Prognostic Impact of Aberrantly Expressed Protein-coding Gene Associated With Gastric Cancer's Regulatory T Cells, Based on Online Databases.

Context: Gastric cancer (GC) remains one of the most prevalent malignancies worldwide, and no effective cure exists for advanced GC. Clinicians believe that molecularly targeted therapy through PCGs may replace surgery, radiotherapy, and other treatments as a breakthrough in curing malignancies. Objective: The study intended to examine the impact of aberrant expression of the protein-coding genes (PCGs) associated with regulatory T cells on the prognosis of patients with gastric cancer (GC). Design: The research team performed a genetic study through research of genetic data in online databases. Setting: The study took place at Zhongda Hospital. Outcome Measures: The research team selected a publicly available dataset, genetic suppressor element 109476 (GSE109476), from the Gene Expression Omnibus (GEO) database for differential gene analysis, gene ontology (GO) analysis, and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis to screen for PCGs associated with regulatory T cells as well as the Gene Expression Profiling Interactive Analysis (GEPIA) database with the Kaplan-Meier Plotter database to analyze the expression of the above PCGs in GC and the prognostic impact on GC. Results: The GEO2R analysis found 315 differentially expressed PCGs in GSE109476, among which nine PCGs were associated with regulatory T cells: (1) chemokine (C-C motif) ligand 19 (CCL19), (2) CCL21, (3) C-C chemokine receptor type 7 (CCR7), (4) cluster of differentiation 70 (CD70), (5) ephrin B3 (EFNB3), (6) early growth response 3 (EGR3), (7) interleukin-7 receptor (IL7R), (8) galectin-1 (LGALS1), and (9) tumor necrosis factor (TNF) receptor superfamily member 13C (TNFRSF13C). The GEPIA database indicated that no significant differences existed between the expression of CCL19, CCL21, CD70, EFNB3, EGR3, IL7R, and TNFRSF13C in stomach adenocarcinoma (STAD) tissues and that in normal tissues (P > .05), while expressions of CCR7 and LGALS1 were significantly elevated in STAD tissues compared to the normal tissues (P < .05). The Kaplan-Meier Plotter database analysis, on the other hand, showed a significant relationship between all of the above-mentioned PCGs, except CCL19, and the prognosis of GC. Conclusions: CCL19, CCL21, CCR7, CD70, EFNB3, EGR3, IL7R, LGALS1, and TNFRSF13C are PCGs are differentially expressed in GC and closely associated with regulatory T cells. They may affect the occurrence and development of GC through a variety of pathways, including regulation of immune infiltration and inflammation, and are of great potential research value.

Bruceine A protects against diabetic kidney disease via inhibiting galectin-1.

Bruceine A is a natural quassinoid compound extracted from the fruit of the Traditional Chinese Medicine Brucea javanica (L.) Merr. that has various types of various biological activities. However, whether the compound has a protective effect on diabetic kidney disease remains unknown. Galectin-1 is actively involved in a variety of chronic inflammation-relevant human diseases including diabetic kidney disease. Here, we identified Bruceine A as a kidney protective molecule against a model of diabetic kidney disease in db/db mice with potent anti-inflammatory activity both in vitro and in vivo. Mechanistically, by selectively binding to the conserved carbohydrate-recognition domain of galectin-1 and disrupting the interaction between galectin-1 and the receptor for activated protein C kinase 1, Bruceine A was found to inhibit galectin-1-mediated inflammatory signal transduction under high glucose stress in rat mesangial HBZY-1 cells. Thus, our findings reveal Bruceine A as an unidentified galectin-1 inhibitor affording significant protection against diabetic kidney disease and may provide novel pharmacological therapeutics for the disease.

Galectin-1 promotes hepatocellular carcinoma and the combined therapeutic effect of OTX008 galectin-1 inhibitor and sorafenib in tumor cells.

BACKGROUND: Galectins are beta-galactose specific binding proteins. In human cancers, including hepatocellular carcinoma (HCC), galectin-1 (Gal-1) is often found to be overexpressed. In order to combat the dismal diagnosis and death rates of HCC, gene silencing and targeted inhibition of Gal-1 was investigated for its improved therapeutic potential. METHODS: Cellular and secretory Gal-1 levels were analyzed using HCC clinical samples. The study of Gal-1 was carried by both knockdown and overexpression approaches. The stable clones were tested by in vitro assays and in vivo experiments. Mass spectrometry was used to identify downstream targets of Gal-1. The upstream regulator of Gal-1, microRNA-22 (miR-22) was characterized by functional assays. The therapeutic effect of inhibiting Gal-1 was also analyzed. RESULTS: Gal-1 overexpression was observed in HCC and correlated with aggressive clinicopathological features and poorer survival. The loss of Gal-1 resulted in hindered cell migration, invasion and anchorage independent growth. This was also observed in the animal models, in that when Gal-1 was knocked down, there were fewer lung metastases. Proteomic profiling of control and Gal-1 knockdown cells identified that the level of retention in endoplasmic reticulum 1 (RER1) was suppressed when Gal-1 level was reduced. The cell motility of Gal-1 knockdown cells was enhanced upon the rescue of RER1 expression. In HCC tissues, Gal-1 and RER1 expressions displayed a significant positive correlation. The upstream regulator of Gal-1, miR-22 was observed to be underexpressed in HCC tissues and negatively correlated with Gal-1. Silencing of miR-22 resulted in the upregulation of Gal-1 and enhanced cell growth, migration and invasion. However, such enhancement was abolished in cells treated with OTX008, an inhibitor of Gal-1. Combinational treatment of OTX008 and sorafenib significantly reduced tumor growth and size. CONCLUSIONS: Gal-1 overexpression was detected in HCC and this played a role in promoting tumorigenic processes and metastasis. The function of Gal-1 was found to be mediated through RER1. The correlations between miR-22, Gal-1 and RER1 expressions demonstrated the importance of miR-22 regulation on Gal-1/RER1 oncogenic activity. Lastly, the combinational treatment of OTX008 and sorafenib proved to be an improved therapeutic option compared to when administering sorafenib alone.

Evaluation of galectin-1 and galectin-3 as prospective biomarkers in keratoconus.

AIMS: To evaluate the expression of ß-galactoside-binding proteins galectin (Gal)-1 and Gal-3 in patients with keratoconus (KC) and postcorneal collagen cross-linking (CXL) treatment in vitro. METHODS: Tear fluid, cornea samples and conjunctival impression cytology specimens from control and KC patients were used to evaluate Gal-1 and Gal-3 expressions. Primary keratocytes were isolated by collagenase digestion from surgically removed corneas of five normal or KC human corneal buttons and cultured in Dulbecco's modified eagle medium/Ham's F12 medium supplemented with 2% fetal bovine serum. These cells were evaluated under two experimental conditions: control and submitted to the application of ultraviolet A light and riboflavin 0.1% (CXL) for 30 min. RESULTS: Patients with KC displayed increased levels of Gal-1 and Gal-3 in conjunctival epithelial cells compared with control. Furthermore, KC corneas were associated with intense expression of Gal-1 in the stroma, released by keratocytes. Ultrastructural analysis of keratocytes showed a marked increase of endogenous Gal-3 levels, but not Gal-1, in KC. In vitro, CXL induced significant release of Gal-1 in keratocyte supernatants (116±18 ng/mL, P<0.05) and decreased inflammatory biomarkers as interleukin (IL)-6, IL-8, matrix metalloproteinase (MMP)-2 and MMP-9. Gal-3 levels were not detected in the keratocyte supernatants. CONCLUSION: Gal-1 and Gal-3 represent new interesting KC biomarkers as revealed by their different expression patterns in KC and control corneal samples. CXL has an immunosuppressive effect on keratocytes by reducing the release of cytokines and MMPs and increased expression of anti-inflammatory protein Gal-1.

Galectin-1-based tumour-targeting for gold nanostructure-mediated photothermal therapy.

PURPOSE: To demonstrate delivery of Au nanocages to cells using the galectin-1 binding peptide anginex (Ax) and to demonstrate the value of this targeting for selective in vitro photothermal cell killing. MATERIALS AND METHODS: Au nanocages were synthesised, coated with polydopamine (PDA), and conjugated with Ax. Tumour and endothelial cell viability was measured with and without laser irradiation. Photoacoustic (PA) mapping and PA flow cytometry were used to confirm cell targeting in vitro and in tissue slices ex vivo. RESULTS: Cell viability was maintained at ≥50% at 100 pM suggesting low toxicity of the nanocage alone. Combining the targeted construct (25 pM) with low power 808 nm laser irradiation for 10-20 min (a duration previously shown to induce rapid and sustained heating of Au nanocages [AuNC] in solution), resulted in over 50% killing of endothelial and tumour cells. In contrast, the untargeted construct combined with laser irradiation resulted in negligible cell killing. We estimate approximately 6 × 104 peptides were conjugated to each nanocage, which also resulted in inhibition of cell migration. Binding of the targeted nanocage reached a plateau after three hours, and cell association was 20-fold higher than non-targeted nanocages both in vitro and ex vivo on tumour tissue slices. A threefold increase in tumour accumulation was observed in preliminary in vivo studies. CONCLUSIONS: These studies demonstrate Ax's potential as an effective targeting agent for Au-based theranostics to tumour and endothelial cells, enabling photothermal killing. This platform further suggests potential for multimodal in vivo therapy via next-generation drug-loaded nanocages.

Immunosensor for the ultrasensitive and quantitative detection of bladder cancer in point of care testing.

An ultrasensitive and real-time impedance based immunosensor has been fabricated for the quantitative detection of Galectin-1 (Gal-1) protein, a biomarker for the onset of multiple oncological conditions, especially bladder cancer. The chip consists of a gold annular interdigitated microelectrode array (3×3 format with a sensing area of 200µm) patterned using standard microfabrication processes, with the ability to electrically address each electrode individually. To improve sensitivity and immobilization efficiency, we have utilized nanoprobes (Gal-1 antibodies conjugated to alumina nanoparticles through silane modification) that are trapped on the microelectrode surface using programmable dielectrophoretic manipulations. The limit of detection of the immunosensor for Gal-1 protein is 0.0078mg/ml of T24 (Grade III) cell lysate in phosphate buffered saline, artificial urine and human urine samples. The normalized impedance variations show a linear dependence on the concentration of cell lysate present while specificity is demonstrated by comparing the immunosensor response for two different grades of bladder cancer cell lysates. We have also designed a portable impedance analyzing device to connect the immunosensor for regular checkup in point of care testing with the ability to transfer data over the internet using a personal computer. We believe that this diagnostic system would allow for improved public health monitoring and aid in early cancer diagnosis.

Integrated Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) and Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) Quantitative Proteomic Analysis Identifies Galectin-1 as a Potential Biomarker for Predicting Sorafenib Resistance in Liver Cancer.

Sorafenib has become the standard therapy for patients with advanced hepatocellular carcinoma (HCC). Unfortunately, most patients eventually develop acquired resistance. Therefore, it is important to identify potential biomarkers that could predict the efficacy of sorafenib. To identify target proteins associated with the development of sorafenib resistance, we applied stable isotope labelling with amino acids in cell culture (SILAC)-based quantitative proteomic approach to analyze differences in protein expression levels between parental HuH-7 and sorafenib-acquired resistance HuH-7 (HuH-7(R)) cells in vitro, combined with an isobaric tags for relative and absolute quantitation (iTRAQ) quantitative analysis of HuH-7 and HuH-7(R) tumors in vivo. In total, 2,450 quantified proteins were identified in common in SILAC and iTRAQ experiments, with 81 showing increased expression (>2.0-fold) with sorafenib resistance and 75 showing decreased expression (<0.5-fold). In silico analyses of these differentially expressed proteins predicted that 10 proteins were related to cancer with involvements in cell adhesion, migration, and invasion. Knockdown of one of these candidate proteins, galectin-1, decreased cell proliferation and metastasis in HuH-7(R) cells and restored sensitivity to sorafenib. We verified galectin-1 as a predictive marker of sorafenib resistance and a downstream target of the AKT/mTOR/HIF-1α signaling pathway. In addition, increased galectin-1 expression in HCC patients' serum was associated with poor tumor control and low response rate. We also found that a high serum galectin-1 level was an independent factor associated with poor progression-free survival and overall survival. In conclusion, these results suggest that galectin-1 is a possible biomarker for predicting the response of HCC patients to treatment with sorafenib. As such, it may assist in the stratification of HCC and help direct personalized therapy.

Modulation of ionotropic glutamate receptor function by vertebrate galectins.

AMPA and kainate receptors are glutamate-gated ion channels whose function is known to be altered by a variety of plant oligosaccharide-binding proteins, or lectins, but the physiological relevance of this activity has been uncertain because no lectins with analogous allosteric modulatory effects have been identified in animals. We report here that members of the prototype galectin family, which are ß-galactoside-binding lectins, exhibit subunit-specific allosteric modulation of desensitization of recombinant homomeric and heteromeric AMPA and kainate receptors. Galectin modulation of GluK2 kainate receptors was dependent upon complex oligosaccharide processing of N-glycosylation sites in the amino-terminal domain and downstream linker region. The sensitivity of GluA4 AMPA receptors to human galectin-1 could be enhanced by supplementation of culture media with uridine and N-acetylglucosamine (GlcNAc), precursors for the hexosamine pathway that supplies UDP-GlcNAc for synthesis of complex oligosaccharides. Neuronal kainate receptors in dorsal root ganglia were sensitive to galectin modulation, whereas AMPA receptors in cultured hippocampal neurons were insensitive, which could be a reflection of differential N-glycan processing or receptor subunit selectivity. Because glycan content of integral proteins can be modified dynamically, we postulate that physiological or pathological conditions in the CNS could arise in which galectins alter excitatory neurotransmission or neuronal excitability through their actions on AMPA or kainate receptors.

Galectin-1 is an interactive protein of selenoprotein M in the brain.

Selenium, an essential trace element for human health, mainly exerts its biological function through selenoproteins. Selenoprotein M (SelM) is one of the highly expressed selenoproteins in the brain, but its biological effect and molecular mechanism remain unclear. Thus, the interactive protein of SelM was investigated in this paper to guide further study. In order to avoid protein translational stop, the selenocysteine-encoding UGA inside the open reading frame of SelM was site-directly changed to the cysteine-encoding UGC to generate the SelM' mutant. Meanwhile, its N terminal transmembrane signal peptide was also cut off. This truncated SelM' was used to screen a human fetal brain cDNA library by the yeast two-hybrid system. A new interactive protein of SelM' was found to be galectin-1 (Gal-1). This protein-protein interaction was further verified by the results of fluorescence resonance energy transfer techniques, glutathione S-transferase pull-down and co-immunoprecipitation assays. As Gal-1 plays important roles in preventing neurodegeneration and promoting neuroprotection in the brain, the interaction between SelM' and Gal-1 displays a new direction for studying the biological function of SelM in the human brain.

Development of a microarray detection method for galectin cancer proteins based on ligand binding.

In this article, we describe the development of a novel detection method for the visualization of ligand-binding proteins. Current proteomic tools, such as the enzyme-linked immunosorbent assay (ELISA), are based on protein abundance rather than protein activity and can result in conflicting data. To address this issue, we developed an assay in which ligand binding is detected using a microarray approach with immobilized antibodies on a porous aluminum oxide matrix. The galectin family of proteins was used as a model system to evaluate the performance of this approach. Galectins selectively bind galactosides and are linked to cancer progression. Our assay employed antibodies directed against different galectins. The antibodies were immobilized on the microarray surface by use of protein A/G. In our example, galectin-1 and galectin-9 were then detected in cell lysates. Lysates were exposed to the anti-galectin surface, followed by washing and quantification with a general fluorescent galectin ligand. The optimal galectin ligand allowed detection of nanogram amounts of galectin using only 1 µg of antibody. Galectin-1 was visualized in HeLa and tumor cell lysates, indicating the potential of the method for a clinical setting.

Atropa belladonna L. water extract: modulator of extracellular matrix formation in vitro and in vivo.

Previously, we found that treatment of cutaneous wounds with Atropa belladonna L. (AB) revealed shortened process of acute inflammation as well as increased tensile strength and collagen deposition in healing skin wounds (Gál et al. 2009). To better understand AB effect on skin wound healing male Sprague-Dawley rats were submitted to one round full thickness skin wound on the back. In two experimental groups two different concentrations of AB extract were daily applied whereas the control group remained untreated. For histological evaluation samples were removed on day 21 after surgery and stained for wide spectrum cytokeratin, collagen III, fibronectin, galectin-1, and vimentin. In addition, in the in vitro study different concentration of AB extract were used to evaluate differences in HaCaT keratinocytes proliferation and differentiation by detection of Ki67 and keratin-19 expressions. Furthermore, to assess ECM formation of human dermal fibroblasts on the in vitro level fibronectin and galectin-1 were visualized. Our study showed that AB induces fibronectin and galectin-1 rich ECM formation in vitro and in vivo. In addition, the proliferation of keratinocytes was also increased. In conclusion, AB is an effective modulator of skin wound healing. Nevertheless, further research is needed to find optimal therapeutic concentration and exact underlying mechanism of action.

Increased galectin-1 expression in muscle of Astragalus polysaccharide-treated Type 1 diabetic mice.

In order to investigate the immunopharmacological function of Astragalus polysaccharide (APS) in Type 1 diabetes mellitus (T1DM), multiple low-dose streptozotocin-induced diabetic mice and normal mice were administered either APS or saline intraperitoneally once daily. The changes in galectin-1 expression in different organs of the mice were detected by ELISA, Real-time fluorescence quantitative RT-PCR and Western blot. The percentages of apoptotic CD8(+) T cells from spleens of APS-treated diabetic mice were measured by flow cytometry. We found that the expression of galectin-1 was increased in serum of APS-treated diabetic mice compared to the non-treated diabetic mice (*p < 0.05). Increased galectin-1 was mainly expressed in the muscle of APS-treated mice. In vitro, APS up-regulated the expression of galectin-1 in muscle cells in a dose-dependent manner. The percentage of apoptotic CD8(+) T cells in spleens of APS-treated mice was positively correlated with the concentration of APS treatment, and the blocking of galectin-1 in vivo by specific antibody reduced the percentage of apoptotic CD8(+) T cells in APS-treated mice. Our findings indicated that APS could up-regulate the expression of galectin-1 in muscle of T1DM mice, resulting in the apoptosis of CD8(+) T cells. This may be an important mechanism by which APS protects ß cells of the pancreatic islets from apoptosis induced by CD8(+) T cells in T1DM in vivo.

Galectin-1-induced down-regulation of T lymphocyte activation protects (NZB x NZW) F1 mice from lupus-like disease.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease characterized by a hyperactive immune system, including activation of autoreactive T and B cells. These studies demonstrate that administration of recombinant galectin-1, a ß-galactose binding protein, to SLE-prone (NZB × NZW) F1 mice reduced lymphocyte activation, inhibited serum anti-double-stranded DNA(dsDNA) IgG antibody production, decreased the incidence of proteinuria, and increased survival rate. In addition, recombinant galectin-1'-treated mice had a higher frequency of Foxp3 expression, which suggested an increase in the percentage of peripheral regulatory T cells. Consistent with the finding that there were fewer activated T lymphocytes, ex vivo T cells from mice treated with recombinant galectin-1 exhibited less proliferation in response to TCR stimulation. Furthermore, these cells were less efficient at lipid raft clustering in response to TCR/CD28 engagement, consistent with published reports that galectin-1 can reorganize the synaptic contact to interfere with TCR signaling and activation to prevent T cell activation. Aged galectin-1-deficient mice had higher serum levels of antibodies against dsDNA, elucidating a role for endogenous galectin-1 in decreasing susceptibility to autoimmunity. Together, the findings highlight galectin-1 as a novel potential therapeutic immune modulator for treatment of lupus-like disease.

Up-regulated proteins in the fluid bathing the tumour cell microenvironment as potential serological markers for early detection of cancer of the breast.

Breast cancer is by far the most common diagnosed form of cancer and the leading cause of cancer death in women today. Clinically useful biomarkers for early detection of breast cancer could lead to a significant reduction in mortality. Here we describe a detailed analysis using gel-based proteomics in combination with mass spectrometry and immunohistochemistry (IHC) of the tumour interstitial fluids (TIF) and normal interstitial fluids (NIF) collected from 69 prospective breast cancer patients. The goal of this study was to identify abundant cancer up-regulated proteins that are externalised by cells in the tumour microenvironment of most if not all these lesions. To this end, we applied a phased biomarker discovery research strategy to the analysis of these samples rather than comparing all samples among each other, with inherent inter and intra-sample variability problems. To this end, we chose to use samples derived from a single tumour/benign tissue pair (patient 46, triple negative tumour), for which we had well-matched samples in terms of epithelial cell numbers, to generate the initial dataset. In this first phase we found 110 proteins that were up-regulated by a factor of 2 or more in the TIF, some of which were confirmed by IHC. In the second phase, we carried out a systematic computer assisted analysis of the 2D gels of the remaining 68 TIF samples in order to identify TIF 46 up-regulated proteins that were deregulated in 90% or more of all the available TIFs, thus representing common breast cancer markers. This second phase singled out a set of 26 breast cancer markers, most of which were also identified by a complementary analysis using LC-MS/MS. The expression of calreticulin, cellular retinoic acid-binding protein II, chloride intracellular channel protein 1, EF-1-beta, galectin 1, peroxiredoxin-2, platelet-derived endothelial cell growth factor, protein disulfide isomerase and ubiquitin carboxyl-terminal hydrolase 5 were further validated using a tissue microarray containing 70 malignant breast carcinomas of various grades of atypia. A significant number of these proteins have already been detected in the blood/plasma/secretome by others. The next steps, which include biomarker prioritization based on the hierarchal evaluation of these markers, antibody and antigen development, assay development, analytical validation, and preliminary testing in the blood of healthy and breast cancer patients, are discussed.

Apoptotic effects of a high performance liquid chromatography (HPLC) fraction of Antrodia camphorata mycelia are mediated by down-regulation of the expressions of four tumor-related genes in human non-small cell lung carcinoma A549 cell.

AIM OF THE STUDY: Antrodia camphorata (niu-chang-chih) is a fungus native to Taiwan which is believed to be effective in preventing diseases. Recent reports demonstrate that Antrodia camphorata products induce the apoptosis of various kinds of tumor cells. In this study we determined the inhibitory effects of alcohol extract and individual fractions of alcohol extract on the proliferation of human non-small cell lung carcinoma A549 cell and clarified the mechanism underlying the anti-cancer activities. MATERIALS AND METHODS: Alcohol extracts of Antrodia camphorata mycelia were prepared by the serial extraction with the solvents with increasing polarity and fractionated using HPLC. Cell viability was determined by MTT assay. Apoptosis detection was carried out by subG(1) analysis and annexin V/propidium iodide staining using flow cytometry. The impacts of HPLC fractions on the expression levels of apoptosis- and cancer-related proteins were evaluated by western blotting. RESULTS: Three HPLC fractions, fractions 5-7, had robust inhibition of human A549 cells and among them fraction 6 (Fr-6) possessed the most potent effectiveness. Apoptotic assay showed that Fr-6-induced human A549 cell apoptosis by triggering the mitochondrial pathway and endothelium reticulum (ER) stress. Immunoblotting results demonstrated that Fr-6 possibly activated ER stress by lowering the expression level of calpain 1/2 small subunit and Fr-6-mediated decrease in cell proliferation might attribute to the suppressive effect on the Erk 1/2 pathway, which arose from Fr-6-derived low galectin-1 expression. Furthermore Fr-6 could diminish Rho GDP dissociation inhibitor alpha (RhoGDI-alpha) expression and subsequently activated c-Jun NH(2)-terminal kinase (JNK) pathway, which is linked to cell apoptosis. Fr-6 also could decrease the production level of eukaryotic translation initiation factor 5A, which is a potential cancer intervention target. CONCLUSION: These results suggested that the anti-cancer activity of Antrodia camphorata might be due to multiple active metabolites, which work together to induce cell apoptosis via various pathways.

Activation of oxidative burst and degranulation of porcine neutrophils by a homologous spleen galectin-1 compared to N-formyl-L-methionyl-L-leucyl-L-phenylalanine and phorbol 12-myristate 13-acetate.

Galectins are a family of animal lectins defined by their beta-galactoside-binding activities and a consensus sequence in their carbohydrate-recognizing domain (CRD). Relevant roles of galectins are described in adaptive immune response, innate immunity and modulation of the acute inflammatory response. We have extended our previous studies on a porcine spleen galectin-1 in relation to its functional roles such as polymorphonuclear neutrophils (PMNs) stimulation compared to well known PMN activators e.g. N-formyl-L-methionyl-L leucyl-L-phenylalanine (fMLP) and phorbol 12-myristate 13-acetate (PMA). Relative to activation of NADPH-oxidase fMLP and PMA are stronger than galectin-1 plus cytochalasin B (CB) when the lectin is employed at low concentrations (gal-1 1 microM, 3.6+0.8 nm O(2)(-)/min/10(7) PMN). Higher doses of galectin-1 (10 microM) plus CB produced a significant activation of NADPH-oxidase (27.9+14.8 nm O(2)(-)/min/10(7) PMN) and stimulated PMN degranulation up to 50%. We propose that local galectin-1 concentrations under physiological conditions might reach suitable levels for pig PMN stimulation, and might be a natural inducer of O(2)(-) formation or degranulation. Porcine galectins might produce enhanced responses in vivo when they stimulate neutrophils in combination with some other stimuli.

Unconventional secretion of fibroblast growth factor 2 is mediated by direct translocation across the plasma membrane of mammalian cells.

Fibroblast growth factor 2 (FGF-2) is a pro-angiogenic mediator that is secreted by both normal and neoplastic cells. Intriguingly, FGF-2 has been shown to be exported by an endoplasmic reticulum/Golgi-independent pathway; however, the molecular machinery mediating this process has remained elusive. Here we introduce a novel in vitro system that functionally reconstitutes FGF-2 secretion. Based on affinity-purified plasma membrane inside-out vesicles, we demonstrate post-translational membrane translocation of FGF-2 as shown by protease protection experiments. This process is blocked at low temperature but apparently does not appear to be driven by ATP hydrolysis. FGF-2 membrane translocation occurs in a unidirectional fashion requiring both integral and peripheral membrane proteins. These findings provide direct evidence that FGF-2 secretion is based on its direct translocation across the plasma membrane of mammalian cells. When galectin-1 and macrophage migration inhibitory factor, other proteins exported by unconventional means, were analyzed for translocation into plasma membrane inside-out vesicles, galectin-1 was found to be transported as efficiently as FGF-2. By contrast, migration inhibitory factor failed to traverse the membrane of inside-out vesicles. These findings establish the existence of multiple distinct secretory routes that are operational in the absence of a functional endoplasmic reticulum/Golgi system.

Structure-activity profiles of complex biantennary glycans with core fucosylation and with/without additional alpha 2,3/alpha 2,6 sialylation: synthesis of neoglycoproteins and their properties in lectin assays, cell binding, and organ uptake.

The consideration of oligosaccharides and glycoconjugates as biopharmaceuticals is an emerging topic in drug design. Chemoenzymatic synthesis of N-glycans was performed to examine the influence of N-glycan core fucosylation on lectin-binding properties and biodistribution. As a first step in a systematic comparison of N-glycans, the core fucose moiety was chemically introduced into a complex-type biantennary heptasaccharide azide. After deprotection and attachment of a spacer, the terminal sections of the N-glycan were elongated enzymatically. Conversion of the amino group in the spacer to an isothiocyanate gave derivatives allowing convenient ligand attachment to bovine serum albumin (BSA). The resulting neoglycoproteins contained an average of 2.9-4.6 chains per carrier molecule. Relative to unsubstituted biantennary complex-type N-glycans, the core fucosylation appears to favor the extended orientation of the alpha 1,6-arm. This was deduced from an up to 5-fold alteration of affinity for lectins in solid-phase assays. Marked differences were also found for cell surface binding of cultured tumor cells, for staining of tumor cells in lung sections, and in organ distribution. In vivo, the alpha 2,6-sialylated neoglycoproteins showed a reduced serum half-life in mice relative to the alpha 2,3-sialylated isomer and the non-fucosylated congeners. These results support the notion that changing the shape of a glycan provides a promising strategy to optimize the affinity of protein-carbohydrate interactions. Overall, our study underscores the importance of chemoenzymatic synthesis to define the effect of chain orientation on the ligand properties of N-glycans.

NMR investigations of protein-carbohydrate interactions: insights into the topology of the bound conformation of a lactose isomer and beta-galactosyl xyloses to mistletoe lectin and galectin-1.

A hallmark of oligosaccharides is their often limited spatial flexibility, allowing them to access a distinct set of conformers in solution. Viewing each individual or even the complete ensemble of conformations as potential binding partner(s) for lectins in protein-carbohydrate interactions, it is pertinent to address the question on the characteristics of bound state conformation(s) in solution. Also, it is possible that entering the lectin's binding site distorts the low-energy topology of a glycosidic linkage. As a step to delineate the strategy of ligand selection for galactosides, a common physiological docking point, we have performed a NMR study on two non-homologous lectins showing identical monosaccharide specificity. Thus, the conformation of lactose analogues bound to bovine heart galectin-1 and to mistletoe lectin in solution has been determined by transferred nuclear Overhauser effect measurements. It is demonstrated that the lectins select the syn conformation of lactose and various structural analogues (Galbeta(1-->4)Xyl, Galbeta(1-->3)Xyl, Galbeta(1-->2)Xyl, and Galbeta(1-->3)Glc) from the ensemble of presented conformations. No evidence for conformational distortion was obtained. Docking of the analogues to the modeled binding sites furnishes explanations, in structural terms, for exclusive recognition of the syn conformer despite the non-homologous design of the binding sites.

Galectin-1 induces partial TCR zeta-chain phosphorylation and antagonizes processive TCR signal transduction.

Galectin-1 is an endogenous lectin with known T cell immunoregulatory activity, though the molecular basis by which galectin-1 influences Ag specific T cell responses has not been elucidated. Here, we characterize the ability of galectin-1 to modulate TCR signals and responses by T cells with well defined hierarchies of threshold requirements for signaling distinct functional responses. We demonstrate that galectin-1 antagonizes TCR responses known to require costimulation and processive protein tyrosine phosphorylation, such as IL-2 production, but is permissive for TCR responses that only require partial TCR signals, such as IFN-gamma production, CD69 up-regulation, and apoptosis. Galectin-1 binding alone or together with Ag stimulation induces partial phosphorylation of TCR-zeta and the generation of inhibitory pp21zeta. Galectin-1 antagonizes Ag induced signals and TCR/costimulator dependent lipid raft clustering at the TCR contact site. We propose that galectin-1 functions as a T cell "counterstimulator" to limit required protein segregation and lipid raft reorganization at the TCR contact site and, thus, processive and sustained TCR signal transduction. These findings support the concept that TCR antagonism can arise from the generation of an inhibitory pp21zeta-based TCR signaling complex. Moreover, they demonstrate that TCR antagonism can result from T cell interactions with a ligand other than peptide/MHC.