Specific interaction of human Tamm-Horsfall gylcoprotein with leucoagglutinin, a lectin from Phaseolus vulgaris (red kidney bean).
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ABSTRACT:
SUBMITTER: Serafini-Cessi F
PROVIDER: S-EPMC1161569 | biostudies-other | 1979 Nov
REPOSITORIES: biostudies-other
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Project description:Tamm-Horsfall protein (THP), also known as uromodulin, is a kidney-specific protein produced by cells of the thick ascending limb of the loop of Henle. Although predominantly secreted apically into the urine, where it becomes highly polymerized, THP is also released basolaterally, toward the interstitium and circulation, to inhibit tubular inflammatory signaling. Whether, through this latter route, THP can also regulate the function of renal interstitial mononuclear phagocytes (MPCs) remains unclear, however. Here, we show that THP is primarily in a monomeric form in human serum. Compared with wild-type mice, THP-/- mice had markedly fewer MPCs in the kidney. A nonpolymerizing, truncated form of THP stimulated the proliferation of human macrophage cells in culture and partially restored the number of kidney MPCs when administered to THP-/- mice. Furthermore, resident renal MPCs had impaired phagocytic activity in the absence of THP. After ischemia-reperfusion injury, THP-/- mice, compared with wild-type mice, exhibited aggravated injury and an impaired transition of renal macrophages toward an M2 healing phenotype. However, treatment of THP-/- mice with truncated THP after ischemia-reperfusion injury mitigated the worsening of AKI. Taken together, our data suggest that interstitial THP positively regulates mononuclear phagocyte number, plasticity, and phagocytic activity. In addition to the effect of THP on the epithelium and granulopoiesis, this new immunomodulatory role could explain the protection conferred by THP during AKI.
| S-EPMC5827593 | biostudies-literature
Project description:A specific enterokinase inhibitor from kidney bean (Phaseolus vulgaris) was purified to homogeneity. It showed a single protein band on sodium dodecyl sulphate/polyacryl-amide-gel electrophoresis in the presence of mercaptoethanol, and the Mr was 31000. Aspartic acid was identified as the N-terminus of the inhibitor. The Mr by gel chromatography on Sephadex G-200 was found to be 60000, indicating the dimeric nature of the inhibitor. The inhibitor was found to be a glycoprotein. The monosaccharide moieties were glucose, mannose, glucuronic acid and glucosamine in the proportions 3.15%, 5.0%, 0.85% and 1.3% respectively. The inhibitor was most active on pig enterokinase, followed by bovine and human enterokinases. Maximal inhibitory activity was elicited by preincubation of the inhibitor with the enzyme for 15 min. Digestion with pepsin resulted in loss of inhibitory activity. The inhibitor was stable to exposure to a wide range of pH values (2-10), and exposure to pH above 10 resulted in loss of inhibitory activity. Modification of arginine residues by cyclohexane 1,2-dione and ninhydrin led to complete loss of enterokinase-inhibitory activity.
| S-EPMC1154059 | biostudies-other
Project description:A two-step method based on an aqueous two-phase system and Sephadex G-75 was used to separate and purify lectin from the seeds of the Zihua snap bean. The preliminary properties and bioactivity of the Zihua snap bean lectin were characterized by different instrumental methods, such as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), liquid chromatography-nano electrospray ionization mass spectrometry (Nano LC-ESI-MS/MS), and Fourier transform infrared spectroscopy (FTIR). The hemagglutinating activity of the Zihua snap bean lectin could not be inhibited by glucose, N-acetyl-D-glucosamine, D-galactose, N-acetyl-D-galactosamine, fructose, sucrose, D-maltose, D-trehalose, and lactose. It was found that the hemagglutinating activity of the lectin showed strong dependence on Mn2+ and Ca2+. The thermal and pH stability of the Zihua snap bean lectin was studied by FTIR and fluorescence spectroscopy. Relatively good stability was observed when the temperature was not higher than 70 °C, as well as in the pH range of 2.0 to 10.0. Digestive stability in vitro was investigated. The untreated lectin was relatively stable to pepsin and trypsin activity, but heat treatment could significantly reduce the digestive stability in vitro. Moreover, the lectin showed an inhibitory effect on the tested bacteria (Staphylococcus aureus (S. aureus), Escherichia coli (E. coli), Bacillus subtilis (B. subtilis)), and it also showed a certain inhibitory effect on the growth of Phytophthora infestans (P. infestans) at higher concentrations.
| S-EPMC6571848 | biostudies-literature
Project description:Both Tamm-Horsfall protein (THP) and collectin-11 (CL-11) are important molecules in acute kidney injury (AKI). In this study, we measured the change of glycosylation of THP in patients with AKI after surgery, using MALDI-TOF MS and lectin array analysis. The amount of high-mannose and core fucosylation in patients with AKI were higher than those in healthy controls. In vitro study showed that THP could bind to CL-11 with affinity at 9.41 × 10-7 mol/L and inhibited activation of complement lectin pathway. The binding affinity decreased after removal of glycans on THP. Removal of fucose completely ablated the binding between the two proteins. While removal of high-mannose or part of the N-glycan decreased the binding ability to 30% or 60%. The results indicated that increase of fucose on THP played an important role via complement lectin pathway in AKI.
| S-EPMC7131921 | biostudies-literature
Project description:A cytogenetic map of common bean was built by in situ hybridization of 35 bacterial artificial chromosomes (BACs) selected with markers mapping to eight linkage groups, plus two plasmids for 5S and 45S ribosomal DNA and one bacteriophage. Together with three previously mapped chromosomes (chromosomes 3, 4, and 7), 43 anchoring points between the genetic map and the cytogenetic map of the species are now available. Furthermore, a subset of four BAC clones was proposed to identify the 11 chromosome pairs of the standard cultivar BAT93. Three of these BACs labelled more than a single chromosome pair, indicating the presence of repetitive DNA in their inserts. A repetitive distribution pattern was observed for most of the BACs; for 38% of them, highly repetitive pericentromeric or subtelomeric signals were observed. These distribution patterns corresponded to pericentromeric and subtelomeric heterochromatin blocks observed with other staining methods. Altogether, the results indicate that around half of the common bean genome is heterochromatic and that genes and repetitive sequences are intermingled in the euchromatin and heterochromatin of the species.
| S-EPMC2886897 | biostudies-literature
Project description:Urinary exosomes have been proposed as starting material for discovery of protein biomarkers of kidney disease. Current protocols for their isolation use a two-step differential centrifugation process. Due to their low density, exosomes are expected to remain in the low-speed (17,000 x g) supernatant and to sediment only when the sample is spun at high speed (200,000 x g). Analysis using western blot and electron microscopy found that urinary exosomes are also present in the low-speed pellet entrapped by polymeric Tamm-Horsfall protein, thus diminishing the procedure's reproducibility. Here we show that addition of dithiothreitol to the low-speed pellet disrupted the polymeric network, presumably by reduction of disulfide bonds linking the monomers. This modification shifted the exosomal proteins from the low- to the high-speed pellet. Also, by shifting the Tamm-Horsfall protein to the high-speed pellet, the use of dithiothreitol makes it feasible to use Tamm-Horsfall protein to normalize excretion rates of exosomal proteins in spot urines. We tested this by western blot, and found that there was a high degree of correlation between exosomal proteins and Tamm-Horsfall protein in the high-speed pellet. Since the yield of exosomes by differential centrifugation can be increased by chemical reduction, Tamm-Horsfall protein may be a suitable normalizing variable for urinary exosome studies when quantitative urine collections are not practical.
| S-EPMC3398710 | biostudies-literature
Project description:BACKGROUND: Legumes are a rich source of proteins but are also potential elicitors of IgE-mediated food allergy. This study aimed to isolate and characterize a major allergen of Phaseolus vulgaris (kidney bean) and determine its allergenicity. METHODOLOGY: Kidney bean allergen was purified using Q Sepharose column (anion exchanger) and eluates with high intensity were pooled to purify protein using Superdex 75 (gel filtration) and C18 column (RP-HPLC). Patients with history of kidney bean allergy were skin prick tested (SPT) with crude kidney bean extract and the purified protein. Specific IgE was estimated in sera by enzyme-linked immunosorbent assay (ELISA). Characterization of purified protein and its cross-reactivity was investigated by immunobiochemical methods. Identification of purified protein was carried out by tandem mass spectrometry. PRINCIPAL FINDINGS: Purified protein appeared as a single band at 31 kDa on SDS-PAGE and showed IgE binding to 88% patients' sera by ELISA and immunoblotting. SPT with purified protein identified 78% hypersensitive patients of kidney bean. Significant release of histamine from sensitized basophils was observed after challenge with purified protein. PAS staining suggested it to be a glycoprotein, but no change in IgE binding was observed after periodate oxidation. The 31 kDa protein remained stable for 60 min on incubation with pepsin. The purified protein had high allergenic potential since it required only 102 ng of self protein for 50% IgE inhibition. Mass spectrometric analysis identified it as Phytohemagglutinin. It also showed hemagglutination with human RBCs. Cross-reactivity was observed with peanut and black gram with IC50 of 185 and 228 ng respectively. CONCLUSION/SIGNIFICANCE: A 31 kDa major allergen of kidney bean was purified and identified as phytohemagglutinin with cross-reactivity to peanut and black gram.
| S-EPMC3650053 | biostudies-literature
Project description:Two protein components were isolated in a highly purified state from a toxic fraction from the navy (haricot) bean (Phaseolus vulgaris). One, a lectin, strongly agglutinated horse erythrocytes and leucocytes, agglutination being readily observable with both types of cell at a lectin concentration of 4mug/ml. The other component (component 1), although possessing some similarity in composition, was thought to be non-agglutinating or, at most, only very weakly so. Component 1 had a mol.wt. of about 143000 and a subunit mol.wt. of about 37000, suggesting a tetrameric structure probably with identical subunits. Alanine was the only N-terminal amino acid identified and the molecule was notable in being devoid of tryptophan and cysteine, low in methionine and high in leucine, glutamic acid and aspartic acid. The lectin was somewhat smaller (mol.wt. about 114000) and apparently also composed of four identical subunits of mol.wt. about 30000. Dansylation showed that arginine occupied the N-terminus of the polypeptide chain. Aspartic acid, serine, threonine and leucine were the predominant amino acids of the lectin, and the sulphur-containing amino acids were entirely absent. Both constituents were glycoproteins and the compositions of the carbohydrate portions (4.9% for component 1 and 8.1% for the lectin) were generally similar, consisting of mannose and glucosamine with smaller amounts of glucose and traces of xylose and arabinose.
| S-EPMC1166299 | biostudies-other
Project description:PURPOSE:Urinary extracellular vesicles (uEVs) are a novel source of biomarkers. However, urinary Tamm-Horsfall Protein (THP; uromodulin) interferes with all vesicle isolation attempts, precipitates with normal urinary proteins, thus, representing an unwanted "contaminant" in urinary assays. Thus, the aim is to develop a simple method to manage THP efficiently. EXPERIMENTAL DESIGN:The uEVs are isolated by hydrostatic filtration dialysis (HFD) and treated with a defined solution of urea to optimize release of uEVs from sample. Presence of uEVs is confirmed by transmission electron microscopy, Western blotting, and proteomic profiling in MS. RESULTS:Using HFD with urea treatment for uEV isolation reduces sample complexity to a great extent. The novel simplified uEV isolation protocol allows comprehensive vesicle proteomics analysis and should be part of any urine analytics to release all sample constituents from THP trap. CONCLUSIONS AND CLINICAL RELEVANCE:The method brings a quick and easy protocol for THP management during uEV isolation, providing major benefits for comprehensive sample analytics.
| S-EPMC6900072 | biostudies-literature
Project description:Arcelin-1 is a lectin-like protein found in the seeds of wild varieties of the kidney bean (Phaseolus vulgaris). This protein displays insecticidal properties, but the mechanism of action is as yet unknown. In the present study we investigated the biochemical and biophysical properties of arcelin-1 from Phaseolus vulgaris cv. RAZ-2. Native arcelin-1 is a dimeric glycoprotein of 60 kDa, built from the non-covalent association of two identical monomers. This dimer resists dissociation by chaotropic agents and is highly resistant to proteolytic enzymes. Each subunit contains 10% (w/w) neutral sugars which belong to the high-mannose and complex-type glycans attached to three glycosylation sites. No interaction of the protein with simple sugars could be detected, but arcelin-1 displays an intrinsic specificity in binding complex glycans. Arcelin-1 therefore differs from the closely related phytohaemagglutinin lectins and alpha-amylase inhibitor in several respects: oligomerization states, sugar-binding affinities and the type and number of glycan chains. These features may be related to the toxicity of arcelin-1.
| S-EPMC1219076 | biostudies-other