Glycoproteins - Hughes R. 1985

Appendix

Below is a brief summary of several important advances published after the completion of the manuscript. A previously unknown carbohydrate sequence has been discovered in the ovine luteinizing hormone molecule, containing an N-acetylgalactosamine residue attached by a ß1→2 glycosidic linkage to a mannose residue, which is in turn linked via an a1→6 bond to the mannose core region (man)3(glcNAc)₃ of N-glycans [104]. The N-acetylgalactosamine residue bears a sulfate group. This is the first well-documented instance of ß-linked N-acetylgalactosamine residues occurring in N-glycans (Section 2.3). A highly valuable addition to the Enzymes listed in Section 2.4 is the broad-spectrum endo-ß-N-acetylglucosaminidase isolated from Flavobacter meningosepticum, which is capable of hydrolyzing both mannose-rich Oligosaccharides and complex N-glycans [105].

Molecules similar to ankyrin and spectrin have been identified not only in erythrocytes but also in various other Cell types [106–108]. This evidently points to a common function of these Proteins, which lies in linking the Actin network to other cytoskeletal elements—such as microtubules—and to membranes (Section 2.6.2).

The assembly process of the primary oligosaccharide-lipid intermediate, discussed in Section 3.3.1, apparently also takes place in the mammary gland [109] and Yeast [110]; the immediate precursor of each a-mannose residue has been unequivocally identified.

Glucosidase II—the enzyme responsible for removing (1→3)-linked glucose residues during N-glycan Processing [112] (Section 3.3.2)—has been isolated in purified form. It does not act on (1→2)-linked glucose units, the Cleavage of which requires a different specific glucosidase. Lectin-resistant lymphoma Cells exhibit a deficiency in glucosidase II, whereas glucosidase III—wait, glucosidase I activity in these same cells remains essentially unchanged [113]. These cells are capable of accumulating Glycoproteins, which underscores the crucial role of glucosidases in The Biosynthesis of "complex" N-glycans. The "late" a-mannosidase (or a-mannosidases), which participates in processing the product formed by N-acetylglucosaminyltransferase I (Section 3.3.3), is strongly and specifically inhibited by swainsonine, an alkaloid isolated from an Australian plant [114]. Other a-mannosidases are not inhibited by this alkaloid. Cells treated with swainsonine are unable to form "complex" N-glycans [115], and feeding the aforementioned plant to animals induces a condition resembling hereditary lysosomal storage disease (mannosidosis, or lysosomal a-mannosidase deficiency), as well as a chronic neurological disorder known as locoism [116].

Two N-acetylglucosaminyltransferases involved in the assembly of highly branched N-glycans have now been identified (Section 3.3.3). One of them is present in large quantities in chick oviducts [117]. This is N-acetylglucosaminyltransferase III, which catalyzes the attachment of a (ß1→4)-linked N-acetylglucosamine residue to the core region containing three mannose residues. This explains the predominance in Ovalbumin of N-glycans containing this linkage (Section 2.3.1). This enzyme utilizes the natural product synthesized by N-acetylglucosaminyltransferase I—namely, glcNAc(man)_s(glcNAc)_a—or the product synthesized by N-acetylglucosaminyltransferase II. This observation helps explain the presence in many glycoproteins, including ovalbumin (Fig. 2.6) and Glycophorin (Fig. 2.19), of "complex" N-glycans containing the glcNAcß1→4 sequence in their core region. The addition of the glcNAcß1→4 unit to the ß-mannose residue in the (man)s(glcNAc)2 Structure prevents processing down to the trimannose stage, or in other words, blocks The formation of the acceptor required for the action of N-acetylglucosaminyltransferase II. Consequently, during the formation of complex N-glycans containing the glcNAcß1→4manß1→4glcNAcß1→4glcNAc sequence, the action of glucosaminyltransferase II must precede that of transferase III. Another specific enzyme, N-acetylglucosaminyltransferase IV [118], catalyzes the attachment of an N-acetylglucosamine residue via a (ß1→4) bond to the mannose residue linked via an (a1→3) bond to the N-glycan core region (Figs. 2.6 and 2.11). The preferred substrate is the (man)3(glcNAc)₂ core sequence in which both a-mannosyl units are substituted with (ß1→2)-linked N-acetylglucosamine residues. Removal of N-acetylglucosamine units from the mannose residues linked to the ß-mannosyl residue via an (a1→6) bond significantly reduces enzymatic activity. The same effect is produced by the substitution with galactose of any (ß1→2)-linked N-acetylglucosamine residue. Thus, the substrate Specificity of these enzymes (glcNAc transferases III and IV) highlights the exceptional sensitivity of Glycosyltransferases toward extended oligosaccharide sequences.

A toxin with unusual properties, gelonin, has been isolated from the plant Gelonium multiflorum [119]. Gelonin appears to be analogous to the toxic A-subunit of ricin or abrin (Section 4.2.3). Gelonin is ineffective when added to intact cells, yet it is highly toxic when conjugated with concanavalin A [119] or with a specific antibody against The Cell-surface antigen Thy-1 [120]. Currently, there is immense interest in developing such chimeric toxins consisting of an enzymatically active component derived from a plant or bacterial toxin attached to another component endowed with binding capacity (e.g., a specific antibody or hormone). Such cell-type-specific chimeric toxins hold potential for the selective elimination of unwanted cells, such as tumor cells.

The surface localization of discoidin has been established [121], supporting the hypothesis (Section 4.5.1) that Lectins are involved in cellular adhesion in slime Molds. Discoidins and pallidins are not integral components of The Plasma Membrane, but instead bind to cell-surface glycoprotein receptors, thereby bridging cells together [122, 123] According to the model shown in Fig. 4.12b. These receptors appear on the cell surface during the developmental cycle, providing compelling evidence that cellular aggregation is governed by the appearance of cell-surface lectins, which in turn is regulated by developmental processes.

There is a growing body of evidence regarding The Role of lectins in bacterial adhesion to animal Tissues. This process is a crucial initial step in bacterial colonization and The Development of pathological states [124]. Many Gram-negative Bacteria, such as Escherichia coli, possess surface projections that bear mannose-binding lectins. The adhesion of these bacteria to mucosal surfaces can be prevented or reversed by the addition of simple mannosides. Although not yet definitively proven, it is possible that the receptors for these bacterial adhesins are N-glycans containing mannose residues. CARBOHYDRATES are also involved in the adhesion of other pathogenic bacteria. For example, Streptococcus sanguis, a saliva-dwelling bacterium responsible for dental caries and periodontal disease, binds to glycoproteins possessing the neuNAca2→3galß1→3galNAc sequence, which are common components of salivary mucins (Section 2.5.1) [125]. Elucidating the mechanisms of bacterial attachment could facilitate the development of effective Vaccines and relatively simple (and inexpensive) carbohydrate-based inhibitors of bacterial colonization and pathogenicity [126].

The potential role of circulating Fibronectin (Cig) (Section 4.5.2) in clearing Connective Tissue debris is indicated by the finding [127] that gelatin-coated particles are phagocytosed only in the presence of fibronectin and heparin.

The hypothesis that circulating fibronectin and cell-associated fibronectin are products of different genes (Section 4.5.2) is supported by structural differences between the two proteins, which can be detected both chemically and immunologically using Monoclonal Antibodies [128–130]. It should be noted, however, that the cell-binding domain of fibronectin is located within a small proteolytic fragment (molecular weight 12,000) whose Primary Structure is likely highly conserved [131].