Plant hydrolytic enzymes (chitinases and β-1,3-glucanases) in root reactions to pathogenic and symbiotic microorganisms

Within the last decade, a great deal of attention has been devoted to the role of chitinases and -1,3-glucanases in plant/microbe interactions. While there is strong evidence that these hydrolases are antifungal proteins, there are also recent indications of roles in both plant morphogenesis and plant/microbe signal perception. This paper reviews recent findings pertinent to root/microbe interactions, and discusses the nature and significance of specific hydrolase isoforms in symbioses with arbuscular mycorrhizal (AM) fungi.     

Synthesis of β(1,3) oligoglucans exhibiting a Dectin-1 binding affinity and their biological evaluation

In this report, we describe the synthesis and biological evaluation of β(1,3) oligosaccharides that contain an aminoalkyl group and their biological evaluation. A 2,3 diol glycoside with a 4,6 benzylidene protecting group was used as an effective glycosyl acceptor for the synthesis of some β(1,3) linked glycosides. The use of a combination of a linear tetrasaccharide and a branched pentasaccharide as glycosyl donors led to the preparation of β(1,3) linear octa- to hexadecasaccharides and branched nona- to heptadecasaccharides in good total yields. Measurements of the competitive effects of the oligosaccharides on the binding of a soluble form of Dectin-1 to a solid-supported Schizophyllan (SPG) revealed that the branched heptadecasaccharide and the linear hexadecasaccharides also have binding activity for Dectin-1. In addition, the two oligosaccharides, both of which contain a β(1,3) hexadecasaccharide backbone, exhibited agonist activity in a luciferase-assisted NF-κB assay. STD-NMR analyses of complexes of Dectin-1 and the linear hexadecasaccharides clearly indicate Dectin-1 specifically recognizes the sugar part of the oligosaccharides and not the aminoalkyl chain.     

Double diastereoselection explains limitations in synthesizing mannose-containing β-(1,3)-glucans

It is known that 3-O-glycosylation of glucosidic acceptors bearing acyl groups in the 4 and 6 positions instead of a 4,6-O-benzylidene ring mainly affords α-glycosides. Described here is an unexpected stereochemical outcome for elongation at glucose O-3 of a β-d-Glcp-(1→3)-α-d-Manp disaccharide using peracetylated ethyl thioglucoside as a donor. This unexpected reaction was correlated with match-mismatch effects, as shown by efficient coupling of the same acceptor by a donor of l-configuration.     

Purification and cellulosomal localization ofClostridium thermocellum mixed linkage β-glucanase LicB (1,3–1,4-β-D-glucanase)

Summary Clostridium thermocellum recombinant -(1,3–1,4)-glucanase LicB was expressed inEscherichia coli K-12 and purified to electrophoretic homogeneity. Specific activity of the final preparation was 12,000 U/mg. Antibodies against recombinant LicB protein reacted with a specific band of purifiedC. thermocellum F7 cellulosomes, suggesting localization in the extracellular multienzyme complex. This indicates that the cellulosome takes part in the hydrolysis of other polysaccharides besides cellulose.     

Effects of adeno-associated virus (AAV) of transforming growth factors β1 and β3 (TGFβ1,3) on promoting synthesis of glycosaminoglycan and collagen type Ⅱ of dedifferentiated nucleus pulposus (NP) cells

The effects of AAV-TGFβ1 and AAV-TGFβ3 on promoting synthesis of glycosaminoglycan and collagen type Ⅱ of dedifferentiated rabbit lumbar disc NP cells were studied in this work. The rabbit lumbar disc NP cells were isolated and cultured. The earlier and later dedifferentiated NP cells were established by subculture. The AAV transfection efficiency to dedifferentiated NP cells was analyzed with AAV-EGFP in vitro. After dedifferentiated NP cells were transfected by AAV-TGFβ1 or AAV-TGFβ3, their biological effects on promoting synthesis of glycosaminoglycan or collagen type Ⅱ were detected and compared by the methods of 35S incorporation or immunoblotting. The experimental results showed that AAV could transfect efficiently the earlier dedifferentiated NP cells, but its transfection rate was shown to be at a low level to the later dedifferentiated NP cells. Both AAV-TGFβ1 and AAV-TGFβ3 could promote the earlier dedifferentiated NP cells to synthesize glycosaminoglycan and collagen type Ⅱ, and the effect of AAV-TGFβ1 was better than that of AAV-TGFβ3. For the later dedifferentiated NP cells, the AAV-TGFβ3 could promote their synthesis, but AAV-TGFβ1 could slightly inhibit their synthesis. Therefore, AAV-TGFβ1 and AAV-TGFβ3 could be used for the earlier dedifferentiated NP cells, and the TGFβ3 could be used as the objective gene for the later dedifferentiated NP cells.     

Monosubstituted γ-lactam and conformationally constrained 1,3-diaminopropan-2-ol transition-state isostere inhibitors of β-secretase (BACE)

The synthesis, evaluation, and structure-activity relationships of a class of γ-lactam 1,3-diaminopropan-2-ol transition-state isostere inhibitors of BACE are discussed. Two strategies for optimizing lead compound 1a are presented. Reducing the overall size of the inhibitors resulted in the identification of γ-lactam 1i, whereas the introduction of conformational constraint on the prime-side of the inhibitor generated compounds such as the 3-hydroxypyrrolidine inhibitor 28n. The full in vivo profile of 1i in rats and 28n in Tg 2576 mice is presented.     

Synthesis and characterization of β-diketiminate germanium(II) and tin(II) bromides

The equimolar reaction of a β-diketiminate lithium salt LLi(OEt₂) [L = HC(CMeNAr)₂; Ar = 2,6-iPr₂C₆H₃] with either GeBr₂ or SnBr₂ in diethyl ether affords the synthetically useful monomeric β-diketiminate-element halides LGeBr (1) and LSnBr (2), respectively. Both are soluble in hydrocarbon solvents, stable in inert atmosphere, and have been characterized by elemental analysis, NMR spectroscopy, and single-crystal X-ray diffraction analysis.     

Characterization and expression of β-1,3-glucanase genes in peach

Beta-1,3-glucanase is one of the pathogenesis-related (PR) proteins involved in plant defense responses. A peach beta-1,3-glucanase gene, designated PpGns1, has been isolated and characterized. The deduced amino acid sequence of the product of PpGns indicates that it is a basic isoform (pI 9.8), and contains a putative signal peptide of 38 amino acids but has no C-terminal extension. Amino acid sequence comparisons revealed that PpGns1 is 69% and 67% identical to citrus and soybean beta-1,3-glucanases, respectively. Southern analysis of total genomic DNA also indicates that at least three genes for beta-1,3-glucanases exist in peach, forming a small gene family. Characterization of four additional clones by PCR has identified a second beta-1,3-glucanase gene, PpGns2. PpGns2 has been partially sequenced, and when compared to PpGns1, it shows high sequence homology, 96% and 99% nucleotide identity in the first and (partial) second exons, respectively. The deduced partial sequence of the PpGns2 product displays only two differences from PpGns1 in the signal peptide and one in the (partial) mature protein (141 amino acids). The 5'-flanking promoter regions of these two genes share 90% identity in nucleotide sequences interrupted by five major gaps (4-109 nt long). The promoter region contains various sequences similar to cis-regulatory elements present in different stress-induced plant genes. In leaves and stems of peach shoot cultures grown in vitro, PpGns1 is induced within 12 h after exposure to a culture filtrate of Xanthomonas campestris pv. pruni or ethephon. However, it is not induced following treatment with mercuric chloride.     

Developmental and hormonal regulation of β-1,3-glucanase in tobacco

A highly sensitive and specific rocket immunoassay was used to measure the content of an endo-type -1,3-glucanase (EC 3.2.1.39) in tissues of Nicotiana tabacum L. cv. Havana 425. We show that the accumulation of -1,3-glucanase in cultured pith-parenchyma tissue is blocked by combinations of the auxin, -naphthaleneacetic acid (NAA), and the cytokinin, kinetin. When tissues pre-incubated for 7 d on complete medium containing 2.0 mg·l^-1 NAA and 0.3 mg·l^-1 kinetin are transferred onto medium without hormones or with either hormone added separately, the -1,3-glucanase content expressed per mg soluble protein increases approx. ten fold over a 7-d period. Under these inductive conditions, up to approx. 5% of the soluble protein is -1,3-glucanase. The induction is inhibited by >90% when tissues are cultured over the same period on medium containing both hormones. This -1,3-glucanase is developmentally regulated in the intact plant. It is a major component of the soluble protien in the lower leaves and roots but is not detectable in leaves near the top of the plant.Abbreviation NAA -naphthaleneacetic acid     

Biosynthesis of (1,3)(1,4)-β-glucan and (1,3)-β-glucan in barley (Hordeum vulgare L.)

Mixed membrane preparations from the coleoptiles and first leaves of young barley (Hordeum vulgare L. cv. Triumph) plants catalysed the synthesis of 55% methanol-insoluble labelled material from UDP[U-¹⁴C]glucose, the main components of which were identified as (1,3)(1,4)-- and (1,3)--D-glucans. The membrane preparations also catalysed the transformation of UDP-glucose into labelled low-molecular-weight products, mainly glucose (by phosphatase action), glucose-1-phosphate (by phosphodiesterase action) and glyco(phospho)lipids (by glycosyltransferase action). The formation of (1,3)(1,4)--glucans, (1,3)--glucans, and the other reactions competing for UDP-glucose, were monitored simultaneously and quantitatively by a novel procedure based on enzymatic analysis, thin-layer chromatography and digital autoradiography. Thus it was possible (i) to optimise conditions to obtain (1,3)(1,4)--glucan synthesis or (1,3)--glucan synthesis in isolation, and (ii) to study the influence of temperature, pH, cofactors, substrate concentration etc. on the (1,3)(1,4) and (1,3)--glucan synthesis reactions even when both occurred together. The synthesis of both -glucans was optimal at 20°C. In Tris-HCl buffer, the pH optima for (1,3)(1,4)--glucan synthesis and (1,3)--glucan synthesis were pH 8.5 and pH 7.0, respectively. Both glucan-synthesis reactions required Mg²⁺: (1,3)--glucan synthesis was optimal at 2 mM, whereas (1,3)(1,4)--glucan synthesis continued to increase up to 200 mM Mg²⁺, when the ion was supplied as the sulphate. (1,3)--Glucan synthesis was Ca²⁺ dependent and this dependence could be abolished by proteinase treatment. The K _m with respect to UDP-glucose was 1.5 mM for (1,3)--glucan synthesis and approximately 1 mM for (1,3)(1,4)--glucan synthesis. The (1,3)(1,4)--glucan formed in vitro had the same ratio of trisaccharide to tetrasaccharide structural blocks irrespective of the experimental conditions used during the synthesis: its enzymatic fragmentation pattern was indistinguishable from that of barley endosperm (1,3)(1,4)--glucan. This indicates either a single synthase enzyme, which is responsible for the formation of both linkage types, or two enzymes which are very tightly coupled functionally.Abbreviations G4G4G3G Glc(1,4)Glc(1,4)Glc(1,3)Glc (-linked) - UDP-Glc uridine-5-diphosphate glucose We are grateful to the Commission of the European Communities for the award of Training Fellowships to Christine Vincent and Martin Becker.     

α-d-allopyranosyl β-d-fructofuranoside (allo-sucrose) and its derivatives

Reduction of 3-ketosucrose (1) with sodium borohydride gave mainly α-d-allopyranosyl β-d-fructofuranoside (2) characterized as its octabenzoate. Using sodium borodeuteride, [3-²H]allo-sucrose (5) and [3-²H]sucrose (6) were obtained in the ratio 12:1. The mixture was fractionated on Dowex-50 X8 resin (Ca²⁺ form), and the [3-²H] derivatives were isolated as their octa-acetates. Inspection of the ¹³C-n.m.r. spectra of 5 and 6 enabled the C-3 signals to be assigned. allo-Sucrose (2) was more readily obtained by oxidation of sucrose with dimethyl sulphoxide-acetic anhydride followed by reduction with sodium borohydride and fractionation on Dowex-50 X8 (Ca²⁺) resin. Tritylation of 2 followed by acetylation gave, after chromatography, the 6,1′,6′-tritrityl ether (9, 10%), the 6,6′-ditrityl ether (10, 26%), and a mixture of monotrityl ethers (20%). Hydrogenolysis of 9 and 10 gave the penta-acetate and hexa-acetate, respectively, with no detectable migration of AcO-4. Treatment of 2 with sulphuryl chloride at -50° gave the 6,6′-dichloride.     

Structural insights into the substrate specificity of Streptococcus pneumoniae β(1,3)-galactosidase BgaC

The surface-exposed β-galactosidase BgaC from Streptococcus pneumoniae was reported to be a virulence factor because of its specific hydrolysis activity toward the β(1,3)-linked galactose and N-acetylglucosamine (Galβ(1,3)NAG) moiety of oligosaccharides on the host molecules. Here we report the crystal structure of BgaC at 1.8 Å and its complex with galactose at 1.95 Å. At pH 5.5–8.0, BgaC exists as a stable homodimer, each subunit of which consists of three distinct domains: a catalytic domain of a classic (β/α)₈ TIM barrel, followed by two all-β domains (ABDs) of unknown function. The side walls of the TIM β-barrel and a loop extended from the first ABD constitute the active site. Superposition of the galactose-complexed structure to the apo-form revealed significant conformational changes of residues Trp-243 and Tyr-455. Simulation of a putative substrate entrance tunnel and modeling of a complex structure with Galβ(1,3)NAG enabled us to assign three key residues to the specific catalysis. Site-directed mutagenesis in combination with activity assays further proved that residues Trp-240 and Tyr-455 contribute to stabilizing the N-acetylglucosamine moiety, whereas Trp-243 is critical for fixing the galactose ring. Moreover, we propose that BgaC and other galactosidases in the GH-35 family share a common domain organization and a conserved substrate-determinant aromatic residue protruding from the second domain.     

Identification of a novel group of putative Arabidopsis thaliana β-(1,3)-galactosyltransferases

To begin biochemical and molecular studies on the biosynthesis of the type II arabinogalactan chains on arabinogalactan-proteins (AGPs), we adopted a bioinformatic approach to identify and systematically characterise the putative galactosyltransferases (GalTs) responsible for synthesizing the beta-(1,3)-Gal linkage from CAZy GT-family-31 from Arabidopsis thaliana. These analyses confirmed that 20 members of the GT-31 family contained domains/motifs typical of biochemically characterised beta-(1,3)-GTs from mammalian systems. Microarray data confirm that members of this family are expressed throughout all tissues making them likely candidates for the assembly of the ubiquitously found AGPs. One member, At1g77810, was selected for further analysis including location studies that confirmed its presence in the Golgi and preliminary enzyme substrate specificity studies that demonstrated beta-(1,3)-GalT activity. This bioinformatic/molecular study of CAZy GT-family-31 was validated by the recent report of Strasser et al. (Plant Cell 19:2278-2292, 2007) that another member of this family (At1g26810; GALT1) encodes a beta-(1,3)-GalT involved in the biosynthesis of the Lewis a epitope of N-glycans in Arabidopsis thaliana.     

Natural products targeting the synthesis of β(1,3)-D-glucan and chitin of the fungal cell wall. Existing drugs and recent findings

Background: During the last three decades systemic fungal infections associated to immunosuppressive therapies have become a serious healthcare problem. Clinical development of new antifungals is an urgent requirement. Since fungal but not mammalian cells are encased in a carbohydrate-containing cell wall, which is required for the growth and viability of fungi, the inhibition of cell wall synthesizing machinery, such as β(1,3)-D-glucan synthases (GS) and chitin synthases (CS) that catalyze the synthesis of β(1-3)-D-glucan and chitin, respectively, represent an ideal mode of action of antifungal agents. Although the echinocandins anidulafungin, caspofungin and micafungin are clinically well-established GS inhibitors for the treatment of invasive fungal infections, much effort must still be made to identify inhibitors of other enzymes and processes involved in the synthesis of the fungal cell wall.Purpose: Since natural products (NPs) have been the source of several antifungals in clinical use and also have provided important scaffolds for the development of semisynthetic analogues, this review was devoted to investigate the advances made to date in the discovery of NPs from plants that showed capacity of inhibiting cell wall synthesis targets. The chemical characterization, specific target, discovery process, along with the stage of development are provided here.Methods: An extensive systematic search for NPs against the cell wall was performed considering all the articles published until the end of 2020 through the following scientific databases: NCBI PubMed, Scopus and Google Scholar and using the combination of the terms “natural antifungals” and “plant extracts” with “fungal cell wall”.Results: The first part of this review introduces the state of the art of the structure and biosynthesis of the fungal cell wall and considers exclusively those naturally produced GS antifungals that have given rise to both existing semisynthetic approved drugs and those derivatives currently in clinical trials. According to their chemical structure, natural GS inhibitors can be classified as 1) cyclic lipopeptides, 2) glycolipids and 3) acidic terpenoids. We also included nikkomycins and polyoxins, NPs that inhibit the CS, which have traditionally been considered good candidates for antifungal drug development but have finally been discarded after enduring unsuccessful clinical trials. Finally, the review focuses in the most recent findings about the growing field of plant-derived molecules and extracts that exhibit activity against the fungal cell wall. Thus, this search yielded sixteen articles, nine of which deal with pure compounds and seven with plant extracts or fractions with proven activity against the fungal cell wall. Regarding the mechanism of action, seven (44%) produced GS inhibition while five (31%) inhibited CS. Some of them (56%) interfered with other components of the cell wall. Most of the analyzed articles refer to tests carried out in vitro and therefore are in early stages of development.Conclusion: This report delivers an overview about both existing natural antifungals targeting GS and CS activities and their mechanisms of action. It also presents recent discoveries on natural products that may be used as starting points for the development of potential selective and non-toxic antifungal drugs.     

Co-ordinated regulation of chitinase and β-1,3-glucanase in bean leaves

Ethylene induced chitinase (EC 3.2.1.14) and -1,3-glucanase (EC 3.2.1.29) to a similar extent in primary leaves of bean seedlings (Phaseolus vulgaris cv. Saxa). Both enzymes were purified from ethylene-treated leaves, and monospecific antibodies were raised aginst them. Ethylene treatments strongly increased the amount of immunore-active chitinase and -1,3-glucanase. Ethylene enhanced synthesis of chitinase in vivo, as tested by immunoprecipitation after pulse-labelling with [³⁵S]methionine. RNA was isolated from bean leaves and translated in a rabbit reticulocyte lysate system in vitro. The chitinase and the -1,3-glucanase antiserum each precipitated a single polypeptide from the translation products. The precipitated polypeptides were 1500 and 4000 daltons larger, respectively, than native chitinase and native -1,3-glucanase, indicating that the two enzymes were synthesized as precursors in vitro. The translatable mRNAs for both enzymes increased at least tenfold within 2 h in response to a treatment with ethylene. When ethylene was withdrawn after 8 h of incubation, the translatable mRNAs for both enzymes decreased somewhat more slowly, reaching the basal level about 25 h later. In all cases, there was a close correlation between the levels of translatable mRNA for chitinase and -1,3-glucanase. A putative -1,3-glucanase cDNA clone, pCH16, was isolated by hybrid-selected translation. The amount of -1,3-glucanase mRNA, as measured by RNA blot analysis using pCH16 as a probe, increased rapidly in response to ethylene and decreased again after withdrawal of ethylene, indicating that the amount of hybridizable RNA and of translatable mRNA for -1,3-glucanase were correlated. In conclusion, the results indicate that chitinase and -1,3-glucanase are regulated co-ordinately at the level of mRNA.Abbreviations poly(A)⁺ RNA polyadenylated RNA - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TCA trichloroacetic acid     

Intracellular transport and processing of a tobacco vacuolar β-1,3-glucanase

The class I β-1,3-glucanases are basic, vacuolar enzymes implicated in the defense of plants against pathogen infection. The tobacco (Nicotiana tabacum L.) enzyme is synthesized as a preproprotein with an N-terminal signal peptide for targeting to the lumen of the endoplasmic reticulum and an N-glycosylated C-terminal extension which is lost during protein maturation. The transport and processing of β-1,3-glucanase in cellsuspension cultures of the tobacco cultivar Havana 425 was investigated by pulse-chase labelling and cell fractionation. We verified that mature β-1,3-glucanase is localized in the vacuole of the suspension-cultured cells. Comparison of the time course of processing in homogenates, the soluble fraction, and membrane fractions indicates that proglucanase is transported from the endoplasmic reticulum via the Golgi compartment to the vacuole. Processing to the mature form occurs in the vacuole. Treatment of cells with tunicamycin, which inhibits N-glycosylation, and digestion of the ³⁵S-labelled processing intermediates with endoglycosidase H indicate that β-1,3-glucanase has a single N-glycan attached to the C-terminal extension. Glycosylation is not required for proteolytic processing or correct targeting to the vacuole.     

Purification and characterization of a novel alkaline β-1,3-1,4-glucanase (lichenase) from thermophilic fungus Malbranchea cinnamomea

A novel alkaline β-1,3-1,4-glucanase (McLic1) from a thermophilic fungus, Malbranchea cinnamomea, was purified and biochemically characterized. McLic1 was purified to homogeneity with a purification fold of 3.1 and a recovery yield of 3.7 %. The purified enzyme was most active at pH 10.0 and 55 °C, and exhibited a wide range of pH stability (pH 4.0–10.0). McLic1 displayed strict substrate specificity for barley β-glucan, oat β-glucan and lichenan, but did not show activity towards other tested polysaccharides and synthetic p-nitrophenyl derivates, suggesting that it is a specific β-1,3-1,4-glucanase. The K _m values for barley β-glucan, oat β-glucan and lichenan were determined to be 0.69, 1.11 and 0.63 mg mL^?1, respectively. Moreover, the enzyme was stable in various non ionic surfactants, oxidizing agents and several commercial detergents. Thus, the alkaline β-1,3-1,4-glucanase may have potential in industrial applications, such as detergent, paper and pulp industries.     

Effects of adeno-associated virus (AAV) of transforming growth factors β_1 and β_3 (TGFβ_(1,3)) on promoting synthesis of glycosaminoglycan and collagen type II of dedifferentiated nucleus pulposus (NP) cells

The effects of AAV-TGFβ1 and AAV-TGFβ3 on promoting synthesis of glycosaminoglycan and collagen type II of dedifferentiated rabbit lumbar disc NP cells were studied in this work. The rabbit lumbar disc NP cells were isolated and cultured. The earlier and later dedifferentiated NP cells were established by subculture. The AAV transfection efficiency to dedifferentiated NP cells was analyzed with AAV-EGFP in vitro. After dedifferentiated NP cells were transfected by AAV-TGFβ1 or AAV-TGFβ3, their biological effects on promoting synthesis of glycosaminoglycan or collagen type II were detected and compared by the methods of 35S incorporation or immunoblotting. The experimental results showed that AAV could transfect efficiently the earlier dedifferentiated NP cells, but its transfection rate was shown to be at a low level to the later dedifferentiated NP cells. Both AAV-TGFβ1 and AAV-TGFβ3 could promote the earlier dedifferentiated NP cells to synthesize glycosaminoglycan and collagen type II, and the effect of AAV-TGFβ1 was better than that of AAV-TGFβ3. For the later dedifferentiated NP cells, the AAV-TGFβ3 could promote their synthesis, but AAV-TGFβ1 could slightly inhibit their synthesis. Therefore, AAV-TGFβ1 and AAV-TGFβ3 could be used for the earlier dedifferentiated NP cells, and the TGFβ3 could be used as the objective gene for the later dedifferentiated NP cells.