Lycopersicon esculentum lectin is a marker of transient amplifying cells in in vitro cultures of isolated limbal stem cells

The maintenance of a healthy corneal epithelium under both normal and wound healing conditions is achieved by a population of stem cells (SCs) located in the basal epithelium at the corneoscleral limbus. In the light of the development of strategies for reconstruction of the ocular surface in patients with limbal stem cell deficiency, a major challenge in corneal SCs biology remains the ability to identify stem cells in situ and in vitro. To date, not so much markers exist for the identification of different phenotypes. CESCs (corneal epithelial stem cells) isolated from limbal biopsies were maintained in primary culture for 14 days and stained with Hoechst and a panel of FITC-conjugated lectins. All lectins, with the exception of Lycopersicon esculentum, labelled CESCs irrespective of the degree of differentiation. Lycopersicon esculentum, that binds N-acetylglucosamine oligomers, labelled intensely only the surface of TACs (single corneal epithelial stem cells better than colonial cells). These results suggest that Lycopersicon esculentum lectin is a useful and easy-to-use marker for the in vitro identification of TACs (transient amplifying cells) in cultures of isolated CESCs.     

Insecticidal properties of Sclerotinia sclerotiorum agglutinin and its interaction with insect tissues and cells

This project studied in detail the insecticidal activity of a fungal lectin from the sclerotes of Sclerotinia sclerotiorum, referred to as S. sclerotiorum agglutinin or SSA. Feeding assays with the pea aphid (Acyrthosiphon pisum) on an artificial diet containing different concentrations of SSA demonstrated a high mortality caused by this fungal lectin with a median insect toxicity value (LC₅₀) of 66 (49–88) μg/ml. In an attempt to unravel the mode of action of SSA the binding and interaction of the lectin with insect tissues and cells were investigated. Histofluorescence studies on sections from aphids fed on an artificial liquid diet containing FITC-labeled SSA, indicated the insect midgut with its brush border zone as the primary target for SSA. In addition, exposure of insect midgut CF-203 cells to 25 μg/ml SSA resulted in a total loss of cell viability, the median cell toxicity value (EC₅₀) being 4.0 (2.4–6.7) μg/ml. Interestingly, cell death was accompanied with DNA fragmentation, but the effect was caspase-3 independent. Analyses using fluorescence confocal microscopy demonstrated that FITC-labeled SSA was not internalized in the insect midgut cells, but bound to the cell surface. Prior incubation of the cells with saponin to achieve a higher cell membrane permeation resulted in an increased internalization of SSA in the insect midgut cells, but no increase in cell toxicity. Furthermore, since the toxicity of SSA for CF-203 cells was significantly reduced when SSA was incubated with GalNAc and asialomucin prior to treatment of the cells, the data of this project provide strong evidence that SSA binds with specific carbohydrate moieties on the cell membrane proteins to start a signaling transduction cascade leading to death of the midgut epithelial cells, which in turn results in insect mortality. The potential use of SSA in insect control is discussed.     

Identification of Novel Contributions to High-affinity Glycoprotein-Receptor Interactions using Engineered Ligands

Engineered receptor fragments and glycoprotein ligands employed in different assay formats have been used to dissect the basis for the dramatic enhancement of binding of two model membrane receptors, dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) and the macrophage galactose lectin, to glycoprotein ligands compared to simple sugars. These approaches make it possible to quantify the importance of two major factors that combine to enhance the affinity of single carbohydrate-recognition domains (CRDs) for glycoprotein ligands by 100-to 300-fold. First, the presence of extended binding sites within a single CRD can enhance interaction with branched glycans, resulting in increases of fivefold to 20-fold in affinity. Second, presentation of glycans on a glycoprotein surface increases affinity by 15-to 20-fold, possibly due to low-specificity interactions with the surface of the protein or restriction in the conformation of the glycans. In contrast, when solution-phase networking is avoided, enhancement due to binding of multiple branches of a glycan to multiple CRDs in the oligomeric forms of these receptors is minimal and binding of a receptor oligomer to multiple glycans on a single glycoprotein makes only a twofold contribution to overall affinity. Thus, in these cases, multivalent interactions of individual glycoproteins with individual receptor oligomers have a limited role in achieving high affinity. These findings, combined with considerations of membrane receptor geometry, are consistent with the idea that further enhancement of the binding to multivalent glycoprotein ligands requires interaction of multiple receptor oligomers with the ligands.     

BDCA-2 signaling inhibits TLR-9-agonist-induced plasmacytoid dendritic cell activation and antigen presentation

Plasmacytoid dendritic cells (PDCs) express Toll-like receptor (TLR) 9, which mediates recognition of microbial DNA during infection or self-DNA in autoimmune diseases. Triggering TLR-9 in PDC induces either maturation (lysosomal TLR-9 triggering) or type I interferon (IFN-I) production (endosomal TLR-9 triggering). PDCs also express BDCA-2 (CD303), a C-type lectin receptor (CLR) unique to these cells. CLRs appear to function in innate immunity and microbial recognition, and may cooperate with TLRs to fine-tune inflammatory responses.It has been shown that anti-BDCA-2 monoclonal antibody is internalized by PDC for antigen presentation and inhibits TLR-9 induced IFN-I expression. Here we investigated the cross-talk between BDCA-2 and TLR-9-signaling during PDC maturation and antigen presentation. We found that BDCA-2-induced signaling in PDCs inhibits up-regulation of CD86 and CD40 molecules in CpG-activated PDCs, but not in CD40L-activated PDCs. Furthermore, triggering of BDCA-2 diminished the ability of CpG- and CD40L-stimulated PDCs to process and present antigen to antigen-specific autologous memory T cells. This study demonstrates that BDCA-2 represents an attractive target for clinical immunotherapy of IFN-I dependent autoimmune diseases influencing both, IFN-I production and antigen-specific T-cell stimulation by PDC.     

Structural basis of carbohydrate transfer activity by human UDP-GalNAc: polypeptide alpha-N-acetylgalactosaminyltransferase (pp-GalNAc-T10)

Mucin-type O-glycans are important carbohydrate chains involved in differentiation and malignant transformation. Biosynthesis of the O-glycan is initiated by the transfer of N-acetylgalactosamine (GalNAc) which is catalyzed by UDP-GalNAc:polypeptide alpha-N-acetylgalactosaminyltransferases (pp-GalNAc-Ts). Here we present crystal structures of the pp-GalNAc-T10 isozyme, which has specificity for glycosylated peptides, in complex with the hydrolyzed donor substrate UDP-GalNAc and in complex with GalNAc-serine. A structural comparison with uncomplexed pp-GalNAc-T1 suggests that substantial conformational changes occur in two loops near the catalytic center upon donor substrate binding, and that a distinct interdomain arrangement between the catalytic and lectin domains forms a narrow cleft for acceptor substrates. The distance between the catalytic center and the carbohydrate-binding site on the lectin beta sub-domain influences the position of GalNAc glycosylation on GalNAc-glycosylated peptide substrates. A chimeric enzyme in which the two domains of pp-GalNAc-T10 are connected by a linker from pp-GalNAc-T1 acquires activity toward non-glycosylated acceptors, identifying a potential mechanism for generating the various acceptor specificities in different isozymes to produce a wide range of O-glycans.     

Transgenic rice expressing Allium sativum leaf lectin with enhanced resistance against sap-sucking insect pests

Mannose binding Allium sativum leaf agglutinin (ASAL) has been shown to be antifeedant and insecticidal against sap-sucking insects. In the present investigation, ASAL coding sequence was expressed under the control of CaMV35S promoter in a chimeric gene cassette containing plant selection marker, hpt and gusA reporter gene of pCAMBIA1301 binary vector in an elite indica rice cv. IR64. Many fertile transgenic plants were generated using scutellar calli as initial explants through Agrobacterium-mediated transformation technology. GUS activity was observed in selected calli and in mature plants. Transformation frequency was calculated to be ~12.1%±0.351 (mean ± SE). Southern blot analyses revealed the integration of ASAL gene into rice genome with a predominant single copy insertion. Transgene localization was detected on chromosomes of transformed plants using PRINS and C-PRINS techniques. Northern and western blot analyses determined the expression of transgene in transformed lines. ELISA analyses estimated ASAL expression up to 0.72 and 0.67% of total soluble protein in T₀ and T₁ plants, respectively. Survival and fecundity of brown planthopper and green leafhopper were reduced to 36% (P<0.01), 32% (P<0.05) and 40.5, 29.5% (P<0.001), respectively, when tested on selected plants in comparison to control plants. Specific binding of expressed ASAL to receptor proteins of insect gut was analysed. Analysis of T₁ progenies confirmed the inheritance of the transgenes. Thus, ASAL promises to be a potential component in insect resistance rice breeding programme.     

Molecular cloning,expression, and cytokinin (6-benzylaminopurine) antagonist activity of peanut (<Emphasis Type="Italic">Arachis hypogaea</Emphasis>) lectin SL-I

Isolation and purification of a α-methyl-mannoside specific lectin (SL-I) of peanut was reported earlier [Singh and Das (1994) Glycoconj J 11:282–285]. Native SL-I is a glycoprotein having ∼31 kDa subunit molecular mass and forms dimer. The gene encoding this lectin is identified from a 6-day old peanut root cDNA library by anti-SL-I antibody and N-terminal amino acid sequence homology to the native lectin. Nucleotide sequence derived amino acid sequence of the re-SL-I shows amino acid sequence homology with the N-terminal and tryptic digests’ amino acid sequence of the native SL-I (nSL-I). Presence of a putative glycosylation (QNPS) site and a hydrophobic adenine-binding (VLVSYDANS) site is also identified in SL-I. Homology modeling of the lectin suggests it to be an archetype of legume lectins. It is expressed as a ~30 kDa apoprotein in E. coli and has the carbohydrate specificity and secondary structure identical to its natural counterpart. The lectin SL-I inhibits cytokinin 6-benzylaminopurine (BA)-induced “delayed leaf senescence” and “cotyledon expansion”. Equilibrium dialysis revealed a single high-affinity binding site for adenine (7.6 × 10⁻⁶ M) and BA (1.09 × 10⁻⁵ M) in the SL-I dimer and thus suggesting that the cytokinin antagonist effect of SL-I is mediated by the direct interaction of SL-I with BA.The nucleotide sequence data reported here are available in the DDBJ/EMBL/GenBank databases under the Accession No. AJ585523     

Anti-HIV Ⅰ/Ⅱ Activity and Molecular Cloning of a Novel Mannose/Sialic Acidbinding Lectin from Rhizome of Polygonatum cyrtonema Hua

The anti-human immunodeficiency virus （HIV） Ⅰ/Ⅱ activity of a mannose and sialic acid binding lectin isolated from rhizomes of Polygonatum cyrtonema Hua was elucidated by comparing its HIV infection inhibitory activity in MT-4 and CEM cells with that of other mannose-binding lectins （MBLs）. The anti-HIV activity of Polygonatum cyrtonema Hua lectin （PCL） was 10- to 100-fold more potent than other tested MBLs, but without significant cytotoxicity towards MT-4 or CEM cells. To amplify cDNA of PCL by 3＇/5＇-rapid amplification of cDNA ends （RACE）, the 30 amino acids of N-terminal were determined by sequencing and the degenerate oligonucleotide primers were designed. The full-length cDNA of PCL contained 693 bp with an open reading frame encoding a precursor protein of 160 amino acid residues, consisting of a 28-residue signal peptide, a 22-residue C-terminal cleavage peptide and a 110-residue mature polypeptide which contained three tandemly arranged subdomains with an obvious sequence homology to the monocot MBL. However, only one active mannose-binding site （QDNVY） was found in subdomain Ⅰ of PCL, that of subdomain Ⅱ and Ⅲ changed to HNNVY and PDNVY, respectively. There was no intron in PCL, which was in good agreement with other monocot MBLs. Molecular modeling of PCL indicated that its three-dimensional structure resembles that of the snowdrop agglutinin. By docking, an active sialic acid-binding site was found in PCL. The instabilization of translation initiation region （TIR） in mRNA of PCL benefits its high expression in rhizomes.     

MBL调控MASP激活补体系统

甘露聚糖结合凝集素(MBL;或甘露聚糖结合蛋白,MBP)是由相同的多肽链组成的寡聚物,它通过结合细胞表面的碳水化合物能够有效地识别侵入体内的多种致病微生物,并激活补体来杀灭病原微生物。MBL能与血清中其相关蛋白酶(MASP)结合,MASP包含3个丝氨酸蛋白酶MASP-1、MASP-2、MASP-3和非酶蛋白MAp19。研究显示,MBL通过2种机理调控MASP-2的活性,在先天性免疫中具有重要的作用。本文简要综述MBL调控MASP激活补体的作用机理。