Nathan Sharon

Purification of Lectins and Determination of Their Carbohydrate Specificity

Publisher Summary This chapter focuses on the purification of lectins and determination of their carbohydrate specificity. A number of procedures employed for the purification of the different lectins are basically similar. A common to virtually all lectin purification schemes is affinity chromatography on immobilized sugars, which exploits the ability of lectins to combine with carbohydrates specifically and reversibly. Hemagglutination is routinely assayed with native or modified erythrocytes from humans or other animals, usually rabbits and sometimes sheep. Blood group-specific lectins are tested with a panel of typed human erythrocytes. In plants, lectins often occur as a group of closely related proteins with the same carbohydrate specificity, or isolectins. Different molecular species of the lectin may also result from intramolecular aggregation, possibly occurring during purification.

Specificity And Affinity

The hallmark of lectins is the ability to bind carbohydrates specifically and reversibly. Understanding the properties and functions of lectins, as well as using them for diverse purposes, requires knowledge of this specificity, which is the major topic of the present chapter. Several lectins combine also with non-carbohydrate ligands, either at their carbohydrate binding sites or at sites distinct from the latter. A few others possess enzymatic activity unrelated to their carbohydrate specificity. These will be discussed briefly at the end of the chapter.

Detection, Occurence And Isolation

Lectins are ubiquitous in nature, and are found in all classes of organism. They are easy to detect and often to isolate. In addition, many are available from commercial suppliers. They are now obtainable also by recombinant techniques. The classic, and still simplest, way to detect the presence of a lectin in a biological material is to prepare an extract from the material and examine its ability to agglutinate erythrocytes (Fig. 3.1) (Rüdiger, 1993). A more refined screening procedure is based on the ability of these proteins to precipitate polysaccharides (Goldstein, 1976) (Fig. 3.2) or glycoproteins. If a positive result is obtained, it is essential to show that the agglutination or precipitation is specifically inhibited by mono- or oligosaccharides, i.e., it is sugar specific (Fig. 3.1). Hemagglutination is commonly assayed by the serial dilution technique using erythrocytes from humans or rabbits. Occasionally erythrocytes that have been treated with trypsin or sialidase are employed, since such cells are often more sensitive to agglutination than the untreated cells (Fig. 3.3). Hemagglutination also serves to monitor and quantify the activity of lectins in the course of purification. Because of the wide use of the agglutination reaction, it deserves some comments (Lis & Sharon, 1986). For agglutination to occur, the lectin must bind to the cells and form cross-bridges between them. There is however no simple relation between the amount of lectin bound and agglutination. Cases are even known where considerable amounts of lectin are bound to cells, without causing agglutination. This is because agglutination is affected by many factors, among them accessibility of receptor sites, membrane fluidity and metabolic state of the cells. It is also influenced by external conditions of the assay, such as temperature, cell concentration, mixing and so on. The relative contribution of the different factors depends on both the lectin and the cells examined. When agglutination does occur and it is inhibited by monoor oligosaccharides, it serves as an indication that carbohydrate structures for which the lectin is specific are present on the surface of the cell. Additional information on the nature of the receptors may be obtained with erythrocytes pretreated with enzymes, in particular glycosidases, or with sugar-modifying reagents, such as periodate. Agglutination with lectins is also of use in following changes on cell surfaces during physiological and pathological processes.