Glycoproteins - Hughes R. 1985

Functions
Catabolism and Clearance
Soluble Glycoproteins

Intracellular protein breakdown during normal growth and differentiation is subject to precise regulation [69]. Part of protein Degradation and Turnover is determined by their intrinsic, characteristic inherited features, such as polypeptide chain conformation. However, compelling evidence indicates that carbohydrate components also participate in regulating glycoprotein Catabolism. For example, in the Liver, Cell/33.html">Plasma Membrane Glycoproteins are renewed faster than non-glycosylated Proteins, whereas in cultured cell lines, plasma membrane glycoproteins are almost completely turned over within a few hours under conditions that essentially prevent growth [18].

A carbohydrate-type signal for glycoprotein catabolism has been discovered, which is likely relevant to the processes under Structure/133.html">Discussion. Specifically, glycoproteins present in the bloodstream are captured by the liver, where their catabolism takes place.

The system described below was discovered serendipitously. Gilbert Ashwell, Anatole Morell, and their coworkers intended to trace The Fate of ceruloplasmin introduced into the bloodstream. Initially, this glycoprotein was treated with neuraminidase to expose galactose residues present within the N-glycans (Fig. 2.5). The galactose residues could then be radiolabeled by first treating them with galactose oxidase to generate an aldehyde group at C-6, followed by reduction with tritiated sodium borohydride. Unexpectedly, it was found that the labeled glycoprotein disappeared from the Blood and appeared in the liver of rats within just a few minutes, whereas the untreated glycoprotein circulated for several days. Subsequent analysis of this intriguing phenomenon revealed that the hepatocyte surface exhibits lectin-like activity, enabling them to bind terminal galactose residues of various asialoglycoproteins and thereby facilitate the uptake of glycoproteins into Lysosomes, where they are hydrolyzed by acid Hydrolases [70]. These findings also help explain earlier observations showing that human blood orosomucoids are almost fully sialylated, exposing very few, if any, galactose residues.

The galactose-binding lectin is a glycoprotein composed of several subunits (most likely two). It is firmly embedded in the hepatocyte membrane and can be solubilized only by Treatment with detergents. The Molecular Weight of a single subunit is 45,000, which differs from that of other galactose-specific Lectins presumably involved in Cell Adhesion (Section 4.5.1). The hepatocyte lectin agglutinates Cells treated with neuraminidase and acts as a mitogen for lymphocytes, i.e., it stimulates DNA Synthesis in resting peripheral lymphocytes. Both properties indicate that a single active lectin molecule possesses Multiple binding sites. The ability for multivalent interaction with the lectin also underlies the binding and uptake of glycoproteins by hepatocytes [71]. Glycoproteins or purified glycopeptides with multiple terminal galactose residues bind with high affinity and are rapidly internalized by The Cell. However, glycoproteins with complex glycans containing only two terminal residues bind weakly and do not undergo endocytosis (Fig. 4.10). For example, asialoorosomucoid with tetraantennary N-glycans (Fig. 2.11) serves as an excellent substrate for hepatocyte endocytosis, whereas serum asialotransferrin, which has only two antennae, is, by contrast, endocytosed very poorly [72]. It should be noted, however, that O-glycans containing N-acetylgalactosamine residues exhibit exceptionally high binding affinity and endocytic capacity (Fig. 4.10).

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Fig. 4.10. Binding and endocytosis of glycoproteins by hepatocytes. O-glycans containing terminal galactose residues behave similarly to glycopeptides from which galactose has been removed. R represents asparagine residues.

Just as efficient binding and initiation of endocytosis by N-glycans require multiple closely spaced antennae terminating in galactose residues, O-glycans must possess closely spaced antennae containing N-acetylgalactosamine residues. Moreover, binding to receptors does not require the terminal N-acetylgalactosamine residues to be substituted with galactose residues.

The high affinity of certain glycoproteins for hepatocytes can be harnessed to clear proteins from the bloodstream into the liver. For instance, glycolipid B (Fig. 4.10) attached to albumin induces rapid clearance of this protein, which is normally non-glycosylated and stable [70].

The binding of glycoproteins to hepatocytes requires the presence of Ca²⁺ ions, and this process is completely inhibited by treating hepatocytes or the purified lectin with neuraminidase. This occurs because the removal of sialic acid from the glycan chains of the lectin induces receptor self-aggregation driven by the interaction of newly exposed galactosyl terminal residues.

The presence of the lectin appears to be restricted to the liver, where it is located On the surface of hepatocytes in direct contact with the bloodstream. This specific localization points to a vital role for the lectin in clearing partially degraded or modified glycoproteins from the blood, and possibly in participating in the Intracellular Transport to the bloodstream of serum glycoproteins synthesized in the liver.

Direct Evidence for the involvement of hepatocyte lectins in in vivo clearance was obtained as follows. It was demonstrated that the simultaneous administration of asialoproteins into the blood along with Antibodies against the purified receptor significantly increases the retention time of these glycoproteins in the Circulation [73]. It was further established that the blood of patients with liver cirrhosis contains higher-than-normal levels of glycoproteins bearing terminal galactose residues. However, at any given time, only about 10% of all receptors reside on the cell surface, with the majority being associated with the smooth membranes of the Golgi apparatus. These membranes may serve as precursors for the vesicles that deliver receptors to the cell surface, which is necessary to continuously replenish cellular receptors lost during endocytosis [74]. It has been estimated, for example, that the entire population of surface receptors is depleted from the cell approximately every 5–10 minutes. Clearly, a substantial intracellular pool of receptors is required to compensate for these losses. Receptor replenishment is likely facilitated by the fact that lysosomal receptors can be recycled, indicating their resistance to lysosomal Enzymes. The mechanism protecting the active sites of the receptors appears to be their inward orientation toward the Cytosol within lysosomes. Presumably, following lysosomal fusion, a reorientation of the receptor molecules occurs within the membrane. For receptors to reappear on the cell surface in the correct orientation, another conformational rearrangement must take place within the membrane system that transports them to the surface. The exact mechanism of this entire process remains unknown.

Fig. 4.11. Uptake of modified glycoproteins by the mammalian liver. R represents the core region of N-glycans.

In the mammalian liver, blood glycoproteins normally contact the surface membranes of both hepatocytes and non-parenchymal cells lining the sinusoids. The latter cell type includes Kupffer cells, which play a crucial role in glycoprotein clearance. If Orosomucoid is treated with neuraminidase and subsequently with ß-galactosidase to expose terminal N-acetylglucosamine residues, the glycoprotein modified in this manner is rapidly internalized by the reticuloendothelial system (Fig. 4.11). Glycoproteins bearing terminal mannose, such as many rat lysosomal enzymes including ß-glucuronidase and ß-N-acetylglucosaminidase, are cleared in a similar fashion, and the simultaneous injection of asialoagalactoorosomucoid blocks this elimination process [75]. It should be kept in mind that the N-glycans of lysosomal enzymes are rich in oligomannoside chains. Consequently, these experiments suggest that recognition molecules on The surface of Kupffer cells bind glycoproteins terminating in N-acetylglucosamine or mannose. This lectin is widespread among phagocytic cells, such as alveolar macrophages, and participates In the second major mechanism of Carbohydrate Catabolism in animal Tissues. Components that bind N-acetylglucosamine and mannose have been isolated and are likely surface lectins of non-parenchymal cells. Specifically, a similar lectin was isolated from rat liver; it proved to be an oligomeric, membrane-associated glycoprotein with a subunit molecular weight of 32,000. No antigenic relationship to the hepatocyte lectin was found, but in both cases, Ca²⁺ ions are required for activity. Glycoproteins with terminal mannose bound somewhat better than those with terminal N-acetylglucosamine, indicating a Specificity similar to that of concanavalin A [76]. It is worth recalling that concanavalin A binds to internal mannose residues even when they are substituted with other sugars, such as N-acetylglucosamine at C-2, although binding affinity is higher for glycoproteins with unsubstituted terminal mannose residues (Fig. 4.1).

The function of hepatocyte and Kupffer cell lectins may also include the removal from circulation of partially degraded serum glycoproteins or lysosomal hydrolases. In particular, the clearance of lysosomal enzymes appears crucial for preventing damage to the surface of cells in contact with blood. However, lectins may perform another essential task, namely, facilitating the destruction of microorganisms, such as Yeasts, which possess abundant oligomannoside chains. For instance, the Digestion of Yeast by alveolar macrophages is inhibited by mannose and mannose-terminated glycoproteins [77].

Unlike mammalian blood glycoproteins, those of birds possess many exposed galactose residues. Therefore, one would expect the receptor system in avian liver cells to differ from that in mammalian hepatocytes. Indeed, the recognition residue in birds is N-acetylglucosamine, and treatment of galactose-terminated glycoproteins with ß-galactosidase triggers the rapid clearance of such modified glycoproteins from the circulation. Such a specific lectin was isolated from chicken liver and was found to be a glycoprotein with a subunit molecular weight of 26,000. Determination of its complete Amino Acid Sequence revealed several unusual features: the N-terminal residue of the chain is acetylated. The adjacent region, spanning residues 25–48, proved to be rich in uncharged Amino Acids with predominantly hydrophobic side chains, which likely participate in anchoring the lectin to The cell membrane. The opposite (carboxyl) end of the peptide chain probably faces the extracellular space and contains a single N-glycan attached to asparagine-67. This long peptide segment also contains seven Cysteine residues, suggesting a highly folded structure. The orientation of the chicken liver lectin in the membrane appears opposite to that of Glycophorin and more closely resembles band 3 glycoprotein, except that the polypeptide chain traverses the membrane only once [78]. The precise localization of the avian lectin is currently unknown, but it is unlikely to be analogous to that of the mammalian Kupffer cell receptor, given that the two proteins differ in molecular weight, cation requirements, and carbohydrate specificity. Thus, after the removal of sialic acid, galactose, and N-acetylglucosamine, orosomucoid—which consequently has a high content of terminal mannose residues—is neither bound by the avian lectin nor taken up by the cells. This indicates that the avian lectin does not recognize terminal mannose residues.