Differential recognition of plant cell walls bymicrobial xylan-specific carbohydrate-binding modulesLesley McCartney*, Anthony W. Blake*, James Flint†, David N. Bolam†, Alisdair B. Boraston‡, Harry J. Gilbert†§,and J. Paul Knox*§

*Centre for Plant Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom; †Institute for Cell and Molecular Biosciences, University ofNewcastle-upon-Tyne, Newcastle-upon-Tyne NE2 4HH, United Kingdom; and ‡Department of Biochemistry and Microbiology, Universityof Victoria, Victoria, BC, Canada V8W 3P6

Edited by Maarten J. Chrispeels, University of California at San Diego, La Jolla, CA, and approved January 17, 2006 (received for review October 11, 2005)

Glycoside hydrolases that degrade plant cell walls have complexmolecular architectures in which one or more catalytic modules areappended to noncatalytic carbohydrate-binding modules (CBMs).CBMs promote binding to polysaccharides and potentiate enzymichydrolysis. Although there are diverse sequence-based families ofxylan-binding CBMs, these modules, in general, recognize bothdecorated and unsubstituted forms of the target polysaccharide,and thus the evolutionary rationale for this diversity is unclear.Using immunohistochemistry to interrogate the specificity of sixxylan-binding CBMs for their target polysaccharides in cell wallshas revealed considerable differences in the recognition of plantmaterials between these protein modules. Family 2b and 15 CBMsbind to xylan in secondary cell walls in a range of dicotyledonspecies, whereas family 4, 6, and 22 CBMs display a more limitedcapability to bind to secondary cell walls. A family 35 CBM, whichdisplays more restricted ligand specificity against purified xylansthan the other five protein modules, reveals a highly distinctivebinding pattern to plant material including the recognition ofprimary cell walls of certain dicotyledons, a feature shared withCBM15. Differences in the specificity of the CBMs toward walls ofwheat grain and maize coleoptiles were also evident. The variationin CBM specificity for ligands located in plant cell walls provides abiological rationale for the repertoire of structurally distinct xylan-binding CBMs present in nature, and points to the utility of thesemodules in probing the molecular architecture of cell walls.

P lant cell walls are highly complex macromolecular structuresthat consist of repertoires of polysaccharides that interact

with each other through extensive networks (1). With the annualrecycling of an estimated 100 billion tons of plant biomass, thedegradation of cell walls by microbial enzymes is an importantbiological and industrial process (2). The intimate association ofpolysaccharides within cell walls limits the access of hydrolyticenzymes that attack these composite structures, and thus thedegradative process is relatively slow (3). To reduce the ‘‘acces-sibility problem,’’ plant cell wall hydrolases have a complexmolecular architecture in which catalytic modules are appendedto noncatalytic carbohydrate-binding modules (CBMs), which,by binding to specific polysaccharides, potentiate the activity ofthese enzymes against insoluble substrates such as cellulose,mannan, and xylan (4). CBMs are grouped into sequence-basedfamilies, although they have also been classified into ‘‘types’’based on the topology of the binding site, which reflects thenature of the target ligand (4). The binding site of type A CBMscomprises a planar hydrophobic surface which recognizes crys-talline polysaccharides such as cellulose, chitin and unsubsti-tuted mannan. The binding site clefts of type B CBMs accom-modate single chains of a variety of polysaccharides, whereas thespatial restriction of the binding site of type C CBMs limits ligandrecognition to mono- or disaccharides. Although the ligandspecificity of type A CBMs is highly conserved, the sugarpolymers recognized by different members of type B families canbe very diverse. For example, the CBM4 and CBM6 families bothcontain members that recognize xylan, �-1,4-glucan and �-1,3-

glucan (5–8), whereas the CBM35 family contains proteins that,currently, have been shown to bind to xylan and mannan (9, 10).

One of the intriguing features of CBMs is the diversity offamilies that contain members which apparently display the sameligand specificity against purified polysaccharides and oligosac-charides. Thus, CBMs located in families 1, 2a, 3a, 5, and 10 bindto crystalline cellulose, whereas CBMs from families 2b, 4, 6, 15,22, 35, and 36 all contain protein modules that recognize xylan(4). Recent in vitro studies have started to unravel the possiblebiological significance for the overlapping specificities displayedby the different CBM families. Carrard et al. (11) showed thatType A CBMs in families 1, 2a, and 3a could target differentregions of purified crystalline cellulose, whereas McLean et al.(12), employing f luorescently tagged CBM4, CBM17, andCBM28, showed that these modules recognize distinct substruc-tures within amorphous cellulose. However, these studies havebeen restricted to defining the specificities of CBMs for purifiedpolysaccharides that have an invariant chemical structure of�-1,4-linked glucose moieties. The specificity of CBMs forpolysaccharides embedded within plant cell walls, the substrateavailable to glycoside hydrolases during the initial rate limitingphase of the degradative process, is currently unknown.

Here we interrogate the ligand specificity of CBMs thatrecognize xylan, a �-1,4-linked xylose polymer, which can bedecorated with glucuronosyl or 4-O-methyl-glucuronosyl resi-dues at O2 and �-arabinofuranosyl or acetyl groups at O2 or O3,with the nature and extent of substitution varying betweenspecies and cell type (1, 13). We have selected six CBMs, locatedin families 2b, 4, 6, 15, 22, and 35, listed in Table 1, which displayspecificity for purified xylans (6, 10, 14–17). The data presentedin this study show that there is significant variation in thespecificity of the different CBMs for plant cell walls, indicatingthat the structure and�or context of the xylans embedded inthese composite structures is a critical specificity determinant forthese protein modules. This variation in specificity for ligandslocated in cell walls provides a biological rationale for therepertoire of structurally distinct xylan-binding CBMs present innature.

Results and DiscussionSpecificity of CBMs for Isolated Polysaccharides. In vitro bindingstudies using isothermal titration calorimetry (ITC) and affinitygel electrophoresis have shown that CBMs from several differentfamilies bind to purified xylans and xylooligosaccharides (refs. 6,10, and 14–19 and Table 2). For a summary of the composition

Conflict of interest statement: No conflicts declared.

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: CBM, carbohydrate-binding module; ITC, isothermal titration calorimetry;OSX, oat spelt xylan; MGX, methylglucuronoxylan; RAX, rye arabinoxylan; GAX, glucu-ronoarabinoxylan.

§To whom correspondence may be addressed. E-mail: h.j.gilbert@ncl.ac.uk or j.p.knox@leeds.ac.uk.

© 2006 by The National Academy of Sciences of the USA

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of the xylans used in this study, see Table 3, which is publishedas supporting information on the PNAS web site. These xylan-binding CBMs are derived from a range of thermophilic andmesophilic prokaryotic xylanases (Table 1). CBM6, CBM15, andCBM22-2 do not distinguish between the largely undecoratedoat spelt xylan (OSX; percentage molar composition: 93% Xyl,6% Ara), xylohexaose, which contains no side chains, and thedecorated xylans methylglucuronoxylan (MGX; 80% Xyl, 18%MeGlcA) and rye arabinoxylan (RAX; 50% Xyl, 50% Ara).However CBM4-2 binds considerably more tightly to xylo-hexaose than decorated xylans and even OSX, which contains alow density of side chains (Table 2). Although CBM2b-1-2displays similar affinity for OSX as CBM4-2, it binds �5- and�10-fold less tightly to RAX and MGX, respectively. CBM35displays no binding to arabinoxylan or methylglucuronoxylan,exhibiting a preference for an unsubstituted form of the poly-saccharide. In addition, this module is unique in displaying noaffinity for xylohexaose. The actual affinity of the six CBMs forpurified xylans (Table 2) reflects their microbial origin; proteinsfrom thermophilic bacteria bind more tightly to their targetligands (CBM4-2, CBM6, and CBM22-2) (6, 16, 17) than indi-vidual CBM2b modules, CBM15 or CBM35, which are derivedfrom mesophilic prokaryotes (10, 15, 17). The relatively highaffinity displayed by CBM2b-1-2 is due to the avidity effect ofjoining two CBMs together that bind to polysaccharides, whichcontain multiple protein binding sites (15, 20). BecauseCfXyn11A contains both CBM2b-1 and CBM2b-2, the avidityeffect displayed by the CBM2b-1-2 construct also occurs in themultimodular enzyme from which they are derived.

The binding profile of the CBMs were also screened by usingmacroarrays in which the polysaccharides are arrayed on nitro-cellulose filters. The specificity of the CBMs for the arrayedpolysaccharides were similar to the ligand specificities deter-mined by ITC (data not shown). Significantly, however, themacroarray screen revealed that CBM35 recognized a sample ofglucuronoarabinoxylan (GAX; 51% Xyl, 30% Ara, 7% GlcA)derived from maize (21). The binding of CBM35 to maize GAX

was quantified by ITC which indicated it had an affinity for GAXthat was similar to OSX (Table 2).

To summarize, although the binding profile of CBM6,CBM15, and CBM22 to decorated xylans such as RAX andMGX and to the largely unsubstituted OSX and xylohexaose arebroadly similar, CBM4, CBM35, and CBM2b-1-2 display signif-icant variation in the specificity for these ligands. Although thebinding profile of the CBMs for purified ligands reveals simi-larities and differences in the specificities of these proteinmodules, the biological significance of these in vitro data areunclear. To address this issue, we have used an immunohisto-chemical approach to interrogate the specificity of xylan-bindingCBMs toward plant materials. This method exploits the anti-genic recognition of the His-tag appended to these proteins asdescribed (22).

Binding of Xylan-Binding CBMs to Dicotyledon Cell Walls. The capac-ity of the xylan-binding CBMs to bind to cell walls in sections ofplant materials from diverse taxonomic groups was assessed byusing immunohistochemistry. In dicotyledons, low substitutedxylans and MGX are abundant in the secondary cell walls ofspecific cell types such as xylem vessels and sclerenchyma fibers(1, 13, 23). The monoclonal antibody LM11, which binds topurified unsubstituted xylans and arabinoxylan, specifically rec-ognizes secondary cell walls in all dicotyledons so far examinedand shows no recognition of dicotyledon primary cell walls (23).In a survey of CBMs binding to sections of a range of plantmaterials CBM2b-1-2 shows a similar binding capability to LM11and bound specifically to secondary cell walls, including those ofxylem vessels and sclerenchyma fibers associated with phloem asshown in Fig. 1.

In contrast to CBM2b-1-2, however, the other xylan-bindingCBMs (except CBM15, see below) showed significant differ-ences in their capacity to bind to cell walls in these same threedicotyledon species when incubated with equivalent sectionsunder the same conditions (Fig. 1). Each CBM bound effectivelyand specifically to secondary cell walls in at least one species, butwas very weak or absent in others. CBM4-2 bound to xylemvessel cell walls of f lax but not to the phloem fibers in this speciesand showed very weak�no recognition of secondary cell walls intobacco and pea (Fig. 1). CBM6 bound effectively to secondarycell walls of both xylem vessels and phloem fibers in flax andxylem cells in pea, but very weakly to tobacco sections. CBM22-2displayed the least recognition of secondary cell walls and boundeffectively to pea stem but only weakly to walls of xylem vesselsof f lax and tobacco stems. The inability of certain CBMs torecognize xylan in some species is likely to reflect the context ofxylan in different cell walls. Family 2b, 4, 6, and 22 CBMs can

Table 1. Origin of the xylan-binding CBMs used in this study

Protein Origin Organism Reference

CfCBM2b-1-2 CfXyn11A C. fimi 15RmCBM4-2 RmXyn10A R. marinus 14CtCBM6 CtXyn11A C. thermocellum 6CjCBM15 CjXyn10C C. japonicus 17CtCBM22-2 CtXyn10B C. thermocellum 16CjCBM35 CjAbf62A C. japonicus 10

Table 2. Binding of CBMs to purified ligands as determined by ITC

Protein OSX RAX MGX GAX Xylohexaose Cellohexaose

CfCBM2b-1-2 51.6 11.4 5.7 – 0.6* �0.01RmCBM4-2 47.1 30.0 39.6 – 270.0 0.7CtCBM6† 5.9 7.0 10.2 – 18.9 0.15CjCBM15‡ 1.3 0.5 0.8 – 2.0 0.2CtCBM22§ 7.6 11.0 – – 15.0 0.08CjCBM35¶ 4.1 NB NB 5.9 NB NB

Values are association constants (M�1 � 104). –, Not determined; NB, no binding detected.*Affinity for xylohexaose of CBM2b-1-2 is the same as that for individual CBM2b-1 or CBM2b-2 proteins. Theenhanced affinity for xylan observed for CBM2b-1-2 is due to cooperative binding, or avidity, through the bind-ing of the linked modules to the same polysaccharide chain. This cannot occur with xylohexaose because themolecule is too small to bind to both CBM molecules at the same time (15).

†Data from ref. 6.‡Data from ref. 17.§Data from ref. 16.¶Data from ref. 10.

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readily recognize isolated xylan polymers (although CBM4-2does display a preference for completely unsubstituted ligandssuch as xylohexaose) and xylan is clearly present in all of thesecondary cell walls in these materials as indicated by the bindingof LM11 and CBM2b-1-2 (Fig. 1).

CBM15 and CBM35 were distinct from the other CBMs in thatthey bound to some dicotyledon primary cell walls. CBM15bound to all secondary cell walls, but also recognized the primarycell walls in the flax section (Fig. 1) and some primary cell wallsin tobacco stem (Fig. 2). CBM35 bound specifically to thesecondary cell walls of pea stem, and to both primary andsecondary cell walls of f lax in a manner similar to CBM15 (Fig.1). However, in tobacco, CBM35 did not bind to all secondarycell walls of xylem vessels but did interact with the primary cellwalls of adjacent parenchyma, indicating clearly contrastingrecognition of cell walls by this CBM in the three dicotyledonspecies. The variation in tobacco stem recognition displayed byCBM2b-1-2, CBM15, and CBM35 is shown for wider areas oftissues in Fig. 2, which demonstrates the secondary cell wallspecificity of CBM2b-1-2 and the binding of CBM15 to primarycell walls in cortical parenchyma in addition to secondary cellwalls. CBM35 displays a highly distinctive pattern of cell recog-nition with strong binding to epidermal cell walls and cellsassociated with regions of internal phloem. Xylans are known tobe a taxonomically variable set of polymers, and biochemicalanalysis have indicated that primary cell walls of dicotyledons

have low levels of xylans that are of the GAX type (24). Therecognition of primary cell walls by CBM15 and CBM35 incertain species and cell types is likely to reflect low levels of GAXin these walls. This finding is consistent with affinity gel elec-trophoresis data showing that both CBM35 and CBM15 bind toGAX in vitro (data not shown).

Binding of Xylan-Binding CBMs to Cell Walls of Graminaceous Mono-cotyledons. Cell walls of the commelinoid monocotyledons,which includes the grasses and cereals, are distinctive in thatabundant heavily substituted GAXs are major components ofprimary cell walls (13, 25). An additional distinctive feature ofcereal grains is the abundance of neutral arabinoxylans in theendosperm.

The xylan-binding CBMs displayed differences in their capac-ity to bind to cell walls when incubated with sections of wheatgrain and maize coleoptiles (Fig. 3). CBM2b-1-2, CBM4-2,CBM6, CBM15, and CBM22-2, but not CBM35, bound to wheatendosperm. This binding likely reflects the in vitro recognitionof arabinoxylan by the CBMs. The CBMs showed differentialrecognition of the cell walls of the aleurone layer and theautofluorescent pericarp cell layers. CBM2b-1-2 and CBM15bound to the aleurone cell walls, whereas the binding ofCBM35 was restricted to a specific cell layer of the inner pericarp(Fig. 3).

A transverse section of the maize coleoptile reveals centraldeveloping leaves surrounded by a sheath of parenchyma cells.CBM4-2 showed no recognition of cell walls in either of theseorgans (Fig. 3). CBM2b-1-2 and CBM35 bound to cell walls ofdeveloping leaves but not to those of the surrounding coleoptilesheath, whereas CBM6 and CBM22-2 bound to all cell walls inboth leaves and sheath, although more effectively to cell walls ofthe developing leaves (Fig. 3). CBM15 displayed strong recog-nition of all primary cell walls in both sheath and leaves.

Topology of CBMs. This study shows that three of the xylan-bindingCBMs (CBM6, CBM15, and CBM22), which display similarspecificities against purified ligands, exhibit considerable varia-tion in their recognition of plant cell walls. Even more intriguingis the observation that although CBM4-2 exhibits tighter bindingto purified xylans than CBM6, CBM15, and CBM22, its capacityto recognize the polysaccharide within plant cell walls is limited.By contrast, CBM2b-1-2, which has a similar binding profile toCBM4-2 for purified undecorated xylan (but binds less tightly toarabinoxylan and MGX), displays more extensive recognition ofplant cell walls than the Rhodothermus marinus protein.

The differences in binding profiles of the CBMs in vivo arelikely to reflect a combination of the accessibility of the targetligands within the cell wall composites and fine details of xylan

Fig. 1. Indirect immunofluorescence microscopy of CBMs and monoclonalantibody LM11 binding to secondary cell walls of vascular tissues in transversesections of tobacco, pea, and flax stems. Binding to phloem sclerenchymafibers is indicated by arrows. X, binding to secondary cell walls of xylem vessels.(Scale bars, 100 �m.)

Fig. 2. Indirect immunofluorescence microscopy of CBM2b-1-2, CBM15, andCBM35 binding to transverse sections of tobacco stem showing cortical pa-renchyma (cp) and pith parenchyma (pp) in addition to vascular tissues. e,epidermis; p, phloem fibers; x, xylem vessels. Arrow pairs indicate files ofdeveloping xylem vessel elements. CBM2b-1-2 binds to these. CBM35 binds tofiles of parenchyma cells between them. Large arrowhead indicates cellsassociated with internal phloem. (Scale bars, 100 �m.)

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structure. The CBMs most suited to bind xylans that are incomplex macromolecular structures are predicted to be thosethat accommodate their respective ligand in shallow clefts,because the exposed binding sites are more likely to be able tointeract with xylan chains that are intimately associated withother cell wall components. Inspection of the 3D structures ofthe five xylan-binding CBMs provides some insight into theinfluence of binding site topology on the access to xylan withincell walls (Fig. 4). Thus, the tandem CBM2b modules thatcomprise CBM2b-1-2, which binds effectively to all dicotyledonsecondary cell walls, have a very shallow xylan binding site (15,18), which places the protein modules at the cusp between typeA CBMs, in which the ligand binding site is a planar hydrophobicsurface, and type B CBMs, where single sugar polymer chains areaccommodated in clefts (4). By contrast, the binding clefts ofCBM4-2, CBM22-2 and CBM6, which display limited recogni-tion of xylan embedded in plant cell walls, are much deeper thanCBM2b-1-2 (6, 16, 26) (Fig. 4).

However, the hypothesis that the depth of the binding cleft isrelated to the capacity of the CBM to bind to xylans embeddedin the plant cell wall is less tenable when applied to CBM15.Although this protein binds to numerous cell walls, the dimen-sions of its xylan-binding site (17) are similar to CBM4-2, CBM6,

and CBM22, which display limited recognition of secondary cellwalls of dicotyledons. Thus, the capacity of CBMs to interactwith cell walls is not exclusively dependent on the depth of thecleft. The binding clefts of CBM2b-1-2 and CBM15 have beendescribed as twisted platforms where single aromatic amino acidside chains in the binding subsites stack only against the � facesof sugars in the polymer, in these cases sugars n and n � 2 (4,15, 17, 18). In contrast, the binding sites of CBM4-2, CBM6, andCBM22 contain a primary binding subsite with two aromaticamino acid side chains that sandwich the same sugar ring, ratherlike ‘‘aromatic tongs,’’ thus interacting with both the � and �faces of a single sugar (6, 16, 26). Studies have shown thatremoval of either of these aromatic residues completely abro-gates xylan binding in CBM2b, 6, 15, and 22 (6, 19, 27, 28). Withinthe complex milieu of the plant cell wall, it is likely that at leastone face of the xylose residues in the xylan backbone interacts

Fig. 3. Indirect immunofluorescence microscopy of xylan-binding CBMsinteracting with sections of wheat grain and maize coleoptiles. Arrows indi-cate recognition of aleurone cell walls. Double arrowheads indicates CBM35recognition of a cell layer of inner pericarp. p, pericarp; e, endosperm; dl,developing leaves; cs, coleoptile sheath. (Scale bars, 100 �m.)

Fig. 4. Three-dimensional structures of xylan-binding CBMs CBM2b-1-2,CBM4-2, CBM6, CBM15, and CBM 22-2. (Left) Top views of the binding cleft ofeach CBM. (Right) The same cleft viewed from the side. Bound oligosaccharideis shown in the clefts of CBM6 and CBM15. Key aromatic residues in thebinding sites of the CBMs are colored green, illustrating the directly opposing‘‘aromatic tongs’’ of CBMs 4, 6 and 22, in contrast to the ‘‘twisted platform’’binding site of CBM2b and -15.

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with other polysaccharides providing an explanation for why theCBMs that contain the pair of aromatic tongs do not bind toxylan when it is embedded in the plant cell wall. By contrast, itis likely that cell wall xylans will remain accessible to CBM2b-1-2and CBM15 as only a single face of the xylose residues stackagainst the two tryptophans in the binding site of these proteins.Thus, we propose a model in which the capacity of CBMs to bindxylans that are intimately associated with other components ofthe cell wall depends on the topological arrangement of thearomatic residues that stack against the sugars in the polysac-charide backbone.

However, the structural basis for the different specificities ofthe CBMs toward cell walls is more subtle than simply the depthof the binding cleft or whether the two key aromatic residuesinvolved in saccharide recognition are adjacent to each other. InCBM22-2 and CBM4-2, which are most restricted in terms ofdicotyledon xylan recognition, the binding site is located on theconcave face of the jelly-roll fold, whereas in CBM6 the ligandis accommodated between the loops connecting the two �-sheetsof the protein, which may impart more flexibility on the bindingsite (6); CBMs are generally considered to be rigid proteins thatdo not undergo substantial conformational change upon ligandbinding (4). However, CBM6 displays considerable structuralf lexibility. For example, when bound to xylopentaose, there is apositional shift in residues 25–27, which brings Ser-26 (shifted by4.1 Å) closer to the sugar at subsite 1 facilitating the interactionbetween the hydroxy amino acid and O4 of the xylose at thissubsite (6). Although such conformational changes are likely toincur an energetic cost, which will lead to a reduction in overallaffinity, the movement of the protein may reflect the heteroge-neity of the xylan decorations. Thus, although the movement ofSer-26 is optimal for binding unsubstituted xylooligosaccharides,this amino acid may need to be more distant from the bindingcleft to accommodate xylans that are decorated or in contactwith other polysaccharides. An explanation for the difference inspecificity of CBM22-2 and CBM4-2 for pea and flax secondarycell walls and wheat and maize coleoptiles is not readily availablebecause the two proteins display the same fold and the bindingsite is conserved, as are the key aromatic residues that stackagainst the bound sugar rings (26, 27). It is apparent that subtledifferences in the binding site of CBMs that target xylans canhave dramatic effects on their capacity to recognize their ligandswithin the complex milieu of cell walls of diverse taxonomicorigin.

CBM35 displays a significant difference in ligand specificity tothe other CBMs evaluated in this study. Although CBM6,CBM4-2, CBM15, and CBM22-2 bind to both unsubstitutedxylan and arabinoxylan, CBM35 displays a strong preference forunsubstituted xylan polymers, compared to MGX and RAX (10)but, in addition, shows significant recognition of GAX (the likelytarget ligand of this module in primary cell walls, see above).Therefore, it is likely that the decorations in GAX are eitherrelatively sparse compared to RAX and MGX, or the substitu-tions are present in discrete blocks thus presenting significantregions of undecorated xylan, although it is also possible thatCBM35 recognizes the specific substitutions in GAX. Althoughthe variation in binding of CBM35 to different cell walls may, inpart, reflect the accessibility of the polysaccharide, it also is likelyto be a consequence of the unusual ligand specificity displayedby this protein.

Conclusion. Although it is well established that xylan-bindingCBMs are located in several different sequence-based families,the biological significance for this structural diversity was un-clear. Here we show that CBMs in different families displaysignificant variation in specificity for xylans in both primary andsecondary cell walls, providing a biological rationale for thisstructural diversity. Different CBMs therefore have the capacity

to target appended catalytic modules to specific cell walls indiverse species. CBM15 and CBM2b-1-2 confer the widestsubstrate specificity, whereas the CBMs derived from thermo-philic bacteria recognize a more restricted range of cell walls. Itis possible that in thermophilic environments the elevatedtemperature mediates the rapid breakdown of the intricatestructure of the plant cell wall increasing the accessibility of thexylan. In view of the variation in specificity displayed by thedifferent CBMs it is interesting that very few xylanases containmultiple xylan-binding CBMs from distinct families. Two xyla-nases�arabinofuranosidases from Caldicelluorupter contain aCBM6 and CBM22-2 (29), whereas a Paenibacillus xylanasecontains a CBM6 and a CBM36 (which also binds to xylan) (30).In contrast, numerous xylanases contain distinct xylan- andcellulose-binding CBMs (10). Thus, in nature, individual xyla-nases may target specific cell walls. However, as wall degradationprogresses, the CBMs will become effective with a wider rangeof substrates as indicated by their recognition of isolated poly-mers. From a biotechnological perspective, the portfolio ofspecificities displayed by CBMs can inform and direct enzymebioengineering strategies. Finally, these CBMs also representvaluable tools with which to probe the intricate architecture ofplant cell walls, because they not only identify xylans but alsoprovide information on the context of these polysaccharideswithin cell walls.

Materials and MethodsProduction of Xylan-Binding CBMs and LM11 Monoclonal Antibody.The CBMs used in this study, which are listed in Table 1, are asfollows: CBM2b-1-2 contains an N-terminal His-10-tag andcomprises CBM2b-1 and CBM2b-2 derived from the Cellulomo-nas fimi xylanase CfXyn11A, which are joined by the enzyme’sproline�threonine linker. The construction of CBM2b-1-2, itsexpression, and its purification were carried out as describedpreviously (15). CBM4-2 contains an N-terminal His-10-tag andthe C-terminal family 4 CBM from the R. marinus xylanaseRmXyn11A and was produced by the method of Abou Hachemet al. (14). CBM15, a component of the Cellvibrio japonicusxylanase CjXyn10C, contains an N-terminal His-10-tag and wasgenerated by following the protocol of Szabo et al. (17). CBM6,CBM22-2, and CBM35 all contain a C-terminal His-6-tag, arederived from Clostridium thermocellum CtXyn11A, Clostridiumthermocellum CtXyn10B, and C. japonicus Xyn10B, respectively,and were produced following the methods of Czjzek et al. (6),Charnock et al. (16), and Bolam et al. (10), respectively. Thegeneration of the anti-xylan monoclonal antibody (LM11), whichrecognizes both decorated and undecorated xylans, was de-scribed (23).

Polysaccharides. 4-O-methyl-D-glucurono-D-xylan and OSX wereobtained from Sigma. Soluble OSX was generated by treating thexylan with alkali followed by neutralization using the method ofGhangas et al. (31). Rye arabinoxylan was obtained fromMegazyme International (Bray, Ireland). Glucuronoarabinoxy-lan (GAX) from maize kernels (21) was kindly provided by LucSaulnier (Institut National de la Recherche Agronomique,Nantes, France).

CBM Assays on Nitrocellulose. Nitrocellulose-based assays of CBMbinding to polysaccharides were carried out as described byMcCartney et al. (22). His-tagged CBMs were incubated withnitrocellulose arrays at 2.5 �g�ml in PBS with 5% (wt�vol) milkprotein (PBS�MP).

ITC. ITC measurements were made at 25°C following standardprocedures (10) using a Microcal Omega titration calorimeter.Proteins were dialyzed extensively against 50 mM sodium phos-phate buffer (pH 7.0), and the ligand was dissolved in the same

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buffer to minimize heats of dilution. During a titration experi-ment, the protein sample (20–200 �M), stirred at 300 rpm in a1.4331-ml reaction cell maintained at 25°C, was injected with 29successive 10-�l aliquots of ligand comprising 1–4 mg�ml ofpolysaccharide or 0.5–10 mM oligosaccharide at 200-s intervals.Integrated heat effects, after correction for heats of dilution,were analyzed by nonlinear regression using a single site bindingmodel (Microcal Origin, version 7.0).

Preparation of Plant Materials and Labeling for CBM and Immunoflu-orescence Microscopy. Pea seedlings (Pisum sativum L.) weregrown at 18°C for 3–4 weeks, and tobacco plants (Nicotianatabacum L.) were grown at 24°C for 6–7 weeks under a regimeof 16 h light and 8 h dark. Regions of stem were excised andimmediately fixed in PEM buffer (50 mM Pipes�5 mM EGTA�5mM MgSO4, pH 6.9) containing 4% paraformaldehyde. Maize(Zea mays L.) kernels were soaked overnight in water andgerminated on moist filter paper at 27°C in darkness. Whencoleoptiles were at least 3 cm long, regions were excised andimmediately fixed as described above. Wheat grain (Triticumaestivum L.) was imbibed overnight and then cut into small cubes

of �8 mm3 to include the aleurone layer and immediately fixedas described above. After fixation, plant materials were embed-ded in wax and sectioned as described (32). Sections of flaxhypocotyls embedded in resin as described (33) were a kind giftfrom Christine Andeme-Onzighi (University of Rouen, Mont-Saint-Aignan, France).

Labeling of sections with LM11 was carried out as described(23). For CBM labeling, sections were incubated with CBMs inthe range of 2.5–5 �g�ml in PBS�MP for 1.5 h. Samples werethen washed in PBS at least three times and incubated with a100-fold dilution of mouse anti-His monoclonal antibody(Sigma) in PBS�MP for 1.5 h. After washing with PBS, anti-mouse IgG FITC (Sigma) was applied for 1.5 h as a 50-folddilution in MP�PBS in darkness. The samples were washed atleast three times, mounted in a glycerol-based anti-fade solution(Citif luor AF1; Agar Scientific), and observed on an OlympusBH-2 microscope equipped with epif luorescence irradiation.Micrographs shown are representative of observations of threeseparate plants for each species.

This work was supported by the U.K. Biotechnology and BiologicalSciences Research Council.

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