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 SerPro₄ 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.     

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。     

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.     

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.     

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.     

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.     

Cloning, sequencing, and expression of the gene encoding Clostridium paraputrificum chitinase ChiB and analysis of the functions of novel cadherin-like domains and a chitin-binding domain.

The Clostridium paraputrificum chiB gene, encoding chitinase B (ChiB), consists of an open reading frame of 2,493 nucleotides and encodes 831 amino acids with a deduced molecular weight of 90,020. The deduced ChiB is a modular enzyme composed of a family 18 catalytic domain responsible for chitinase activity, two reiterated domains of unknown function, and a chitin-binding domain (CBD). The reiterated domains are similar to the repeating units of cadherin proteins but not to fibronectin type III domains, and therefore they are referred to as cadherin-like domains. ChiB was purified from the periplasm fraction of Escherichia coli harboring the chiB gene. The molecular weight of the purified ChiB (87,000) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, was in good agreement with the value (86,578) calculated from the deduced amino acid sequence excluding the signal peptide. ChiB was active toward chitin from crab shells, colloidal chitin, glycol chitin, and 4-methylumbelliferyl beta-D-N,N'-diacetylchitobioside [4-MU-(GlcNAc)2]. The pH and temperature optima of the enzyme were 6.0 and 45 degrees C, respectively. The Km and Vmax values for 4-MU-(GlcNAc)2 were estimated to be 6.3 microM and 46 micromol/min/mg, respectively. SDS-PAGE, zymogram, and Western blot analyses using antiserum raised against purified ChiB suggested that ChiB was one of the major chitinase species in the culture supernatant of C. paraputrificum. Deletion analysis showed clearly that the CBD of ChiB plays an important role in hydrolysis of native chitin but not processed chitin such as colloidal chitin.     

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.     

苏云金芽孢杆菌chiA,chiB全基因的克隆、表达及其序列分析

以苏云金芽孢杆菌科默尔亚种15A3菌株基因组DNA为模版,用touchdown PCR方法扩增几丁质酶ChiA和ChiB的全基因序列(GenBank登录号:EF103273和DQ512474)。将PCR产物连接pUCm-T克隆载体,获得重组质粒pUCm-chiA和pUCm-chiB,分别转化E.coliXL-Blue。克隆的几丁质酶基因可以利用本身的启动子异源表达各自的蛋白,不需要几丁质作为诱导物。表达的几丁质酶能够分泌到胞外。证明15A3菌株可组成型表达2种几丁质酶。经核苷酸及氨基酸序列分析证明,chiA基因全长1426bp,含有343bp的上游非编码区和1083bp的ORF,编码360个氨基酸。推测成熟蛋白分子量为36kD,只有一个几丁质酶催化域。chiB基因全长2279bp,含有248bp的上游非编码区和2031bp的ORF,编码676个氨基酸。推测成熟蛋白分子量约为70.6kD,具有三个功能域。核苷酸序列分析显示chiA和chiB的启动子所处的位置及转录起始碱基都不相同,-35区相同,而-10区有两个碱基不同,SD序列也不完全一致。     

苏云金芽孢杆菌chiA,chiB全基因的克隆、表达及其序列分析

以苏云金芽孢杆菌科默尔亚种15A3菌株基因组DNA为模版,用touchdown PCR方法扩增几丁质酶ChiA和ChiB的全基因序列(GenBank登录号:EF103273和DQ512474)。将PCR产物连接pUCm-T克隆载体,获得重组质粒pUCm-chiA和pUCm-chiB,分别转化E.coliXL-Blue。克隆的几丁质酶基因可以利用本身的启动子异源表达各自的蛋白,不需要几丁质作为诱导物。表达的几丁质酶能够分泌到胞外。证明15A3菌株可组成型表达2种几丁质酶。经核苷酸及氨基酸序列分析证明,chiA基因全长1426bp,含有343bp的上游非编码区和1083bp的ORF,编码360个氨基酸。推测成熟蛋白分子量为36kD,只有一个几丁质酶催化域。chiB基因全长2279bp,含有248bp的上游非编码区和2031bp的ORF,编码676个氨基酸。推测成熟蛋白分子量约为70.6kD,具有三个功能域。核苷酸序列分析显示chiA和chiB的启动子所处的位置及转录起始碱基都不相同,-35区相同,而-10区有两个碱基不同,SD序列也不完全一致。     

Characterization and sequence of tomato 2S seed albumin: a storage protein with sequence similarities to the fruit lectin

We found a 2S storage albumin from the seed of tomato ( Lycopersicon esculentum L. cv. Cherry) that cross-reacted with antiserum to the fruit lectin, and named it Lec2SA. According to its size and basicity, Lec2SA was classified into four isoforms. These isoforms have an M(r) of approximately 12,000, and are composed of a small subunit (M(r) 4,000) and a large subunit (M(r) 8,000) linked by disulfide bonds. The complete amino acid sequence of Lec2SA was determined. The small subunit was composed of 32 amino acids, whereas the large subunit contained 70 amino acids with a pyroglutamine as the N-terminal residue. The sequence of Lec2SA was similar to that of 2S albumins from different plants, such as Brazil nut and castor beans. Furthermore, a sequence similarity was found between the large subunit of Lec2SA and the peptide sequence from tomato lectin. Although these similarities were found, Lec2SA did not show hemagglutinating activity or sugar-chain-binding activity, indicating that Lec2SA lacks the carbohydrate-binding domain. These results suggest that tomato lectin is a chimeric lectin sharing the seed storage protein-like domain that is incorporated into the gene encoding tomato lectin through gene fusion.     

Molecular cloning, characterization and expression analysis of thrombospondin gene from Penaeus monodon

In present study, a thrombospondin gene was obtained from the ovary and neurosecretory organ in eyestalk cDNA library of black tiger prawn (Penaeus monodon). The full-length P. monodon thrombospondin (PmTSP) cDNA contained a 5' untranslated region (UTR) of 9 bp, an open reading frame (ORF) of 2778 bp encoding a polypeptide of 925 amino acids with molecular mass 100.57 kDa, and a 3'UTR of 99 bp. ScanProsite analysis indicated that PmTSP contained four chitin-binding type-II domains, an EGF-like domain, eight thrombospondin type-III repeats and one thrombospondin C-terminal domain. Homology analysis of the deduced amino acid sequence of the PmTSP with other known TSP sequences by MatGAT software revealed that the PmTSP shows very high homology with the sequences of Fennerpenaeus chinensis (89.9% similarity, 83.8% identity). Analysis of the tissue expression pattern of the PmTSP gene showed that the PmTSP mRNA was expressed in all tested tissues, including hepatopancreas, ovary, muscle, intestine, neurosecretory organ in eyestalk, neurosecretory organ in brain, stomach, and heart, with highest level in the ovary. Furthermore, the PmTSP expression was found to be of high level in six development stages of the ovary. The results indicated that PmTSP might play an important role in ovarian development.