Lectin is a kind of glycoprotein or sugar-binding protein purified from various plants, invertebrates and higher animals, so it is named lectin because it can agglutinate red blood cells (including blood group substances). See table 6- 1 for its common types. Phytoagglutinin (PNA) is commonly used, and it is usually named after the plant from which it is extracted, such as sword bean A (ConA), Wheat germ agglutinin, WGA), Peanut agglutinin, PNA) and Soybean agglutinin, SBA). Lectins are not the products of origin or immune reaction. They are included in this book because of their "affinity" characteristics, which can be applied by immunocytochemistry techniques. Therefore, Ponder( 1983) suggested that it should be called "lectin histochemistry" instead of "lectin immunohistochemistry".
I. Characteristics of lectin
Lectin has many characteristics, and here we only briefly mention some of its characteristics related to the application of immunocytochemistry technology. We know that biofilm contains a certain amount of sugar, mainly in the form of glycoprotein and glycolipid. The biggest feature of lectins is that they can recognize the complex carbohydrate structure in glycoprotein and glycopeptide, especially in cell membrane, that is, the carbon lipid determinant on the surface of cell membrane. A lectin has the ability to specifically bind to a specific sugar group, such as sword bean binding to α-D-mannose; Maltin is combined with N-acetyl glucosamine; Bean agglutinin binds to N-acetyllactosamine (see Table 6-1in this chapter). Therefore, lectin can be used as a probe to study specific sugar groups on cell membranes. On the other hand, lectin has multivalent binding ability, and can bind with tracers such as fluorescein, biotin, enzyme, colloidal gold and ferritin, thus showing its binding site at the level of light microscope and/or electron microscope.
Second, the application of lectin
It is generally believed that specific glycosyl groups on cell membrane can be used to distinguish cell types and reflect the changes of cells in differentiation, maturation and tumor cell degeneration. Only in some special cases, the cell binding capacity of lectin can be estimated in advance, such as the specificity of lentinin to blood group A and the specificity of Vitex negundo agglutinin to blood group O 2-L-fucose. However, in most cases, the types of carbohydrate determinants recognized by lectins and the properties and functions of molecules carrying determinants are completely discovered by experimental experience.
1.As a marker of cell differentiation and maturation, lectin is used as a marker of cell differentiation, and the application report in this respect is the most, and the research focuses on the grouping of blood cells, especially lymphocytes. For example, Rose( 1980) found that immature T lymphocytes in the thymus cortex of mice were PNA-positive, and about 20% of PNA-positive cells were also found in the germinal center of small intestinal lymph nodes in mice. Whether the latter belonged to immature T lymphocytes is a question worthy of further study. Newman et al. (1979) labeled lectin PNA with fluorescein, and found that it showed different fluorescence intensity at different differentiation stages of rat mammary epithelium. In immature rat mammary epithelial cells, the fluorescence is weak or absent, and the fluorescence intensity of mammary epithelial cells gradually increases from sexual maturity to pregnancy, while the fluorescence intensity reaches the peak during lactation. In the process of differentiation and maturation of skin keratinocytes from basement to surface, the distribution and properties of carbohydrates on the cell surface are changing. Brabed et al. (198 1) applied the skin of newborn rats, and the results showed that the cells in each layer of the skin combined with different lectins respectively. Maltin binds to keratinocytes, ricin binds to spinous cells and basal cells, and Vitex negundo agglutinin is labeled on the surface of spinous cells. During the differentiation and maturation of myeoblast, Winaod and Luzzati( 1975) noticed similar skin changes.
2. Kivela and Farkkanen( 1987), as special types of cells, were found in human retina. PNA labeled cone cells but not rod cells. In breast, breast epithelial cells showed PNA positive reaction, while myoepithelial cells and interstitial cells showed PNA negative reaction. The renal tissue sections of mice, rats and rabbits were stained with various lectins. The results showed that sword bean A and ricin existed in all parts of the kidney, PNA and double-flower lentil lectin (DBA) mainly distributed in epithelial cells of distal convoluted tubules and collecting tubules, Vitex negundo lectin (UEA) mainly distributed in vascular endothelial cells, while maltol was distributed in glomerulus. The study of mice with RIII and DDK strains by DBA showed that DBA was mainly bound to capillary endothelial cells in various tissues, and electron microscope observation showed that DBA was bound to the surface of endothelial cells. Interestingly, the endothelial cells in some tissues of mice with RIII strains showed positive DBA negative reaction, indicating that there were also tissue-specific differences in vascular endothelial cells of the same genus. Streit and Kreutzberg( 1987) found that Griffonia Simplicifolia agglutinin specifically labeled microglia in facial ganglion, and other types of glial cells such as astrocyte showed negative reaction. After the facial nerve was cut off, the response of proliferating microglia to Griffonia Simplicifolia agglutinin was strengthened. The observation of immunoelectron microscope showed that agglutinin was mainly deposited on the surface of cell membrane or axonal membrane of microglia, and the specific binding sugar group was α-D-galactose. The location of lectin receptors in human embryos and various normal tissues was systematically studied by using 12 lectins (Table 6- 1) in the immunopathology room of the tumor hospital affiliated to Shanghai Medical University. The results showed that there was no definite rule in the distribution of lectin receptors. For example, the main cell of gastric mucosa is PNA receptor, while the parietal cell is BSL receptor, and the double-flowered lentil receptor (DBA) mainly appears in the large intestine.
3. Changes in lectin binding in tumors Tumor cells are accompanied by changes in cell membranes, and sugar groups on cell membranes will also change accordingly, which can be detected by lectins. A large number of studies have found that lectin can be used as a marker of tumor tissue origin, tumor specific diagnosis, tumor malignancy and differentiation of different tumors. For example, Zhang Huazhong et al. (1987) reported 1 15 cases of gastric cancer, the positive rate of PHA was as high as 90.43%, while the normal gastric mucosa was basically negative, so PHA was considered as a diagnostic marker of gastric cancer. The positive rate of BSA for breast malignant tumor was 79%, but it was negative for benign lesions, suggesting that BSA may be a related marker of breast malignant tumor. Lectin is also helpful to distinguish the histological types of tumors, such as ConA positive for astrocytoma of nervous system, negative for microglioma, UEA 1 negative for renal adenocarcinoma and positive for clear cell carcinoma.
Third, the classification of lectins
Lectins can be classified according to sugar specificity, molecular structure, binding sites and their functions. Animal lectins are classified into C- type lectins, S- type lectins, P- type lectins, I- type lectins and Pentraxins according to their molecular structures. C- type lectin is Ca2+ dependent lectin; S- type lectin is a lectin that specifically recognizes β -galactoside bond; P- type lectin is a lectin that specifically recognizes mannose 6 phosphate; Type I lectin is similar to immunoglobulin; Pentraxins is a lectin with five subunits.
Fourth, the nature of lectin
So far, lectins found in invertebrates are glycoproteins, and sugars are bound into lectins in the form of valence bonds. The types of sugars mainly include mannose, glucosamine and galactose, but xylose and arabinose are rare. The types of sugar contained in animal lectins are different from those contained in plant and microbial lectins. The protein part of lectins is mainly composed of aspartic acid, serine and threonine, and sulfur-containing amino acids are rare. The metal ions related to some lectin activities are often Ca2+ and Mg2+, which are necessary for many sugars to combine or agglutinate. The existence of Ca2+ is an essential condition for agglutination of many lectins (such as type C lectins). In the lectin (Limulin), it is necessary to expand C and carry out physiological activities in the form of calmodulin. In Anthocidariscr assispina, it is Ca2+ that affects the molecular configuration of lectin; Ca2+ affects oyster lectin by changing the protein configuration, rather than directly participating in ligand binding. Some people think that Ca2+ interacts with spindle through ionic bonds to stabilize the structure and enhance the interaction between hydrogen bonds and hydrophobic groups.
The agglutination reaction of lectins is often inhibited by monosaccharides, while some require some disaccharides, trisaccharides or polysaccharides, and the sensitivity of inhibition is quite different. Some typical specific lectins are easily inhibited by some sugars in the corresponding blood group substances, such as the specific lectin of blood antigen A is inhibited by N- acetyl -D-galactose; O-type specific lectin was inhibited by L-fucose. The specificity range of lectin binding sugar is different. The binding range of a few lectins is quite narrow. Mild treatment of lectin with protease, such as trypsin and pronase, can improve the sensitivity of agglutination activity, and some additives and metal ions also affect lectin activity.
Fifth, study the significance of lectin
Lectins exist widely in animals and plants. The biggest feature of lectin is that it can recognize the complex carbohydrate structure in glycoprotein and glycolipid, especially in cell membrane, that is, the sugar group on the surface of cell membrane. A lectin has the ability to bind specifically to only one specific sugar group. Therefore, lectin can be used as a probe to study the structure of cell membrane. Lectin has a variety of biological activities in invertebrate blood, which can selectively agglutinate various cells and has specific agglutination effect on tumor cells. It is one of the important humoral factors of immune defense. On the other hand, lectin has multivalent binding ability, which can bind with fluorescein, enzyme, biotin, ferritin and colloidal gold without affecting its biological activity. It can be used in immunocytochemistry research at the level of light microscope or electron microscope, and has certain value in exploring the biological evolution process of cell differentiation, proliferation and malignant transformation, displaying tumor-related antigen substances, and diagnosing and evaluating tumors. In addition, plant lectins also play an important role in plants, such as in the process of seed germination; As a mitogenic factor of plant embryo cells; The protective function in crop pest control and so on. Studying the specificity of lectin is helpful to understand life phenomena or pathological changes at Molecular or atomic level.