Molecular structure and properties of lectin from tomato fruit

A cDNA encoding tomato fruit lectin was cloned from an unripe cherry-tomato fruit cDNA library. The isolated lectin cDNA contained an open reading frame encoding 365 amino acids, including peptides that were sequenced. The deduced sequence consisted of three distinct domains: (i) an N-terminal short extensin-like domain; (ii) a Cys-rich carbohydrate binding domain composed of four almost identical chitin-binding domains; (iii) an internal extensin-like domain of 101 residues containing 15 SerPro(4) motifs inserted between the first and second chitin-binding domains. The molecular weight of the lectin was 65,633 and that of the deglycosylated lectin was 32,948, as determined by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). This correlated with the estimated molecular weight of the deduced sequence. Recombinant tomato lectin expressed in Pichia pastoris possessed chitin-binding but not hemagglutinating activity. These findings confirmed that the cDNA encoded tomato lectin.     

Potato lectin: an updated model of a unique chimeric plant protein

A complete cDNA encoding a potato tuber lectin has been identified and sequenced. Based on the deduced amino acid sequence, the still enigmatic molecular structure of the classical chimeric potato lectin could eventually be determined. Basically, the potato lectin consists of two nearly identical chitin-binding modules, built up of two in-tandem arrayed hevein domains that are interconnected by an extensin-like domain of approximately 60 amino acid residues. Although this structure confirms the 'canonical' chimeric nature of the Solanaceae lectins, it differs fundamentally from all previously proposed models. The new insights in the structure are also discussed in view of the physiological role of the Solanaceae lectins.     

The gene for stinging nettle lectin (Urtica dioica agglutinin) encodes both a lectin and a chitinase.

Chitin-binding proteins are present in a wide range of plant species, including both monocots and dicots, even though these plants contain no chitin. To investigate the relationship between in vitro antifungal and insecticidal activities of chitin-binding proteins and their unknown endogenous functions, the stinging nettle lectin (Urtica dioica agglutinin, UDA) cDNA was cloned using a synthetic gene as the probe. The nettle lectin cDNA clone contained an open reading frame encoding 374 amino acids. Analysis of the deduced amino acid sequence revealed a 21-amino acid putative signal sequence and the 86 amino acids encoding the two chitin-binding domains of nettle lectin. These domains were fused to a 19-amino acid "spacer" domain and a 244-amino acid carboxyl extension with partial identity to a chitinase catalytic domain. The authenticity of the cDNA clone was confirmed by deduced amino acid sequence identity with sequence data obtained from tryptic digests, RNA gel blot, and polymerase chain reaction analyses. RNA gel blot analysis also showed the nettle lectin message was present primarily in rhizomes and inflorescence (with immature seeds) but not in leaves or stems. Chitinase enzymatic activity was found when the chitinase-like domain alone or the chitinase-like domain with the chitin-binding domains were expressed in Escherichia coli. This is the first example of a chitin-binding protein with both a duplication of the 43-amino acid chitin-binding domain and a fusion of the chitin-binding domains to a structurally unrelated domain, the chitinase domain.     

Potato lectin: a modular protein sharing sequence similarities with the extensin family, the hevein lectin family, and snake venom disintegrins (platelet aggregation inhibitors)

Potato (Solanum tuberosum) lectin, is a chimeric chitin-binding protein comprised of a lectin domain fused to a hydroxyproline-rich glycoprotein domain. Here peptide sequence information from both domains is presented. A partial sequence of a major tryptic peptide T2: Leu-Pro-Ser-Hyp-Hyp-Hyp-Hyp-Hyp-Hyp-(His)-Hyp-Ser-Hyp-Hyp-Hyp-Hyp-Ser-Hyp-Hyp-Ser-Hyp-Hyp-Hyp-Hyp-Ser-Hyp-Hyp- was similar to the ‘P3’ type extensin major repetitive sequence: Ser-Hyp-Hyp-Hyp-Hyp-Ser-Hyp-Ser-Hyp-Hyp-Hyp-Hyp-suggesting common evolutionary origins for the extensins and the hydroxyproline-rich glycoprotein (HRGP) domain of potato lectin. Furthermore, alignment of three chymotryptic peptides from potato lectin, C1: Cys-Gly-Thr-Thr-Ser-Asp-Tyr, C2: Cys-Ser-Pro-Gly-Tyr, and C8: Thr-Gly-Glu-Cys-Cys-Ser-Ile with similar sequences from the hevein lectin family indicates that they have homologous chitin-binding domains, and hence have common evolutionary origins. Finally, all plant chitin-binding domains examined bore a remarkable sequence similarity, particularly in the spacing of Cys residues, to the disintegrins (platelet aggregation inhibitors) which occur in crotalid and viperid snake venoms. As such, sequence similarities not only identify potato lectin as a member of both the hevein and extensin families of plant proteins, but also suggest that an archetypal polypeptide module gave rise to both the plant chitin-binding domain and the reptile disintegrins.     

A novel gene of tomato preferentially expressed in fruit encodes a protein with a Ca2+-dependent lipid-binding domain

A cDNA clone, called CLB1, was isolated from a cDNA library from tomato (Lycopersicon esculentum) and characterized. The CLB1 cDNA contains an open reading frame of 1518 bp, and encodes a putative protein of 506 amino acids with a predicted molecular mass of 54 633 Da. The deduced CLB1 amino acid sequence contains a domain that exhibits from 26% to 37% identity with the Ca²⁺-dependent lipid-binding domains of cytosolic phospholipase A₂, protein kinase C, Rabphilin-3A, and Synaptotagmin I of animals. Southern blot analysis indicates that the CLB1 gene belongs to a small gene family in the tomato genome. The CLB1 mRNA is preferentially expressed in fruit tissues.     

Ripening-related occurrence of phosphoenolpyruvate carboxykinase in tomato fruit

Phosphoenolpyruvate carboxykinase (PEPCK) is present in ripening tomato fruits. A cDNA encoding PEPCK was identified from a PCR-based screen of a cDNA library from ripe tomato fruit. The sequence of the tomato PEPCK cDNA and a cloned portion of the genomic DNA shows that the complete cDNA sequence contains an open reading frame encoding a peptide of 662 amino acid residues in length and predicts a polypeptide with a molecular mass of 73.5 kDa, which corresponds to that detected by western blotting. Only one PEPCK gene was identified in the tomato genome. PEPCK is shown to be present in the pericarp of ripening tomato fruits by activity measurements, western blotting and mRNA analysis. PEPCK abundance and activity both increased during fruit ripening, from an undetectable amount in immature green fruit to a high amount in ripening fruit. PEPCK mRNA, protein and activity were also detected in germinating seeds and, in lower amounts, in roots and stems of tomato. The possible role of PEPCK in the pericarp of tomato fruit during ripening is discussed.     

Purification,cDNA cloning,and characterization of LysM-containing plant chitinase from horsetail (Equisetum arvense)

Chitinase-A (EaChiA), molecular mass 36 kDa, was purified from the vegetative stems of a horsetail (Equisetum arvense) using a series of column chromatography. The N-terminal amino acid sequence of EaChiA was similar to the lysin motif (LysM). A cDNA encoding EaChiA was cloned by rapid amplification of cDNA ends and polymerase chain reaction. It consisted of 1320 nucleotides and encoded an open reading frame of 361 amino acid residues. The deduced amino acid sequence indicated that EaChiA is composed of a N-terminal LysM domain and a C-terminal plant class IIIb chitinase catalytic domain, belonging to the glycoside hydrolase family 18, linked by proline-rich regions. EaChiA has strong chitin-binding activity, however, no antifungal activity. This is the first report of a chitinase from Equisetopsida, a class of fern plants, and the second report of a LysM-containing chitinase from a plant.     

Purification,cDNA cloning,and characterization of plant chitinase with a novel domain combination from lycophyte Selaginella doederleinii

Chitinase-A from a lycophyte Selaginella doederleinii (SdChiA), having molecular mass of 53 kDa, was purified to homogeneity by column chromatography. The cDNA encoding SdChiA was cloned by rapid amplification of cDNA ends and polymerase chain reaction. It consisted of 1477 nucleotides and its open reading frame encoded a polypeptide of 467 amino acid residues. The deduced amino acid sequence indicated that SdChiA consisted of two N-terminal chitin-binding domains and a C-terminal plant class V chitinase catalytic domain, belonging to the carbohydrate-binding module family 18 (CBM18) and glycoside hydrolase family 18 (GH18), respectively. SdChiA had chitin-binding ability. The time-dependent cleavage pattern of (GlcNAc)₄ by SdChiA showed that SdChiA specifically recognizes the β-anomer in the + 2 subsite of the substrate (GlcNAc)₄ and cleaves the glycoside bond at the center of the substrate. This is the first report of the occurrence of a family 18 chitinase containing CBM18 chitin-binding domains.

Abbreviations: AtChiC: Arabidopsis thaliana class V chitinase; CBB: Coomassie brilliant blue R250; CBM: carbohydrate binding module family; CrChi-A: Cycas revolute chitinase-A; EaChiA: Equisetum arvense chitinase-A; GH: glycoside hydrolase family, GlxChi-B: gazyumaru latex chitinase-B; GlcNAc: N-acetylglucosamine; HPLC: high performance liquid chromatography; LysM; lysin motif; MtNFH1: Medicago truncatula ecotypes R108-1 chitinase; NCBI: national center for biotechnology information; NF: nodulation factor; NtChiV: Nicotiana tabacum class V chitinase; PCR: polymerase chain reaction; PrChi-A: Pteris ryukyuensis chitinase-A; RACE: rapid amplification of cDNA ends; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SdChiA: Selaginella doederleinii chitinase-A.     

A leucine-rich repeat region is conserved in pollen extensin-like (Pex) proteins in monocots and dicots

We previously isolated a pollen-specific gene encoding a pollen tube wall-associated glycoprotein with a globular domain and an extensin domain from maize (mPex1). To evaluate which protein domains might be important for function, we isolated a second monocot gene (mPex2) and a dicot gene (tPex). Each gene encodes a signal sequence, an N-terminal globular domain comprised of a variable region, a leucine-rich repeat (LRR) with an adjacent cysteine-rich region, a transition region and an extensin-like C-terminal domain. The LRRs of the maize and tomato Pex proteins are highly conserved. Although the extensin domains in the maize and tomato proteins vary in length and in amino acid sequence, they are likely to be structurally conserved. Additional putative Pex gene sequences were identified by either GenBank search (Arabidopsis) or PCR (sorghum and potato); all encode conserved LRRs. The presence of a conserved LRR in the known and potential Pex proteins strongly suggests that this motif is involved in the binding of a specific ligand during pollen tube growth. Gene expression studies using RNA and protein blotting as well as promoter-reporter gene fusions in transient and stable transformation indicate that the tomato Pex gene is pollen-specific.     

A new type of plant chitinase containing LysM domains from a fern (Pteris ryukyuensis): roles of LysM domains in chitin binding and antifungal activity

Chitinase-A (PrChi-A), of molecular mass 42 kDa, was purified from the leaves of a fern (P. ryukyuensis) using several column chromatographies. The N-terminal amino acid sequence of PrChi-A was similar to the lysin motif (LysM). A cDNA encoding PrChi-A was cloned by rapid amplification of cDNA ends and polymerase chain reaction. It consisted of 1459 nucleotides and encoded an open-reading frame of 423-amino-acid residues. The deduced amino acid sequence indicated that PrChi-A is composed of two N-terminal LysM domains and a C-terminal catalytic domain, belonging to the group of plant class IIIb chitinases, linked by proline, serine, and threonine-rich regions. Wild-type PrChi-A had chitin-binding and antifungal activities, but a mutant without LysM domains had lost both activities. These results suggest that the LysM domains contribute significantly to the antifungal activity of PrChi-A through their binding activity to chitin in the cell wall of fungi. This is the first report of the presence in plants of a family-18 chitinase containing LysM domains.     

Tobacco-expressed Brassica juncea chitinase BjCHI1 shows antifungal activity in vitro

We have previously isolated a Brassica juncea cDNA encoding BjCHI1, a novel chitinase with two chitin-binding domains, and have shown that its mRNA is induced by wounding and methyl jasmonate treatment (K.-J. Zhao and M.-L. Chye, Plant Mol. Biol. 40 (1999) 1009–1018). By the presence of two chitin-binding domains, BjCHI1 resembles the precursor of UDA (Urtica dioica agglutinin) but, unlike UDA, BjCHI1 retains its chitinase catalytic domain after post-translational processing. Here, we indicate the role of BjCHI1 in plant defense by demonstrating its mRNA induction upon Aspergillus niger infection or caterpillar Pieris rapae (L.) feeding. To further investigate the biological properties of BjCHI1, we transformed tobacco with a construct expressing the BjCHI1 cDNA from the CaMV 35S promoter. Subsequently, we purified BjCHI1 from the resultant transgenic R₀ plants using a regenerated chitin column followed by fast protein liquid chromatography (FPLC). Also, the significance of the second chitin-binding domain in BjCHI1 was investigated by raising transgenic tobacco plants expressing BjCHI2, a deletion derivative of BjCHI1 lacking one chitin-binding domain. Colorimetric chitinase assays at 25 °C, pH 5, showed no significant differences between the activities of BjCHI1 and BjCHI2, suggesting that chitinase activity, due to the catalytic domain, is not enhanced by the presence of a second chitin-binding domain. Both BjCHI1 and BjCHI2 show in vitro anti-fungal activity toward Trichoderma viride, causing reductions in hyphal diameter, hyphal branching and conidia size.     

Domain conservation in several volvocalean cell wall proteins

Based on our previous work demonstrating that (SerPro)_x epitopes are common to extensin-like cell wall proteins in Chlamydomonas reinhardtii, we looked for similar proteins in the distantly related species C. eugametos. Using a polyclonal antiserum against a (SerPro)₁₀ oligopeptide, we found distinct sets of stage-specific polypeptides immunoprecipitated from in vitro translations of C. eugametos RNA. Screening of a C. eugametos cDNA expression library with the antiserum led to the isolation of a cDNA (WP6) encoding a (SerPro)_x-rich multidomain wall protein. Analysis of a similarly selected cDNA (VSP-3) from a C. reinhardtii cDNA expression library revealed that it also coded for a (SerPro)_x-rich multidomain wall protein. The C-terminal rod domains of VSP-3 and WP6 are highly homologous, while the N-terminal domains are dissimilar; however, the N-terminal domain of VSP-3 is homologous to the globular domain of a cell wall protein from Volvox carteri. Exon shuffling might be responsible for this example of domain conservation over 350 million years of volvocalean cell wall protein evolution.     

小地老虎几丁质酶基因的克隆与序列分析

利用昆虫几丁质酶对几丁质的调控作用破坏几丁质新陈代谢的平衡来防治害虫, 在生物防治策略中具有很大的发展潜力。从处于预蛹期的小地老虎Agrotls ipsilon (Hufnagel)体中肠内提取总的RNA, 经反转录, 利用cDNA末端快速扩增技术（RACE）获得了几丁质酶基因的cDNA序列。该基因序列已经登录GenBank并获得登录号为EU035316。该序列长度为2 823个碱基, 含有一个1 674个碱基的开放读码框。开放读码框编码558个氨基酸残基, 预测的分子量为62.5 kDa, 等电点5.12。推导得到的氨基酸序列含有2个N-位糖基化位点,20个O-位糖基化位点, 含有2个几丁质酶所具有的保守序列：N-端的催化区和C-端的几丁质结合区。氨基酸序列与其他昆虫, 特别是鳞翅目昆虫的几丁质酶高度同源。     

Molecular cloning and expression of cDNA encoding a galactose/N-acetylgalactosamine-specific lectin on mouse tumoricidal macrophages.

We previously reported that the mouse macrophage galacose and N-acetylgalactosamine-specific lectin (MMGL) may participate in the binding of the macrophages to tumor cells [Oda, S., Sato, M., Toyoshima, S., & Osawa, T. (1989) J. Biochem. 105, 1040-1043]. We now report the cloning and characterization of a cDNA encoding MMGL. The MMGL gene encoded a protein consisting of 304 amino acid residues with a molecular weight of 34,595. The deduced amino acid sequence indicated that MMGL had a single membrane-spanning region, three leucine zipper-like domains, and a carbohydrate recognition domain. Two N-glycosylation sites were found in the extracellular region of MMGL, corresponding to the heavy N-glycosylation in the native MMGL. Comparison of the amino acid sequence of MMGL with those of rat hepatic lectins revealed a high overall sequence homology. The sequence homology was especially high in the putative membrane-spanning region and carbohydrate recognition domain. There was, however, a region of 25 amino acids which did not exist on hepatic lectins. The MMGL cDNA without the region encoding the putative membrane-spanning region and intracellular region was expressed in Escherichia coli. The expressed protein had galactose-binding activity and its sugar-binding specificity was same as that of the native lectin.     

Molecular characterization of tomato fruit polygalacturonase

Summary Using the expression vector gt11 and immunological detection, cDNA clones of an endopolygalacturonase gene of tomato (Lycopersicon esculentum Mill.) were isolated and sequenced. The 1.6 kb cDNA sequence predicts a single open reading frame encoding a polypeptide of 457 amino acids. The PG2A isoform of tomato fruit endopolygalacturonase was purified and 80% of the amino acid sequence determined. The amino acid sequence predicted by the cDNA sequence was identical to the amino acid sequence of the PG2A isoform. The position of the codon for the N-terminal amino acid of mature PG2A in the open reading frame indicates the presence of a 71 amino acid N-terminal signal peptide which is post-translationally processed. The C-terminus of purified PG2A occurred 13 amino acids before the stop codon in the cDNA suggesting that C-terminal processing of PG2A may also occur. The nucleotide and amino acid sequence data predict a mature protein of 373 amino acids and a polypeptide molecular weight of 40279. The sequence contains four potential glycosylation sites. Northern analysis detected endopolyga-lacturonase mRNA in stage 3 (turning) and stage 6 (red) ripening fruit, but not in leaves, roots, or green fruit of normal cultivars or in mature fruit of the rin mutant.     

Molecular cloning and characterization of a mannose-binding lectin gene from <Emphasis Type="Italic">Pinellia cordata</Emphasis>

Using RNA extracted from Pinellia cordata young leaves and primers designed according to the conserved regions of Araceae lectins, the full-length cDNA of Pinellia cordata agglutinin (PCL) was cloned by rapid amplification of cDNA ends (RACE). The full-length cDNA of pcl was 1,182 bp and contained a 768 bp open reading frame (ORF) encoding a lectin precursor of 256 amino acids. Through comparative analysis of pcl gene and its deduced amino acid sequence with those of other Araceae species, it was found that pcl encoded a precursor lectin with signal peptide. PCL is a mannose-binding lectin with three mannose-binding sites. Semi-quantitative RT-PCR analysis revealed that pcl is expressed in all tested tissues including leaf, stem and bulbil, but with the highest expression in bulbil. PCL protein was successfully expressed in Escherichia coli with the molecular weight expected.     

Molecular cloning and expression of an expansin-like gene in ‘Navel’ orange fruit during postharvest stresses

In a search for differentially expressed genes in peel pitting of ‘Navel’ orange fruit (Citrus sinensis L. Osbeck), a cDNA subtraction library was constructed and a sequence encoding expansin-like gene was isolated and identified as pitting related gene. Based on sequence information derived from this fragment, a full-length cDNA (CsEXP, GenBank accession no. FJ769424) of 1,083 nucleotides encoding expansin was isolated from ‘Navel’ orange by RACE approaches. CsEXP encoded a protein of 254 amino acid residues with an open reading frame located in the region between 52 and 816 bp. The calculated molecular weight of the mature protein was 27.05 kDa and theoretical isoelectric point was 7.93. The deduced protein contained conserved domains of expansin: the histidine-phenylalanine-aspartate motif in central portion, cysteine residues in N-terminus, and tryptophan residues in C-terminal region. The expression of CsEXP was higher in pitting than the control. Exposure of fruit to stresses, including wounding, anoxia, low temperature (4°C), and treatment with ethylene, increased CsEXP mRNA levels in comparison with the control untreated fruit, whereas high temperature (40°C) decreased its mRNA levels. Since low temperature, low oxygen and wounding were suspected factors inducing peel pitting of citrus fruit. The present results provided us a clue that CsEXP may play a role in response to peel pitting related stresses.     

Molecular cloning and expression analysis of a F-type lectin gene from Japanese sea perch (<Emphasis Type="Italic">Lateolabrax japonicus</Emphasis>)

The techniques of homology cloning and anchored PCR were used to clone the fucose-binding lectin (F-type lectin) gene from Japanese sea perch (Lateolabrax Japonicus). The full-length cDNA of sea perch F-lectin (JspFL) contained a 5′ untranslated region (UTR) of 39 bp, an ORF of 933 bp encoding a polypeptide of 310 amino acids with an estimated molecular mass of 10.82 kDa and a 3′ UTR of 332 bp. The searches for nucleotides and protein sequence similarities with BLAST analysis indicated that the deduced amino acid sequence of JspFL was homological to the Fucose-binding lectin in other fish species. In the JspFL deduced amino acid sequence, two tandem domains that exhibit the eel carbohydrate-recognition sequence motif were found. The temporal expressions of gene in the different tissues were measured by real-time PCR. And the mRNA expressions of the gene were constitutively expressed in tissues including spleen, head-kidney, liver, gill, and heart. The JspFL expression in spleen was different during the stimulated time point, 2 h later the expression level became up-regulated, and 6 h later the expression level became down-regulated. The result indicated that JspFL was constitutive and inducible expressed and could play a critical role in the host-pathogen interaction.