OsGLU1, A Putative Membrane-bound Endo-1,4-ß-D-glucanase from Rice, Affects Plant Internode Elongation

A dwarf mutant glu was identified from screening of T-DNA tagged rice population. Genetic analysis of the T1 generation of glu revealed that a segregation ratio of wild-type:dwarf phenotype was 3:1, suggesting that the mutated phenotype was controlled by a single recessive nuclear locus. The mutated gene OsGLU1, identified by Tail-PCR, encodes a putative membrane-bound endo-1,4-β-D-glucanase, which is highly conserved between mono- and dicotyledonous plants. Mutation of OsGLU1 resulted in a reduction in cell elongation, and a decrease in cellulose content but an increase in pectin content, suggesting that OsGLU1 affects the internode elongation and cell wall components of rice plants. Transgenic glu mutants harboring the OsGLU1 gene complemented the mutation and displayed the wild-type phenotype. In addition, OsGLU1 RNAi plants showed similar phenotype as the glu mutant has. These results indicate that OsGLU1 plays important roles in plant cell growth. Gibberellins and brassinosteroids induced OsGLU1 expression. In rice genome, endo-1,4-β-D-glucanases form a multiple gene family with 15 members, and each may have a distinct expression pattern in different organs. These results indicate that endo-1, 4-β-D-glucanases may play diverse roles in growth and developmental process of rice plants. Hua-Lin Zhou, Si-Jie He: These authors contributed equally to this work     

The Pepper Extracellular Xyloglucan-Specific Endo-β-1,4-Glucanase Inhibitor Protein Gene,CaXEGIP1, Is Required for Plant Cell Death and Defense Responses

Plants produce various proteinaceous inhibitors to protect themselves against microbial pathogen attack. A xyloglucan-specific endo-β-1,4-glucanase inhibitor1 gene, CaXEGIP1, was isolated and functionally characterized in pepper (Capsicum annuum) plants. CaXEGIP1 was rapidly and strongly induced in pepper leaves infected with avirulent Xanthomonas campestris pv vesicatoria, and purified CaXEGIP1 protein significantly inhibited the hydrolytic activity of the glycoside hydrolase74 family xyloglucan-specific endo-β-1,4-glucanase from Clostridium thermocellum. Soluble-modified green fluorescent protein-tagged CaXEGIP1 proteins were mainly localized to the apoplast of onion (Allium cepa) epidermal cells. Agrobacterium tumefaciens-mediated overexpression of CaXEGIP1 triggered pathogen-independent, spontaneous cell death in pepper and Nicotiana benthamiana leaves. CaXEGIP1 silencing in pepper conferred enhanced susceptibility to virulent and avirulent X. campestris pv vesicatoria, accompanied by a compromised hypersensitive response and lowered expression of defense-related genes. Overexpression of dexamethasone:CaXEGIP1 in Arabidopsis (Arabidopsis thaliana) enhanced resistance to Hyaloperonospora arabidopsidis infection. Comparative histochemical and proteomic analyses revealed that CaXEGIP1 overexpression induced a spontaneous cell death response and also increased the expression of some defense-related proteins in transgenic Arabidopsis leaves. This response was also accompanied by cell wall thickening and darkening. Together, these results suggest that pathogen-inducible CaXEGIP1 positively regulates cell death-mediated defense responses in plants.Plant cell walls provide a physical barrier that separates challenging pathogens from the internal contents of plant cells. Additionally, the cell walls regulate cell expansion and differentiation (York et al., 2004; Flors et al., 2007; Cantu et al., 2008). Polysaccharides, such as cellulose, hemicellulose, and pectic polysaccharides, are the main components of primary cell walls. Xyloglucan (XG), the most abundant hemicellulose in the primary cell wall, plays a structural role by forming strong hydrogen bonds with cellulose microfibrils (Carpita and Gibeaut, 1993). The primary structure of XG contains a common β-(1→4)-d-glucan backbone, which is repeatedly substituted with α(1→6)-d-xylopyranosyl residues. Depolymerization of XG is proposed to play an important role during both cell wall expansion and pathogen invasion (Bourquin et al., 2002; Qin et al., 2003; Baumann et al., 2007). During cell wall expansion, plant XG endotransglycosylases cut and rejoin XG chains to allow the cellulose microfibrils to move apart. From a pathogen point of view, the carbon-rich complex represents a useful energy source for pathogen growth. To facilitate penetration into the plant tissues and to catabolize carbon sources, many plant pathogens secrete a mixture of cell wall-degrading enzymes, such as polygalacturonases, pectin methyl esterases, pectin/pecatae lyases, xylanases, and endoglucanases (Valette-Collet et al., 2003; DeBoy et al., 2008). Some microbial glycoside hydrolase (GH) family proteins, including GH5, GH12, and GH74, reportedly hydrolyze plant-derived XG (Martinez-Fleites et al., 2006; Gloster et al., 2007).To inhibit pathogen-derived cell wall-degrading enzymes, plants secrete a mixture of inhibitor proteins into the cell wall (Qin et al., 2003; An et al., 2008; Xie et al., 2008). Some of the best characterized inhibitor proteins are polygalacturonase-inhibiting proteins (PGIPs; Albersheim and Anderson, 1971; De Lorenzo and Ferrari, 2002; Federici et al., 2006). In bean (Phaseolus vulgaris), two pairs of PGIPs, PvPGIP1/PvPGIP2 and PvPGIP3/PvPGIP4, are present in the genome. These genes may have originated from independent gene duplication events (D’Ovidio et al., 2004a). PvPGIP2 strongly inhibits polygalacturonases from Fusarium phyllophilum and Aspergillus niger via three conserved Asp residues (Spinelli et al., 2009). PGIPs reduce the hydrolytic activity of polygalacturonases to favor the accumulation of long-chain oligogalacturonides, known as elicitors of a variety of defense responses (Côté and Hahn, 1994; D’Ovidio et al., 2004b). Furthermore, transgenic expression of pear (Pyrus communis) PGIP in transgenic tomato (Solanum lycopersicum) plants limited fungal colonization, suggesting a role of PGIPs in plant defense (Powell et al., 2000).The proteinaceous inhibitor of the cell wall-degrading enzyme xyloglucan-specific endo-β-1,4-glucanase (XEG) was identified from suspension-cultured tomatoes (Qin et al., 2003). The purified xyloglucan-specific endo-β-1,4-glucanase inhibitor protein (XEGIP) strongly inhibited XEG activity through the formation of a 1:1 protein:protein complex with XEG of Aspergillus aculeatus. More recently, two putative XEGIPs were isolated from Nicotiana benthamiana based on conserved regions found in plant XEGIP genes, and these genes were functionally characterized using virus-induced gene silencing (VIGS; Xie et al., 2008). VIGS of NbXEGIP1 strongly enhanced the wilting symptoms exhibited following infection by virulent Pseudomonas syringae pv tabaci. This finding supports the notion that NbXEGIP1 may act as an inhibitor of bacterial cell wall-degrading enzymes in N. benthamiana plants.Programmed cell death (PCD) has been extensively characterized in plants (Lam, 2004). The hypersensitive response (HR), a well-known form of plant PCD, is one of the most efficient and immediate resistance reactions of plants. The HR is characterized by the rapid death of cells in the local region surrounding an infection site. As a result, the growth and spread of the pathogen is restricted or confined. During HR cell death development, cell wall strengthening occurs. Histochemical analyses of cells involved in melon (Cucumis melo)-powdery mildew (Podosphaera fusca) interactions demonstrate the reinforcement of the cell wall compartment as part of HR cell death-mediated resistance (Romero et al., 2008). Treatment of suspension-cultured tobacco (Nicotiana tabacum) cells with cryptogein, a 10-kD protein secreted by the oomycete Phytophthora cryptogea, induces a HR on tobacco leaves, accompanied by induced strengthening of the cell wall (Kieffer et al., 2000). However, the role of cell wall strengthening in HR cell death is poorly understood. A second type of PCD is thought to be associated with the differentiation of procambium into tracheary elements in the xylem of vascular plants (Fukuda, 2000; Lam, 2004). During the early formation of mature tracheary elements, vacuoles accumulate degradation enzymes and the cell wall is remodeled into a highly reticulated form. A similar phenomenon occurs during some plant developmental processes, including senescence and aerenchyma formation in roots (Jones, 2001).In this study, we have isolated and functionally characterized a pepper (Capsicum annuum) xyloglucan-specific endo-β-1,4-glucanase inhibitor-protein1 gene (CaXEGIP1). Expression of CaXEGIP1 was strongly induced in pepper leaves infected with avirulent Xanthomonas campestris pv vesicatoria (Xcv) strain Bv5-4a. The purified CaXEGIP1 protein inhibited the hydrolytic activity of GH74 family XEG from the thermophilic bacterium Clostridium thermocellum. The soluble-modified GFP (smGFP)-fused CaXEGIP1 protein was localized in the external and intercellular regions of onion epidermal cells. Importantly, Agrobacterium tumefaciens-mediated transient expression of CaXEGIP1 induced the hypersensitive cell death response in pepper and N. benthamiana leaves. VIGS of CaXEGIP1 significantly enhanced the growth of virulent and avirulent Xcv in pepper leaves, accompanied by compromised HR cell death and lowered expression of CaPR1 (pathogenesis-related protein1 [PR1]) and CaDEF1 (defensin [DEF1]). We also investigated the role of CaXEGIP1 in plant cell death and defense responses using transgenic Arabidopsis (Arabidopsis thaliana) plants harboring the dexamethasone (DEX)-inducible CaXEGIP1 transgene. Overexpression of CaXEGIP1 triggered spontaneous cell death and modification of the cell wall compartment in Arabidopsis plants. Together, these results suggest that the pathogen-responsive CaXEGIP1 is involved in plant cell death-mediated defense signaling.     

MppA,a Periplasmic Binding Protein Essential for Import of the Bacterial Cell Wall Peptide l-Alanyl-γ-d-Glutamyl-meso-Diaminopimelate

Mutants of a diaminopimelic acid (Dap)-requiring strain of Escherichia coli were isolated which failed to grow on media in which Dap was replaced by the cell wall murein tripeptide, l-alanyl-γ-d-glutamyl-meso-diaminopimelate. In one such mutant, which is oligopeptide permease (Opp) positive, we have identified a new gene product, designated MppA (murein peptide permease A), that is about 46% identical to OppA, the periplasmic binding protein for Opp. A plasmid carrying the wild-type mppA gene allows the mutant to grow on tripeptide. Two other mutants that failed to grow on tripeptide were resistant to triornithine toxicity, indicating a defect in the opp operon. An E. coli strain whose entire opp operon was deleted but which carried the mppA locus was unable to grow on murein tripeptide unless it was provided with oppBCDF genes in trans. Our data suggest a model whereby the periplasmic MppA binds the murein tripeptide, which is then transported into the cytoplasm via membrane-bound and cytoplasmic OppBCDF. In assessing the affinity of MppA for non-cell wall peptides, we have found that proline auxotrophy can be satisfied with the peptide Pro-Phe-Lys, which utilizes either MppA or OppA in conjunction with OppBCDF for its uptake. Thus, MppA, OppA, and perhaps the third OppA paralog revealed by the E. coli genome sequence may each bind a particular family of peptides but interact with common membrane-associated components for transport of their bound ligands into the cell. As to the physiological function of MppA, the possibility that it may be involved in signal transduction pathway(s) is discussed.During growth, Escherichia coli breaks down over one-third of its cell wall each generation and efficiently reutilizes the tripeptide therefrom for synthesis of new murein in a sequence of events termed the recycling pathway (9, 11, 32; see reference 33 for a review). In this pathway, murein is degraded to N-acetylglucosaminyl-1,6-anhydro-N-acetylmuramyl-l-alanyl- γ-d-glutamyl-meso-diaminopimelate (GlcNAc-anhMurNAc-tripeptide) by the combined action of lytic transglycosylases, endopeptidases, and d,d- and l,d-carboxypeptidases which are present in the periplasm (39). The muropeptide, GlcNAc- anhMurNAc-tripeptide, presumably is transported into the cytoplasm via the membrane-bound AmpG permease (20, 24). The tripeptide is then released from the muropeptide by AmpD anhydro-N-acetylmuramyl-l-alanine amidase (19, 21). Surprisingly, almost all murein tripeptide for recycling is transported into the cell as GlcNAc-anhMurNAc-tripeptide via the AmpG permease and is then released by the cytoplasmic AmpD amidase (20, 32), rather than being transported as the free tripeptide via the oligopeptide permease (Opp) as was originally proposed (10). Direct utilization of the tripeptide for cell wall synthesis was assumed to depend on a hypothetical ligase which would attach tripeptide to UDP-MurNAc, thereby reintroducing it into the biosynthetic pathway for wall synthesis (9, 20, 33). In fact, the enzyme responsible for this activity has recently been identified, and the gene, mpl, was shown to be the open reading frame (ORF) yifG at 96 min on the E. coli map (29). An mpl null mutant was completely devoid of ligase activity, and cells of this mutant were viable and accumulated tripeptide in their cytoplasm (29).During a search for mutants lacking this murein peptide ligase activity, four mutants were isolated from a pool of mutagenized diaminopimelic acid (Dap)-negative (dap) parental cells in a screen that assayed the growth of cells on free tripeptide as a source of Dap. In this report, we describe the isolation and initial characterization of one such mutant. A new genetic locus, mppA, has been identified which codes for a periplasmic binding protein required for uptake of murein peptides. Two other mutants, one with a mutation in oppB and the other with a mutation in groESL (unpublished), were found to be defective in Opp function because of their resistance to triornithine toxicity. The oppB mutation indicates that murein tripeptide is transported from MppA into the cytoplasm via membrane components of Opp, and the groE mutation suggests that the chaperonin is involved in the proper folding and assembly of the components of the peptide transport system.     

Purification and Characterization of Two Endo-β-1,4-glucanases from Mollusca, Ampullaria crossean

Two novel endo-β-1,4-glucanases, EG45 and EG27, were isolated from the gastric juice of mollusca, Ampullaria crossean, by anion exchange, hydrophobic interaction, gel filtration and a second round of anion exchange chromatography. The purified proteins EG45 and EG27 appeared as a single band on sodium dodecylsulfate polyacrylamide gel electrophoresis with a molecular mass of 45 kDa and 27 kDa, respectively. The optimum pH for CMC activity was 5.5 for EG45 and 4.4-4.8 for EG27. The optimum temperature range for EG27 was broad, between 50℃ and 60 ℃; for EG45 it was 50 ℃. The analysis on the stability of these two endo-β-1,4-glucanases showed that EG27 was acceptably stable at pH 3.0-11.0 even when the incubation time was prolonged to 24 h at 30 ℃, whereas EG45 remained relatively stable at pH 5.0-8.0. About 85% of the activity of EG27 could be retained upon incubation at 60 ℃ for 24 h. However, less than 10% residual activity of EG45 was detected at 50 ℃. Among different kinds of substrates, both enzymes showed a high preference for carboxymethyl cellulose. EG45, in particular, showed a carboxymethyl cellulose hydrolytic activity of 146.5 IU/mg protein. Both enzymes showed low activities to xylan （from oat spelt） and Sigmacell 101, and they were inactive to p-nitrophenyl-β-D-cellobioside, salicin and starch.     

Distribution and Properties of Endo-β-1,4-glucanase from a Lower Termite,Coptotermes formosanus (Shiraki)

A multi-enzyme distribution of endo-β-1,4-glucanase activity was found in the digestive system of a worker caste of the lower termite Coptotermes formosanus (Shiraki) by zymogram analysis. Its distribution analysis demonstrated that about 80% of this activity was localized in salivary glands from where only one component (EG-E) was secreted into the digestive tract.

EG-E was isolated by a combination of chromatographic and electrophoretic techniques. Its molecular mass, optimal pH and temperature, isoelectric point, and K _m were 48 kDa, 6.0, 50°C, 4.2, and 3.8 (mg/ml on carboxymethylcellulose), respectively. EG-E hydrolyzed cellooligosaccharides with a degree of polymerization of 4 and larger, and had low activity on crystalline cellulose. Main reaction products from low molecular weight cellulose were cellobiose and cellotriose. The N-terminal amino acid sequence of EG-E has similarity with fungal endo-β-1,4-glucanases and cellobiohydrolases of the glycosyl hydrolase family 7 rather than the other insect endo-β-1,4-glucanases of family 9.     

Identification and Deletion of Tft1, a Predicted Glycosyltransferase Necessary for Cell Wall β-1,3;1,4-Glucan Synthesis in Aspergillus fumigatus

Aspergillus fumigatus is an environmental mold that causes severe, often fatal invasive infections in immunocompromised patients. The search for new antifungal drug targets is critical, and the synthesis of the cell wall represents a potential area to find such a target. Embedded within the main β-1,3-glucan core of the A. fumigatus cell wall is a mixed linkage, β-D-(1,3;1,4)-glucan. The role of this molecule or how it is synthesized is unknown, though it comprises 10% of the glucans within the wall. While this is not a well-studied molecule in fungi, it has been studied in plants. Using the sequences of two plant mixed linkage glucan synthases, a single ortholog was identified in A. fumigatus (Tft1). A strain lacking this enzyme (tft1Δ) was generated along with revertant strains containing the native gene under the control of either the native or a strongly expressing promoter. Immunofluorescence staining with an antibody against β-(1,3;1,4)-glucan and biochemical quantification of this polysaccharide in the tft1Δ strain demonstrated complete loss of this molecule. Reintroduction of the gene into the knockout strain yielded reappearance in amounts that correlated with expected expression of the gene. The loss of Tft1 and mixed linkage glucan yielded no in vitro growth phenotype. However, there was a modest increase in virulence for the tft1Δ strain in a wax worm model. While the precise roles for β-(1,3;1,4)-glucan within A. fumigatus cell wall are still uncertain, it is clear that Tft1 plays a pivotal role in the biosynthesis of this cell wall polysaccharide.     

Amino Acid Sequence and Stereoselective Hydrolytic Reaction of an Endo-1,4-β-glucanase from a Bacillus Strain

Bacillus sp. KSM-522 produces three different extracellular endo-l,4-β-glucanases [EGs; Okoshi et al., Agric. Biol. Chem., 54, 83–89 (1990)]. Here, we report the molecular cloning and sequencing of the gene for the fourth EG (EG-IV) of the organism and the mechanism of its hydrolytic reaction. The structural gene contained an open reading frame of 1911 bp, corresponding to 636 amino acids, the amino acid sequence of which was very close to that of an EG of Clostridium cellulovorans, belonging to the cellulase family E2. The molecular mass of the extracellular mature enzyme (Ser²⁶ through Lys⁶³⁶) was calculated to be 69,076 Da, a value close to the 69.2 kDa measured for the recombinant EG-IV expressed in Bacillus subtilis. The optimum pH and temperature for activity of the recombinant enzyme were pH 8.0 and 50°C, respectively. By ¹H-NMR spectroscopy, we demonstrated that the hydrolysis of p-nitrophenyl β-d-cellotrioside by EG-IV proceeded with inversion of the anomeric configuration.     

Identification and Characterization of a Halotolerant,Cold-Active Marine Endo-β-1,4-Glucanase by Using Functional Metagenomics of Seaweed-Associated Microbiota

A metagenomic library was constructed from microorganisms associated with the brown alga Ascophyllum nodosum. Functional screening of this library revealed 13 novel putative esterase loci and two glycoside hydrolase loci. Sequence and gene cluster analysis showed the wide diversity of the identified enzymes and gave an idea of the microbial populations present during the sample collection period. Lastly, an endo-β-1,4-glucanase having less than 50% identity to sequences of known cellulases was purified and partially characterized, showing activity at low temperature and after prolonged incubation in concentrated salt solutions.     

Klotho is a substrate for α-, β- and γ-secretase

Klotho is an anti-aging protein with different functions of the full-length membrane protein and the secreted hormone-like form. Using overexpression and knock-down approaches as well as embryonic fibroblasts of knock-out mice we present evidence that Klotho is shedded by the α-secretases ADAM10 and 17 as well as by the β-secretase β-APP cleaving enzyme 1. The remaining membrane-bound fragment is a substrate for regulated intramembrane proteolysis by γ-secretase. Our data suggest that therapeutic approaches targeting these proteases should be carefully analyzed for potential side effects on Klotho-mediated physiological processes.     

Determining the Subcellular Location of Synthesis and Assembly of the Cell Wall Polysaccharide (1,3; 1,4)-β-d-Glucan in Grasses

The current dogma for cell wall polysaccharide biosynthesis is that cellulose (and callose) is synthesized at the plasma membrane (PM), whereas matrix phase polysaccharides are assembled in the Golgi apparatus. We provide evidence that (1,3;1,4)-β-d-glucan (mixed-linkage glucan [MLG]) does not conform to this paradigm. We show in various grass (Poaceae) species that MLG-specific antibody labeling is present in the wall but absent over Golgi, suggesting it is assembled at the PM. Antibodies to the MLG synthases, cellulose synthase-like F6 (CSLF6) and CSLH1, located CSLF6 to the endoplasmic reticulum, Golgi, secretory vesicles, and the PM and CSLH1 to the same locations apart from the PM. This pattern was recreated upon expression of VENUS-tagged barley (Hordeum vulgare) CSLF6 and CSLH1 in Nicotiana benthamiana leaves and, consistent with our biochemical analyses of native grass tissues, shown to be catalytically active with CSLF6 and CSLH1 in PM-enriched and PM-depleted membrane fractions, respectively. These data support a PM location for the synthesis of MLG by CSLF6, the predominant enzymatically active isoform. A model is proposed to guide future experimental approaches to dissect the molecular mechanism(s) of MLG assembly.     

PtrCel9A6, an Endo-l,4-β-Glucanase,Is Required for Cell Wall Formation during Xylem Differentiation in Populus

ABSTRACT Endo-l,4-β-glucanases （EGases） are involved in many aspects of plant growth. Our previous study found that an EGase, PtrCel9A6, is specifically expressed in differentiating xylem cells during Populus secondary growth. In this study, the xylem-specific PtrCel9A6 was characterized for its role in xylem differentiation. The EGase is localized on the plasma membrane with catalytic domain toward the outside cell wall, hydrolyzing amorphous cellulose. Suppression of PtrCel9A6 expression caused secondary cell wall defects in xylem cells and significant cellulose reduction in Populus. Heterologous expression of PtrCelgA6 in Arabidopsis enhanced plant growth as well as increased fiber cell length. In addition, introduction of PtrCel9A6 into Arabidopsis resulted in male sterility due to defects in anther dehiscence. Together, these results demonstrate that PtrCel9A6 plays a critical role in remodeling the 1,4-β-glucan chains in the wall matrix and is required for cell wall thickening during Populus xylem differentiation.     

An α-Amylase Homologue,aah3, Encodes a GPI-Anchored Membrane Protein Required for Cell Wall Integrity and Morphogenesis in Schizosaccharomyces pombe

Glycosylphosphatidylinositol (GPI)-anchored proteins are essential for normal cellular morphogenesis and have an additional role in mediating cross-linking of glycoproteins to cell wall glucan in yeast cells. Although many GPI-anchored proteins have been characterized in Saccharomyces cerevisiae, none have been reported for well-characterized GPI-anchored proteins in Schizosaccharomyces pombe to date. Among the putative GPI-anchored proteins in S. pombe, four α-amylase homologs (Aah1p-Aah4p) have putative signal sequences and C-terminal GPI anchor addition signals. Disruption of aah3 ⁺ resulted in a morphological defect and hypersensitivity to cell wall-degrading enzymes. Biochemical analysis showed that Aah3p is an N-glycosylated, GPI-anchored membrane protein localized in the membrane and cell wall fractions. Conjugation and sporulation were not affected by the aah3 ⁺ deletion, but the ascal wall of aah3Δ cells was easily lysed by hydrolases. Expression of aah3 alleles in which the conserved aspartic acid and glutamic acid residues required for hydrolase activity were replaced with alanine residues failed to rescue the morphological and ascal wall defects of aah3Δ cells. Taken together, these results indicate that Aah3p is a GPI-anchored protein and is required for cell and ascal wall integrity in S. pombe.     

Purification and Partial Characterization of an Endo-(1→3, 1→4)-β-glucanase from Rice,Oryza sativa L.

(1→3, 1→4)-β-Glucanase (EC 3.2.1.73), with a molecular weight of 34, 000 and an isoelectric point of 4.9, was purified to homogeneity from extracts of fresh rice bran. The enzyme specifically hydrolyzed (1→3, 1→4)-β-glucans such as barley β-glucan and lichenans, but laminarins and CM-cellulose were not substrates. Endproduct analysis using barley β-glucan as the substrate suggested that the enzyme is an endo-type (1→3, 1→4)-β-glucanase.     

Recombinant expression,characterization, and pulp prebleaching property of a Phanerochaete chrysosporium endo-β-1,4-mannanase

A Phanerochaete chrysosporium cDNA predicted to encode endo-1,4-β-d-mannanase, man5D, was cloned and expressed in Aspergillus niger. The coding region of the gene man5D was predicted to contain, in order from the N-terminal: a secretory signal peptide, cellulose-binding domain, linker region, and glycosyl hydrolase family 5 catalytic site. The enzyme was purified from culture filtrate of A. niger transformants that carried the recombinant man5D. Recombinant Man5D had an apparent molecular size of about 65 kDa by SDS-PAGE, and optimal activity at pH 4.0–6.0 and 60 °C. It was stable from pH 4.0 to 8.0 and up to 60 °C. The enzyme showed affinity for Avicel cellulose, suggesting that the predicted cellulose-binding domain is biologically functional. The specific activities of Man5D on mannan, galactomannan, and glucomannan at pH 5 and 60 °C ranged from 160 to 460 μmol/(min mg), with apparent K_m values from 0.54 to 2.3 mg/mL. Product analysis results indicated that Man5D catalyzes endo-cleavage, and appears to have substantial transglycosylase activity. When used to treat softwood kraft pulp, Man5D hydrolyzed mainly glucomannan and exhibited a positive effect as a prebleaching agent. Compared to a commercial prebleaching with xylanase, the prebleaching effect of Man5D was weaker but with reduced loss of fibre yield as determined by the release of solubilized sugars.     

The N-Linked Outer Chain Mannans and the Dfg5p and Dcw1p Endo-α-1,6-Mannanases Are Needed for Incorporation of Candida albicans Glycoproteins into the Cell Wall

A biochemical pathway for the incorporation of cell wall protein into the cell wall of Neurospora crassa was recently proposed. In this pathway, the DFG-5 and DCW-1 endo-α-1,6-mannanases function to covalently cross-link cell wall protein-associated N-linked galactomannans, which are structurally related to the yeast outer chain mannans, into the cell wall glucan-chitin matrix. In this report, we demonstrate that the mannosyltransferase enzyme Och1p, which is needed for the synthesis of the N-linked outer chain mannan, is essential for the incorporation of cell wall glycoproteins into the Candida albicans cell wall. Using endoglycosidases, we show that C. albicans cell wall proteins are cross-linked into the cell wall via their N-linked outer chain mannans. We further demonstrate that the Dfg5p and Dcw1p α-1,6-mannanases are needed for the incorporation of cell wall glycoproteins into the C. albicans cell wall. Our results support the hypothesis that the Dfg5p and Dcw1p α-1,6-mannanases incorporate cell wall glycoproteins into the C. albicans cell wall by cross-linking outer chain mannans into the cell wall glucan-chitin matrix.     

The endo-1,4-β-glucanase Korrigan exhibits functional conservation between gymnosperms and angiosperms and is required for proper cell wall formation in gymnosperms

The evolution of compositional polymers and their complex arrangement and deposition in the cell walls of terrestrial plants included the acquisition of key protein functions. A membrane-bound endoglucanase, termed Korrigan (KOR), has been shown to be required for proper cellulose synthesis. To date, no extensive characterization of the gymnosperm KOR has been undertaken. Characterization of the white spruce (Picea glauca) gene encoding KOR (PgKOR) shows conserved protein features such as polarized targeting signals and residues predicted to be essential for catalytic activity. The rescue of the Arabidopsis thaliana kor1-1 mutant by the expression of PgKOR suggests gene conservation, providing evidence for functional equivalence. Analyses of endogenous KOR expression in white spruce revealed the highest expression in young developing tissues, which corresponds with primary cell wall development. Additionally, RNA interference of the endogenous gymnosperm gene substantially reduced growth and structural glucose content, but had no effect on cellulose ultrastructure. Partial functional conservation of KOR in gymnosperms suggests that its role in cell wall synthesis dates back to 300 million yr ago (Mya), predating angiosperms, which arose 130 Mya, and shows that proteins contributing to proper cellulose deposition are important conserved features of vascular plants.