Purification and characterization of a beta-galactoside-binding soluble lectin from rat and bovine brain.

A beta-galactoside-binding activity has been detected in mammalian brain extracts using a hemagglutination test and a nerve cell aggregation assay. Inhibition studies suggested the involvement of lectin-carbohydrate interactions in these processes. In an attempt to explore further the biological role of brain lectins, the beta-galactoside-binding activity has been purified to apparent homogeneity from bovine and rat brain by salt extraction of the brain tissue and affinity chromatography on asialofetuin-agarose. The molecular weights determined by gel filtration, under native conditions on Ultrogel AcA-34, were 30,000 for the bovine brain lectin and 32,000 for the rat brain lectin; polyacrylamide gel electrophoresis in SDS gave molecular weights of 15,000 and 16,000, respectively, suggesting that the two brain lectins are dimers. Both lectins have an isoelectric point of 3.9. Amino acid composition data indicate that both lectins contain high proportions of glycine and acidic amino acids. The lectins are specific for beta-D-galactosides and related sugars and the configuration of carbon atoms 1, 2 and 4 seems of primary importance. Moreover, the nerve cell aggregation-promoting activity of the purified lectin is 300-fold that of the crude extracts.

Purification and some characteristics of a beta-galactoside binding soluble lectin from amphibian ovary.

Soluble extracts of Bufo ovaries agglutinate sialidase-treated rabbit erythrocytes. Unlike other amphibian lectins this agglutination activity does not require the presence of calcium ions. It is specifically inhibited by D-galactose and its derivatives. Thiodi-D-galactoside is the most potent saccharide inhibitor followed by lactose and methyl-beta-D-galactoside, respectively. D-Fucose, D-glucose and D-mannose do not inhibit the activity at concentrations at or above 100 mM. The lectin has been purified 500-fold to apparent homogeneity from the ovaries by salt extraction and affinity chromatography on lactose-aminophenyl-agarose, with a yield of about 0.2%. The molecular mass determined by gel filtration under native conditions was 30 kDa; polyacrylamide gel electrophoresis in SDS gave a molecular mass of 15 kDa, suggesting that the lectin is a dimer. The lectin has an isoelectric point of 40 and contains a high proportion of acidic amino acids.

The role of T-agglutinin in the disappearance of erythrocytes artificially aged by desialylation.

The chance discovery of two mouse strains, one with and one without a high presence of serum T-agglutinin, permitted the investigation of the role of this antibody in the disappearance of desialylated erythrocytes, which may be regarded as a model for ageing. The proportional relationship between the quantity of sialic acid removed and the diminution of half-life is not affected by the presence or absence of T-agglutinin. Opsonization by T-agglutinin would therefore appear to be an improbable mechanism. Other possible theories are discussed.

Does red blood cell size correlate with red blood cell age in mouse?

Mouse red blood cells (RBC) can be fractionated according to their size by counterflow centrifugation. The mean corpuscular hemoglobin content and the enzyme activities (ASAT, LDH, PK and acetylcholinesterase) increase when the mean corpuscular volume (MCV) rises. However, the in-vivo survival of size-separated RBC is similar whatever their MCV is; thus, counterflow centrifugation is not a suitable procedure to achieve an age fractionation of mouse RBC. Moreover, RBC subpopulations collected by counterflow centrifugation are different from those obtained when RBC are fractionated according to their density.

Purification and characterization of natural antibodies that recognize a human brain lectin.

We have recently identified oligoclonal IgG antibodies that are related to a human brain lectin (HBL14) from serum and cerebrospinal fluid of patients with neurological disorders. They were termed lectin-like IgG (L-IgG) (Joubert-Caron et al., 1994a,b). In this paper, the occurrence of antibodies reactive both towards HBL14 and L-IgG was investigated. Binding of antibodies to HBL14 was demonstrated by solid-phase ELISA and chromatography on immobilized HBL14. Fab fragments of these antibodies were also shown to bind to HBL14. The specificity of the antibodies towards HBL14 was studied using a panel of different antigens. Our data show that individual sera from healthy people as well as a pool of immunoglobulins from 80 blood donors contain an IgG autoreactivity to HBL14, while no IgM autoreactivity was detected. Anti-HBL14 antibodies from sera were purified using affinity chromatography on immobilized HBL14. Affinity chromatography further allowed us to demonstrate that the binding of anti-HB14 antibodies was mediated through their Fab fragments. A higher amount of anti-HBL14 antibodies was purified using a L-IgG-depleted fraction of sera. The binding of anti-HBL14 antibodies to L-IgG was confirmed by ELISA. Finally, anti-HBL14 antibodies were found to be polyreactive. These results indicate the occurrence of a novel class of natural antibodies reactive towards a human brain lectin and suggest that these antibodies may participate in immunoregulatory mechanisms probably though idiotypic/anti-idiotypic interaction.

?-Galactoside soluble lectin from human brain: complete amino acid sequence.

A soluble lectin from human brain, specific for ?-galactoside-containing glycoconjugates, has been sequenced and compared with a similar protein purified from human placenta. The sequence was established from the analysis of the peptides obtained by chemical and proteolytic cleavage. The brain lectin has a complete homology with that of placenta. This and similar results observed in other mammals strongly suggest that there is only one gene, per species, encoding for this protein.

Oligoclonal beta-galactoside-specific gamma immunoglobulins allow the immunocytochemical detection of cellular antigenic determinants expressed in mouse brain after surgical injury.

Histological brain sections were probed with human oligoclonal lectin-like IgGs (L-IgG) purified from normal serum. In intact brain, antigenic determinants for these IgG were restricted to some blood vessel endothelial cells. By contrast, during the inflammatory reaction following a surgical injury, these determinants were detected at the cell surface of different cell types, within and near the lesion site. The cells reacting with L-IgG consisted of endothelial cell, mature astrocytes, activated microglial and ependymal cells.

Brain lectin-mediated agglutinability of dissociated cells from embryonic and postnatal mouse brain.

Brain extracts contain a soluble lectin which enables the agglutination of dissociated mouse brain cells via saccharidic receptors. The ability of the brain cells to be agglutinated depends on their stage of development in vivo. Furthermore, after birth, the mechanism of the lectin-promoted agglutination is complicated by the appearance of a self-aggregation of the dissociated cells. Lactose and galactosides are inhibitors of lectin-mediated agglutination as well as of the dissociated cells' self-aggregation.

Human brain lectin immunoreactive material in cerebrospinal fluids determined by enzyme immunoassay (EIA).

A sensitive enzyme immunoassay (EIA) micromethod is described which can measure levels of a 14 kDa human brain lectin (HBL) in the cerebrospinal fluid (CSF) of patients submitted to CSF examination. The assay is based on the use of a polyclonal antibody to HBL and the simultaneous application of biotinylated and unlabeled HBL. Biotin was then reacted with a streptavidin-peroxidase (Strep-HRP) conjugate and the bound enzyme quantified with the substrate orthophenylenediamine (OPD). The assay requires only 50 microliters of CSF and is very sensitive: as little as 6 ng/ml of HBL 14 can be detected. In a blind-test screening, the mean (+/- SEM) concentration of the HBL immunoreactive material (HIM) in CSF was determined to be 72.4 +/- 6.6 ng/ml. Our results indicate that EIA measurement of HIM levels in the CSF may find useful applications in elucidating the involvement of HBL in the physiopathology of human nervous system (NS).

Anti-galectin-1 autoantibodies in serum of patients with neurological diseases.

The presence of autoantibodies to human brain galectin-1 was investigated in serum from patients with multiple sclerosis, patients with or without evidence of other neurological disorders, and healthy controls, using an ELISA on purified brain galectin-1. Levels of autoantibodies to galectin-1 were significantly higher in patients than in healthy controls. Comparison of levels of anti-galectin-1 and anti-idiotypic antibodies mimicking human brain galectin-1 (L-IgG) showed that the highest levels of autoantibodies were present in patients with low levels of L-IgG. This finding can be explained by hypothesizing that the concentration of autoantibodies to galectin-1 is possibly associated with impairment of the regulation of the immune system.

Changes in carbohydrate content of surface membranes of human erythrocytes during ageing.

The carbohydrate content (neutral hexoses, fucose, sialic acid, and hexosamine) of human erythrocytes is significantly higher in young than in old erythrocytes. The differences observed in the carbohydrate content of surface glycopeptides, obtained after incubation with either Pronase or trypsin, is still more striking, suggesting important modifications of the carbohydrate moeties of the erythrocyte membrane during in vivo ageing.

Individual variations of the seven carbohydrate components of human erythrocyte membrane during aging in vivo.

The contents of fucose, mannose, galactose, glucose, 2-acetamido-2-deoxy-D-glucose and -D-galactose, and sialic acid, when the results were expressed as nmol per mg of membrane dry-weights, were found to be significantly lower in the membranes of old erythrocytes than in the membranes of young ones. No significant difference was found between young and old membranes when the compositions were expressed as residues per one hundred carbohydrate residues, suggesting that a homogeneous decrease of the carbohydrate moieties may occur during aging in vivo.

In vivo ageing of human erythrocytes and cell-surface labeling by D-galactose oxidase and sodium borotritide.

Young and old, human erythrocytes, separated in vitro according to their age in vivo, were radioactively labeled at the cell-surface D-galactosyl and 2-acetamido-2-deoxy-D-galactosyl residues by treatment with D-galactose oxidase, followed by reduction with sodium borotritide. The labeling was quantitatively determined for each type of erythrocyte by measuring the molar amounts of borohydride necessary for the complete reduction of the oxidized residues. The number of surface residues per blood-group A+ erythrocyte was found to be 37.6 +/- 1.8 x 10(6) (n = 8) for young, 21.8 +/- 4.9 x 10(6) (n = 8) for old, and 24.8 +/- 6.4 x 10(6) (n = 8) for middle-aged erythrocytes, indicating a significant decrease of the residues during ageing.

N-Acetyl-beta-D-glucosaminyl-binding properties of the envelope glycoprotein of human immunodeficiency virus type 1.

The effect of carbohydrate structures on the adsorption of HIV-1 or of recombinant envelope glycoprotein gp 160 (rgp 160) to cells of the CEM line was investigated with an indirect immunofluorescence assay using gp 120-specific mouse monoclonal antibodies (mAbs) directed to envelope gp 120. The beta-D-galactosyl, alpha-D-mannosyl, beta-D-glucosyl, N-acetyl-beta-D-glucosaminyl, sialosyl, and L-fucosyl derivatives tested had no effect on this binding. However, preincubation of HIV-1 (or rgp 160) with the neoglycoprotein, beta-D-GlcNAc47-BSA, specifically inhibited the labeling, by some of the mAb used, of HIV-1 (or rgp 160) bound at the cell membrane. This inhibition occurred only with mAbs that were specific for the immunodominant "neutralizing" third variable region (V3) of gp 120. Competition for the binding to rgp 160 between beta-D-GlcNAc47-BSA and mAb was further demonstrated by use of affinity matrices substituted with one of the relevant mAb (110-4), or with beta-D-GlcNAc47-BSA. Besides beta-D-GlcNAc47-BSA-Sepharose, rgp 160 also bound with low affinity, but high specificity, to two other N-acetyl-beta-D-glucosaminyl affinity matrices, beta-D-GlcNAc-divinylsulfone-agarose and asialoagalactothyroglobulin-agarose. Conversely, beta-D-[125I]GlcNAc47-BSA bound specifically to gp 160-Sepharose. These results indicated that rgp 160 behaves as a N-acetyl-beta-D-glucosaminyl-binding protein for GlcNAc residues presented at high density on a carrier, the carbohydrate-binding site of which is close to, or located on the V3 region of gp 120.

[Interest of the determination of reference values for the monosaccharide composition of the human erythrocyte membrane (author's transl)]. / Intérêt de l'établissement de valeurs de référence pour la composition en monosaccharides de la membrane d'hématie humaine.

The carbohydrate composition of the human erythrocyte membrane was just established ten years ago. Carbohydrates have crucial functions in the membrane structures and the determination of this composition may have an important semeiological signification. In this way, reference values are necessary. The existence of possible variations of the composition with two physiological parameters: the groups in the ABO system and the age of erythrocytes have been investigated and are reported. Their repercussion on the determination of reference values is discussed.

Ageing in vivo and neuraminidase treatment of rabbit erythrocytes: influence on half-life as assessed by 51Cr labelling.

Old and young rabbit erythrocytes, separated by centrifugation, contained different respective activities of glucose-6-phosphate dehydrogenase, pyruvate kinase and acetylcholinesterase, and different quantities of stromal sialic acid. A systematic study of the survival rate of young and old erythrocytes incubated with different amounts of Vibrio cholerae neuraminidase is described. The half-life of intact old erythrocytes is significantly shorter than that of young erythrocytes with a similar sialic acid content.

Lecithin content estimate of human alveolar lining layer: comparison with mouse, rat and rabbit.

Saturated as well as total lecithins recovered by lung lavage are linearly correlated with the species body weight and with their respiratory rate. The value of the ratio:amount of saturated lecithins recovered by lung lavage to theoretical minimum amount needed to form a monomolecular film over the alveolar surface at functional residual capacity decreases linearly according to the respiratory rate of the species examined. In man this ratio is lower than 1 (0.45 +/- 0.11) and suggests the possible existence of a discontinuous mono molecular film of lipidic surfactant.