Glycobiology Advance Access originally published online on August 10, 2006
Glycobiology 2006 16(12):1171-1180; doi:10.1093/glycob/cwl038
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Engineered xyloglucan specificity in a carbohydrate-binding module
2 Department of Immunotechnology, Lund University, BMC D13, SE-221 84 Lund, Sweden;
3 School of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Center, SE-106 91 Stockholm, Sweden;
4 Institute for Cell and Molecular Biosciences, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne NE2 4HH, UK; and
5 Department of Biotechnology, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden
1 To whom correspondence should be addressed; e-mail: mats.ohlin{at}immun.lth.se
Received on November 24, 2005; revised on August 7, 2006; accepted on August 8, 2006
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Abstract |
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The field of plant cell wall biology is constantly growing and consequently so is the need for more sensitive and specific probes for individual wall components. Xyloglucan is a key polysaccharide widely distributed in the plant kingdom in both structural and storage tissues that exist in both fucosylated and non-fucosylated variants. Presently, the only xyloglucan marker available is the monoclonal antibody CCRC-M1 that is specific to terminal
-1,2-linked fucosyl residues on xyloglucan oligo- and polysaccharides. As a viable alternative to searches for natural binding proteins or creation of new monoclonal antibodies, an approach to select xyloglucan-specific binding proteins from a combinatorial library of the carbohydrate-binding module, CBM4-2, from xylanase Xyn10A of Rhodothermus marinus is described. Using phage display technology in combination with a chemoenzymatic method to anchor xyloglucan to solid supports, the selection of xyloglucan-binding modules with no detectable residual wild-type xylan and ß-glucan-binding ability was achieved. Key words: binding specificity / carbohydrate-binding module / molecular engineering / phage display / xyloglucan
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
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Plant cell walls, specifically wood and fibers, have been used for millennia as a versatile raw material and continue to attract scientific attention because of their excellent mechanical properties and status as a renewable resource (Ball, 2005
; Klemm et al., 2005). The main load-bearing component of the cell wall is the cellulose microfibril, which is composed of paracrystalline aggregates of ß(1
4) glucan held together by hydrogen bonding and hydrophobic interactions. In the cell wall, microfibrils associate with other polymers, including anionic and neutral polysaccharides, aromatic hydrocarbon polymers, and structural proteins, to form a strong, flexible biocomposite (Carpita and McCann, 2000). Although the details of the biosynthesis and assembly of the various cell wall components are slowly beginning to be revealed through a combination of genetic, (bio)chemical, and microscopic approaches, a full understanding lies far in the future (Rose et al., 2004; Somerville et al., 2004; Dhugga, 2005). An important element of ongoing efforts to unravel the mechanisms of cell wall morphogenesis is the development of sensitive and specific probes for individual wall components. Traditionally, antibodies have been the primary workhorses for the spacial localization of cell wall polysaccharides (Puhlmann et al., 1994; Knox, 1997; Willats et al., 2000; McCartney et al., 2005), and currently nearly 30 monoclonal antibodies directed toward specific arabinan, galactan, xylan, galacturonan, fucosylated xyloglucan (FucXG), and cell wall glycoprotein epitopes are available through academic and commercial sources [NSF Monoclonal Antibody Project, Complex Carbohydrate Research Center (CCRC), Athens, GA, http://cell.ccrc.uga.edu/~mao/wallmab/Home/Home.php; Carbosource, CCRC, http://www.ccrc.uga.edu/~carbosource/CSS_home.html; PlantProbes, Leeds, UK, http://www.plantprobes.co.uk/].
Despite their established effectiveness, the leading role antibodies play in cell wall analysis is beginning to be challenged by other specific carbohydrate-binding proteins such as catalytically inactive glycosidase variants (Adams et al., 2004; Beaugrand et al., 2005) and non-catalytic carbohydrate-binding modules (CBMs) (Hilden et al., 2003; McCartney et al., 2004). In Nature, CBMs play a key role in the degradation of biomass by targeting microbial glycosidases to their associated substrate within the complex polysaccharide architecture that comprises the plant cell wall. Over 40 sequence-related CBM families are currently known (http://afmb.cnrs-mrs.fr/CAZY/), which encompass a diversity of binding specificities toward crystalline, amorphous, and soluble polysaccharides (Boraston et al., 2004; Hilden and Johansson, 2004). CBMs are particularly attractive as cell wall probes because of their individual, intrinsic specificities toward polysaccharides, small molecular footprints (typically 5–20 kDa, binding 1–6 consecutive glycosyl units, versus 150 kDa in the case of immunoglobulins), ease of heterologous expression, and convenience of modification with peptide and small molecule detection tags (Hilden et al., 2003; Lehtiö et al., 2003).
A considerable amount of attention in the field of plant cell wall biology has been paid to xyloglucan, a key polysaccharide responsible for crosslinking cellulose microfibrils in plant cell walls (Fry, 1989; Hayashi, 1989; Pauly et al., 1999) that is also found as a predominant storage carbohydrate in some seeds (Reid, 1985; Gidley et al., 1991). In particular, the composition of the xyloglucan–cellulose network is currently believed to be one of the main factors affecting cell wall extensibility (Rose and Bennett, 1999; Darley et al., 2001; Thompson, 2005), which has motivated detailed study of the enzymes responsible for the biosynthesis (Reiter, 2002) and in muro manipulation of xyloglucan (Rose et al., 2002; Fry, 2004; Johansson et al., 2004).
For the analysis of xyloglucan in plant tissues, the only marker that exists today is the monoclonal antibody CCRC-M1 that is specific to terminal -1,2-linked fucosyl residues on xyloglucan oligo- and polysaccharides (Puhlmann et al., 1994). This epitope is typically found in xyloglucans from primary walls (Carpita and McCann, 2000; Reiter, 2002) but is absent in those from seeds including the extensively studied Tamarindus indica xyloglucan (York et al., 1990), and wall xyloglucans from the Oleaceae, Poaceae, and Solanaceae (Vincken et al., 1997; Vierhuis et al., 2001; Tine et al., 2003). Thus, there is a need to diversify the toolbox of xyloglucan-binding probes to address biological questions involving other prevalent xyloglucan variants.
Interestingly, no xyloglucan-specific CBMs have been reported in the literature, although promiscuous CBMs with xyloglucan affinity, such as CBM9–2 from Thermotoga maritima Xyn10A (Boraston et al., 2001), are known. More recently, it has been shown that xyloglucan affinity is a common feature of a number of CBMs which bind unsubstituted ß-glucans (Najmudin et al., 2006). The potential of altering the binding specificity of CBMs has been demonstrated (Smith et al., 1998; Lehtiö et al., 2000; Simpson et al., 2000), including work in our laboratory (Cicortas Gunnarsson et al., 2004) on CBM4-2 of xylanase Xyn10A from Rhodothermus marinus (Nordberg Karlsson et al., 1997). This CBM binds preferentially to xylan (Abou Hachem et al., 2000) by establishing specific interactions between the helical secondary structure of the polysaccharide and several surface residues in the binding cleft (Simpson et al., 2002). We have previously constructed a combinatorial library by limited substitution of 12 residues within the xylan-binding site of CBM4-2 and successfully selected variants specific to two other polysaccharides and a glycoprotein using phage display technology (Cicortas Gunnarsson et al., 2004).
In this work, we have used the combinatorial library from our previous studies and selected for xyloglucan binding modules (XGBMs) to overcome the lack of appropriate reagents to detect xyloglucan. The outcome of these selections was further characterized, and we identified two proteins that have a high specificity for xyloglucan and preferentially bind to the non-fucosylated variant of the polysaccharide.
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Results |
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Preparation of fucosylated and non-fucosylated xyloglucan–biotin conjugates
Biotin conjugates for the specific immobilization of xyloglucans were prepared by exploiting the ability of the plant enzyme xyloglucan endo-transglycosylase (XET) to incorporate well-defined, chemically modified xyloglucan oligosaccharides (XGOs) into high molar mass polysaccharide chains (Brumer et al., 2004
; Zhou et al., 2006). T. indica and Rubus fruticosus xyloglucans were chosen as representative non-fucosylated xyloglucans (XG) (seed) and FucXG (primary cell wall), respectively, to allow selection of evolved XGBMs specific to either polysaccharide variant. Under the agency of XET catalysis, XGO–biotin conjugates were incorporated into both XG and FucXG to produce xyloglucans specifically biotinylated at their reducing ends. The enzyme reaction, which results in a net reduction of xyloglucan chain length, was carefully controlled to ensure that XG–biotin and FucXG–biotin had comparable molecular mass distributions (Figure 1). Owing to the specific XET-based biotinylation procedure used, these conjugates do not present unnatural structures, which would otherwise be obtained by modification of the polysaccharide backbone or branching residues (Priem et al., 1997) and which would confound the selection process. Furthermore, only xyloglucans are biotinylated by this method, thus eliminating the possibility that other polysaccharide contaminants in xyloglucan preparations from plant sources (Chambat et al., 2005) might be captured on the solid supports used in the selection of binding proteins.
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Selection of XGBMs from CBM4–2 combinatorial library
A combinatorial library with 1.6 x 106 clones created in our previous work (Cicortas Gunnarsson et al., 2004
) was in this study used for selection of XGBMs by phage display. Phage selections were performed in two ways, by incubating the phages with beads carrying immobilized XG, either in the absence or in the presence of soluble xylan in the selection buffer. Addition of xylan to the selection system was used to remove those CBM variants in the phage library that still carried the wild-type specificity for xylan, allowing facile selection of specific XGBMs. Using the selection ratio (output phages/input phages) as a measurement of the selection process, we could follow the enrichment of XG-specific CBMs. The presence of xylan in the selection buffer clearly increased the selection pressure leading to a decrease of the selection ratios (first 1/1,000,000, second 1/1000, and third 1/440) compared with those from the selections performed in the absence of xylan (first 1/15,000 and second 1/30).
Sequence analysis
Clones from selections in the presence and absence of xylan were sequenced, and 21 unique sequences were found (Figure 2) (complete gene sequences are available at GenBank, Accession numbers DQ279262
[GenBank]
–DQ279282
[GenBank]
). Interestingly, these sequences showed that variation was allowed in position 69 of XG-selected clones, in contrast to earlier findings that 25 of 27 CBM variants selected toward other carbohydrates had tryptophan in that position (Cicortas Gunnarsson et al., 2004). A major difference between clones selected in the presence or absence of xylan was that leucin was found in position 76 in all 11 variants selected only in the presence of xylan, whereas three of eight CBMs selected only in the absence of xylan allowed other amino acids in that position (Figure 2). Sequence analysis also revealed that of the 12 residues targeted with mutations in the combinatorial library, a specific amino acid was selected in position 72, 112, 115, and 146, in addition to leucin in position 76. The conserved wild-type amino acids E112, R115, and H146 and the two substitutions, E72H and F76L, are likely to be key residues affecting the binding affinity to XG.
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Specificity of selected clones
Binding experiments were performed to investigate the specificity of selected CBMs. Phage stocks displaying CBM variants were incubated directly with insoluble Avicel, XG–biotin immobilized on paramagnetic beads, or soluble carbohydrates adsorbed onto polystyrene surfaces. Some of the clones had a broad specificity, whereas others, in particular XG-34 and XG-35, which were selected in the presence of competing soluble xylan, bound well to XG but poorly or not at all to birchwood xylan (substrate of wild-type CBM4-2) (Figure 3A), Avicel, arabinoxylan, and barley ß-glucan (Figure 4). Under these assay conditions, XG-34 and XG-35 were also able to discriminate between the selection target, XG, and FucXG, which contains 4.5 mol % Fuc, corresponding to fucosylation of one in three Gal side chain residues (Figure 3B).
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Binding studies
Two of the XGBMs selected in the presence of xylan, XG-34 and XG-35, were produced in soluble form yielding
50 mg/L from shake flask cultures. Binding to xylan, XG, and ß-glucan was assessed by affinity electrophoresis (AE) (Figure 5), and KD values were calculated from gels with varying concentrations of the XG (Figure 6, Table I). These data showed that the wild-type CBM4-2 had a previously unknown ability to bind to XG that was significantly increased in the XG-34 and XG-35 variants (5- and 4-fold, respectively). Binding of the wild-type CBM4-2 and XG-34 to xyloglucan was also evident from isothermal titration calorimetry (ITC) but too low to quantify. However, XG-34 in contrast to the wild-type module displayed no detectable affinity at all for xylopentaose, as determined by ITC (data not shown), or for xylan and ß-glucan, as determined by AE (Figure 5). The same specificity was observed for XG-35 in the AE experiments, implicating that the XGBMs had been evolved by retaining the affinity for xyloglucan while loosing the capacity to bind xylan and ß-glucan. Furthermore, as binding to XG could not be inhibited by the presence of a large molar excess of either xylopentaose or cellopentaose (data not shown), this was obviously not just a result of an evolution of a module requiring a carbohydrate terminus that would be present only in low amounts in the high-molecular-weight xylan and ß-glucan preparations. Thus, the XGBMs had a high degree of selectivity on the polysaccharides tested without showing any affinity for the non-reducing end of sugars.
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To compare the specificity of the XGBM variant XG-34 to that of the commercially available monoclonal antibody CCRC-M1 that recognizes terminal
-1,2-linked fucosyl residues in FucXG, we used these proteins in competitive enzyme-linked immunosorbent assay (ELISA) experiments. Both CCRC-M1 and XG-34 were pre-incubated with different concentrations of either FucXG–biotin or XG–biotin to inhibit binding to biotinylated xyloglucans immobilized on plates (Figure 7). In agreement with previous studies (Puhlmann et al., 1994), it was not possible to inhibit the binding of CCRC-M1 to FucXG with XG (Figure 7A). XGBM variant XG-34, on the contrary, could only be inhibited from binding immobilized XG by XG, whereas FucXG had no inhibition effect (Figure 7B). Thus, XGBMs selected from the combinatorial library based on CBM4-2 extend the bioanalytical toolbox available to us by providing a specificity profile very different from that of the single existing xyloglucan-specific probe.
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Discussion |
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The wild-type protein used in this study, the CBM4-2 of R. marinus xylanase Xyn10A, is a so-called type B CBM (Boraston et al., 2004
) with a groove-shaped binding site accommodating soluble polysaccharide chains. The preferred ligand of CBM4-2 is xylan, but it can also bind to other carbohydrates such as ß-glucan (Abou Hachem et al., 2000) and, as demonstrated here for the first time, xyloglucan. This complex specificity pattern makes the wild-type CBM4-2 unsuitable for many assay applications requiring high ligand specificity. Building upon our earlier work using this module as a scaffold to generate specific binding proteins and the powerful phage display technology (Cicortas Gunnarsson et al., 2004), we have used a rather small combinatorial library (1.6 x 106 clones) to successfully evolve highly specific XGBMs. The variants with the best specificity profile, XG-34 and XG-35, have slightly increased affinities for XG, were able to discriminate between XG and FucXG, and, most importantly, have lost the ability of the wild-type protein to bind to xylan and ß-glucan. The ability of these CBMs to bind XG but not xylan and ß-glucan appeared not to be a consequence of the presence of a large chain-end to mass ratio in the relatively low-molecular-weight XG preparation as neither xylopentaose nor cellopentaose at molar concentrations 100-fold higher than those of soluble XG that inhibited binding of XG-34 and XG-35 to immobilized XG affected the binding activity. In conclusion, the overall process identified molecules with new binding properties.
The addition of soluble xylan in the selection strategy was a key to select against clones with residual specificity for the natural polysaccharide ligand, thus ensuring that only variants with a high XG/xylan selectivity were captured on the surface-immobilized XG–biotin. The exact determinants that result in the loss of xylan affinity of the selected XGBMs are not clear from the deduced protein sequence data. For example, the frequently occurring mutation of residue 69 may contribute to the lack of binding of XG-34 and XG-35 to xylan. However, it is not likely to be the sole determining factor as W69Y or F only reduces the affinity for xylan of the wild-type CBM4-2 by 3-fold (Cicortas Gunnarsson et al., manuscript in preparation). Rather, it is a combination of residues that modulates the XG/xylan selectivity. This implies that rational design of such selective binders may be difficult to achieve, whereas a combinatorial library and selection approach succeeded in this case.
Xyloglucan structures vary depending upon the species and tissue of origin but are united by a linear ß(14) glucan backbone that is extensively decorated at C-6 with -linked xylopyranosyl residues (Carpita and McCann, 2000). To describe the highly branched structure of the component oligosaccharides, a shorthand is widely used where G represents an unbranched Glc(ß14) unit, X = [Xyl(16)]Glc(ß14), L = [Gal(ß12)Xyl(16)]Glc(ß14), and F = [Fuc(12) Gal(ß12)Xyl(16)]Glc(ß14) (Fry et al., 1993). In general, two repeating backbone branching patterns are observed: XXXG or XXGG (Vincken et al., 1997; Vierhuis et al., 2001; Zhou et al., 2006) where the xylose residues may be further substituted to yield L and F units (Figure 8); O-acetylation of Gal is also observed (Kiefer et al., 1989; Vincken et al., 1997). The successful selection of phages bearing the XGBMs XG-34 and XG-35 was performed using the seed storage xyloglucan from T. indica that is composed of XXXG, XXLG, XLXG, and XLLG oligosaccharides (1.4:3:1:5.4) and is thus devoid of fucosyl residues (York et al., 1990). Interestingly, binding of these XGBMs, recombinantly expressed as free proteins, to T. indica xyloglucan was not inhibited by the FucXG from the primary walls of Rubus fructicosus. This FucXG contains the XXFG (Glc4Xyl3Gal1Fuc1) oligosaccharide block in addition to XXXG (Glc4Xyl3) (Joseleau et al., 1992), the latter of which is common to T. indica seed xyloglucan. It is therefore tempting to speculate that the observed selectivity for XG over FucXG perhaps resides in the binding of these XGBMs to side chain galactose epitopes which are masked or otherwise non-existent in R. fructicosus FucXG. Regardless, the evolved XGBMs provide a complimentary set of binding proteins to the monoclonal antibody CCRC-M1, which is specific to FucXGs. Curiously, attempts to select CBM variants which bound specifically only to FucXG were unsuccessful (data not shown), indicating that selection was dominated by interactions with structures common to both FucXG and XG in these experiments, much in analogy to the immunodominance of specific epitopes in many immune responses.
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Xyloglucan binds at the same binding site as xylan does in the wild-type protein, which was confirmed by the inhibition of binding to immobilized XG with xylopentaose (data not shown). Binding with a measurable affinity in the binding cleft of the wild-type CBM4-2 requires an oligosaccharide backbone length of at least five units (Simpson et al., 2002
). A similar demand for length of the backbone seems to exist for the interaction of XGBMs with XG, as they show no binding to a mixture of XGOs with a backbone length of four units and as selections performed on these short oligosaccharides did not generate any specific binders either (data not shown). This suggests that the ligand has to occupy the whole binding cleft and consequently interact with many residues for a selectable binding to occur. Also, the fact that the conserved residues found in the selected clones are distributed over a large part of the binding site (Figure 9) indicates that the entire cleft is involved in binding to XG.
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The low affinities displayed by the XGBMs for XG were not surprising considering the small size of the combinatorial library, 1.6 x 106 clones, from which they were selected. Natural xyloglucan-binding CBMs were reported to have somewhat higher affinities for XG with KA-values of 103–105/M (Najmudin et al., 2006
) but far from the same specificity as the selected XGBMs. Most biotechnological applications demand a high specificity of the binding proteins, which favors the selected modules, such as XG-34. Further engineering of the XGBMs can also be carried out to improve their affinities if needed for their use in different assays.
The XGBMs presented in this work and affinity-maturated variants of these modules comprise new tools for the detection of xyloglucans in plant tissues. It is anticipated that they will find application as histochemical reagents complimentary to CCRC-M1 antibody (Puhlmann et al., 1994) widely used for the spacial analysis of FucXGs in cell walls (Bourquin et al., 2002; Freshour et al., 2003). In particular, combining the use of CCRC-M1 and an engineered XGBM could generate a more complete picture of the distribution of FucXG and XG throughout plant tissues. We will report on the applications of these new XGBMs, as well as detailed protein structure–function studies, in due course.
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Materials and methods |
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Polysaccharides
Tamarind (T. indica) XG (Glc:Xyl:Gal:Ara = 45:35:16:4), barley ß-glucan, arabinoxylan, and xylopentaose were purchased from Megazyme (Bray, Ireland). Birchwood xylan, oat spelt xylan, and cellopentaose were from Sigma-Aldrich (St Louis, MO), and Avicel was purchased from Merck (Darmstadt, Germany). FucXG from 24-day-old suspension-cultured R. fructicosus (blackberry) cells (Chambat et al., 1997
) was a kind gift from Gérard Chambat (CERMAV-CNRS, Grenoble, France). The sample provided was produced as described in Chambat and others (2005) and had the following monosaccharide composition (mol %): Glc, 40; Xyl, 34; Gal, 15; Fuc, 4.5; Ara, 3.0; Man, 3.5 [the presence of Man is because of contaminating galactoglucomannan (Chambat et al., 2005)].
Xyloglucan–biotin conjugates
A mixture of XGO–biotin conjugates (composed of XXXG, XLXG, XXLG, and XLLG in the molar ratio 15:7:32:46) was prepared from tamarind XG via endoglucanase digestion, reductive amination, and N-acylation, as described previously (Brumer et al., 2004). Xyloglucan–biotin conjugates were subsequently prepared by XET-mediated incorporation of XGO–biotin into both Tamarind and Rubus xyloglucan using heterologously expressed XET 16A from Populus tremula x tremuloides (Bollok et al., 2005; Kallas et al., 2005), essentially as described in Brumer et al. (2004). A sample (50 mL total volume) containing a mixture of Tamarind XG (1 g/L, Mw 4.5 x 105, Mw/Mn 1.5, where Mw is the weight average molecular weight and Mn is the number average molecular weight), XGO–biotin (0.5 g/L), and XET (100 U) in sodium acetate buffer (20 mM, pH 5.5) was incubated at 30°C for 60 min. The reaction was terminated by heating (80oC, 10 min), and the denatured XET was removed by centrifugation at 12,000 x g for 20 min. Then, 100 mL of ethanol was added to precipitate XG–biotin, while the XGO–biotin remained in the supernatant. After filtration with a Whatman GF/A glass microfiber filter, the precipitate was dried under vacuum, redissolved in water, and lyophilized. The XG–biotin produced in this manner had an Mw value of 1.57 x 104 (Mw/Mn 1.6). The FucXG–biotin conjugate (Mw 1.74 x 104, Mw/Mn 1.4) was prepared in an identical manner from Rubus XG (Mw 2.63 x 104, Mw/Mn 1.7), except that the enzyme reaction time was 30 min. Gel permeation chromatography was performed as described by Brumer and others (2004).
Strains and vectors
Escherichia coli strain Top10F’ (Invitrogen, Paisley, UK) was used as host for phage display work together with a variant of the phagemid pFab5c.His (Engberg et al., 1996) carrying the part of gene III encoding only the final C-terminal domain of M13 protein 3. Genes coding for selected CBMs were inserted in the expression vector pET-22b(+) (Novagen, Madison, WI) and then transformed into a cloning host, E. coli strain XL1-Blue (Stratagene, La Jolla, CA). Production of recombinant protein was achieved using E. coli strain BL21(DE3) (Novagen).
Phage selections
Before the selections, phage stocks were prepared using helper phages VCSM13 (Stratagene), as described previously (Cicortas Gunnarsson et al., 2004). The XG–biotin (around 2 µg) was first bound to 3.4 x 106 streptavidin-coated Dynabeads (Dynal A/S, Oslo, Norway) by incubation in 500 µL of blocking buffer [phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA) and 0.05% (v/v) Tween 20] for 30 min on an end-over-end rotator at room temperature. During this time, 500 µL of the phage stock (around 1011 phage particles, displaying different variants of the CBM molecule) was pre-incubated with the same amount of streptavidin-coated Dynabeads and 500 µL of 2x blocking buffer, in some cases containing the soluble part of 8 mg birchwood xylan (Sigma-Aldrich) to remove any potential streptavidin-binding CBMs and to block xylan-binding CBMs from being selected. The XG–biotin-coated beads were then washed four times using blocking buffer before the phage containing supernatant from the pre-incubation was added. The mixture was incubated for 1 h on an end-to-end rotator at room temperature, and the beads were subsequently washed four times with blocking buffer and twice with PBS. Bound phages were eluted using 100 µL of trypsin (0.5 mg/mL) (Invitrogen) (Johansen et al., 1995) by incubation for 30 min at room temperature with shaking. After removing the beads, the activity of trypsin was neutralized by addition of 100 µL of aprotinin (0.1 mg/mL) (Roche Diagnostic Corporation, Indianapolis, IN). A few microliters of the elution solution was saved for titration experiments, whereas the rest was used to transfect exponentially growing E. coli Top10F’ (about 10 mL of OD600 
0.5) for preparation of new phage stocks. Both input and output phages were titrated on exponentially growing E. coli Top10F’. The selection process was repeated three times before clones were picked for analysis.
Protein production and purification
Genes encoding CBM variants were cloned in-between the NdeI and XhoI sites found in the pET-22b(+) vector. Production of CBM carrying a C-terminal hexa-histidine tag in BL21(DE3) and purification of the soluble protein using metal-ion-affinity chromatography have been described earlier (Cicortas Gunnarsson et al., 2004). The concentration of each purified protein was calculated from A280 measurements using extinction coefficients calculated for each CBM variant (Gill and von Hippel, 1989).
Enzyme-linked immunosorbent assay
Phage stocks of individual clones from the selections were prepared and used in ELISA experiments to screen the specificity of the selected CBM variants. Ninety-six-well microtiter plates were coated with soluble carbohydrates in PBS or with 1 µg/mL of streptavidin in PBS at 4°C overnight. After washing the plates with washing buffer [154 mM NaCl and 0.05% (v/v) Tween 20 in PBS], 0.5 µg/mL of XG–biotin or FucXG–biotin in PBS was added to the streptavidin-coated plates, and these were then incubated for 45 min at 37°C. A second washing step was followed by addition of phages diluted in blocking buffer, and the plates were incubated for 2 h at room temperature. Plates were washed, and horseradish peroxidase (HRP)-conjugated anti-M13 antibody (Amersham Pharmacia Biotech Inc., Piscataway, NJ) diluted in blocking buffer was added to the wells and incubated for 1 h at room temperature. For detection, 0.67 mg/mL of o-phenylenediamine and 0.012% H2O2 in 35 mM citrate and 67 mM phosphate buffer, pH 5, was added. The reaction was stopped by addition of H2SO4 to a final concentration of 0.6 M, and A490 was measured.
Competitive ELISA was performed by incubating the soluble CBM or the CCRC-M1 antibody (CarboSource, Athens, GA) with different concentrations of soluble biotinylated XG or FucXG or with xylopentaose or cellopentaose for 2 h at 37°C before the addition to the plates coated with streptavidin and saturated with XG–biotin or FucXG–biotin (see former paragraph). HRP-conjugated anti-His6 antibody (Roche Diagnostic Corporation) was used to detect the CBM that had bound, whereas HRP-conjugated rabbit anti-mouse immunoglobulins (DAKO A/S, Glostrup, Denmark) were used to detect binding of CCRC-M1 to the plate. The detection method followed the one described above.
Affinity electrophoresis
The AE method was run in the Bio-Rad (Hercules, CA) mini-gel apparatus as earlier described for the wild-type CBM4-2 (Abou Hachem et al., 2000). Purified CBM variants (3 µg per gel) were separated on native gels, containing none or different concentrations of tamarind seed XG, barley ß-glucan, or oat spelt xylan. The gels were run at an ambient temperature of 4°C, which resulted in a temperature of 22–25°C in the inner chamber of the gel apparatus. A kaleidoscope pre-stained standard (Bio-Rad) was included in each gel, and the proteins were detected by staining with Simply Blue Safe stain (Invitrogen). The concentration of XG in the gels ranged from 0.2 to 2 g/L, and affinity constants were calculated for this ligand according to the theory of AE as described by Takeo (1984). The relative mobilities of a CBM variant, r (in the presence of ligand) and R (in the absence of ligand), versus BSA used as a negative, non-interacting, control were calculated from the migration distances, the distances between the major protein band and the migration front of the gel. The –1/KD value was obtained as the x-intercept of a straight line in a plot of 1/(R – r) versus 1/c according to the relationship:
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where c is the ligand concentration in the gel.
Isothermal titration calorimetry
ITC measurements were made at 25°C following standard procedures (Flint et al., 2004) using a Microcal Omega titration calorimeter. Proteins were extensively dialyzed against 20 mM of Hepes–HCl buffer, pH 8.0, and the ligands [xylopentaose and xyloglucan (Megazyme)] were dissolved in the same buffer to minimize the heat of dilution. During a titration experiment, the protein sample (50 µM), stirred at 300 rpm in a 1.4331-mL reaction cell, was injected with a single 1 µL aliquot, followed by 27 successive 10 µL aliquots of ligand comprising 0.7 mM of xylopentaose or 0.5% xyloglucan at 300-s intervals. Integrated heat effects, after correction for heats of dilution, were analyzed by nonlinear regression using a single-site binding model (Microcal Origin, version 7.0).
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Conflict of interest statement |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
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None declared.
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Acknowledgments |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
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The study was supported by a grant from the Swedish Research Council. The CCRC-M1 antibody was made available to us in part supported by NSF grant RCN-0090281. H.B. is a Fellow (Rådforskare) of the Swedish Research Council.
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Abbreviations |
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AE, affinity electrophoresis; CBM, carbohydrate-binding module; ELISA, enzyme-linked immunosorbent assay; FucXG, fucosylated xyloglucan; HRP, horseradish peroxidase; IPTG, isopropyl-ß-D-thiogalactoside; ITC, isothermal titration calorimetry; Mn, number average molecular weight; Mw, weight average molecular weight; PBS, phosphate-buffered saline; XET, xyloglucan endo-transglycosylase; XG, non-fucosylated xyloglucan; XGBM, xyloglucan-binding module; XGO, xyloglucan oligosaccharide
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References |
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