The carbohydrate-binding module (CBM)-like sequence is crucial for rice CWA1/BC1 function in proper assembly of secondary cell wall materials

We recently reported that the cwa1 mutation disturbed the deposition and assembly of secondary cell wall materials in the cortical fiber of rice internodes. Genetic analysis revealed that cwa1 is allelic to bc1, which encodes glycosylphosphatidylinositol (GPI)-anchored COBRA-like protein with the highest homology to Arabidopsis COBRA-like 4 (COBL4) and maize Brittle Stalk 2 (Bk2). Our results suggested that CWA1/BC1 plays a role in assembling secondary cell wall materials at appropriate sites, enabling synthesis of highly ordered secondary cell wall structure with solid and flexible internodes in rice. The N-terminal amino acid sequence of CWA1/BC1, as well as its orthologs (COBL4, Bk2) and other BC1-like proteins in rice, shows weak similarity to a family II carbohydrate-binding module (CBM2) of several bacterial cellulases. To investigate the importance of the CBM-like sequence of CWA1/BC1 in the assembly of secondary cell wall materials, Trp residues in the CBM-like sequence, which is important for carbohydrate binding, were substituted for Val residues and introduced into the cwa1 mutant. CWA1/BC1 with the mutated sequence did not complement the abnormal secondary cell walls seen in the cwa1 mutant, indicating that the CBM-like sequence is essential for the proper function of CWA1/BC1, including assembly of secondary cell wall materials.Key words: carbohydrate-binding module, COBRA-LIKE, CWA1/BC1, glycosylphosphatidylinositol-anchored protein, secondary cell wall formationThe main function of carbohydrate-binding modules (CBMs) of microbes and plants is to attach the enzyme to a variety of cell surface glycans and thereby increase the local concentration of substrate, leading to more efficient catalysis.^1–4 Almost all CBMs studied to date contain surface-exposed aromatic rings, which have been shown to be the main sites of interaction with polysaccharides. These residues form face-to-face hydrophobic stacking interactions in which a Trp residue or ring of a Tyr residue interacts with the non-polar face of a sugar ring.^5–9 CBMs have been classified into families based on amino acid sequence similarity. Currently, there are 59 defined families of CBMs and these CBMs display substantial variation in ligand specificity (http://www.cazy.org/Carbohydrate-Binding-Modules.html). Among these CBM families, the large family of CBM2 has been further classified into two subgroups, CBM2a and 2b, which have shown to bind cellulose and xylan, respectively.^10–12 CBM2a characteristically possess three exposed Trp residues,¹³ whereas CBM2b have two Trp residues,¹⁴ which are conserved among the CBM2 members (Fig. 1A).Open in a separate windowFigure 1Sequence alignment of the CBM-like sequence of CWA1/BC1, the BC1L proteins and bacterial CBM2 members. (A) Sequence alignment between bacterial CBM2a, 2b and CWA1/BC1. The three surface-exposed Trp residues of CBM2a members are indicated by asterisks and W. The CBM sequences of CBM2a are: CfiCenA, Cellulomonas fimi endo-1,4-glucanase; CfiCex, C. fimi exo-beta-1,4-glucanase. Those of CBM2b are: CfiXylD1, C. fimi endo-1,4-beta-xylanase D; CfiXylD2, C. fimi endo-1,4-beta-xylanase. CWA1/BC1 shows weak similarity to CBM2, and some Trp residues are conserved with bacterial CBM2 members. (B) Sequence alignment of CWA1/BC1, the BC1L proteins and CWA1/BC1 orthologs, Zea maiz Brittle Stalk 2 (ZmBk2) and Arabidopsis thaliana COBRA-LIKE 4 (AtCOBL4). The CBM-like sequence of CWA1/BC1, especially the Trp residues, is highly conserved among the analyzed sequences. Substituted Trp (W) residues to Val (V) in CWA1/BC1 are indicated by closed triangles. Numbers at the left are the positions of the amino acids in each protein, with gaps (dashes) included to maximize alignments. Identical and similar amino acids are shaded and gray, respectively.Our recent study showed that the defect of the rice CWA1/BC1 (CELL WALL ARCHITECTURE 1/BRITTLE CULM 1) gene induced abnormal secondary cell wall formation with amorphous and bulky structures at the cytoplasm side and CWA1/BC1 encodes one of COBRA-like glycosylphosphatidylinositol (GPI)-anchored proteins, which are specifically found in plants, suggesting that CWA1/BC1 regulates assembly of secondary cell wall materials in rice sclerenchyma. Furthermore, several reports have shown that the N-terminus of rice CWA1/BC1 and other COBRA-like GPI-anchored proteins in Arabidopsis (12 members) and maize Brittle Stalk 2 (Bk2) share weak similarity to a CBM2 in several bacterial cellulases.^15,16 However, the importance of CBM-like sequence in COBRA family members has not been clarified. To investigate the nature of CWA1/BC1, we compared the CBM-like sequence in rice CWA1/BC1 with bacterial CBM2, 10 members of the BC1-like (BC1L) protein in rice and CWA1/BC1 orthologs, Arabidopsis COBL4 and maize Bk2. Furthermore, we constructed three-point mutated CWA1/BC1, in which three conserved Trp residues in CBM-like sequence were substituted for Val residues (CWA1/BC1^W→V), and introduced it into the cwa1 mutant to evaluate the necessity of the CBM-like sequence for proper function of CWA1/BC1. We discuss a putative explanation, based on our results, of the properties and possible functions of CWA1/BC1.     

Structure and characterization of amidase from Rhodococcus sp. N-771: Insight into the molecular mechanism of substrate recognition

In this study, we have structurally characterized the amidase of a nitrile-degrading bacterium, Rhodococcus sp. N-771 (RhAmidase). RhAmidase belongs to amidase signature (AS) family, a group of amidase families, and is responsible for the degradation of amides produced from nitriles by nitrile hydratase. Recombinant RhAmidase exists as a dimer of about 107 kDa. RhAmidase can hydrolyze acetamide, propionamide, acrylamide and benzamide with kcat/Km values of 1.14 ± 0.23 mM^− 1s^− 1, 4.54 ± 0.09 mM^− 1s^− 1, 0.087 ± 0.02 mM^− 1s^− 1 and 153.5 ± 7.1 mM^− 1s^− 1, respectively. The crystal structures of RhAmidase and its inactive mutant complex with benzamide (S195A/benzamide) were determined at resolutions of 2.17 Å and 2.32 Å, respectively. RhAmidase has three domains: an N-terminal α-helical domain, a small domain and a large domain. The N-terminal α-helical domain is not found in other AS family enzymes. This domain is involved in the formation of the dimer structure and, together with the small domain, forms a narrow substrate-binding tunnel. The large domain showed high structural similarities to those of other AS family enzymes. The Ser-cis Ser-Lys catalytic triad is located in the large domain. But the substrate-binding pocket of RhAmidase is relatively narrow, due to the presence of the helix α13 in the small domain. The hydrophobic residues from the small domain are involved in recognizing the substrate. The small domain likely participates in substrate recognition and is related to the difference of substrate specificities among the AS family amidases.     

Protein disulfide isomerase-P5, down-regulated in the final stage of boar epididymal sperm maturation,catalyzes disulfide formation to inhibit protein function in oxidative refolding of reduced denatured lysozyme

In mammalian spermiogenesis, sperm mature during epididymal transit to get fertility. The pig sharing many physiological similarities with humans is considered a promising animal model in medicine. We examined the expression profiles of proteins from boar epididymal caput, corpus, and cauda sperm by two-dimensional gel electrophoresis and peptide mass fingerprinting. Our results indicated that protein disulfide isomerase-P5 (PDI-P5) human homolog was down-regulated from the epididymal corpus to cauda sperm, in contrast to the constant expression of protein disulfide isomerase A3 (PDIA3) human homolog. To examine the functions of PDIA3 and PDI-P5, we cloned and sequenced cDNAs of pig PDIA3 and PDI-P5 protein precursors. Each recombinant pig mature PDIA3 and PDI-P5 expressed in Escherichia coli showed thiol-dependent disulfide reductase activities in insulin turbidity assay. Although PDIA3 showed chaperone activity to promote oxidative refolding of reduced denatured lysozyme, PDI-P5 exhibited anti-chaperone activity to inhibit oxidative refolding of lysozyme at an equimolar ratio. SDS-PAGE and Western blotting analysis suggested that disulfide cross-linked and non-productively folded lysozyme was responsible for the anti-chaperone activity of PDI-P5. These results provide a molecular basis and insights into the physiological roles of PDIA3 and PDI-P5 in sperm maturation and fertilization.     

Purification,Biochemical Characterization,and Cloning of Phospholipase D from <Emphasis Type="Italic">Streptomyces racemochromogenes</Emphasis> Strain 10-3

We previously isolated Streptomyces racemochromogenes strain 10-3, which produces a phospholipase D (PLD) with high transphosphatidylation activity. Here, we purified and cloned the PLD (PLD103) from the strain. PLD103 exerted the highest hydrolytic activity at a slightly alkaline pH, which is in contrast to the majority of known Streptomyces PLDs that have a slightly acidic optimum pH. PLD103 shares only 71–76% amino acid sequence identity with other Streptomyces PLDs that have a slightly acidic optimum pH; thus, the diversity in the primary structure might explain the discrepancy observed in the optimum pH. The purified PLD displayed high transphosphatidylation activity in the presence of glycerol, l-serine, and 2-aminoethanol hydrochloride with a conversion rate of 82–97% in a simple one-phase system, which was comparable to the rate of other Streptomyces PLDs in a complicated biphasic system.     

Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells

Intestinal stem cells, characterized by high Lgr5 expression, reside between Paneth cells at the small intestinal crypt base and divide every day. We have carried out fate mapping of individual stem cells by generating a multicolor Cre-reporter. As a population, Lgr5(hi) stem cells persist life-long, yet crypts drift toward clonality within a period of 1-6 months. We have collected short- and long-term clonal tracing data of individual Lgr5(hi) cells. These reveal that most Lgr5(hi) cell divisions occur symmetrically and do not support a model in which two daughter cells resulting from an Lgr5(hi) cell division adopt divergent fates (i.e., one Lgr5(hi) cell and one transit-amplifying [TA] cell per division). The cellular dynamics are consistent with a model in which the resident stem cells double their numbers each day and stochastically adopt stem or TA fates. Quantitative analysis shows that stem cell turnover follows a pattern of neutral drift dynamics.     

Cloning and Expression of a Novel NADP(H)-Dependent Daidzein Reductase,an Enzyme Involved in the Metabolism of Daidzein,from Equol-Producing Lactococcus Strain 20-92

Equol is a metabolite produced from daidzein by enteric microflora, and it has attracted a great deal of attention because of its protective or ameliorative ability against several sex hormone-dependent diseases (e.g., menopausal disorder and lower bone density), which is more potent than that of other isoflavonoids. We purified a novel NADP(H)-dependent daidzein reductase (L-DZNR) from Lactococcus strain 20-92 (Lactococcus 20-92; S. Uchiyama, T. Ueno, and T. Suzuki, international patent WO2005/000042) that is involved in the metabolism of soy isoflavones and equol production and converts daidzein to dihydrodaidzein. Partial amino acid sequences were determined from purified L-DZNR, and the gene encoding L-DZNR was cloned. The nucleotide sequence of this gene consists of an open reading frame of 1,935 nucleotides, and the deduced amino acid sequence consists of 644 amino acids. L-DZNR contains two cofactor binding motifs and an 4Fe-4S cluster. It was further suggested that L-DZNR was an NAD(H)/NADP(H):flavin oxidoreductase belonging to the old yellow enzyme (OYE) family. Recombinant histidine-tagged L-DZNR was expressed in Escherichia coli. The recombinant protein converted daidzein to (S)-dihydrodaidzein with enantioselectivity. This is the first report of the isolation of an enzyme related to daidzein metabolism and equol production in enteric bacteria.Isoflavones are flavonoids present in various plants and are known to be abundant in soybeans and legumes. These compounds have been called phytoestrogens because their chemical structure is similar to that of the female sex hormone, estrogen. Isoflavones have an ability to bind to estrogen receptors and show protection against or improvement in several sex hormone-dependent diseases, such as breast cancer, prostate cancer, menopausal disorder, lower bone density, and hypertension, due to their weak agonistic or antagonistic effects (1, 19, 27).Daidzein is one of the main soy isoflavonoids produced from daidzin by the glucosidase of intestinal bacteria (17). Equol is a metabolite produced from daidzein by the enterobacterial microflora (5). Recently, equol has attracted a great deal of attention because its estrogenic activity is more potent than that of other isoflavonoids, including daidzein (27). It is well known that individual variation exists in the ability of these enteric microflora to produce equol and that less than half the human population is capable of producing equol after ingesting soy isoflavones (3). Therefore, to increase the production of equol in the enteric environment of each individual, the development of probiotics using safe bacteria which have the ability to produce equol from daidzein is ongoing.Lactococcus strain 20-92 (Lactococcus 20-92; 30a) is an equol-producing lactic acid bacterium isolated from the feces of healthy humans by Uchiyama et al. (30). This bacterium is spherical and Gram positive and is a strain of L. garvieae. The application of Lactococcus 20-92 in probiotics is advantageous because L. garvieae is not pathogenic or toxic to humans.To date, other bacterial strains that are capable of transforming daidzein to dihydrodaidzein or equol have been isolated (9, 21, 22, 23, 29, 32, 36, 37). Daidzein is thought to be metabolized by human intestinal bacteria to equol or to O-desmethylangolensin via dihydrodaidzein and tetrahydrodaidzein (14, 15, 22, 32); however, neither the enzymes involved in the metabolism of daidzein to equol nor even the metabolic pathway has been clarified fully for equol-producing bacteria.In this study, we purified an enzyme from Lactococcus 20-92 that assisted in the conversion of daidzein to dihydrodaidzein. Furthermore, we cloned the L-DZNR gene and expressed the active recombinant enzyme in E. coli.     

Mechanical forces regulate elastase activity and binding site availability in lung elastin

Many fundamental cellular and extracellular processes in the body are mediated by enzymes. At the single molecule level, enzyme activity is influenced by mechanical forces. However, the effects of mechanical forces on the kinetics of enzymatic reactions in complex tissues with intact extracellular matrix (ECM) have not been identified. Here we report that physiologically relevant macroscopic mechanical forces modify enzyme activity at the molecular level in the ECM of the lung parenchyma. Porcine pancreatic elastase (PPE), which binds to and digests elastin, was fluorescently conjugated (f-PPE) and fluorescent recovery after photobleach was used to evaluate the binding kinetics of f-PPE in the alveolar walls of normal mouse lungs. Fluorescent recovery after photobleach indicated that the dissociation rate constant (k_off) for f-PPE was significantly larger in stretched than in relaxed alveolar walls with a linear relation between k_off and macroscopic strain. Using a network model of the parenchyma, a linear relation was also found between k_off and microscopic strain on elastin fibers. Further, the binding pattern of f-PPE suggested that binding sites on elastin unfold with strain. The increased overall reaction rate also resulted in stronger structural breakdown at the level of alveolar walls, as well as accelerated decay of stiffness and decreased failure stress of the ECM at the macroscopic scale. These results suggest an important role for the coupling between mechanical forces and enzyme activity in ECM breakdown and remodeling in development, and during diseases such as pulmonary emphysema or vascular aneurysm. Our findings may also have broader implications because in vivo, enzyme activity in nearly all cellular and extracellular processes takes place in the presence of mechanical forces.     

Estimating stand volume in broad-leaved forest using discrete-return LiDAR: plot-based approach

Quantitative assessment of forests is important at a variety of scales for forest planning and management. This study investigated the use of small-footprint discrete-return lidar for estimating stand volume in broad-leaved forest at plot level. Field measurements were conducted at 20 sample plots in the study area in western Japan, composed of temperate broad-leaved trees. Five height variables and two density variables were derived from the lidar data: 25th, 50th, 75th, and 100th percentiles, and mean of laser canopy heights as height variables (h ₂₅, h ₅₀, h ₇₅, h ₁₀₀, h _mean); and ground fraction and only-and-vegetation fraction (d _GF, d _OVF) as density variables, defined respectively as the proportion of laser returns that reached the ground, and the proportion of only echoes (i.e., single pulse returns for which the first and last pulses returned from the same point) within vegetation points. In addition, the normalized difference vegetation index (NDVI), which is often used as an estimator for leaf area index (LAI) and above-ground biomass, was derived from multispectral digital imagery as an alternative density variable (d _NDVI). Nonlinear least-square regression with cross-validation analysis was performed with single variables and combinations; a total of 23 models were studied. The best prediction was found when h ₇₅ and d _OVF were used as independent variables, resulting in adjusted R ² of 0.755 and root-mean-square error (RMSE) of 41.90 m³ ha⁻¹, corresponding to 16.4% of the mean stand volume, better than or comparable to the prediction models of previous studies.     

Impacts of land-use history on the diversity of a riparian forest landscape in warm-temperate Kyushu,southern Japan

We examined the impacts of land-use history on the species composition and diversity of a warm-temperate riparian forest landscape in Kyushu, southern Japan, focusing on the relationship between evergreen oaks and deciduous trees in natural and seminatural forests. The species composition of 59 plots was classified into four types (A to D). Type A, which showed a significant bias towards sites not subject to nonforest land use since 1947, had high species diversity consisting of (1) many lucidophyllous components of the region, including the rare indigenous oak Quercus hondae, and (2) summergreen tree species of varying dominance and number representing unique or locally rare elements of the riparian landscape in this warm-temperate region. Type B was dominated by a common species of oak, Q. glauca, and displayed less clear distribution bias with land-use history. In contrast to types A and B, types C and D, which were characterized by high dominance of deciduous trees, had negative bias away from sites that had been under forest land use in 1947. Presumably, intensive anthropogenic disturbances associated with nonforest land uses had expanded the habitats for deciduous trees. This phenomenon was represented by the establishment of forests (type D) dominated by Ulmus davidiana var. japonica (UDJ) after it had been released from the suppression of evergreen forest trees during a period of nonforest land use that prevents the successful recovery of evergreen trees. From these results we conclude that the impacts of land-use history on the diversity of warm-temperate riparian forest landscape are multiphased: a period of nonforest land use has a strong negative impact on lucidophyllous forest trees represented by the rare indigenous oak Q. hondae; release from the suppressive effects of the lucidophyllous species then encourages establishment of locally rare deciduous tree flora represented by UDJ, which continue to persist for decades after abandonment of nonforest land use.