Iben Damager

Arabidopsis thaliana RGXT1 and RGXT2 Encode Golgi-Localized (1,3)-α-d-Xylosyltransferases Involved in the Synthesis of Pectic Rhamnogalacturonan-II

Two homologous plant-specific Arabidopsis thaliana genes, RGXT1 and RGXT2, belong to a new family of glycosyltransferases (CAZy GT-family-77) and encode cell wall (1,3)-α-d-xylosyltransferases. The deduced amino acid sequences contain single transmembrane domains near the N terminus, indicative of a type II membrane protein structure. Soluble secreted forms of the corresponding proteins expressed in insect cells showed xylosyltransferase activity, transferring d-xylose from UDP-α-d-xylose to l-fucose. The disaccharide product was hydrolyzed by α-xylosidase, whereas no reaction was catalyzed by β-xylosidase. Furthermore, the regio- and stereochemistry of the methyl xylosyl-fucoside was determined by nuclear magnetic resonance to be an α-(1,3) linkage, demonstrating the isolated glycosyltransferases to be (1,3)-α-d-xylosyltransferases. This particular linkage is only known in rhamnogalacturonan-II, a complex polysaccharide essential to vascular plants, and is conserved across higher plant families. Rhamnogalacturonan-II isolated from both RGXT1 and RGXT2 T-DNA insertional mutants functioned as specific acceptor molecules in the xylosyltransferase assay. Expression of RGXT1- and RGXT2-enhanced green fluorescent protein constructs in Arabidopsis revealed that both fusion proteins were targeted to a Brefeldin A–sensitive compartment and also colocalized with the Golgi marker dye BODIPY TR ceramide, consistent with targeting to the Golgi apparatus. Taken together, these results suggest that RGXT1 and RGXT2 encode Golgi-localized (1,3)-α-d-xylosyltransferases involved in the biosynthesis of pectic rhamnogalacturonan-II.

First Principles Insight into the α-Glucan Structures of Starch: Their Synthesis, Conformation, and Hydration

Carbohydrates constitute the most abundant group of organic compounds found in nature. Oxygenic photosynthesis, the process energizing carbon dioxide fixation in the biosphere, is estimated to 1011 tons of dry weight biomass per year, most of it being carbohydrate.(1) For human consumption, the abundance of starch and the possibility to carry out large-scale purification, derivatization and processing provide unique and straightforward options to design starch crops harboring new valuable functionalities offering diversified uses in the food and nonfood sectors.2,3 These include raw materials for the design of advanced and healthy foods to combat obesity and other lifestyle-related diseases(4) or to replace gelatin.(5) Today, starch constitutes a major raw material in the bioethanol production6,7 and in the future starch is expected to play an important role in providing resources for the increasing demand for CO2-neutral energy. The global annual starch production by man approximates 3000 million tons and the industrial production of pure, refined starch now exceeds 60 million tons.(8) n nThe simple and compact structure of starch and its human analogue glycogen has proven to be very successful for providing energy to living organisms and as energy storage reservoirs in biological systems. The metabolism and architecture of these two polymers are highly dependent on the presence of water. Understanding of the detailed structure and molecular models of complex α-glucans in an aqueous environment would be useful tools in the attempt to provide science-based recommendations in our efforts to build a bio-based society where starches play a major role as bulk polymers. Advances within these areas are dependent on the availability of complex α-glucans of defined chemical structures that mimic the key features of starch and other complex α-glucans and thus offer the opportunity to gain detailed knowledge of the molecular structure of hydrated starch and α-glucan systems. This review provides an overview of this rapidly expanding and challenging field of research with main focus on starch structure and hydration. n nStarch9−11 and glycogen12−14 are synthesized by sets of specific enzyme activities that directly determine their molecular structures and physical properties. The extent of crystallinity, aggregation and hydration is of fundamental importance for starch and its human analogue glycogen. Starch is deposited in the plant as a stable form in highly organized, semicrystalline granules15,16 (Figure u200b(Figure1)1) having specific crystalline polymorphs (Figure u200b(Figure2)2) as determined by powder X-ray crystallography.(17) Glycogen is not crystalline, but the importance of correctly structured glycogen granules12−14 can be exemplified by the occurrence of specific Mendelian inherited glycogen-dependent disorders,(18) such as the epileptic Lafora disease(19) or the Cori disease.(20) These two diseases are characterized by deposition of aberrant “starch-like” glycogen structures resulting in the inability to properly store and mobilize deposited glycogen. n n n n n n nOpen in a separate window n n nFigure 1 n n nPrinciple of the “top-down” strategy of starch analysis. (A) A cross section of a wheat starch granule (Confocal microscopic image by Mikkel A. Glaring). (B) A schematic drawing of the layered structure of amylopectin. Alternating amorphous and crystalline lamellae are repeated with 9 nm spacing

Functional characterisation of a putative rhamnogalacturonan II specific xylosyltransferase

An Arabidopsis thaliana gene, At1g56550, was expressed in Pichia pastoris and the recombinant protein was shown to catalyse transfer of d‐xylose from UDP‐α‐d‐xylose onto methyl α‐l‐fucoside. The product formed was shown by 1D and 2D 1H NMR spectroscopy to be Me α‐d‐Xyl‐(1,3)‐α‐l‐Fuc, which is identical to the proposed target structure in the A‐chain of rhamnogalacturonan II. Chemically synthesized methyl l‐fucosides derivatized by methyl groups on either the 2‐, 3‐ or 4 position were tested as acceptor substrates but only methyl 4‐O‐methyl‐α‐l‐fucopyranoside acted as an acceptor, although to a lesser extent than methyl α‐l‐fucoside. At1g56550 is suggested to encode a rhamnogalacturonan II specific xylosyltransferase.

Residue Specific Hydration of Primary Cell Wall Potato Pectin Identified by Solid-State 13C Single-Pulse MAS and CP/MAS NMR Spectroscopy

Hydration of rhamnogalacturonan-I (RG-I) derived from potato cell wall was analyzed by (13)C single-pulse (SP) magic-angle-spinning (MAS) and (13)C cross-polarization (CP) MAS nuclear magnetic resonance (NMR) and supported by (2)H SP/MAS NMR experiments. The study shows that the arabinan side chains hydrate more readily than the galactan side chains and suggests that the overall hydration properties can be controlled by modifying the ratio of these side chains. Enzymatic modification of native (NA) RG-I provided samples with reduced content of arabinan (sample DA), galactan (sample DG), or both side chains (sample DB). Results of these samples suggested that hydration properties were determined by the length and character of the side chains. NA and DA exhibited similar hydration characteristics, whereas DG and DB were difficult to hydrate because of the less hydrophilic properties of the rhamnose-galacturonic acid (Rha-GalA) backbone in RG-I. Potential food ingredient uses of RG-I by tailoring of its structure are discussed.

Chemical synthesis of 6‴-α-maltotriosyl-maltohexaose as substrate for enzymes in starch biosynthesis and degradation

A branched nonasaccharide 6-alpha-maltotriosyl-maltohexaose was synthesised in 40 steps from D-glucose and maltose. Phenyl O-(2,3,4,6-tetra-O-benzyl-alpha-D-glucopyranosyl)-(1-->4)-O- (2,3,6-tri-O-benzyl-alpha-D-glucopyranosyl)-(1-->4)-2,3-di-O-benzyl-1-th io- beta-D-glucopyranoside and O-(2,3,4,6-tetra-O-benzyl-alpha-D-glucopyranosyl)-(1-->4)-O-(2,3,6-tri- O-benzyl-alpha-D-glucopyranosyl)-(1-->4)-2,3,6-tri-O-benzyl-alpha, beta-D-glucopyranosyl trichloroacetimidate were coupled by a general condensation reaction to form the per-O-benzylated branched hexasaccharide phenyl thioglycoside. The phenylthio group of this compound was converted into a trichloroacetimidate, which was coupled with phenyl O-(2,3,6-tri-O-benzyl-alpha-D-glucopyranosyl)-(1-->4)-O-(2,3,6-tri-O- benzyl-alpha-D-glucopyranosyl)-(1-->4)-2,3,6-tri-O-benzyl-1-thio-beta-D- glucopyranoside to afford the per-O-benzylated branched nonasaccharide phenyl thioglycoside. Replacement of the phenylthio group with a free OH-group followed by hydrogenolysis gave the desired product. The synthons reported for this synthesis constitute a versatile tool for the chemical synthesis of other complex carbohydrates.

Chemical Synthesis of a Dual Branched Malto-Decaose: A Potential Substrate for α-Amylases

A convergent block strategy for general use in efficient synthesis of complex α‐(1→4)‐ and α‐(1→6)‐malto‐oligosaccharides is demonstrated with the first chemical synthesis of a malto‐oligosaccharide, the decasaccharide 6,6′′′′‐bis(α‐maltosyl)‐maltohexaose, with two branch points. Using this chemically defined branched oligosaccharide as a substrate, the cleavage pattern of seven different α‐amylases were investigated. α‐Amylases from human saliva, porcine pancreas, barley α‐amylase 2 and recombinant barley α‐amylase 1 all hydrolysed the decasaccharide selectively. This resulted in a branched hexasaccharide and a branched tetrasaccharide. α‐Amylases from Asperagillus oryzae, Bacillus licheniformis and Bacillus sp. cleaved the decasaccharide at two distinct sites, either producing two branched pentasaccharides, or a branched hexasaccharide and a branched tetrasaccharide. In addition, the enzymes were tested on the single‐branched octasaccharide 6‐α‐maltosyl‐maltohexaose, which was prepared from 6,6′′′′‐bis(α‐maltosyl)‐maltohexaose by treatment with malt limit dextrinase. A similar cleavage pattern to that found for the corresponding linear malto‐oligosaccharide substrate was observed.

Chemical synthesis of methyl 6′-α-maltosyl-α-maltotrioside and its use for investigation of the action of starch synthase II

Abstract The branched pentasaccharide methyl 6′-α-maltosyl-α-maltotrioside was chemically synthesised and investigated as a primer for particulate starch synthase II (SSII) using starch granules prepared from the low-amylose pea mutant lam as the enzyme source. For chemical synthesis, the trichloroacetimidate activation method was used to synthesise methyl O -(2,3,4,6-tetra- O -benzyl-α- d -glucopyranosyl)-(1→4)- O -(2,3,6-tri- O -benzyl-α- d -glucopyranosyl)-(1→6)- O -[(2,3,4,6-tetra- O -benzyl-α- d -glucopyranosyl-(1→4)]- O -(2,3-di- O -benzyl-α- d -glucopyranosyl)-(1→4)-2,3,6-tri- O -benzyl-α- d -glucopyranoside, which was then debenzylated to provide the desired branched pentasaccharide methyl 6′-α-maltosyl-α-maltotrioside as documented by 1 H and 13 C NMR spectroscopy. Using a large excess of the maltoside, the pentasaccharide was tested as a substrate for starch synthase II (SSII). Both of the non-reducing ends of methyl 6′-α-maltosyl-α-maltotrioside were extended equally resulting in two hexasaccharide products in nearly equal amounts. Thus, SSII catalyses an equimolar and non-processive elongation reaction of this substrate. Accordingly, the presence of the α-1,6 linkages does not dictate a specific structure of the pentasaccharide in which only one of the two non-reducing ends are available for extension.

Effect of nanocoating with rhamnogalacturonan-I on surface properties and osteoblasts response.

Long-term stability of titanium implants are dependent on a variety of factors. Nanocoating with organic molecules is one of the methods used to improve osseointegration. Therefore, the aim of this study is to evaluate the in vitro effect of nanocoating with pectic rhamnogalacturonan-I (RG-I) on surface properties and osteoblasts response. Three different RG-Is from apple and lupin pectins were modified and coated on amino-functionalized tissue culture polystyrene plates (aminated TCPS). Surface properties were evaluated by scanning electron microscopy, contact angle measurement, atomic force microscopy, and X-ray photoelectron spectroscopy. The effects of nanocoating on proliferation, matrix formation and mineralization, and expression of genes (real-time PCR) related to osteoblast differentiation and activity were tested using human osteoblast-like SaOS-2 cells. It was shown that RG-I coatings affected the surface properties. All three RG-I induced bone matrix formation and mineralization, which was also supported by the finding that gene expression levels of alkaline phosphatase, osteocalcin, and collagen type-1 were increased in cells cultured on the RG-I coated surface, indicating a more differentiated osteoblastic phenotype. This makes RG-I coating a promising and novel candidate for nanocoatings of implants.

Osteoblastic response to pectin nanocoating on titanium surfaces

Osseointegration of titanium implants can be improved by organic and inorganic nanocoating of the surface. The aim of our study was to evaluate the effect of organic nanocoating of titanium surface with unmodified and modified pectin Rhamnogalacturonan-Is (RG-Is) isolated from potato and apple with respect to surface properties and osteogenic response in osteoblastic cells. Nanocoatings on titanium surfaces were evaluated by scanning electron microscopy, contact angle measurements, atomic force microscopy, and X-ray photoelectron spectroscopy. The effect of coated RG-Is on cell adhesion, cell viability, bone matrix formation and mineralization was tested using SaOS-2 cells. Nanocoating with pectin RG-Is affected surface properties and in consequence changed the environment for cellular response. The cells cultured on surfaces coated with RG-Is from potato with high content of linear 1.4-linked galactose produced higher level of mineralized matrix compared with control surfaces and surfaces coated with RG-I with low content of linear 1.4-linked galactose. The study showed that the pectin RG-Is nanocoating not only changed chemical and physical titanium surface properties, but also specific coating with RG-Is containing high amount of galactan increased mineralized matrix formation of osteoblastic cells in vitro.

Affecting osteoblastic responses with in vivo engineered potato pectin fragments

Pectins, complex plant-derived polysaccharides, are novel candidates for biomaterial nanocoatings. Pectic rhamnogalacturonan-I regions (RG-I) can be enzymatically treated to so-called modified hairy regions (MHR). We surveyed the growth and differentiation of murine preosteoblastic MC3T3-E1 cells on Petri dishes coated with RG-Is from native or genetically engineered potato tubers. Uncoated tissue culture polystyrene (TCPS) and aminated (AMI) dishes served as controls. MHRPTR_GAL sample was depleted of galactose (9 mol % galactose; 23 mol % arabinose) and MHRPTR_ARA of arabinose (61 mol % galactose; 6 mol % arabinose). Wild-type (modified hairy region from potato pectin (MHRP)_WT) fragment contained default amounts (58 mol % galactose; 13 mol % arabinose) of both sugars. Focal adhesions (FAs) indicating cellular attachment were quantified. Reverse transcriptase polymerase chain reaction (RT-PCR) of alkaline phosphatase and osteocalcin genes indicating osteoblastic differentiation was performed along with staining the produced calcium with tetracycline as an indicator of osteoblastic differentiation. Osteoblasts proliferated on all the samples to some extent. The control surfaces performed better than any of the pectin samples, of which the MHRP_WT seemed to function best. FA length was greater on MHRPTR_GAL than on other pectin samples, otherwise the mutants did not significantly deviate. RT-PCR results indicate that differences between the samples at the gene expression level might be even subtler. However, tetracycline-stained calcium-containing mineral was detected merely only on uncoated TCPS. These results indicate the possibility to affect bone cell growth with in vivo-modified pectin fragments, consecutively providing information on the significance of certain monosaccharides on the biocompatibility of these polysaccharides.