Functional Characterisation of a Putative Rhamnogalacturonan II Specific Xylosyltransferase
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View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector FEBS Letters 582 (2008) 3217–3222 Functional characterisation of a putative rhamnogalacturonan II specific xylosyltransferase Jack Egelunda,1,2, Iben Damagera,2, Kirsten Fabera, Carl-Erik Olsenb, Peter Ulvskova, Bent Larsen Petersena,* a Institute of Genetics and Biotechnology, Faculty of Agricultural Sciences, University of Aarhus and Center for Pro-Active Plants (VKR), Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark b Department of Natural Sciences, Bioorganic Chemistry, Faculty of Life Sciences, University of Copenhagen, DK-1871 Frederiksberg C, Denmark Received 23 July 2008; revised 13 August 2008; accepted 15 August 2008 Available online 26 August 2008 Edited by Ulf-Ingo Flu¨gge onosyltransferase and a xylosyltransferase (XylT) were shown Abstract An Arabidopsis thaliana gene, At1g56550, was ex- pressed in Pichia pastoris and the recombinant protein was to be involved in synthesis of HG [16] and XGA [17], respec- shown to catalyse transfer of D-xylose from UDP-a-D-xylose tively, and two XylTs, designated RhamnoGalacturonan spe- onto methyl a-L-fucoside. The product formed was shown by cific Xylosyltransferase 1 and -2 (RGXT1 and -2), have been 1 1D and 2D H NMR spectroscopy to be Me a-D-Xyl-(1,3)-a- implicated in the xylosylation of the internal L-fucose of the L-Fuc, which is identical to the proposed target structure in the A-chain of RG II yielding an a-(1,3)-linkage between the xy- A-chain of rhamnogalacturonan II. Chemically synthesized lose and fucose moiety [18]. The two RGXTs, which originally methyl L-fucosides derivatized by methyl groups on either the were identified among 27 unclassified GTs in Arabidopsis 2-, 3- or 4 position were tested as acceptor substrates but only thaliana [19], are members of a small sub-family of four genes methyl 4-O-methyl-a-L-fucopyranoside acted as an acceptor, in A. thaliana classified in the Carbohydrate Active enZyme although to a lesser extent than methyl a-L-fucoside. (CAZy) database [20] as GT-family-77 clade B. In the pres- At1g56550 is suggested to encode a rhamnogalacturonan II spe- cific xylosyltransferase. ent study, we have determined biochemical properties Ó 2008 Federation of European Biochemical Societies. Pub- in vitro of a member of this clade and hence a putative member lished by Elsevier B.V. All rights reserved. of the RGXT family, RGXT3 (At1g56550), in A. thaliana. A method for obtaining regio-chemical information of the Keywords: Rhamnogalacturonan II; Pectin; GT-family-77; specificity of a GT, based on methyl derivatives of accep- Xylosyltransferase; Pichia pastoris tor substrate analogues, is presented as part of the enzymatic characterisation. 1. Introduction 2. Materials and methods Plant cell walls (CWs) are composed of complex polysaccha- 2.1. Accessions and sequence analysis ride structures, glycoproteins and proteoglycans, where the Accessions in Fig. 1 are found at the National Center for Biotech- nology Information (NCBI): A. thaliana (At4g01770, At4g01750 and major polysaccharides cellulose and hemicelluloses are embed- At4g01220), Oryza sativa (Os05g03869, AAV31334) and Linum usita- ded in a matrix of pectin [1]. The pectic polysaccharides consist tissimum (AAZ94713). Prefix Selmo is used for Selaginella moellendorf- of homogalacturonan (HG), which can be substituted to yield fii and Phypa for Physcomitrella patens. These sequences were retrieved xylogalacturonan (XGA) and the more complex rhamnogalac- from the DOE Joint Genome Institute (JGI). The phylogenetic tree of clade B was generated by feeding an alignment of the entire CAZy GT- turonan II (RG II) [2]. HG is in turn attached to rhamnogalac- family-77 generated by Muscle [21] to protdist, neighborjoin and draw- turonan I (RG I) [3]. Only a dozen of the presumed more than tree modules of the Phylip package [22]. 200 non-cellulosic plant cell wall glycosyltransferases (GTs) have been successfully heterologously expressed and biochem- 2.2. Plant material and growth condition ically characterised. Functional characterization of GTs with A. thaliana ecotype Columbia (Col-0) and promoter::GUS transfor- roles in hemicellulose biosynthesis have been reported [4–13]. mants were grown in soil as described in [23]. Although the overall structural composition of the pectic poly- saccharides are known [1,2,14,15], the biosynthetic machinery 2.3. Cloning of RGXT3 RGXT3 (At1g56550) cDNA was PCR amplified from a kYES responsible for their synthesis is less well resolved. A galactur- cDNA library [24] using the primers, 50-atggcgcagcagagccaacgtcc-30 and 50-ctagggtaacttgtgttttttac-30. The soluble part of the deduced pro- tein, i.e. amino acid residues 41–383, was PCR amplified using the primers 50-acatggatccagaattcatggattaacaaggaaggacgacgacgacaagcac- *Corresponding author. Fax: +45 35 28 25 89. gtgacaaaatcttctttgttcatgtttcc-30 (Flag-Tag encoding nucleotides are E-mail address: [email protected] (B.L. Petersen). italicized) and 50-ctatgcggccgcgagctcctagggtaacttgtgttttttacc-30 (restric- tion sites EcoRI, PmlI, NotI are underlined) and full-length RGXT3 as 1Current address: University of Copenhagen, Department of Molec- template. Soluble N-terminal Flag tagged RGXT3 was cloned into the ular Biology, Building 4-2-15, Ole Maaløes Vej 5, 2200 Copenhagen, pPICZaA expression vector (Invitrogenä, UK), using the EcoRI and Denmark. NotI sites. All PCR-products were sub-cloned in pCRÒ2.1-TOPO 2Equal contribution. (Invitrogen A/S, Denmark) and sequenced. 0014-5793/$34.00 Ó 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2008.08.015 3218 J. Egelund et al. / FEBS Letters 582 (2008) 3217–3222 2.7. Product analysis by TLC Enzyme reaction mixtures (50 ll) contained 25 mM NH4HCO2,pH 14 7.5, 10 mM MnCl2, 100 lM UDP-a-D-xylose, 2.0 lM UDP-[ C]-a-D- xylose, 5 mM monosaccharide (1, methyl 2-O-methyl-a-L-fucopyrano- side; 2, methyl 3-O-methyl-a-L-fucopyranoside or 3, methyl 4-O- methyl-a-L-fucopyranoside) and 10 ll P. pastoris derived RGXT3 which were incubated for 3 days at 30 °C. As only minute amounts of compound 3 derived product was seen at this time point (data not shown), subsequently, 200 lM UDP-a-D-xylose, 0.5 lM UDP-a-D- [14C]-xylose and 5 ll enzyme were added and this treatment was re- peated every second day for 2 weeks. Reaction mixtures were separated on TLC plates (Gel 60F254, 0.2-mm thickness, E. Merck, Darmstadt, Germany, eluent system: 0.1% pyridine in MeOH:CH2Cl2 (20:80)), twice. Radiolabeled compounds were visualized using a Phospho- ImagerFAQ (Storm 860, Molecular Dynamics). 2.8. Product analysis by mass spectrometry and NMR For preparative purposes an up-scaled (a final total volume of 5 ml) XylT assay containing 1 mM UDP-a-D-xylose and 1 mM methyl a-L- Fuc was used. Isolation of disaccharide product by HPLC–MS guided fractionation (mass of sodium adduct ([M+Na]+)atm/z 333), and NMR analysis are described in [18]. The recovered amount of material Fig. 1. Phylogenetic relationship of genes in clade B of CAZy GT- did not allow recording of 1D 13C or DEPT spectra; all chemical shifts family-77. AtXgY, designates Arabidopsis thaliana accessions; prefix were extracted from the 2D spectra (COSY, HSQC and JRES). The Os, designates Oryza sativa.LUisLinum usitatissimum, Selmo is number of scans per increment in HSQC was 384. Selaginella moellendorffii and Phypa is Physcomitrella patens. 2.9. Generation of RGXT3-promoter::GUS plants The promoter region, defined as ca. 2.0 kb upstream of the RGXT3 start codon, was PCR amplified from gDNA using the primers: 50- atatggatccggtaccttcaatttatttccttatag-30 and 50-atatgtcgacccatggggaaac- 2.4. Expression of secreted, soluble RGXT3 in Pichia pastoris aaatttggaaagaa-30, cloned into pCambia 1301, using NcoI and KpnI, The highest yielding transformants, as determined by western anal- and transformed into A. thaliana ecotype Columbia (Col-0) plants. ysis, were grown at 20 °C, RPM P 280, in 60 ml cultures and induc- Transformants were selected and tested for b-glucuronidase (GUS) tion of expression was obtained by adding 0.5% (v/v) MeOH each activity using the protocol from [23]. All experiments were performed day for 5 days. Media fractions were dialyzed against 25 mM in triplicates with 10 independent transgenic lines. NH4HCO2, pH 7.0, and stored at À80 °C. P. pastoris transformants, expressing BSA and Flag-tagged soluble RGXT3 were in the GS115 strain and the KM71 strain, respectively. Endoglycosidase digestion of RGXT3 was performed using the PROzyme Enzymatic Deglycosy- 3. Results lation Kit as described by the manufacturer (Glyko GK80110, Reac- tionlab AS, Lynge, Denmark). Identical control-samples were 3.1. A phylogenetic distinct family of RhamnoGalacturonan II incubated without peptide-N-glycanase and O-glycanase. Aliquots of XylosylTransferases (RGXT) in CAZy GT-family-77 each sample were analyzed by immunoblot analysis and tested in xylo- In GT-family-77, the XylTs, RGXT1 and -2, are members of a syltransferase assays. Immunoblots were performed as described in [25] using a mouse Anti Flag M2 monoclonal antibody (F3165, Sigma–Al- small, distinct sub-group (clade B) of higher plant sequences drich, Denmark) (1:1000), and a rabbit anti mouse peroxidase-conju- consisting of four A. thaliana accessions, one O. sativa accession gated secondary antibody (P0162, Dako Denmark A/S) (1:1000). and one L. usitatissimum accession [23] (Fig. 1). Expressed se- quence tags, putatively orthologous to members of the clade B 2.5. Enzyme assay genes, have so far only been found in angiosperms (data not The free sugar assay (FSA) was performed according to Bru¨ckner shown). At1g56550, which is characterized in the present study, et al. [26] with the following modifications. Reaction mixtures (50 ll) and At4g01220 are highly identical to RGXT1 and RGXT2 (68– contained 25 mM NH4HCO2, pH 7.5, 10 mM MnCl2, 100 lM NDP- 14 75% at the amino acid level) and share the same overall type II sugar, 0.5 lM NDP-[ C]-sugar (200–300 mCi/mmol, 10 nCi/ll, 9.8 GBq/mmol, 1 nCi = 37 Bq 1.5 · 103 cpm), 0.5 M monosaccha- protein structure typical of Golgi-localized GTs [19].