Production and characterization of a monoclonal antibody able to discriminate galectin-1 from galectin-2 and galectin-3.

Antisera raised against galectin-1 exhibit crossreactivities with other galectins or related molecules. In order to overcome this problem, a monoclonal antibody to human brain galectin-1 was obtained by selecting clones without reactivity toward galectin-3. This mAb specifically bound galectin-1 of various animal origins but neither galectin-2 nor galectin-3. Western-blotting analysis of soluble human brain extracts after 2D gel electrophoresis revealed only the two most acidic isoforms of galectin-1. The ability of this mAb to bind galectin-1/asialofetuin complexes indicates that its epitope is not localized in the carbohydrate recognition domain of galectin-1. This particularity induces with efficiency its monospecificity.

Identification of different galectins by immunoblotting after two-dimensional polyacrylamide get electrophoresis with immobilized pH gradients.

Vertebrate soluble beta-galactoside-binding lectins form a growing protein family that recently have been named galectins. Seven different galectins have been sequenced and characterized in mammals, and there is compelling evidence for the existence of other members of this lectin family. Three among six galectins are homodimers with (i) an identical subunit of a relative molecular mass of about 14500, and (ii) amino acid sequence homologies giving rise to possible immunochemical cross-reactivities. They are indistinguishable from each other by conventional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), even when followed by immunoblotting. However, their different isoelectric points allow their identification using isoelectric focusing and two-dimensional (2-D) polyacrylamide gel electrophoresis. A strategy was developed to identify these galectins in crude extracts from cells and tissues, based on the two-dimensional electrophoresis with immobilized pH gradient (IPG-Dalt) analysis of the specific spots of purified galectins and of the spots of crude extracts, after silver staining. In addition, 2-D immunoblotting using anti-galectin 1 (Gal-1) and anti carbohydrate-binding protein 15 (CPB15) antibodies were performed on brain and leukemia cells (HL60) allowing an identification of related polypeptides. Our results indicate that the use of IPG-Dalt provides a suitable reproducibility and allows the detection of galectins or other galactoside-binding proteins even at basic pIs.

Changes in S-type lectin localization in neuroblastoma cells (N1E115) upon differentiation.

The distribution of a 14.4 kDa S-type lectin was examined in murine neuroblastoma cells, either undifferentiated or after differentiation induced by dibutyryl-cyclic adenosine monophosphate. In undifferentiated cells the immunoreactivity was detected extracellularly, associated with the plasma membrane and in bulges released into the extracellular milieu. Important modifications of the lectin localization were associated with the differentiation process that induced an increased cytosolic expression and a decreased externalization. Possible functions for the lectin expressed intracellularly in the differentiated cells are also considered.

Use of an immobilized human endogenous lectin for the purification of complementary ligands.

1. A galactoside-specific endogenous lectin isolated from human brain was covalently immobilized on divinylsulfone-activated agarose. This highly selective affinity adsorbent proved to be useful in purifying soluble protein ligands. 2. The maximum binding capacity of the adsorbent for complementary proteins was calculated to be 618 micrograms per g of gel (wet resin). 3. Sequential elutions using 0.1 M lactose, 0.3 M lactose and 0.5 M NaCl, and competition assays using incorporation in the presence 0.1 M lactose revealed differences in lectin-ligand interactions.