Glycoproteins - Hughes, R. 1985

Functions
Cell Adhesion
Cell-to-Cell Adhesion

Cell adhesion plays a crucial role in The formation of Tissues and Organs during Embryogenesis. During embryonic development, totipotent Cells undergo differentiation, sorting into organized clusters of similar cells, followed by cell translocation and the final formation of specialized organs. These processes depend on specific intercellular recognition and selective cell adhesion. Classic experiments with cells taken from embryos or differentiated organs (such as The Heart, Kidneys, Liver, etc.) indicate that adhesion Specificity is determined by the differentiated cells themselves. Cells derived from different organs and subsequently suspended together exhibit The ability to reaggregate according to a tissue-specific principle; for instance, heart tissue cells exclude liver cells during aggregation.

As already noted, disruptions in the normal glycosylation process frequently prevent development, particularly in sea urchin embryos or certain mammalian systems (e.g., the Kidney). Therefore, Glycoproteins are attributed an important role in many cell interactions that govern normal development.

Cell adhesion is most likely driven by reactions between complementary molecules. Such interactions may fall into the following two types, provided that carbohydrate components of glycoproteins are indeed involved: 1) reactions between carbohydrate chains, analogous to the interactions between chains of many linear Polysaccharides [4], and 2) Reactions between a carbohydrate component and a carbohydrate-binding protein, i.e., a lectin. Recently, the second possibility (Fig. 4.12) has been favored, since direct interactions between glycoprotein glycans have not been detected. On the other hand, carbohydrate-binding Proteins with the necessary specificity have now been discovered, and their localization on The Cell surface has been established. In A number of cases, these proteins appear precisely at the time when specific changes occur during development, supporting the hypothesis of protein-carbohydrate interactions. The latter apparently take place between molecules integrated into the cell surface membranes of the responding cells (Fig. 4.12, a). Alternatively, aggregation factors may also participate in these processes (Fig. 4.12, b, c). These could be substances secreted into the intercellular space by one of the interacting cells or by a cell located at a distance from the site of cell adhesion. Such a cell can obviously induce a response regarding the aggregation of other cells—a phenomenon frequently encountered during differentiation.

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Fig. 4.12. Carbohydrate-Structure/156.html">Protein Interactions in cell adhesion. CHO — carbohydrate component, s — carbohydrate-binding site.

Aggregation factors have been isolated from a variety of tissues, notably from marine Sponges—the Organism in which specific cell adhesion mechanisms were first studied. Early in this century, H. V. Wilson demonstrated that sponges dissociate into single cells when placed in fresh Water and reaggregate upon transfer back to sea water. This effect is regulated by salt composition, specifically the calcium content in sea water. If cells from two different sponges are dissociated, mixed, and incubated in sea water, the cells of each sponge form separate aggregates devoid of cells from the other species. This phenomenon is easily observed by mixing sponges with different pigmentation due to endogenous chromophores (e.g., sponges with red and green cells). This results in the formation of exclusively red or green aggregates, rather than mixed ones (Fig. 4.13).

Fig. 4.13. Dissociation and specific reaggregation of cells.

Aggregation factors released by sponge cells upon dissociation can be easily separated from the cells themselves by centrifugation. They are high-molecular-weight glycoproteins with an unusual composition [82]. In addition to galactose, mannose, and N-acetylhexosamine, they contain sulfuric and uronic acids. The latter is an important component, since The addition of free glucuronic acid inhibits cell aggregation in the presence of the aggregation factor. It has been established that aggregation factors possess the necessary specificity; for example, the factor from *Microciona parthena* is unable to induce cell aggregation in other, unrelated sponge species such as *Haliclona*.

A similar system has also been discovered in the sponge *Geodia cydonium*. Interestingly, the partially purified aggregation factor exhibits glucuronyltransferase activity. This implies that The conversion of a non-aggregating system into an aggregating one can occur via the extracellular addition of glucuronic acid residues, provided that the necessary activated sugar derivative is available [83]. Aggregation factors have also been isolated from the culture media of higher organism cells, such as primary cultures of chick embryo retina cells [84]. This factor induces the aggregation of retinal cells but has no effect on cells from other tissues. It is a glycoprotein containing Monosaccharides characteristic of N-glycans. Another cell aggregation factor is secreted by cultured mouse teratoma ascites cells, and its aggregating activity is suppressed by Treatment with β-galactosidase or the addition of free galactose [85]. Clearly, the galactose residues contained within the aggregation factor are critically important for initiating cellular interactions in this system (Fig. 4.12, c).

Fig. 4.14. Interaction of cell surface CARBOHYDRATES and protein receptors in cell adhesion. Adhesion is enhanced by treatment of cells with neuraminidase, which exposes an additional terminal galactose residue. Treatment with β-galactosidase reduces adhesion.

Other evidence supporting the involvement of glycoproteins and galactose residues in cell adhesion, while indirect, is nevertheless substantial. Individual baby hamster kidney cells (BHK line) readily form aggregates in suspension in the presence of Calcium Ions. This process is stimulated by preliminary Treatment of the cells with neuraminidase and blocked by treatment with galactose oxidase, which converts the hydroxyl group at C-6 of galactose into an aldehyde group. Similar results were obtained in experiments with other cells, where in one case β-galactosidase caused a significant decrease in the aggregation rate. These results indicate an essential role in adhesion for complex N-glycans possessing the following terminal sequence: neuNAc-gal-glcNAc→R, where R denotes the core region (Fig. 4.14). Other indirect indications come from studying the adhesive Properties of the aforementioned ricin-resistant mutants (Section 4.2.4) of baby hamster kidney cells (BHK line). Many of these resistant cell lines aggregate poorly. It has been shown that this phenomenon is associated with blocked glycosylation and a reduced number of surface carbohydrates that bind ricin. Finally, cells are able to attach to agarose particles covalently coupled with galactose residues, whereas particles with other sugar residues prove ineffective [86].

All these data taken together point to the existence of direct interactions (Fig. 4.12, a) between galactose-rich carbohydrate chains and carbohydrate-binding proteins located On the surface of adjacent cells (Fig. 4.14). The first hypothesis concerning The Nature of the carbohydrate-binding proteins involved in adhesion processes was put forward by Roseman [87]. He pointed out the very high specificity of Glycosyltransferases, making them highly probable candidates for participating in adhesive interactions, which also proceed with a high degree of specificity. It is unlikely that activated sugar precursors are present in the intercellular space under normal conditions. Therefore, glycosyltransferases, being located on the cell surface, could participate in stable interactions with corresponding carbohydrate chains acting as acceptors in the Reactions Catalyzed by these Enzymes. Such a model offers certain advantages, as it indicates how the dissociation of clumped cells is controlled. If activated sugars reach the contact sites between cells, The transfer of sugar residues will occur concomitantly with the dissociation of the transferase-carbohydrate complex. Initial attempts to detect glycosyltransferases on the cell surface yielded ambiguous results and were therefore severely criticized. However, recent experiments support the hypothesis that The surface of certain cells possesses sialyltransferase activity. Of course, it remains to be proven that these transferases actually participate in adhesion. Another possibility is based on the fact that cell-surface glycosidases are capable of interacting non-hydrolytically with the carbohydrate chains of adjacent cells. Lysosomal enzymes, which have a relatively low pH optimum, can form stable bonds because the extracellular pH around the cell is presumably much higher than the pH values within Lysosomes—that is, higher than those required for optimal catalytic activity. Direct support for this hypothesis comes from the detection of large amounts of lysosomal enzymes on the cell surface, as well as the fact that such cells are able to adhere to an inert Glass surface coated with neuraminidase, β-galactosidase, and α-mannosidase. These experiments still require further confirmation and Discussion.

The most likely candidates for The Role of carbohydrate-binding molecules are Lectins, which reportedly lack any enzymatic activity. Several such lectins have been successfully isolated. For instance, in *Dictyostelium discoideum*, a lectin appears when the unicellular amoeboid form of this slime mold suffers from food deprivation. Under these conditions, the cells acquire the ability to form aggregates that subsequently differentiate into Multicellular Organisms consisting of a stalk and a fruiting body containing numerous spores. The isolated lectin is inhibited by N-acetylglucosamine, galactose, and D-fucose, i.e., sugars of D-galactan. The slime mold apparently possesses at least two such lectins, designated as discoidins (I and II). Similar lectins (purpurins, pallidins) have been found in other slime Molds—*Dictyostelium purpureum* and *Polyspondylium pallidum*. All these lectins exhibit a defined specificity toward homologous organisms. The aggregation process likely involves carbohydrate chains whose sequences are specific to each given slime mold, and the various lectins possess a corresponding ability to recognize them, although simple galactosides at high concentrations can inhibit cell aggregation. Clearly, these very sugars serve as the primary determinant in all cases. Discoidins I and II and purpurins appear at different periods of embryonic development, evidently indicating that the developmental process is accompanied by subtle changes in The structure of surface carbohydrates, each recognized by a corresponding lectin that arises in a specific regulated order on the cell surface. Each of the lectins is possibly essential for normal development, since mutant *Dictyostelium discoideum* cells incapable of aggregation lack active discoidin I molecules while retaining surface receptors for discoidins isolated from normal organisms. Slime mold lectins are Oligomeric Proteins with subunits of a molecular mass of approximately 25,000 [89].

A different class of lectins has been isolated from various mammalian and avian tissues. Their molecular mass is lower—around 13,000—and their ability to induce cell agglutination is inhibited by galactosides. In the absence of reducing agents, lectin subunits aggregate, possibly through the formation of Disulfide Bonds, thereby losing activity. These lectins are not integral components of cell membranes, as they are solubilized when tissues are incubated in solutions of simple galactosides, such as lactose solution. Consequently, these lectins function as extracellular aggregation factors that mediate cell clumping by reacting with corresponding integral membrane glycoproteins (Fig. 4.12, b).

There are several lines of Evidence indicating the involvement of these galactoside-binding lectins in the differentiation process. Chick pectoral Muscle lectin appears between days 8 and 16 of embryonic life—a period of active muscle differentiation. In Cells of the myoblastic Lineage (L6), lectin activity increases during the fusion of myoblasts into myotubes. Fusion is invariably preceded by cell adhesion, and lectin has been detected on the surface of myoblasts by immunofluorescence. Paradoxically, however, the bulk of the lectin is located intracellularly. It is possible that this intracellular localization is necessary for the continuous replenishment of surface lectin during the fusion process. Alternatively, it is conceivable that the lectin may have multiple distinct functions, some of which are related to the Intracellular Transport of glycoproteins. In any case, however, the specific interaction of the lectin with surface galactose residues during the Cytology/cytology/16.html">Early stages of muscle differentiation can be considered established [89].

Another lectin is present in embryonic chick skeletal Muscles, and its activity also changes during Muscle Development. Its cell-agglutinating ability is strongly inhibited by heparan and heparan sulfate, which is an important constituent of the Extracellular matrix. This lectin is secreted by differentiated muscle cells into the extracellular space in culture and may play a more important role in cell attachment to the supporting matrix (Section 4.5.2) than in cell-cell adhesion [89].

Erythrocyte development represents another interesting system in which galactose-specific lectins also play a major role. Erythroblasts bear on their surface a typical lectin with a molecular mass of 13,000, which is capable of clumping these cells into "erythroblastic islands" surrounding a nurse macrophage. The amount of lectin on the cell surface decreases as the erythroblasts mature and reticulocytes—the direct precursors of erythrocytes—are released from the aggregates [90]. During erythroid differentiation, marked changes occur in the galactose-containing carbohydrate chains. At least this is observed in human cells, according to data obtained using Reagents that bind to Ii antigenic determinants (Chap. 3). Lectins similar to those located on the erythroblast surface are capable of reacting with Ii determinants [91]. It would be highly interesting to uncover a parallel between the appearance of the galactose-binding lectin and the modulations of Ii determinants in developing systems.

Finally, mention should be made of a galactose-binding lectin that is released from the Brain cells of newborn rat pups upon the addition of lactose. It is apparently analogous to the other aforementioned lectins that also bind galactose. The brain lectin appears in 10-day-old rat pups during the peak period of synapse formation, and it may play an important role, for example, in synaptogenesis during cortical development [92].