Oxidized cellulose binding to allergens with a carbohydrate-binding module attenuates allergic reactions.

Grass and mite allergens are of the main causes of allergy and asthma. A carbohydrate-binding module (CBM) represents a common motif to groups I (ß-expansin) and II/III (expansin-like) grass allergens and is suggested to mediate allergen-IgE binding. House dust mite group II allergen (Der p 2 and Der f 2) structures bear strong similarity to expansin's CBM, suggesting their ability to bind carbohydrates. Thus, this study proposes the design of a carbohydrate-based treatment in which allergen binding to carbohydrate particles will promote allergen airway clearance and prevent allergic reactions. The aim of the study was to identify a polysaccharide with high allergen-binding capacities and to explore its ability to prevent allergy. Oxidized cellulose (OC) demonstrated allergen-binding capacities toward grass and mite allergens that surpassed those of any other polysaccharide examined in this study. Furthermore, inhalant preparations of OC microparticles attenuated allergic lung inflammation in rye grass-sensitized Brown Norway rats and OVA-sensitized BALB/c mice. Fluorescently labeled OC efficiently cleared from the mouse airways and body organs. Moreover, long-term administration of OC inhalant to Wistar rats did not result in toxicity. In conclusion, many allergens, such as grass and dust mite, contain a common CBM motif. OC demonstrates a strong and relatively specific allergen-binding capacity to CBM-containing allergens. OC's ability to attenuate allergic inflammation, together with its documented safety record, forms a firm basis for its application as an alternative treatment for prevention and relief of allergy and asthma.

Carbohydrate binding modules: biochemical properties and novel applications.

Polysaccharide-degrading microorganisms express a repertoire of hydrolytic enzymes that act in synergy on plant cell wall and other natural polysaccharides to elicit the degradation of often-recalcitrant substrates. These enzymes, particularly those that hydrolyze cellulose and hemicellulose, have a complex molecular architecture comprising discrete modules which are normally joined by relatively unstructured linker sequences. This structure is typically comprised of a catalytic module and one or more carbohydrate binding modules (CBMs) that bind to the polysaccharide. CBMs, by bringing the biocatalyst into intimate and prolonged association with its substrate, allow and promote catalysis. Based on their properties, CBMs are grouped into 43 families that display substantial variation in substrate specificity, along with other properties that make them a gold mine for biotechnologists who seek natural molecular "Velcro" for diverse and unusual applications. In this article, we review recent progress in the field of CBMs and provide an up-to-date summary of the latest developments in CBM applications.

Expression and Regulation of the Arabidopsis thaliana Cel1 Endo 1,4 beta Glucanase Gene During Compatible Plant-Nematode Interactions.

The root-knot nematode Meloidogyne incognita is an obligate endoparasite of plant roots and stimulates elaborate modifications of selected root vascular cells to form giant cells for feeding. An Arabidopsis thaliana endoglucanase (Atcel1) promoter is activated in giant cells that were formed in Atcel1::UidA transgenic tobacco and Arabidopsis plants. Activity of the full-length Atcel1 promoter was detected in root and shoot elongation zones and in the lateral root primordia. Different 5' and internal deletions of regions of the 1,673 bp Atcel1 promoter were each fused to the UidA reporter gene and transformed in tobacco, and roots of the transformants were inoculated with M. incognita to assay for GUS expression in giant cells and noninfected plant tissues. Comparison of the Atcel1 promoter deletion constructs showed that the region between -1,673 and -1,171 (fragment 1) was essential for Atcel1 promoter activity in giant cells and roots. Fragment 1 alone, however, was not sufficient for Atcel1 expression in giant cells or roots, suggesting that cis-acting elements in fragment 1 may function in consort with other elements within the Atcel1 promoter. Root-knot nematodes and giant cells developed normally within roots of Arabidopsis that expressed a functional antisense construct to Atcel1, suggesting that a functional redundancy in endoglucanase activity may represent another level of regulatory control of cell wall-modifying activity within nematode feeding cells.

Modification of polysaccharides and plant cell wall by endo-1,4-beta-glucanase and cellulose-binding domains.

Cellulose is one of the most abundant polymers in nature. Different living systems evolved simultaneously, using structurally similar proteins to synthesize and metabolize polysaccharides. In the growing plant, cell wall loosening, together with cellulose biosynthesis, enables turgor-driven cell expansion. It has been postulated that endo-1,4-beta-glucanases (EGases) play a central role in these complex activities. Similarly, microorganisms use a consortium of lytic enzymes to convert cellulose into soluble sugars. Most, if not all, cellulases have a modular structure with two or more separate independent functional domains. Binding to cellulose is mediated by a cellulose-binding domain (CBD), whereas the catalytic domain mediates hydrolysis. Today, EGases and CBDs are known to exist in a wide range of species and it is evident that both possess immense potential in modifying polysaccharide materials in-vivo and in-vitro. The hydrolytic function is utilized for polysaccharide degradation in microbial systems and cell wall biogenesis in plants. The CBDs exerts activity that can be utilized for effective degradation of crystalline cellulose, plant cell wall relaxation, expansion and cell wall biosynthesis. Applications range from modulating the architecture of individual cells to an entire organism. These genes, when expressed under specific promoters and appropriate trafficking signals can be used to alter the nutritional value and texture of agricultural crop and their final products. EGases and CBDs may also find applications in the modification of physical and chemical properties of composite materials to create new materials possessing improved properties.

Loosening xyloglucan accelerates the enzymatic degradation of cellulose in wood.

In order to create trees in which cellulose, the most abundant component in biomass, can be enzymatically hydrolyzed highly for the production of bioethanol, we examined the saccharification of xylem from several transgenic poplars, each overexpressing either xyloglucanase, cellulase, xylanase, or galactanase. The level of cellulose degradation achieved by a cellulase preparation was markedly greater in the xylem overexpressing xyloglucanase and much greater in the xylems overexpressing xylanase and cellulase than in the xylem of the wild-type plant. Although a high degree of degradation occurred in all xylems at all loci, the crystalline region of the cellulose microfibrils was highly degraded in the xylem overexpressing xyloglucanase. Since the complex between microfibrils and xyloglucans could be one region that is particularly resistant to cellulose degradation, loosening xyloglucan could facilitate the enzymatic hydrolysis of cellulose in wood.

Xyloglucan for generating tensile stress to bend tree stem.

In response to environmental variation, angiosperm trees bend their stems by forming tension wood, which consists of a cellulose-rich G (gelatinous)-layer in the walls of fiber cells and generates abnormal tensile stress in the secondary xylem. We produced transgenic poplar plants overexpressing several endoglycanases to reduce each specific polysaccharide in the cell wall, as the secondary xylem consists of primary and secondary wall layers. When placed horizontally, the basal regions of stems of transgenic poplars overexpressing xyloglucanase alone could not bend upward due to low strain in the tension side of the xylem. In the wild-type plants, xyloglucan was found in the inner surface of G-layers during multiple layering. In situ xyloglucan endotransglucosylase (XET) activity showed that the incorporation of whole xyloglucan, potentially for wall tightening, began at the inner surface layers S1 and S2 and was retained throughout G-layer development, while the incorporation of xyloglucan heptasaccharide (XXXG) for wall loosening occurred in the primary wall of the expanding zone. We propose that the xyloglucan network is reinforced by XET to form a further connection between wall-bound and secreted xyloglucans in order to withstand the tensile stress created within the cellulose G-layer microfibrils.

Expression of endo-1,4-beta-glucanase (cel1) in Arabidopsis thaliana is associated with plant growth, xylem development and cell wall thickening.

Arabidopsis thaliana CEL1 protein was detected in young expanding tissues. Immunostaining revealed that CEL1 accumulated mostly in xylem cells. The primary, as well as the secondary xylem showed considerable CEL1 staining. CEL1 was also observed in young epidermal cells, in which the thicker lateral and tangential walls stained more intensely than the inner walls. In newly formed cell walls, the lateral tangential walls were labeled more intensively than the inner walls. Cellulase activity was found to be significantly higher in growing tissue compared to mature parts of the plant. Cel1 expression concurrently with cellulase activity could be restored in detached matured leaves by sucrose treatment after 48 h in the culture medium.

Abnormal 'wrinkled' cell walls and retarded development of transgenic Arabidopsis thaliana plants expressing endo-1,4-beta-glucanase (cell) antisense.

Transgenic Arabidopsis thaliana plants expressing cel1 antisense exhibit reduced levels of cel1 mRNA and protein compared with wild-type plants. The former display significant alterations in their phenotype. cel1 antisense plants have shorter stems and roots and are mechanically weaker than their wild-type counterparts. In cel1 antisense plants, the cell wall structure is markedly disrupted: both fluorescent confocal microscopy and scanning electron microscopy revealed 'wrinkled' cell walls, thus indicating that CEL1 plays an important role in cell wall relaxation during cell growth and expansion. In cel1 antisense plants, the number of xylem elements per bundle is smaller than in the wild-type. In addition, both xylem elements and interfascicular fibers are significantly less lignified in the former. It is suggested that in A. thaliana, abnormal cell wall deposition affected by CEL1 depletion is associated not only with cell growth, but also with the differentiation process in the vascular and supporting tissues.

The promoter of the Arabidopsis thaliana Cel1 endo-1,4-beta glucanase gene is differentially expressed in plant feeding cells induced by root-knot and cyst nematodes.

SUMMARY Transgenic tobacco and Arabidopsis thaliana carrying the Arabidopsis endo-1,4-beta-glucanase (EC 3.2.1.4) Cel1 promoter fused to the beta-glucuronidase (GUS) reporter gene were infected with the root-knot nematode, Meloidogyne incognita, and either the tobacco cyst nematode, Globodera tabacum (tobacco), or beet cyst nematode, Heterodera schachtii (Arabidopsis). Cel1-driven GUS expression was detected in cell elongation zones of noninfected plants and within feeding sites (giant-cells) induced in roots of both plant hosts by M. incognita. The first detectable signs of Cel1 expression within developing giant-cells occurred at the onset of giant-cell formation and continued throughout the M. incognita life cycle. UidA (Gus) transcripts were detectable within giant-cells induced in tobacco roots at 11-13 days postinoculation with M. incognita as determined by in situ mRNA hybridization. By contrast, expression of the Cel1 promoter was not detected within developing syncytia induced in tobacco or Arabidopsis roots by G. tabacum and H. schachtii, respectively, at any time point. The results demonstrate specific regulation of cell wall-degrading enzymes that may be required for cell wall modifications during feeding cell formation by sedentary endoparasitic nematodes. Differential expression of Cel1 by cyst and root-knot nematodes further supports underlying mechanistic differences in giant-cell and syncytium formation.