Structural and Functional Evolution of Chitinase&#X02010;Like Proteins from Plants

Proteomics 2015, 15, 1693–1705 DOI 10.1002/pmic.201400421 1693

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 Received: August 31, 2014 chitinases referred to as chitinase-like proteins (CLPs). Although CLPs share high sequence Revised: January 16, 2015 and structural homology with chitinases of glycosyl hydrolase 18 (TIM barrel domain) and 19 Accepted: February 24, 2015 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 plu- rifunctional 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.

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

1 Introduction 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 Nature has equipped plants with chitinases to protect them GH19 family. The classes III and V show very less homol- from chitin-containing pathogens. Chitinases are also ex- ogy with each other and no sequence similarity to enzymes pressed in response to abiotic stress and during developmen- of GH19 family. The chitinases of GH18 family adopt ␤/␣ tal processes of plants [1–3]. These proteins are primarily barrel fold, whereas the GH19 chitinases have a high helical categorized into glycosyl hydrolase (GH) 18 and 19 families. content because of the presence of nonpolar residues in the The cDNA-deduced sequence of the proteins from both the core region [8]—and show structural similarity to chitosanase families shows that the chitinases are composed of an N- and lysozyme [9]. Both families exhibit diversity in their se- terminal signal peptide of variable lengths [4–6]. As per the quences, domain orientation, and hydrolytic mechanisms. revised chitinase gene classiﬁcation, they are mainly grouped 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 Correspondence: Dr. Pravindra Kumar, Department of Biotechnol- ogy, Indian Institute of Technology Roorkee, Roorkee, UK 247667, (CLPs). The CLPs share high sequence and structural similar- India ity with chitinases of GH18 and 19 families; but they may lack E-mail: [email protected] the binding/catalytic activity due to the presence of substitu- Fax: +91-1332-273560 tions in the chitin-binding domain (CBD) or CatD. Molecular genetic analysis reveals that gene duplication events followed Abbreviations: AFPs, antifreeze proteins; CBD, chitin-binding do- by mutation in the existing chitinase gene have resulted in main; CHRK, chitinase-related receptor-like kinase; CLP, chitinase- like proteins; CTL, chitinase-like; GH, glycosyl hydrolase; Glc- the loss of activity [10]. NAc, N-acetylglucosamine; PPL2, Parkia platycephala lectin 2; PR, CLPs have evolved through two different evolutionary pathogenesis related; TCLL, tamarind CTL lectin; XAIP, xylanase pathways. In the ﬁrst category, a mutation has led to loss and ␣-amylase inhibitor protein; XIP-I, xylanase inhibitor protein I of catalytic potential “Glu” residue. These CLPs do not

C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com 1694 P. Kesari et al. Proteomics 2015, 15, 1693–1705 possess catalytic activity; however polysaccharide-binding has a barrel fold, but the first helix of the fold is replaced ability remains intact. The second pathway is the one in with a loop region; also ␤1and␤2 strands are connected which the catalytic activity is retained while the molecule by an extended loop comprising of two ␤-turns. Addition- has also developed other functional domains. Thus, despite ally, an extended antiparallel ␤-strand ␤2 is placed parallel being homologous to chitinases, the individual gene has to ␣3and␣4; a short ␣-helix situated between ␤7and␣7 evolved independently and has led to adaptive functional and 310 helix between ␤8 and C-terminal helix has also been diversification. observed. Only one cysteine residue has been reported in Sequence analysis shows that many lectins have evolved to narbonin as compared to other plant-type chitinases. ConB interact with N-acetylglucosamine (GlcNAc), thus progress- has an extra disulfide bond Cys41–Cys93 in comparison to ing from chitinase to CLPs. Such CLPs are known for their narbonin, this bond is engaged in stabilization of L␤2␣2and ability to agglutinate erythrocytes and are thus referred to L␤3␣3. Morrison et al. showed presence of a firmly bound as chi-lectins. Chi-lectins adopt the folds similar to chiti- zinc ion, NADPH, and flavin mononucleotide [15]. ConB has nase, but the catalytic Glu residues are substituted, lead- an extended loop between the ␤2and␣2-helix [16]. Sequence ing to loss of catalytic activity. Thus chi-lectin can interact showed a glycosylation site (Asn284) in ConB [17]. with chitin but cannot hydrolyze the subunits. The struc- XIP is the first example of a protein able to inhibit mem- tural comparison of CTL lectins and chitinase gene shows bers of GH10, GH11, and GH13 families [18, 19]. The struc- that lectins have evolved CBD from some ancestral chitinase ture of XIP-I has two disulfide bridges connecting ␣1and genes [11]. ␣2(Cys25andCys66)andL␤5␣5andL␤6␣6 (Cys164 and The CLPs are known to inhibit the fungal growth by in- Cys195) and three cis-peptides. One cis-proline (Pro167) is ob- hibiting fungal xylanases [12, 13]. In such proteins the struc- served in L␤5␣5 and others occur between Ser36 and Phe37 tural scaffold is preserved, but the classic “Glu” residue is and between Trp256 and Asp257. The residues Phe37 and engaged in intermolecular H-bond formation with surround- Trp256 emerging from ␤2andendof␤8 strand, respectively, ing residue, thus abolishing the chitin hydrolyzing ability. In protrude into the groove on the top of the ␤-barrel and form other such proteins the CBD has evolved to recognize chitin- the rigid central part of the XIP-I. The XIP-I mimics the inter- like molecules, thus playing an important role in growth and action of xylanase enzyme with oligosaccharides and occludes developmental processes. its enzyme’s active site. Complex of XIP-I with GH10 and 11 shows that the protein adopts two independent conforma- tions for inhibiting two structurally and functionally different 2 GH18 family class III CLPs classes of enzymes [18]. The L␣4␤5 of XIP-I helps in inter-

action with GH11 xylanase wherein the residue Arg149XIP-I 2.1 Overall structure interacts with the residues Glu85 and Glu176 of GH11. The GH10 xylanase interacts with XIP-I through the ␣7 (232–245)

The GH18 class III chitinases are characterized by (␤/␣)8 of XIP-I that blocks four subsites in the substrate-binding barrel topology; an active site containing two Asp and one groove of GH10 enzyme. The Lys234 of ␣7XIP-I interacts with Glu acid residues separated by phenylalanine and isoleucine Glu131 and Glu239 of GH10. The XIP-I inhibitor is specific (DxDxE) and belongs to pathogensis-related (PR) 8 family for fungal and bacterial xylanases from GH family. It has a proteins. Hevamine isolated from the rubber plant (Hevea putative site for N-linked glycosylation (Asn89 and Asn265) brasiliensis) is considered as the prototype of this family as [20]. The XAIP inhibits GH11 xylanase and GH13 ␣-amylase it exhibits both lysozyme and chitinase activity [9]. In CLPs, [13]. The barrel fold of XAIP consists of an extra helix located substitution of catalytic Glu residue with any other residue is between ␤8and␣8. Although XAIP shares high sequence ho- related to lack of hydrolytic activity. Some of the CLPs whose mology with hevamine (48%) and ConB (39%), the disulfide crystal structures are available include ConB from Canavalia linkages in XAIP are identical to those of XIP-I. The XAIP has ensiformis, narbonin from Vicia narbonensis, xylanase in- a novel L␣3␤4 that protrudes sharply away from the surface hibitor protein I (XIP-I) from Triticum aestivum (var. Sois- of the protein and has Pro–Pro dipeptide, which disrupts the son), xylanase and ␣-amylase inhibitor protein (XAIP) from conformation of ␣3 located at the C-terminal. The inhibition Scadoxus multiflorus, Parkia platycephala lectin 2 (PPL2), and of GH11 xylanase and G13 ␣-amylase takes place through tamarind CTL lectin (TCLL) from Tamarindus indica [12–17] L␣4␤4andL␤6␣6 (along with ␣7) of XAIP [13].

(Fig. 1). These proteins are also composed of (␤/␣)8 topol- Plant lectins have also evolved to adopt a GlcNac-binding ogy, two consensus regions equivalent to the third and fourth ability. PPL2 exhibits a GlcNac-dependent hemagglutination barrel strands (some mutations exist in these regions) and and endo-chitinase ability [21]. The N-terminal (42 residues) an elongated ␤2␣2 loop (L␤2␣2) having one antiparallel ␤- showed high similarity with hydrolases of GH18 family. The hairpin (Fig. 2). The structure is stabilized by disulfide bridges ␤6␣6 cleft region and L␤2␣2andL␤7␣7 of PPL2 are different and three nonproline cis-peptide bonds localized in the cen- as compared to other plant chitinases. It has five cis-peptide tral groove, later interacts with the pyranose rings of GlcNAc. bonds and three disulfide bonds. A CTL lectin from T. indica Narbonin exhibits weak yet clear similarity to endo-p-N- (TCLL) also shows hemagglutination but has no endochiti- acetylglucosaminidase H from S. plicatus [14]. The protein nase activity. It has two novel sites for GlcNAc binding, which

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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, 2.2 CBD and CatD architecture SI-CLP, SPS-40, SPG-40, SPC-40, YKL-39, Ym1, and IDGF-2 [22–30]). The ␤1strand,␣3-helix, an antiparallel ␤-hairpin Class III hydrolyzes chitin by a double-displacement mech- in the L␤2␣2 region, and ␤-sheet insertion in the L␤3␣3are anism that involves two catalytic residues and proceeds unique features of TCLL. The insertion of ␣1(20–23) and ␣8 through a geometrically distorted oxocarbonium intermedi- (258–264) disrupts the ␤␣␤ fold of the protein. Such insertion ate [32,33]. The catalytic site is formed by the C-terminal ends has been reported in several (␤␣)8 barrel topologies [16, 31]. of the ␤ strands and the loops linking them with the subse- The sequence results from LC MALDI TOF-TOF and ESI- quent ␣-helices [34]. The carboxyl side chain of Glu127 acts Trap analysis showed that N-linked glycosylation (residues as a proton donor to the scissile glycosidic-bond between the 62 and 146) site exist on TCLL. sugar residues bound at the −1and+1 subsites. Apart from

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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 ing a wider cavity. Also Ser19TCLL along with the residues carbonyl oxygen of the N-acetyl group near the C1 atom of of extra ␣1-helix (Asp20-Glu23) in subsite −4 occludes the the substrate and help in the stabilization of the oxazolinium substrate entry. In hevamine, NH group of the main chain of intermediate [35,36]. Thus, the catalytic triad is composed of Ile82 (␤3␣3) is involved in H-bonding with the substrate at Asp125, Glu127, and Tyr183 (Hevamine). This is different subsite −2. This loop region of subsite −2 protrudes away in inactive chitinases such as narbonin (His130, Glu132, and from the core barrel in TCLL. In the subsite −1, Tyr187 Gln191), ConB (Asp129, Gln131, and Tyr189), XIP-I (Phe126, protrudes into the cleft and reduces GlcNAc-binding ability. Glu128, and Tyr183), and XAIP (His123, Glu125, and Tyr181 The conformation of the loop containing Leu227 (Ala224 of in XAIP) [12, 13, 16, 37]. Hevamine) is rigid and is lying away from the barrel, restrain- Apart from this, other factors that contribute to lack of ing the interaction of Leu227 with O6 of GlcNAc. Moreover, activity are the substitutions in the CBD. In XAIP, the pres- the catalytic triad of TCLL is composed of Ala128, Val130, and ence of the side chains of residues Phe13, Pro77, Lys78, and Phe186. Neither the Ala128 nor Phe186 can interact with Glc- Trp253 ﬁlls the pocket leaving no place for chitin [13]. The NAc to stabilize the intermediate. The catalytic Glu residue Glu132 of narbonin and Glu128 of XIP-I are engaged in formis replaced by a nonpolar valine at the catalytic site. The gly- ing salt bridge with Arg87 (narbonin) and Arg187 and Arg163 cosylation in TCLL is different than observed in mammalian residues in XIP-I, respectively [9, 12, 37]. These salt bridges chi-lectins. It has been reported that CBD of chi-lectins such prevent proteins from acting as active chitinases. Moreover, as HCgp-39 and YKL-39 has conserved tryptophan residues or Gly81 of hevamine, which forms H-bond with substrate, is SPG-40 and SI-CLP have aromatic residues. These residues replaced with Tyr80 in XIP-I [35, 36]. The Tyr80 shields the help in hydrophobic interactions and H-bonding with sug- catalytic Glu128. ars. The TCLL–GlcNac complex contains one GlcNac moiety In PPL2, the catalytic triad (Asp125, Glu127, Tyr182) and in subsites S1 and S2 each. The site S1 is formed by L␣3␤4 catalytic mechanism is well conserved. TCLL has a unique and L␣4␤5and␣2. The sugar interacts with Glu119 and GlcNAc-binding site (S1 and S2). The subsites −4and−3lie Arg152 directly and with Gln74, Tyr121,and Asp123 via wa- close to one another (C␣ distances between Gly18 and Gln16 ter molecules. The site S2 is formed by L␤4␣4andL␤5␣5and of TCLL and hevamine are 5.5 and 6.6 A,˚ respectively) form- ␣5. This pocket of TCLL is longer in comparison to PPL2. In

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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 tinin and glycan-binding activity [11], showed no sequence with Tyr168 and Lys171 through water. The S1 and S2 sites similarity with black locust bark lectin I (RPbAI) [40] or any are composed of similar polar residues. 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) 3 GH18 family class V CLPs [39]. The structure of RobpsCRA (homology modeling using human chitotriosidase hMChi [42]) showed a conserved TIM The class V chitinases include the core domain and an inser- barrel domain along with an additional hairpin loop formed tion domain arranged in a manner that active site is formed by three antiparallel strands (Fig. 3B). The DxDxE (Asp112, in between to accommodate the chitin chain through stack- Asp114, and Glu116) motif and the hydrophobic environment ing interaction between the pyranose rings of the GlcNAc of CatD is also conserved [38]. These structural features show units and hydrophobic residues lining the cleft [38]. The core that RobpsCRA can cleave the glycosidic bond linking chitin. domain is homologous to GH18 family and adopts a TIM However, biochemical experiment showed that RobpsCRA barrel fold, whereas the insertion domain, which is embed- does not act as a chitinase. The solvent-exposed CBD of hM- ded into the L␤9␣8, is composed of ﬁve antiparallel ␤-sheets Chi [42], NtChiV [39], and AtChiC [41] is mostly composed ﬂanked by one ␣-helix on one side and long loop on the other of hydrophobic residues (mainly Trp residues), whereas in side. The catalytic site located at one end of the cleft is strong RobpsCRA hydrophilic residues (Lys-3, Ser-45, Gly-75, and electronegatively. Other conserved structural motifs include Asp-191) are present, leading to changes in the overall con- canonical catalytic acidic DxDxE residues, YD motif, and a Ser formation and physicochemical properties (hydrophilicity, residue Ser69 (numbering according to NtChiV, Fig. 3A) [39]. charges) of the CBD. Also unlike all class V chitinases The N-terminal region of RobpsCRA, a lectin from the that are monomeric proteins, RobpsCRA is a homodimer. bark of black locust (Robinia pseudoacacia) with hemagglu- The subunits contain some structural features that allow

C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com 1698 P. Kesari et al. Proteomics 2015, 15, 1693–1705 dimerization and formation of a divalent carbohydrate- Another group of CTL proteins, GhCTL are produced in binding protein. However, since no chitinase activity has been the cotton (Gossypium hirsutum L.), pea, Arabidopsis,and proposed for RobpsCRA, it can be viewed as an intermediary rice [59, 60]. These proteins show a high sequence similar- in this evolutionary pathway that will eventually yield lectins ity with GH19. GhCTL group lack a chitin-binding region, with an increased affinity for glycans. 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 4 GH19 family CLPs and CBD. The residues in two functional sites (His66, Glu89, Ser120, and His121-barley chitinase) were mutated to non- 4.1 Overall structure similar amino acids (Ser68 and Glu91, Tyr124, and Trp125, respectively). Moreover substitution of Glu67 with Lys lead to Seed chitinases were initially thought to be confined to “fam- the abolition of activity of both the CLPs. Phylogenetic analy- ily 19” [43]. The family 19 contains classes I, II, IV, and sis showed that such substitutions are conserved within the VI chitinases. The class I contains cysteine-rich hevein do- GhCTL family. The GhCTL2 have insertions (2-5 a.a.) in the main, a glycine/proline/threonine-rich hinge region at its N- loop regions (43–44, 103–104, and 235–239). terminal [44], and a COOH-terminal vacuolar-targeting motif [45], whereas the same are absent in class II enzymes. Class 4.2 CBD and CatD architecture IV chitinases resemble class I chitinase of GH19 family, but are small in comparison to class I hevein domain due to The catalytic mechanism of GH19 family protein requires a deletions of residues. The hevein domain is 43 residues long Glu residue that acts as an acid catalyst to attack C1 atom of and is stabilized by four disulfide bridges. These bridges are the chitin fragment and a base to polarize the water molecule. also essential for chitin binding [46]. The members of this The base is around 22 residues downstream of Glu. The CatD family contain a highly conserved “signature sequence” QT- is lined by polar residues similar to chitosanase and lysozyme SHETTGW, the “chitinase consensus sequence,” which is in [61]. The nonpolar residues are involved in controlling the fold the active site within 6A of bound substrate consisting of Asn, of the core region. The site-directed mutagenesis experiment Ser, and Tyr and two catalytic active Glu (E) residues [47–51]. shows that mutation of Glu212 and Glu234 leads to loss of A group of CLPs (HbCLP1 and HbCLP2) encoded by latex enzyme activity [53–55, 62]. and leaves of H. brasiliensis are the first examples of naturally The structures of HbCLPs isoforms lack the acidic Glu occurring plant CLPs belonging to the GH19 family that residues (Ala117 in HbCLP1 and Ala147 in HbCLP2). exhibit strong chitin- and chitotriose-binding abilities [52]. Mutation of these Ala residues to Glu in both the isoforms The HbCLP1 has a hevein-like CBD and one CatD, both recovered the chitin catalytic activity [52]. Residues involved separated by a linker region of ten residues rich in glycine. in carbohydrate recognition are His116, Tyr146, and Phe207. The CBD of HbCLP1 shows the characteristic fold of GH19 Interactions between the CatD of HbCLP1 and (GlcNAc) family and has four conserved disulfide bonds (Cys3/Cys18, 6 involve loop III (residues Tyr146, Gln168, Ser170, Trp171, Cys12/Cys24, Cys17/Cys31, Cys35/Cys38; Fig. 4A). The Tyr173, and Asn174) and cis-peptide (between Phe213 and residues Ser19, Tyr21, Trp23, and Tyr30 that are involved Pro214 in loop IV). These residues are conserved between in chitin binding are also conserved. The CatD is composed active chitinases [58, 61] and the HbCLPs. The HbCLPs have of ten ␣-helices connected by five loops (Fig. 4B) stabilized one tryptophan (Trp23) in the CBD and two tryptophan by three disulfide bridges (Cys73/Cys135, Cys147/Cys155, residues (Trp122 and Trp171 in HbCLP1, Trp152 and Trp201 Cys254/Cys286). The HbCLP1 also has two short antiparallel in HbCLP2) in the CatD that participate in interaction with ␤-strands, two ␣-helices, and an intrinsically disordered the ligand. ␣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 5 Role of CLPs class II of GH19 family [53–57]. However, loops III, IV, and V exhibit an intermediate conformation in comparison to the 5.1 Antifungal and insecticidal apo- and substrate-bound structure of GH19 family (Fig. 4C). The loop III contains the carboxylate Glu139 (Glu 89 of barley The CLPs display insecticidal and antifungal activities [63,64]. chitinase), residue essential for catalysis [55, 57, 58]. Interest- Literature suggests that CLPs are overexpressed in response ingly, the structure of HbCLP2 was novel, since it has one and to pathogenic attacks [63]. These proteins either act as “at- a half CBDs (CBD (Glu1–Val43), first linker (Glu44–Gly53), tack” molecules and damage the pathogen [65, 66] or act as peptide that corresponds to the second half of the CBD “defense” molecules to protect plant cells from the molec- domain (Cys54–Val73), and a second linker (Gly74–Gly83). ular attack of pathogens [65–69]. The seeds of leguminous The long CBD domain of HbCLP2 does not show homology plants have been a rich source of CLP having antifun- to any protein; however, it exhibits 99% identity with Hev b gal activity. Two CLPs homologous to class III chitinases 11.0102 if residues of the first linked region are removed. (acidic pH) and class I chitinases (basic pH) from seeds of

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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 lectin (Urtica dioica agglutinin) encoded a precursor protein rabiei [70]. Dolichin (field bean) has a unique N-terminal se- (UDA) with a lectin and a chitinase activity [6]. UDA can abol- quence and exhibits 83% similarity with Canavalia ensiformis ish the hyphal growth of several phytopathogenic and sapro- lectin. The unique sequence has Asn8, Leu13, and Gln22- phytic chitin-containing fungi. Processing of UDA leads to a Glu25 [71]. The LKHRND, GFYTY, and AFITA blocks of se- 8.5-kDa small-sized isolectin that has antifungal activity and quence are well conserved. It also exhibits antiviral activity. can inhibit fungal hyphal growth in coordination with chiti- Other proteins that show antiviral and antifungal activities nase [77]. These isolectins can penetrate the plasma mem- include Delandia, phaseinA, 28-kDa CLP from cowpea, and brane of the pathogen and block cell-wall morphogenesis 36-kDa CLP from inner shoots of chive [72–75]. Dolichin, [78]. Mature UDA is true lectin and can agglutinate erythro- delandin, 15-kDa antifungal protein from roots of Panax no- cytes. It also acts as a superantigen and induces IFN-␥ in toginseng, and 36-kDa CLP from inner shoots of chive exhibit human lymphocytes, inhibiting the cytopathicity introduced antifungal response against Fusarium oxysporum, Coprinus co- by human viruses [79]. Like UDA, SN-HLPf from mature el- matus, Mycosphaerella arachidicola, Botrytis cinerea,andRhi- derberry fruits is another chimeric protein that has antifungal zoctonia solani [71, 72, 75, 76]. The gene for stinging nettle activity [80].

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HbCLPs isoforms are expressed in response to pathogenic 5.3 Growth and development stress [52]. Although the natural-occurring CLPs are inactive, their mutant forms show selective inhibition of A. alternate In the process of evolution, many CLPs have lost their [52]. The EgChit1-1 and EgChit5-1 genes in oil palm roots ex- hydrolytic activity while retaining their chitin-binding abil- pressed two CLPs when treated with either Ganoderma boni- ities, which has given rise to scaffolds with new functions. nense or Trichoderma harzianum, and both [81]. The EgChit1-1 Evidences suggest that GhCTL are essential for cellulose shares homology with class I chitinases while EgChit5-1 be- synthesis in primary and secondary cell walls [60]. The elp1 longs to class V chitinases. mutation within the Arabidopsis AF422179 CTL (AtCTL1) XIPs are potential defense molecules that can control the protein leads to ectopic lignin deposition, elevated ethylene degradation of plant cell wall by fungal xylanases. A CTL production, variation in root and shoot architecture, and in- XIP, a class III CLP from leaves of coffee (Coffea arabica), complete primary cell walls in pith tissue [90]. Similarly hot2 showed 60% inhibition of xylanases from Acrophialophora mutant in Arabidopsis (AtCTL1) was identified in response nainiana [67]. It also prevents spore germination in Phakop- to thermotolerance and pleiotropic abnormal phenotypes sora pachyrhizi (Asian rust) in soybean. OsCLP, a class III (semidwarfism, ethylene overproduction, and aberrant cell xylanase inhibitor from rice is differentially expressed during shape with incomplete cell walls) [91]. The mutant showed various developmental stages and under stress conditions an abnormal tolerance to salinity and drought stresses, and [66]. The high dosage of OsCLP shows a strong chitinase accumulated high levels of Na+ in cells under either normal activity against R. solani. XIP-I [69] and rice (RIXI) [82] do not or NaCl stress conditions. LusCTL1 and 2 from flax were inhibit plants endogenous xylanase, however they are potent found to be expressed more strongly in xylem tissue than against xylanases from fungal and bacterial sources [68]. in any other tissue [92]. The sequence showed similarity to Mulatexin (MLX56) and latex-abundant proteins a and b AtCTL2 of A. thaliana and GhCTL1, GhCTL2 of G. hirsutum. (LA-a and LA-b) from plants’ latex have been found to play a These CLPs are essentially involved in wall thickening. critical role in defense against pathogenic insects. MLX56, a Rodrıguez et al. reported that CLPs CTL1/POM1 and CTL2 56-kDa chimeric protein, has Solanaceae lectin-like structure mediate the movement of cellulose synthase [93]. at its N-terminal consisting of two hevein-like CBDs, an The brittle culm BC15/OsCTL1 (for CTL1) gene encodes extensin domain followed by CTL domains at the C-terminal the classII CLP, which affects the cellulose content and [83]. It is highly toxic to Sclerotinia ricini and Mamestra mechanical strength without noticeable alterations in plant brassicae, however showed no toxicity to Bombyx mori.The growth [94]. BC15 is devoid of hevein domain and the MLX56 is exclusively found only in the latex of mulberries chitinase activity motif (H-E-T-T) but has an N-terminal (Morus bombycis, M. alba,andM. australis). The LA-a and transmembrane domain. In Carribean pine, the CLPs have LA-b are approximately 50- and 46-kDa proteins. They are shown to bind to arabinogalactan protein fraction from glycosylated and showed insecticidal activities against larvae embryogenic tissues [95]. of Drosophila melanogaster [84]. These defense proteins are resistant to proteolytic degradation and are active under 5.4 Chitinase-related receptor-like kinase (CHRK) 1 alkaline pH (midgut luminal pH). CHRK1 has an enzymatically inactive class V CatD in the 5.2 Antifreeze proteins extracellular region [96]. The C-terminal kinase domain of CHRK1 is similar to serine/threonine protein kinases. The The overwintering plants express antifreeze proteins (AFPs) discovery of CHRK1 reveals that CLPs can bind oligosaccha- to tolerate the extracellular ice formation (during cold accli- rides and can act as regulators of responses that are normally mation in apoplast) [85]. Interestingly, these AFPs or their mediated through oligosaccharide-dependent signals. The ki- corresponding genes are homologous to PR proteins. The nase domain exhibited autophosphorylation, suggesting that cellular localization of AFPs and pathogen-induced CLPs are CHRK1 upon chitin recognition of the extracellular domain similar. This signifies that in plants common pathway is used activates an intracellular serine/threonine kinase domain and for penetration and propagation of pathogens and ice [86]. triggers signal transduction. AFPs are localized in various parts including rhizomes, seeds, stems, crowns, barks, branches, buds, petioles leaf blades, flowers, berries, roots, and tubers. 5.5 Nodule development Three Solanum thermal hysteresis proteins (29, 47, and 64 kDa) from the stem of winter bittersweet nightshade (S. Srchi24, a novel early nodulin, plays a role in nodule dulcamara) possess antifreeze activity and chitinase activity development process [97]. During nodulation, the CLPs [87,88]. Interestingly STHP-64 also has conserved glutamine- interact with the rhizoidal Nod-factors that are the main rich region, zinc finger motif, an acidic domain, and another signal molecules to trigger the onset of nodulation. The zinc finger motif. These features are present in transcription immunolocalization and in situ hybridization experiments factors (WRKY proteins) [89] that regulate the expression of demonstrate that Srchi24 is localized in the outermost PR proteins in plants. cortical cell layers of the developing nodules. The Srchi24

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Ta b l e 1 . CLPs from plants and their function

Source Function Ref.

An acidic CLP and basic CLP from seeds of Antifungal [70] chickpea (C. arietinum) Dolichin from field beans (D. lablab) Antifungal and HIV-1 reverse-transcriptase inhibition [71] Delandin from rice beans (D. umbellata) Antifungal, HIV-1 reverse-transcriptase inhibition and [72] mitogenic activities Phasein A from pinto bean (P.vulgaris cv. Pinto) Antifungal, HIV-1 reverse-transcriptase inhibition [73] 28-kDa CLP from cowpea (V. unguiculata) Antifungal and antiviral activities [74] 36-kDa CLP from Chive Antifungal, antiviral, cytotoxic to breast cancer cells [75] 15-kDa CLP from roots of Panax notoginseng Antifungal [76] Stinging nettle lectin (U. dioica agglutinin) Antifungal [77] SN-HLPf from mature elderberry fruits Antifungal [80] HbCLPs from rubber tree (H. brasiliensis) Antifungal and allergen [52] EgChit1-1 and EgChit5-1 genes in oil palm roots Antifungal [81] CTL XIP isolated from leaves of coffee XIPs [67] XIP-I from wheat XIPs [69] OsCLP from Rice XIPs [66] RIXI from rice XIPs [82] XAIP from S. multiflorus XAIP [13] PPL2 Antifungal [21] Mulatexin (MLX56) Insecticidal [83] LA-a and LA-b from mulberry (Morus sp.) Insecticidal [84] Thermal hysteresis proteins (29, 47, and 64 Antifreeze [87, 88] kDa) from S. dulcamara GhCTL from secondary cotton cells Cellulose biosynthesis [60] elp1 mutation within AtCTL1 from Arabidopsis Ectopic lignin deposition, elevated ethylene [90] production, variation in root and shoot architecture hot2 mutation within AtCTL1 from Arabidopsis Salt tolerant [91] LusCTL1 and 2 of flax Cell wall thickening [92] CTL1/POM1 and CTL2 from Arabidopsis Cellulose biosynthesis [93] Brittle culm BC15/OsCTL1 in rice Cellulose content and mechanical strength [94] CLPs from C. pine Development of embryogenic tissues [95] CHRK1 from tobacco Receptor-like kinase [96] CLP from banana Storage proteins [98] Srchi24 from Sesbania rostrata Nodule development [97] resembles class III chitinases, but catalytic Glu is replaced also show antibacterial, antiviral, antiproliferative, and mito- by Lys. The protein contains an ER-targeting signal peptide genic activities that can be further exploited for medicinal at the N-terminal and 35-residue-long C-terminal extension. usage and can be of interest to the biomedical and pharmaceutical industries. CLPs are being currently exploited for their usage in food 5.6 Storage proteins industry. XIP-I and XAIP inhibit different types of xylanases utilized in food processing industry for enzymatic modifi- A 30-kDa inactive homolog CRP belonging to class III acidic cation [101]. They are used in bread making, beer making, chitinases was seen during the fruit ripening process in ba- brewing, and animal feeding [19]. AFPs can also be used for nana [98]. It serves as a source of amino acids for the synthesis producing GM crops having increased freeze tolerance. AFPs of ripening-associated proteins. 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 6 Potential applications of CLPs [102–107]. Table 1 lists CLPs from plants involved in various functions. 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 7 Conclusion hs2 gene encoding a CLP in a triploid Dutch elm disease resistant American elm (Ulmus americana NPS-3-487) has been The CLPs have evolved into a new class of proteins with func- used to genetically modify creeping bentgrass (Agrostis palus- tional diversification. CLPs have undergone adaptive func- tris Huds.) to provide resistance against R. solani [100]. CLPs tional diversification in response to various biotic and abiotic

C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com 1702 P. Kesari et al. Proteomics 2015, 15, 1693–1705 stress conditions, which has either lead to plurifunctionality chitinase gene in Brassica oleracea vat. capitata. Cell Res. or neofunctionalization. Since each CLP has evolved inde- 1996, 6, 65–73. pendently during this process of evolution, the functional [5] Regalado, A. P., Pinheiro, C., Vidal, S., Chaves, I. et al., mechanism of many of them is still unknown. They share The Lupinus albus class-III chitinase gene, IF3, is consti- structural features with chitinases belonging to GH18 and tutively expressed in vegetative organs and developing 19 families. In many CLPs, the catalytic Glu or essential seeds. Planta 2002, 210, 543–550. residues of the CatD/CBD of chitinases are replaced with [6] Does, M. P., Houterman, P.M., Dekker, H. L., Cornelissen, B. similar residues—leading to loss of chitinases’ binding and J. C., Processing, targeting, and antifungal activity of sting- hydrolyzing abilities. In other cases, even if a Glu residue is ing nettle agglutinin in transgenic tobacco. Plant Physiol. present, it is engaged in the salt-bridge formation and other 1999, 120, 421–432. interactions. [7] Neuhaus, J. M., Fritig, B., Linthorst, H. J. M., Meins, F.et al., The structural features of CLPs are such that it can resist A revised nomenclature for chitinase genes. Plant Mol. Biol. abiotic stresses and can survive hostile environments. These Rep. 1996, 14, 102–104. CLPs have retained their chitin-binding ability while the hy- [8] Robertas, J. D., Monzingo, A. F., The structure and action of drolyzing ability is lost. They have acquired the ability to medi- chitinases. EXS 1999, 87, 125–135. ate a number of biological processes such as stress responses, [9] Terwisscha van Scheltinga, A. C., Hennig, M., Dijkstra, B. W., growth, and developmental operations. 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