Structural and functional evolution of chitinase-like proteins from plants.

Proteomics 2015, 00, 1–13

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DOI 10.1002/pmic.201400421

REVIEW

Structural and functional evolution of chitinase-like proteins from plants Pooja Kesari, Dipak Narhari Patil, Pramod Kumar, Shailly Tomar, Ashwani Kumar Sharma and Pravindra Kumar Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, India The plant genome contains a large number of sequences that encode catalytically inactive chitinases referred to as chitinase-like proteins (CLPs). Although CLPs share high sequence and structural homology with chitinases of glycosyl hydrolase 18 (TIM barrel domain) and 19 families, they may lack the binding/catalytic activity. Molecular genetic analysis revealed that gene duplication events followed by mutation in the existing chitinase gene have resulted in the loss of activity. The evidences show that adaptive functional diversification of the CLPs has been achieved through alterations in the flexible regions than in the rigid structural elements. The CLPs plays an important role in the defense response against pathogenic attack, biotic and abiotic stress. They are also involved in the growth and developmental processes of plants. Since the physiological roles of CLPs are similar to chitinase, such mutations have led to plurifunctional enzymes. The biochemical and structural characterization of the CLPs is essential for understanding their roles and to develop potential utility in biotechnological industries. This review sheds light on the structure–function evolution of CLPs from chitinases.

Received: August 31, 2014 Revised: January 16, 2015 Accepted: February 24, 2015

Keywords: Chitinase-like proteins / Glycosyl hydrolase family / N-Acetylglucosamine / Plant proteomics / TIM barrel

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Introduction

Nature has equipped plants with chitinases to protect them from chitin-containing pathogens. Chitinases are also expressed in response to abiotic stress and during developmental processes of plants [1–3]. These proteins are primarily categorized into glycosyl hydrolase (GH) 18 and 19 families. The cDNA-deduced sequence of the proteins from both the families shows that the chitinases are composed of an Nterminal signal peptide of variable lengths [4–6]. As per the revised chitinase gene classification, they are mainly grouped Correspondence: Dr. Pravindra Kumar, Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, UK 247667, India E-mail: [email protected] Fax: +91-1332-273560 Abbreviations: AFPs, antifreeze proteins; CBD, chitin-binding domain; CHRK, chitinase-related receptor-like kinase; CLP, chitinaselike proteins; CTL, chitinase-like; GH, glycosyl hydrolase; GlcNAc, N-acetylglucosamine; PPL2, Parkia platycephala lectin 2; PR, pathogenesis related; TCLL, tamarind CTL lectin; XAIP, xylanase and ␣-amylase inhibitor protein; XIP-I, xylanase inhibitor protein I

C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

into six classes I to VI [7]. The classes III and V belong to the GH18, whereas classes I, II, IV, and VI belong to the GH19 family. The classes III and V show very less homology with each other and no sequence similarity to enzymes of GH19 family. The chitinases of GH18 family adopt ␤/␣ barrel fold, whereas the GH19 chitinases have a high helical content because of the presence of nonpolar residues in the core region [8]—and show structural similarity to chitosanase and lysozyme [9]. Both families exhibit diversity in their sequences, domain orientation, and hydrolytic mechanisms. Along with active chitinases, the plant genome also consists of a large number of sequences that encode catalytically inactive chitinases also referred to as chitinase-like (CTL) proteins (CLPs). The CLPs share high sequence and structural similarity with chitinases of GH18 and 19 families; but they may lack the binding/catalytic activity due to the presence of substitutions in the chitin-binding domain (CBD) or CatD. Molecular genetic analysis reveals that gene duplication events followed by mutation in the existing chitinase gene have resulted in the loss of activity [10]. CLPs have evolved through two different evolutionary pathways. In the first category, a mutation has led to loss of catalytic potential “Glu” residue. These CLPs do not

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possess catalytic activity; however polysaccharide-binding ability remains intact. The second pathway is the one in which the catalytic activity is retained while the molecule has also developed other functional domains. Thus, despite being homologous to chitinases, the individual gene has evolved independently and has led to adaptive functional diversification. Sequence analysis shows that many lectins have evolved to interact with N-acetylglucosamine (GlcNAc), thus progressing from chitinase to CLPs. Such CLPs are known for their ability to agglutinate erythrocytes and are thus referred to as chi-lectins. Chi-lectins adopt the folds similar to chitinase, but the catalytic Glu residues are substituted, leading to loss of catalytic activity. Thus chi-lectin can interact with chitin but cannot hydrolyze the subunits. The structural comparison of CTL lectins and chitinase gene shows that lectins have evolved CBD from some ancestral chitinase genes [11]. The CLPs are known to inhibit the fungal growth by inhibiting fungal xylanases [12, 13]. In such proteins the structural scaffold is preserved, but the classic “Glu” residue is engaged in intermolecular H-bond formation with surrounding residue, thus abolishing the chitin hydrolyzing ability. In other such proteins the CBD has evolved to recognize chitinlike molecules, thus playing an important role in growth and developmental processes.

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GH18 family class III CLPs

2.1 Overall structure The GH18 class III chitinases are characterized by (␤/␣)8 barrel topology; an active site containing two Asp and one Glu acid residues separated by phenylalanine and isoleucine (DxDxE) and belongs to pathogensis-related (PR) 8 family proteins. Hevamine isolated from the rubber plant (Hevea brasiliensis) is considered as the prototype of this family as it exhibits both lysozyme and chitinase activity [9]. In CLPs, substitution of catalytic Glu residue with any other residue is related to lack of hydrolytic activity. Some of the CLPs whose crystal structures are available include ConB from Canavalia ensiformis, narbonin from Vicia narbonensis, xylanase inhibitor protein I (XIP-I) from Triticum aestivum (var. Soisson), xylanase and ␣-amylase inhibitor protein (XAIP) from Scadoxus multiflorus, Parkia platycephala lectin 2 (PPL2), and tamarind CTL lectin (TCLL) from Tamarindus indica [12–17] (Fig. 1). These proteins are also composed of (␤/␣)8 topology, two consensus regions equivalent to the third and fourth barrel strands (some mutations exist in these regions) and an elongated ␤2␣2 loop (L␤2␣2) having one antiparallel ␤hairpin (Fig. 2). The structure is stabilized by disulfide bridges and three nonproline cis-peptide bonds localized in the central groove, later interacts with the pyranose rings of GlcNAc. Narbonin exhibits weak yet clear similarity to endo-p-Nacetylglucosaminidase H from S. plicatus [14]. The protein C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim


has a barrel fold, but the first helix of the fold is replaced with a loop region; also ␤1 and ␤2 strands are connected by an extended loop comprising of two ␤-turns. Additionally, an extended antiparallel ␤-strand ␤ 2 is placed parallel to ␣3 and ␣4; a short ␣-helix situated between ␤7 and ␣7 and 310 helix between ␤8 and C-terminal helix has also been observed. Only one cysteine residue has been reported in narbonin as compared to other plant-type chitinases. ConB has an extra disulfide bond Cys41–Cys93 in comparison to narbonin, this bond is engaged in stabilization of L␤2␣2 and L␤3␣3. Morrison et al. showed presence of a firmly bound zinc ion, NADPH, and flavin mononucleotide [15]. ConB has an extended loop between the ␤2 and ␣2-helix [16]. Sequence showed a glycosylation site (Asn284) in ConB [17]. XIP is the first example of a protein able to inhibit members of GH10, GH11, and GH13 families [18, 19]. The structure of XIP-I has two disulfide bridges connecting ␣1 and ␣2 (Cys25 and Cys66) and L␤5␣5 and L␤6␣6 (Cys164 and Cys195) and three cis-peptides. One cis-proline (Pro167) is observed in L␤5␣5 and others occur between Ser36 and Phe37 and between Trp256 and Asp257. The residues Phe37 and Trp256 emerging from ␤2 and end of ␤8 strand, respectively, protrude into the groove on the top of the ␤-barrel and form the rigid central part of the XIP-I. The XIP-I mimics the interaction of xylanase enzyme with oligosaccharides and occludes its enzyme’s active site. Complex of XIP-I with GH10 and 11 shows that the protein adopts two independent conformations for inhibiting two structurally and functionally different classes of enzymes [18]. The L␣4␤5 of XIP-I helps in interaction with GH11 xylanase wherein the residue Arg149XIP-I interacts with the residues Glu85 and Glu176 of GH11. The GH10 xylanase interacts with XIP-I through the ␣7 (232–245) of XIP-I that blocks four subsites in the substrate-binding groove of GH10 enzyme. The Lys234 of ␣7XIP-I interacts with Glu131 and Glu239 of GH10. The XIP-I inhibitor is specific for fungal and bacterial xylanases from GH family. It has a putative site for N-linked glycosylation (Asn89 and Asn265) [20]. The XAIP inhibits GH11 xylanase and GH13 ␣-amylase [13]. The barrel fold of XAIP consists of an extra helix located between ␤8 and ␣8. Although XAIP shares high sequence homology with hevamine (48%) and ConB (39%), the disulfide linkages in XAIP are identical to those of XIP-I. The XAIP has a novel L␣3␤4 that protrudes sharply away from the surface of the protein and has Pro–Pro dipeptide, which disrupts the conformation of ␣3 located at the C-terminal. The inhibition of GH11 xylanase and G13 ␣-amylase takes place through L␣4␤4 and L␤6␣6 (along with ␣7) of XAIP [13]. Plant lectins have also evolved to adopt a GlcNac-binding ability. PPL2 exhibits a GlcNac-dependent hemagglutination and endo-chitinase ability [21]. The N-terminal (42 residues) showed high similarity with hydrolases of GH18 family. The ␤6␣6 cleft region and L␤2␣2 and L␤7␣7 of PPL2 are different as compared to other plant chitinases. It has five cis-peptide bonds and three disulfide bonds. A CTL lectin from T. indica (TCLL) also shows hemagglutination but has no endochitinase activity. It has two novel sites for GlcNAc binding, which www.proteomics-journal.com

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Figure 1. Structures of hevamine (A), narbonin (B), ConB (C), XIP-I (D), XAIP (E), PPL2(F), and TCLL(G) generated with PyMol.

is different from plant and animal CLPs (HCgp-39, MGP-40, SI-CLP, SPS-40, SPG-40, SPC-40, YKL-39, Ym1, and IDGF-2 [22–30]). The ␤1 strand, ␣3-helix, an antiparallel ␤-hairpin in the L␤2␣2 region, and ␤-sheet insertion in the L␤3␣3 are unique features of TCLL. The insertion of ␣1 (20–23) and ␣8 (258–264) disrupts the ␤␣␤ fold of the protein. Such insertion has been reported in several (␤␣)8 barrel topologies [16, 31]. The sequence results from LC MALDI TOF-TOF and ESITrap analysis showed that N-linked glycosylation (residues 62 and 146) site exist on TCLL. C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

2.2 CBD and CatD architecture Class III hydrolyzes chitin by a double-displacement mechanism that involves two catalytic residues and proceeds through a geometrically distorted oxocarbonium intermediate [32,33]. The catalytic site is formed by the C-terminal ends of the ␤ strands and the loops linking them with the subsequent ␣-helices [34]. The carboxyl side chain of Glu127 acts as a proton donor to the scissile glycosidic-bond between the sugar residues bound at the −1 and +1 subsites. Apart from www.proteomics-journal.com

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Figure 2. The sequence alignment of hevamine, ConB, narbonin, XIP-I, XAIP, PPL2, and TCLL generated with ClustalW and ESpript. The conserved residues are represented in black background; disulfide bonds, arrow; catalytic site residues, circle; and residues forming a cis-proline bond, triangle.

this, Asp125 (L␤4␣4) and Tyr183 (L␤6␣6) interact with the carbonyl oxygen of the N-acetyl group near the C1 atom of the substrate and help in the stabilization of the oxazolinium intermediate [35, 36]. Thus, the catalytic triad is composed of Asp125, Glu127, and Tyr183 (Hevamine). This is different in inactive chitinases such as narbonin (His130, Glu132, and Gln191), ConB (Asp129, Gln131, and Tyr189), XIP-I (Phe126, Glu128, and Tyr183), and XAIP (His123, Glu125, and Tyr181 in XAIP) [12, 13, 16, 37]. Apart from this, other factors that contribute to lack of activity are the substitutions in the CBD. In XAIP, the presence of the side chains of residues Phe13, Pro77, Lys78, and Trp253 fills the pocket leaving no place for chitin [13]. The Glu132 of narbonin and Glu128 of XIP-I are engaged in forming salt bridge with Arg87 (narbonin) and Arg187 and Arg163 residues in XIP-I, respectively [9, 12, 37]. These salt bridges prevent proteins from acting as active chitinases. Moreover, Gly81 of hevamine, which forms H-bond with substrate, is replaced with Tyr80 in XIP-I [35, 36]. The Tyr80 shields the catalytic Glu128. In PPL2, the catalytic triad (Asp125, Glu127, Tyr182) and catalytic mechanism is well conserved. TCLL has a unique GlcNAc-binding site (S1 and S2). The subsites −4 and −3 lie close to one another (C␣ distances between Gly18 and Gln16 ˚ respectively) formof TCLL and hevamine are 5.5 and 6.6 A, C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

ing a wider cavity. Also Ser19TCLL along with the residues of extra ␣1 -helix (Asp20-Glu23) in subsite −4 occludes the substrate entry. In hevamine, NH group of the main chain of Ile82 (␤3␣3) is involved in H-bonding with the substrate at subsite −2. This loop region of subsite −2 protrudes away from the core barrel in TCLL. In the subsite −1, Tyr187 protrudes into the cleft and reduces GlcNAc-binding ability. The conformation of the loop containing Leu227 (Ala224 of Hevamine) is rigid and is lying away from the barrel, restraining the interaction of Leu227 with O6 of GlcNAc. Moreover, the catalytic triad of TCLL is composed of Ala128, Val130, and Phe186. Neither the Ala128 nor Phe186 can interact with GlcNAc to stabilize the intermediate. The catalytic Glu residue is replaced by a nonpolar valine at the catalytic site. The glycosylation in TCLL is different than observed in mammalian chi-lectins. It has been reported that CBD of chi-lectins such as HCgp-39 and YKL-39 has conserved tryptophan residues or SPG-40 and SI-CLP have aromatic residues. These residues help in hydrophobic interactions and H-bonding with sugars. The TCLL–GlcNac complex contains one GlcNac moiety in subsites S1 and S2 each. The site S1 is formed by L␣3␤4 and L␣4␤5 and ␣2. The sugar interacts with Glu119 and Arg152 directly and with Gln74, Tyr121,and Asp123 via water molecules. The site S2 is formed by L␤4␣4 and L␤5␣5 and ␣5. This pocket of TCLL is longer in comparison to PPL2. In www.proteomics-journal.com


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Figure 3. (A) Sequence alignment of RobpsCRA with class V chitinase from N. tabaccum (3ALF), A. thaliana (3AQU), and human chitotriosidase (1GUV) generated with ClustalW and ESpript. The conserved residues are represented in black background; hydrophobic residues of catalytic groove, triangle; hydrophilic residues, asterisk; catalytic site residues, square; and catalytic Ser residue, circle. (B) The modeled structure of RobpsCRA shows the characteristic fold of GH18 family.

S2 site, sugar interacts with Glu132 and Tyr167 directly and with Tyr168 and Lys171 through water. The S1 and S2 sites are composed of similar polar residues.

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GH18 family class V CLPs

The class V chitinases include the core domain and an insertion domain arranged in a manner that active site is formed in between to accommodate the chitin chain through stacking interaction between the pyranose rings of the GlcNAc units and hydrophobic residues lining the cleft [38]. The core domain is homologous to GH18 family and adopts a TIM barrel fold, whereas the insertion domain, which is embedded into the L␤9␣8, is composed of five antiparallel ␤-sheets flanked by one ␣-helix on one side and long loop on the other side. The catalytic site located at one end of the cleft is strong electronegatively. Other conserved structural motifs include canonical catalytic acidic DxDxE residues, YD motif, and a Ser residue Ser69 (numbering according to NtChiV, Fig. 3A) [39]. The N-terminal region of RobpsCRA, a lectin from the bark of black locust (Robinia pseudoacacia) with hemagglu-


tinin and glycan-binding activity [11], showed no sequence similarity with black locust bark lectin I (RPbAI) [40] or any other legume lectin, but readily aligned with the N-terminal of a plant class V chitinase from Arabidopsis thaliana (AtChiC) [41] and tobacco (NtChiV, 54% identical and 80% similarity) [39]. The structure of RobpsCRA (homology modeling using human chitotriosidase hMChi [42]) showed a conserved TIM barrel domain along with an additional hairpin loop formed by three antiparallel strands (Fig. 3B). The DxDxE (Asp112, Asp114, and Glu116) motif and the hydrophobic environment of CatD is also conserved [38]. These structural features show that RobpsCRA can cleave the glycosidic bond linking chitin. However, biochemical experiment showed that RobpsCRA does not act as a chitinase. The solvent-exposed CBD of hMChi [42], NtChiV [39], and AtChiC [41] is mostly composed of hydrophobic residues (mainly Trp residues), whereas in RobpsCRA hydrophilic residues (Lys-3, Ser-45, Gly-75, and Asp-191) are present, leading to changes in the overall conformation and physicochemical properties (hydrophilicity, charges) of the CBD. Also unlike all class V chitinases that are monomeric proteins, RobpsCRA is a homodimer. The subunits contain some structural features that allow


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dimerization and formation of a divalent carbohydratebinding protein. However, since no chitinase activity has been proposed for RobpsCRA, it can be viewed as an intermediary in this evolutionary pathway that will eventually yield lectins with an increased affinity for glycans.

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GH19 family CLPs

4.1 Overall structure Seed chitinases were initially thought to be confined to “family 19” [43]. The family 19 contains classes I, II, IV, and VI chitinases. The class I contains cysteine-rich hevein domain, a glycine/proline/threonine-rich hinge region at its Nterminal [44], and a COOH-terminal vacuolar-targeting motif [45], whereas the same are absent in class II enzymes. Class IV chitinases resemble class I chitinase of GH19 family, but are small in comparison to class I hevein domain due to deletions of residues. The hevein domain is 43 residues long and is stabilized by four disulfide bridges. These bridges are also essential for chitin binding [46]. The members of this family contain a highly conserved “signature sequence” QTSHETTGW, the “chitinase consensus sequence,” which is in the active site within 6A of bound substrate consisting of Asn, Ser, and Tyr and two catalytic active Glu (E) residues [47–51]. A group of CLPs (HbCLP1 and HbCLP2) encoded by latex and leaves of H. brasiliensis are the first examples of naturally occurring plant CLPs belonging to the GH19 family that exhibit strong chitin- and chitotriose-binding abilities [52]. The HbCLP1 has a hevein-like CBD and one CatD, both separated by a linker region of ten residues rich in glycine. The CBD of HbCLP1 shows the characteristic fold of GH19 family and has four conserved disulfide bonds (Cys3/Cys18, Cys12/Cys24, Cys17/Cys31, Cys35/Cys38; Fig. 4A). The residues Ser19, Tyr21, Trp23, and Tyr30 that are involved in chitin binding are also conserved. The CatD is composed of ten ␣-helices connected by five loops (Fig. 4B) stabilized by three disulfide bridges (Cys73/Cys135, Cys147/Cys155, Cys254/Cys286). The HbCLP1 also has two short antiparallel ␤-strands, two ␣-helices, and an intrinsically disordered ␣3␣4 loop. Structurally, it is similar to other members of GH19 family such as rice, mustard, and papaya chitinases belonging to the class I GH19 family and barley and rye of class II of GH19 family [53–57]. However, loops III, IV, and V exhibit an intermediate conformation in comparison to the apo- and substrate-bound structure of GH19 family (Fig. 4C). The loop III contains the carboxylate Glu139 (Glu 89 of barley chitinase), residue essential for catalysis [55, 57, 58]. Interestingly, the structure of HbCLP2 was novel, since it has one and a half CBDs (CBD (Glu1–Val43), first linker (Glu44–Gly53), peptide that corresponds to the second half of the CBD domain (Cys54–Val73), and a second linker (Gly74–Gly83). The long CBD domain of HbCLP2 does not show homology to any protein; however, it exhibits 99% identity with Hev b 11.0102 if residues of the first linked region are removed. C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim


Another group of CTL proteins, GhCTL are produced in the cotton (Gossypium hirsutum L.), pea, Arabidopsis, and rice [59, 60]. These proteins show a high sequence similarity with GH19. GhCTL group lack a chitin-binding region, a hinge region, and a vacuole-targeting motif. Two 35-kDa CLPs (GhCTL1 and GhCTL2) encoded by genes from cotton leaves and fiber possess novel consensus sequences in CatD and CBD. The residues in two functional sites (His66, Glu89, Ser120, and His121-barley chitinase) were mutated to nonsimilar amino acids (Ser68 and Glu91, Tyr124, and Trp125, respectively). Moreover substitution of Glu67 with Lys lead to the abolition of activity of both the CLPs. Phylogenetic analysis showed that such substitutions are conserved within the GhCTL family. The GhCTL2 have insertions (2-5 a.a.) in the loop regions (43–44, 103–104, and 235–239). 4.2 CBD and CatD architecture The catalytic mechanism of GH19 family protein requires a Glu residue that acts as an acid catalyst to attack C1 atom of the chitin fragment and a base to polarize the water molecule. The base is around 22 residues downstream of Glu. The CatD is lined by polar residues similar to chitosanase and lysozyme [61]. The nonpolar residues are involved in controlling the fold of the core region. The site-directed mutagenesis experiment shows that mutation of Glu212 and Glu234 leads to loss of enzyme activity [53–55, 62]. The structures of HbCLPs isoforms lack the acidic Glu residues (Ala117 in HbCLP1 and Ala147 in HbCLP2). Mutation of these Ala residues to Glu in both the isoforms recovered the chitin catalytic activity [52]. Residues involved in carbohydrate recognition are His116, Tyr146, and Phe207. Interactions between the CatD of HbCLP1 and (GlcNAc)6 involve loop III (residues Tyr146, Gln168, Ser170, Trp171, Tyr173, and Asn174) and cis-peptide (between Phe213 and Pro214 in loop IV). These residues are conserved between active chitinases [58, 61] and the HbCLPs. The HbCLPs have one tryptophan (Trp23) in the CBD and two tryptophan residues (Trp122 and Trp171 in HbCLP1, Trp152 and Trp201 in HbCLP2) in the CatD that participate in interaction with the ligand.

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Role of CLPs

5.1 Antifungal and insecticidal The CLPs display insecticidal and antifungal activities [63,64]. Literature suggests that CLPs are overexpressed in response to pathogenic attacks [63]. These proteins either act as “attack” molecules and damage the pathogen [65, 66] or act as “defense” molecules to protect plant cells from the molecular attack of pathogens [65–69]. The seeds of leguminous plants have been a rich source of CLP having antifungal activity. Two CLPs homologous to class III chitinases (acidic pH) and class I chitinases (basic pH) from seeds of www.proteomics-journal.com


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Figure 4. (A) Sequence alignment of HbCLP1 with barley chitinase (PDB ID 1CNS), rice chitinase (2DKV), mustard chitinase (2Z38), papaya chitinase (3CQL), and rye chitinase (4DWX) generated with ClustalW and ESpript. The conserved residues are represented in black background; loop ␣3␣4, box; catalytic Glu residue, triangle; and disulfide bridges, circles. (B) The CatD of HbCLP1 shows the characteristic GH19 fold. (C) Structure alignment of flexible L␣3␣4 of HbCLP1 with barley, rice, mustard, papaya, and rye chitinase is generated with PyMol.

chickpea showed strong antifungal activity against Ascochyta rabiei [70]. Dolichin (field bean) has a unique N-terminal sequence and exhibits 83% similarity with Canavalia ensiformis lectin. The unique sequence has Asn8, Leu13, and Gln22Glu25 [71]. The LKHRND, GFYTY, and AFITA blocks of sequence are well conserved. It also exhibits antiviral activity. Other proteins that show antiviral and antifungal activities include Delandia, phaseinA, 28-kDa CLP from cowpea, and 36-kDa CLP from inner shoots of chive [72–75]. Dolichin, delandin, 15-kDa antifungal protein from roots of Panax notoginseng, and 36-kDa CLP from inner shoots of chive exhibit antifungal response against Fusarium oxysporum, Coprinus comatus, Mycosphaerella arachidicola, Botrytis cinerea, and Rhizoctonia solani [71, 72, 75, 76]. The gene for stinging nettle


lectin (Urtica dioica agglutinin) encoded a precursor protein (UDA) with a lectin and a chitinase activity [6]. UDA can abolish the hyphal growth of several phytopathogenic and saprophytic chitin-containing fungi. Processing of UDA leads to a 8.5-kDa small-sized isolectin that has antifungal activity and can inhibit fungal hyphal growth in coordination with chitinase [77]. These isolectins can penetrate the plasma membrane of the pathogen and block cell-wall morphogenesis [78]. Mature UDA is true lectin and can agglutinate erythrocytes. It also acts as a superantigen and induces IFN-␥ in human lymphocytes, inhibiting the cytopathicity introduced by human viruses [79]. Like UDA, SN-HLPf from mature elderberry fruits is another chimeric protein that has antifungal activity [80].


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HbCLPs isoforms are expressed in response to pathogenic stress [52]. Although the natural-occurring CLPs are inactive, their mutant forms show selective inhibition of A. alternate [52]. The EgChit1-1 and EgChit5-1 genes in oil palm roots expressed two CLPs when treated with either Ganoderma boninense or Trichoderma harzianum, and both [81]. The EgChit1-1 shares homology with class I chitinases while EgChit5-1 belongs to class V chitinases. XIPs are potential defense molecules that can control the degradation of plant cell wall by fungal xylanases. A CTL XIP, a class III CLP from leaves of coffee (Coffea arabica), showed 60% inhibition of xylanases from Acrophialophora nainiana [67]. It also prevents spore germination in Phakopsora pachyrhizi (Asian rust) in soybean. OsCLP, a class III xylanase inhibitor from rice is differentially expressed during various developmental stages and under stress conditions [66]. The high dosage of OsCLP shows a strong chitinase activity against R. solani. XIP-I [69] and rice (RIXI) [82] do not inhibit plants endogenous xylanase, however they are potent against xylanases from fungal and bacterial sources [68]. Mulatexin (MLX56) and latex-abundant proteins a and b (LA-a and LA-b) from plants’ latex have been found to play a critical role in defense against pathogenic insects. MLX56, a 56-kDa chimeric protein, has Solanaceae lectin-like structure at its N-terminal consisting of two hevein-like CBDs, an extensin domain followed by CTL domains at the C-terminal [83]. It is highly toxic to Sclerotinia ricini and Mamestra brassicae, however showed no toxicity to Bombyx mori. The MLX56 is exclusively found only in the latex of mulberries (Morus bombycis, M. alba, and M. australis). The LA-a and LA-b are approximately 50- and 46-kDa proteins. They are glycosylated and showed insecticidal activities against larvae of Drosophila melanogaster [84]. These defense proteins are resistant to proteolytic degradation and are active under alkaline pH (midgut luminal pH). 5.2 Antifreeze proteins The overwintering plants express antifreeze proteins (AFPs) to tolerate the extracellular ice formation (during cold acclimation in apoplast) [85]. Interestingly, these AFPs or their corresponding genes are homologous to PR proteins. The cellular localization of AFPs and pathogen-induced CLPs are similar. This signifies that in plants common pathway is used for penetration and propagation of pathogens and ice [86]. AFPs are localized in various parts including rhizomes, seeds, stems, crowns, barks, branches, buds, petioles leaf blades, flowers, berries, roots, and tubers. Three Solanum thermal hysteresis proteins (29, 47, and 64 kDa) from the stem of winter bittersweet nightshade (S. dulcamara) possess antifreeze activity and chitinase activity [87,88]. Interestingly STHP-64 also has conserved glutaminerich region, zinc finger motif, an acidic domain, and another zinc finger motif. These features are present in transcription factors (WRKY proteins) [89] that regulate the expression of PR proteins in plants. C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim


5.3 Growth and development In the process of evolution, many CLPs have lost their hydrolytic activity while retaining their chitin-binding abilities, which has given rise to scaffolds with new functions. Evidences suggest that GhCTL are essential for cellulose synthesis in primary and secondary cell walls [60]. The elp1 mutation within the Arabidopsis AF422179 CTL (AtCTL1) protein leads to ectopic lignin deposition, elevated ethylene production, variation in root and shoot architecture, and incomplete primary cell walls in pith tissue [90]. Similarly hot2 mutant in Arabidopsis (AtCTL1) was identified in response to thermotolerance and pleiotropic abnormal phenotypes (semidwarfism, ethylene overproduction, and aberrant cell shape with incomplete cell walls) [91]. The mutant showed an abnormal tolerance to salinity and drought stresses, and accumulated high levels of Na+ in cells under either normal or NaCl stress conditions. LusCTL1 and 2 from flax were found to be expressed more strongly in xylem tissue than in any other tissue [92]. The sequence showed similarity to AtCTL2 of A. thaliana and GhCTL1, GhCTL2 of G. hirsutum. These CLPs are essentially involved in wall thickening. Rodrı guez et al. reported that CLPs CTL1/POM1 and CTL2 mediate the movement of cellulose synthase [93]. The brittle culm BC15/OsCTL1 (for CTL1) gene encodes the classII CLP, which affects the cellulose content and mechanical strength without noticeable alterations in plant growth [94]. BC15 is devoid of hevein domain and the chitinase activity motif (H-E-T-T) but has an N-terminal transmembrane domain. In Carribean pine, the CLPs have shown to bind to arabinogalactan protein fraction from embryogenic tissues [95]. 5.4 Chitinase-related receptor-like kinase (CHRK) 1 CHRK1 has an enzymatically inactive class V CatD in the extracellular region [96]. The C-terminal kinase domain of CHRK1 is similar to serine/threonine protein kinases. The discovery of CHRK1 reveals that CLPs can bind oligosaccharides and can act as regulators of responses that are normally mediated through oligosaccharide-dependent signals. The kinase domain exhibited autophosphorylation, suggesting that CHRK1 upon chitin recognition of the extracellular domain activates an intracellular serine/threonine kinase domain and triggers signal transduction. 5.5 Nodule development Srchi24, a novel early nodulin, plays a role in nodule development process [97]. During nodulation, the CLPs interact with the rhizoidal Nod-factors that are the main signal molecules to trigger the onset of nodulation. The immunolocalization and in situ hybridization experiments demonstrate that Srchi24 is localized in the outermost cortical cell layers of the developing nodules. The Srchi24 www.proteomics-journal.com

9

Proteomics 2015, 00, 1–13 Table 1. CLPs from plants and their function

Source

Function

Ref.

An acidic CLP and basic CLP from seeds of chickpea (C. arietinum) Dolichin from field beans (D. lablab) Delandin from rice beans (D. umbellata)

Antifungal

[70]

Antifungal and HIV-1 reverse-transcriptase inhibition Antifungal, HIV-1 reverse-transcriptase inhibition and mitogenic activities Antifungal, HIV-1 reverse-transcriptase inhibition Antifungal and antiviral activities Antifungal, antiviral, cytotoxic to breast cancer cells Antifungal Antifungal Antifungal Antifungal and allergen Antifungal XIPs XIPs XIPs XIPs XAIP Antifungal Insecticidal Insecticidal Antifreeze

[71] [72]

Phasein A from pinto bean (P. vulgaris cv. Pinto) 28-kDa CLP from cowpea (V. unguiculata) 36-kDa CLP from Chive 15-kDa CLP from roots of Panax notoginseng Stinging nettle lectin (U. dioica agglutinin) SN-HLPf from mature elderberry fruits HbCLPs from rubber tree (H. brasiliensis) EgChit1-1 and EgChit5-1 genes in oil palm roots CTL XIP isolated from leaves of coffee XIP-I from wheat OsCLP from Rice RIXI from rice XAIP from S. multiflorus PPL2 Mulatexin (MLX56) LA-a and LA-b from mulberry (Morus sp.) Thermal hysteresis proteins (29, 47, and 64 kDa) from S. dulcamara GhCTL from secondary cotton cells elp1 mutation within AtCTL1 from Arabidopsis hot2 mutation within AtCTL1 from Arabidopsis LusCTL1 and 2 of flax CTL1/POM1 and CTL2 from Arabidopsis Brittle culm BC15/OsCTL1 in rice CLPs from C. pine CHRK1 from tobacco CLP from banana Srchi24 from Sesbania rostrata

Cellulose biosynthesis Ectopic lignin deposition, elevated ethylene production, variation in root and shoot architecture Salt tolerant Cell wall thickening Cellulose biosynthesis Cellulose content and mechanical strength Development of embryogenic tissues Receptor-like kinase Storage proteins Nodule development

resembles class III chitinases, but catalytic Glu is replaced by Lys. The protein contains an ER-targeting signal peptide at the N-terminal and 35-residue-long C-terminal extension.

5.6 Storage proteins A 30-kDa inactive homolog CRP belonging to class III acidic chitinases was seen during the fruit ripening process in banana [98]. It serves as a source of amino acids for the synthesis of ripening-associated proteins.

6

Potential applications of CLPs

CLPs can be used in various areas of biotechnology. The genes of CLPs possessing fungicidal and insecticidal properties can be utilized for the development of transgenic plants [99]. The hs2 gene encoding a CLP in a triploid Dutch elm disease resistant American elm (Ulmus americana NPS-3-487) has been used to genetically modify creeping bentgrass (Agrostis palustris Huds.) to provide resistance against R. solani [100]. CLPs C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

[73] [74] [75] [76] [77] [80] [52] [81] [67] [69] [66] [82] [13] [21] [83] [84] [87, 88] [60] [90] [91] [92] [93] [94] [95] [96] [98] [97]

also show antibacterial, antiviral, antiproliferative, and mitogenic activities that can be further exploited for medicinal usage and can be of interest to the biomedical and pharmaceutical industries. CLPs are being currently exploited for their usage in food industry. XIP-I and XAIP inhibit different types of xylanases utilized in food processing industry for enzymatic modification [101]. They are used in bread making, beer making, brewing, and animal feeding [19]. AFPs can also be used for producing GM crops having increased freeze tolerance. AFPs can help in improving quality and shelf life of frozen foods and vegetables; cyropresevation of tissues and organs such as oocytes, embryos, sperms, and platelets; and cryosurgery [102–107]. Table 1 lists CLPs from plants involved in various functions.

7

Conclusion

The CLPs have evolved into a new class of proteins with functional diversification. CLPs have undergone adaptive functional diversification in response to various biotic and abiotic www.proteomics-journal.com

10

P. Kesari et al.

stress conditions, which has either lead to plurifunctionality or neofunctionalization. Since each CLP has evolved independently during this process of evolution, the functional mechanism of many of them is still unknown. They share structural features with chitinases belonging to GH18 and 19 families. In many CLPs, the catalytic Glu or essential residues of the CatD/CBD of chitinases are replaced with similar residues—leading to loss of chitinases’ binding and hydrolyzing abilities. In other cases, even if a Glu residue is present, it is engaged in the salt-bridge formation and other interactions. The structural features of CLPs are such that it can resist abiotic stresses and can survive hostile environments. These CLPs have retained their chitin-binding ability while the hydrolyzing ability is lost. They have acquired the ability to mediate a number of biological processes such as stress responses, growth, and developmental operations. Many class II CLPs are induced by salt stress. The class I proteins can tolerate a high range of temperature. Since plants possess common pathways for the penetration and propagation of pathogens and ice, the evolutionary event has resulted in chitinase with antifreeze activity. In CHRK1 the evolutionary event has resulted in fusion of an enzymatically inactive CatD with a kinase domain. Thus, the protein can bind oligosaccharides and triggers the signal transduction process. In the process of evolution, many lectins have developed a CBD and are anticipated to acquire novel functions. There could be a possibility that these have evolved to recognize glycan moieties present on the cell surface involved in signaling. The various roles of CLPs have been proposed and discussed, however the vast applications need to be further exploited for their potential usage in agriculture, food, pharmaceutical, and biotech industries. In future, structure-based protein engineering can be employed to design CLPs with novel functions. PK, AKS, and ST thank the Department of Science and Technology (DST ref no. SR/SO/BB-0026/2009), New Delhi, India, for the financial support for this work. PK also thanks DRDO, Government of India, for financial support. The authors have declared no conflict of interest.

8

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