Paper electrochemical immunosensor for the rapid screening of Galectin-3 patients with heart failure.

A paper electrochemical immunosensor for the combined binding and quantification of the heart failure (HF) biomarker Galectin-3 has been developed. The simple design of the new sensor is comprised of paper material that is decorated with gold nanostructures, to maximize its electroactive surface area, and functionalized with target-specific recognition molecules to selectively bind the protein from aqueous solutions. The binding of the protein caused the blockage of the electron flow to the sensor electroactive surface, thus causing its oxidation potential to shift and the corresponding current to reduce quantitatively with the increase in the protein concentration within the working range of 0.5ng/mL-8ng/mL (LOQ-0.5 ng/mL). This novel sensor was able to quantify Galectin-3 concentration in saliva samples from HF patients and healthy controls within 20 min with good reproducibility (RSD = 3.64%), without the need for complex sample processing steps. The electrochemical measurements of the patient samples were cross validated by ELISA where the percent agreement between the two methods was found to be 92.7% (RSD = 7.20%). Therefore, the new paper immunosensor sensor has a strong potential for rapid and cost-effective screening of the Galectin 3 biomarker at points of care, thus supporting the timely diagnosis of heart failure.

Membrane organization by tetraspanins and galectins shapes lymphocyte function.

Immune receptors are not randomly distributed at the plasma membrane of lymphocytes but are segregated into specialized domains that function as platforms to initiate signalling, as exemplified by the B cell or T cell receptor complex and the immunological synapse. 'Membrane-organizing proteins' and, in particular, tetraspanins and galectins, are crucial for controlling the spatiotemporal organization of immune receptors and other signalling proteins. Deficiencies in specific tetraspanins and galectins result in impaired immune synapse formation, lymphocyte proliferation, antibody production and migration, which can lead to impaired immunity, tumour development and autoimmunity. In contrast to conventional ligand-receptor interactions, membrane organizers interact in cis (on the same cell) and modulate receptor clustering, receptor dynamics and intracellular signalling. New findings have uncovered their complex and dynamic nature, revealing shared binding partners and collaborative activity in determining the composition of membrane domains. Therefore, immune receptors should not be envisaged as independent entities and instead should be studied in the context of their spatial organization in the lymphocyte membrane. We advocate for a novel approach to study lymphocyte function by globally analysing the role of membrane organizers in the assembly of different membrane complexes and discuss opportunities to develop therapeutic approaches that act via the modulation of membrane organization.

Purification and Characterization of a Novel D-Galactose Binding Lectin from Seeds of Meizotropis buteiformis.

Plant lectins are carbohydrate-binding proteins that are ubiquitously found in almost all plant species and have different structures and functions depending on the sources. Purifying lectins from their plant sources and determining their sugar specificity become an important goal for evaluating their potential biomedical applications. Here, we report the affinity purification of a Dgalactose specific lectin from the seeds of Meizotropis buteiformis Voigt., and its physicochemical parameters, and LC-MS/MS (tandem mass spectrometry) analysis. Isolation and purification of this lectin were performed by simple successive steps of lectin extraction, ammonium sulphate fractionation, and affinity chromatography using lactose-linked Sepharose-4B chromatography column. The affinity-purified lectin has a native molecular weight of 75 kDa and is found to be a heterodimer (molecular weight of 36 and 38 kDa). The LC-MS/MS results suggested that the purified lectin had not been reported earlier. AIM: The main aim of the present study is to find out the novelty and characteristics of a lectin purified from the plant Meizotropis buteiformis. BACKGROUND: Lectins are proteins that possess the ability to specifically bind glycans of glycoconjugates. Plants are considered rich sources of lectins and the determination of sugar specificity of a purified plant lectin is an important aspect in order to evaluate its potential area of application. In the present study, a novel D-Galactose specific lectin is purified from Meizotropis buteiformis through affinity chromatography and examined for its various physical and biochemical characteristics. OBJECTIVE: The objective of the present study is to purify a novel lectin up to its homogeneity from the seeds of Meizotropis buteiformis and characterization of its various physical and biochemical properties. METHODS: The lectin was purified by simple successive steps of lectin extraction, ammonium sulphate fractionation, and affinity chromatography. Activity of the purified lectin was determined by hemagglutination assay. Some physicochemical parameters of the purified protein were also determined along with identification of protein by LC-MS/MS and the spectra analysis using Mascot sequence matching software (Matrix Science) with the NCBI database. RESULTS: From the current investigation, it was found that the purified lectin has a native molecular weight of 75 kDa. Among the various sugars and sugar derivatives tested, lactose and D-galactose were found to be potent inhibitors of its activity. Its optimum pH range was found to be from 6.5 to 7.5 and also it exhibited full activity at a temperature from 0ºC to 50ºC. The purified lectin does not show any effects on its activities for metal ions tested. The protein view report of the LC-MS/MS result analysis showed a 50% sequence similarity with that of the lectin beta-chain of the Butea monosperma. CONCLUSION: In the present study, a novel D-Galactose specific lectin is purified from Meizotropis buteiformis by affinity chromatography using Sepharose 4B. The purified lectin is found to be heterodimeric and metal ion independent. The LC-MS/MS results suggested that the purified lectin has not been reported earlier.

Visualization of Cytosolic Galectin Accumulation Around Damaged Vesicles and Organelles.

Galectins are animal lectins that recognize ß-galactoside and bind glycans. Recent studies have indicated that cytosolic galectins recognize cytosolically exposed glycans and accumulate around endocytic vesicles or organelles damaged by various disruptive substances. Accumulated galectins engage other cytosolic proteins toward damaged vesicles, leading to cellular responses, such as autophagy. Disruptive substances include bacteria, viruses, particulate matters, and protein aggregates; thus, this process is implicated in the pathogenesis of various diseases. In this chapter, we describe methods for studying three disruptive substances: photosensitizers, Listeria monocytogenes, and Helicobacter pylori. We summarize the tools used for the detection of cytosolic galectin accumulation around damaged vesicles.

Galectin-3: A Novel Marker for the Prediction of Stroke Incidence and Clinical Prognosis.

Stroke, whether ischemic or haemorrhagic, is one of the main causes of mortality and disability all over the world, which entails huge burdens in both healthcare environments as well as social and economic aspects of life. Therefore, there is a continuous search for novel reliable biomarkers that can enhance the recognition of stroke events in a timely manner and predict the clinical outcomes following a stroke event. Galectins are a group of proteins expressed by many types of cells and tissues including vasculature, certain immune cells, fibroblasts, and gastrointestinal epithelial cells. These proteins vary in their structure and configuration according to their type and have a diversity of functions according to the type of tissue they are expressed in. Among these proteins, a few studies investigated mainly the roles played by galectin-1 (Gal-1) and galectin-3 (Gal-3) in the molecular mechanisms of atherosclerosis and in brain tissue remodeling after a stroke event. In this review, we present an updated overview of the current understanding of Gal-3's functions and implications in stroke occurrence and the response of the brain tissue to stroke events, which may be a key to its utility as a predictor of stroke incidence and clinical prognosis in the future.

Splenic CD4+ and CD8+ T-cells highly expressed PD-1 and Tim-3 in cirrhotic patients with HCV infection and portal hypertension.

Introduction: The spleen plays an important role in regulating the immune response to infectious pathogens. T-cells dysfunction and exhaustion have been reported in patients with hepatitis B/C virus (HBV/HCV) infection, which contributes to persistent virus infection. The aims of this study were to investigate spleen-related evidence of immunosuppression and immune tolerance in HCV cirrhotic patients with portal hypertension (PH). Methods: The expression of programmed cell death 1 (PD-1), T-cell immunoglobulin domain and mucin domain-containing molecule-3 (Tim-3) and its ligand PD-L1/2, and Galectin-9 in the spleens and livers of HCV cirrhotic patients (n = 15) was analyzed using real-time PCR and immunohistochemistry. Flow cytometry was used to evaluate the expression of PD-1 and Tim-3 on splenic T-cells and the peripheral blood T-cells before and after splenectomy (n = 8). Results: Spleens from patients with PH showed significantly increased mRNA levels of PD-L2, Tim-3, Galectin-9, CD80, and CD86, and decreased levels of CD28 compared to control spleens (spleens removed due to traumatic injury) (all p < 0.05). Additionally, protein expression of inhibitory signaling molecules was significantly increased in both the spleens and livers of cirrhotic patients compared with controls (all p < 0.05). Peripheral blood and splenic CD4+ and CD8+ T-cells also expressed higher protein levels of PD-1, Tim-3, and CTLA-4 in cirrhotic patients as compared with healthy controls (all p < 0.05). The proportion of PD-1+CD4+T lymphocytes (26.2% ± 7.12% vs. 21.0% ± 9.14%, p = 0.0293) and Tim-3+CD8+ T lymphocytes (9.4% ± 3.04% vs. 6.0% ± 2.24%, p = 0.0175) in peripheral blood decreased followed splenectomy. Conclusion: The CD4+ and CD8+ T-cells in spleen and peripheral blood highly expressed PD-1 and Tim-3 in HCV-infected and cirrhotic patients with portal hypertension. Highly expressed PD-1 and Tim-3 in peripheral blood T-lymphocytes can be partly reversed following splenectomy.

Imitating evolution's tinkering by protein engineering reveals extension of human galectin-7 activity.

Wild-type lectins have distinct types of modular design. As a step to explain the physiological importance of their special status, hypothesis-driven protein engineering is used to generate variants. Concerning adhesion/growth-regulatory galectins, non-covalently associated homodimers are commonly encountered in vertebrates. The homodimeric galectin-7 (Gal-7) is a multifunctional context-dependent modulator. Since the possibility of conversion from the homodimer to hybrids with other galectin domains, i.e. from Gal-1 and Gal-3, has recently been discovered, we designed Gal-7-based constructs, i.e. stable (covalently linked) homo- and heterodimers. They were produced and purified by affinity chromatography, and the sugar-binding activity of each lectin unit proven by calorimetry. Inspection of profiles of binding of labeled galectins to an array-like platform with various cell types, i.e. sections of murine epididymis and jejunum, and impact on neuroblastoma cell proliferation revealed no major difference between natural and artificial (stable) homodimers. When analyzing heterodimers, acquisition of altered properties was seen. Remarkably, binding properties and activity as effector can depend on the order of arrangement of lectin domains (from N- to C-termini) and on the linker length. After dissociation of the homodimer, the Gal-7 domain can build new functionally active hybrids with other partners. This study provides a clear direction for research on defining the full range of Gal-7 functionality and offers the perspective of testing applications for engineered heterodimers.

Galectin-3 in septic acute kidney injury: a translational study.

BACKGROUND: Galectin-3 (Gal-3) is a pleiotropic glycan-binding protein shown to be involved in sepsis and acute kidney injury (AKI). However, its role has never been elucidated in sepsis-associated AKI (S-AKI). We aimed to explore Gal-3's role and its potential utility as a therapeutic target in S-AKI. METHODS: In 57 patients admitted to the intensive care unit (ICU) with sepsis, serum Gal-3 was examined as a predictor of ICU mortality and development of AKI. In a rat model of S-AKI induced by cecal ligation and puncture (CLP), 7-day mortality and serum Gal-3, Interleukin-6 (IL-6), and creatinine were examined at 2, 8, and 24 hours (h) post-CLP. Two experimental groups received the Gal-3 inhibitor modified citrus pectin (P-MCP) at 400 mg/kg/day and 1200 mg/kg/day, while the control group received water only (n = 18 in each group). RESULTS: Among 57 patients, 27 developed AKI and 8 died in the ICU. Serum Gal-3 was an independent predictor of AKI (OR = 1.2 [95% CI 1.1-1.4], p = 0.01) and ICU mortality (OR = 1.4 [95% CI 1.1-2.2], p = 0.04) before and after controlling for age, AKI, and acute physiology and chronic health evaluation (APACHE II) score. In the CLP rat experiment, serum Gal-3 peaked earlier than IL-6. Serum Gal-3 was significantly lower in both P-MCP groups compared to control at 2 h post-CLP (400 mg: p = 0.003; 1200 mg: p = 0.002), and IL-6 was significantly lower in both P-MCP groups at all time points with a maximum difference at 24 h post-CLP (400 mg: p = 0.015; 1200 mg: p = 0.02). In the Gal-3 inhibitor groups, 7-day mortality was significantly reduced from 61% in the control group to 28% (400 mg P-MCP: p = 0.03) and 22% (1200 mg P-MCP: p = 0.001). Rates of AKI per RIFLE criteria were significantly reduced from 89% in the control group to 44% in both P-MCP groups (400 mg: p = 0.007; 1200 mg: p = 0.007). CONCLUSIONS: This translational study demonstrates the importance of Gal-3 in the pathogenesis of S-AKI, and its potential utility as a therapeutic target.

The Role of Cell Blocks and Immunohistochemistry in Thyroid Atypia of Undetermined Significance/Follicular Lesion of Undetermined Significance Bethesda Category.

INTRODUCTION: Thyroid cytology is a widely accepted tool in the clinical triaging of nodular lesions. Cell blocks (CBs) can help in the diagnosis of atypical lesions, namely, thyroid Bethesda category of Atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS). METHODS: In a series of 224 AUS/FLUS thyroid samples with CB, we studied CB cellularity and feasibility of 3 immunohistochemical markers (cytokeratin 19 [CK19], HBME-1, and galectin-3) apart and in combination. RESULTS: The CBs were non-diagnostic in 34 cases. Twenty-four CBs contained <10 cells, 45 CBs 10-50 cells, and 121 CBs >50 cells. Notably, more cellularity was found in CBs performed by plasma-thrombin and in-house techniques (p < 0.001). The diagnostic accuracy to detect malignancy was 65.1% for CK19, 72.1% for HBME-1, and 70.3% for galectin-3. CONCLUSION: In conclusion, CB cellularity is essential for successful immunohistochemistry application and further diagnostic workup of AUS/FLUS cases.

The role of Tim-3/Galectin-9 pathway in T-cell function and prognosis of patients with human papilloma virus-associated cervical carcinoma.

The interaction between Tim-3 on T cell and its ligand, Galectin-9, negatively regulates cellular immune responses. However, the role of Tim-3/Galectin-9 pathway in the immune evasion of cervical cancer remains unknown. This study is to investigate the expression, function, and regulation of Tim-3/Galectin-9 signaling pathway in human papilloma virus (HPV) positive cervical cancer. Flow cytometry showed that Tim-3 expression on T cell and Galectin-9 expression on monocytes in HPV positive cervical cancer patients were significantly higher compared to cervical intraepithelial neoplasia and benign uterine fibroids Tim-3 + CD4+ Th1 cells and Tim-3 + CD8+ T cells in HPV positive cervical cancer patients were significantly reduced after surgery. Serum TGF-ß and IL-10 levels were positively correlated with Tim-3 + Treg cells, while IFN-γ and IL-2 were negatively correlated with Tim-3 + Th1 cells. Additionally, Tim-3 + CD4+ T cells were positively correlated with Galectin-9 + monocytes. Survival curve analysis showed that Tim-3 + CD4+ T cells were negatively correlated with patient survival, and closely related to FIGO stage, degree of differentiation, and lymph node metastasis of HPV positive cervical cancer. In vitro experiments showed that by blocking the Tim-3/Galectin-9 pathway, the proliferation of T cells and their ability to express IFN-γ, IL-2, perforin, and granzyme B was significantly restored. In conclusion, high levels of Tim-3 and Galectin-9 in HPV positive cervical cancer patients play roles in the progression of disease by promoting Treg cells to inhibit the cytotoxic function of Th1 and CD8+ T cells. Tim-3/Galectin-9 may serve as a new immunotherapy target for patients with HPV positive cervical cancer.

Galectin-9 as a biomarker of disease severity.

Galectin-9 (Gal-9) is a ß-galactoside binding lectin known for its immunomodulatory role in various microbial infections. Gal-9 is expressed in all organ systems and localized in the nucleus, cell surface, cytoplasm and the extracellular matrix. It mediates host-pathogen interactions and regulates cell signalling via binding to its receptors. Gal-9 is involved in many physiological functions such as cell growth, differentiation, adhesion, communication and death. However, recent studies have emphasized on the elevated levels of Gal-9 in autoimmune disorders, viral infections, parasitic invasion, cancer, acute liver failure, atopic dermatitis, chronic kidney disease, type-2 diabetes, coronary artery disease, atherosclerosis and benign infertility-related gynecological disorders. In this paper we have reviewed the potential of Gal-9 as a reliable, sensitive and non-invasive biomarker of disease severity. Tracking changes in Gal-9 levels and its implementation as a biomarker in clinical practice will be an important tool to monitor disease activity and facilitate personalized treatment decisions.

Global Longitudinal Strain of the Systemic Ventricle Is Correlated with Plasma Galectin-3 and Predicts Major Cardiovascular Events in Adult Patients with Congenital Heart Disease.

Backround and Objective: We sought to assess in adult congenital heart disease (ACHD) patients the prognostic value of plasma galectin-3 (Gal-3) levels and systemic ventricular global longitudinal strain (SV GLS) as well as their association with NTproBNP and arrhythmogenesis. Materials and Methods: We studied 58 patients (26 men, mean age 37 ± 16.8 years) with various congenital heart diseases. Patients underwent echocardiogram, 24 h ambulatory ECG monitoring, while NTproBNP and Gal-3 were measured. They were followed up (median of 790.5 days -IQR 350.3 days) and major cardiovascular events (MACE) were recorded. Results. Mean Gal-3 levels were 17.07 ± 6.38 ng/m. Plasma Gal-3 was correlated with LogNTproBNP (r = 0.456, p = 0.001).Gal-3 levels associated with supraventricular tachycardia (SVT) (p < 0.001) and ventricular tachycardia (VT) (p < 0.001), but was not associated with MACE (HR 1.018, 95% CI 0.944-1.098, p = 0.641).Mean SVGLS in patients with systemic left ventricle was -15.91% ± 4.09%, which was significantly lower compared to patients with systemic right ventricle and patients with single ventricle (-11.42% ± 3.37% and -11.9% ± 5.06%, respectively, p = 0.021).SV GLS correlated with plasma Gal-3 (r = 0.313, p = 0.027) and logNTproBNP (r = 0.479, p < 0.001). SVGLS correlated with VT arrhythmias (p = 0.004). NTproBNP predicted MACE (AUC 0.750, p = 0.03). SVGLS also predicted MACE (AUC 0.745, p = 0.03. In multivariate analysis, SVGLS and logNTproBNP maintained their predictive value (p = 0.004 and p = 0.009, respectively) Conclusion: In ACHD patients, SV GLS was found to predict MACE independently from NTproBNP and correlated with VT. Gal-3 correlated with NTproBNP and SVGLS as well as SVT and VT, but has not been shown to bear significant prognostic potential.

Different Expression Pattern of TIM-3 and Galectin-9 Molecules by Peripheral and Peritoneal Lymphocytes in Women with and without Endometriosis.

Endometriosis is a gynecological condition that is associated with chronic pelvic inflammation, pain, and infertility. Although substantial evidence supports that immunological alterations contribute to its pathogenesis and we previously posed a pivotal role of Galectin-9 (Gal-9) in this disorder, the involvement of the TIM-3/Gal-9 pathway in the development of endometriosis-associated immunological abnormalities is not yet known. In the present study, multicolor flow cytometry was used to compare the immunophenotype and cell surface expression of TIM-3 and Gal-9 molecules on peripheral blood (PB) and peritoneal fluid (PF) lymphocytes of women with and without endometriosis. We found an altered distribution of different lymphocyte subpopulations, a markedly decreased TIM-3 labeling on all T and NK subsets and a significantly increased Gal-9 positivity on peripheral CD4+ T and Treg cells of the affected cohort. Furthermore, a significantly increased TIM-3 expression on CD4+T-cells and elevated Gal-9 labeling on all T and NK subsets was also revealed in the PF of the examined patients. In conclusion, our results suggest a persistent activation and disturbed TIM-3/Gal-9-dependent regulatory function in endometriosis, which may be involved in the impaired immune surveillance mechanisms, promotes the survival of ectopic lesions, and aids the evolution of reproductive failures in endometriosis.

Role of Human Galectins in Inflammation and Cancers Associated with Endometriosis.

Galectins are a family of ß-galactoside-binding proteins that contribute to multiple cellular functions, including immune surveillance and apoptosis. Human galectins are also important regulators of inflammation, making them a research target for various inflammatory diseases and tumorigenesis associated with pro-inflammatory conditions. This review focuses on the involvement of human galectins in modulation of inflammation and in the pathophysiology of endometriosis and endometriosis-associated neoplasms. Endometriosis is a chronic inflammatory disease with unknown etiology. Galectins -1, -3 and -9 were found to be overexpressed in ectopic and eutopic endometrium of females with endometriosis compared to those without endometriosis. These findings suggest galectins' role in the progression on endometriotic lesions and their potential use as diagnostic biomarkers and/or targets for therapeutic approaches. Galectins -1, -3, and -9 have also been implicated in the development of endometriosis-associated neoplasms. Furthermore, galectin-3 has been shown to interact with KRAS protein and contribute to cellular growth, proliferation, inflammation, and the uptake of nutrients in endometriotic lesions and may be involved in the maintenance and propagation of endometriosis. These galectins have been shown to be upregulated in certain forms of cervical, ovarian, endometrial, and colon cancer associated with endometriosis and have become a potential target for anti-cancer therapies.

High-Affinity N-(2-Hydroxypropyl)methacrylamide Copolymers with Tailored N-Acetyllactosamine Presentation Discriminate between Galectins.

N-Acetyllactosamine (LacNAc; Galß4GlcNAc) is a typical disaccharide ligand of galectins. The most abundant members of these human lectins, galectin-1 (Gal-1) and galectin-3 (Gal-3), participate in a number of pathologies including cancerogenesis and metastatic formation. In this study, we synthesized a series of fifteen N-(2-hydroxypropyl)methacrylamide (HPMA)-based glycopolymers with varying LacNAc amounts and presentations and evaluated the impact of their architecture on the binding affinity to Gal-1 and Gal-3. The controlled radical reversible addition-fragmentation chain transfer copolymerization technique afforded linear polymer precursors with comparable molecular weight (Mn ≈ 22,000 g mol-1) and narrow dispersity (D̵ ≈ 1.1). The precursors were conjugated with the functionalized LacNAc disaccharide (4-22 mol % content in glycopolymer) prepared by enzymatic synthesis under catalysis by ß-galactosidase from Bacillus circulans. The structure-affinity relationship study based on the enzyme-linked immunosorbent assay revealed that the type of LacNAc presentation, individual or clustered on bi- or trivalent linkers, brings a clear discrimination (almost 300-fold) between Gal-1 and Gal-3, reaching avidity to Gal-1 in the nanomolar range. Whereas Gal-1 strongly preferred a dense presentation of individually distributed LacNAc epitopes, Gal-3 preferred a clustered LacNAc presentation. Such a strong galectin preference based just on the structure of a multivalent glycopolymer type is exceptional. The prepared nontoxic, nonimmunogenic, and biocompatible glycopolymers are prospective for therapeutic applications requiring selectivity for one particular galectin.

Galectin-9: A Predictive Biomarker Negatively Regulating Immune Response in Glioma Patients.

BACKGROUND: Glioma is the most frequent primary brain tumor. Immunotherapy is one of the most promising therapeutic approaches for gliomas. T cell immunoglobulin domain and mucin domain-3 can induce the malignancy of gliomas. The function of galectin-9 (GAL-9), as one of the ligands of T cell immunoglobulin domain and mucin domain-3, in glioma has remained elusive. The aim of this study was to characterize the expression of GAL-9 in patients with glioma. METHODS: This study enrolled 1292 patients with glioma from the GSE 16011 array set, the Chinese Glioma Genome Atlas, and The Cancer Genome Atlas datasets. Kaplan-Meier analysis was undertaken to explore the prognostic value of GAL-9. Graphpad software and R language were used for statistical analysis. RESULTS: Expression of GAL-9 was highly correlated with major clinical and molecular features. Patients with high expression of GAL-9 were more susceptible to development of malignant tumors. Gene Ontology analysis revealed that expression of GAL-9 was closely associated with function of immune response in glioma. Clinically, the results of Kaplan-Meier analysis showed that expression of GAL-9 was negatively associated with overall survival in all grades of glioma including high-grade gliomas. High expression of GAL-9 was an independent indicator of poor prognosis. CONCLUSIONS: Our results highlight the pivotal role of GAL-9 in regulation of immune suppressive features of gliomas and indicate that GAL-9 is a promising target for cancer immunotherapy and may lead to development of further therapies.

Charcot-Leyden crystal protein/galectin-10 is a surrogate biomarker of eosinophilic airway inflammation in asthma.

Aim: Eosinophilic asthma is associated with more exacerbations and differential responses to treatment. The aim of this study was to assess if CLC/Gal-10 and MBP-1 are surrogate biomarkers of eosinophilic inflammation in asthma. Methods & results: Sputum induction was performed in patients with asthma and in healthy controls. Sputum analysis revealed higher (p < 0.001) levels of CLC/Gal-10 and MBP-1 in asthmatics versus healthy controls. CLC/Gal-10 levels were highly correlated (rs = 0.74; p < 0.001) with sputum eosinophils; MBP-1 approached significance (r = 0.44; p = 0.07). Conclusion: Increased CLC/Gal-10 and MBP-1 levels in the sputum were strongly correlated with sputum eosinophils in patients with asthma. CLC/Gal-10 and MBP-1 may be useful biomarkers for differentiation of eosinophilic airway inflammation in asthma.

Resetting the ligand binding site of placental protein 13/galectin-13 recovers its ability to bind lactose.

Placental protein 13/galectin-13 (Gal-13) is highly expressed in placenta, where its lower expression is related to pre-eclampsia. Recently, the crystal structures of wild-type Gal-13 and its variant R53H at high resolution were solved. The crystallographic and biochemical results showed that Gal-13 and R53H could not bind lactose. Here, we used site-directed mutagenesis to re-engineer the ligand binding site of wild-type Gal-13, so that it could bind lactose. Of six newly engineered mutants, we were able to solve the crystal structures of four of them. Three variants (R53HH57R, R53HH57RD33G and R53HR55NH57RD33G had the same two mutations (R53 to H, and H57 to R) and were able to bind lactose in the crystal, indicating that these mutations were sufficient for recovering the ability of Gal-13 to bind lactose. Moreover, the structures of R53H and R53HR55N show that these variants could co-crystallize with a molecule of Tris. Surprisingly, although these variants, as well as wild-type Gal-13, could all induce hemagglutination, high concentrations of lactose could not inhibit agglutination, nor could they bind to lactose-modified Sepharose 6b beads. Overall, our results indicate that Gal-3 is not a normal galectin, which could not bind to ß-galactosides. Lastly, the distribution of EGFP-tagged wild-type Gal-13 and its variants in HeLa cells showed that they are concentrated in the nucleus and could be co-localized within filamentary materials, possibly actin.