mouse embryos as well as in a variety of cancer cells and stem cells, including embryonic stem cells and a hematopoietic stem cell-enriched fraction. NS is generally down-regulated in the early stage of differentiation before exit from the cell cycle. In addition, knockdown of NS significantly inhibits proliferation of cortical stem cells and cancer cells. These initial observations led to suggestions that NS is involved in multipotency in stem cells as well as in the regulation of cancer and stem cell proliferation (2).Recent work, however, has demonstrated that NS is in fact widely expressed in many types of normal proliferating cells at levels similar to those in malignant cells. For instance, NS is expressed in normal kidney cells and renal carcinoma cells at comparable levels as detected in histological sections (3). The expression of NS is significantly up-regulated when normal T lymphocytes are activated by concanavalin A (3) and when bone marrow stem cells are stimulated by fibroblast growth factor 2 (4). Cells in NS-null mouse embryos fail to enter the S phase, resulting in embryonic death at the blastocyst stage (5, 6). In early Xenopus embryos NS is also expressed in the sites of active cell proliferation and local depletion of NS results in a decrease in proliferating neural progenitor cells (6). Based on these observations, it was proposed that expression of NS is more closely linked with cell proliferation than with the malignant state or differentiation status of a cell.Several studies have provided evidence that the p53 signaling pathway is involved in the G₁ arrest of the cell cycle induced by the down-regulation of NS. Physical interaction between NS and p53 was initially reported by Tsai and McKay (2). Later, it was shown that the G₁ arrest requires the presence of p53 (7). In the most recent study Dai et al. (8) showed that knockdown of NS enhances the interaction between the p53-binding protein MDM2 and the ribosomal protein L5 or L11, preventing MDM2 from inducing ubiquitylation-based p53 degradation. However, other studies have also suggested that NS may have a p53-independent role in the regulation of cell proliferation. For instance, the depletion of p53 from NS-null blastocysts did not rescue them from the embryonic lethality (6). In addition, NS partial loss-of-function in mouse fibroblasts did not result in any change in the p53 level (5). Furthermore, knockdown of L5 and L11 only partially rescued the G₁ arrest in NS knockdown cells (8). Finally, the fact that NS is primarily localized in the nucleolus, whereas the p53-mediated mechanism occurs in the nucleoplasm, suggests that NS might have an additional role more directly relevant to nucleolar functions.To identify novel functions of NS, we purified an endogenous NS complex from HeLa cell extract and investigated whether NS interacts with other proteins not described previously. Identification of the components of this complex and the alterations of the expression level of NS in HeLa cells led us to uncover a novel role of NS in the processing of rRNA. Our findings not only provide supporting evidence for the hypothesis that NS has a p53-independent function but also demonstrate that NS is critical for ribosome biogenesis, one of the most fundamental processes common for all cell types.     

Four-electron Reduction of Dioxygen by a Multicopper Oxidase, CueO, and Roles of Asp112 and Glu506 Located Adjacent to the Trinuclear Copper Center

The mechanism of the four-electron reduction of dioxygen by a multicopper oxidase, CueO, was studied based on reactions of single and double mutants with Cys⁵⁰⁰, a type I copper ligand, and the noncoordinating Asp¹¹² and Glu⁵⁰⁶, which form hydrogen bonds with the trinuclear copper center directly and indirectly via a water molecule. The reaction of C500S containing a vacant type I copper center produced intermediate I in an EPR-silent peroxide-bound form. The formation of intermediate I from C500S/D112N was restricted due to a reduction in the affinity of the trinuclear copper center for dioxygen. The state of intermediate I was realized to be the resting form of C500S/E506Q and C500S of the truncated mutant Δα5–7CueO, in which the 50 amino acids covering the substrate-binding site were removed. Reactions of the recombinant CueO and E506Q afforded intermediate II, a fully oxidized form different from the resting one, with a very broad EPR signal, g < 2, detectable only at cryogenic temperatures and unsaturated with high power microwaves. The lifetime of intermediate II was prolonged by the mutation at Glu⁵⁰⁶ involved in the donation of protons. The structure of intermediates I and II and the mechanism of the four-electron reduction of dioxygen driven by Asp¹¹² and Glu⁵⁰⁶ are discussed.CueO is a multicopper oxidase involved in a copper efflux system of Escherichia coli (1–3). In contrast to other multicopper oxidases such as laccase and ascorbate oxidase (4), CueO exhibits strong activity toward cuprous ion but does not show activity toward most organic substrates such as 2,6-dimethoxyphenol, catechol, and guaiacol, except considerably low levels toward 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS)² and p-phenylenediamine. This substrate specificity, unique to CueO, originates in the methionine-rich helical region covering the substrate-binding site (5–7). Nevertheless, CueO has the same catalytic copper centers as other multicopper oxidases: a type I copper that mediates electron transfer and a trinuclear copper center comprised of a type II copper and a pair of type III copper atoms, where dioxygen is reduced to two water molecules (5, 7). The type I copper is responsible for the intense charge transfer band at 610 nm due to Cys(S^-)_π → Cu²⁺ and the bands at 430, ∼500, and ∼750 nm due to the charge transfers His(N) → Cu²⁺ and Cys(S^-)_σ → Cu²⁺ and d-d transitions, respectively (4). The type III copper atoms bridged with a hydroxide ion afford an intense charge transfer band, OH^- → Cu²⁺ at ∼330 nm, whereas the type II copper does not give a conspicuous band in the visible region. The type I and II coppers give rise to EPR signals with the hyperfine splitting of small (6.7 milliteslas (mT)) and normal (18.5 mT) magnitudes, respectively, whereas the type III copper atoms are EPR-silent because of the strong anti-ferromagnetic interaction (7–9).Special attention has been paid to the four-electron reduction of dioxygen by multicopper and terminal oxidases because activated oxygen species such as superoxide, peroxide, etc. are not formed or, if formed, are effectively converted into water molecules without damage to protein molecules. Therefore, this four-electron reduction of dioxygen by multicopper oxidases has been expected to be applicable to biofuel cells (10–12). Two reaction intermediates have been detected during reactions of some multicopper oxidases. One of them, intermediate I, could be trapped by the following modified multicopper oxidases so as to interrupt the electron transfer from the type I copper: a plant laccase whose type I copper was substituted with mercury (13); a mixed valent laccase in which the type I copper was oxidized, but the trinuclear copper center was reduced (14); and a Cys → Ser mutant of bilirubin oxidase (15) and Fet3p (16) whose type I copper center became vacant. Although the trinuclear copper center must be fully reduced to produce intermediate I, it has been considered to be a two-electron reduced form and, therefore, also called the peroxide intermediate (13, 16). Another reaction intermediate, II, also called the native intermediate, has been detected at the final stage of a single turnover (15, 17–19). Four electrons have already been transferred to dioxygen in this intermediate, and accordingly, intermediate II is in a fully oxidized form to give the g < 2 EPR signal at cryogenic temperatures. Under catalytic conditions, intermediate II is not detected because of its prompt conversion to the fully reduced form for the next enzyme cycle without decaying to the resting form. Both intermediates have a half-life in the order of seconds to minutes, but information to directly show their structures has not been obtained yet. They afford analogous absorption bands at ∼330–350, 450–470, and 680 nm, of which the former two bands have been assigned to the charge transfer from a certain oxygen group to Cu²⁺ (σ and π transitions) and the latter to the d-d transitions of the trinuclear copper center in the cupric state. The d-d transitions of intermediate II are masked by strong absorption due to the oxidized type I copper (13–19).In the present study, we succeeded in trapping intermediates I and II from reactions of a recombinant form of CueO (rCueO) and mutants altered at Cys⁵⁰⁰, a ligand to the type I copper, and at Asp¹¹² and Glu⁵⁰⁶ located adjacent to the trinuclear copper center to modify the dioxygen reduction process. The Asp residue is conserved in every multicopper oxidase except for ceruloplasmin, which has Glu instead (Fig. 1). According to the x-ray crystal structures of rCueO (5) and the truncated mutant, Δα5–7CueO, missing the 50 amino acids covering the substrate-binding site (Fig. 2) (7, 20), Asp¹¹² forms a hydrogen bond with His⁴⁴⁸, a ligand to a type III copper, and indirectly with the water molecule coordinating the type II copper through an ordered water molecule. In a preliminary study on the Asp¹¹² mutants (21), we showed that this acidic amino acid functions in the binding of dioxygen at the trinuclear copper center and may also be involved in the donation of protons to the reaction intermediate(s). On the other hand, one to three acidic amino acids are present in the spacers to connect the copper ligands of multicopper oxidases, His-Cys-His-XXX-His-XXXX-Met-(Leu/Phe). Fig. 2 shows that Glu⁵⁰⁶ of CueO in this spacer is directly hydrogen-bonded with the His¹⁴³ ligand to one of the type III copper atoms and indirectly with the hydroxide ion bridged between the type III copper atoms through an ordered water molecule. Therefore, Glu⁵⁰⁶ is also speculated to play a crucial role in the reduction of dioxygen. We singly and doubly mutated Cys⁵⁰⁰, Asp¹¹², and Glu⁵⁰⁶ of CueO to trap intermediates I and II and to elucidate the mechanism behind the four-electron reduction of dioxygen.Open in a separate windowFIGURE 1.Homology of amino acid sequence around the copper binding sites of multicopper oxidase. The numbers 1, 2, and 3 represent the type I, II, and III copper ligands, respectively. BO, Myrothecium verrucaria bilirubin oxidase; RvLc, Rhus vernicifera laccase; CpAO, Cucurbita pepo ascorbate oxidase; TvLc, Trametes versicolor laccase; CcLc, Coprinus cinereus laccase; Fet3p, multicopper oxidase from Saccharomyces cerevisiae; CumA, multicopper oxidase from Pseudomonas putida; CotA, multicopper oxidase from Bacillus subtilis; SLAC, small laccase from Streptomyces coelicolor; hCp, human ceruloplasmin. The single asterisk represents the conserved acidic amino acid residue in all multicopper oxidases, and the double asterisk represents Glu⁵⁰⁶ in CueO, which forms a hydrogen bond with a His residue coordinating a type III copper and the hydroxide ion bridged between type III coppers.Open in a separate windowFIGURE 2.Structure around the active site of the truncated mutant of CueO (7). Type I, II, and III coppers are represented as spheres. Small spheres, oxygen atoms. The two networks of hydrogen bonds lead to the exterior of the protein molecule, forming the pathway to let protons in and water molecules out. Mutated amino acid residues, Cys⁵⁰⁰, Glu⁵⁰⁶, and Asp¹¹², and the networks of hydrogen bonds are indicated.     

Identification of the clonal complexes of Staphylococcus aureus strains by determination of the conservation patterns of small genomic islets

Aims:  To investigate the clonality of Staphylococcus aureus isolates, it is important to identify their clonal complexes (CCs) with multilocus sequence typing (MLST). However, it is expensive to carry out MLST analyses for many isolates. The aim of this study, therefore, was to develop a cost-effective method to identify CCs by determining the conservation pattern of 'small genomic islets' (SGIs). SGIs are nonconserved regions between strains and have single or multiple open-reading frames (ORFs).
Methods and Results:  The whole-genome sequences of nine strains were compared in order to select 16 SGIs. The conservation patterns of the 16 SGIs (islet patterns) were investigated in 136 S. aureus isolates, which were classified into 21 CCs. The islet patterns (IPs) exhibited a one-to-one correspondence with the CCs, except for isolates belonging to CC1, CC5 and CC8. The IPs typical of strains belonging to CC1, CC5 and CC8 differed between those of sequence type 1 (ST1) and ST188 (CC1), ST5 and ST6 (CC5) and ST8 and ST239 (CC8).
Significance and Impact of the Study:  The CCs of many isolates can be identified in an easy and inexpensive manner by detecting these 16 SGIs. Emergent clones, particularly methicillin-resistant ones, can be identified by examining numerous islets by IP analysis.     

What makes the satellite-based EVI–GPP relationship unclear in a deciduous broad-leaved forest?

Recent studies have suggested that gross primary production (GPP) of terrestrial vegetation can be estimated directly with the satellite-based Enhanced Vegetation Index (EVI). However, the reported EVI–GPP relationships showed wide variability, with the regression functions showing widely scattered data. In the present study, we examined the possible reasons for this variability in the EVI–GPP relationship using daily EVI values from satellite and field measurements and daily flux-based GPP in a cool-temperate deciduous broad-leaved forest in Japan. The variability appears to be caused by noise due to cloud contamination in the satellite data as well as the different seasonality of EVI and GPP, especially during the leaf-expansion period. Our findings indicate that improvement of cloud screening and consideration of the leaf-expansion period are critical when applying the EVI–GPP relationship.     

Effects of seasonal and interannual variations in leaf photosynthesis and canopy leaf area index on gross primary production of a cool-temperate deciduous broadleaf forest in Takayama, Japan

Revealing the seasonal and interannual variations in forest canopy photosynthesis is a critical issue in understanding the ecological mechanisms underlying the dynamics of carbon dioxide exchange between the atmosphere and deciduous forests. This study examined the effects of temporal variations of canopy leaf area index (LAI) and leaf photosynthetic capacity [the maximum velocity of carboxylation (V _cmax)] on gross primary production (GPP) of a cool-temperate deciduous broadleaf forest for 5 years in Takayama AsiaFlux site, central Japan. We made two estimations to examine the effects of canopy properties on GPP; one is to incorporate the in situ observation of V _cmax and LAI throughout the growing season, and another considers seasonality of LAI but constantly high V _cmax. The simulations indicated that variation in V _cmax and LAI, especially in the leaf expansion period, had remarkable effects on GPP, and if V _cmax was assumed constant GPP will be overestimated by 15%. Monthly examination of air temperature, radiation, LAI and GPP suggested that spring temperature could affect canopy phenology, and also that GPP in summer was determined mainly by incoming radiation. However, the consequences among these factors responsible for interannual changes of GPP are not straightforward since leaf expansion and senescence patterns and summer meteorological conditions influence GPP independently. This simulation based on in situ ecophysiological research suggests the importance of intensive consideration and understanding of the phenology of leaf photosynthetic capacity and LAI to analyze and predict carbon fixation in forest ecosystems.     

Identification of FABP7 in fibroblastic reticular cells of mouse lymph nodes

Fatty acids and their metabolites regulate immune cell function. The present study was undertaken to examine the detailed distribution of fatty acid binding proteins (FABPs), the cytosolic chaperones of fatty acids, in mouse peripheral immune organs. Using immunohistochemistry, FABP7 was localized to the alpha-smooth muscle actin (SMA)⁺ fibroblastic reticular cells, which construct the stromal reticula in the T cell areas of the peripheral lymph nodes and spleen. Immunoelectron microscopy showed that FABP7⁺ cells enclosed the collagen fibers, forming a conduit system, which transport lymph and associated low-molecular-mass proteins. In contrast, FABP5⁺ cells were distributed throughout the lymph node and contained well-developed lysosome and phagocytic materials within the cytoplasm. The mesenteric lymph nodes of FABP7 knockout mice showed normal histological features, but the percentage of CD4⁺ cells was significantly increased compared with that in wild-type mice. These data indicate that FABP7 may be involved in T cell homeostasis, possibly by modulating lipid metabolism in fibroblastic reticular cells within the peripheral lymph nodes.     

Lineage-specific positive selection at the merozoite surface protein 1 (msp1) locus of Plasmodium vivax and related simian malaria parasites

Background

The 200 kDa merozoite surface protein 1 (MSP-1) of malaria parasites, a strong vaccine candidate, plays a key role during erythrocyte invasion and is a target of host protective immune response. Plasmodium vivax, the most widespread human malaria parasite, is closely related to parasites that infect Asian Old World monkeys, and has been considered to have become a parasite of man by host switch from a macaque malaria parasite. Several Asian monkey parasites have a range of natural hosts. The same parasite species shows different disease manifestations among host species. This suggests that host immune responses to P. vivax-related malaria parasites greatly differ among host species (albeit other factors). It is thus tempting to invoke that a major immune target parasite protein such as MSP-1 underwent unique evolution, depending on parasite species that exhibit difference in host range and host specificity.

Results

We performed comparative phylogenetic and population genetic analyses of the gene encoding MSP-1 (msp1) from P. vivax and nine P. vivax-related simian malaria parasites. The inferred phylogenetic tree of msp1 significantly differed from that of the mitochondrial genome, with a striking displacement of P. vivax from a position close to P. cynomolgi in the mitochondrial genome tree to an outlier of Asian monkey parasites. Importantly, positive selection was inferred for two ancestral branches, one leading to P. inui and P. hylobati and the other leading to P. vivax, P. fieldi and P. cynomolgi. This ancestral positive selection was estimated to have occurred three to six million years ago, coinciding with the period of radiation of Asian macaques. Comparisons of msp1 polymorphisms between P. vivax, P. inui and P. cynomolgi revealed that while some positively selected amino acid sites or regions are shared by these parasites, amino acid changes greatly differ, suggesting that diversifying selection is acting species-specifically on msp1.

Conclusions

The present results indicate that the msp1 locus of P. vivax and related parasite species has lineage-specific unique evolutionary history with positive selection. P. vivax and related simian malaria parasites offer an interesting system toward understanding host species-dependent adaptive evolution of immune-target surface antigen genes such as msp1.     

Introgression between native and prehistorically naturalized (archaeophytic) wild pear (<Emphasis Type="Italic">Pyrus</Emphasis> spp.) populations in Northern Tohoku,Northeast Japan

Hybridization between native and cultivated species is a concern in conservation biology. However, detecting such hybridization and distinguishing true natives from prehistorically naturalized species based on phenotypic characteristics is difficult. Here, we report on introgression between native and prehistorically introduced pear (Pyrus) species in Northern Tohoku (northern end of Honshu Island), Japan. We analyzed 20 microsatellites in 226 wild, seemingly wild, or cultivated materials. Phenetic analysis showed that wild Japanese populations of P. ussuriensis var. ussuriensis in Northern Tohoku, previously considered true natives based on morphology and phytogeography, differed from those in continental Asia, confirming their nativeness. However, Bayesian inference of population structures showed that Japanese P. ussuriensis was genetically admixed with two genetic clusters: true native P. ussuriensis var. ussuriensis and prehistorically introduced P. pyrifolia. Even in the Kitakami Mountains, where true native populations of var. ussuriensis are believed to persist, most wild trees were at least somewhat admixed. Prehistorically introduced then naturalized plants are treated as natives in Japan’s conservation management, and some are considered endangered. However, introgression of prehistorically naturalized P. pyrifolia into threatened native P. ussuriensis var. ussuriensis has occurred. This paper examines the implications for conservation management.