Glycoproteins - Hughes R. 1985

Functions
Cell adhesion
Attachment of cells to the substrate

All Cells, with the exception of freely circulating ones (such as erythrocytes or circulating peripheral lymphocytes), are surrounded by or embedded in a carbohydrate-rich layer of substance. This layer is referred to as the glycocalyx, Cell coat, or Extracellular matrix. Connective Tissues, such as bone, Cartilage, or tendons, represent an extreme case of such a highly differentiated acellular matrix deposited in certain areas by specialized cells located within or in close contact with it. The extracellular matrix protects The Cell surface (e.g., against damaging agents), maintains a buffer layer that compensates for sudden Changes in the COMPOSITION OF THE fluid bathing the cells, and serves as a relatively rigid Structure that Supports body shape. Furthermore, it plays a vital role in controlling cell growth and organizing cell masses into tissues. The Fine Structure of the extracellular matrix varies depending on the type of cell that produces it. However, its main component is Collagen, accompanied by varying amounts of Proteoglycans and various Glycoproteins. The glycoprotein component appears to play a crucial role as a mediator in the adhesion of cells to the extracellular matrix, particularly to collagen, thereby participating in the Regulation of cellular METABOLISM and growth.

There are at least five genetically distinct types of collagens, each with its characteristic localization. A specific cell type has been found to be associated with each of them (Table 4.2). To date, several glycoproteins that determine cell attachment to collagen have been identified (Table 4.2). These adhesion factors are secreted by cells, and at least some of them likely possess separate binding sites for collagen and for the cell surface. The preferential combination of different cell types with a specific collagen is apparently explained precisely by the Specificity of these binding sites. A number of the factors under Discussion are also present in serum, which is typically added to in vitro cell culture media. These factors can passively adsorb onto plastic tissue culture ware, thereby facilitating cell attachment even in the absence of an underlying collagen layer.

Class="center">Table 4.2. Collagens and glycoproteins promoting Cell Adhesion to collagen

Fig. 4.15. Cell adhesion to the substrate. A — glycoprotein factor binds to the substrate. B — cell attachment. C — cell spreading.

Cell adhesion to a collagen substrate or plastic surface involves three distinct stages: 1) binding of the glycoprotein factor to the plastic or collagen surface, 2) attachment of cells to the surface-bound factor, and 3) multiplication of interactions between the cell surface and glycoprotein molecules, coupled with the reorganization of the intracellular Cytoskeleton—specifically microfilaments—resulting in the spreading of the Cell Cytoplasm across the substrate (Fig. 4.15). Mitotic cells or cells released from a monolayer culture by Trypsin appear as spherical particles whose surfaces are covered with numerous folds, blebs, and microvilli of varying lengths. The longest microvilli, or filopodia, make initial contact with the substrate and apparently bear surface-localized molecules at their distal ends that are capable of probing the appropriate adhesive substrate and subsequently interacting with it [93]. Filopodia are rich in cytoplasmic microfilaments that form bundles and flat strands upon cell stretching, and then interact with other cytoskeletal components to anchor the stable structure characteristic of a firmly attached cell.

All stages of cell attachment to the substrate proceed rapidly (within 1–2 h). This process requires neither de novo protein nor nucleic acid synthesis, but it does necessitate the presence of divalent cations (Ca²⁺ and Mg²⁺). During prolonged culturing, The addition of an exogenous adhesion factor may become unnecessary if the cells themselves are capable of producing and secreting it. For instance, the attachment of fibroblasts to a plastic or collagen surface requires the addition of fibronectin for about two hours, whereas within 12–16 h these cells synthesize sufficient fibronectin for their own adhesion even in the absence of exogenous fibronectin. The addition of cytochalasin B, which inhibits Actin polymerization, hinders cell attachment, indicating a specific role for microfilaments in this process.

a) Fibronectin. Fibronectin was the first discovered among the factors promoting adhesion to collagen (Table 4.3) [94, 95]. In the 1940s, John Edsall and his coworkers isolated a protein from blood that was termed cold-insoluble globulin, or Cig, because it is the major component of the precipitate formed during the cold-induced clotting of blood. Later, it was demonstrated that fibronectin possesses a binding affinity for Fibrinogen and fibrin, which accounts for its presence in the fibrin clot. Subsequently, researchers successfully isolated fibronectin from the blood and tissues of numerous vertebrates, as well as from the culture medium and various mammalian and avian cells cultured as monolayers, particularly fibroblast cultures. In intact tissues, fibronectin is primarily located in fibrils in close contact with collagen. It is present in the basement lamina adjacent to both epithelial cell layers and nearby Connective Tissue, in the stroma of lymphoid tissue, and around smooth muscle cells and striated muscle fibers. The exact cells within these tissues that produce fibronectin are not yet entirely clear. However, In addition to cultured fibroblasts, fibronectin is also synthesized by myoblasts, astroglial cells, endothelial cells, macrophages, mast cells, and epithelial cells, frequently accumulating in large amounts within the extracellular matrix.

Table 4.3. Properties of fibronectins

The circulating form of the fibronectin molecule is a dimer constructed from two similar, yet non-identical in size (one is slightly larger than the other) and structure, subunits linked by disulfide bonds. Cell-associated molecules consist of two subunits with very similar molecular weights (approximately 225,000) and can be polymerized via The formation of multiple disulfide bonds. The dimeric form can be extracted from cells and cell secretions. All these Various Forms of fibronectin appear very similar in their genetic, chemical, and functional properties, yet they are not identical. A simplified diagram of The structure of fibronectin molecules is shown in Fig. 4.16. This protein is a glycoprotein containing about four N-glycans whose structures are similar to those of serum glycoproteins, such as Orosomucoid. The carbohydrate component probably exerts only an indirect influence on biological activity; it may protect the molecule from extracellular proteolysis during Synthesis and Secretion. As clearly demonstrated by electron micrographs [96] as well as by chemical Cleavage, dimeric fibronectin molecules consist of functionally distinct domains separated by flexible, non-globular Regions of the peptide chain that are particularly sensitive to proteolysis. Active fragments were thus obtained and shown to exhibit distinct biological activities. For instance, a fragment with a Molecular Weight of 40,000 (40K) that binds to collagen is located within the molecule but close to the N-terminal portion of the polypeptide chain. This fragment, similar to the intact fibronectin molecule, binds to native fibrillar collagen, particularly during the precipitation of collagen fibrils [97]. This reaction proceeds more efficiently with denatured type I–IV collagens. A distinct preference for type I and III interstitial collagens is observed during fibroblast adhesion. The segment of the collagen polypeptide chain to which the 40K fibronectin domain attaches represents a highly conserved region that is also recognized by mammalian collagenase. In early studies, Stephen Hauschka showed that myoblast differentiation into muscle is promoted by this same collagen fragment. Since fibronectin is synthesized by myoblasts and in turn promotes myoblast adhesion to collagen, The Mechanism of this process can be considered established.

Fig. 4.16. Simplified Cytology/cytology/92.html">SCHEMATIC STRUCTURE OF fibronectin. Sites of Limited proteolysis and active domains are indicated. ? — glycans.

The bond between fibronectin and collagen is relatively weak and is stabilized by A large number of interactions between each cell and the substrate. For instance, Cells of the BHK line form about 50,000 such contacts [86]. Furthermore, these interactions can be stabilized by the secretion of heparan sulfate—a proteoglycan typically found on the undersurface of substrate-attached cells—by the attached cells. The primary region of fibronectin responsible for heparan sulfate attachment is located near the collagen-binding domain, but does not overlap with it.

It has been suggested that Plasma Membrane components participate in the reaction of attachment to a fibronectin-coated surface, and this information is transmitted directly or indirectly from the outside of the cell into the cytoplasm, triggering microfilament reorganization. Cytoplasmic Proteins that anchor microfilaments or provide attachment "anchors" are located on the inner side of The Plasma Membrane. Two proteins take part in this process: vinculin (molecular weight 130,000) and α-actinin (molecular weight 94,000). Antibodies against vinculin stain regions on the lower surface of substrate-attached cells that do not always contain fibronectin, whereas α-actinin is localized alongside fibronectin molecules and appears to be most actively involved in the cell spreading process stimulated by a fibronectin-coated surface [98]. However, the true Nature of the plasma membrane component(s) that interact directly with fibronectin has not yet been established. It is possible that the carbohydrate components of Glycolipids [94] or glycoproteins [86, 99] are involved, and it is entirely plausible that multiple factors rather than a single one participate (Fig. 4.17).

b) Laminin. Although fibronectin can mediate the attachment and spreading of many cell types, it is not effective in all cases. One compelling example is the fact that various epithelial cells are capable of attaching and spreading on a collagen layer even in the absence of fibronectin [94]. Another glycoprotein, laminin, accelerates the attachment of such cells and evidently acts as a partner to fibronectin in its effects on cells of epithelial origin. Laminin is a multimeric glycoprotein with a molecular weight of 800,000, composed of subunits with molecular weights of 200,000 and 400,000. Unlike fibronectin, it exhibits high specificity toward cells that attach to type IV collagen, which is present in basement membranes and, as a rule, in contact with epithelial cells in the Organism. Laminin is produced exclusively by cells of epithelial origin. However, fibronectin can also be produced by these same cells, and it apparently mediates the in vitro attachment of certain epithelial cells to interstitial collagens, whereas laminin participates in their attachment to basement membrane collagen. Laminin, similar to fibronectin, binds to heparan and heparan sulfate [100]. Since the latter is present in many basement membranes, such interactions may play a specific role in matrix Organization and adhesion to it.

Fig. 4.17. Molecules involved in cell spreading.

Other factors recently isolated from basement laminae may also participate in cell attachment to basement membrane collagens. One of these is entactin, a sulfated glycoprotein with a molecular weight of 158,000 that is secreted by cultured endodermal cells and localized at the basal surface of cells facing the basement membrane in tissues [101].

c) Chondronectin. Highly specialized cells—chondrocytes—also require a specific glycoprotein factor for adhesion to collagen (in this case, cartilage type II collagen) [94]. The molecular weight of chondronectin is 180,000; like fibronectin, it is present in serum, which can therefore promote chondrocyte adhesion on its own. Chondronectin specifically binds to type II collagen found in cartilage. Interestingly, chondrocytes can respond to both fibronectin and chondronectin. For instance, rounded cells attached to type II collagen by chondronectin can be transformed into a fibroblast-like Morphology with the help of fibronectin. The cells then cease to produce cartilage-specific collagen as well as proteoglycans and revert to the fibroblast phenotype [102]. Therefore, in such a system, fibronectin suppresses specific Cell Differentiation in culture. This evidently implies that The production of fibronectin by chondrocytes or other cells modulates tissue development. Such modulation may be of great importance in the formation of myogenic regions within cartilaginous tissues and points to a regulatory role of the supporting matrix in directing cell growth and differentiation.

From this Brief Overview of intensively developing research fields, one can envision the primary unresolved problems concerning glycoprotein functions: 1) What is The Role of glycoproteins in regulating the ORGANIZATION OF THE extracellular matrix? 2) Does glycoprotein-mediated specific cell adhesion promote special relationships between differentiated cell types and specialized extracellular matrices? 3) Which cell surface-associated glycoproteins, glycolipids, or proteoglycans are involved in close contacts between cells and the extracellular matrix? 4) By what pathway do such interactions promote, for example, cell movement across an adhesive matrix or the migration of malignant cells from the primary tumor site followed by infiltration and colonization in other regions of the extracellular matrix?

Both these and other questions concerning glycoproteins are currently under active investigation and, along with other problems discussed in this book, testify to a very high level of interest in the Structure and Biosynthesis of these important biological macromolecules [103].