Vladimír Farkaš

Pectic homogalacturonan masks abundant sets of xyloglucan epitopes in plant cell walls.

BackgroundMolecular probes are required to detect cell wall polymers in-situ to aid understanding of their cell biology and several studies have shown that cell wall epitopes have restricted occurrences across sections of plant organs indicating that cell wall structure is highly developmentally regulated. Xyloglucan is the major hemicellulose or cross-linking glycan of the primary cell walls of dicotyledons although little is known of its occurrence or functions in relation to cell development and cell wall microstructure.ResultsUsing a neoglycoprotein approach, in which a XXXG heptasaccharide of tamarind seed xyloglucan was coupled to BSA to produce an immunogen, we have generated a rat monoclonal antibody (designated LM15) to the XXXG structural motif of xyloglucans. The specificity of LM15 has been confirmed by the analysis of LM15 binding using glycan microarrays and oligosaccharide hapten inhibition of binding studies. The use of LM15 for the analysis of xyloglucan in the cell walls of tamarind and nasturtium seeds, in which xyloglucan occurs as a storage polysaccharide, indicated that the LM15 xyloglucan epitope occurs throughout the thickened cell walls of the tamarind seed and in the outer regions, adjacent to middle lamellae, of the thickened cell walls of the nasturtium seed. Immunofluorescence analysis of LM15 binding to sections of tobacco and pea stem internodes indicated that the xyloglucan epitope was restricted to a few cell types in these organs. Enzymatic removal of pectic homogalacturonan from equivalent sections resulted in the abundant detection of distinct patterns of the LM15 xyloglucan epitope across these organs and a diversity of occurrences in relation to the cell wall microstructure of a range of cell types.ConclusionThese observations support ideas that xyloglucan is associated with pectin in plant cell walls. They also indicate that documented patterns of cell wall epitopes in relation to cell development and cell differentiation may need to be re-considered in relation to the potential masking of cell wall epitopes by other cell wall components.

Versatile High Resolution Oligosaccharide Microarrays for Plant Glycobiology and Cell Wall Research

Background: Microarrays of plant-derived oligosaccharides are potentially powerful tools for the high throughput discovery and screening of antibodies, enzymes, and carbohydrate-binding proteins. Results: Oligosaccharide microarrays were produced, and their utility was demonstrated in several applications. Conclusion: A new generation of oligosaccharide microarrays will make an important contribution to plant glycomic research. Significance: High throughput screening technology enables the more effective production of carbohydrate active enzymes and molecular probes. Microarrays are powerful tools for high throughput analysis, and hundreds or thousands of molecular interactions can be assessed simultaneously using very small amounts of analytes. Nucleotide microarrays are well established in plant research, but carbohydrate microarrays are much less established, and one reason for this is a lack of suitable glycans with which to populate arrays. Polysaccharide microarrays are relatively easy to produce because of the ease of immobilizing large polymers noncovalently onto a variety of microarray surfaces, but they lack analytical resolution because polysaccharides often contain multiple distinct carbohydrate substructures. Microarrays of defined oligosaccharides potentially overcome this problem but are harder to produce because oligosaccharides usually require coupling prior to immobilization. We have assembled a library of well characterized plant oligosaccharides produced either by partial hydrolysis from polysaccharides or by de novo chemical synthesis. Once coupled to protein, these neoglycoconjugates are versatile reagents that can be printed as microarrays onto a variety of slide types and membranes. We show that these microarrays are suitable for the high throughput characterization of the recognition capabilities of monoclonal antibodies, carbohydrate-binding modules, and other oligosaccharide-binding proteins of biological significance and also that they have potential for the characterization of carbohydrate-active enzymes.

Cleavage of xyloglucan by nasturtium seed xyloglucanase and transglycosylation to xyloglucan subunit oligosaccharides

Oligosaccharide subunits were prepared from xyloglucan (XG) by partial hydrolysis with cellulase and added back at micro- to millimolar concentrations to XG in the presence of nasturtium seed xyloglucanase (XG-ase). The oligosaccharides (0.2 mM) stimulated the capacity of this XG-ase to reduce the viscosity of XG solutions by 10- to 20-fold. Purification and fractionation of seed XG-ase activity by gel permeation fast protein liquid chromatography produced a single peak that was much more active in the presence than absence of added XG oligosaccharide. [14C]Fucose-labeled XG nonasaccharide was synthesized by pea fucosyltransferase and shown to be incorporated into polymeric XG in the presence of seed XG-ase without the net production of new reducing chain ends, even while the loss of XG viscosity and XG depolymerization were enhanced. It is concluded that in vitro seed XG-ase can transfer cleavage products of XG to XG oligosaccharides via endotransglycosylation reactions, thereby reducing XG M(r) without hydrolysis. Since this is the only XG-cleaving enzyme that develops in nasturtium seeds during germination, it may be that its transglycosylase and hydrolase capacities are both necessary to account for the rapid and complete depolymerization of XG that takes place.

A Barley Xyloglucan Xyloglucosyl Transferase Covalently Links Xyloglucan, Cellulosic Substrates, and (1,3;1,4)-β-D-Glucans

Molecular interactions between wall polysaccharides, which include cellulose and a range of noncellulosic polysaccharides such as xyloglucans and (1,3;1,4)-β-d-glucans, are fundamental to cell wall properties. These interactions have been assumed to be noncovalent in nature in most cases. Here we show that a highly purified barley xyloglucan xyloglucosyl transferase HvXET5 (EC 2.4.1.207), a member of the GH16 group of glycoside hydrolases, catalyzes the in vitro formation of covalent linkages between xyloglucans and cellulosic substrates and between xyloglucans and (1,3;1,4)-β-d-glucans. The rate of covalent bond formation catalyzed by HvXET5 with hydroxyethylcellulose (HEC) is comparable with that on tamarind xyloglucan, whereas that with (1,3; 1,4)-β-d-glucan is significant but slower. Matrix-assisted laser desorption ionization time-of-flight mass spectrometric analyses showed that oligosaccharides released from the fluorescent HEC:xyloglucan conjugate by a specific (1,4)-β-dglucan endohydrolase consisted of xyloglucan substrate with one, two, or three glucosyl residues attached. Ancillary peaks contained hydroxyethyl substituents (m/z 45) and confirmed that the parent material consisted of HEC covalently linked with xyloglucan. Similarly, partial hydrolysis of the (1,3;1,4)-β-d-glucan:xyloglucan conjugate by a specific (1,3;1,4)-β-d-glucan endohydrolase revealed the presence of a series of fluorescent oligosaccharides that consisted of the fluorescent xyloglucan acceptor substrate linked covalently with 2-6 glucosyl residues. These findings raise the possibility that xyloglucan endo-transglucosylases could link different polysaccharides in vivo and hence influence cell wall strength, flexibility, and porosity.

Assembly of the Yeast Cell Wall Crh1p AND Crh2p ACT AS TRANSGLYCOSYLASES IN VIVO AND IN VITRO

The cross-linking of polysaccharides to assemble new cell wall in fungi requires mechanisms by which a preexisting linkage is broken for each new one made, to allow for the absence of free energy sources outside the plasma membrane. Previous work showed that Crh1p and Crh2p, putative transglycosylases, are required for the linkage of chitin to β(1–3)glucose branches of β(1–6)glucan in the cell wall of budding yeast. To explore the linking reaction in vivo and in vitro, we used fluorescent sulforhodamine-linked laminari-oligosaccharides as artificial chitin acceptors. In vivo, fluorescence was detected in bud scars and at a lower level in the cell contour, both being dependent on the CRH genes. The linking reaction was also shown in digitonin-permeabilized cells, with UDP-N-acetylglucosamine as the substrate for nascent chitin production. Both the nucleotide and the Crh proteins were required here. A gas1 mutant that overexpresses Crh1p showed very high fluorescence both in intact and permeabilized cells. In the latter, fluorescence was still incorporated in patches in the absence of UDP-GlcNAc. Isolated cell walls of this strain, when incubated with sulforhodamine-oligosaccharide, also showed Crhp-dependent fluorescence in patches, which were identified as bud scars. In all three systems, binding of the fluorescent material to chitin was verified by chitinase digestion. Moreover, the cell wall reaction was inhibited by chitooligosaccharides. These results demonstrate that the Crh proteins act by transferring chitin chains to β(1–6)glucan, with a newly observed high activity in the bud scar. The importance of transglycosylation for cell wall assembly is thus firmly established.

Induction of conidiation by endogenous volatile compounds in Trichoderma spp.

Light and starvation are two principal environmental stimuli inducing conidiation in the soil micromycete Trichoderma spp. We observed that volatiles produced by conidiating colonies of Trichoderma spp. elicited conidiation in colonies that had not been induced previously by exposure to light. The inducing effect of volatiles was both intra- and interspecific. Chemical profiles of the volatile organic compounds (VOCs) produced by the nonconidiated colonies grown in the dark and by the conidiating colonies were compared using solid-phase microextraction of headspace samples followed by tandem GC-MS. The conidiation was accompanied by increased production of eight-carbon compounds 1-octen-3-ol and its analogs 3-octanol and 3-octanone. When vapors of these compounds were applied individually to dark-grown colonies, they elicited their conidiation already at submicromolar concentrations. It is concluded that the eight-carbon VOCs act as signaling molecules regulating development and mediating intercolony communication in Trichoderma.

Extracellular β-glucanases of the yeast, Saccharomyces cerevisiae

Abstract Fractionation of proteins secreted by protoplasts and intact cells of Saccharomyces cerevisiae by DEAE-cellullose chromatography in 0.05 M phosphate buffer (pH 7) using a linear gradient of NaCl revealed the presence of at least three different fractions hydrolyzing laminarin (β-1,3-glucan). The first fraction, not retained by the column, appeared to consist of two enzymes exhibiting activity for laminarin, p- nitrophenyl -β- d -glucopyranoside , pustulan (β-1,6-glucan) and a low but detectable activity for laminarin treated with NaIO4. The second fraction, eluted from the DEAE-cellulose column at 0.23 M NaCl possessed the substrate specificity of an endo-β-1,3-glucanase. It exhibited the maximum activity at pH 5.5 in 0.1 M acetate buffer. The third fraction, eluted from the column at 0.35 M NaCl, was an unspecific exo-β-glucanase hydrolyzing terminally both laminarin and pustulan. The pH optimum of the latter enzyme was in the range 4.5–5.0 with laminarin, p- nitrophenyl -β- d -glucopyranoside or pustulan as substrates. The β-glucanases found extracellularly were compared with those occurring in protoplast lysates and cell-free extracts of intact cells.

Multiple enzyme forms in the cellulase system of Trichoderma reesei during its growth on cellulose

Abstract The pattern of multiple molecular forms of endo-1,4-β- d -glucanase (EC 3.2.1.4) and of β-glucosidase (EC 3.2.1.21) components has been followed by means of analytical isoelectric focusing during submerged growth of the fungus Trichoderma reesei QM 9414 on microcrystalline cellulose, both in buffered and in non-buffered medium. It has been found that multiple molecular forms of the cellulase enzymes appear very early in the course of cultivation. It is concluded that the presence of multiple enzyme forms is an inherent property of the cellulase system of Trichoderma and cannot be considered solely as the result of proteolytic postsecretional modification of secreted extracellular enzymes.

Structure and biosynthesis of fungal cell walls: methodological approaches.

Fungal cell walls possess a characteristic chemical composition differentiating fungal cells from other cell types. For this reason, the mechanisms involved in cell-wall formation represent a potential target for selective antifungal drugs. Understanding the structure and biosynthesis of fungal cell walls opens the ways for design of effective drugs for treating fungal diseases. This article reviews the history methods employed in chemical and structural analysis of fungal cell walls and in studies concerning their formation.

Biosynthesis of β-glucans catalyzed by a particulate enzyme preparation from yeast

The /3-glucan represents one of the major components of the yeast cell walls. In Succharomyces cerevisiae cell walls the glucan has been found to be composed of two fractions a high mol. wt. fi( l-+3) linked glucan containing small proportion of /3(1+6) interchain linkages [ 11. The minor fraction is a polymer consisting of predominantly /3( l-+6) linked glucosyl units containing about 20% of /3( 1+3) interchain and inter-residue linkages [2]. The biosynthesis of fungal cell wall glucans in a cell free system has been studied to some extent in Phytophtora cinnamomi [3] and Cochliobolus miyabeanus [4,5]. In yeast, where the glucan represents some 30-50% of dry weight of the cell walls the molecular mechanism of its biosynthesis is so far unknown, while the biosynthesis of other yeast cell wall polysaccharides mannan and chitin has been subject of numerous studies [6-91. This paper describes isolation of an enzyme system from yeast, which catalyzes the transfer of glucosyl units from GDP[U-‘“Cl glucose and UDP[U-‘“Cl glucose into polymers tentatively characterized as P-glucans containing both /3( l-+3) and /3( 1+6) glycosidic linkages.