concanavalin-a and Gaucher-Disease

Topics: Adolescent; Binding Sites; Brain; Child; Concanavalin A; Fucose; Galactose; Gangliosidoses; Gaucher Disease; Glycoproteins; Hexosamines; Humans; Infant, Newborn; Leukodystrophy, Globoid Cell; Leukodystrophy, Metachromatic; Lipidoses; Lysosomes; Mannose; Middle Aged; Protein Binding; Sialic Acids; Sphingolipidoses

Ferritin is the major iron-storage glycoprotein found in all tissues. Ferritin glycosylation can be assessed by the differential affinities of ferritin glycoforms for Concanavalin A (ConA), a lectin. The fraction of serum ferritin bound to ConA is called "glycosylated ferritin" (GF). Low GF reflects macrophagic activation and is an essential biomarker used in adult-onset Still's disease (AOSD), macrophage activation syndrome (MAS) and Gaucher disease diagnosis and therapeutic management. To date, no complete assay description and method validation according to the ISO 15189 standard has been published. This study aimed to describe and validate our method used for GF measurement and describe GF values observed in patients.. Ferritin glycoforms were separated based on their affinities for ConA using commercially available TRIS-barbital buffer, Sepharose and ConA/Sepharose 4B gels. Ferritin concentrations were measured on the Siemens Dimension Vista 1500®. We analysed 16,843 GF values obtained between 2000 and 2021 from our database of patients.. Optimal separation of ferritin glycoforms was obtained by 15-min incubation of serum with ConA/Sepharose at pH 8. The optimized volume were 0.4 mL for total serum ferritin (TSF) 30-1000 µg/L and 0.5 mL for TSF 1000-2500 µg/L. Serum with higher TSF should be pre-diluted in the TRIS-barbital buffer. Reproducibility of ferritin measurement in the TRIS-barbital buffer matrix was excellent (intra-assay CV < 1%; inter-assay CV < 4%). Reproducibility of GF assay was good (intra-assay CV < 10% for low and high ferritin samples, respectively; and inter-assay CV < 10%). Inter-operator variability was 21.6% for GF < 20%. Ferritin was stable for up to 3 days in the TRIS-barbital buffer. An inter-laboratory exchange program conducted with another French hospital showed good agreement between results. In our database, <20% GF levels were scarce, compatible with the low prevalence of Still's disease, MAS, and Gaucher disease. The 95% confidence interval for GF was [26-58]%, lower than values described in the literature for healthy individuals.. Thanks to good performances, this technique can become readily available for laboratories servicing patients with AOSD, MAS (including severe COVID-19 patients) and Gaucher disease patients.

Topics: Biomarkers; Chemistry Techniques, Analytical; Concanavalin A; Ferritins; Gaucher Disease; Humans; Macrophage Activation Syndrome; Protein Binding; Still's Disease, Adult-Onset

Gaucher spleen sphingolipid activator protein 2 was fractionated into concanavalin A binding- and non-binding fractions. These fractions each contained several bands on non-denaturing polyacrylamide gel electrophoresis (PAGE). The two fractions were further fractionated by electroblotting the proteins from preparative gels onto nitrocellulose, staining with Ponceau S to locate the bands of protein and then eluting the protein components from the nitrocellulose. A total of ten fractions, each containing only one or two major components, was collected. All of these subfractions activated beta-glucocerebrosidase and sphingomyelinase and most subfractions also activated beta-galactocerebrosidase. The structural relationship of the bands was investigated using endoglycosidase digestions. The results indicated that the two bands with the fastest mobility on non-denaturing PAGE did not contain any carbohydrate. The remaining bands showed only limited or partial digestion with endoglycosidase H and endoglycosidase D, but were readily hydrolysed with endoglycosidase F. The products of these digestions included bands with similar mobilities to the non-carbohydrate containing bands.

Topics: Concanavalin A; Electrophoresis, Polyacrylamide Gel; Galactosylceramidase; Gaucher Disease; Glucosylceramidase; Glycoproteins; Glycoside Hydrolases; Humans; Saposins; Sphingolipid Activator Proteins; Sphingomyelin Phosphodiesterase; Spleen

Gaucher's disease (GD) which is a congenital defect characterized by the accumulation of glucocerebroside in cells of the reticulo endothelial system, is associated with malignancies of the lymphoid system such as multiple myeloma, chronic lymphatic leukemia, as well as acute leukemia of childhood. We report the immunological profile of the T-cell system in GD patients and in GD carriers. We found a variable response to lectins stimulation (PHA, Con A, PWM in various concentrations) which supports the hypothesis that a persistent antigenic stimulation in GD affects all components of the immune system of those patients. A highly significant decrease in the number of NK cells was also found, which might be a predisposing factor to the increase in malignant B and other lymphoid cell proliferations described in patients with GD.

Topics: B-Lymphocytes; Concanavalin A; Gaucher Disease; Humans; Killer Cells, Natural; Lymphocyte Activation; Phytohemagglutinins; Pokeweed Mitogens; T-Lymphocytes; T-Lymphocytes, Helper-Inducer; T-Lymphocytes, Regulatory

Plastic-embedded bone marrow biopsies from four patients with Gaucher's disease have been studied histochemically. Concanavalin A (ConA) was found to bind to cytoplasmic inclusions of Gaucher cells; the binding was prevented by lipid extraction or beta-glucosidase digestion. This suggests that glucocerebrosides stored in Gaucher cells are responsible for ConA binding; ConA staining combined with lipid extraction and beta-glucosidase digestion tests may be taken as a tool for the demonstration of Gaucher's cerebrosides of possible practical importance in diagnosis and investigation of Gaucher's disease. An excess of vic-glycol groups with respect to ConA binding-sugar residues and not extractable by lipid solvents are demonstrable in Gaucher cells. Vic-glycols appear to be regularly arranged at the electron microscopy level within Gaucher cell lysosomes along typical Gaucher "tubules", where some kind of interaction between lipid and protein should occur. Acid phosphatase might be one protein species involved in such interaction.

Topics: Acid Phosphatase; beta-Glucosidase; Bone Marrow; Concanavalin A; Gaucher Disease; Glucosylceramides; Histocytochemistry; Humans; Inclusion Bodies; Lysosomes; Methenamine; Microscopy, Electron; Periodic Acid; Silver; Staining and Labeling

'Acid' beta-glucosidase of human spleen, from either normal controls or patients with type 1 (adult) Gaucher disease, was incorporated into phosphatidylcholine liposomes. The non-incorporated (soluble) Gaucher-enzyme had a higher apparent molecular weight than had the corresponding control. Liposomal 'acid' beta-glucosidase prepared from Gaucher-spleen was more thermostable than was the corresponding normal enzyme; it was also stimulated by acidic lipids to a much lesser extent. The results suggest that the genetic mutation in type 1 (adult) Gaucher disease has multiple effects on the glycoprotein form of 'acid' beta-glucosidase.

Topics: beta-Glucosidase; Cell Membrane; Concanavalin A; Gaucher Disease; Glucosidases; Glucosides; Hot Temperature; Humans; Hydrogen-Ion Concentration; Hymecromone; Kinetics; Liposomes; Molecular Weight; Phosphatidylcholines; Solubility; Spleen

Membrane-bound 'acid' beta-glucosidase of human spleen was solubilized with either sodium cholate or 'Cutscum'. The solubilized enzyme in type 1 (adult) Gaucher disease was less heat-stable than the normal enzyme, and when precipitated by ammonium sulphate it had a higher apparent molecular weight than the corresponding normal enzyme. The normal beta-glucosidase was activated by taurocholate, whereas the Gaucher enzyme was inhibited. The decrease in 'acid' beta-glucosidase activity in Gaucher disease was associated with a profound deficiency of that form of the enzyme which bound to Concanavalin A. The results are consistent with faulty processing of newly synthesized 'acid' beta-glucosidase in type 1 Gaucher disease.

Topics: beta-Glucosidase; Chemical Phenomena; Chemistry, Physical; Concanavalin A; Gaucher Disease; Glucosidases; Humans; Kinetics; Molecular Weight; Solubility; Spleen; Taurocholic Acid

Topics: Chromatography, Affinity; Concanavalin A; Female; Gaucher Disease; Glucosidases; Glucosylceramidase; Humans; Immunodiffusion; Liver; Organ Specificity; Placenta; Pregnancy; Spleen