Carbohydrate Recognition Specificity of Trans-sialidase Lectin Domain from Trypanosoma congolense

Fourteen different active Trypanosoma congolense trans-sialidases (TconTS), 11 variants of TconTS1 besides TconTS2, TconTS3 and TconTS4, have been described. Notably, the specific transfer and sialidase activities of these TconTS differ by orders of magnitude. Surprisingly, phylogenetic analysis of the catalytic domains (CD) grouped each of the highly active TconTS together with the less active enzymes. In contrast, when aligning lectin-like domains (LD), the highly active TconTS grouped together, leading to the hypothesis that the LD of TconTS modulates its enzymatic activity. So far, little is known about the function and ligand specificity of these LDs. To explore their carbohydrate-binding potential, glycan array analysis was performed on the LD of TconTS1, TconTS2, TconTS3 and TconTS4. In addition, Saturation Transfer Difference (STD) NMR experiments were done on TconTS2-LD for a more detailed analysis of its lectin activity. Several mannose-containing oligosaccharides, such as mannobiose, mannotriose and higher mannosylated glycans, as well as Gal, GalNAc and LacNAc containing oligosaccharides were confirmed as binding partners of TconTS1-LD and TconTS2-LD. Interestingly, terminal mannose residues are not acceptor substrates for TconTS activity. This indicates a different, yet unknown biological function for TconTS-LD, including specific interactions with oligomannose-containing glycans on glycoproteins and GPI anchors found on the surface of the parasite, including the TconTS itself. Experimental evidence for such a scenario is presented.     

Cooperativity of catalytic and lectin-like domain of Trypanosoma congolense trans-sialidase modulates its catalytic activity

Trans-sialidases (TS) represent a multi-gene family of unusual enzymes, which catalyse the transfer of terminal sialic acids (Sia) from sialoglycoconjugates to terminal galactose or N-acetylgalactosamine residues of oligosaccharides without the requirement of CMP-Neu5Ac, the activated Sia used by typical sialyltransferases. Enzymes comprise a N-terminal catalytic domain (CD) followed by a lectin-like domain (LD). Most work on trypanosomal TS has been done on enzymatic activities focusing on the CD of TS from Trypanosoma cruzi (causing Chagas disease in Latin America), subspecies of Trypanosoma brucei, (causing human sleeping sickness in Africa) and Trypanosoma congolense (causing African Animal Trypanosomosis in livestock). Previously, we demonstrated that T. congolense TS (TconTS)-LD binds to several carbohydrates, such as 1,4-β-mannotriose. In this study we investigated the influence of TconTS3-LD on Sia transfer efficiency of TconTS1a-CD by swapping domains. in silico analysis on structure models of TconTS enzymes revealed the potential of domain swaps between TconTS1a and TconTS3 without structural disruptions of the enzymes overall topologies. Recombinant domain swapped TconTS1a/TS3 showed clear Sia transfer activity, when using fetuin and lactose as Sia donor and acceptor substrates, respectively. While Sia transfer activity remained unchanged from the level of TconTS1a, hydrolytic release of free Neu5Ac as a side product was suppressed resulting in increased transfer efficiency. Presence of 1,4-β-mannotriose during TS reactions modulates enzyme activities enhancing transfer efficiency possibly due to occupation of the binding site in TconTS1a-LD. Interestingly this effect was in the same range as that observed when swapping TconTS1a-CD and TconTS3-LD. In summary, this study demonstrate the proof-of-principle for swapping CDs and LDs of TconTS and that TconTS3-LD influences enzymatic activity of TconTS1a-CD providing evidence that LDs play pivotal roles in modulating activities and biological functions of TconTS and possibly other TS.     

Identification of a second catalytically active trans-sialidase in Trypanosoma brucei

The procyclic stage of Trypanosoma brucei is covered by glycosylphosphatidylinositol (GPI)-anchored surface proteins called procyclins. The procyclin GPI anchor contains a side chain of N-acetyllactosamine repeats terminated by sialic acids. Sialic acid modification is mediated by trans-sialidases expressed on the parasite’s cell surface. Previous studies suggested the presence of more than one active trans-sialidases, but only one has so far been reported. Here we cloned and examined enzyme activities of four additional trans-sialidase homologs, and show that one of them, Tb927.8.7350, encodes another active trans-sialidase, designated as TbSA C2. In an in vitro assay, TbSA C2 utilized α2-3 sialyllactose as a donor, and produced an α2-3-sialylated product, suggesting that it is an α2-3 trans-sialidase. We suggest that TbSA C2 plays a role in the sialic acid modification of the trypanosome cell surface.     

Trans-sialidase-like sequences from Trypanosoma congolense conserve most of the critical active site residues found in other trans-sialidases

Trypanosoma congolense is the agent of Nagana, the trypanosomiasis in African ruminants. Trypanosomes express an enzyme called trans-sialidase, which is believed to play an important role in maintaining pathogenicity of the parasites. Thus far, only two complete trans-sialidase sequences have been characterised, one from the American trypanosome T. cruzi and one from the African trypanosome T. brucei brucei. Although the crystal structure of T. cruzi trans-sialidase has recently been published [Buschiazzo et al., Mol. Cell 10 (2002), pp. 757-768], a number of questions concerning the exact transfer mechanism remain unanswered. The availability of further trans-sialidase sequences will ensure a better understanding of how transfer activity can be achieved and will provide the opportunity to develop highly specific, structure-based trans-sialidase inhibitors. Utilising a PCR-based approach two different trans-sialidase gene copies from T. congolense were identified, which share only 50% identity with each other, but show significant similarity with known viral, bacterial and trypanosomal sialidases and trans-sialidases. In both partial sequences most of the critical active site residues common to other trypanosomal sialidases and trans-sialidases are conserved. This is further illustrated by modelling the active site of the longer of the two partial gene sequences.     

Costimulation of host T lymphocytes by a trypanosomal trans-sialidase: involvement of CD43 signaling

Trans-sialidase is a membrane-bound and shed sialidase from Trypanosoma cruzi, the protozoan parasite responsible for Chagas disease. We investigated the role of soluble trans-sialidase on host CD4+ T cell activation. Trans-sialidase activated naive CD4+ T cells in vivo. Both enzymatically active and inactive recombinant trans-sialidases costimulated CD4+ T cell activation in vitro. Costimulation resulted in increased mitogen-activated protein kinase activation, proliferation, and cytokine synthesis. Furthermore, active and inactive trans-sialidases blocked activation-induced cell death in CD4+ T cells from T. cruzi-infected mice. By flow cytometry, inactive trans-sialidase bound the highly sialylated surface Ag CD43 on host CD4+ T cells. Both costimulatory and antiapoptotic effects of trans-sialidases required CD43 signaling. These results suggest that trans-sialidase family proteins are involved in exacerbated host T lymphocyte responses observed in T. cruzi infection.     

Protein kinase A catalytic subunit interacts and phosphorylates members of trans-sialidase super-family in Trypanosoma cruzi

Protein kinase A (PKA) has been suggested as a regulator of stage differentiation in Trypanosoma cruzi. Using a yeast two-hybrid system we have begun to characterize the downstream substrates of T. cruzi PKA. We identified several members of the trans-sialidase super family by this approach. Immunoprecitation demonstrated that a TcPKAc monoclonal antibody was able to pull-down proteins recognized by trans-sialidase antibodies as well as a SA85-1.1 antibody and vice versa. An in vitro phosphorylation assay demonstrated that PKA phosphorylated the recombinant protein of an active trans-sialidase. In addition, a phospho-(Ser/Thr) PKA substrate antibody detected bands on immunoblot analysis of trans-sialidase antibody precipitated proteins from parasite lysate and the media of L₆E₉ myoblasts infected with trypomastigotes as well as from a SA85-1.1 antibody precipitated proteins from parasite lysate. Immunofluorescence analysis suggested that some TcPKAc localizes to the plasma membrane surface of trypomastigotes. The identified trans-sialidases have PKA consensus phosphorylation sites located near the endoplasmic reticulum retention motif in the N-terminal. These data support that PKA phosphorylates trans-sialidase super family members in vivo.     

Enzymatically inactive trans-sialidase from Trypanosoma cruzi binds sialyl and beta-galactopyranosyl residues in a sequential ordered mechanism

Host/parasite interaction mediated by carbohydrate/lectin recognition results in the attachment to and invasion of host cells and immunoregulation, enabling parasite replication and establishment of infection. Trypanosoma cruzi, the protozoan responsible for Chagas disease, expresses on its surface a family of enzymatically active and inactive trans-sialidases. The parasite uses the active trans-sialidase for glycoprotein sialylation in an unusual trans-glycosylation reaction. Inactive trans-sialidase is a sialic acid-binding lectin that costimulates host T cells through leucosialin (CD43) engagement. The co-mitogenic effect of trans-sialidase can be selectively abrogated by N-acetyllactosamine, suggesting the presence of an additional carbohydrate binding domain for galactosides, in addition to that for sialic acid. Here we investigated the interaction of inactive trans-sialidase in the presence of beta-galactosides. By using NMR spectroscopy, we demonstrate that inactive trans-sialidase has a beta-galactoside recognition site formed following a conformational switch induced by sialoside binding. Thus prior positioning of a sialyl residue is required for the beta-galactoside interaction. When an appropriate sialic acid-containing molecule is available, both sialoside and beta-galactoside are simultaneously accommodated in the inactive trans-sialidase binding pocket. This is the first report of a lectin recognizing two distinct ligands by a sequential ordered mechanism. This uncommon binding behavior may play an important role in several biological aspects of T. cruzi/host cell interaction and could shed more light into the catalytic mechanism of the sialic acid transfer reaction of enzymatically active trans-sialidase.     

Relevance of the diversity among members of the Trypanosoma cruzi trans-sialidase family analyzed with camelids single-domain antibodies

The sialic acid present in the protective surface mucin coat of Trypanosoma cruzi is added by a membrane anchored trans-sialidase (TcTS), a modified sialidase that is expressed from a large gene family. In this work, we analyzed single domain camelid antibodies produced against trans-sialidase. Llamas were immunized with a recombinant trans-sialidase and inhibitory single-domain antibody fragments were obtained by phage display selection, taking advantage of a screening strategy using an inhibition test instead of the classic binding assay. Four single domain antibodies displaying strong trans-sialidase inhibition activity against the recombinant enzyme were identified. They share the same complementarity-determining region 3 length (17 residues) and have very similar sequences. This result indicates that they likely derived from a unique clone. Probably there is only one structural solution for tight binding inhibitory antibodies against the TcTS used for immunization. To our surprise, this single domain antibody that inhibits the recombinant TcTS, failed to inhibit the enzymatic activity present in parasite extracts. Analysis of individual recombinant trans-sialidases showed that enzymes expressed from different genes were inhibited to different extents (from 8 to 98%) by the llama antibodies. Amino acid changes at key positions are likely to be responsible for the differences in inhibition found among the recombinant enzymes. These results suggest that the presence of a large and diverse trans-sialidase family might be required to prevent the inhibitory response against this essential enzyme and might thus constitute a novel strategy of T. cruzi to evade the host immune system.     

Procyclic Trypanosoma brucei expresses separate sialidase and trans-sialidase enzymes on its surface membrane

The procyclic stage of Trypanosoma brucei in the insect vector expresses a surface-bound trans-sialidase (TbTS) that transfers sialic acid from glycoconjugates in the environment to glycosylphosphatidylinositol-anchored proteins on its surface membrane. RNA interference against TbTS abolished trans-sialidase activity in procyclic cells but did not diminish sialidase activity, suggesting the presence of a separate sialidase enzyme for hydrolyzing sialic acid. A search of the T. brucei genome sequence revealed seven other putative genes encoding proteins with varying similarity to TbTS. RNA interference directed against one of these proteins, TbSA C, greatly decreased the sialidase activity but had no effect on trans-sialidase activity. The deduced amino acid sequence of TbSA C shares only 40% identity with TbTS but conserves most of the relevant residues required for catalysis. However, the sialidase has a tryptophan substitution for a tyrosine at position 170 that is crucial in binding the terminal galactose that accepts the transferred sialic acid. When this same tryptophan substitution in the sialidase was placed into the recombinant trans-sialidase, the mutant enzyme lost almost all of its trans-sialidase activity and increased its sialidase activity, further confirming that the gene and protein identified correspond to the parasite sialidase. Thus, in contrast to all other trypanosomes analyzed to date that express either a trans-sialidase or a sialidase but not both, T. brucei expresses these two enzymatic activities in two separate proteins. These results suggest that African trypanosomes could regulate the amount of critical sialic acid residues on their surface by modulating differential expression of each of these enzymes.     

Probing molecular function of trypanosomal sialidases: single point mutations can change substrate specificity and increase hydrolytic activity

Sialidases are present on the surface of several trypanosomatid protozoan parasites. They are highly specific for sialic acid linked in alpha-(2,3) to a terminal beta-galactose and include the strictly hydrolytic enzymes and trans-sialidases (sialyl-transferases). Based on the structural comparison of the sialidase from Trypanosoma rangeli and the trans-sialidase from T. cruzi (the agent of Chagas' disease in humans), we have explored the role of specific amino acid residues sought to be important for substrate specificity. The substitution of a conserved tryptophanyl residue in the two enzymes, Trp312/313-Ala, changed substrate specificity, rendering the point mutants capable to hydrolyze both alpha-(2,3)- and alpha-(2,6)-linked sialoconjugates. The same mutation abolished sialyl-transferase activity, indicating that transfer (but not hydrolysis) requires a precise orientation of the bound substrate. The exchange substitution of another residue that modulates oligosaccharide binding, Gln284-Pro, was found to significantly increase the hydrolytic activity of sialidase, and residue Tyr119 was confirmed to be part of a second binding site for the acceptor substrate in trans-sialidase. Together with the structural information, these results provide a consistent framework to account for the unique enzymatic properties of trypanosome trans-sialidases.     

A nonradioactive 96-well plate assay for screening of trans-sialidase activity

Trans-sialidase (E.C. 3.2.1.18) catalyzes the transfer of preferably alpha2,3-linked sialic acid to another glycan or glycoconjugate, forming a new alpha2,3 linkage to galactose or N-acetylgalactosamine. Here, we describe a nonradioactive 96-well plate fluorescence test for monitoring trans-sialidase activity with high sensitivity, specificity, and reproducibility using sialyllactose and 4-methylumbelliferyl-beta-D-galactoside as donor and acceptor substrates, respectively. The assay conditions were optimized using the trans-sialidase from Trypanosoma congolense and its general applicability was confirmed with recombinant trans-sialidase from Trypanosoma cruzi. Using this procedure, a large number of samples can be tested quickly and reliably, for instance in monitoring trans-sialidase during enzyme purification and the production of monoclonal antibodies, for enzyme characterization, and for identifying potential substrates and inhibitors. The trans-sialidase assay reported here was capable of detecting trans-sialidase activity in the low-mU range and may be a valuable tool in the search for further trans-sialidases in various biological systems.     

The high resolution structures of free and inhibitor-bound Trypanosoma rangeli sialidase and its comparison with T. cruzi trans-sialidase

The structure of the recombinant Trypanosoma rangeli sialidase (TrSA) has been determined at 1.6A resolution, and the structures of its complexes with the transition state analog inhibitor 2-deoxy-2,3-dehydro-N-acetyl-neuraminic acid (DANA), Neu-5-Ac-thio-alpha(2,3)-galactoside (NATG) and N-acetylneuraminic acid (NANA) have been determined at 1.64A, 2.1A and 2.85A, respectively. The 3D structure of TrSA is essentially identical to that of the natural enzyme, except for the absence of covalently attached sugar at five distinct N-glycosylation sites. The protein exhibits a topologically rigid active site architecture that is unaffected by ligand binding. The overall binding of DANA to the active site cleft is similar to that observed for other viral and bacterial sialidases, dominated by the interactions of the inhibitor carboxylate with the conserved arginine triad. However, the interactions of the other pyranoside ring substituents (hydroxyl, N-acetyl and glycerol moieties) differ between trypanosomal, bacterial and viral sialidases, providing a structural basis for specific inhibitor design. Sialic acid is found to bind the enzyme with the sugar ring in a distorted (half-chair or boat) conformation and the 2-OH hydroxyl group at hydrogen bonding distance of the carboxylate of Asp60, substantiating a direct catalytic role for this residue. A detailed comparison of TrSA with the closely related structure of T.cruzi trans-sialidase (TcTS) reveals a highly conserved catalytic center, where subtle structural differences account for strikingly different enzymatic activities and inhibition properties. The structure of TrSA in complex with NATG shows the active site cleft occupied by a smaller compound which could be identified as DANA, probably the product of a hydrolytic side reaction. Indeed, TrSA (but not TcTS) was found to cleave O and S-linked sialylated substrates, further stressing the functional differences between trypanosomal sialidases and trans-sialidases.     

Only some members of a gene family in Trypanosoma cruzi encode proteins that express both trans-sialidase and neuraminidase activities.

Trypomastigotes, the blood stage form of the human parasite Trypanosoma cruzi, contain an enzyme on their surface, trans-sialidase, which catalyses the transfer of sialic acid from host glycoconjugates to acceptors on its own cell surface. At least a subset of the sialic acid-bearing acceptor molecules are involved in parasite invasion of host cells, an essential step in the life cycle of the parasite. Another trypomastigote surface enzyme that affects host cell invasion is neuraminidase and recent evidence suggests that both trans-sialidase and neuraminidase activities may be expressed by the same proteins on the parasite surface. We describe here the isolation and expression of several members of a trans-sialidase--neuraminidase gene family from T.cruzi. One of the isolated genes does indeed encode a protein with both trans-sialidase and neuraminidase activities, while other members of the gene family encode closely related proteins that express neither enzymatic activity. Chimeric protein constructs combining different portions of active and inactive genes identified a region of the gene necessary for enzymatic activity. Sequence analysis of this portion of the gene revealed a limited number of amino acid differences between the predicted active and inactive gene products.     

Sialidases play a key role in infection and anaemia in Trypanosoma congolense animal trypanosomiasis

Animal African trypanosomiasis is a major constraint to livestock productivity and has an important impact on millions of people in developing African countries. This parasitic disease, caused mainly by Trypanosoma congolense, results in severe anaemia leading to animal death. In order to characterize potential targets for an anti-disease vaccine, we investigated a multigenic trans-sialidase family (TcoTS) in T. congolense. Sialidase and trans-sialidase activities were quantified for the first time, as well as the tightly regulated TcoTS expression pattern throughout the life cycle. Active enzymes were expressed in bloodstream form parasites and released into the blood during infection. Using genetic tools, we demonstrated a significant correlation between TcoTS silencing and impairment of virulence during experimental infection with T. congolense. Reduced TcoTS expression affected infectivity, parasitaemia and pathogenesis development. Immunization-challenge experiments using recombinant TcoTS highlighted their potential protective use in an anti-disease vaccine.     

Molecular engineering of the cellulosome complex for affinity and bioenergy applications

The cellulosome complex has evolved to degrade plant cell walls and, as such, combines tenacious binding to cellulose with diverse catalytic activities against amorphous and crystalline cellulose. Cellulolytic microorganisms provide an extensive selection of domains; those with affinity for cellulose, cohesins and their dockerin binding partners that define cellulosome stoichiometry and architecture, and a range of catalytic activities against carbohydrates. These robust domains provide the building blocks for molecular design. This review examines how protein modules derived from the cellulosome have been incorporated into chimaeric proteins to provide biosynthetic tools for research and industry. These applications include affinity tags for protein purification, and non-chemical methods for immobilisation and presentation of recombinant protein domains on cellulosic substrates. Cellulosomal architecture provides a paradigm for design of enzymatic complexes that synergistically combine multiple catalytic subunits to achieve higher specific activity than would be obtained using free enzymes. Multimeric enzymatic complexes may have industrial applications of relevance for an emerging carbon economy. Biocatalysis will lead to more efficient utilisation of renewable carbon-fixing energy sources with the added benefits of reducing chemical waste streams and reliance on petroleum.     

Overproduction of,and interaction within,bifunctional domains from the amino- and carboxy-termini of the pentafunctional AROM protein of Aspergillus nidulans

The pentafunctional AROM protein in Aspergillus nidulans and other fungi catalyses five consecutive enzymatic steps leading to the production of 5-enolpyruvylshikimate 3-phosphate (EPSP) in the shikimate pathway. The AROM protein has five separate enzymatic domains that have previously been shown to display a range of abilities to fold and function in isolation as monofunctional enzymes. In this communication, we report (1) the stable overproduction of a bifunctional protein containing the 3-dehydroquinate (DHQ) synthase and EPSP synthase activities in Escherichia coli to around 10% of the total cell protein; (2) that both the DHQ synthase and EPSP synthase activities in the over-produced fragment are enzymatically active as judged by their ability to complement aroA and aroB mutants of E. coli; (3) that the EPSP synthase domain is only enzymatically active when covalently attached to the DHQ synthase domain (the cis arrangement). When DHQ synthase and EPSP synthase are produced concomitantly by transcribing sequences encoding the individual domains from separate plasmids in the same bacterial cell (the trans arrangement) no overproduction or enzyme activity can be detected for the EPSP synthase domain; (4) the EPSP synthase domain can be stably overproduced as a fusion protein with glutathione S-transferase (GST), however the EPSP synthase in this instance is enzymatically inactive; (5) a protein containing an enzymatically inactive DHQ synthase domain in the cis arrangement with EPSP synthase domain is stably overproduced with enzymatically active EPSP synthase; (6) the two C-terminal domains of the AROM protein specifying the 3-dehydroquinase and shikimate dehydrogenase domains can be overproduced in A. nidulans using a specially constructed expression vector. This same bi-domain fragment however is not produced in E. coli when identical coding sequences are transcribed from a prokaryotic expression vector. These data support the view that multifunctional/multidomain proteins do not solely consist of independent units covalently linked together, but rather that certain individual domains interact to varying degrees to stabilise enzyme activity.     

Identification of Trans-Sialidases as a Common Mediator of Endothelial Cell Activation by African Trypanosomes

Understanding African Trypanosomiasis (AT) host-pathogen interaction is the key to an “anti-disease vaccine”, a novel strategy to control AT. Here we provide a better insight into this poorly described interaction by characterizing the activation of a panel of endothelial cells by bloodstream forms of four African trypanosome species, known to interact with host endothelium. T. congolense, T. vivax, and T. b. gambiense activated the endothelial NF-κB pathway, but interestingly, not T. b. brucei. The parasitic TS (trans-sialidases) mediated this NF-κB activation, remarkably via their lectin-like domain and induced production of pro-inflammatory molecules not only in vitro but also in vivo, suggesting a considerable impact on pathogenesis. For the first time, TS activity was identified in T. b. gambiense BSF which distinguishes it from the subspecies T. b. brucei. The corresponding TS were characterized and shown to activate endothelial cells, suggesting that TS represent a common mediator of endothelium activation among trypanosome species with divergent physiopathologies.     

Purification of cholesterol-esterifying enzymes from rat liver cytosol by high-performance liquid chromatography

Three enzymes esterifying cholesterol with long-chain fatty acids were purified approximately 31 000-fold to apparent homogeneity from the cytosol of normal rat liver. The enzymatic activity was tested by incubation of active fractions with tritiated cholesterol and separation of newly formed esters from non-reacted cholesterol by a passage through silica gel cartridges with subsequent assay for radioactivity by liquid scintillation. For the purification of enzymes, active proteins were precipitated by (NH₄)₂SO₄ to 35% saturation. The bulk of inactive proteins was removed by size-exclusion chromatography on TSK G3000 SW. The active fraction was subsequently separated on Separon HEMA BIO 1000 DEAE in gradients of 0–500 mM KCl into three enzymatic activities differing in their retention and these proteins were finally purified by affinity HPLC on columns of cholesterol immobilized on HEMA BIO 1000 E-H. Final purified enzymes showed the same single band in polyacrylamide gel electrophoresis corresponding to 16.5 kDa. Combination of individual enzymes did not increase the overall yield of cholesteryl esters but the reaction-rate was significantly accelerated. These proteins are apparently subunits of a larger complex (M_r 65 000) that can be demonstrated by electrophoresis in the absence of 2-mercaptoethanol. Results presented in this paper indicate that because of good and rapid separation of active proteins, HPLC may be a method of choice for enzyme purifications.     

Jacalin domain in wheat jasmonate-regulated protein Ta-JA1 confers agglutinating activity and pathogen resistance

Ta-JA1 is a jacalin-like lectin from wheat (Triticum aestivum) plants. To date, its homologs are only observed in the Gramineae family. Our previous experiments have demonstrated that Ta-JA1 contains a modular structure consisting of an N-terminal dirigent domain and a C-terminal jacalin-related lectin domain (JRL) and this protein exhibits a mannose-specific lectin activity. The over-expression of Ta-JA1 gene provides transgenic plants a broad-spectrum resistance to diseases. Here, we report the differential activities of the dirigent and JRL domains of Ta-JA1. In vitro assay showed that the recombinant JRL domain could effectively agglutinate rabbit erythrocytes and pathogen bacteria Pseudomonas syringe pv tabaci. These hemagglutination activities could be inhibited by mannose but not by galactose. In contrast, the recombinant dirigent domain did not show agglutination activity. Corresponding to these differentiations of activities, similar to full-length of Ta-JA1, the over-expression of JRL domain in transgenic plants also increased resistance to the infection of P. syringe. Unlike JRL, the over-expression of dirigent domain in transgenic plants led to alteration of the seedling sensitivity to salts. In addition, a d_N/d_S ratio analysis of Ta-JA1 and its related proteins showed that this protein family functionally limited to a few crop plants, such as maize, rice and wheat.