Interrelationship among Lectins, Antigens and Antibodies with Plasma Membrane

Lectins:

Originally, the term lectin (from the Latin legeres, which means to select) referred to a special class of carbohydrate-binding proteins present in plant seeds that could differentially agglutinate human blood cells according to a person’s blood type.

Since their original discovery in plant cells by H. Stilmark some 90 years ago (at which time they were referred to as phytohemagglutinins), lectins have been identified in many other biological sources, including bacteria, molds, al­gae, sponges, snails, crabs, and fishes.

Recently, lec­tins have been identified in the membranes of mam­malian cells. Today, the term lectin has been expanded to include all carbohydrate-binding proteins that are of non-immune origin (see below).

Lectins agglutinate cells by interacting with the sugars of membrane glycoproteins. The affinity and interaction of the lectin with the surface sugar is rem­iniscent of the manner in which an enzyme combines with its substrate or an antibody combines with an an­tigen. Because the interaction of the lectin with sugar is specific (see Table 15-4), lectins can be used to map the distribution of sugars on the cell surface.

Lectin molecules contain two or more sugar-binding sites, and when large numbers of lectins bind simulta­neously to sugars on the surfaces of separate cells (thereby cross-linking the cells), the result is aggluti­nation (Fig. 15-17). It should be noted that binding of the lectin to the cell surface sugars can occur without ensuing agglutination if no cross-linking takes place.

The presence and extent of cross-linking is dependent on the balance between the lectin concentration and the numbers of surface sugars. In this respect, the lectin-sugar interaction is much like that of an antibody-antigen reaction (see below). However, un­like antibodies, which chemically are very similar pro­teins, lectins are of diverse structure, or­ganization, and size. Lectins will bind free sugars as well as sugars at­tached to cell membranes. Consequently, lectin- induced cell agglutination can be blocked by prelimi­nary addition of the appropriate free sugar to a suspension of cells.

Lectins have been used to verify that the plasma membranes of malignant cells and normal cells differ. Malignant cells are much more readily agglutinated by lectins than the normal cells from which they are derived, that is, the malignant cells can be caused to agglutinate at much lower lectin concentrations than are required to agglutinate normal cells.

It has been found that the increased agglutinability of the malig­nant cells results from increased glycoprotein mobil­ity in the lipid bilayer of the plasma membrane. Be­cause the malignant cell membrane is more fluid, lectins are able to cluster the glycoproteins in the membrane (i.e., draw them together) and thereby make it possible to form greater numbers of cross- bridges.

How lectins can be used to determine the composi­tion of the sugar chains of surface carbohydrate is il­lustrated in Figure 15-18. The lectins that are bound by unmodified cell membranes establish the nature of the terminal sugars; these may then be cleaved from the remaining carbohydrate using a specific enzyme.

The newly exposed terminal sugars are now examined for their lectin-binding characteristics, following which another round of enzymatic sugar removal is carried out. Repetition of this sequence of treatments progressively reveals the order of sugars. Lectins may also be combined with fluorescent labels or radioac­tive tracers and then be used as surface probes to ex­plore the types, quantities, and distributions of sugars in the plasma membranes of cells.

Recently, lectins have been used in affinity chroma­tography to separate different kinds of cells from one another. A column is filled with sup­porting medium (e.g., cross-linked dextrans) to which specific lectins have been chemically bound.

When a cell suspension is then percolated through the column, cells whose surface sugars interact with the lectin are retained by the column, whereas unbound cells are eluted. Although a large number of lectins have been iden­tified and their sugar-binding properties studied, the physiological role of the lectins in the cells that pro­duce them still remains unknown. Speculations on their roles include sugar storage and transport or, perhaps, some protective function.

Antigens and Antibodies:

An antigen may be loosely defined as any molecule that has the capacity to stimulate antibody production by the immune system of higher animals. Typically, antigens are glycoproteins in the membranes of cells or in other particles foreign to the animal.

For exam­ple, the antigens present in the membranes of bacte­rial cells or in the coats of viruses act to stimulate an­tibody production by the immune system of the infected animal. The antibodies or immunoglobulin’s produced in response to the presence of the antigen combine with the antigen to form a complex, and this is followed by a series of reactions in which the antigen-bearing agents (e.g., the bacteria) are destroyed.

Antibodies are synthesized by lymphocytes, a class of white blood cells. Two subpopulations of lympho­cytes may be identified: T lymphocytes and B lympho­cytes. T lymphocytes are produced by the thymus gland (i.e., “T” stands for “thymus”) and other or­gans (e.g., lymph nodes and spleen) that are “seeded” by cells migrating from the thymus during develop­ment. In birds, B lymphocytes originate in an out- pocket of the gut called the bursa of Fabricius (i.e., “B” stands for “bursa”), but in man and other mam­mals they are thought to originate in the hemopoietic tissues (i.e., the liver in the developing fetus and the bone marrow in children and adults).

The first anti­bodies produced by newly formed B lymphocytes be­come constituents of the cell’s plasma membrane, where they act as receptors for antigens; additional antibody molecules produced by B lymphocytes are secreted into the surrounding blood plasma. T lym­phocytes do not secrete antibodies instead antibody­like proteins are incorporated into the cell’s plasma membrane.

The reaction between antibody and antigen is very specific, with a particular antibody combining with only one type of antigen. An enormous variety of B and T lymphocytes are present in the body’s tissues, each capable of manufacturing only a single antibody type (and therefore capable of reacting with a single type of antigen or foreign cell).

In the case of B lymphocytes, binding of antigen by surface antibody leads to the proliferation of plasma cells, which then secrete large quantities of antibody. Following the initial reaction on the lymphocyte cell surface, subsequent antigen-antibody reactions may not involve B lymphocytes directly.

Instead, the se­creted antibodies react with either free antigens or more likely with antigens in the invading cells’ mem­branes. The antibody like proteins present in the mem­branes of T lymphocytes permit these cells to attach to the surfaces of foreign cells containing the appro­priate antigen.

This is then followed by the secretion of cytotoxic substances into the foreign cell causing its destruction. The involvement of either lymphocyte membranes or foreign cell membranes (or both) in the antigen-antibody reaction makes these molecules es­pecially valuable tools for studying the properties of the cell surface.

It should be clear that the surface antibodies of the lymphocytes of one animal can serve as antigens if these lymphocytes are transferred to the blood stream of another animal. The transferred lympho­cytes will be treated much like any other foreign cell and serve to stimulate the production of anti-­immunoglobulin antibodies (AIA). AIA has been es­pecially useful in probing the distribution of glycoproteins in the cell membrane.

M. C. Raff and S. dePetris prepared AIA that they then coupled with ferritin. Ferritin is a liver protein rich in iron and readily discernible as dark spots by transmission electron microscopy (i.e., the iron ren­ders the ferritin electron-dense). When ferritin- coupled AIA is added to suspensions of lymphocytes, it combines with their surface immunoglobulin’s, thereby assisting in their identification.

Raff and de­Petris showed that when lymphocytes are incubated at 4°C with ferritin-coupled AIA, the electron-dense spots are distributed all over the membrane surface, whereas at 20°C, the ferritin-coupled AIA is clus­tered together at one pole of the cell.The AIA is able to clus­ter the surface immunoglobulin at 20°C but not at 4°C because at the higher temperature the plasma mem­branes of the cell are more fluid.