Phosphorylation of the beta-galactoside-binding protein galectin-3 modulates binding to its ligands

The beta-galactoside-binding protein galectin-3 has pleiotropic biological functions and has been implicated in cell growth, differentiation, adhesion, RNA processing, apoptosis, and malignant transformation. Galectin-3 may be phosphorylated at N-terminal Ser(6), but the role of phosphorylation in determining interactions of this endogenous lectin with its ligands remains to be elucidated. We therefore studied the effect of phosphorylation on binding of galectin-3 to two of its reported ligands, laminin and purified colon cancer mucin. Human recombinant galectin-3 was phosphorylated in vitro by casein kinase I, and separated from the native species by isoelectric focusing for use in solid phase binding assays. Non-phosphorylated galectin-3 bound to laminin and asialomucin in a dose-dependent manner with half-maximal binding at 1.5 microg/ml. Phosphorylation reduced saturation binding to each ligand by >85%. Ligand binding could be fully restored by dephosphorylation with protein phosphatase type 1. Mutation of galectin-3 at Ser(6) (Ser to Glu) did not alter galectin ligand binding. Metabolic labeling or separation by isoelectric focusing confirmed the presence of phosphorylated galectin-3 species in vivo in the cytosol of human colon cancer cells from which ligand mucin was purified. Phosphorylation significantly reduces the interaction of galectin-3 with its ligands. The process by which phosphorylation modulates protein-carbohydrate interactions has important implications for understanding the biological functions of this protein, and may serve as an "on/off" switch for its sugar binding capabilities.     

Binding of urokinase-type plasminogen activator receptor (uPAR) to the mannose 6-phosphate/insulin-like growth factor II receptor: contrasting interactions of full-length and soluble forms of uPAR

Urokinase-type plasminogen activator receptor (uPAR) binding by the mannose 6-phosphate/insulin-like growth factor II receptor (Man-6-P/IGF2R) is considered important to Man-6-P/IGF2R tumor suppressor function via regulation of cell surface proteolytic activity. Our goal was to map the uPAR binding site of the Man-6-P/IGF2R by analyzing the uPAR binding characteristics of a panel of minireceptors containing different regions of the Man-6-P/IGF2R extracytoplasmic domain. Coimmunoprecipitation assays revealed that soluble recombinant uPAR (suPAR) bound the Man-6-P/IGF2R at two distinct sites, one localized to the amino-terminal end of the Man-6-P/IGF2R extracytoplasmic domain (repeats 1-3) and the other to the more carboxyl-terminal end (repeats 7-9). These sites correspond with the positions of the two Man-6-P binding domains of Man-6-P/IGF2R. Indeed, the suPAR-Man-6-P/IGF2R interaction was inhibited by Man-6-P, and binding-competent su-PAR species represented a minor percentage (8-30%) of the suPAR present. In contrast, Man-6-P/IGF2R binding of endogenous, full-length uPAR solubilized from plasma membranes of the prostate cancer cell line, PC-3, was not inhibited by Man-6-P. Further studies showed that very little (<5%) endogenous uPAR was Man-6-P/IGF2R binding-competent. We conclude that, contrary to previous reports, the interaction between uPAR and Man-6-P/IGF2R is a low percentage binding event and that suPAR and full-length uPAR bind the Man-6-P/IGF2R by different mechanisms.     

Solution structure of interleukin-13 and insights into receptor engagement.

The complex and interrelated function of the interleukin cytokines relies on a range of pro-inflammatory and anti-inflammatory immune responses mediated by an array of receptors, and there is considerable cross-reactivity for related cytokines. Recent findings continue to elucidate the expression patterns of interleukin receptors associated with a range of diseases, including cancer. We report here the first experimentally determined high-resolution structure of human interleukin-13 (IL-13). The experimental structure is significantly different from an earlier homology model, which could have led to improper estimation of receptor interaction surfaces and design of mutational experiments. Similarities between the presented IL-13 structure and the homologous interleukin-4 (IL-4) are discussed. Additionally, mutation data for IL-4 and IL-13 are analyzed and combined with a detailed structural analysis of the IL-4/IL4Ralpha interface that leads us to postulate interactions at the IL-13/receptor interface. The structural comparison is used to interpret the different affinities for various receptors and establishes the basis for further mutational experiments and antagonist design.     

Stimulation of allergen-loaded macrophages by TLR9-ligand potentiates IL-10-mediated suppression of allergic airway inflammation in mice

BackgroundPreviously, we demonstrated that OVA-loaded macrophages (OVA-Mφ) partially suppress OVA-induced airway manifestations of asthma in BALB/c mice. In vitro studies showed that OVA-Mφ start to produce IL-10 upon interaction with allergen-specific T cells, which might mediate their immunosuppressive effects. Herein, we examined whether IL-10 is essential for the immunosuppressive effects of OVA-Mφ in vivo, and whether ex vivo stimulation of the IL-10 production by OVA-Mφ could enhance these effects.MethodsPeritoneal Mφ were loaded with OVA and stimulated with LPS or immunostimulatory sequence oligodeoxynucleotide (ISS-ODN) in vitro. The increase of IL-10 production was examined and, subsequently, ex vivo stimulated OVA-Mφ were used to treat (i.v.) OVA-sensitized mice. To further explore whether Mφ-derived IL-10 mediates the immunosuppressive effects, Mφ isolated from IL-10^-/- mice were used for treatment.ResultsWe found that stimulation with LPS or ISS-ODN highly increased the IL-10 production by OVA-Mφ (2.5-fold and 4.5-fold increase, respectively). ISS-ODN stimulation of OVA-Mφ significantly potentiated the suppressive effects on allergic airway inflammation. Compared to sham-treatment, ISS-ODN-stimulated OVA-Mφ suppressed the airway eosinophilia by 85% (vs. 30% by unstimulated OVA-Mφ), IL-5 levels in bronchoalveolar lavage fluid by 80% (vs. 50%) and serum OVA-specific IgE levels by 60% (vs. 30%). Importantly, IL-10^-/-Mφ that were loaded with OVA and stimulated with ISS-ODN ex vivo, failed to suppress OVA-induced airway inflammation.ConclusionsThese results demonstrate that Mφ-derived IL-10 mediates anti-inflammatory responses in a mouse model of allergic asthma, which both can be potentiated by stimulation with ISS-ODN.     

Leaf cavity CO2 concentrations and CO2 exchange in onion,Allium cepa L.

Onion (Allium cepa L.) plants were examined to determine the photosynthetic role of CO₂ that accumulates within their leaf cavities. Leaf cavity CO₂ concentrations ranged from 2250 L L^–1 near the leaf base to below atmospheric (<350 L L^–1) near the leaf tip at midday. There was a daily fluctuation in the leaf cavity CO₂ concentrations with minimum values near midday and maximum values at night. Conductance to CO₂ from the leaf cavity ranged from 24 to 202 mol m^–2 s^–1 and was even lower for membranes of bulb scales. The capacity for onion leaves to recycle leaf cavity CO₂ was poor, only 0.2 to 2.2% of leaf photosynthesis based either on measured CO₂ concentrations and conductance values or as measured directly by ¹⁴CO₂ labeling experiments. The photosynthetic responses to CO₂ and O₂ were measured to determine whether onion leaves exhibited a typical C₃-type response. A linear increase in CO₂ uptake was observed in intact leaves up to 315 L L^–1 of external CO₂ and, at this external CO₂ concentration, uptake was inhibited 35.4±0.9% by 210 mL L^–1 O₂ compared to 20 mL L^–1 O₂. Scanning electron micrographs of the leaf cavity wall revealed degenerated tissue covered by a membrane. Onion leaf cavity membranes apparently are highly impermeable to CO₂ and greatly restrict the refixation of leaf cavity CO₂ by photosynthetic tissue.Abbreviations C_a external CO₂ concentration - C_i intercellular CO₂ concentration - CO₂ compensation concentration - PPFR photosynthetic photon fluence rate