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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 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 (Invitrogen , UK), using the EcoRI and Denmark. NotI sites. All PCR-products were sub-cloned in pCR 2.1-TOPO 2Equal contribution. (Invitrogen A/S, Denmark) and sequenced.
0014-5793/$34.00 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 80 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]. ride and 5 ll P. pastoris supernatant. Assays using UDP-a-D-xylose, as donor-substrate are designated xylosyltransferase (XylT) assays. 3.2. Tissue specific expression of RGXT3 Approximate K values towards UDP-a-D-xylose of Pichia expressed m The expression pattern of RGXT3 was studied using a RGXT1, RGXT2 and RGXT3 were determined using the standard XylT assay with UDP-a-D-xylose in concentrations (lM) of 1, 1.25, RGXT3 promotor-b-glucuronidase A (GUS) reporter gene con- 14 2.5, 5, 10, 20, 100, 200, 1.0 lM UDP-a-D-[ C]-xylose, 0.5 M L-Fucose struct, which was transformed into A. thaliana. GUS activity and 5 ll enzyme which was incubated at 30 C for 1, 1 and 2 h for was only observed in mature plants and was absent in seedlings RGXT1, RGXT2 and RGXT3, respectively. Cloning and initial char- (data not shown). Weak GUS activity was found in rosette acterisation of soluble N-terminal Flag-tagged RGXT1 and RGXT2 are described in [26]. Ten millimolar acceptor and 10 ll P. pastoris leaves (Supplementary Fig. 1A), mainly concentrated around supernatant were used in the L-fucoside derivate acceptor studies. trichome support cells in the adaxial epidermis (Supplemen- Chemical synthesis of methyl a-L-fucoside, methyl b-L-fucoside and tary Fig. 1C, F and G). GUS activity was found in cauline methyl a-L-Fuc-(1,4)-b-L-Rha is described in [18]. Chemical synthesis leaves of bolting plants (Supplementary Fig. 1D and E), in pet- of the disaccharides as well as methyl 2-O-methyl-a-L-fucopyranoside, als (Supplementary Fig. 1B), and in both the proximal and dis- methyl 3-O-methyl-a-L-fucopyranoside and methyl 4-O-methyl-a-L- fucopyranoside is described in [27]. tal ends of siliques (Supplementary Fig. 1B). Expression of RGXT1 and 2 was determined with PCR [18] and thus did 2.6. Product analysis by a- and b-xylosidase treatment not address expression on the cellular level. Expression of a- and b-xylosidase treatments of the methyl a-D-xylosyl-L-fucoside, RGXT3 generally overlaps at the organ level with that ob- were performed as described in [18]. served for RGXT2. J. Egelund et al. / FEBS Letters 582 (2008) 3217–3222 3219
3.3. At1g56550 is a xylosyltransferase with L-fucose as acceptor amounts as a hyper glycosylated protein in the medium substrate (Fig. 2). Using Flag-tagged BSA of known concentration as When expressed as an N-terminal Flag-tagged soluble se- internal standards in immunoblots, At1g56550 was estimated creted protein in P. pastoris, At1g56550 accumulated in ample to accumulate to 0.07–0.14 lg/ll of medium (Fig. 2). In the free sugar assay (FSA) At1g56550 catalyzed transfer 14 14 14 of D-[ C]-xylose (D-[ C]-Xyl) from UDP-a-D-[ C]-Xyl onto L-fucose (L-Fuc) (Fig. 3A) and to a significantly lesser extent BSA RGXT3 on to D-arabinopyranose (D-Ara). In contrast, none of Time (hours) 120 0 24 48 72 96 120 the other combinations of donor NDP-[14C]-sugars 14 14 (UDP-a-D-[ C]-glucose, UDP-a-D-[ C]-galactose, UDP-a-D- 14 14 [ C]-N-acetyl-glucosamine, UDP-a-D-[ C]-glucuronic acid, 14 14 kDa GDP-b-L-[ C]-fucose and GDP-a-D-[ C]-mannose) and monosaccharide acceptors L-arabinose (L-Ara), D-glucose (D-Glc), D-galactose (D-Gal), L-rhamnose (L-Rha), D-xylose (D-Xyl), D-mannose (D-Man), L-Fuc and N-acetyl-D-glucosa- mine (D-GlcNAc) resulted in formation of radioactive prod- ucts (data not shown, all sugar tested were on the pyranose form). The minor XylT activity towards D-Ara, which was also reported for the RGXT1 and -2 enzymes [18], may be rational- ized in terms of L-Fuc differing from D-Ara only by the pres- ence of a methyl group at C5. As arabinose in plants is only found on the L-form (L-Ara), we ascribe this as a non-physio- logical irrelevant side activity. These initial activities are simi- lar to those reported for RGXT1 and RGXT2, which were implicated in the xylosylation of the internal fucose of the A- Fig. 2. Immunoblot analysis of At1g56550 (RGXT3) expressed in Pichia pastoris. Samples were collected each day for 5 days and chain of RG II [18]. At1g56550 is thus proposed to encode a subjected to immunoblot analysis as described in Section 2. A non- third member of the RG II specific XylT (RGXT) family and tagged BSA construct was used as negative control. designated RGXT3 accordingly.
6000 A
5000
4000
3000 CPM
2000
1000
0 LD-- GlcD- GalL- RhaD- GlcNAcD- Xyl ManL- AraL- Fuc B 3500
3000
2500
2000 CPM 1500
1000
500
0 L-Fuc Me α - L-Fuc Me β - L-Fuc Me-α-L-Fuc-β-L-Rha
Fig. 3. At1g56550 (RGXT3) possesses xylosyltransferase activity. RGXT3 assayed in the free sugar assay (FSA) using labelled and unlabelled UDP- a-D-xylose as donor and the depicted mono- and disaccharide acceptors. Gray bar: dialysed media fraction containing Pichia pastoris expressed soluble N-terminal Flag-tagged RGXT3. Black bar: BSA control (n = 2). (A) The FSA using 0.5 M of the depicted monosaccharides. (B) The FSA using 10 mM of each of the depicted acceptor substrates. 3220 J. Egelund et al. / FEBS Letters 582 (2008) 3217–3222
Although RGXT3 in P. pastoris (Fig. 2) is expressed at sig- nificantly higher levels than the Baculo virus expressed OMe OMe OMe H C H C RGXT1 and -2 enzymes ([18] and data not shown), RGXT3 H3C O 3 O 3 O OH OH has on a lltoll basis less than 20% of the activity of Baculo OCH3 HO OH HO OCH3 CH3O OH virus expressed RGXT1 and RGXT2. We speculate that the low activity of RGXT3 may result from incorrect folding and/or excessive glycosylation of the protein (Fig. 2). Several methyl L-fucoside derivates were tested as acceptor molecules in order to attain further insight in the acceptor specificity of RGXT3 (Fig. 3B). Methyl a-L-fucoside (Me a- L-Fuc) was found to be an approximately five times more effi- cient acceptor than L-Fuc, whereas Me b-L-Fuc did not work Me Xyl*- 4MeFuc 14 as an acceptor (Fig. 3). The level of D-[ C]-Xyl incorporation onto methyl a-L-fucosyl-(1,4)-b-L-rhamnoside (Me a-L-Fuc- (1,4)-b-L-Rha), which represent a fragment of the RG II A- Xyl* chain, was one third of the incorporation obtained with methyl a-L-Fuc (Fig. 3B), exactly as observed for RGXT1 and RGXT2 [18]. A search at the Salk Institute Genomic Analysis Labora- toryÕs homepage (http://signal.salk.edu/cgi-bin/tdnaexpress) did not identify T-DNA insertional mutant lines in RGXT3.
RG II isolated from T-DNA insertional mutants of RGXT1 UDP-Xyl* and -2 (rgxt-1 and -2), in which a minor proportion seemingly lacks the 2-O-Me-D-Xyl residue, have been shown to function Fig. 4. TLC analysis of radiolabeled compounds of the XylT assays as acceptor substrate for the Baculo virus expressed RGXT1 14 containing RGXT3 incubated with UDP-[ C]-a-D-xylose and the and -2 enzymes [18]. However, RGXT3 did not incorporate acceptors; water, methyl 2-O-methyl-a-L-fucopyranoside (1), methyl 3- 14 detectable amounts of D-[ C]-Xyl into RG II of mutant O-methyl-a-L-fucopyranoside (2) and methyl 4-O-methyl-a-L-fucopyr- rgxt-1 or -2 perhaps because of the low activity of RGXT3 anoside (3). or because the hyper-glycosylated enzyme may impose steric hindrance when incubated together with a large and complex acceptor molecule such as RG II. RGXT3 was therefore sub- fucopyranoside were therefore synthesized [27] and tested as jected to enzyme mediated deglycosylation, using peptide:N- acceptor substrates in the XylT assay (1, 2 and 3, see Fig. 4). glycanase and O-glycanase, which resulted in a clear shift in TLC analysis revealed that only the methyl 4-O-methyl-a-L- mobility from the high MW smear in to a more congregating fucopyranoside (compound 3) functioned as an acceptor band of ca. 5 kDa above the predicted MW (Supplemental (Fig. 4), and although this acceptor was ca. 40-fold less effi- Fig. 2), but also in a five fold loss of activity (data not shown), cient compared to methyl L-fucoside, (data not shown), these rendering enzyme assays containing RG II of mutant rgxt-1 results suggest that position 4 is an unlikely acceptor site. An and -2 unfeasible (RGXT1 on the other hand, which is fully identical pattern was obtained for Baculo virus expressed deglycosylated under the same conditions, retain 96% of the RGXT1 and -2 (data not shown). activity, data not shown). It thus remains unresolved whether The Me a-D-Xylp-a-L-Fucp disaccharide was produced in RG II from mutant rgxt-1 or -2 may serve as acceptor for a up-scaled XylT assays, purified (Fig. 5A and B) and analysed less glycosylated form of RGXT3. In order to investigate by NMR. 1H NMR, COSY and HSQC spectra were obtained whether the low activity of RGXT3 resulted from a poor affin- and all carbon- and proton-signals were found to be identical ity towards the UDP-donor substrate the apparent Km values to those previously published for the disaccharide product of towards UDP-a-D-xylose of P. pastoris expressed N-terminal RGXT2 where the stereochemistry was determined to be an 1 13 Flag-tagged soluble RGXT1, RGXT2 and RGXT3 were a-(1,3) linkage (Fig. 5D) [18]. The signals (x2- H, x1- C: determined. The Km values towards UDP-a-D-xylose: approx- (4.8,100.5), (3.65,74), Fig. 5C) of the Me a-D-Xylp-(1,3)-a-L- imately 60, 140 and 110 lM for RGXT1, RGXT2 and Fucp containing fraction (RT 6.1 min, Fig. 5A and B) may RGXT3, respectively, were not sufficiently different to account be ascribed to a contamination by the close following fraction for the low activity of RGXT3. (RT 6.8 min, major compound [M+Na] at m/z 261), as evi- denced by an HSQC NMR spectrum of this fraction in D2O, 3.4. Linkage of the disaccharide product which was super imposable (data not show) with the impurities Treatment of radiolabelled Me a-L-Fuc-D-Xyl with either a- seen in Fig. 5C. or b-xylosidase followed by size-exclusion chromatography, revealed that only a-xylosidase released radiolabelled D-Xyl (Supplementary Fig. 3) thus demonstrating that RGXT3 is a 4. Discussion retaining a-XylT. Methyl L-fucoside derivatives derivatized by a methyl group 4.1. RGXT3 – an additional member of the RGXT gene family in on either the 2-, 3- or 4-position were considered as acceptor A. thaliana substrate analogues that could reveal the regiospecificity of RGXT3 was shown to possess biochemical activities in vitro the XylT. Methyl 2-O-methyl-a-L-fucopyranoside, methyl similar to those earlier reported for RGXT1 and RGXT2 [18]. 3-O-methyl-a-L-fucopyranoside and methyl 4-O-methyl-a-L- NMR analysis of the disaccharide product formed showed full J. Egelund et al. / FEBS Letters 582 (2008) 3217–3222 3221
Intens. 5 given compound with a blocked hydroxyl group functioned as
x10 . A an acceptor, a site or sites other than the derivatized position is 20 15 likely to be the acceptor site(s). The strategy, appeared to rule 10 out position 4, but it fell short of discriminating between posi- 5 0 tions 2 and 3. This approach, however, do not necessitate puri- 10 B fication steps and requires only nano moles of enzyme product 8 formed. Furthermore, the significantly lower acceptor effi- 6 4 ciency of the position 4 derivative, which is the site of attach- 2 0 ment of the glucoronic acid on the RG II A-chain Me Fuc, as 8 10 compared to that of Me a-L-Fuc may hint that the glucoronic acid is added to the RG II A-chain Me Fuc after the addition C of the (Me) xylose.
4.2. The need for multiple isoforms? The presence of another compensating gene product may further rationalize the earlier reported feeble phenotype of the T-DNA single mutants rgxt1 and rgxt2 with the hardly more than 1–2% of variant RG II in the mutants [18]. Our re- sults indicate, that a small gene-family is responsible for the xylosylation of the internal fucose moiety of the RG II A-chain in A. thaliana and hence suggest that At4g01220 may also en- code an RGXT. Recruitment of a family of paralogous genes in Arabidopsis appears also to be the case in homogalacturo- nan synthesis [28] but whether this is a common theme in the biosynthesis of RG II or of other cell wall polymers, remains an open question. Interestingly, it appears that there is only D one rice ortholog to the RG II specific A. thaliana XylTs.
Acknowledgements: This work was supported by The Danish National Research Foundation, The Carlsberg Foundation, The Ministry of Science, Technology and Innovation, The Villum Kann Rasmussen Foundation and The Marie Curie Fellowship of the European Com- munity FP6 Program.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.febslet.2008. 08.015.
References
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