Effects of chelators (EGTA, EDTA) and calcium ions on nematode capture by the nematode-trapping fungusArthrobotrys ellipsospora

The nematode-trapping fungusArthrobotrys ellipsospora developed an adhesive knob and trapped nematodes when cultured on a low-nutrient medium. It also trapped polystyrene beads in the same way. The adhesive knob produced mucus that was stained with alcian blue, while mycelium of the fungus was stained with periodic acid/Schiff (PAS). The amount of mucus increased with in days after culturing in the low-nutrient media. The fungus completely lost its ability to trap nematodes when treated with EDTA and EGTA, but it recovered the ability after incubation in the presence of a low concentration of Ca (10⁻⁶–10⁻⁷ M) for 1 h. Calmodulin inhibitor W-7 also inhibited the trapping ability of the fungus, and there was a significant (p<0.05) difference between the effects of W-7 and W-5. Ca-binding protein was also detected in the fungus.     

少孢节丛孢中CRISPR/Cas9基因编辑系统的建立及6-甲基水杨酸合酶新活性位点的发现

以捕食线虫真菌少孢节丛孢Arthrobotrys oligospora YMF 1.03170为研究材料,通过优化sgRNA 表达驱动体系 tRNA^Gly,构建CRISPR/Cas9基因编辑系统,成功获得基因定点编辑菌株。将该CRISPR/Cas9系统与同源重组相结合,可精确地对两个目的氨基酸编码基因同时进行定点置换。结合代谢图谱及前体化合物饲喂实验,发现6-甲基水杨酸合酶编码蛋白新的活性位点Arg17、Arg18、His33和His34。本研究将CRISPR/Cas9基因编辑系统应用在少孢节丛孢中,并成功建立基因编辑精细体系,为快速构建少孢节丛孢的遗传转化体系和研究该菌的基因功能提供有效方法。     

Purification of a lectin from Eugenia uniflora L. seeds and its potential antibacterial activity

Aims: The aim of this work was to analyse the antimicrobial properties of a purified lectin from Eugenia uniflora L. seeds. Methods and Results: The E. uniflora lectin (EuniSL) was isolated from the seed extract and purified by ion‐exchange chromatography in DEAE‐Sephadex with a purification factor of 11·68. The purified lectin showed a single band on denaturing electrophoresis, with a molecular mass of 67 kDa. EuniSL agglutinated rabbit and human erythrocytes with a higher specificity for rabbit erythrocytes. The haemagglutination was not inhibited by the tested carbohydrates but glycoproteins exerted a strong inhibitory action. The lectin proved to be thermo resistant with the highest stability at pH 6·5 and divalent ions did not affect its activity. EuniSL demonstrated a remarkable nonselective antibacterial activity. EuniSL strongly inhibited the growth of Staphylococcus aureus, Pseudomonas aeruginosa and Klebsiella sp. with a minimum inhibitory concentration (MIC) of 1·5 μg ml^?1, and moderately inhibited the growth of Bacillus subtilis, Streptococcus sp. and Escherichia coli with a MIC of 16·5 μg ml^?1. Conclusions: EuniSL was found to be effective against bacteria. Significance and Impact of the Study: The strong antibacterial activity of the studied lectin indicates a high potential for clinical microbiology and therapeutic applications.     

Mannose binding lectin and lung collectins interact with Toll-like receptor 4 and MD-2 by different mechanisms

Background

We have previously shown that lung collectins, surfactant protein A (SP-A) and surfactant protein D, interact with Toll-like receptor (TLR) 2, TLR4, or MD-2. Bindings of lung collectins to TLR2 and TLR4/MD-2 result in the alterations of signaling through these receptors, suggesting the immunomodulatory functions of lung collectins. Mannose binding lectin (MBL) is another collectin molecule which has structural homology to SP-A. The interaction between MBL and TLRs has not yet been determined.

Methods

We prepared recombinant MBL, and analyzed its bindings to recombinant soluble forms of TLR4 (sTLR4) and MD-2.

Results

MBL bound to sTLR4 and MD-2. The interactions were Ca²⁺-dependent and inhibited by mannose or monoclonal antibody against the carbohydrate-recognition domain of MBL. Treatment of sTLR4 or MD-2 by peptide N-glycosidase F significantly decreased the binding of MBL. SP-A bound to deglycosylated sTLR4, and this property did not change in chimeric molecules of SP-A/MBL in which Glu¹⁹⁵–Phe²²⁸ or Thr¹⁷⁴–Gly¹⁹⁴ of SP-A were replaced with the corresponding MBL sequences.

General Significance

These results suggested that MBL binds to TLR4 and MD-2 through the carbohydrate-recognition domain, and that oligosaccharide moieties of TLR4 and MD-2 are important for recognition by MBL. Since our previous studies indicated that lung collectins bind to the peptide portions of TLRs, MBL and lung collectins interact with TLRs by different mechanisms. These direct interactions between MBL and TLR4 or MD-2 suggest that MBL may modulate cellular responses by altering signals through TLRs.     

GISP: a novel brain-specific protein that promotes surface expression and function of GABA(B) receptors

Synaptic transmission depends on the regulated surface expression of neurotransmitter receptors, but many of the cellular processes required to achieve this remain poorly understood. To better define specific mechanisms for the GABA(B) receptor (GABA(B)R) trafficking, we screened for proteins that bind to the carboxy-terminus of the GABA(B1) subunit. We report the identification and characterization of a novel 130-kDa protein, GPCR interacting scaffolding protein (GISP), that interacts directly with the GABA(B1) subunit via a coiled-coil domain. GISP co-fractionates with GABA(B)R and with the postsynaptic density and co-immunoprecipitates with GABA(B1) and GABA(B2) from rat brain. In cultured hippocampal neurons, GISP displays a punctate dendritic distribution and has an overlapping localization with GABA(B)Rs. When co-expressed with GABA(B)Rs in human embryonic kidney cells, GISP promotes GABA(B)R surface expression and enhances both baclofen-evoked extracellular signal-regulated kinase (ERK) phosphorylation and G-protein inwardly rectifying potassium channel (GIRK) currents. These results suggest that GISP is involved in the forward trafficking and stabilization of functional GABA(B)Rs.     

Purification, physicochemical characterization, saccharide specificity, and chemical modification of a Gal/GalNAc specific lectin from the seeds of Trichosanthes dioica

A new galactose-specific lectin has been purified from the extracts of Trichosanthes dioica seeds by affinity chromatography on cross-linked guar gum. The purified lectin (T. dioica seed lectin, TDSL) moved as a single symmetrical peak on gel filtration on Superose-12 in the presence of 0.1 M lactose with an M(r) of 55 kDa. In the absence of ligand, the movement was retarded, indicating a possible interaction of the lectin with the column matrix. In SDS-PAGE, in the presence of beta-mercaptoethanol, two non-identical bands of M(r) 24 and 37 kDa were observed, whereas in the absence of beta-mercaptoethanol, the lectin yielded a single band corresponding to approximately 55,000 Da, indicating that the two subunits of TDSL are connected by one or more disulfide bridges. TDSL is a glycoprotein with about 4.9% covalently bound neutral sugar. Analysis of near-UV CD spectrum by three different methods (CDSSTR, CONTINLL, and SELCON3) shows that TDSL contains 13.3% alpha-helix, 36.7% beta-sheet, 19.4% beta-turns, and 31.6% unordered structure. Among a battery of sugars investigated, TDSL was inhibited strongly by beta-d-galactopyranosides, with 4-methylumbelliferyl-beta-d-galactopyranoside being the best ligand. Chemical modification studies indicate that tyrosine residues are important for the carbohydrate-binding and hemagglutinating activities of the lectin. A partial protection was observed when the tyrosine modification was performed in the presence of 0.2 M lactose. The tryptophan residues of TDSL appear to be buried in the protein interior as they could not be modified under native conditions, whereas upon denaturation with 8 M urea two Trp residues could be selectively modified by N-bromosuccinimide. The subunit composition and size, secondary structure, and sugar specificity of this lectin are similar to those of type-2 ribosome inactivating proteins, suggesting that TDSL may belong to this protein family.     

Fusion of a soybean cysteine protease inhibitor and a legume lectin enhances anti-insect activity synergistically

Abstract 1 The soybean cysteine protease inhibitor soyacystatin N (scN) and Griffonia simplicifolia lectin II (rGSII) have defense functions against the coleopteran cowpea bruchid beetle Callosobruchus maculatus. However, the ability of the insect to activate scN‐insensitive digestive proteases and the relatively low potency of rGSII have hindered their practical application in plant protection. 2 Recent research suggests that defense proteins may achieve increased toxicity and durability when used in combination. Based on the structures of several natural toxin molecules, we hypothesized that covalently linked scN and rGSII could exhibit greater anti‐insect efficacy than the mixture containing individual proteins. 3 To test this hypothesis, a recombinant scN‐rGSII fusion protein that retained both protease inhibitor and lectin functions was constructed. 4 When fed to cowpea bruchid, this new protein showed a synergistic delay in insect development, whereas a mixture of the separate proteins only showed an additive effect. 5 Our results suggest that tethering digestive protease inhibitors to gut epithelium‐interacting lectins could give plant protection superior to strategies based on single genes or mixtures of single gene products.     

从进口早熟禾种子中检出的一种腥黑粉菌与其近似种的比较

早熟禾是优良的草坪草和牧草,近年来,天津出入境检验检疫局多次从进境的草地早熟禾、加拿大早熟禾及一年生早熟禾上截获一种腥黑粉菌,暂名为Tilletia sp.。针对T. sp.、早熟禾上的形态相近的T. sterilis、T. togwateei,以及同属近似种雀麦腥黑粉菌T. bromi和禾草腥黑粉菌T. fusca在冬孢子形态特征、萌发特性及细胞学性状等方面进行了系统比较。结果表明,Tilletia sp.与T. sterilis、T. togwateei存在较大差异,与T. bromi和T. fusca差别不明显;截获菌与Tilletia sp.在菌瘿、冬孢子形态特征上与T. bromi更为接近,根据研究结果将Tilletia sp.定名为T. bromi。     

Localization of peanut (Arachis hypogaea) root lectin (PRA II) on root surface and its biological significance

The glucose-specific peanut root lectin, PRA II, is localized on the surface of 7-day-old peanut seedling root and in root cortical parenchymatous cells. The lectin is eluted from intact roots upon washing with buffer containing glucose. Rabbit erythrocytes bind to the root surface and the cortical cells; the binding is inhibited by antibodies raised against PRA II, peanut-specificRhizobium cells and by glucose. Lipopolysaccharides isolated from host-specificRhizobium strain inhibit the haemagglutinating activity of PRA II and are precipitated by the lectin. Our results suggest that PRA II might be involved in recognition ofRhizobium by peanut roots.     

The endoplasmic reticulum‐associated protein,OS‐9, behaves as a lectin in targeting the immature calcium‐sensing receptor

The mechanisms responsible for the processing and quality control of the calcium‐sensing receptor (CaSR) in the endoplasmic reticulum (ER) are largely unknown. In a yeast two‐hybrid screen of the CaSR C‐terminal tail (residues 865–1078), we identified osteosarcoma‐9 (OS‐9) protein as a binding partner. OS‐9 is an ER‐resident lectin that targets misfolded glycoproteins to the ER‐associated degradation (ERAD) pathway through recognition of specific N‐glycans by its mannose‐6‐phosphate receptor homology (MRH) domain. We show by confocal microscopy that the CaSR and OS‐9 co‐localize in the ER in COS‐1 cells. In immunoprecipitation studies with co‐expressed OS‐9 and CaSR, OS‐9 specifically bound the immature form of wild‐type CaSR in the ER. OS‐9 also bound the immature forms of a CaSR C‐terminal deletion mutant and a C677A mutant that remains trapped in the ER, although binding to neither mutant was favored over wild‐type receptor. OS‐9 binding to immature CaSR required the MRH domain of OS‐9 indicating that OS‐9 acts as a lectin most likely to target misfolded CaSR to ERAD. Our results also identify two distinct binding interactions between OS‐9 and the CaSR, one involving both C‐terminal domains of the two proteins and the other involving both N‐terminal domains. This suggests the possibility of more than one functional interaction between OS‐9 and the CaSR. When we investigated the functional consequences of altered OS‐9 expression, neither knockdown nor overexpression of OS‐9 was found to have a significant effect on CaSR cell surface expression or CaSR‐mediated ERK1/2 phosphorylation.     

Cloning of a lectin cDNA and seasonal changes in levels of the lectin and its mRNA in the inner bark ofRobinia pseudoacacia

A cDNA clone encoding a lectin was isolated by immunological screening of an expression library prepared from poly(A)⁺ RNA from the inner bark ofRobinia pseudoacacia. The cDNA clone (RBL104) had an open reading frame of 858 bp that encoded a polypeptide with a predicted molecular weight of 31210. This molecular weight corresponded closely to that of a polypeptide immunoprecipitated from products of translationin vitro of the poly(A)⁺ RNA. Thus, RBL104 appeared to be a full-length cDNA. The N-terminal amino acid sequence of the purified lectin protein matched a portion of the predicted amino acid sequence. It appeared that the lectin was synthesized as a precursor that consisted of a putative signal peptide of 31 amino acids and a mature polypeptide of 255 amino acids. Southern blot analysis of the genomic DNA revealed that the lectin was encoded by a small multigene family. The lectin was mostly localized in the axial and ray parenchymal cells of the inner bark. A small amount of lectin was also found in the axial and ray parenchymal cells of the xylem. The lectin accumulated in the inner bark in September, remained at high levels during the winter and disappeared in May. The mRNA for the lectin was detected from August to the following March. The appearance and disappearance of the mRNA were observed prior to those of the lectin protein.     

Cathepsin D interacts with adenosine A2A receptors in mouse macrophages to modulate cell surface localization and inflammatory signaling

Adenosine A_2A receptor (A_2AR)–dependent signaling in macrophages plays a key role in the regulation of inflammation. However, the processes regulating A_2AR targeting to the cell surface and degradation in macrophages are incompletely understood. For example, the C-terminal domain of the A_2AR and proteins interacting with it are known to regulate receptor recycling, although it is unclear what role potential A_2AR-interacting partners have in macrophages. Here, we aimed to identify A_2AR-interacting partners in macrophages that may effect receptor trafficking and activity. To this end, we performed a yeast two-hybrid screen using the C-terminal tail of A_2AR as the “bait” and a macrophage expression library as the “prey.” We found that the lysosomal protease cathepsin D (CtsD) was a robust hit. The A_2AR–CtsD interaction was validated in vitro and in cellular models, including RAW 264.7 and mouse peritoneal macrophage (IPMΦ) cells. We also demonstrated that the A_2AR is a substrate of CtsD and that the blockade of CtsD activity increases the density and cell surface targeting of A_2AR in macrophages. Conversely, we demonstrate that A_2AR activation prompts the maturation and enzymatic activity of CtsD in macrophages. In summary, we conclude that CtsD is a novel A_2AR-interacting partner and thus describe molecular and functional interplay that may be crucial for adenosine-mediated macrophage regulation in inflammatory processes.     

A new chitooligosaccharide specific lectin from snake gourd (Trichosanthes anguina) phloem exudate. Purification, physico-chemical characterization and thermodynamics of saccharide binding

A new lectin has been purified to homogeneity from the phloem exudate of snake gourd (Trichosanthes anguina) by affinity chromatography on chitin. The snake gourd phloem lectin (SGPL) specifically binds chitooligosaccharides and their inhibitory potency increased with increase in size. PAGE and SDS-PAGE studies indicate that SGPL is a heterodimer, in which the two subunits (48 and 53 kDa) are joined by disulfide bonds. Consistent with this, electrospray-ionization mass spectrum yielded the exact mass of the protein as 104,621.8 Daltons. CD studies showed that SGPL contains about 9% α-helix, 39% β-sheet, 20% β-turns and 32% unordered structures and that saccharide binding does not significantly affect its secondary and tertiary structures. Titration calorimetric studies indicate that the dimeric lectin binds two ligand molecules [(GlcNAc)_3–6] with association constants determined at 25 °C being 1.7 × 10⁵ and 3.6 × 10⁵ M⁻¹, for chitotriose and chitohexaose, respectively. Binding of all the chitooligosaccharides is governed by enthalpic forces, whereas the contribution from binding entropies was unfavorable. These results suggest that the SGPL-saccharide interaction is stabilized by hydrogen bonding and van der Waals’ interactions. Enthalpy–entropy compensation was observed for the SGPL-chitooligosaccharide interaction, suggesting that water molecules play a key role in the binding process.     

A viral lectin encoded in Cotesia plutellae bracovirus and its immunosuppressive effect on host hemocytes

An endoparasitoid wasp, Cotesia plutellae, induces immunosuppression of the host diamondback moth, Plutella xylostella. To identify an immunosuppressive factor, the parasitized hemolymph of P. xylostella was separated into plasma and hemocyte fractions. When nonparasitized hemocytes were overlaid with parasitized plasma, they showed significant reduction in bacterial binding efficacy. Here, we considered a viral lectin previously known in other Cotesia species as a humoral immunosuppressive candidate in C. plutellae parasitization. Based on consensus regions of the viral lectins, the corresponding lectin gene was cloned from P. xylostella parasitized by C. plutellae. Its cDNA is 674 bp long and encodes 157 amino acid residues containing a signal peptide (15 residues) and one carbohydrate recognition domain. Open reading frame is divided by one intron (156 bp) in its genomic DNA. Amino acid sequence shares 80% homology with that of C. ruficrus bracovirus lectin and is classified into C-type lectin. Southern hybridization analysis indicated that the cloned lectin gene was located at C. plutellae bracovirus (CpBV) genome. Both real-time quantitative RT-PCR and immunoblotting assays indicated that CpBV-lectin showed early expression during the parasitization. A recombinant CpBV-lectin was expressed in a bacterial system and the purified protein significantly inhibited the association between bacteria and hemocytes of nonparasitized P. xylostella. In the parasitized P. xylostella, CpBV-lectin was detected on the surface of parasitoid eggs after 24 h parasitization by its specific immunostaining. The 24 h old eggs were not encapsulated in vitro by hemocytes of P. xylostella, compared to newly laid parasitoid eggs showing no CpBV-lectin detectable and easily encapsulated. These results support an existence of a polydnaviral lectin family among Cotesia-associated bracovirus and propose its immunosuppressive function.     

Role of glycosylation in structure and stability of Erythrina corallodendron lectin (EcorL): a molecular dynamics study

The effect of glycosylation on structure and stability of glycoproteins has been a topic of considerable interest. In this work, we have investigated the solution conformation of the oligosaccharide and its effect on the structure and stability of the glycoprotein by carrying out a series of long Molecular dynamics (MD) simulations on glycosylated Erythrina corallodendron lectin (EcorL) and nonglycosylated recombinant Erythrina corallodendron lectin (rEcorL). Our results indicate that, despite the similarity in overall three dimensional structures, glycosylated EcorL has lesser nonpolar solvent accessible surface area compared to nonglycosylated EcorL. This might explain the experimental observation of higher thermodynamic stability for glycosylated EcorL compared to nonglycosylated EcorL. Analysis of the simulation results indicates that, dynamic view of interactions between protein residues and oligosaccharide is entirely different from the static picture seen in the crystal structure. The oligosaccharide moiety had dynamically stable interactions with Lys 55 and Tyr 53, both of which are separated in sequence from the site of glycosylation, Asn 17. It is possible that glycosylation helps in forming long-range contacts between amino acids, which are separated in sequence and thus provides a folding nucleus. Thus our simulations not only reveal the conformations sampled by the oligosaccharide, but also provide novel insights into possible molecular mechanisms by which glycosylation can help in folding of the glycoprotein by formation of folding nucleus involving specific contacts with the oligosaccharide moiety.     

Synchronous modulation of cell surface lectin and its receptor in a homologous cell population: a novel mechanism of cellular regulation

Testicular immotile sperm undergo maturation during epididymal transit when these cells pass through caput, corpus, and cauda-epididymal regions. Maturing goat spermatozoa specifically at the distal corpus epididymal stage show head-to-head autoagglutination when incubated in vitro in a modified Ringer's solution. Here, we show the biochemical mechanism of autoagglutination event and its functional significance. A lectin-like molecule located on sperm surface specifically interacts with its receptor of the neighboring homologous cells to cause autoagglutination. Lectin is a Ca++-dependent galactose-specific protein. Failure of the pre- and post-distal corpus sperm to show autoagglutination is due to lack of lectin-like molecule and its receptors, respectively. Maturing sperm at distal corpus stage acquire lectin-like molecule followed by sharp disappearance of its receptor, and this event is synchronously associated with the initiation of sperm forward motility that is essential for fertilization in vivo. Lectin and its receptor isolated from sperm plasma membrane showed high efficacy for blocking autoagglutination phenomenon. The data are consistent with the view that synchronous modulation of homologous cell surface lectin and their receptors constitutes a novel mechanism for cellular regulation by generating waves of signals by manipulating lectin-sugar-dependent "self-talk" and cell-cell "cross-talk".     

Matrilysin (MMP‐7) cleaves C‐type lectin domain family 3 member A (CLEC3A) on tumor cell surface and modulates its cell adhesion activity

Matrilysin (MMP‐7) plays important roles in tumor progression. Previous studies have suggested that MMP‐7 binds to tumor cell surface and promotes their metastatic potential. In this study, we identified C‐type lectin domain family 3 member A (CLEC3A) as a membrane‐bound substrate of MMP‐7. Although this protein is known to be expressed specifically in cartilage, its message was found in normal breast and breast cancer tissues as well as breast and colon cancer cell lines. Because few studies have been done on CLEC3A, we overexpressed its recombinant protein in human cancer cells. CLEC3A was found in the cell membrane, extracellular matrix (ECM), and culture medium of the CLEC3A‐expressing cells. CLEC3A has a basic sequence in the NH₂‐terminal domain and showed a strong heparin‐binding activity. MMP‐7 cleaved the 20‐kDa CLEC3A protein, dividing it to a 15‐kDa COOH‐terminal fragment and an NH₂‐terminal fragment with the basic sequence. The 15‐kDa fragment no longer had heparin‐binding activity. Treatment of the CLEC3A‐expressing cells with MMP‐7 released the 15‐kDa CLEC3A into the culture supernatant. Furthermore, the 20‐kDa CLEC3A promoted cell adhesion to laminin‐332 and fibronectin substrates, but this activity was abrogated by the cleavage by MMP‐7. These results suggest that CLEC3A binds to heparan sulfate proteoglycans on cell surface, leading to the enhancement of cell adhesion to integrin ligands on ECM. It can be speculated that the cleavage of CLEC3A by MMP‐7 weakens the stable adhesion of tumor cells to the matrix and promotes their migration in tumor microenvironments. J. Cell. Biochem. 106: 693–702, 2009. © 2009 Wiley‐Liss, Inc.