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Xyloglucan Calactosyl- and Fucosyltransferase Activities from Pea Epicotyl Microsomes' Plant Physiol. (1 997) 1 14: 245-254 Xyloglucan Calactosyl- and Fucosyltransferase Activities from Pea Epicotyl Microsomes' Ahmed Faik, Chinga Chileshe, Jason Sterling, and Cordon Maclachlan* Department of Biology, McGill University, 1205 Avenue Docteur Penfield, Montreal, Quebec, Canada H3A 1 B1 important enzyme activity is a recent thesis by Chileshe Microsomal membranes from growing tissue of pea (Pisum sa- (1995), which provided the starting point for this paper. tivum L.) epicotyls were incubated with the substrate UDP- XGs are composed of chains of the repeating subunit ['4C]galactose (Cal) with or without tamarind seed xyloglucan heptasaccharide Glc,Xyl,, more precisely designated (XC) as a potential galactosyl acceptor. Added tamarind seed XC -XXXG- (Fry et al., 1993), where X = xylosylglucose (isopri- enhanced incorporation of ['4C]Cal into high-molecular-weight meverose) and G is Glc at the reducing end. Gal is attached products (eluted from columns of Sepharose CL-6B in the void to one or both of the two Xyl units closest to the reducing volume) that were trichloroacetic acid-soluble but insoluble in end to form two possible monogalactosylated octasaccha- 67% ethanol. These products were hydrolyzed by cellulase to ride subunits (-XLXG- or -XXLG-) and/or a digalactosy- fragments comparable in size to XC subunit oligosaccharides. lated nonasaccharide (-XLLG-). Although elongation of the XC-dependent galactosyltransferase activity could be solubilized, along with XC fucosyltransferase, by the detergent 3-[(3- XG backbone can proceed in vitro without concurrent cholamidopropyl)-dimethylammonio]-1 -propanesulfonate. When galactosylation (Gordon and Maclachlan, 1989), the incor- this enzyme was incubated with tamarind (Tamarindus indica poration of Gal in vivo appears to follow soon after chain 1.) seed XC or nasturtium (Tropaeolum majus 1.) seed XC that elongation in the frans-Golgi cisternae (Brummell et al., had been partially degalactosylated with an XC-specific 1990; Zhang and Staehelin, 1992). The addition of Gal /3-galactosidase, the rates of Cal transfer increased and fucose interferes with self-association by chains of the XG back- transfer decreased compared with controls with native XC. lhe bone and prevents its precipitation from aqueous solution reaction products were hydrolyzed by cellulase to 14C fragments (Reid et al., 1988). Galactosylation may be completed be- that were analyzed by gel-filtration and high-performance liquid fore the final decoration with Fuc takes place in the frans- chromatography fractionation with pulsed amperometric detec- Golgi network (Zhang and Staehelin, 1992). The addition of tion. lhe major components were XC subunits, namely one of the Fuc generates subunits with side chains containing three two possible monogalactosyl octasaccharides (-XXLC-) and di- sugars, a structure that facilitates binding of XG to cellulose galactosyl nonasaccharide (-XLLC-), whether the predominant oc- microfibrils (Levy et al., 1991, 1996). tasaccharide in the acceptor was XXLC (as in tamarind seed XG) or XLXC (as in nasturtium seed XC). It is concluded that the first PXG was first thought to be composed almost exclu- xylosylglucose from the reducing end of the subunits was the Cal sively of alternating heptasaccharide and nonasaccharide acceptor locus preferred by the solubilized pea transferase. These in a repeating dimer (-XXXG.XXFG-), (Hayashi and Ma- observations are incorporated into a model for the biosynthesis of clachlan, 1984a), since this dimer was the chief intermedi- cell wall XCs. ate that was formed transiently during cellulase hydrolysis, and equal amounts of the hepta- and nonasaccharides are always major components in complete digestion mixtures. Recent studies (Guillén et al., 1995) have also detected the A11 XG chains analyzed to date contain 1,2-P-linked Gal presence of small amounts of other subunits, including two attached to particular Xyl side chains that are linked 1,6-a- octasaccharides and the decasaccharide -XLFG-. Thus, pea to a 1,4-P-glucan (cellulosic) backbone. These Gal units microsomes must contain XG galactosyltransferase activity may terminate the side chains as in the storage XG of many that preferentially galactosylates the nascent XG backbone seeds, e.g. tamarind (Tamarindus indica L.) and nasturtium at every second heptasaccharide to form a transient repeat- (Tropaeolum majus L.), or they may be fucosylated by 1,2- ing unit (-XXXG.XXLG-) in the backbone, which is then a-linked Fuc, as in the structural XG of many dicot primary fucosylated. walls, e.g. pea (Pisum sativum L.), soybean, and sycamore. Studies of pea microsomes (Gordon and Maclachlan, In this paper we focus on the detection and solubilization 1989; White et al., 1993) have confirmed results from earlier of XG galactosyltransferase activity from microsomes of studies of soybean and French bean microsomes (Hayashi growing regions of the pea epicotyl. To our knowledge, the only published report of the direct assay of this potentially Abbreviations: CHAPS, 3-[(3-~holamidopropyl)-dimethylam- moniol-1-propanesulfonate;PXG, TXG, NXG, xyloglucans derived from growing pea epicotyl, tamarind seed, and nasturtium seed, ' This work was funded by the Natural Sciences and Engineer- respectively; TFA, trifluoroacetic acid; V,, void volume of a chro- ing Research Council of Canada. matography column; V,, total volume of a chromatography col- * Corresponding author; e-mail [email protected]; umn; XG, xyloglucan. Oligosaccharide subunits of XG are abbre- fax 1-514-398-5069. viated according to the nomenclature proposed by Fry et al. (1993). 245 246 Fa'ik et al. Plant Physiol. Vol. 114, 1997 and Matsuda, 1981; Campbell et al., 1988), which demon- DEAE-cellulose, dextrans, P-galactosidase (E.C. 3.2.1.23) strated that continued elongation of the XG backbone to from various organisms, leupeptin, PMSF, and pronase form a structure with repeating subunits requires cooper- were from Sigma. Purified (HPLC grade) NaOH (50% ativity between XG glucosyl- and xylosyltransferases. [w/w]) was from Fisher Scientific, and anhydrous sodium Some XG chain elongation may occur in the presence of acetate was from Fluka. Sephadex G-10, Sepharose CL-6B, UDP-Glc alone, with subsequent xylosyl transfer to the and CM-Sephadex column supports were from Pharma- P-glucan extension (Ray, 1980), but the fact that Glc incor- cia. CarboPac PA-100 columns for HPLC were supplied poration is enhanced by UDP-Xy1 and vice versa implies by Dionex (Sunnyvale, CA). Partially purified cellulase that their transferases act in vitro concurrently and inter- from Trichoderma sp. (1%suspension in dilute [NH,],SO,) dependently to generate the observed regularity of the pea and TXG were purchased from Megazyme (Sydney, Aus- XG backbone. It is not known whether XG galactosyl trans- tralia). NXG was prepared from Tropaeolum majus L. var fer is coupled to the process of chain elongation in vivo. Climbing Giant Tal1 Forest, as described by Edwards et al. It is established, however, that PXG fucosyltransferase (1985). can act independently of other XG glycosyltransferases to add Fuc to preformed acceptor XG chains in the medium in which microsomes are suspended (Camirand and Ma- Plant Materiais clachlan, 1986). PXG fucosyltransferase has been solubi- lized from microsomal extracts with the detergent CHAPS, A11 studies were conducted with microsomes isolated using added TXG (Hanna et al., 1991) or a cellulase- from seedlings of garden pea (Pisum sativum L. var Alaska) resistant trimer from TXG (Maclachlan et al., 1992) as a purchased from Rogers Bros. Seed Co. (Boise, ID). Seeds fucosyl acceptor. Free XG monomer subunits do not act as were washed in 10% (v/v) commercial NaClO, for 10 min, fucosyl acceptors, but, on the contrary, as competitive in- rinsed in water, and soaked overnight. They were germi- hibitors of fucosyl transfer to longer chains (Farkas and nated in moist vermiculite in the dark for 7 to 8 d at 22°C Maclachlan, 1988). This last study also showed that fuco- until the third internodes were 3 to 5 cm long. The apical2 syltransferase activity in pea microsomes was distinctly to 3 cm of internodes cut just below the hook were used as enhanced when UDP-Gal was added to the reaction me- the source of microsomes. dium, which may mean that galactosylation of nascent PXG could take place in these preparations to form new sites where further fucosylation could then occur. Membrane Preparation and Solubilization To demonstrate directly that [14C]Gal is incorporated from UDP-[14C]Gal into XG, a high-M, product must be Pea microsomal membranes were prepared using pro- identified that is hydrolyzed by purified endo-1,4-P- cedures described previously with minor changes (Cam- glucanase (E.C. 3.2.1.4) to authentic XG subunit oligosac- irand and Maclachlan, 1986; Gordon and Maclachlan, charides. These can be detected tentatively by their elution 1989). Stem segments were weighed and homogenized volumes after gel filtration, e.g. on Bio-Gel columns (Ha- with a blender (twice for 30 s) in cold extraction buffer (2 yashi and Maclachlan, 1984a; Maclachlan et al., 1992; volumes of 0.1 M Pipes-KOH, pH 6.2, 0.4 M SUC,10 mM Guillén et al., 1995), and, more precisely, by their elution MgCl,, 10 mM MnCl,, 5 mM DTT, 1 PM leupeptin, 0.1% patterns from HPLC columns (McDougall and Fry, 1990; [w/v] BSA, and 1 mM PMSF dissolved in n-propanol). The Buckeridge et al., 1992; Vincken et al., 1995) and their NMR mixture was filtered through nylon cloth and the filtrate spectra (York et al., 1990, 1993). was centrifuged (5000g for 10 min) to remove cell wall In the present study the optimal detergent leve1 that debris and unbroken cells. The supernatant was then solubilized PXG fucosyltransferase activity (0.3% [w / v] microcentrifuged (model L8-80, Beckman) at high speed CHAPS) also extracted XG galactosyltransferase activity. (100,OOOg) with angled (80 Ti) or swinging bucket (SW41) However, TXG, although an effective acceptor for fucosyl rotors for 60 min at 4°C.
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