Pea lectin receptor-like kinase promotes high salinity stress tolerance in bacteria and expresses in response to stress in planta.

The plant lectin receptor-like kinases (LecRLKs) are involved in various signaling pathways but their role in salinity stress tolerance has not heretofore been well described. Salinity stress negatively affects plant growth/productivity and threatens food security worldwide. Based on functional gene-mining assay, we have isolated 34 salinity tolerant genes out of one million Escherichia coli (SOLR) transformants containing pea cDNAs grown in 0.8 M NaCl. Sequence analysis of one of these revealed homology to LecRLK, which possesses N-myristilation and N-glycosylation sites thus corroborating the protein to be a glycoconjugate. The homology based computational modeling of the kinase domain suggested high degree of conservation with the protein already known to be stress responsive in plants. The NaCl tolerance provided by PsLecRLK to the above bacteria was further confirmed in E. coli (DH5alpha). In planta studies showed that the expression of PsLecRLK cDNA was significantly upregulated in response to NaCl as compared to K(+) and Li(+) ions, suggesting the Na(+) ion specific response. Transcript of the PsLecRLK gene accumulates mainly in roots and shoots. The purified 47 kDa recombinant PsLecRLK-KD (kinase domain) protein has been shown to phosphorylate general substrates like MBP and casein. This study not only suggests the conservation of the cellular response to high salinity stress across prokaryotes and plant kingdom but also provides impetus to develop novel concepts for better understanding of mechanism of stress tolerance in bacteria and plants. It also opens up new avenues for studying practical aspects of plant salinity tolerance for enhanced agricultural productivity.

Glycosylation-dependent interaction of Jacalin with CD45 induces T lymphocyte activation and Th1/Th2 cytokine secretion.

Jacalin, an alpha-O-glycoside of the disaccharide Thomsen-Friedenreich antigen (galactose beta1-3 N-acetylgalactosamine, T-antigen)-specific lectin from jackfruit seeds, has been shown to induce mitogenic responses and to block infection by HIV-1 in CD4+ T lymphocytes. The molecular mechanism underlying Jacalin-induced T cell activation has not been elucidated completely yet. In the present study, protein tyrosine phosphatase (PTPase) CD45 was isolated from a Jurkat T cell membrane fraction as a major receptor for Jacalin through affinity chromatography and mass spectrometry. CD45, which is highly glycosylated and expressed exclusively on the surface of lymphocytes, is a key regulator of lymphocyte signaling, playing a pivotal role in activation and development. We found that the lectin induced significant IL-2 production by a CD45-positive Jurkat T cell line (JE6.1) and primary T cells. However, this effect did not occur in a CD45-negative Jurkat T cell line (J45.01) and was blocked completely by a specific CD45 PTPase inhibitor in Jurkat T (JE6.1) and primary T cells. Furthermore, we also observed that Jacalin caused a marked increase in IL-2 secretion in response to TCR ligation and CD28 costimulation and contributed to Th1/Th2 cytokine production by activating CD45. Jacalin increased CD45 tyrosine phosphatase activity, which resulted in activation of the ERK1/2 and p38 MAPK cascades. Based on these findings, we propose a new, immunoregulatory model for Jacalin, wherein glycosylation-dependent interactions of Jacalin with CD45 on T cells elevate TCR-mediated signaling, which thereby up-regulate T cell activation thresholds and Th1/Th2 cytokine secretion.

Isolation and characterization of the potential receptor for wheat germ agglutinin from human neutrophils.

Neutrophils participate in host protection and central to this process is the regulation of oxidative mechanisms. We purified by affinity chromatography the receptor for the GlcNAc-specific WGA from CD14+ CD16+ cell lysates (WGAr). The receptor is a 141 kDa glycoprotein constituted by two subunits of 78 and 63 kDa. It is mainly composed of Ser, Asx, and Gly, and, in a minor proportion, His, Cys, and Pro. Its glycan portion contains GlcNAc, Gal, and Man; NeuAc and GalNAc were identified in a minor proportion. The amino acid sequence of the WGA receptor was predicted from tryptic peptides by MALDI-TOF, both subunits showed homology with cytokeratin type II (26 and 29% for the 78 and 63 kDa subunits, respectively); the 78 kDa subunit showed also homology with the human transferrin receptor (24%). Antibodies against WGAr induce higher oxidative burst than WGA, determined by NBT reduction; however, this effect was inhibited (p < 0.05) with GlcNAc suggesting that WGAr participates as mediator in signal transduction in neutrophils.

Expression of discoidin domain receptor 2 (DDR2) extracellular domain in pichia pastoris and functional analysis in synovial fibroblasts and NIT3T3 cells.

Discoidin domain receptor 2 (DDR2) is a kind of protein tyrosine kinases associated with cell proliferation and tumor metastasis, and collagen, identified as a ligand for DDR2, up-regulates matrix metallloproteinase 1 (MMP-1) and MMP-2 expression in cellular matrix. To investigate the roles of DDR2 in destruction of cartilage in rheumatoid arthritis (RA) and tumor metastasis, we tried to express extracellular domain of DDR2 fused with a His tag to increase protein solubility and facilitate purification (without signal peptide and transmembrane domain, designated DR) in Pichia pastoris, purify the expressed protein, and characterize its function, for purpose of future application as a specific DDR2 antagonist. Two clones of relative high expression of His-DR were obtained. After purification by a Ni-NTA (nitric-tri-acetic acid) chromatographic column, soluble fused His-DR over 90% purity were obtained. Competitive binding inhibition assay demonstrated that expressed His-DR could block the binding of DDR2 and natural DDR2 receptors on NIT3T3 and synovial cell surfaces. Results of RT-PCR, Western blotting, and gelatinase zymography showed that His-DR was capable of inhibiting MMP-1 and MMP-2 secretion from NIT3T3 cells and RA synoviocytes stimulated by collagen II. For MMP-1, the inhibitory effect was displayed at the levels of mRNA and protein, whereas for MMP-2 it was demonstrated at the level of protein physiological activity. All these findings suggested that the fused expressed His-DR inhibited the activity of natural DDR2, and relevant MMP-1 and MMP-2 expression in synoviocytes and NIH3T3 cells provoked by collagen II.

Isolation of the receptor for the Amaranthus leucocarpus lectin from human T lymphocytes.

Amaranthus leucocarpus lectin (ALL) is specific for GalNAc, and recognizes human T cells. The receptor for ALL was purified from T cells using biotin-labeled lectin and avidin-agarose as affinity matrix. It is a 70-kDa glycoprotein, constituted mainly by serine, glycine, and glutamic acid; its glycosidic portion contains mainly GalNAc; galactose, sialic acid, mannose, and GlcNAc were identified at a lower proportion. By ionic strength chromatography, as well as double dimension electrophoresis, we identified four isoforms of the ALL-receptor. N-terminal amino acid was blocked both in the ALL-receptor and its isoforms, therefore, tryptic peptides of ALL-receptor, analyzed through MALDI-TOF, were compared with the relative values obtained from the NCBInr (ProFound 2004/06/01) database. Our results indicated that the tryptic peptides obtained showed 54% homology with a DnaK-core molecular chaperone, 47% with human KIAA protein, and 44% with heat shock protein 8. The most frequent phenotype of the CD4 or CD8 ALL+ T cells was CD45RA+ CD27+; 26% of ALL+ T cells were CD25+ and 13% were CD69+, indicating that the glycoprotein recognized by ALL is present mainly on naive or quiescent T cells.

Three-dimensional interplay among the ligand-binding domains of the urokinase-plasminogen-activator-receptor-associated protein, Endo180.

Endo180, also known as the urokinase plasminogen activator receptor (uPAR)-associated protein (uPARAP), is one of the four members of the mannose receptor family, and is implicated in extracellular-matrix remodelling through its interactions with collagens, sugars and uPAR. The extracellular portion of Endo180 contains an amino-terminal cysteine-rich domain, a single fibronectin type II domain and eight C-type lectin-like domains. We have purified a soluble version of Endo180 and analysed it by single-particle electron microscopy to obtain a three-dimensional structure of the N-terminal part of the protein at a resolution of 17 A and reveal, for the first time, the interactions between non-adjacent domains in the mannose receptor family. We show that for Endo180, the cysteine-rich domain contacts the second C-type lectin-like domain, thus providing structural insight into how modulation of its several ligand interactions may regulate Endo180 receptor function.

Differential expression of a 70 kDa O-glycoprotein on T cells: a possible marker for naive and early activated murine T cells.

We purified a 70 kDa O-glycoprotein that binds to the GalNAc specific lectin from Amaranthus leucocarpus (ALLr) and determined its expression pattern on T lymphocytes from different murine lymphoid organs. High level of ALLr expression was demonstrated in 95-98% of both CD4(+)8(+) and CD4(-)8(+) thymocytes, and in 80-95% of CD8(+) T cells from peripheral blood, lymph nodes, and spleen, whereas a minor fraction of CD4(+)8(-) thymocytes (46-67%) and peripheral CD4(+) T cells (9-40%) showed low ALLr expression. Peripheral CD19(+) B cells were ALLr negative and most of the peripheral ALL(+) T cells showed a CD62L(hi)CD45RB(hi)CD44(lo/-) phenotype, indicating features of naive cells. Mitogenic activation of peripheral T cells increased 3-fold the number of ALL(+)CD4(+) T cells 24 h after stimulation, as opposed to a >80% decrease in CD8(+) T cells 72 h after stimulation. Our results suggest that ALL detects a non-described surface O-glycoprotein selectively expressed by naive CD8(+) T cells and by early activated CD4(+) T cells.

DCIR acts as an inhibitory receptor depending on its immunoreceptor tyrosine-based inhibitory motif.

Major histocompatibility complex class II positive cells, namely dendritic cells, monocytes/macrophages, and B cells, are categorized as antigen-presenting cells. Dendritic cells, so-called professional antigen-presenting cells, use distinct sets of surface receptors before and after maturation: those to capture antigens and those to interact with T cells, respectively. But there remain many surface molecules whose functions are still unknown. In this study, we isolated dendritic cell immunoreceptor from mouse bone-marrow-derived mature dendritic cells. Dendritic cell immunoreceptor is a recently reported C-type lectin receptor characteristic with cytoplasmic immunoreceptor tyrosine-based inhibitory motif. Expression of mouse dendritic cell immunoreceptor mRNA was observed specifically in spleen and lymph node, slightly increased with dendritic cell maturation during in vitro culture of bone marrow cells, and was not detected in cultured natural killer cells. Surface expression of mouse dendritic cell immunoreceptor protein was observed in splenic antigen-presenting cells including B cells, monocytes/macrophages, and dendritic cells, but not in T cells. To reveal the downregulating capacity of dendritic cell immunoreceptor in antigen-presenting cells, the change of B-cell-receptor-mediated signals after coligation with a chimeric Fcgamma receptor IIB containing the cytoplasmic portion of mouse dendritic cell immunoreceptor was examined. As a result, we detected two distinct inhibitory effects of cytoplasmic dendritic cell immunoreceptor minus sign inhibition of B-cell-receptor-mediated Ca2+ mobilization and protein tyrosine phosphorylation minus sign and both of these effects required the tyrosine residue inside the immunoreceptor tyrosine-based inhibitory motif. This report presents immunoreceptor tyrosine-based inhibitory motif-dependent negative regulatory function of dendritic cell immunoreceptors. In conclusion, mouse dendritic cell immunoreceptor expressed on antigen-presenting cells can exert two distinct inhibitory signals depending on its immunoreceptor tyrosine-based inhibitory motif tyrosine residue.

Isolation of the receptor for Amaranthus leucocarpus lectin from murine naive thymocytes.

From murine medullary thymocytes we purified the receptor for the Amaranthus leucocarpus lectin (ALL) using a complex with the biotin-labeled lectin and avidin-agarose as the affinity matrix. Most ALL(+)thymocytes (83%) are naive cells with the CD4(+)CD8(-)CD45RB(+)phenotype. The receptor for this lectin is a 70 kDa glycoprotein that contains 20% of sugar by mass. It is constituted mainly by aspartic and glutamic acids, serine, proline, and glycine; its glycosidic portion contains mainly O-glycosidically linked glycans with Gal, GalNAc and NeuAc residues as well as one N-glycosidically linked glycan per molecule. Ionic strength chromatography revealed that the ALL-thymocyte receptor (ALLTr) is made up by three isoforms, which possess similar amino acid composition but show slight differences in their sugar composition. The N-terminal amino acid residues are blocked both in the receptor and its purified isoforms. Analyses of the receptor's peptides, obtained by trypsin digestion with MALDI-TOF (matrix assisted laser desorption ionization-time of flight), were compared with the relative values obtained from the NCBInr (Swiss-Prot 10/01/99) database. Our results indicate that the peptides of ALLTr show low homology (<17%) with the human KIIA protein, the Fas-associated death domain protein, and the transforming growth factor-beta type II receptor. Our results suggest that the ALL thymocyte receptor could be considered a novel phenotypic marker specific for naive T cells.

Interaction of Azospirillum lipoferum with wheat germ agglutinin stimulates nitrogen fixation.

In vitro, the nitrogen fixation capability of A. lipoferum is efficiently increased in the presence of wheat germ agglutinin (WGA). A putative WGA-binding receptor, a 32-kDa protein, was detected in the cell capsule. The stimulatory effect required N-acetyl-D-glucosamine dimer (GlcNAcdi) terminated sugar side chains of the receptor and was dependent on the number of GlcNAcdi links involved in receptor-WGA interface. Binding to the primary sugar binding sites on WGA had a larger stimulatory effect than binding to the secondary sites. The WGA-receptor complex generated stimulus led to elevated transcription of the nifH and nifA genes and of the glnBA gene cluster but not of the glnA gene from its own promoter. There may well be a signalling cascade contributing to the regulation of nitrogen fixation.

Isolation of the receptor for Amaranthus leucocarpus lectin from murine peritoneal macrophages.

The receptor for Amaranthus leucocarpus lectin from CD-1 resident macrophages was purified with affinity chromatography with biotin labeled A. leucocarpus lectin and using avidin-agarose as affinity matrix. The receptor is a glycoprotein of 70 kDa that contains 18% of sugar by weight; it is mainly composed of galactose and N-acetyl-D-galactosamine in its saccharidic portion, and lacks sialic acid; the protein is rich in glycine, serine and alanine and lacks cysteine residues. The amino terminus of the receptor is blocked. By ionic strength chromatography on a mono P column in anionic form we purified three isoforms from the affinity purified receptor, each showing quantitative differences in glycosylation. The A. leucocarpus lectin receptor is identified only in resting, not activated, macrophages suggesting that it plays a role in activation mechanisms of macrophages.

Neuropilin-1 is expressed by endothelial and tumor cells as an isoform-specific receptor for vascular endothelial growth factor.

Vascular endothelial growth factor (VEGF), a major regulator of angiogenesis, binds to two receptor tyrosine kinases, KDR/Flk-1 and Flt-1. We now describe the purification and the expression cloning from tumor cells of a third VEGF receptor, one that binds VEGF165 but not VEGF121. This isoform-specific VEGF receptor (VEGF165R) is identical to human neuropilin-1, a receptor for the collapsin/semaphorin family that mediates neuronal cell guidance. When coexpressed in cells with KDR, neuropilin-1 enhances the binding of VEGF165 to KDR and VEGF165-mediated chemotaxis. Conversely, inhibition of VEGF165 binding to neuropilin-1 inhibits its binding to KDR and its mitogenic activity for endothelial cells. We propose that neuropilin-1 is a novel VEGF receptor that modulates VEGF binding to KDR and subsequent bioactivity and therefore may regulate VEGF-induced angiogenesis.

Differential reactivities of the Arachis hypogaea (peanut) and Vicia villosa B4 lectins with human ovarian carcinoma cells, grown either in vitro or in vivo xenograft model.

PNA and VVA B4 recognize the tumor-associated T antigen and its immediate precursor Tn, respectively. We found that both lectins are highly reactive in vitro, with human ovarian carcinoma cell lines, but only VVA B4 bound significantly to breast and oral cancer cells. This binding is inhibited by specific monosaccharides. The lectin binding receptors were purified, revealing a glycoprotein of 32 kDa for PNA, and two glycoproteins of 35 and 38 kDa for VVA B4. In vivo localization of PNA was almost exclusive (except for the kidneys) to the ovarian tumor xenografts. VVA B4 showed wider tissue biodistribution being preferentially accumulated in the tumors and ovaries.

Rat sperm galactosyl receptor: purification and identification by polyclonal antibodies raised against multiple antigen peptides.

We have previously reported the purification of rats testis galactosyl receptor, an equivalent to the Ca(2+)-dependent (C-type) minor variant of rat hepatic lectin-2/3 (RHL-2/3). We now report the purification of galactosyl receptor from rat sperm and its immunolocalization in the intact rat testis and sperm by polyclonal antibodies prepared using multiple antigen peptides (MAP) as immunogens. Two MAP antigens (designated 27-mer and 28-mer), corresponding to amino acid sequences of the carbohydrate-recognition domain (galactose) and adjacent Ca(2+)-binding sites of RHL-2/3, were used for immunization. Anti-RHL-2/3, anti-p27, and anti-p28 sera crossreacted with rat hepatocyte RHL-2/3 and its rat testis and sperm equivalent, galactosyl receptor, purified by chromatofocusing followed by galactose-Hydropore-EP affinity chromatography. Neither anti-p27 nor anti-p28 sera cross-reacted with the major hepatocyte variant, RHL-1. A RHL-1-equivalent was not detected in rat testis and sperm. Immunofluorescence studies demonstrated that anti-p27 and anti-p28 sera recognize galactosyl receptor sites at the Sertoli cell-spermatogenic cell interface and on the dorsal surface of the sperm head, overlying the acrosome. The characteristic crescent-shaped immunoreactive pattern in sperm was lost after induction of the acrosome reaction. Further studies should determine whether antisera to MAP antigens 27-mer and 28-mer, corresponding to specific protein motifs, can serve as immunological probes for examining cell-cell interaction events during spermatogenesis and at fertilization.

Identification of peanut agglutinin-binding glycoproteins on lizard lymphocytes.

The expression of PNA-binding glycoproteins on lizard lymphocytes was investigated by studying the reactivity of FITC-PNA towards lizard lymphocytes obtained from the different lymphoid organs. Direct immunofluorescence assays have demonstrated that the majority of lizard thymocytes (70%) and only a fraction of lymphocytes in the spleen, peripheral blood and bone marrow were PNA-positive. This positivity was selectively inhibited by galactose as well as lactose, indicating the specificity of binding. Putative PNA receptors were purified from lizard thymocytes and splenocytes by affinity chromatography on a PNA-Sepharose 4B column and resulted in fractions enriched 1,792-fold and 3,141-fold for the PNA-binding component expressed on lizard thymocytes and splenocytes, respectively. Analysis on reducing and non-reducing SDS-PAGE revealed that both thymic and splenic PNA-binding glycoproteins migrated as a single component of 35 KDa, with no evidence for the association into higher multimers in both tissues. Analyses for amino acid and carbohydrate compositions indicated that the thymic and splenic glycoproteins have similar amino acid composition and differed in the content of neutral and amino-sugars as well as sialic acid. The content of the latter residue was relatively higher in the splenic form of the receptor compared to its thymic counterpart, and was inversely correlated with the content of galactosyl residues in both forms of the receptor. The functional significance of PNA-binding glycoproteins during vertebrate evolution is discussed.

Interaction of the c-fes proto-oncogene product with the interleukin-4 receptor.

Interleukin-4 (IL-4) regulates proliferation and differentiation of a variety of hematopoietic cell lineages through binding to the IL-4 receptor (IL-4R). Recently, we have demonstrated that IL-4 induces tyrosine phosphorylation of several proteins including the IL-4R and also induces association of phosphatidylinositol-3 kinase with the IL-4R. Since IL-4 induces tyrosine phosphorylation, we speculated that some tyrosine kinase may associate with the IL-4R. In this study, we demonstrate that IL-4 induces tyrosine phosphorylation of a protein closely related to, or identical to the c-fes protooncogene-encoded protein tyrosine kinase (FES). Furthermore, tyrosine phosphorylated FES or the closely related protein is found associated with the IL-4R. We also demonstrate that FES associates with the IL-4R in COS7 cells transfected with cDNA expression plasmids encoding these two proteins and in transfected COS7 cells IL-4 augments association of FES with the IL-4R and tyrosine phosphorylation of both FES and the IL-4R. Through a deletion analysis of the IL-4R we have identified a putative FES-binding site in the cytoplasmic domain of the IL-4R.

Tumor necrosis factor upregulates interleukin-4 receptors on murine sarcoma cells.

We have previously shown that murine solid tumor cells express high affinity IL-4 receptors (IL-4R) which are internalized after binding to ligand. In the present study, we have examined the regulation of IL-4R by TNF. We demonstrate that TNF upregulated the expression of IL-4R on murine MCA-106 sarcoma cells. Maximum upregulation of IL-4R surface expression occurred after 24 h, whereas, maximum elevation in IL-4R mRNA levels was observed after only 4 hours of TNF treatment. This increase in mRNA levels for IL-4R occurred in a dose dependent manner. As little as 0.83 ng/ml of TNF significantly upregulated mRNA levels, whereas maximum effect was obtained with 83 ng/ml TNF. IL-4 receptor density was increased in response to TNF, no effect on IL-4R affinity was observed. Cycloheximide and Actinomycin D treatment decreased the surface expression of IL-4R by 50% in about 2 h and 7 h, respectively, in both TNF treated and untreated cells indicating the half life for the IL-4R protein expression. These studies may help understand the mechanism of cytokine interaction on tumor cells.

Intracellular lectin-binding sites in symbiont-bearing Crithidia species.

Crithidia oncopelti, C. deanei, and C. desouzai are flagellates of the Trypanosomatidae family that present bacterium-like endosymbionts in their cytoplasm. Direct and indirect lectin-gold labeling techniques were used at the electron microscopic level in Lowicryl K4M-embedded cells to demonstrate the presence of intracellular lectin-binding sites. We used the lectins Ulex europaeus I, Griffonia simplicifolia II, Ricinus communis I, Arachis hypogaea, G. simplicifolia I, Wistaria floribunda, Limulus polyphemus, and Canavalia ensiformis, which recognize alpha-L-fucose, alpha- and beta-N-acetylglucosamine, beta-galactose and beta-N-acetylgalactosamine, beta-galactose, alpha-galactose, beta-N-acetylgalactosamine, sialic acid and alpha-D-mannose, and alpha-D-glucose residues, respectively. The nucleus was the cellular structure most frequently labeled by the lectins. The Golgi complex was seldom labeled, whereas the endoplasmic reticulum and the flagellar pocket presented a large number of binding sites. Symbionts had their two unit membranes weakly labeled by the different lectins but displayed no labeling of the space between the membranes.