Xylosyl Units Research Articles

Novel 4-O-β-d-xylopyranosyl-3,6-anhydro-l-galactopyranosyl disaccharide units in a polysaccharide from the red alga Pyrophyllon subtumens

Thalli of the endemic epiphytic New Zealand red seaweed Pyrophyllon subtumens are known to contain a high level of xylose and a notable amount of arabinose but the extracted polysaccharide has not been characterised.The linkage/substitution of individual sugars within the water-soluble polysaccharide extract and various derivatives were determined by chemical and spectroscopic methods. No 3-linked sugars nor any d-galactose were found, which excluded agar-, carrageenan- or mixed 3-linked/4-linked β-d-xylan-type polysaccharides found in many other red macroalgae. Instead, the polysaccharide backbone contained predominantly 4-linked β-d-xylopyranosyl, 4-linked 3,6-anhydro-l-galactopyranosyl and 4-linked l-galactopyranosyl units. Some of each type of sugar were sulfated at various positions. Some xylosyl units were substituted at the 2- or 3-position with l-arabinosyl units. The polysaccharide is complex and likely contains a range of structures. However, partial sequencing was successfully used to recover and identify a novel disaccharide 4-O-d-xylopyranosyl-3,6-anhdydro-l-galactopyranose, which indicates a unique →4)-β-d-Xylp-(1 → 4)-3,6-anhydro-l-Galp-(1 → repeat unit in the polysaccharide.

Two Subgroups within the GH43_36 α-l-Arabinofuranosidase Subfamily Hydrolyze Arabinosyl from Either Mono-or Disubstituted Xylosyl Units in Wheat Arabinoxylan.

Fungal arabinofuranosidases (ABFs) catalyze the hydrolysis of arabinosyl substituents (Ara) and are key in the interplay with other glycosyl hydrolases to saccharify arabinoxylans (AXs). Most characterized ABFs belong to GH51 and GH62 and are known to hydrolyze the linkage of α-(1→2)-Ara and α-(1→3)-Ara in monosubstituted xylosyl residues (Xyl) (ABF-m2,3). Nevertheless, in AX a substantial number of Xyls have two Aras (i.e., disubstituted), which are unaffected by ABFs from GH51 and GH62. To date, only two fungal enzymes have been identified (in GH43_36) that specifically release the α-(1→3)-Ara from disubstituted Xyls (ABF-d3). In our research, phylogenetic analysis of available GH43_36 sequences revealed two major clades (GH43_36a and GH43_36b) with an expected substrate specificity difference. The characterized fungal ABF-d3 enzymes aligned with GH43_36a, including the GH43_36 from Humicola insolens (HiABF43_36a). Hereto, the first fungal GH43_36b (from Talaromyces pinophilus) was cloned, purified, and characterized (TpABF43_36b). Surprisingly, TpABF43_36b was found to be active as ABF-m2,3, albeit with a relatively low rate compared to other ABFs tested, and showed minor xylanase activity. Novel specificities were also discovered for the HiABF43_36a, as it also released α-(1→2)-Ara from a disubstitution on the non-reducing end of an arabinoxylooligosaccharide (AXOS), and it was active to a lesser extent as an ABF-m2,3 towards AXOS when the Ara was on the second xylosyl from the non-reducing end. In essence, this work adds new insights into the biorefinery of agricultural residues.

Differential bacterial capture and transport preferences facilitate co-growth on dietary xylan in the human gut.

Metabolism of dietary glycans is pivotal in shaping the human gut microbiota. However, the mechanisms that promote competition for glycans among gut commensals remain unclear. Roseburia intestinalis, an abundant butyrate-producing Firmicute, is a key degrader of the major dietary fibre xylan. Despite the association of this taxon to a healthy microbiota, insight is lacking into its glycan utilization machinery. Here, we investigate the apparatus that confers R. intestinalis growth on different xylans. R. intestinalis displays a large cell-attached modular xylanase that promotes multivalent and dynamic association to xylan via four xylan-binding modules. This xylanase operates in concert with an ATP-binding cassette transporter to mediate breakdown and selective internalization of xylan fragments. The transport protein of R. intestinalis prefers oligomers of 4-5 xylosyl units, whereas the counterpart from a model xylan-degrading Bacteroides commensal targets larger ligands. Although R. intestinalis and the Bacteroides competitor co-grew in a mixed culture on xylan, R. intestinalis dominated on the preferred transport substrate xylotetraose. These findings highlight the differentiation of capture and transport preferences as a possible strategy to facilitate co-growth on abundant dietary fibres and may offer a unique route to manipulate the microbiota based on glycan transport preferences in therapeutic interventions to boost distinct taxa.

Biochemical characterization of rice xylan O-acetyltransferases.

Rice xylan is predominantly monoacetylated at O-2 and O-3, and 14 rice DUF231 proteins were demonstrated to be xylan acetyltransferases. Acetylated xylans are the principal hemicellulose in the cell walls of grass species. Because xylan acetylation impedes the conversion of cellulosic biomass into biofuels, knowledge on acetyltransferases catalyzing xylan acetylation in grass species will be instrumental for a better utilization of grass biomass for biofuel production. Xylan in rice (Oryza sativa) is predominantly monoacetylated at O-2 and O-3 with a total degree of acetylation of 0.19. In this report, we have characterized 14 rice DUF231 proteins (OsXOAT1 to OsXOAT14) that are phylogenetically grouped together with Arabidopsis xylan acetyltransferases ESK1 and its close homologs. Complementation analysis demonstrated that the expression of OsXOAT1 to OsXOAT7 in the Arabidopsis esk1 mutant was able to rescue its defects in 2-O- and 3-O-monoacetylation and 2,3-di-O-acetylation. Activity assay of recombinant proteins revealed that all 14 OsXOATs exhibited acetyltransferase activities capable of transferring acetyl groups from acetyl-CoA to the xylohexaose acceptor with 10 of them having high activities. Structural analysis of the OsXOAT-catalyzed products showed that the acetylated structural units consisted mainly of 2-O- and 3-O-monoacetylated xylosyl residues with a minor amount of 2,3-di-O-acetylated xylosyl units, which is consistent with the acetyl substitution pattern of rice xylan. Further kinetic studies revealed that OsXOAT1, OsXOAT2, OsXOAT5, OsXOAT6 and OsXOAT7 had high affinity toward the xylohexaose acceptor. Our results provide biochemical evidence indicating that OsXOATs are acetyltransferases involved in xylan acetylation in rice.

Structural insights into the inhibition of cellobiohydrolase Cel7A by xylo-oligosaccharides.

The filamentous fungus Hypocrea jecorina (anamorph of Trichoderma reesei) is the predominant source of enzymes for industrial saccharification of lignocellulose biomass. The major enzyme, cellobiohydrolase Cel7A, constitutes nearly half of the total protein in the secretome. The performance of such enzymes is susceptible to inhibition by compounds liberated by physico-chemical pre-treatment if the biomass is kept unwashed. Xylan and xylo-oligosaccharides (XOS) have been proposed to play a key role in inhibition of cellobiohydrolases of glycoside hydrolase family 7. To elucidate the mechanism behind this inhibition at a molecular level, we used X-ray crystallography to determine structures of H. jecorina Cel7A in complex with XOS. Structures with xylotriose, xylotetraose and xylopentaose revealed a predominant binding mode at the entrance of the substrate-binding tunnel of the enzyme, in which each xylose residue is shifted ~ 2.4 Å towards the catalytic center compared with binding of cello-oligosaccharides. Furthermore, partial occupancy of two consecutive xylose residues at subsites -2 and -1 suggests an alternative binding mode for XOS in the vicinity of the catalytic center. Interestingly, the -1 xylosyl unit exhibits an open aldehyde conformation in one of the structures and a ring-closed pyranoside in another complex. Complementary inhibition studies with p-nitrophenyl lactoside as substrate indicate mixed inhibition rather than pure competitive inhibition. The atomic coordinates and structure factors are available in the Protein Data Bank under accession number 4D5I (H. jecorina Cel7A E212Q variant, complex with xylotriose), 4D5J (H. jecorina Cel7A E217Q variant, complex with xylotriose), 4D5O (H. jecorina Cel7A E212Q variant, complex with xylopentaose), 4D5P (H. jecorina Cel7A E217Q variant, complex with xylopentaose), 4D5Q (wild-type H. jecorina Cel7A, complex with xylopentaose) and 4D5V (H. jecorina Cel7A E217Q variant, complex with xylotetraose).

Aqueous processing of Pinus pinaster wood: Kinetics of polysaccharide breakdown

Aqueous treatments of Pinus pinaster wood were carried out in a pressurized reactor under non-isothermal conditions. The kinetics of the hydrolytic degradation of hemicelluloses has been studied. Hemicelluloses are formed by two major polymers (glucomannan and heteroxylan) made up of a number of structural components (chains of β-linked mannosyl, glucosyl, and xylosyl units, with acetyl, galactosyl, and arabinosyl substituents). The hydrolytic breakdown of polymers was assumed to proceed through a number of first order reactions. The backbones of the hemicellulosic polymers were split into oligomers, whereas substituents were hydrolyzed into the corresponding monomers. The resulting model provided a satisfactory interpretation of the experimental results.

NMR Spectroscopic Analysis Reveals Extensive Binding Interactions of Complex Xyloglucan Oligosaccharides with the Cellvibrio japonicus Glycoside Hydrolase Family 31 α‐Xylosidase

The study of the interaction of glycoside hydrolases with their substrates is fundamental to diverse applications in medicine, food and feed production, and biomass-resource utilization. Recent molecular modeling of the α-xylosidase CjXyl31A from the soil saprophyte Cellvibrio japonicus, together with protein crystallography and enzyme-kinetic analysis, has suggested that an appended PA14 protein domain, unique among glycoside hydrolase family 31 members, may confer specificity for large oligosaccharide fragments of the ubiquitous plant polysaccharide xyloglucan (J. Larsbrink, A. Izumi, F.M. Ibatullin, A. Nakhai, H.J. Gilbert, G.J. Davies, H. Brumer, Biochem. J. 2011, 436, 567-580). In the present study, a combination of NMR spectroscopic techniques, including saturation transfer difference (STD) and transfer NOE (TR-NOE) spectroscopy, was used to reveal extensive interactions between CjXyl31A active-site variants and xyloglucan hexa- and heptasaccharides. The data specifically indicate that the enzyme recognizes the entire cello-tetraosyl backbone of the substrate and product in positive enzyme subsites and makes further significant interactions with internal pendant α-(1→6)-linked xylosyl units. As such, the present analysis provides an important rationalization of previous kinetic data on CjXyl31A and unique insight into the role of the PA14 domain, which was not otherwise obtainable by protein crystallography.

Chemical structure and antiviral activity of the sulfated heterorhamnan isolated from the green seaweed Gayralia oxysperma

A homogeneous sulfated heterorhamnan was obtained by aqueous extraction, then by ultrafiltration from the green seaweed Gayralia oxysperma. Besides α- l-rhamnose it contains glucuronic and galacturonic acids, xylose and glucose. The structure was established by methylation analyses of the carboxyl-reduced, carboxyl-reduced/desulfated, carboxyl-reduced/Smith-degraded, and carboxyl-reduced/Smith-degraded/desulfated products and 1D, 2D NMR spectroscopy analyses. The heterorhamnan backbone is constituted by 3- and 2-linked rhamnosyl units (1.00:0.80), the latter being ∼50% substituted at C-3 by side chains containing 2-sulfated glucuronic and galacturonic acids and xylosyl units. The 3- and 2-linked rhamnosyl units are unsulfated (20%), disulfated (16%), and mostly monosulfated at C-2 (27%) and C-4 (37%). The branched and sulfated heterorhamnan had high and specific activity against herpes simplex virus.

Glycosynthase‐Assisted Synthesis of Xylo‐Gluco‐Oligosaccharide Probes for α‐Xylosyltransferases.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF.

Glycosynthase‐Assisted Synthesis of Xylo‐Gluco‐Oligosaccharide Probes for α‐Xylosyltransferases

AbstractAn efficient chemo‐enzymatic synthesis involving glycosylation catalyzed by E197A nucleophile mutant of the retaining endo‐cellulase Cel7B from Humicola insolens afforded well defined xylo‐gluco‐oligosaccharides. The synthesis of these complex oligosaccharides requires the use of an enzymatic protection/deprotection concept to allow a single‐step condensation of donors onto acceptors. These molecules were tested as potential acceptor substrates for two Arabidopsis thaliana putative xyloglucan α‐xylosyltransferases expressed in insect cells. Both AtXT1 and AtXT2 catalyzed the incorporation of xylosyl unit(s) onto all of these substrates, but with various efficiencies. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007)

New 4- O-substituted xylosyl units in the xyloglucan from leaves of Hymenaea courbaril

A homogeneous fucogalactoxyloglucan, isolated from the leaves of Hymenaea courbaril, was analysed by methylation-GC–MS. These procedures involved derived partially O-methylated alditol acetates and acetylated aldononitriles, which demonstrated the presence of both 2- O- and 4- O-substituted Xyl p units in the side-chains. The presence of the unusual, latter structure was confirmed by 2D NMR spectroscopy with a correlated HMQC C-4/H-4 signal at δ 77.8/3.73. A similar 4- O-substituted xylosyl structure was present in a decasaccharide Glc 4Xyl 3Gal 2Fuc obtained via endo-glucanase treatment of the polysaccharide, which gave rise to a molecular ion with m/ z 1555 (ESI-MS, Na + form).

Changes in cell wall polysaccharides in developing barley (Hordeum vulgare) coleoptiles

Cell wall polysaccharides in developing barley coleoptiles were examined using acetic acid-nitric acid extraction, alditol acetate and methylation analyses and enzymatic digestion. The coleoptile cell wall from imbibed grain was rich in pectic polysaccharides (30 mol%), arabinoxylan (25 mol%), cellulose (25 mol%) and xyloglucan (6 mol%), but contained only low levels of (1-->3,1-->4)-beta-D-glucan (1 mol%). During 5 days of coleoptile growth, pectic polysaccharides decreased steadily to about 9 mol%, while (1-->3,1-->4)-beta-D-glucan increased to 10 mol%. Following the cessation of growth of the coleoptiles at about 5 days, (1-->3,1-->4)-beta-D-glucan content rapidly decreased to 1 mol%. The cellulose content of the walls remained at about 35-40 mol% throughout coleoptile growth. Similarly, arabinoxylan content remained essentially constant at 25-30 mol% during growth, although the ratio of substituted to unsubstituted 4-linked xylosyl units decreased from about 4:1 to 1:1. Xyloglucan content ranged from 6 mol% to 10 mol% and the oligosaccharide profile determined using a xyloglucan-specific endoglucanase and MALDI-TOF mass spectrometry indicated that the oligosaccharides XXGG and XXGGG were the principal components, with one and two acetyl groups, respectively, Thus, dramatic changes in wall composition were detected during the growth of barley coleoptiles, both with respect to the relative abundance of individual wall constituents and to the fine structure of the arabinoxylans.

Generation of polyclonal and monoclonal antibodies against arabinoxylans and their use for immunocytochemical location of arabinoxylans in cell walls of endosperm of wheat

Polyclonal and monoclonal antibodies (pAbs and mAbs) against arabinoxylans (AX) were produced and used in addition to a mAb against (1→3,1→4)-β-d-glucan to investigate cell-wall structure in endosperm of wheat by light and electron microscopy. pAbs were raised against a xylotetraose BSA conjugate (pAbs anti-X3) while mAbs were produced from a mixture of arabino-xylo-oligosaccharides conjugated to keyhole limpet hemocyanin (KLH) (mAbs anti-AX1). The pAbs anti-X3 and the mAb anti-AX1 recognised unsubstituted (1→4)-β-d-xylosyl sequences but differed in capacity to bind arabinosylated xylo-oligosaccharides. The pAb anti-X3 did not bind to xylotetrasaccharides with the third xylosyl unit bearing arabinose at C(0)3 or at C(0)3 and C(0)2 while the mAb anti-AX1 did. The pAbs anti-X3 reacted strongly with the aleurone and central starchy endosperm cell walls and less strongly with subaleurone cell walls. The mAb anti-AX1 bound strongly and equally to all the cell walls of the endosperm. The distribution pattern of the AX epitopes was distinct according to cell types. (1→3,1→4)-β-d-Glucans were abundant in the aleurone and subaleurone cell walls but were not detected in the junction region between aleurone cells at the nucellus epidermis interface. The epitopes occurred in discreet domains of the walls of central cells.

Evaluation of the Structure of the Polysaccharides from Chondria macrocarpa and Ceramium rubrum as Determined by 13C NMR Spectroscopy

The polysaccharides from Chondria macrocarpa and Ceramium rubrum were examined by 13C NMR spectroscopy and their spectra were analysed bymeans of set theory. The spectra of the polysaccharides from Chondria macrocarpa collected in autumn were in accord with previous chemical analysis but polysaccharides from seaweed collected in spring comprise a higher level of agar precursors, much lower levels of galactose 2-sulfate, and the polysaccharide is separable into two components, the predominant one of which is essentially free of anhydrogalactose. The signal subset due to the xylosyl unit substituted at the 3-position of 4-linked l-galactose could be assigned from the spectrum of desulfated polysaccharide from Chondria macrocarpa. The extract from Ceramium rubrum consisted of a backbone of 3-linked d-galactosyl units alternating with the 4-linked units 3, 6- anhydro-l-galactosyl and l-galactosyl in a ratio of 2:1 respectively, to which substitution by xylose occurred at approximately 8% of the total sugar units. The natural polymer had moderate levels of sulfate ester, with galactose-6-sulfate being the predominant unit, some of which survives alkaline treatment and was consistent with being 3-linked. There is also evidence of low levels of 3-linked galactose-2-sulfate.

Insight into multi-site mechanisms of glycosyl transfer in (1→4)β- d-glycans provided by the cereal mixed-linkage (1→3),(1→4)β- d-glucan synthase

Synthases of cellulose, chitin, hyaluronan, and all other polymers containing (1→4)β-linked glucosyl, mannosyl and xylosyl units have overcome a substrate orientation problem in catalysis because the (1→4)β-linkage requires that each of these sugar units be inverted nearly 180° with respect to its neighbors. We and others have proposed that this problem is solved by two modes of glycosyl transfer within a single catalytic subunit to generate disaccharide units, which, when linked processively, maintain the proper orientation without rotation or re-orientation of the synthetic machinery in 3-dimensional space. A variant of the strict (1→4)β- d-linkage structure is the mixed-linkage (1→3),(1→4)β- d-glucan, a growth-specific cell wall polysaccharide found in grasses and cereals. β-Glucan is composed primarily of cellotriosyl and cellotetraosyl units linked by single (1→3)β- d-linkages. In reactions in vitro at high substrate concentration, a polymer composed of almost entirely cellotriosyl and cellopentosyl units is made. These results support a model in which three modes of glycosyl transfer occur within the synthase complex instead of just two. The generation of odd numbered units demands that they are connected by (1→3)β-linkages and not (1→4)β-. In this short review of β-glucan synthesis in maize, we show how such a model not only provides simple mechanisms of synthesis for all (1→4)β- d-glycans but also explains how the synthesis of callose, or strictly (1→3)β- d-glucans, occurs upon loss of the multiple modes of glycosyl transfer to a single one.

Crystallographic evidence for substrate ring distortion and protein conformational changes during catalysis in cellobiohydrolase Ce16A from trichoderma reesei.

Cel6A is one of the two cellobiohydrolases produced by Trichoderma reesei. The catalytic core has a structure that is a variation of the classic TIM barrel. The active site is located inside a tunnel, the roof of which is formed mainly by a pair of loops. We describe three new ligand complexes. One is the structure of the wild-type enzyme in complex with a nonhydrolysable cello-oligosaccharide, methyl 4-S-beta-cellobiosyl-4-thio-beta-cellobioside (Glc)(2)-S-(Glc)(2), which differs from a cellotetraose in the nature of the central glycosidic linkage where a sulphur atom replaces an oxygen atom. The second structure is a mutant, Y169F, in complex with the same ligand, and the third is the wild-type enzyme in complex with m-iodobenzyl beta-D-glucopyranosyl-beta(1,4)-D-xylopyranoside (IBXG). The (Glc)(2)-S-(Glc)(2) ligand binds in the -2 to +2 sites in both the wild-type and mutant enzymes. The glucosyl unit in the -1 site is distorted from the usual chair conformation in both structures. The IBXG ligand binds in the -2 to +1 sites, with the xylosyl unit in the -1 site where it adopts the energetically favourable chair conformation. The -1 site glucosyl of the (Glc)(2)-S-(Glc)(2) ligand is unable to take on this conformation because of steric clashes with the protein. The crystallographic results show that one of the tunnel-forming loops in Cel6A is sensitive to modifications at the active site, and is able to take on a number of different conformations. One of the conformational changes disrupts a set of interactions at the active site that we propose is an integral part of the reaction mechanism.

Active-site binding of glycosides by Thermomonospora fusca endocellulase E2.

The determination of the high-resolution structure of the Thermomonospora fusca endocellulase E2 catalytic domain makes it ideal for exploring cellulase structure-function relationships. Here we present binding parameters (Kd, DeltaH degrees, and DeltaS degrees) describing the interaction of E2 with 4-methylumbelliferyl glycosides, determined by titrating the quenching of ligand fluorescence in equilibrium binding experiments. Quenched MU(Glc)2/E2 complexes were used as indicators in displacement titrations to measure the binding of natural glycosides and also of a nonhydrolyzable cellotetraose analogue. Binding of MU(Glc)2 and cellotriose were also determined by titration calorimetry. The results show that E2 binds glycosides exclusively in its active-site cleft, with high affinity and specificity. The observed patterns of ligand hydrolysis and the results with MU(Glc)2 as a substrate indicated that ligands bound to E2 with their nonreducing ends in position -2, consistent with the position of cellobiose in the E2cd structure. Polymerase chain reaction (PCR) mutagenesis of the conserved residue Tyr 73 (in E2 binding subsite -1) to Phe and Ser produced enzymes with lower activity but higher binding affinities, indicating that the volume of the subsite -1 binding pocket is crucial for enzyme function. Similarly, MUXylGlc (with its xylosyl unit located in position -1) bound with 100-fold higher affinity than MU(Glc)2. These results are similar to those for the related Trichoderma reesei exocellulase CBH II. The binding data were compared with that previously reported for CBH II and interpreted in terms of the functional differences between endo- and exocellulases.

Infrared study of arabinoxylans

AbstractThe influence of substitution of the (1 → 4)‐β‐D‐xylan backbone with α‐L‐arabinofuranosyl units on the infrared spectra has been studied using the freeze‐dried form of related oligo‐ and polysaccharide models. The peak intensity ratios at 1164 and 990 cm‐1 can be suggested as an index of arabinofuranosyl contribution. A valid correlation was found between the substitution of the xylosyl units at the position O‐3 by arabinosyl units and the intensity of the above‐mentioned absorption bands. The results are valuable for the identification of arabinoxylan type polysaccharides, as well as, for studies of the xylan component behaviour of plant materials in various technical and biotcch‐nological processes.

Novel (rhamnosyl and ribosyl) and uncommon (xylosyl) monosaccharide residues are present in asparagine-linked oligosaccharides of the trypanosomatid Blastocrithidia culicis.

Blastocrithidia culicis is a trypanosomatid protozoon that transfers Man6GlcNAc2 in protein N-glycosylation. Compounds containing mannosyl, xylosyl, and rhamnosyl residues were found among the endo-beta-N-acetylglucosaminidase H-sensitive oligosaccharides of whole cell glycoproteins of this parasite. The compositions of some of them were as follows: Man5GlcNAc2, Man6GlcNAc2, Rha1Man5GlcNAc2, Rha2Man6GlcNAc2, Xyl1Rha2Man6-GlcNAc2, Xyl1Rha3Man6GlcNAc2, and Xyl2Rha3Man6-GlcNAc2. On the other hand, oligosaccharides containing mannosyl, xylosyl, rhamnosyl, and ribosyl units were liberated from endo-beta-N-acetylglucosaminidase-resistant glycopeptides upon treatment with N-glycanase. This is the first report on the presence of ribosyl units in eukaryote glycoconjugates, of rhamnosyl residues in asparagine-linked oligosaccharides, and of xylosyl units in high mannose-type compounds.

Highly uneven distribution of O-acetyl groups in the acidic d-xylan of Mimosa scabrella (bracatinga)

The acidic O-acetyl- d-xylan of Mimosa scabrella contains xylose, 4- O-methylglucuronic acid, and O-acetyl groups in the molar ratios of 60:7:33 and, when subjected to controlled Smith degradation, provided xylosyl-, xylobiosyl-, xylotriosyl-, and xylotetraosyl-glycerol, as well as glycerol. Their proportions differed greatly from that which would arise from xylopyranosyl units substituted regularly with O-acetyl groups. In order to avoid ambiguities arising from resistance of some of the xylosyl units by substitution with 4- O-methylglucuronic acid, the O-acetylxylan was converted into a partly O-methylated polysaccharide having O-methyl groups with similar positions of substitution. Two successive Smith degradations were carried out, and the two sets of partly O-methylated derivatives resulting were identified by f.a.b.-m.s. The highly uneven distribution of O-acetyl groups along the xylan main-chain was confirmed, with the highest number of consecutive O-acetyl and 4- O-methylglucuronic acid substituents being six, and five O-acetyl substituents alone. 4- O-Methylglucuronic acid units were not substituted.