Myoinositol kinase: partial purification and identification of product.

Myoinositol kinase found in plant, animal, and microbial extracts has been partially purified by densitygradient centrifugation. The product of the enzymic reaction has been tentatively identified by paper chromatography. as myoinositol-1-phosphate.

Identification of the 1H-NMR spectra of complex oligosaccharides with artificial neural networks.

Artificial networks can be used to identify hydrogen nuclear magnetic resonance (1H-NMR) spectra of complex oligosaccharides. Feed-forward neural networks with back-propagation of errors can distinguish between spectra of oligosaccharides that differ by only one glycosyl residue in twenty. The artificial neural networks use features of the strongly overlapping region of the spectra (hump region) as well as features of the resolved regions of the spectra (structural reporter groups) to recognize spectra and efficiently recognized 1H-NMR spectra even when the spectra were perturbed by minor variations in their chemical shifts. Identification of spectra by neural network-based pattern recognition techniques required less than 0.1 second. It is anticipated that artificial neural networks can be used to identify the structures of any complex carbohydrate that has been previously characterized and for which a 1H-NMR spectrum is available.

Requirement of borate cross-linking of cell wall rhamnogalacturonan II for Arabidopsis growth.

Turgor-driven plant cell growth depends on wall structure. Two allelic l-fucose-deficient Arabidopsis thaliana mutants (mur1-1 and 1-2) are dwarfed and their rosette leaves do not grow normally. mur1 leaf cell walls contain normal amounts of the cell wall pectic polysaccharide rhamnogalacturonan II (RG-II), but only half exists as a borate cross-linked dimer. The altered structure of mur1 RG-II reduces the rate of formation and stability of this cross-link. Exogenous aqueous borate rescues the defect. The reduced cross-linking of RG-II in dwarf mur1 plants indicates that plant growth depends on wall pectic polysaccharide organization.

Substitution of L-fucose by L-galactose in cell walls of Arabidopsis mur1.

An Arabidopsis thaliana mutant (mur1) has less than 2 percent of the normal amounts of L-fucose in the primary cell walls of aerial portions of the plant. The survival of mur1 plants challenged the hypothesis that fucose is a required component of biologically active oligosaccharides derived from cell wall xyloglucan. However, the replacement of L-fucose (that is, 6-deoxy-L-galactose) by L-galactose does not detectably alter the biological activity of the oligosaccharides derived from xyloglucan. Thus, essential structural and conformational features of xyloglucan and xyloglucan-derived oligosaccharides are retained when L-galactose replaces L-fucose.

The Complex Carbohydrate Structure Database.

The Complex Carbohydrate Structure Database (CCSD) and CarbBank, an IBM PC/AT (or compatible) database management system, were created to provide an information system to meet the needs of people interested in carbohydrate science. The CCSD, which presently contains more than 2000 citations, is expected to double in size in the next two years and to include, soon thereafter, all of the published structures of carbohydrates larger than disaccharides.

Pectic Cell Wall Fragments Regulate Tobacco Thin-Cell-Layer Explant Morphogenesis.

Pectic fragments of cell wall polysaccharides, released from the walls of suspension-cultured sycamore cells by treatment with endopolygalacturonase, were tested for morphogenesis-regulating activity in a modified tobacco thin-cell-layer explant (TCL) bioassay (D. Mohnen, S. Eberhard, V. Marfa, N. Doubrava, P. Toubart, D. J. Gollin, T.A. Gruber, W. Nuri, P. Albersheim, and A. Darvill, manuscript submitted). The pectic fragments inhibited the formation of roots on TCLs grown on a root-inducing medium containing 15 micromolar indole-3-butyric acid and 0.5 micromolar kinetin. Addition of the pectic fragments to a root-inducing medium containing 7 micromolar indole-3-butyric acid and 0.15 micromolar kinetin caused roots to form on the basal end of TCLs. TCLs cultured on this medium in the absence of added pectic fragments formed roots along their entire length. The pectic fragments induced polar tissue enlargement and the formation of flowers on TCLs cultured on transition medium. The flower-inducing activity was stable to heat treatment and proteolytic digestion. Pectic fragments isolated from the walls of suspension-cultured tobacco cells were as effective as those from the walls of sycamore cells in inducing de novo flower formation in the TCLs. These results support the hypothesis that oligosaccharins from plant cell walls regulate morphogenesis.

Some phytotoxic glycopeptides from Ceratocystis ulmi, the Dutch Elm Disease pathogen.

Ceratocystis ulmi, the causal agent of Dutch Elm Disease, produces phytotoxic glycopeptides in culture. A mixture of phytotoxic glycopeptides has been prepared by affinity chromatography on a concanavalin A-Sepharose column and collectively they have been termed the toxin. The polydisperse component that makes up the majority of toxin (80%) by weight has a molecular weight of about 2.7.10(5). The large molecular weight component (less than 5%) elutes at the void volume of a Bio-Gel A 50 m column. The other component (15%) appears as a trailing peak on the edge of the major component and has an approximate molecular weight of 7.10(4). The toxin is composed of 83% sugar residues, primarily rhamnose and mannose, and 7% amino acid residues. Methylation analysis coupled with mild acid hydrolysis indicates that the backbone of the polysaccharide portion of the toxin is composed of alpha -1,6-linked mannosyl residues with a 3-linked terminal rhamnosyl residue linked to C-3 of almost every mannosyl residue. The carbohydrate portion of the molecule is linked to the peptide via O-glycosidic linkages to both threonyl and seryl residues. All three components of the toxin are capable of causing wilt in stem cuttings of American elm.

Developmental and Tissue-Specific Structural Alterations of the Cell-Wall Polysaccharides of Arabidopsis thaliana Roots.

The plant cell wall is a dynamic structure that plays important roles in growth and development and in the interactions of plants with their environment and other organisms. We have used monoclonal antibodies that recognize different carbohydrate epitopes present in plant cell-wall polysaccharides to locate these epitopes in roots of developing Arabidopsis thaliana seedlings. An epitope in the pectic polysaccharide rhamnogalacturonan I is observed in the walls of epidermal and cortical cells in mature parts of the root. This epitope is inserted into the walls in a developmentally regulated manner. Initially, the epitope is observed in atrichoblasts and later appears in trichoblasts and simultaneously in cortical cells. A terminal [alpha]-fucosyl-containing epitope is present in almost all of the cell walls in the root. An arabinosylated (1->6)-[beta]-galactan epitope is also found in all of the cell walls of the root with the exception of lateral root-cap cell walls. It is striking that these three polysaccharide epitopes are not uniformly distributed (or accessible) within the walls of a given cell, nor are these epitopes distributed equally across the two walls laid down by adjacent cells. Our results further suggest that the biosynthesis and differentiation of primary cell walls in plants are precisely regulated in a temporal, spatial, and developmental manner.

Purification, cloning and characterization of two xylanases from Magnaporthe grisea, the rice blast fungus.

Magnaporthe grisea, the fungal pathogen that causes rice blast disease, secretes two endo-beta-1,4-D-xylanases (E. C. 3.2.1.8) when grown on rice cell walls as the only carbon source. One of the xylanases, XYN33, is a 33-kD protein on sodium dodecyl sulfate-polyacrylamide gel and accounts for approximately 70% of the endoxylanase activity in the culture filtrate. The second xylanase, XYN22, is a 22-kD protein and accounts for approximately 30% of the xylanase activity. The two proteins were purified, cloned, and sequenced. XYN33 and XYN22 are both basic proteins with calculated isoelectric points of 9.95 and 9.71, respectively. The amino acid sequences of XYN33 and XYN22 are not homologous, but they are similar, respectively, to family F and family G xylanases from other microorganisms. The genes encoding XYN33 and XYN22, designated XYN33 and XYN22, are single-copy in the haploid genome of M. grisea and are expressed when M. grisea is grown on rice cell walls or on oatspelt xylan, but not when grown on sucrose.

Fungal polygalacturonases exhibit different substrate degradation patterns and differ in their susceptibilities to polygalacturonase-inhibiting proteins.

Polygalacturonic acid (PGA) was hydrolyzed by polygalacturonases (PGs) purified from six fungi. The oligogalacturonide products were analyzed by HPAEC-PAD (high performance anion exchange chromatography-pulsed amperimetric detection) to assess their relative amounts and degrees of polymerization. The abilities of the fungal PGs to reduce the viscosity of a solution of PGA were also determined. The potential abilities of four polygalacturonase-inhibiting proteins (PGIPs) from three plant species to inhibit or to modify the hydrolytic activity of the fungal PGs were determined by colorimetric and HPAEC-PAD analyses, respectively. Normalized activities of the different PGs acting upon the same substrate resulted in one of two distinct oligogalacturonide profiles. Viscometric analysis of the effect of PGs on the same substrate also supports two distinct patterns of cleavage. A wide range of susceptibility of the various PGs to inhibition by PGIPs was observed. The four PGs that were inhibited by all PGIPs tested exhibited an endo/exo mode of substrate cleavage, while the three PGs that were resistant to inhibition by one or more of the PGIPs proceed by a classic endo pattern of cleavage.

Polygalacturonase-inhibiting proteins can function as activators of polygalacturonase.

The interaction between fungal endopolygalacturonases (EPGs) and polygalacturonase-inhibiting proteins (PGIPs) found in plant cell walls has been well established. The typical EPG/PGIP interaction is characterized by high affinity, reversibility, and a 1:1 stoichiometry that results in lowering the catalytic rate of a particular endopolygalacturonase by up to 99.7%. Various EPG and PGIP isoforms and glycoforms have been isolated and characterized, and combinations of EPGs and PGIPs demonstrate a range of enzyme inhibition. EPG/PGIP interactions have prompted many researchers to suspect the involvement of these proteins in the production of specific signals (oligosaccharins) during plant pathogenesis. We have recently reported on initial studies in our laboratory indicating that, for certain EPG/PGIP combinations, the specific activity of EPG is increased beyond that characteristic of the enzyme alone. In this paper, we present a detailed analysis of the product of the interaction of native Phaseolus vulgaris PGIP-2 with five EPGs from Aspergillus niger, namely PGI, PGII, PGA, PGB, and PGC in the presence of homogalacturonan. We demonstrate that for PGA and PGC, the interaction with PGIP-2 may result in either inhibition or activation in a manner that is pH dependent. This data suggests the need for a reevaluation of the conventional description applied to PGIPs; suggestions include polygalacturonase-binding protein and polygalacturonase-modulating protein.

Preservation of the delayed-type hypersensitivity response to alloantigen by xyloglucans or oligogalacturonide does not correlate with the capacity to reject ultraviolet-induced skin tumors in mice.

Chronic exposure to ultraviolet radiation suppresses T cell-mediated immune responses and induces the formation of suppressor T lymphocytes that prevent the rejection of highly antigenic ultraviolet-induced skin cancers in mice. Tamarind seed xyloglucans and pectinic oligogalacturonides prevent suppression of delayed-type hypersensitivity immune responses in mice to Candida albicans and alloantigen caused by a single exposure of ultraviolet radiation. We therefore investigated the ability of these poly/oligosaccharides to prevent suppression of T cell-mediated immune responses and suppressor cell induction during chronic ultraviolet irradiation and to preserve the capacity of ultraviolet-irradiated mice to reject a transplanted, highly antigenic, ultraviolet-induced tumor. C3H/HeN mice were treated 3x per week for 12 wk with 15 kJ per m2 ultraviolet B radiation followed by application of the polysaccharides/ oligosaccharides. The delayed-type hypersensitivity responses to C. albicans and alloantigen were measured after 1, 6, and 12 wk of treatment. Following the 12th wk of treatment the remaining mice were injected with the highly antigenic ultraviolet-induced, syngeneic tumor cell line UV5497-5. The polysaccharides/oligosaccharides protected delayed-type hypersensitivity responses to C. albicans but not contact hypersensitivity responses to dinitrofluorobenzene for up to 6 wk of ultraviolet radiation after which protection declined and suppressor cells were observed. In contrast, the delayed-type hypersensitivity response to alloantigen was preserved for the entire 12 wk of ultraviolet irradiation. Despite protection of immunity to alloantigen, the transplanted tumor cells grew equally well in all ultraviolet-irradiated animals. These results indicate that delayed-type hypersensitivity responses are heterogeneous and that delayed-type hypersensitivity to alloantigen is not a surrogate marker for rejection of ultraviolet-induced skin tumors.

Inhibition of UV-induced immune suppression and interleukin-10 production by plant oligosaccharides and polysaccharides.

Application of Aloe barbadensis poly/oligosaccharides to UV-irradiated skin prevents photosuppression of delayed-type hypersensitivity (DTH) responses in mice. We tested the hypothesis that these carbohydrates belong to a family of biologically active, plant-derived polysaccharides that can regulate responses to injury in animal tissues. C3H mice were exposed to 5 kJ/m2 UVB from unfiltered FS40 sunlamps and treated with between 1 pg and 10 micrograms tamarind xyloglucans or control polysaccharides methylcellulose or dextran in saline. The mice were sensitized 3 days later with Candida albicans. Tamarind xyloglucans and purified Aloe poly/oligosaccharides prevented suppression of DTH responses in vivo and reduced the amount of interleukin (IL)-10 observed in UV-irradiated murine epidermis. Tamarind xyloglucans were immunoprotective at low picogram doses. In contrast, the control polysaccharides methylcellulose and dextran had no effect on immune suppression or cutaneous IL-10 at any dose. Tamarind xyloglucans and Aloe poly/oligosaccharides also prevented suppression of immune responses to alloantigen in mice exposed to 30 kJ/m2 UVB radiation. To assess the effect of the carbohydrates on keratinocytes, murine Pam212 cells were exposed to 300 J/m2 UVB radiation and treated for 1 h with tamarind xyloglucans or Aloe poly/oligosaccharides. Treatment of keratinocytes with immunoprotective carbohydrates reduced IL-10 production by approximately 50% compared with the cells treated with UV radiation alone and completely blocked suppressive activity of the culture supernatants in vivo. The tamarind xyloglucans also blocked UV-activated phosphorylation of SAPK/JNK protein but had no effect on p38 phosphorylation. These results indicate that animals, like plants, may use carbohydrates to regulate responses to environmental stimuli.

Structural characterization of two oligosaccharide fragments formed by the selective cleavage of rhamnogalacturonan II: evidence for the anomeric configuration and attachment sites of apiose and 3-deoxy-2-heptulosaric acid.

Evidence for the anomeric configurations and attachment sites of 3-deoxy-D-lyxo-2-heptulosaric acid (DHA) and apiosyl residues has been obtained through the characterization of two oligoglycosyl fragments isolated from rhamnogalacturonan II (RG-II). One of the oligoglycosyl fragments, a pentaglycosyl aldonic acid generated by Smith degradation of RG-II, was composed of four D-galactopyranosyluronic acid residues, a DHA residue, and a threonic acid residue (derived from a D-galactopyranosyluronic acid residue). The structural analysis of the pentaglycosyl aldonic acid established the beta-D-configuration for the DHA residue. Furthermore, it established that a previously identified diglycosyl side chain, 5-O-(beta-L-arabinofuranosyl)-DHA was directly attached to O-3 of a D-galactopyranosyluronic acid residue in the backbone of RG-II. The second oligoglycosyl fragment, a peralkylated diglycosyl hex-1-enitol, was generated by hex-5-enose degradation of permethylated and carboxyl-reduced RG-II. The structure of the peralkylated diglycosyl hex-1-enitol, beta-L-Rhap-(1----3')-beta-D-Apif-(1----5)-hex-1-enitol++ +, was determined by a combination of glycosyl-linkage composition analysis and n.m.r. spectroscopy. The n.m.r. data indicated the beta-configuration for the D-apiosyl residue. The isolation and characterization of the diglycosyl hex-1-enitol also established that a previously identified heptaglycosyl side chain was directly attached to O-2 of a D-galactopyranosyluronic acid in the backbone of RG-II.

Isolation and structural characterization of beta-D-glucosyluronic acid and 4-O-methyl beta-D-glucosyluronic acid-containing oligosaccharides from the cell-wall pectic polysaccharide, rhamnogalacturonan I.

Rhamnogalacturonan I (RG-I), a pectic polysaccharide isolated from the walls of suspension-cultured sycamore cells, was shown by glycosyl-residue composition analysis to contain D-glucosyluronic acid (GlcpA) residues (1 mol%) and 4-O-methyl-D-glucosyluronic acid (4-O-Me-GlcpA) residues (0.5 mol%). These monosaccharides were shown, by glycosyl-linkage analysis, to be present in RG-I as terminal nonreducing residues. The glycosyl sequences containing GlcpA and 4-O-Me-GlcpA were determined by structurally characterizing the acidic oligosaccharides released by partial acid hydrolysis of RG-I. Six acidic oligosaccharides were purified by semipreparative high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and characterized by glycosyl-residue and glycosyl-linkage composition analyses, GLC-CIMS, GLC-EIMS, electrospray MS (ESMS), and 1H NMR spectroscopy. We propose that three of the acidic oligosaccharides characterized, 4-O-Me-beta-D-GlcpA-(1-->6)-D-Gal, beta-D-GlcpA-(1-->6)-D-Gal, and beta-D-GlcpA-(1-->4)-D-Gal, originate from the galactosyl-containing side chains of RG-I. The three other acidic oligosaccharides characterized, alpha-D-GalpA-(1-->2)-L-Rha, alpha-D-GalpA-(1-->2)-alpha-L-Rhap-(1-->4)-alpha-D-GalpA+ ++-(1-->2)-alpha-L-Rha, and alpha-D-GalpA-(1-->2)-alpha-L-Rhap-(1-->4)-alpha-D-GalpA+ ++-(1-->2)-alpha-L- Rhap-(1-->4)-alpha-D-GalpA-(1-->2)-alpha-L-Rha, were generated by partial hydrolysis of the RG-I backbone. No evidence was obtained for the presence of galactosyluronic acid in the side chains of RG-I.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural characterization of endo-glycanase-generated oligoglycosyl side chains of rhamnogalacturonan I.

Rhamnogalacturonan I (RG-I) has been isolated from the walls of suspension-cultured sycamore cells (Acer pseudoplatanus), and additional structural features of the polysaccharide were elucidated. Treatment of RG-I with a purified endo-(1-->5)-alpha-L-arabinanase released a series of arabinose-containing oligosaccharides with degrees of polymerization (dp's) between 2 and 20. These oligosaccharides were shown, by glycosyl-linkage composition analysis, to contain terminal, 5-, and (3-->5)-linked Araf residues. These results provide evidence that a branched arabinan is attached to the backbone of RG-I. RG-I was freed of 95% of its arabinosyl residues by treating the polysaccharide with a combination of endo-(1-->5)-alpha-L-arabinanase and alpha-L-arabinosidase. No galacturonic acid was released by these enzymes, which is evidence that the arabinosyl-containing portions of the side chains do not contain galactosyluronic acid residues. The galactose-containing portions of the side chains of RG-I were not fragmented by an endo-(1-->4)-beta-D-galactanase. However, approximately 85% of the galactose and small amounts of galacturonic acid were released by digestion of arabinose-depleted RG-I with a combination of endo- and exo-beta-D-galactanases. The galacturonic acid may have been released by small amounts of an exo-alpha-galactosyluronidase contaminating the galactanases. Treatment of RG-I with this mixture of endo- and exo-glycanases resulted in a relatively size-homogeneous, almost side chain-free backbone composed of the O-acetylated diglycosyl repeating unit -->4)-alpha-D-GalpA-(1-->2)-alpha-L-Rhap. A combination of 1H NMR spectroscopy and periodate oxidation established that the backbone repeating unit contained a single O-acetyl substituent on C-2 or C-3 of each galactosyluronic acid residue.

Structural analysis of xyloglucan oligosaccharides by 1H-n.m.r. spectroscopy and fast-atom-bombardment mass spectrometry.

A method to determine rapidly the identities and proportions of the oligosaccharide repeating-units in plant cell-wall xyloglucans by 1D 1H-n.m.r. spectroscopy was developed. Six of the most commonly found xyloglucan oligosaccharide subunits (including three subunits that had not been fully characterized previously) were prepared by endo-(1----4)-beta-D-glucanase digestion of xyloglucans from various plant species. The oligosaccharides were reduced to the corresponding oligoglycosyl-alditols, purified, and characterized by glycosyl composition and linkage analysis, 1H-n.m.r. spectroscopy, and f.a.b.-mass spectrometry. Correlations between the 1H-n.m.r. spectra and the structures of the oligoglycosyl-alditols can be used to identify oligoglycosyl-alditols derived from xyloglucans of unknown structure. The identities and relative amounts of the oligosaccharide subunits of xyloglucans isolated from tamarind seed and rapeseed hulls were determined on this basis.

Characterization of seven xyloglucan oligosaccharides containing from seventeen to twenty glycosyl residues.

The complete primary structures of seven oligosaccharide subunits of the xyloglucan secreted by suspension-cultured Acer pseudoplatanus cells were determined. The oligosaccharides, ranging in size from 17 to 20 glycosyl residues, were generated by treatment of the xyloglucan with an endo-beta-(1----4)-glucanase. The oligosaccharide components of a fraction obtained by Bio-Gel P-2 chromatography of enzyme-treated xyloglucan were further purified by normal-phase h.p.l.c. and then converted to the corresponding oligoglycosyl alditols by reduction with NaBH4. The oligoglycosyl alditols, after purification to near homogeneity by reversed-phase h.p.l.c., were structurally characterized by 1H-n.m.r. spectroscopy, fast-atom bombardment mass spectrometry (f.a.b.-m.s.), and analysis of their glycosyl-residue and glycosyl-linkage compositions. Novel structural elements of xyloglucans were observed in this study, including beta-D-xylopyranosyl and alpha-L-arabinofuranosyl-(1----3)-beta-D-xylopyranosyl sidechains. The results also extend our list of correlations between 1H-n.m.r. resonances and specific structural features of xyloglucans and thus enhance our ability to determine the structures of xyloglucans from various sources.

Structural analysis of tamarind seed xyloglucan oligosaccharides using beta-galactosidase digestion and spectroscopic methods.

The borohydride-reduced forms (oligoglycosyl alditols) of two isomeric octasaccharides (Glc4Xyl3Gal) that are released from xyloglucans of various plant species upon treatment with a fungal endo-(1-->4)-beta-glucanase were isolated and structurally characterized. A mixture of oligosaccharides that is released from tamarind seed xyloglucan by the endo-(1-->4)-beta- glucanase was digested with a commercially available beta-galactosidase (Aspergillus niger). The beta-galactosidase selectively hydrolyzed the galactosyl residue of one of the two isomeric octasaccharides present in the mixture. A homogeneous preparation of the beta-galactosidase-resistant octasaccharide was prepared by high-resolution gel-permeation chromatography of the enzyme-digestion products. Spectroscopic characterization of the oligoglycosyl alditol prepared by reduction of this octasaccharide confirmed the previously proposed structure that had been based on analysis of the mixture of isomeric octasaccharides. The availability of large amounts of the pure, reduced octasaccharide and of a pure, reduced pentasaccharide (Glc3Xyl2) made it possible to completely assign their 1H and 13C NMR spectra. In addition, the borohydride-reduced form of the beta-D-galactosidase-susceptible octasaccharide isomer was purified by high pH anion-exchange chromatography of the endo-(1-->4)-beta-glucanase-released octasaccharides from rape-seed xyloglucan (no beta-galactosidase treatment), and its 1H and 13C NMR spectra were assigned. Additional correlations between specific structural features of xyloglucan oligoglycosyl alditols and the positions of specific resonances in their NMR spectra were deduced and added to the extensive list that we have compiled. The effects of recording the NMR spectra of the xyloglucan oligoglycosyl alditols in the presence of borate salts, which could lead to incorrect structural assignments, are also described.