Emergence of a subfamily of xylanase inhibitors within glycoside hydrolase family 18 Anne Durand 1, *, Richard Hughes 1, *, Alain Roussel 2 , Ruth Flatman 1, *, Bernard Henrissat 2 and Nathalie Juge 1,3 1 Institute of Food Research (IFR), Norwich, UK 2 Architecture et Fonction des Macromole ´ cules Biologiques, UMR6098, CNRS et Universite ´ s d’Aix-Marseille I et II, Marseille, France 3 Institut Me ´ diterrane ´ en de Recherche en Nutrition, UMR INRA 1111, Faculte ´ des Sciences et Techniques de St Je ´ ro ˆ me, Marseille, France Recently two classes of plant proteins, designated as XIP (xylanase inhibitor protein) [1] and TAXI ( Triti- cum aestivum xylanase inhibitor) [2] have been shown to inhibit xylanases. XIP-I from wheat (Triticum aesti- vum) represents the prototype of a novel class of (b ⁄ a) 8 inhibitors that inhibits reversibly xylanases belonging to glycoside hydrolase families (GHs) 10 and 11 (CAZY database http://afmb.cnrs-mrs.fr/CAZY/) [3]. The structural features essential for xylanase inhibi- tion were recently largely unravelled by the resolution of the crystal structures of XIP-I in complex with a GH10 xylanase from Aspergillus nidulans and a GH11 xylanase from Penicillium funiculosum [4]. The inhibi- tion mechanism is novel since XIP-I possesses two inde- pendent enzyme-binding sites, allowing binding to two glycoside hydrolases with different folds [4]. XIP-I belongs to a large protein family (GH18) that contains mostly chitinases and proteins of unknown function. The crystal structure of XIP-I confirmed the structural resemblance to GH18 chitinases [5]. In XIP- I, however, clear structural differences in the region corresponding to the active site of chitinases account for its lack of enzymatic activity towards chitin [5–7]. XIP-type proteins were also isolated from rye, durum wheat, barley and maize [8], but sequence infor- mation is limited and the only clones available are those encoding XIP-I (GenPept, AN: CAD19479) and XIP-II (GenPept, AN: CAC87260), the other putative XIP-type inhibitor from wheat (Triticum turgidum ssp. Durum). The widespread representation of XIP-type inhibi- tors in cereals questions further the place ⁄ evolution of Keywords chitinase; evolution; family 18 glycoside hydrolase; proteinaceous xylanase inhibitors; rice *Present address John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK (Received 16 December 2004, revised 3 February 2005, accepted 9 February 2005) doi:10.1111/j.1742-4658.2005.04606.x The xylanase inhibitor protein I (XIP-I), recently identified in wheat, inhib- its xylanases belonging to glycoside hydrolase families 10 (GH10) and 11 (GH11). Sequence and structural similarities indicate that XIP-I is related to chitinases of family GH18, despite its lack of enzymatic activity. Here we report the identification and biochemical characterization of a XIP-type inhibitor from rice. Despite its initial classification as a chitinase, the rice inhibitor does not exhibit chitinolytic activity but shows specificities towards fungal GH11 xylanases similar to that of its wheat counterpart. This, together, with an analysis of approximately 150 plant members of glycosidase family GH18 provides compelling evidence that xylanase inhibi- tors are largely represented in this family, and that this novel function has recently emerged based on a common scaffold. The plurifunctionality of GH18 members has major implications for genomic annotations and pre- dicted gene function. This study provides new information which will lead to a better understanding of the biological significance of a number of GH18 ‘inactivated’ chitinases. Abbreviations E:I 50 , molar ratio enzyme–inhibitor that gives 50% of inhibition; GH, glycoside hydrolase; pRIXI, putative rice xylanase inhibitor; RIXI, rice xylanase inhibitor; rXIP-I, recombinant XIP-I produced in Pichia pastoris; SPR, surface plasmon resonance; XIP-I, xylanase inhibitor protein I; XYNC, Penicillium funiculosum xylanase C. FEBS Journal 272 (2005) 1745–1755 ª 2005 FEBS 1745 this new class of protein within GH18. The existence of several classes of GH18 chitinases in plants was pre- viously suggested [9]. However the general impression was that gene duplications, gene losses and perhaps also translocations resulted in rather unreliable rela- tionships for deriving evolutionary conclusions [10]. In contrast to the abundant genetic information produced from recent sequencing programmes of plant organ- isms (rice and Arabidopsis), relatively little is known about the enzymatic and structural properties of GH18 plant chitinases. An emergent proportion of sequences appear to encode plant inactivated chitin- ases, such as narbonin and concanavalin B, the recep- tor-like kinase Chrk1, and XIP [11]. Based on the recent structural data obtained on XIP-I, can we ana- lyze family GH18 and find other proteins with the same function as XIP? This has implications for an improved annotation of plant genes or ESTs and is particularly important as there is no apparent relation- ship between the old function (chitinase) and the newly evolved one (xylanase inhibitor), despite sequence and structural similarity. No XIP-type protein was so far identified in rice. Among the GH18 sequences isolated from the rice genome [at least 23 – data from the Carbohydrate- active enzymes database, http://afmb.cnrs-mrs.fr/ CAZY/ accessed 11 January 2005)], only two cDNA sequences were shown to encode recombinant proteins having chitinase activity [12] while others were classified as putative rice class III chitinase(s) based on sequence homology only [13]. In particular the (GenPept data- bank; AN: BAA23810.1) clone shares higher similarity with XIP-I than with ‘active’ chitinases and was thus selected as a putative rice xylanase inhibitor (pRIXI). In this work, we report for the first time the func- tional identification of a rice ortholog of the wheat XIP, originally classified as a rice class III chitinase and analyze the features that allow discriminating the subfamily of xylanase inhibitors within the large GH18 family. Results Production and structural characterization of pRIXI The pRIXI clone (GenPept databank; AN: BAA23810.1) is expected to encode a protein of 304 residues with a predicted relative molecular mass of 33 946.8 and a theoretical pI of 9.33 [13]. In order to address the functionality of this potential inhibitor, its cDNA sequence and that of XIP-I were expressed in conditions similar to those used for the production of active basic chitinase in Pichia pastoris GS115 strain [12], e.g. under the control of the alcohol oxidase pro- moter and with an His-tag tail at the C-terminus. Both recombinant XIP-I (rXIP-I) and pRIXI were produced in P. pastoris with a high secretion yield of approxi- mately 250 mgÆL )1 . The recombinant proteins were purified to apparent homogeneity from the culture supernatant as a C-terminal tag fusion protein using one step affinity chromatography. The purified pro- teins migrated on SDS ⁄ PAGE as a 33 and 37 kDa single bands for pRIXI and rXIP-I, respectively. The relative molecular mass of pRIXI, obtained by ESI- MS, was 33 446 Da, thus in total agreement with the predicted calculated mass including the myc epitope and His-tag in C-terminal. In contrast, rXIP-I showed an apparent relative molecular mass of 37 000 Da on SDS ⁄ PAGE, thus higher than the size of the native protein from wheat (34 076 Da). Native XIP-I has been reported to be weakly glycosylated and the two N-glycosylation sites (Asn89 and Asn265) are occupied [3,5,6]. These glycosylation sites are not present in the rice homologue. The relative molecular mass discrep- ancy between the native and recombinant proteins may be explained by hyperglycosylation of the rXIP-I in P. pastoris, as confirmed by mass spectrometry, where five main peaks were identified (37 529, 37 692, 37 853, 37 873 and 38 015 Da). Isoelectric focusing revealed that rXIP-I consisted of three molecular iso- forms of pI 6.8–7.2–8.2 with a main band at pI 7.2. This value differs from native XIP-I of pI 8.7–8.9 [6], due to the insertion of the myc epitope and His-tag in C-terminal. The recombinant pRIXI showed a pI close to pH 9, thus in agreement with the calculated pI of 8.7. The predominant N-terminal sequences, EAEAEFAGGK for rXIP-I and EFGPAMAAGK for pRIXI indicated that the two proteins were correctly processed at the Kex2 and Ste13 signal cleavage sites, respectively. Both recombinant proteins were recog- nized by antibodies raised against His-tag. However, although the rXIP-I was recognized by antibodies raised against native XIP-I, there was no cross-reaction with pRIXI (data not shown). Functionality of the recombinant proteins The recombinant proteins were tested for their chitinase activity using two different size substrates. Using chitin azure, a long and insoluble substrate, no chitinase activ- ity could be detected at pH 5.5 and 8.0 for both pRIXI and rXIP-I, confirming the lack of chitinase activity pre- viously reported for native XIP-I at pH 5.5 in the same conditions [6]. Interestingly, no activity could be detec- ted using this substrate with the recombinant basic A novel GH18 xylanase inhibitor identified in rice A. Durand et al. 1746 FEBS Journal 272 (2005) 1745–1755 ª 2005 FEBS chitinase (GenPept databank; AN: BAA22266.1) al- though Streptomyces griseus chitinase, used as control, was active. The activity of the proteins were then further tested on a short and soluble substrate, 4-nitrophenyl b-d-N,N¢,N¢¢-triacetylchitotriose [p-nitrophenol-(Glc- NAc) 3 ]. Using this substrate, the recombinant basic chi- tinase showed a specific activity of 31.3 and 9.9 UÆmg )1 at pH 5.5 and 8.0, respectively. However, neither pRIXI nor rXIP-I showed any evidence for chitinase activity even in a presence of a molar excess of inhibitors, 3.5 : 1 and 10 : 1 (inhibitor–basic chitinase) for pRIXI and rXIP-I, respectively. The specificity of pRIXI towards fungal and bacter- ial GH10 and GH11 xylanases was compared to that of rXIP-I (Table 1). The pattern of inhibition of rXIP- I towards GH11 xylanases was similar to that previ- ously reported for the native inhibitor (E : I 50 values, Table 1). All the fungal GH11 xylanases were inhibited by both pRIXI and rXIP-I up to a molar ratio E : I of 1 : 30 (Table 1), although the E : I 50 of pRIXI were higher than those of rXIP-I. The lowest molar ratio (1 : 6.5) was obtained for the Trichoderma longibrachi- atum (M3) xylanase. Indeed, for the GH11 Aspergil- lus niger xylanase the value of the E : I 50 is greater than 1 : 52, in these conditions 34% of inhibition was observed. As for native XIP-I, no inhibition was observed for pRIXI and rXIP-I against two bacterial GH11 xylanases from Bacillus subtilis and rumen microorganism (M6) (Table 1). In contrast to both native and recombinant XIP-I, none of the GH10 xy- lanases from A. niger, A. aculeatus and A. nidulans (fungal) or from Cellvibrio japonicus (bacterial) were inhibited by pRIXI (Table 1). Interaction of the inhibitors with xylanases The relative affinities and pH dependencies of the inter- action of XIP-I with xylanases were studied using titra- tion curves. The GH11 XYNC from P. funiculosum interacted with both pRIXI and rXIP-I across the entire range of pH (Fig. 1B). However, although GH11 A. niger (Fig. 1C) and GH10 A. nidulans (Fig. 1D) xy- lanases interacted with rXIP-I, no complex formation was observed with pRIXI (Fig. 1C,D), in agreement with the activity assays data, suggesting that pRIXI is a weaker inhibitor than XIP-I. The interaction between rXIP-I and A. niger xylanase occurred across a narrow range of pH, as previously demonstrated with native XIP-I [3]. In contrast, no complex was observed with bacterial GH11 xylanases from rumen microorganism M6 and B. subtilis (data not shown) in agreement with the reported specificity of XIP-type inhibitors. The molecular interaction between xylanases and pRIXI or rXIP-I was further studied in real time by using a biosensor based on surface SPR. The inhibitor proteins were immobilized as a ligand on the dextran surface of a chip whereas the P. funiculosum XYNC xylanase was used as an analyte over the surface. The sensorgrams for the interaction with XYNC are shown in Fig. 2. The increase of RU from the baseline repre- sents the binding of the xylanase to the surface-bound inhibitor. The plateau line represents the steady- state ⁄ equilibrium phase of the XYNC–inhibitor inter- action, whereas the decrease in RU from the plateau represents the dissociation phase. The slow dissociation phase observed on the SPR sensorgrams for the com- plex between rXIP-I and XYNC suggests that the interaction is stronger than the one previously reported between native XIP-I and the A. niger xylanase [3], in agreement with the inhibition constants reported for these enzymes (K i ¼ 3.4 nm and 317 nm for XYNC and A. niger xylanases, respectively) [3]. XYNC exhib- ited a faster dissociation with pRIXI compared to rXIP-I, in agreement with the lower value obtained from E : I 50 for pRIXI (1 : 45 for pRIXI against 1 : 2.3 for rXIP-I and 1 : 1.6 for native XIP-I). SPR analysis thus demonstrated that a faster dissociation rate probably accounts for the weaker interaction between pRIXI and XYNC compared to XIP-I. Taken together, these data demonstrate that pRIXI is not a chitinase but a novel XIP-type inhibitor in rice and herein named RIXI for ‘RIce xylanase inhibitor’. Table 1. Xylanase inhibition specificity of XIP-I and RIXI towards xylanases. RIXI XIP-I Recombinant Recombinant Native Family 11 xylanases Fungal A. niger Yes a 1:3.6 b 1 : 2.1 XYNC P. funiculosum 1 : 45 1 : 2.3 1 : 1.6 T. longibrachiatum (M3) 1 : 6.5 1 : 2.3 1 : 1.1 Bacterial B. subtilis No c No No Rumen microorganism (M6) No No No Family 10 xylanases Fungal A. nidulans No Yes 1 : 0.6 d A. niger No Yes 1 : 0.7 A. aculeatus No No No Bacterial P. fluorescens No No No a Inhibition observed within the limit defined earlier (> 10% inhibi- tion at E : I molar ratio up to 1 : 30 maximum [3]). b E:I 50 , molar ratio of enzyme to inhibitor that gives 50% of inhibition. c No inhibi- tion within the detection limit described in a . d From [3]. A. Durand et al. A novel GH18 xylanase inhibitor identified in rice FEBS Journal 272 (2005) 1745–1755 ª 2005 FEBS 1747 Discussion RIXI is a novel xylanase inhibitor from rice Our data clearly show that the rice putative chitinase sequence (GenPept databank; AN: BAA23810.1) in fact encodes a xylanase inhibitor. The previous lack of detection of xylanase inhibitor in rice extracts can be explained by the methodology used in the previous reports [14,15]. Indeed the absence of detec- tion by Western blotting is due to the lack of cross- reactivity between purified RIXI and anti-XIP-I Igs [14]. Furthermore, the weak interaction between RIXI and GH11 A. niger xylanase explains why affinity chromatography failed to interact with the rice inhibitor [15] and why no xylanase inhibitor activity was detected in rice extracts using the same enzyme [14]. The observed weaker interaction is not A B C D Fig. 1. Interaction of RIXI and rXIP-I with xylanases. (A) Titration curves showing the inhibitors. (i) RIXI; (ii) rXIP-I. (B) Titration cur- ves showing the interaction between GH11 XYNC from P. funiculosum and the two rec- ombinant inhibitors. (i) XYNC; (ii) a mixture of XYNC and RIXI; (iii) a mixture of XYNC and rXIP-I. (C) Titration curves showing the interaction between GH11 A. niger xylanase and the two inhibitors. (i) A. niger xylanase; (ii) a mixture of A. niger xylanase and RIXI; (iii) a mixture of A. niger xylanase and rXIP-I. (D) Titration curves showing the interaction between GH10 A. nidulans xylanase and the two inhibitors. (i) A. nidulans xylanase; (ii) a mixture of A. nidulans xylanase and RIXI; (iii) a mixture of A. nidulans xylanase and rXIP-I. For each experiment the molar ratio E : I was identical (1 : 1) and 116 pmol of each protein were loaded on the gel. A novel GH18 xylanase inhibitor identified in rice A. Durand et al. 1748 FEBS Journal 272 (2005) 1745–1755 ª 2005 FEBS expected to be due to the lack of glycosylation of RIXI, as glycosylation in XIP-I does not affect inhi- bition specificity [4,5]. The presence of xylanase inhibitor in rice is not surprising as hemicellulose in the cell walls of rice cells is composed mainly of arabinoxylan [16] and the ability to degrade xylan represents an important attribute for a rice pathogen to infect plant tissues. Indeed, secretion of xylanases by rice pathogens was reported for Magnaporthe grisea, the fungal pathogen that causes rice blast disease [17], and Xanthomonas oryzae pv. oryzae, the causal agent of bacterial leaf blight, a serious disease in rice [17–19]. The recent demonstration that xylanases secreted by rice patho- gens are important factors of their virulence agrees with a potential role of RIXI in plant defence, as proposed for XIP-I [20]. This hypothesis is reinforced by the homology of RIXI with chitinases, which are known to act in response to invading pathogens by degrading polysaccharides of their cell wall. Class III chitinases have been classified into pathogenesis-rela- ted proteins (PR-8) because of their inducible expres- sion upon infection by pathogens [21,22]. Plant chitinases exhibit rapid evolution by acting as prime targets for the coevolution of plant–pathogen interac- tions. XIP-type proteins could have evolved from chitinases as part of the plant defence pathway to act both on the xylanases secreted by pathogens and on the pathogen itself. In both cases, the function is ori- entated towards a general role in plant defence and the production of inhibitors prevents the plant to undergo unnecessary metabolic costs. XIP-type inhibitors represent a subfamily of GH18 GH18 includes chitinases from various species, inclu- ding bacteria, fungi, nematodes, insects plants, and mammals, but also a growing number of nonchitinase proteins, the latter making genome and ESTs annota- tions particularly unreliable (for instance RIXI was thought to be a basic chitinase). Sequence-based famil- ies such as those in CAZy, PFAM, etc., group together proteins that have sometimes different functions. Here the case is particularly tricky as the novel function has been acquired relatively recently (in such a case, only functional and structural characterization can help building the necessary knowledge to enable prediction methods). In the present work, novel biochemical and structural information of XIP-type inhibitors are used to test whether it is possible to better predict function- ality within the GH18 family. Although the overall sequence similarity between GH18 chitinases is not particularly high (average pair- wise 21%), their active site regions contain many residues that are fully or highly conserved. The most prominent motif dictating chitinase activity is DxxDxDxE that includes the glutamate acting as the catalytic acid. The GH18 members devoid of chitinase or known enzymatic activity, all have nonconservative substitutions of one of the acidic amino acid residues in the catalytic region (Fig. 3). The XIP-type inhibitors all have the third aspartic acid DxxDxDxE mutated into an aromatic residue (Phe126 XIP-I ) whereas the cat- alytic glutamate residue is only conserved in XIP-I (Glu128 XIP-I ). The substitution of the critical Asp aci- dic amino acid by a bulky residue thus is a major determinant for the lack of chitinase activity reported for XIP-I and RIXI. This suggests that another GH18 sequence (GenPept, AN: BAC10141.1) could be an additional xylanase inhibitor in rice. The inhibition specificity of RIXI can be explained on the basis of the recently solved 3-D structure of XIP-I in complex with a GH10 xylanase from A. nidu- lans and a GH11 xylanase from P. funiculosum [4]. The inhibition of GH10 xylanase occurs through extensive interactions between the two proteins. XIP-I a7 helix (232–245) interacts with the loops forming the xylanase groove; side chains emerging from the helix point into the heart of the cleft and occupy the four central subsites: )1 (Lys234 XIP-I ), +1 (Asn235 XIP-I ), )2 (His232 XIP-I ), and +2 (Tyr238 XIP-I ), whereas Lys246 XIP-I sterically blocks access to subsite )3. Two additional regions (loop b 6 a 6 from residue 193–205 and a8 helix 268–272) make contact with the enzyme. These three regions are determinants for the inhibitory activity. Although a7 and a8 helixes are pretty well Fig. 2. SPR sensorgrams showing the interaction between XYNC ⁄ RIXI (A) and XYNC ⁄ rXIP-I (B). In both panels, XYNC (14 l M)was injected at a flow rate of 50 lLÆmin )1 . The signal is indicated in resonance units (RU) and time 0 corresponds to the injection of XYNC. A. Durand et al. A novel GH18 xylanase inhibitor identified in rice FEBS Journal 272 (2005) 1745–1755 ª 2005 FEBS 1749 conserved, the lack of inhibition activity of RIXI against GH10 xylanases can be explained by differ- ences in the loop b 6 a 6 , a region at the interface between XIP-I and A. nidulans xylanase (Fig. 4A). This loop contains two strictly conserved residues, Cys195 (involved in a disulfide bond with Cys164) and Trp205 separated by a variable number of amino acids (see alignment, Fig. 3). In both RIXI and hevamine, an active GH18 chitinase from Hevea brasiliensis, the loop contains 13 amino acids, as compared to nine in XIP-I (see alignment, Fig. 3). When the b 6 a 6 loop of hevamine for which the three-dimensional structure is known (pdb code 2HVM), is superimposed on the cor- responding loop of XIP-I, the extra residues introduce steric clashes in the interaction with GH10 xylanases, thus preventing binding with the xylanase (Fig. 4A). A similar situation is thus expected to occur for RIXI but also for XIP-II, the other wheat XIP-type inhib- itor, which contains also four additional amino acids in b 6 a 6 loop as compared to XIP-I. In contrast, the BAC10141.1 sequence shows a shorter loop, suggesting a possible inhibition of GH10, although a clash result- ing of an insertion before the Cys195 residue cannot be ruled out. The XIP-I strategy for inhibition of GH11 xylana- ses consists of an inhibitory head (the P-shaped La 4 b 5 loop, amino acids 148–153) blocking the entrance to the active site. The main inhibition determinant involves a functional arginine side-chain (Arg149 XIP-I ) projecting into the glycone subsites of the enzyme’s active site. The top of the inhibitor loop is slightly twisted, allowing the GGP(150–152) segment to extend closely parallel to the )3 subsite plane whereas the main-chain segment 149–150 occupies subsite )2. This loop is three residues shorter in hevamine, pre- venting an interaction with xylanase (Fig. 4B) but of similar length in RIXI, allowing interaction of the rice inhibitor with GH11 xylanases. However the important determinant RGG(149–151) in XIP-I is replaced by MYR(149–151) in RIXI, which might explain the observed weaker interaction (Fig. 3). The same characteristic feature is also observed in XIP-II, predicting interaction with GH11 xylanases, although weaker. However, in the BAC10141.1 sequence, the loop is shorter, which might prevent binding to GH11 xylanases, as also observed with hevamine. The additional interacting regions of XIP-I; the C-ter- minal extremity helix a2 and the loops a 5 b 6 and a 6 b 7 , corresponding to residues 64–70, 179–185 and 213–217 [4] are well conserved among the sequences (Fig. 3). The structural data thus agree with the inhibition specificity pattern observed for RIXI and predict BAC10141.1 to be another xylanase inhibitor from rice with no chitinase activity and a specificity pattern opposite to that of RIXI, inhibiting GH10 but not GH11 xylanases. Fig. 3. Amino acid sequence alignment of selected GH18 plant members: XIP-I (CAD19479.1); BAC (BAC10141.1); RIXI (BAA23810.1); XIP-II (CAC87260.1); basic chitinase (BAA22266.1); hevamine (CAA07608.1). Residue numbering is given for the XIP-I sequence [5]. Grey shading shows residues conserved in all sequences. Boxes show consensus residues involved in chitinase activity and residues involved in complex- ing with a GH10 or GH11 xylanase [4]. A novel GH18 xylanase inhibitor identified in rice A. Durand et al. 1750 FEBS Journal 272 (2005) 1745–1755 ª 2005 FEBS XIP-type inhibitors have evolved from a common scaffold A phylogenetic tree is presented for the known plant members of the GH18 family (Fig. 5). The sequences were retrieved from the CAZy database [23]. Clearly, four major subfamilies can be distinguished: (a) hevamine-type chitinases; (b) putative chitinases; (c) narbonins; and (d) putative chitinases. Out of the four major subfamilies that appear from the phylogenetic analysis, only the one that contains hevamine actually contains enzymes of demonstrated activity. XIP-type inhibitors emerged from the hevamine cluster along with concanavalin B, another GH18 member with no enzymatic function. A large number of the related sequences are chitinases, but the evolutionary tree also includes receptor-like kinase (Chrk1) and individual sequences with no catalytic residue. Based on the pre- sent work, we suggest that the proteins emerging from cluster (a) also have xylanase inhibitor activity. The tree clearly shows that the xylanase inhibitors appeared after the emergence of the various subfamilies of chitinases from their common ancestor. In this respect, the xylanase inhibitors are a relatively new invention, and so far no protein has been reported to display both xylanase inhibition and chitinase activities. The assignment of sequences to large disparate multifunctional glycosidase families such as GH18 is useful to unravel ancient evolutionary events, but is of limited use for ORF function prediction based solely on sequence similarity. The present study adds to the growing body of evidence that sequence similarity alone would result in a wrong (and self-propagating) functional assignment, and that biochemical characteri- zation is required to establish protein function. The GH18 xylanase inhibitors are an example of evolution- ary inventions from pre-existing proteins. The xylanase inhibitor and its chitinase ancestors are believed to be produced by the plant as part of their defence system against fungi. It is tempting to speculate that the novel function emerged based on a class of proteins whose synthesis was already triggered by fungal attack, and that evolution has kept the existing signal recognition and expression-regulation pathways [24]. Additional biochemical and structural characterization of plant GH18 ‘chitinase’ sequences will clarify some features of the evolution of this family of chitinases. Experimental procedures Materials and strains The cDNA clone encoding a putative rice class III chitinase (DNA Data Bank of Japan; AN: D55712 corresponding to the protein AN: BAA23810.1 in the GenPept databank) was a kind gift of T. Sasaki (National Institute of Agrobio- logical Resources, Tsukba, Japan) [13]. The full-length cDNA encoding XIP-I (GenPept databank; AN: CAD19479.1) was from in house collection [7]. The P. pas- toris clone encoding a basic active rice chitinase (GenBank; AN: AB003195) was a kind gift from S M. Park (Basic Y189 C164 C195 W205 L143 L158 R149 GH10 GH11 A B Fig. 4. (A) Close-up view of the interaction between XIP-I (in purple) or hevamine (in blue) and GH10 A. nidulans xylanase. The hevamine structure (pdb code 2HVM) was superimposed onto the XIP-I struc- ture in complex with A. nidulans xylanase (pdb code 1TA3) using TURBO-FRODO [33]. The loop between residues 195 and 205 in XIP-I (in purple) is in close contact with the GH10 xylanase. The cor- responding loop in hevamine (in blue) is four residues longer. This insertion may induce steric clashes with the enzyme. (B) Close-up view of the interaction between XIP-I (in purple) or hevamine (in blue) and GH11 P. funiculosum xylanase. The hevamine structure (pdb code 2HVM) was superimposed onto the XIP-I structure in complex with P. funiculosum xylanase (pdb code 1TE1) using TURBO- FRODO [33]. The Arg149 located in the P-shape loop from residues 143–158 (in purple) plays an important role for the inhibitory activity of XIP-I. Such interaction is not possible in the case of hevamine because the corresponding loop (in blue) is two residues smaller. A. Durand et al. A novel GH18 xylanase inhibitor identified in rice FEBS Journal 272 (2005) 1745–1755 ª 2005 FEBS 1751 Science Research Institute, Chonbuk National University, Korea) [12]. The GH11 xylanases from P. funiculosum (XYNC) and A. niger were from in house [25,26]. The GH10 xylanases from B. subtilis and A. aculeatus, A. niger, C. japonicus, and A. nidulans, were from L. Saulnier (INRA, Nantes, France), K. Gebruers (Laboratory of Food Chemistry, University of Leuven, Belgium), H. Gilbert (University of Newcastle, United Kingdom), and P. Man- zanares (Instituto de Agroquı ´ mica y Technologı ´ a de Ali- mentos, Valencia, Spain), respectively. The native XIP-I was purified from wheat as described previously [3,6]. Low viscosity arabinoxylan, T. longibrachiatum M3 and rumen microorganism (M6) xylanases were from Megazyme Inter- national Ireland Ltd (Co. Wicklow, Ireland). The P. pastor- is expression kit containing the pPICZaA vector and the anti-[His(C-term)-HRP] Ig were from Invitrogen (San Diego, CA, USA). The HisTrap purification kit was from Amersham Pharmacia Biotech (Uppsala, Sweden). PmeI was from New England Biolabs (Beverly, MA, USA) and the other restriction endonucleases and DNA modifying enzymes from Promega (Madison WI, USA). Escheri- chia coli DH5a (supE44, hsdR17, recA1, endA1, gyrA96, thi-1, relA1) was used for DNA subcloning. Chitin azure, dinitrosalicylic acid (DNS), 4-nitrophenyl b-d-N,N¢,N¢¢-tri- acetylchitotriose and Streptomyces griseus chitinase were from Sigma Chemical Co. (St. Louis, MO, USA). Oligo- nucleotides were synthesized as high purity salt-free oligos by Sigma-Genosys Ltd. (Pampisford, UK). Cloning and expression in P. pastoris The cDNAs encoding XIP-I and the putative rice xylanase inhibitor (pRIXI) were amplified by PCR using the fol- lowing primers: 5¢-CCG GAATTCGCGGGGGGAAAG-3¢ (forward primer) and 5¢-GC TCTAGAGCGGCGTAGTAC TT-3¢ (reverse primer) for XIP-I and 5¢-CCG GAATTCGG CCCGGCGATG-3¢ (forward primer) and 5¢-GC TCTAGA GCAGCCCAGTACTT-3¢ (reverse primer) for pRIXI. EcoRI and XbaI restriction sites (underlined) were intro- duced in 5¢ and 3¢ of these primers, respectively, to facilitate subsequent cloning steps. DNA amplification was carried out through 25 cycles of denaturation (1 min at 94 ° C), annealing (30 s at 61 °C) and extension (1.5 min at 72 °C) in a DNA thermocycler (Perkin Elmer GeneAmp PCR system 2400) using Pfu polymerase (Stratagene, UK) and Vent polymerase New England Biolabs (Beverly, MA, USA) for amplification of RIXI and XIP-I cDNAs, respectively. The gel-purified fragments and pPICZaA vector were digested with EcoRI and XbaI. The digested cDNA fragments were purified and ligated into pPICZaA vector at 16 °C overnight using T4 DNA ligase. After transformation into E. coli DH5a by heat-shock and plating the cells on low salt LB agar contain- ing 25 lgÆmL )1 zeocin (Invitrogen), positive clones were se- quenced to check the integrity of the insert. The recombinant plasmids (10 lg) were linearized by PmeI and used to trans- form P. pastoris strain (his4) GS115 [27] using an adaptation of the spheroplast method [28]. Transformed colonies were selected on RDB plates containing zeocin (100 lgÆmL )1 ) and histidin (4 mgÆmL )1 ). After incubation at 30 °C for 4–5 days, 50 clones of each transformation were screened on minimum methanol medium (MM) agar plates and on minimal dextrose medium (MD) agar plates at 30 °C. After 3 days, the transformants growing on MD medium and slowly on MM medium were screened for protein secretion. Single zeocin-resistant colonies were grown in rich nonbuffered gly- cerol complex medium (10 mL) at 30 °C with shaking (250 r.p.m.). After 48 h, the cells were centrifuged and resuspended in the induction medium (nonbuffered rich methanol complex medium). The secretion of the recombin- ant proteins in the supernatants was analysed after 48 and 96 h of methanol induction on 12% SDS ⁄ PAGE gels and the proteins revealed using Coomassie blue staining or Fig. 5. Unrooted phylogenetic tree for plant members of family GH18. The scale bar ind- icates the number of substitutions per posi- tion following alignment with MUSCLE [34] and bootstrap analysis by CLUSTAL W [35]. The tree was displayed with TREEVIEW [36]. The thick lines identify the various clusters discussed in the text. The accession num- bers (GenBank or SwissProt) of the isolated sequences are given on the figure. Acces- sion numbers of representative members of the various subfamilies: subfamily 1, CA- A07608; subfamily 2, AAO15366; subfamily 3, O81862; subfamily 4, Q41675. A novel GH18 xylanase inhibitor identified in rice A. Durand et al. 1752 FEBS Journal 272 (2005) 1745–1755 ª 2005 FEBS transferred onto nitrocellulose membranes for immuno-reve- lation. Clones secreting the highest level of the recombinant proteins were chosen for large-scale expression. Protein production and purification For large-scale production of the recombinant proteins, P. pastoris transformants were grown in 200 mL of rich nonbuffered glycerol complex medium in 1 L flasks at 30 °C with shaking (170 r.p.m.) for 2 days. After centrifu- gation (2500 g, 10 min, room temperature), the pellet was resuspended in the induction medium (40 mL). The cells were shaken in 250 mL flask for 72 h at 30 °C and 170 r.p.m. Purification was performed using a one-step nickel affinity-chromatography using the HisTrap kit. Fol- lowing centrifugation (2500 g, 10 min, 4 °C), the pH of the supernatant was adjusted to pH 7.1–7.2 with 1 m sodium phosphate buffer pH 7.4 and filtered through 0.45-lm filter. The HiTrap chelating HP column was charged with Ni 2+ ions according to the manufacturer instructions and equili- brated with phosphate buffer pH 7.4 (10 mL) containing 0.5 m NaCl and 10 mm imidazole using a peristaltic pump. The sample was loaded at a 1.5 mLÆmin )1 flow rate. The histidine-tagged proteins were eluted using phosphate buffer containing increasing concentrations of imidazole (20, 50, 100, 300 and 500 mm). The A 280 of the collected fractions was measured and the proteins analysed by SDS ⁄ PAGE. Fractions containing pure protein were pooled and dialysed overnight against McIlvaine buffer pH 5.5 at 4 °C. Purification of the recombinant basic chitinase (GenPept databank; AN: BAA22266.1) was performed as described previously [12] with the exception of the last chromatogra- phy step on con-A agarose. Enzyme assays Xylanase inhibition activity was measured using the dinitro- salicylic acid (DNS) assay [29] in McIlvaine’s buffer (pH 5.5) at 30 °C for varying times depending on the enzyme used. One unit of xylanase activity was defined as the amount of protein that released 1 lmol xylose per min at 30 °C and pH 5.5. Enzyme sample (2–4 lL) was added to 10 mgÆmL )1 low viscosity arabinoxylan (145 lL) dis- solved in McIlvaine’s buffer pH 5.5. The reaction was ter- minated by the addition of (DNS) reagent (150 lL), and the samples were boiled for 5 min. After centrifugation at 13000 g for 5 min, the supernatant (200 lL) was transferred to a microtitre plate and the absorbance at 550 nm meas- ured relative to a xylose standard curve (0–180 lgÆmL )1 ). For determination of the inhibition parameters, the activ- ity of the enzymes was measured at 30 °C, in McIlvaine’s buffer pH 5.5 using low viscosity arabinoxylan substrate. The E : I 50 value corresponded to the molar ratio of enzyme– inhibitor required to inhibit xylanase activity by 50%. The inhibitor was preincubated with substrate for 10 min at 30 °C. The reaction was initiated by the addition of the enzymes: A. niger xylanase (53 pmol), XYNC (30 pmol) and T. longibrachiatum M3 xylanase (40 pmol) and carried out at 30 °C for 10 min for A. niger xylanase or for 5 min for XYNC and T. longibrachiatum M3 xylanases. The E : I 50 was calculated with the sigma plot program. The chitinase activity assay was performed at two differ- ent pH using McIlvaine buffer pH 5.5 or 100 mm Tris ⁄ HCl pH 8.0 and with two different size substrates. The assay using insoluble chitin azure was performed as previously described [6]. 4-Nitrophenyl b-d-N,N¢,N¢¢-triacetylchitotri- ose [p-nitrophenol-(GlcNAc) 3 ] was used following the method previously described [30]. Briefly, the purified pro- teins were incubated at 37 °C with 5 lL of substrate (3 mgÆmL )1 dissolved in sterile demineralized water) and the volume adjusted to 70 lL with buffer. After 2.5 h of incubation, the reaction was terminated by adding 0.4 m Na 2 CO 3 (50 lL) and the absorbance was measured at 410 nm. The amount of p-nitrophenol released was deter- mined from a standard curve. Protein assays and protein sequencing Total protein concentration was calculated using an extinc- tion coefficient at 280 nm of 73090 m )1 Æcm )1 for recombin- ant XIP-I and 62990 m )1 Æcm )1 for recombinant pRIXI based on the amino acid composition derived from the pri- mary structure (http://www.expasy.ch/). N-Terminal sequencing was performed at the Protein and Nucleic Acid Chemistry Facility, University of Cambridge using an ABI 491 Procise sequencer. Electrospray mass spectrometry (ESI-MS) Electrospray mass spectra were performed at the Depart- ment of Chemistry, University of Cambridge, on an ABI QSTAR pulsar mass spectrometer (Applied Biosystems) equipped with a nanospray ion source. The sample (10 lm) was put into the nanospray needle and 1000 V was applied to start spraying. The declustering potential was 30. The scans were summed and the raw data was analysed using the instrument’s analyst software. Gel electrophoresis and immunoblotting SDS ⁄ PAGE was performed using 12% homogeneous Novex Tris ⁄ Glycine gels (Invitrogen) according to the manufacturer’s instructions using Mark12 unstained stand- ard as markers (Invitrogen). The samples were reduced with dithiothreitol and boiled before loading on the gels. For immunodetection, proteins were transferred onto nitrocellulose membrane by semidry blotting (Bio-Rad). The blots were probed with 1 : 5000 dilution of the anti- [His(C-term)-HRP] Ig, after the washing steps they were A. Durand et al. A novel GH18 xylanase inhibitor identified in rice FEBS Journal 272 (2005) 1745–1755 ª 2005 FEBS 1753 developed using enhanced chemiluminescent detection rea- gents (ECL Plus Detection Kit, Amersham Pharmacia Bio- tech, Uppsala, Sweden). The blots were probed with a 1 : 5000 dilution of polyclonal antiserum raised in rabbits against XIP-I purified from wheat [14]. Immunoreactive proteins were visualized using a horseradish peroxidase anti-rabbit secondary Ig (Sigma, 1 : 2000) together with the chemiluminescent detection as above. Isoelectric focusing gels were run using the Bio-Rad sys- tem and performed using the Novex IEF gel from Invitro- gen following the instructions manual. Electrophoretic titration Titration curves of inhibitors alone or in complex with dif- ferent xylanases were performed using the Phast system (Amersham Pharmacia Biotech, Uppsala, Sweden) as descri- bed previously [3,31]. Prior to loading, inhibitors (1–2 lL) were preincubated with xylanases (0.5–1.8 lL) in McIlva- ine’s buffer pH 5.5 for 10 min at room temperature in a total volume of 4 lL (E : I molar ratio, 1 : 1 using 116 pmol of each protein). Surface plasmon resonance BIAcore X system, Hepes-buffered saline buffer [10 mm Hepes (pH 7.4) ⁄ 0.15 m NaCl ⁄ 3.4 mm of EDTA ⁄ 0.005% of surfactant P20], CM5 sensor chips and amine coupling kit were from BIAcore AB (Uppsala, Sweden). RIXI (1 lm) and rXIP-I (0.9 lm )in10mm sodium acetate buffer (pH 5.5) were immobilized by the amine coupling method [32] at a flow rate of 10 lLÆmin )1 , using Hepes-buffered saline as running buffer. Briefly, equal volumes of N-hy- droxysuccinimide (0.06 m in water) and N-ethyl-N¢-(3- di- ethylaminopropyl)carbodiimide (0.2 m in water) were mixed and injected on to a CM5 sensor chip to activate the carb- oxymethylated dextran surface. The volume used was adjus- ted to achieve immobilization levels of inhibitors giving 2000–3000 resonance units (RU); 1 RU is defined as 1 pg of bound protein per mm 2 . After injection of inhibitor (40 lL), the residual N-hydroxysuccinimide esters were deactivated by the injection of 35 lL of ethanolamine (1 m, pH 8.5). Flow cell 2 was used to immobilize inhibitors, and control flow cell 1 was treated identically but without inhib- itor. XYNC (80 lL, 14 lm)in10mm sodium acetate (pH 5.5) was injected at a flow rate of 50 lLÆmin )1 , using 10 mm sodium acetate (pH 5.5) as running buffer. Acknowledgements We thank Dr Takuji Sasaki (Japan) for the kind gift of the cDNA clone of the putative rice class III chitinase, Dr Seung-Moon Park (Korea) for providing the Pichia pastoris strain expressing the basic active chitinase and Dr Caroline Furniss, Dr Tariq Tahir, Dr Kurt Gebr- uers, Professor Harry Gilbert, Dr Paloma Manzanares and Dr Luc Saulnier for providing xylanases. This study has been carried out with the financial sup- port from the Commission of the European Communi- ties, under the specific programme for RTD and Demonstration on ‘Quality of Life and management of living resources’, Key Action 1-Food, Nutrition and Health, Contract: QLK1-2000–00811 GEMINI ‘Solving the problem of glycosidase inhibitors in food processing’. References 1 Juge N, Payan F & Williamson G (2004) XIP-I, a xyla- nase inhibitor protein from wheat: a novel protein func- tion. Biochim Biophys Acta 1696, 203–211. 2 Gebruers K, Brijs K, Courtin CM, Fierens K, Goesaert H, Rabijns A, Raedschelders G, Robben J, Sansen S, Sorensen JF, Van Campenhout S & Delcour JA (2004) Properties of TAXI-type endoxylanase inhibitors. 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