Isolectins from wax bean with differential agglutination of normal and transformed mammalian cells

doi:10.1016/0005-2795(73)90030-5

Biochimica et Biophysica Acta (BBA) - Protein Structure

Volume 310, Issue 1, 17 May 1973, Pages 273-277

https://doi.org/10.1016/0005-2795(73)90030-5 Get rights and content

Abstract

Wax bean agglutinin prepared according to Liener was separated by chromatography on hydroxylapatite into three fractions, two of which were biologically active. Both fractions are glycoproteins with essentially identical amino acid composition and molecular weight, each consisting of four subunits, molecular weight 30 000. The isolectins agglutinated transformed cells at a concentration about 100 times lower than that required to agglutinate normal cells. Agglutination was inhibited by fetuin but not by any of the simple sugars tested.

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TwoTwo lectins, GNL-1 and 2, were isolated from extracts of Great Northern bean powder through fractionation with ammonium sulfate, ion-exchange chromatographies on CM- and DEAE-celluloses, and gel filtration chromatography on Sephacryl S-200 HR. These lectins were shown to be homogenous by gel electrophoresis, gel filtration, and isoelectric focusing. The lectins (GNL-1 and 2) have molecular masses of 175 and 145 kDa on gel filtration, respectively. They yield three bands having the respective same molecular masses on SDS-PAGE (GNL-1; α-subunit of 34.5 kDa, β of 37.0, and γ pf 39.0; GNL-2; α′ of 34.5 kDa, β′ of 37.0, and γ′ of 39.0). Two lectins are shown to be glycoproteins and the carbohydrate contents of GNL-1 and 2 are 5.1 and 4.5%, respectively. The isoelectric points are 5.5 and 5.1 and the extinction coefficients (A_1cm^1% at 280 nm are 11.37 and 11.45, respectively. These lectins are nonspecific in agglutination for rabbit and any types of human erythrocytes. Inhibition study show no specificity against mono and disaccharides. On the other hand, bindingu assay of horseradish peroxidase-glycoproteins to the bands electroblotted onto PVDF membrane reveales that all of the subunits can bind to sugar moieties in fetuin, asialofetuin, and porcineu thyroglobulin specifically. Moreover, assay of mitogenic activity shows that GNL-1 is a strong mitogen, but GNL-2 is lack of the activity.
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Purified lectin proteins from the bean cultivar ‘Tendergreen’ were separated on S-P Sephadex C-50. The five major fractions obtained corresponded to the postulated five tetrameric forms: E₄, E₃L₁, E₂L₂, E₁L₃ and L₄, composed of an erythro-agglutinating subunit (E) and a leuco-agglutinating subunit (L). Further separation using one- and two-dimensional electrophoresis showed greater complexity for the polypeptide composition of these fractions. In particular, two major polypeptides differing from the major erythro-agglutinating polypeptide were found in the L₄, E₁L₃ and E₂L₂ fractions. Also, two minor polypeptides were present in the E₄, E₃L₁, E₂L₂ and E₁L₃ fractions.
Physical, chemical, and nutritional properties of common bean (phaseolus) proteins
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This chapter discusses that the family Leguminosae includes approximately 600 genera, with about 13,000 species. In legume seeds, the cotyledons form the bulk of the seed and synthesize most of the proteins. Research on identification and quantification of the storage proteins in beans (Phaseolus) goes back several decades. These investigators referred to three proteins in Phaseolus (phaseolin, phaselin, and conphaseolin). The chapter discusses that bean proteins were first studied by Osborne. He found that the extractable proteins were mostly globulins, a fact which was later confirmed and emphasized by other investigators, who found three globulins in Phaseolus seeds. The biological value of bean proteins in general is low as compared to most other food proteins. According to Boyd, hemagglutinating activity was first described by Stillmark in 1888 in extracts of castor bean (Ricinus communis) and the first plant lectin found to be blood group specific was from lima bean (Phaseolus lunatus). Proteins that inhibit a-amylases from animals and insects have been reported in a variety of plants. Common beans (Phaseolus vulgaris) are normally harvested with about 20% moisture in the seeds and dried to about 10% water content before storage or consumption. The chapter discusses that common beans contain from 18% to 35% protein, however, their biological value is much lower than expected on a protein and amino acid composition basis.
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Wax bean agglutinin (WBA) was found to mimic the activities of insulin in mediating glucose oxidation and antilipolysis. In contrast, soybean and peanut agglutinins do not exert any of these activities. Unlike concanavalin A and wheat germ agglutinin that were reported previously to exhibit insulin-like activites, WBA neither enhances nor competes with the [ $^{125} I$ ]insulin binding at relatively high concentrations. Moreover, mild trypsinization of adipocytes, a treatment which greatly diminishes the binding and bioactivity of insulin in fat cells, only slightly affects glucose oxidation induced by WBA. ED₅₀ values for WBA mediated glucose oxidation and antilipolysis are 9.3 μg and 40.0 μg, respectively, compared with the nearly identical concentrations required for 50% of maximal effect of both glucose oxidation and antilipolysis, mediated by wheat germ agglutinin. The present studies suggest that these two activities may be triggered by WBA via surface glycoproteins that are distinct from the binding site of insulin.
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The Lectins: Carbohydrate-Binding Proteins of Plants and Animals
1978, Advances in Carbohydrate Chemistry and Biochemistry
Lectins play an important role in the development of immunology. Lectins also find application in serological laboratories for typing blood and determining secretor status, separating leucocytes from erythrocytes, and agglutinating cells from blood in the preparation of plasma. They serve as reagents for the detection, isolation, and characterization of carbohydrate-containing macromolecules, including blood-group antigens. In their interaction with saccharides, lectins serve as models for carbohydrate-specific antibodies, with the important advantage to purify lectins in gram quantities. Lectins are classified according to their carbohydrate-binding specificity that includes D-mannose(D-glucose)-binding lectins and 2-acetamido-2-deoxy-D-glucose-binding lectins. The chapter considers only those lectins that have been purified to homogeneity, and studied with regard to their biophysical, biochemical, and carbohydrate-binding specificity. The chapter also describes the cell-binding and biological properties of lectins. The chapter concludes with the description of several glycopeptide structures showing the carbohydrate-binding loci with which various lectins interact.

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Isolectins from wax bean with differential agglutination of normal and transformed mammalian cells

Abstract

Biochim. Biophys. Acta

Biochim. Biophys. Acta

Biochim. Biophys. Acta

Biochim. Biophys. Acta

Arch. Biochem. Biophys.

Science

Nature