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Xylose, xylo-oligosaccharides with a degree of polymerization (DP) from 2 to 6, xylo-oligosaccharides carrying a single arabinofuranosyl, glucosyluronic acid, or 4- O -methyl glucosyluronic acid residues and 4-β- d -Xyl p -(1 → 4)- d -Xyl p -(1 → 3)-α- l -Rha p -(1 → 2)-α- d -Gal p A-(1 → 4)- d -Xyl p were labeled at their reducing ends with 2-aminobenzamide (2AB) in the presence of sodium cyanoborohydride (NaBH 3 CN). These derivatives were then analyzed by high-performance anion-exchange chromatography (HPAEC) and structurally characterized by electrospray-ionization mass spectrometry (ESI-MS) and by 1 H and 13 C NMR spectroscopy. Reacting Xyl 3 -Xylitol-2AB with UDP-Xyl in the presence of rice microsomes resulted in the formation of small amounts of Xyl 4–6 -Xylitol-2AB showing that the 2AB-labeled compound is an acceptor for xylosyltransferase. The 2AB-labeled xylo-oligosaccharides and the 2AB-labeled xylo-oligosaccharides carrying 4- O -methyl glucuronic acid are fragmented by xylanase and α-glucuronidase present in the culture filtrate of Fomitopsis palustris FFPRI 0507. Thus, the 2AB-labeled xylo-oligosaccharides are useful for studying enzymes involved in xylan degradation and biosynthesis.

The biosynthesis of arabinoxylan was investigated using microsomal membranes isolated from wheat seedlings. Incubation of the microsomes with UDP-[ 14 C]-D-xylose resulted in incorporation of radioactivity into polymeric product. Incorporation reached a maximum at day 3 during development of the seedlings. Treatment of the radiolabeled product with Aspergillus niger xylanase A released about 70% of the radioactivity to a 65% ethanol-soluble fraction. The released radioactivity was shown to be in the form of xylobiose, xylotriose and xylotetraose, which are the expected hydrolysis products of endo-xylanases. The synthesized xylan had an apparent molecular mass larger than 500 kDa.

The opportunistic yeast Cryptococcus neoformans causes serious disease in humans and expresses a prominent polysaccharide capsule that is required for its virulence. Little is known about how this capsule is synthesized. We previously identified a beta1,2-xylosyltransferase (Cxt1p) with in vitro enzymatic activity appropriate for involvement in capsule synthesis. Here, we investigate C. neoformans strains in which the corresponding gene has been deleted (cxt1Delta). Loss of CXT1 does not affect in vitro growth of the mutant cells or the general morphology of their capsules. However, NMR structural analysis of the two main capsule polysaccharides, glucuronoxylomannan (GXM) and galactoxylomannan (GalXM), showed that both were missing beta1,2-xylose residues. There was an approximately 30% reduction in the abundance of this residue in GXM in mutant compared with wild-type strains, and mutant GalXM was almost completely devoid of beta1,2-linked xylose. The GalXM from the mutant strain was also missing a beta1,3-linked xylose residue. Furthermore, deletion of CXT1 led to attenuation of cryptococcal growth in a mouse model of infection, suggesting that the affected xylose residues are important for normal host-pathogen interactions. Cxt1p is the first glycosyltransferase with a defined role in C. neoformans capsule biosynthesis, and cxt1Delta is the only strain identified to date with structural alterations of the capsule polysaccharide GalXM.

Human xylosyltransferase II (EC 2.4.2.26, XT-II) represents an isoform of xylosyltransferase I (XT-I). Recently, we and others provided first evidence that XT-II is capable of initiating the biosynthesis of glycosaminoglycan chains in proteoglycans. Here, a soluble form of human XT-II was expressed in the yeast Pichia pastoris and the substrate specificity for various acceptors was investigated, pointing to a modified bikunin peptide to be the optimal XT-II acceptor (KM = 1.9 μM). Furthermore, biochemical characterization of XT-II showed that this enzyme was strongly inhibited by nucleotides and glycosaminoglycans. Its temperature optimum, stability, and ion dependency were further examined, demonstrating necessity for Mg2+ or Mn2+ ions for its enzymatic activity. Our data show for the first time that XT-I and XT-II are xylosyltransferases with similar but not identical properties, pointing to their potential role in modulating the cellular proteoglycan pool.

Xylan, cellulose and lignin are the three major components of secondary walls in wood, and elucidation of the biosynthetic pathway of xylan is of importance for potential modification of secondary wall composition to produce wood with improved properties. So far, three Arabidopsis glycosyltransferases, FRAGILE FIBER8, IRREGULAR XYLEM8 and IRREGULAR XYLEM9, have been implicated in glucuronoxylan (GX) biosynthesis. In this study, we demonstrate that PARVUS, which is a member of family GT8, is required for the biosynthesis of the tetrasaccharide primer sequence, beta-D-Xyl-(1 --> 3)-alpha-l-Rha-(1 --> 2)-alpha-D-GalA-(1 --> 4)-D-Xyl, located at the reducing end of GX. The PARVUS gene is expressed during secondary wall biosynthesis in fibers and vessels, and its encoded protein is predominantly localized in the endoplasmic reticulum. Mutation of the PARVUS gene leads to a drastic reduction in secondary wall thickening and GX content. Structural analysis of GX using (1)H-nuclear magnetic resonance (NMR) spectroscopy revealed that the parvus mutation causes a loss of the tetrasaccharide primer sequence at the reducing end of GX and an absence of glucuronic acid side chains in GX. Activity assay showed that the xylan xylosyltransferase and glucuronyltransferase activities were not affected in the parvus mutant. Together, these findings implicate a possible role for PARVUS in the initiation of biosynthesis of the GX tetrasaccharide primer sequence and provide novel insights into the mechanisms of GX biosynthesis.

The stereoselective synthesis of the spacer-armed disaccharide α- D-Xyl-(1→3)-β- D-Glc- O-(CH 2 ) 3 NH 2 and trisaccharide α- D-Xyl-(1→3)-α- D-Xyl-(1→3)-β- D-Glc- O-(CH 2 ) 3 NH 2 , as well as of their N-octanoyl derivatives, was performed for their subsequent use as acceptors and reference samples in xylosyltransferase assays. These structures are found as O-linked glycans on epidermal growth factor (EGF) domains of several blood-clotting factors and Notch. The target products were prepared by glycosylation with a 3-O-acetylated xylosyl donor that was previously found to be an effective α-xylosylating agent. However, in this paper we show that the structure of the glycosyl acceptor could influence the stereochemical results of the glycosylation and even lead to reversed stereoselectivity in the case of one acceptor.

Xylan is the second most abundant polysaccharide in dicot wood, and thus elucidation of the xylan biosynthetic pathway is required to understand the mechanisms controlling wood formation. Genetic and chemical studies in Arabidopsis have implicated three genes, FRAGILE FIBER8 (FRA8), IRREGULAR XYLEM8 (IRX8) and IRREGULAR XYLEM9 (IRX9), in the biosynthesis of glucuronoxylan (GX), but the biochemical functions of the encoded proteins are not known. In this study, we determined the effect of the fra8, irx8 and irx9 mutations on the activities of xylan xylosyltransferase (XylT) and glucuronyltransferase (GlcAT). We show that microsomes isolated from the stems of wild-type Arabidopsis exhibit XylT and GlcAT activities in the presence of exogenous 1,4-linked beta-d-xylooligomers. Xylooligomers ranging in size from two to six can be used as acceptors by XylT to form xylooligosaccharides with up to 12 xylosyl residues. We provide evidence that the irx9 mutation results in a substantial reduction in XylT activity but has no discernible effect on GlcAT activity. In contrast, neither XylT nor GlcAT activity is affected by fra8 and irx8 mutations. Our results provide biochemical evidence that the irx9 mutation results in a deficiency in xylan XylT activity, thus leading to a defect in the elongation of the xylan backbone.

The basic biochemical mechanisms underlying many heritable human polycystic diseases are unknown despite evidence that most cases are caused by mutations in members of several protein families, the most prominent being the polycystin gene family, whose products are found on the primary cilia, or due to mutations in posttranslational processing and transport. Inherited polycystic kidney disease, the most prevalent polycystic disease, currently affects ≈500,000 people in the United States. Decreases in proteoglycans (PGs) have been found in tissues and cultured cells from patients who suffer from autosomal dominant polycystic kidney disease, and this PG decrease has been hypothesized to be responsible for cystogenesis. This is possible because alterations in PG concentrations would be predicted to disrupt many homeostatic mechanisms of growth, development, and metabolism. To test this hypothesis, we have generated mice lacking xylosyltransferase 2 (XylT2), an enzyme involved in PG biosynthesis. Here we show that inactivation of XylT2 results in a substantial reduction in PGs and a phenotype characteristic of many aspects of polycystic liver and kidney disease, including biliary epithelial cysts, renal tubule dilation, organ fibrosis, and basement membrane abnormalities. Our findings demonstrate that alterations in PG concentrations can occur due to loss of XylT2, and that reduced PGs can induce cyst development.

Peptide O-xylosyltransferase (EC 2.4.2.26) is the first enzyme required for the generation of chondroitin and heparan sulfate glycosaminoglycan chains of proteoglycans. Cloning of cDNAs has previously shown that, whereas invertebrates generally have a single xylosyltransferase gene, vertebrate genomes encode two similar proteins, xylosyltransferase I and II (XT-I and XT-II). To date, enzymatic activity has only been demonstrated for the human XT-I, Caenorhabditis SQV-6, and Drosophila OXT isoforms. In the present study, we demonstrate that a soluble form of human XT-II expressed in the xylosyltransferase-deficient pgsA-745 (S745) Chinese hamster ovary cell line is indeed capable of catalyzing the transfer of xylose to a variety of peptide substrates; its enzyme activity was also proven using a Pichia-expressed form of XT-II. Its pH, temperature, and cation dependences are similar to those of XT-I expressed in either mammalian cells or yeast. Our data suggest that XT-I and XT-II are, at least in vitro, functionally identical.

Human xylosyltransferase I (XT-I) initiates the biosynthesis of the glycosaminoglycan (GAG) linkage tetrasaccharide in proteoglycans. Xylosyltransferase II (XT-II) is a protein homologous to XT-I but with hitherto unknown activity or physiological function. Here, we report the enzymatic activity of XT-II and provide evidence that XT-II initiates the biosynthesis of both heparan sulfate and chondroitin sulfate GAGs. Transfection of the xylosyltransferase-deficient Chinese hamster ovary mutant pgsA-745 with XT-I or XT-II coding cDNA completely restored GAG biosynthesis. GAG disaccharide analysis revealed that XT-I- and XT-II-transfected pgsA-745 cells produced similar amounts of chondroitin sulfate and heparan sulfate. Furthermore, a high xylosyltransferase activity was measured after transfection with cDNAs encoding either isozyme. Analysis of the enzyme activity revealed that XT-II catalyzes the transfer of xylose to similar peptide acceptors as XT-I but with different efficiency. The optimal XT-II acceptor was observed using a bikunin-related peptide (Km 5.2 μm). Analysis of XT-I and XT-II mRNA expression in murine tissues showed a differential expression pattern for both enzymes. In particular, XT-II is highly expressed in liver tissue, where XT-I transcripts were not detected. This is the first report on the enzyme activity of XT-II and its involvement in chondroitin sulfate and heparan sulfate biosynthesis.

A unique O-glycan structure, Xylalpha1-3Xylalpha1-3Glcbeta1-O-Ser is found on the consensus sequence C-X-S-X-P-C (X denotes any amino acid) in epidermal growth factor (EGF)-like domains of plasma proteins such as clotting factor VII and IX. One of the enzymes involved in the biosynthesis of this trisaccharide, UDP-d-xylose:beta-d-glucoside 1,3-d-xylosyltransferase has been identified in HepG2 cells (Omichi, K., Aoki, K., Minamida, S., and Hase, S. Eur. J. Biochem. 245, 143-146 [1997]). Here, we report that this enzyme activity can be detected in bovine liver and that the enzyme has been purified from the microsomal fraction. The enzyme was purified 6200-fold in terms of specific activity and ran as a single band on native-PAGE and isoelectric focusing gel electrophoresis. The best acceptor substrate of those tested was the EGF-like domain of bovine factor IX carrying beta-glucoside at Ser53. The Km value for this substrate was 34 muM. Comparison of initial velocity with various acceptor substrates shows that this xylosyltransferase recognizes not only the glucose moiety to which xylose is transferred but also the tertiary structure of the EGF-like domain. With regard to the donor substrate, the enzyme does not recognize UDP-d-glucose but does recognize UDP-d-xylose.

Mesenchymal stem cells are multipotent progenitor cells that can differentiate into the chondrogenic lineage. To date, only limited knowledge about the formation and remodeling of the cartilaginous extracellular matrix is available. We recently analyzed the coordinated expression of proteins involved in the biosynthesis of proteoglycans and collagens, the two major components of cartilage matrix, to understand matrix formation and to provide potential tools to improve the quality of tissue-engineered cartilage.

O-xylosyltransferase (Oxt) is a transmembrane glycosyltransferase that initiates the first step in heperan and chrondroitin sulfate (HS and CS) proteoglycan biosynthesis. HSPGs and CSPGs are abunda...

The genus Pseudomonas is a group of gram-negative, motile, rod-shaped bacteria known for their metabolic versatility. One of the species is Pseudomonas fluorescens, which has an efficient system for detoxification of industrial waste. Other aspects include, catabolic versatility, excellent root colonizing abilities and capacity to produce a wide range of antifungal metabolites. They are also known for their resistance and survival in the presence of several organic and inorganic pollutants. P. fluorescens has also been isolated from metal polluted water and soils but the elucidation of proteins responsible for its survival is still not clear. The aim of the study was to elucidate the differential protein expression of this bacterium when exposed to heavy metal stress, using two-dimensional electrophoresis. The proteins spo VG and enolase showed upregulation during the bacterial exposure to lead and copper. Hypothetical protein showed downregulation when bacterium was exposed to cobalt. Some proteins like xylosyltransferase, ORF 18 phage phi KZ, OMP H1 and translational elongation factor EF-Tu appeared only during their exposure to cobalt. These were absent in the control condition. Analysis of the differentially expressed proteins as well as the newly synthesized proteins along with the results obtained growth and enzyme activity indicate the involvement of all these factors in the survival of this organism in the presence of heavy metals.

Xyloglucan (XyG) is the principal hemicellulose found in the primary cell walls of most plants. XyG is composed of a beta-(1,4)-glucan backbone that is substituted in a regular pattern with xylosyl residues, which are added by at least one and likely two or three xylosyltransferase (XT) enzymes. Previous work identified seven Arabidopsis thaliana candidate genes, one of which (AtXT1) was shown to encode a protein with XT activity (Faik, A., Price, N. J., Raikhel, N. V., and Keegstra, K. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 7797-7802). We expressed both AtXT1 and a second closely related gene, now called AtXT2, in insect cells and demonstrated that both have XT activity for cellopentaose and cellohexaose acceptor substrates. Moreover, we showed that cellohexaose was a significantly better acceptor substrate than cellopentaose. Product structural characterization showed that AtXT1 and AtXT2 preferentially added the first xylosyl residue to the fourth glucosyl residue from the reducing end of both acceptors. Furthermore, when the ratio of UDP-xylose to cellohexaose and the reaction time were increased, both AtXT1 and AtXT2 added a second xylosyl residue adjacent to the first, which generated dixylosylated cellohexaose. On the basis of these results, we concluded that AtXT1 and AtXT2 have the same acceptor specificities and generate the same products in vitro. The implications of these results for understanding in vivo XyG biosynthesis are considered.

Chondroitin and heparan sulphates have key functions in animal development and their synthesis is initiated by the action of UDP-alpha-D-xylose:proteoglycan core protein beta-D-xylosyltransferase (EC 2.4.2.26). cDNAs encoding this enzyme have been previously cloned from mammalian species; this in turn facilitated identification of corresponding Caenorhabditis elegans (sqv-6) and Drosophila melanogaster (oxt) genes. In the present study, we report the expression in Pichia pastoris and subsequent assay using either MALDI-TOF MS or RP-HPLC of recombinant forms of the Caenorhabditis xylosyltransferase SQV-6 and the human xylosyltransferase I, in addition to extending our previous studies on the xylosyltransferase from Drosophila. The enzyme activities were tested with a number of peptide substrates based on portions of the human bikunin, human perlecan and Drosophila syndecan core peptides. Whereas a variant of the latter, containing two Ser-Gly motifs was only modified on one of these motifs, the perlecan peptide with three Ser-Gly motifs could be multiply modified in vitro. Using this substrate, we could for the first time follow, by mass spectrometry, the xylosylation of a peptide with multiple xylosyltransferase acceptor motifs.

The processing of N-linked oligosaccharides in the secretory pathway requires the sequential action of a number of glycosidases and glycosyltransferases. We studied the spatial distribution of several type II membrane-bound enzymes from Glycine max, Arabidopsis thaliana, and Nicotiana tabacum. Glucosidase I (GCSI) localized to the endoplasmic reticulum (ER), α-1,2 mannosidase I (ManI) and N-acetylglucosaminyltransferase I (GNTI) both targeted to the ER and Golgi, and β-1,2 xylosyltransferase localized exclusively to Golgi stacks, corresponding to the order of expected function. ManI deletion constructs revealed that the ManI transmembrane domain (TMD) contains all necessary targeting information. Likewise, GNTI truncations showed that this could apply to other type II enzymes. A green fluorescent protein chimera with ManI TMD, lengthened by duplicating its last seven amino acids, localized exclusively to the Golgi and colocalized with a trans-Golgi marker (ST52-mRFP), suggesting roles for protein–lipid interactions in ManI targeting. However, the TMD lengths of other plant glycosylation enzymes indicate that this mechanism cannot apply to all enzymes in the pathway. In fact, removal of the first 11 amino acids of the GCSI cytoplasmic tail resulted in relocalization from the ER to the Golgi, suggesting a targeting mechanism relying on protein–protein interactions. We conclude that the localization of N-glycan processing enzymes corresponds to an assembly line in the early secretory pathway and depends on both TMD length and signals in the cytoplasmic tail.

Maize is considered a promising alternative production system for pharmaceutically relevant proteins. However, like in all other plant species asparagine-linked oligosaccharides of maize glycoproteins are modified with beta1,2-xylose and core alpha1,3-fucose sugar residues, which are considered to be immunogenic in mammals. This altered N-glycosylation when compared to mammalian cells may reduce the potential of maize as a production system for heterologous glycoproteins. Here we report the cloning and characterization of the cDNA sequences coding for the maize enzymes beta1,2-xylosyltransferase (XylT) and core alpha1,3-fucosyltransferase (FucT). The cloned XylT and FucT cDNAs were shown to encode enzymatically active proteins, which were independently able to convert a mammalian acceptor glycoprotein into an antigen binding anti-plant N-glycan antibodies. The complete sequence of the XylT gene was determined. Evidence for the presence of at least three XylT and FucT gene loci in the maize genome was obtained. The identification of the two enzymes and their genes will allow the targeted downregulation or even elimination of beta1,2-xylose and core alpha1,3-fucose addition to recombinant glycoproteins produced in maize.

In vitro differentiation of mesenchymal stem cells (MSCs) into chondrogenic cells and their transplantation is promising as a technique for the treatment of cartilaginous defects. But the regulation of extracellular matrix (ECM) formation remains elusive. Therefore, the objective of this study was to analyze the regulation of proteoglycan (PG) biosynthesis during the chondrogenic differentiation of MSCs. In different stages of chondrogenic differentiation, we analyzed mRNA and protein expression of key enzymes and PG core proteins involved in ECM development. For xylosyltransferase I (XT-I), we found maximum mRNA levels 48 hours after chondrogenic induction with a 5.04 +/- 0.58 (mean +/- SD)-fold increase. This result correlates with significantly elevated levels of enzymatic XT-I activity (0.49 +/- 0.03 muU/1 x 10(6) cells) at this time point. Immunohistochemical staining of XT-I revealed a predominant upregulation in early chondrogenic stages. The highly homologous protein XT-II showed 4.7-fold (SD 0.6) increased mRNA levels on day 7. To determine the differential expression of heparan sulfate (HS), chondroitin sulfate (CS), and dermatan sulfate (DS) chains, we analyzed the mRNA expression of EXTL2 (alpha-4-N-acetylhexosaminyltransferase), GalNAcT (beta-1,4-N-acetylgalactosaminyltransferase), and GlcAC5E (glucuronyl C5 epimerase). All key enzymes showed a similar regulation with temporarily downregulated mRNA levels (up to -87-fold) after chondrogenic induction. In accordance to previous studies, we observed a similar increase in the expression of PG core proteins. In conclusion, we could show that key enzymes for CS, DS, and HS synthesis, especially XT-I, are useful markers for the developmental stages of chondrogenic differentiation.

Background: Pseudoxanthoma elasticum (PXE) is a heritable connective tissue disorder caused by mutations in the ABCC6 gene. Fragmentation of elastic fibres and deposition of proteoglycans result in a highly variable clinical picture. The altered proteoglycan metabolism suggests that enzymes from this pathway function as genetic co-factors in the severity of PXE. Therefore, we propose the XYLT genes encoding xylosyltransferase I (XT-I) as the chain-initiating enzyme in the biosynthesis of proteoglycans and the highly homologous XT-II as potential candidate genes. Methods: We screened all XYLT exons in 65 German PXE patients using denaturing high performance liquid chromatography and analysed the influence of the variations on clinical characteristics. Results: We identified 22 variations in the XYLT genes. The missense variation p.A115S (XT-I) is associated with higher serum XT activity (p = 0.005). The amino acid substitution p.T801R (XT-II; c.2402C>G) occurs with significantly higher frequency in patients under 30 years of age at diagnosis (43% v 26%; p = 0.04); all PXE patients with this variation suffer from skin lesions compared to only 75% of the wild type patients (p = 0.002). c.166G>A, c.1569C>T, and c.2402C>G in the XYLT-II gene were found to be more frequent in patients with higher organ involvement (p = 0.04, p = 0.01, and p = 0.02, respectively). Conclusions: Here we show for the first time that variations in the XYLT-II gene are genetic co-factors in the severity of PXE. Furthermore, the higher XT activity in patients with the exchange p.A115S (XT-I) indicates that this polymorphism is a potential marker for increased remodelling of the extracellular matrix.

Expression of decorin using the vaccinia virus/T7 expression system resulted in secretion of two distinct glycoforms: a proteoglycan substituted with a single chondroitin sulfate chain and N-linked oligosaccharides and a core protein glycoform substituted with N-linked glycans but without a glycosaminoglycan chain. In this report, we have addressed two distinct questions. What is the rate-limiting step in glycosaminoglycan synthesis? Is glycosylation with either N-linked oligosaccharides or glycosaminoglycan required for secretion of decorin? N-terminal sequencing of the core protein glycoform, the addition of benzyl-β-d-xyloside, and a UDP-xylose: core protein β-d-xylosyltransferase activity assay show that xylosylation is a rate-limiting step in chondroitin sulfate biosynthesis. Decorin can be efficiently secreted with N-linked oligosaccharides alone or with a single chondroitin sulfate chain alone; however, there is severely impaired secretion of core protein devoid of any glycosylation. A decorin core protein mutant devoid of N-linked oligosaccharide attachment sites will not be secreted by Chinese hamster ovary cells deficient in xylosyltransferase or by parental Chinese hamster ovary wild type cells if the xylosyltransferase recognition sequence is disrupted. This finding suggests that quality control mechanisms sensitive to an absence of N-linked oligosaccharides can be abrogated by interaction of the core protein with the glycosaminoglycan synthetic machinery. We propose a model of regulation of decorin secretion that has several components, including appropriate substitution with N-linked oligosaccharides and factors involved in glycosaminoglycan synthesis.

In recent years, sulfated fucans have emerged as an important class of natural biopolymers. In this study, the anti-adhesive activity of a fucan from the brown seaweed Spatoglossum schröederi was analyzed using tumorigenic cells: wild-type Chinese hamster ovary cells (CHO-K1) and the mutant type deficient in xylosyltransferase (CHO-745). Fibronectin (FN) was used as substrate for cell attachment. For both cell types, this fucan has shown a dose-dependent anti-adhesive effect, reaching saturation at around 400 mug/mL. This effect was abolished by desulfation of the fucan. In addition, this polymer exhibited the highest inhibitory effect in comparison to other sulfated polysaccharides. The fucan was biotinylated and used as a probe to identify its action sites. Biotinylated fucan was detected in the extracellular matrix environment by confocal microscopy and flow cytometric analysis, but not at the cell surface. The results suggest that the fucan shows anti-adhesive activity by binding directly to FN, and blocking FN sites that are recognized by cell surface ligands, possibly the integrin family.

A β-(1→4)-xylosyltransferase (XylTase; EC 2.4.2.24) participating in the synthesis of arabinoxylans was investigated using microsomal membranes prepared from developing barley (Hordeum valgare L.) endosperms. The microsomal fraction transferred Xyl from uridine 5'-diphosphoxylose (UDP-Xyl) into exogenous β-(1→4)-xylooligosaccharides derivatized at their reducing ends with 2-aminopyridine. HPLC analysis showed chain elongation of pyridylaminated β-(1→4)-xylotriose (Xyl 3 -PA) by repeated attachment of one to five single xylosyl residues depending on the reaction time, leading to the formation of Xyl 4-8 -PA. Methylation analysis and enzymatic digestions with βxylosidase (EC 3.2.1.37) and emdo-β-(1→4)-xylanase (EC 3.2.1.8) confirmed that the transfer of xylosyl residues into the newly synthesized products occurred through β-(1→4)-linkages. The activity of the XylTase was maximal at pH 6.8 and 20°C and most enhanced in the presence of 0.5% Triton X-100 and 5 mM MnCl 2 . The apparent Michaelis constant and maximal velocity of the enzyme for Xyl 3 -PA were 2.1 mM and 25400 pmol min -1 mg protein -1 , respectively. The enzyme also transferred [ 14 C]Xyl from UDP-[ 14 C]Xyl into higher β-(1→4)-xylooligosaccharides and birchwood xylans through β-(1→4)-linkages. The enzyme activity varied according to the stage of development (7-35 days after flowering) of the endosperms. Maximal activity occurred at 13-16 days; no activity was detectable in mature seeds. A comparison of endosperms from 10 different cultivars of barley harvested 11-22 days after flowering showed no correlation between enzyme activity and the amount of Xyl in the cell walls.

Human xylosyltransferase I (XT-I) is the initial enzyme involved in the biosynthesis of the glycosaminoglycan linker region in proteoglycans. Here, we tested the importance of the DXD motifs at positions 314-316 and 745-747 for enzyme activity and the nucleotide binding capacity of human XT-I. Mutations of the 314DED316 motif did not have any effect on enzyme activity, whereas alterations of the 745DWD747 motif resulted in reduced XT-I activity. Loss of function was observed after exchange of the highly conserved aspartic acid at position 745 with glycine. However, mutation of Asp745 to glutamic acid retained full enzyme activity, indicating the importance of an acidic amino acid at this position. Reduced substrate affinity was observed for mutants D747G (Km=6.9 microm) and D747E (Km=4.4 microm) in comparison with the wild-type enzyme (Km=0.9 microm). Changing the central tryptophan to a neutral, basic, or acidic amino acid resulted in a 6-fold lower Vmax, with Km values comparable with those of the wild-type enzyme. Despite the major effect of the DWD motif on XT-I activity, nucleotide binding was not abolished in the D745G and D747G mutants, as revealed by UDP-bead binding assays. Ki values for inhibition by UDP were determined to be 1.9-24.6 microm for the XT-I mutants. The properties of binding of XT-I to heparin-beads, the Ki constants for noncompetitive inhibition by heparin, and the activation by protamine were not altered by the generated mutations.

Human xylosyltransferase I (XT-I) catalyzes the transfer of xylose from UDP–xylose to consensus serine residues of proteoglycan core proteins. Expression of a soluble form of recombinant histidine-tagged XT-I (rXT-I-HIS) was accomplished at a high level with High Five /pCG255-1 insect cells in suspension culture. The recombinant protein was purified to homogeneity by a combination of heparin affinity chromatography and metal (Ni 2+ ) chelate affinity chromatography. Using the modern technique of perfusion chromatography, a rapid procedure for purification of the rXT-I-HIS from insect cell culture supernatant was developed. The purified, biologically active enzyme was homogeneous on SDS–PAGE, was detected with anti-XT-I-antibodies, and had the expected tryptic fragment mass spectrum. N-terminal amino acid sequencing demonstrated that the N-terminal signal sequence of the expressed protein was quantitatively cleaved. The total yield of the enzyme after purification was 18% and resulted in a specific XT-I activity of 7.9 mU/mg. The K m of the enzyme for recombinant [Val 36 ,Val 38 ] δ1 ,[Gly 92 ,Ile 94 ] δ2 bikunin was 0.8 μM. About 5 mg purified enzyme could be obtained from 1 L cell culture supernatant. The availability of substantial quantities of active, homogeneous enzyme will be of help in future biochemical and biophysical characterization of XT-I and for the development of a immunological XT-I assay.

In mutants defective in any of eight Caenorhabditis elegans sqv (squashed vulva) genes, the vulval extracellular space fails to expand during vulval morphogenesis. Strong sqv mutations result in maternal-effect lethality, caused in part by the failure of the progeny of homozygous mutants to initiate cytokinesis and associated with the failure to form an extracellular space between the egg and the eggshell. Recent studies have implicated glycosaminoglycans in these processes. Here we report the cloning and characterization of sqv-2 and sqv-6. sqv-6 encodes a protein similar to human xylosyltransferases. Transfection of sqv-6 restored xylosyltransferase activity to and rescued the glycosaminoglycan biosynthesis defect of a xylosyltransferase mutant hamster cell line. sqv-2 encodes a protein similar to human galactosyltransferase II. A recombinant SQV-2 fusion protein had galactosyltransferase II activity with substrate specificity similar to that of human galactosyltransferase II. We conclude that C. elegans SQV-6 and SQV-2 likely act in concert with other SQV proteins to catalyze the stepwise formation of the proteoglycan core protein linkage tetrasaccharide GlcAbeta1,3Galbeta1, 3Galbeta1,4Xylbeta-O-(Ser), which is common to the two major types of glycosaminoglycans in vertebrates, chondroitin and heparan sulfate. Our results strongly support a model in which C. elegans vulval morphogenesis and zygotic cytokinesis depend on the expression of glycosaminoglycans.

In mammals, two active xylosyltransferase isoenzymes (EC 2.4.2.16) exist. Both xylosyltransferases I and II (XT-I and XT-II) catalyze the transfer of xylose from UDP-xylose to select serine residues in the proteoglycan core protein. Altered XT activity in human serum was found to correlate directly with various diseases such as osteoarthritis, systemic sclerosis, liver fibrosis, and pseudoxanthoma elasticum. To interpret the significance of the enzyme activity alteration observed in disease states it is important to know which isoenzyme is responsible for the XT activity in serum. Until now it was impossible for a specific measurement of XT-I or XT-II activity, respectively, because of the absence of a suitable enzyme substrate. This issue has now been solved and the following experimental study demonstrates for the first time, via the enzyme activity that XT-II is the predominant isoenzyme responsible for XT activity in human serum. The proof was performed using natural UDP-xylose as the xylose donor, as well as the artificial compound UDP-4-azido-4-deoxyxylose, which is a selective xylose donor for XT-I.

Garden asparagus (Asparagus officinalis L.) is a commercially important crop species utilized for its excellent source of vitamins, minerals and dietary fiber. However, after harvest the tissue hardens and its quality rapidly deteriorates because spear cell walls become rigidified due to lignification and substantial increases in heteroxylan content. This latter observation prompted us to investigate the in vitro xylan xylosyltransferase (XylT) activity in asparagus. The current model system for studying heteroxylan biosynthesis, Arabidopsis, whilst a powerful genetic system, displays relatively low xylan XylT activity in in vitro microsomal preparations compared with garden asparagus therefore hampering our ability to study the molecular mechanism(s) of heteroxylan assembly. Here, we analyzed physiological and biochemical changes of garden asparagus spears stored at 4 °C after harvest and detected a high level of xylan XylT activity that accounts for this increased heteroxylan. The xylan XylT catalytic activity is at least thirteen-fold higher than that reported for previously published species, including Arabidopsis and grasses. A biochemical assay was optimized and up to seven successive Xyl residues were incorporated to extend the xylotetraose (Xyl4) acceptor backbone. To further elucidate the xylan biosynthesis mechanism, we used RNA-seq to generate an Asparagus reference transcriptome and identified five putative xylan biosynthetic genes (AoIRX9, AoIRX9-L, AoIRX10, AoIRX14_A, AoIRX14_B) with AoIRX9 having an expression profile that is distinct from the other genes. We propose that Asparagus provides an ideal biochemical system to investigate the biochemical aspects of heteroxylan biosynthesis and also offers the additional benefit of being able to study the lignification process during plant stem maturation.

Microsomal membranes catalyze the formation of xyloglucan from UDP-Glc and UDP-Xyl by cooperative action of α-xylosyltransferase and β-glucan synthase activities. Here we report that etiolated pea microsomes contain an α-xylosyltransferase that catalyzes the transfer of xylose from UDP-[ 14 C]xylose onto β(1,4)-linked glucan chains. The solubilized enzyme had the capacity to transfer xylosyl residues onto cello-oligosaccharides having 5 or more glucose residues. Analysis of the data from these biochemical assays led to the identification of a group of Arabidopsis genes and the hypothesis that one or more members of this group may encode α-xylosyltransferases involved in xyloglucan biosynthesis. To evaluate this hypothesis, the candidate genes were expressed in Pichia pastoris and their activities measured with the biochemical assay described above. One of the candidate genes showed cello-oligosaccharide-dependent xylosyltransferase activity. Characterization of the radiolabeled products obtained with cellopentaose as acceptor indicated that the pea and the Arabidopsis enzymes transfer the 14 C-labeled xylose mainly to the second glucose residue from the nonreducing end. Enzymatic digestion of these radiolabeled products produced results that would be expected if the xylose was attached in an α(1,6)-linkage to the glucan chain. We conclude that this Arabidopsis gene encodes an α-xylosyltransferase activity involved in xyloglucan biosynthesis.

Wood, derived from plant secondary growth, is a commercially important material. Both cellulose and lignin assembly have been well studied during wood formation (xylogenesis), but heteroxylan biosynthesis is less well defined. Elucidation of the heteroxylan biosynthetic pathway is crucial to understand the mechanism of wood formation. Here, we use Neolamarckia cadamba, a fast-growing tropical tree, as a sample to analyze heteroxylan formation at the biochemical and molecular levels during wood formation. Analysis of the non-cellulosic polysaccharides isolated from N. cadamba stems shows that heteroxylans dominate non-cellulosic polysaccharides and increase with xylogenesis. Microsomes isolated from stems of 1-year-old N. cadamba exhibited UDP-Xyl synthase and xylosyltransferase activities with the highest activity present in the middle and basal stem regions. To further understand the genetic basis of heteroxylan synthesis, RNA sequencing (RNA-seq) was used to generate transcriptomes of N. cadamba during xylogenesis. The RNA-seq results showed that genes related to heteroxylan synthesis had higher expression levels in the middle and basal part of the stem compared to the apical part. Our results describe the heteroxylan distribution and heteroxylan synthesis trait in N. cadamba and give a new example for understanding the mechanism of heteroxylan synthesis in tropical tree species in future.

O-xylosyltransferase (Oxt) is a Golgi transmembrane glycosyltransferase that initiates the first step in the heperan and chrondroitin sulfate biosynthesis on core proteoglycans (HSPG and CSPG). HSP...

Several lines of evidence support the hypothesis of the involvement of altered proteoglycan deposition in the development of lung diseases. UDP-d-xylose: core protein β-d-xylosyltransferase (UDP-xylosyltransferase; EC 2.4.2.26) is a key enzyme for the glycosylation of proteoglycan core proteins. This study examined the catalytic activity of UDP-xylosyltransferase in lung tissue and in isolated fibroblasts, as well as the deposition of the proteoglycans versican, biglycan and decorin in rat lung tissue during bleomycin-induced lung injury.Rats were given, endotracheally, a single dose of bleomycin. Deposition of proteoglycans in lung tissue was assessed by immunohistochemistry and the catalytic activity of xylosyltransferase was determined with an acceptor peptide of the sequence Q-E-E-E-G-S-G-G-G-Q-G-G as a substrate.The results show coincidence of increasing xylosyltransferase activities in lung tissue with accumulation of versican at alveolar entrance rings and in fibrotic regions in close proximity to α-smooth muscle actin-positive cells. In contrast, no changes in biglycan and decorin deposition in fibrotic lungs were observed, except for decorin in alveolar type II pneumocytes and alveolar macrophages. Bleomycin treatment of isolated rat lung fibroblasts resulted in a concentration-dependent increase of xylosyltransferase activity up to 2 mU bleomycin·mL−1.The data suggest a participation of myofibroblasts with increased xylosyltransferase activities in accumulation of versican in fibrotic foci of injured lung tissue at the early stages of development of lung fibrosis.

A particulate preparation from 6-day-old seedlings of wheat (Triticum aestivum L.) was found to contain a xylosyltransferase (XylTase) which incorporated xylose (Xyl) from UDP-xylose into exogenous beta-(1--4)-xylooligosaccharides with 2-aminopyridine-derivatized reducing end groups. High-performance liquid chromatographic analysis showed that the chain elongation of pyridylaminated beta-(1--4)-xylotriose (Xyl3-PA) occurred by attachment of a series of one, two, or three xylosyl residues, depending on substrate concentrations and reaction times. Methylation analysis and beta-xylosidase digestion of the newly synthesized Xyl4-PA confirmed that the xylosyl residues were incorporated through beta-(1--4)-linkages. The enzyme was maximally active at pH 6.8 and 20 degrees C, and required Triton X-100, which enhanced activity 5-fold at a concentration of 0.05-2%. Divalent ions, including Mn2+ and Mg2+, did not affect activity. Enzyme activity increased with increasing polymerization of xylosyl residues of the acceptor substrates: for instance, Xyl5-PA was almost 7 times as efficient as Xyl2-PA. The apparent Michaelis constants of the enzyme for Xyl3-PA and UDP-xylose were 13.5 and 7.9 mM, respectively. The enzyme also catalyzed incorporation of radioactive sugars (Xyl together with a small portion of L-arabinose) from UDP-[14C]xylose into higher beta-(1--4)-xylooligosaccharides (degree of polymerization7) with or without (4-O-methyl-)glucuronosyl side chains at activities comparable to those observed for pyridylaminated xylooligosaccharides, and into several heteroxylans but with much lower efficiency. Enzymatic hydrolysis of the product with a beta-xylanase degraded it into mainly xylobiose, providing further evidence that the xylosyl residues are incorporated through beta-(1--4)-linkages.

UDP-xylosyltransferase (UDP-D-xylose:proteoglycan core protein beta-D-xylosyltransferase EC 2.4.2.26) initiates the formation of chondroitin sulfate in the course of proteoglycan biosynthesis. The enzyme catalyzes the transfer of D-xylose from UDP-D-xylose to specific serine residues in the core protein. A procedure for purification of xylosyltransferase from rat ear cartilage was developed which includes ammonium sulfate fractionation, chromatography on heparin-agarose, on Sephacryl S300 and finally a substrate affinity chromatography applying the dodeca peptide Q-E-E-E-G-S-G-G-G-Q-G-G. The specific activity of the purified enzyme was about 420 mU per mg protein. The purification factor was about 26.000 with 27% yield. In SDS-polyacrylamide gel electrophoresis, the highly purified enzyme is homogeneous and yields only a single distinct band of 78 kDa. An apparent molecular mass of 71 kDa was determined for the native enzyme. These data suggest a monomeric structure for the enzyme. Xylosyltransferase activity was found to depend essentially on the presence of divalent metal ions. The K(m) value for UDP-D-xylose was determined to 6.5 micromol/l and for the dodeca peptide Q-E-E-E-G-S-G-G-G-Q-G-G as xylose acceptor to 8 micromol/l.

The transfer of xylose from UDP-xylose to the core β-linked mannose of N-linked oligosaccharides by β1,2-xylosyltransferase (XylT) is a widespread feature of plant glycoproteins which renders them immunogenic and allergenic in man. Here, we report the isolation of the Arabidopsis thaliana XylT gene, which contains two introns and encodes a 60.2 kDa protein with a predicted type II transmembrane protein topology typical for Golgi glycosyltransferases. Upon expression of A. thaliana XylT cDNA in the baculovirus/insect cell system, a recombinant protein was produced that exhibited XylT activity in vitro. Furthermore, the recombinant enzyme displayed XylT activity in vivo in the insect cells, as judged by the acquired cross-reaction of cellular glycoproteins with antibodies against the β1,2-xylose epitope. The cloned XylT cDNA as well as the recombinant enzyme are essential tools to study the role of β1,2-xylose in the immunogenicity and allergenicity of plant glycoproteins at the molecular level.

Zeatin is the most active and ubiquitous form of the naturally occurring cytokinins. Glycosyl conjugates of zeatin are found in many plant tissues and are considered important for storage and protection against degradative enzymes. Two enzymes catalyzing the formation of O-glycosyl derivatives of zeatin have been characterized,O-glucosyltransferase andO-xylosyltransferase, occurring in seeds of lima bean (Phaseolus lunatus) and bean (Phaseolus vulgaris), respectively. Recently, the ZOG1 gene (zeatinO-glucosyltansferase) was isolated from P. lunatis (Martin et al., 1999). Based on the ZOG1 sequence, the ZOX1 gene (zeatinO-xylosyltransferase) was cloned from P. vulgaris. ZOX1contains an open reading frame of 1362 bp that codes for a 454-amino acid peptide of 51 kD. The recombinant protein has properties identical to the native enzyme: it catalyzes O-xylosylzeatin formation with UDP-Xyl as a glycosyl donor but does not recognize UDP-Glucose as a substrate. The ZOX1 andZOG1 genes exhibit 93% identity at the nucleotide level and 90% similarity at the amino acid level. Neither gene contains introns. These zeatin-specific genes and their promoters will be useful for studies of the regulation of active versus storage forms of cytokinins. Comparison of sequences encoding similar enzymes with distinct substrate specificity may lead to identification of epitopes specific to cytokinin and glycosyl donor molecules.

We have developed a rapid, high throughput screening assay for compounds that alter the assembly of glycosaminoglycan chains in Chinese hamster ovary cells. The assay uses autoradiography to measure the binding of newly synthesized [35S]proteoglycans and [35S]glycosaminoglycans to a positively charged membrane. Screening over 1000 extracts from a random plant collection obtained from the Amazon rain forest yielded five plants that stimulated glycosaminoglycan assembly in both wild-type cells and a mutant cell line defective in xylosyltransferase (the first committed enzyme involved in glycosaminoglycan biosynthesis). Fractionation of an extract of Maieta guianensis by silica gel and reverse-phase chromatography yielded two pure compounds with stimulatory activity. Spectroscopic analysis by NMR and mass spectrometry revealed that the active principles were xylosides of dimethylated ellagic acid. One of the compounds also contained a galloyl group at C-3 of the xylose moiety. These findings suggest that plants and other natural products may be a source of agents that can potentially alter glycosaminoglycan and proteoglycan formation in animal cells.

Acceptor affinities of UDP-D-xylose:proteoglycan core protein beta-D-xylosyltransferase (XT) recognition signals in synthetic L-APP and L-APLP2 homologous peptides were determined. The Michaelis-Menten constants (KM) of the L-APP peptide TENEGSGLTNIK and the L-APLP2 peptide SENEGSGMAEQK were 20.1 and 18.9 microM, respectively. Therefore, the peptides proved to be as good acceptors for XT as the bikunin aminoterminus homologous peptide (KM = 22 microM). Due to the occurrence of L-APP and L-APLP2 transcripts in human brain tissue, XT activity was measured in human liquor cerebrospinalis. Mean values were calculated as 0.22 mU/L in males and 0.47 mU/L in females without disturbance of blood brain barrier. In addition, in homogenized rat brain tissue a mean XT activity of 0.75 mU/L was determined. Furthermore, XT activity was investigated in 21 different human cell lines. In 7 cell lines an enzyme activity was not detected in either extracellular space or cytoplasma. Our findings indicate that XT is not ubiquitously expressed in human cell types.

Production of pharmaceutical glycoproteins, such as therapeutic antibodies and cytokines, in plants has many advantages in safety and reduced costs. However, plant-made glycoproteins have N -glycans with plant-specific sugar residues (core β-1,2-xylose and α-1,3-fucose) and a Lewis a (Le a ) epitope, Galβ(1–3)[Fucα(1–4)]GlcNAc. Because it is likely that these sugar residues and glycan structures are immunogenic, many attempts have been made to delete them. Previously, we reported the simultaneous deletion of the plant-specific core α-1,3-fucose and α-1,4-fucose residues in Le a epitopes by repressing the GDP- d -mannose 4,6-dehydratase ( GMD ) gene, which is associated with GDP- l -fucose biosynthesis, in Nicotiana benthamiana plants (rGMD plants, renamed to ΔGMD plants) (Matsuo and Matsumura, Plant Biotechnol. J., 9, 264–281, 2011). In the present study, we generated a core β-1,2-xylose residue-repressed transgenic N. benthamiana plant by co-suppression of β-1,2-xylosyltransferase (ΔXylT plant). By crossing ΔGMD and ΔXylT plants, we successfully generated plants in which plant-specific sugar residues were repressed (ΔGMDΔXylT plants). The proportion of N -glycans with deleted plant-specific sugar residues found in total soluble protein from ΔGMDΔXylT plants increased by 82.41%. Recombinant mouse granulocyte/macrophage-colony stimulating factor (mGM-CSF) and human monoclonal immunoglobulin G (hIgG) harboring N -glycans with deleted plant-specific sugar residues were successfully produced in ΔGMDΔXylT plants. Simultaneous repression of the GMD and XylT genes in N. benthamiana is thus very useful for deleting plant-specific sugar residues.

Zeatin is rapidly metabolized to O-xylosylzeatin in Phaseolus vulgaris seeds. The zeatin O-xylosyltransferase mediating this conversion, a 50 kDa protein, occurs mainly in the endosperm, both in the cytoplasm and the nuclei. A monoclonal antibody specific to the enzyme was used to isolate cDNAs from an expression library derived from P. vulgaris seeds. Two highly homologous, full-length cDNAs were isolated. The ORFs encode proteins of 69 and 67 kDa, respectively, with 90% homology at the amino acid level. cDNA-encoded protein obtained from in vitro transcription/translation was processed to protein of 50 kDa by bean endosperm extract. Transgenic tobacco plants harboring the larger ORF under the control of the CaMV35S promoter were more sensitive to the auxin NAA than control plants. The symptoms included leaf chlorosis, restriction of root elongation, and eventual cessation of growth. The antigenic preprotein was processed, and labeled zeatin was converted to O-xylosylzeatin in transgenic plants grown on NAA-containing medium. Analyses of independently transformed families indicated that the presence of the transgene coincided with the increased auxin sensitivity and protein processing correlated with the manifestation of auxin-induced damage. These results suggest that posttranslational processing regulates enzyme activity, and offer the possibility that cytokinin-auxin balance may be affected by stimulation of cytokinin metabolic enzyme activity by auxin.

HepG2 cells were examined for the presence of UDP-D-xylose: beta-D-glucoside alpha-1,3-xylosyltransferase activity by using 2-[(2-pyridyl)amino]ethyl beta-D-glucopyranoside (Glc beta-R) as a fluorogenic acceptor. UDP-D-xylose and the acceptor were incubated with the HepG2 microsomal fraction, and the enzymatic reaction mixture was analyzed by HPLC. Structural analysis of the fluorogenic product by alpha-D-xylosidase digestion, FAB-MS, and Smith degradation revealed that it was Xyl alpha 1-3Glc beta-R, thus demonstrating the existence of beta-D-glucoside alpha-1,3-xylosyltransferase. A solubilization study of the enzyme from the microsomal fraction indicated that it differed from UDP-D-xylose: alpha-D-xylodie alpha-1,3-xylosyltransferase of the HepG2 microsomal fraction producing Xyl alpha 1-3Xyl alpha 1-3Glc-R' [R', (2-pyridyl)amino-] from UDP-D-xylose and Xyl alpha 1-3Glc-R' [Minamida, S., Aoki, K., Natsuka, S., Omichi, K., Fukase, K., Kusumoto, S. & Hase, S. (1996) J. Biochem. (Tokyo) 120, 1002-1006]. The newly detected enzyme appears to be involved in the biosynthesis of the Xyl alpha 1-3Glc beta-Ser structure of glycoproteins such as human blood coagulation factors VII and IX.

Membranes from etiolated pea epicotyls were fractionated by discontinuous sucrose-density-gradient centrifugation into endoplasmic reticulum, Golgi apparatus and plasma membrane. The Golgi apparatus was further fractionated on a shallow, continuous sucrose density gradient into Golgi subtractions of low, medium and high density. Xylosyl- and glucuronyltransferases were both found to be concentrated in the Golgi apparatus. The glucuronyltransferase was concentrated in the low-density Golgi membranes, but xylosyltransferases were found in all three Golgi subfractions. The multiple location of xylosyltransferases within the Golgi was found in both younger and older regions of the epicotyl, and xylan was the major product of the xylosyltransferase in the low- and medium-density subfractions. In the presence of UDPglucose, xylose was also incorporated into xyloglucan, but this activity was concentrated in the highdensity Golgi membranes.

Objectives: Serum xylosyltransferase I (XT-I) is a marker for the determination of tissue remodeling in systemic sclerosis. Here, we investigated whether renal insufficiency affects XT-I levels in blood. Methods: We measured serum XT-I activity in 236 patients with different serum creatinine levels. Results: XT-I activities in cohorts with increased creatinine levels were not significantly altered compared to controls. Conclusions: Serum XT-I activity is applicable as a fibrosis marker independent from renal function.

Glycine–serine (GS) linkers are commonly used in recombinant proteins to connect domains. Here, we report the posttranslational O-glycosylation of a GS linker in a novel fusion protein. The structure of the O-glycan moiety is a xylose-based core substituted with hexose and sulfated hexauronic acid residues. The total level of O-xylosylation was approximately 30% in the material expressed in HEK-293 cell lines. There was an approximate 10-fold reduction in O-xylosylation levels when the material was expressed in Chinese hamster ovary cell lines. Similar O-glycan structures have been reported for human urinary thrombomodulin and represent the initial building block for proteoglycans such as chondroitin sulfate and heparin. The sites of attachment, determined by electron transfer dissociation mass spectrometry, were localized to serine in the linker regions of the recombinant fusion protein. This attachment could be attributed, in part, to the inherent xylosyltransferase motif present in GS linkers. Elimination of the O-glycan moiety was achieved with modified linkers containing only glycine residues. The aggregation and fragmentation behavior of the GGG construct were comparable to the GSG-linked material during thermal stress. The O-xylosylation reported has implications for the manufacturing consistency of recombinant proteins containing GS linkers. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:3920–3924, 2013

Xylan is the second most abundant polysaccharide on Earth, and represents a major component of both dicot wood and the cell walls of grasses. Much knowledge has been gained from studies of xylan biosynthesis in the model plant, Arabidopsis. In particular, the irregular xylem (irx) mutants, named for their collapsed xylem cells, have been essential in gaining a greater understanding of the genes involved in xylan biosynthesis. In contrast, xylan biosynthesis in grass cell walls is poorly understood. We identified three rice genes Os07g49370 (OsIRX9), Os01g48440 (OsIRX9L), and Os06g47340 (OsIRX14), from glycosyltransferase family 43 as putative orthologs to the putative β-1,4-xylan backbone elongating Arabidopsis IRX9, IRX9L, and IRX14 genes, respectively. We demonstrate that the over-expression of the closely related rice genes, in full or partly complement the two well-characterized Arabidopsis irregular xylem (irx) mutants: irx9 and irx14. Complementation was assessed by measuring dwarfed phenotypes, irregular xylem cells in stem cross sections, xylose content of stems, xylosyltransferase (XylT) activity of stems, and stem strength. The expression of OsIRX9 in the irx9 mutant resulted in XylT activity of stems that was over double that of wild type plants, and the stem strength of this line increased to 124% above that of wild type. Taken together, our results suggest that OsIRX9/OsIRX9L, and OsIRX14, have similar functions to the Arabidopsis IRX9 and IRX14 genes, respectively. Furthermore, our expression data indicate that OsIRX9 and OsIRX9L may function in building the xylan backbone in the secondary and primary cell walls, respectively. Our results provide insight into xylan biosynthesis in rice and how expression of a xylan synthesis gene may be modified to increase stem strength. © 2013 Chiniquy, Varanasi, Oh, Harholt, Katnelson, Singh, Auer, Simmons, Adams, Scheller and Ronald.

特徴のあるコンドロイチン硫酸グリコサミノグリカン鎖の合成は、受容体コアタンパク質の、あるセリン残基の水酸基へUDP-キシロースからキシロースが転移されることから始まり、その反応はキシロース転移酵素により触媒される。キシロース付加がプロテオグリカン生合成の特異的な制御機構の重要なポイントであることが、いくつかの方面の証拠により示唆される: コアタンパク質修飾の最初のステップとしてのその反応の位置; コアタンパク質とキシロース転移酵素の互いに調和する発現; キシロース転移酵素と、合成経路においてその次の反応を担う酵素であるガラクトース転移酵素Iとの特異的な相互作用; 生成されたUDP-キシロース生産のフィードバック阻害; そして、コアタンパク質の合成とグリコサミノグリカン鎖の残りの部分の付加反応の間に細胞内の区画を移動することの必要性。本総説では、キシロース転移酵素の性質、作用の仕方、細胞内小器官での局在、及びそれが触媒する反応を中心に述べる。

LARGE-dependent modification enables α-dystroglycan (α-DG) to bind to its extracellular matrix ligands. Mutations in the LARGE gene and several others involved in O-mannosyl glycan synthesis have been identified in congenital and limb-girdle muscular dystrophies that are characterized by perturbed glycosylation and reduced ligand-binding affinity of α-DG. LARGE is a bifunctional glycosyltransferase that alternately transfers xylose and glucuronic acid, thereby generating the heteropolysaccharides on α-DG that confer its ligand binding. Although the LARGE paralog LARGE2 (also referred to as GYLTL1B) has likewise been shown to enhance the functional modification of α-DG in cultured cells, its enzymatic activities have not been identified. Here, we report that LARGE2 is also a bifunctional glycosyltransferase and compare its properties with those of LARGE. By means of a high-performance liquid chromatography-based enzymatic assay, we demonstrate that like LARGE, LARGE2 has xylosyltransferase (Xyl-T) and glucuronyltransferase (GlcA-T) activities, as well as polymerizing activity. Notably, however, the pH optima of the Xyl-T and GlcA-T of LARGE2 are distinct from one another and also from those of LARGE. Our results suggest that LARGE and LARGE2 catalyze the same glycosylation reactions for the functional modification of α-DG, but that they have different biochemical properties.

Triterpene saponins are a diverse group of biologically functional products in plants. Saponins usually are glycosylated, which gives rise to a wide diversity of structures and functions. In the group A saponins of soybean (Glycine max), differences in the terminal sugar species located on the C-22 sugar chain of an aglycone core, soyasapogenol A, were observed to be under genetic control. Further genetic analyses and mapping revealed that the structural diversity of glycosylation was determined by multiple alleles of a single locus, Sg-1, and led to identification of a UDP-sugar-dependent glycosyltransferase gene (Glyma07g38460). Although their sequences are highly similar and both glycosylate the nonacetylated saponin A0-αg, the Sg-1(a) allele encodes the xylosyltransferase UGT73F4, whereas Sg-1(b) encodes the glucosyltransferase UGT73F2. Homology models and site-directed mutagenesis analyses showed that Ser-138 in Sg-1(a) and Gly-138 in Sg-1(b) proteins are crucial residues for their respective sugar donor specificities. Transgenic complementation tests followed by recombinant enzyme assays in vitro demonstrated that sg-1(0) is a loss-of-function allele of Sg-1. Considering that the terminal sugar species in the group A saponins are responsible for the strong bitterness and astringent aftertastes of soybean seeds, our findings herein provide useful tools to improve commercial properties of soybean products.