This chapter explores degradation and turnover of glycans in lysosomes, especially with respect to human genetic disorders, with representative glycans, illustrating features unique to different pathways. Degradation of oligomannosyl N-glycans removed from misfolded, newly synthesized glycoproteins is covered in Chapters 39 and 45.

LYSOSOMAL ENZYMES

Most glycans are degraded in lysosomes by highly ordered pathways using endo- and exoglycosidases, sometimes aided by noncatalytic proteins. Insights that unraveled these complex pathways emerged from studies of rare human genetic disorders called lysosomal storage diseases. In each disease, undigested molecules accumulate in lysosomes. Clever experiments combining enzymology with glycan structural analyses revealed the steps of the pathways and also unlocked the mechanisms of lysosomal enzyme targeting (Chapter 33).

Lysosomes contain approximately 50 to 60 soluble hydrolases that degrade various macromolecules. Most of the glycan-degrading enzymes (endo- and exoglycosidases and sulfatases) have pH optima between 4 and 5.5, but a few have higher pH optima, nearing neutral. Exoglycosidases cleave the glycosidic linkage of terminal sugars from the nonreducing end of glycans (the outermost left end of glycans for figures in this book, e.g., Figure 44.1). Exoglycosidases recognize only one monosaccharide (rarely two) in a specific anomeric linkage, and are much less particular about the structure of the molecule beyond that glycosidic linkage. This lack of specificity allows these enzymes to act on a broad range of substrates. However, exoglycosidases do not usually work unless all of the hydroxyl groups of the terminal sugar are unmodified. Acetate, sulfate, or phosphate groups usually have to be removed before action of the glycosidases. Esterases cleave acetyl groups and specific sulfatases remove the sulfate groups on glycosaminoglycans (GAGs) and N- or O-linked glycans. Endoglycosidases cleave internal glycosidic linkages of larger chains. These enzymes are often more tolerant of modifications of the glycan; in some cases, they require a modified sugar for optimal cleavage.

FIGURE 44.1.

Degradation of complex N-glycans. The lysosomal degradation pathway of glycoproteins carrying complex-type glycans proceeds simultaneously on both the protein and glycan moieties. The N-glycans are sequentially degraded by the indicated exoglycosidases (more...)

Even though the lysosomal glycosidases perform similar reactions, their amino acid sequences are only ∼15%–20% identical to each other. Thus, there are no highly conserved glycosidase catalytic domains. Lysosomal enzymes are all N-glycosylated and most are targeted to the lysosome by the mannose-6-phosphate pathway (Chapter 33), share aspects of the recognition marker for assembly of mannose-6-phosphate on N-linked glycans, and have affinity for mannose-6-phosphate receptors. The concentration of enzymes within the lysosome is difficult to determine. Proteinases such as cathepsins are estimated to be ∼1 mm; glycosidases are probably present at much lower concentrations.

GENETIC DEFECTS IN LYSOSOMAL DEGRADATION OF GLYCANS

About 50 known inherited diseases impair lysosomal degradation of macromolecules. Although each one is rare, together, their occurrence is about one in every 5000 to 10,000 births. Loss of a single lysosomal hydrolase leads to the accumulation of its substrate as undegraded fragments in tissues and the appearance of related fragments in urine. Many of the human disorders have animal models. Tables 44.1, 44.2, and 44.3 show some of the major clinical symptoms of diseases associated with the degradation of three classes of glycans. Many of the diseases share overlapping symptoms, and yet each disease has unique clinical features that allow it to be diagnosed. The advent of exome and genome sequencing has shortened the diagnostic waiting period for concerned families. Many of the diseases also present with a range of severities. Usually, an infantile onset is the most severe and the juvenile or adult onsets have milder symptoms. The later-onset forms may even affect organ systems distinct from those affected by early onset forms. Hundreds of mutations have been mapped in the different disorders. The severity usually depends on the combination of mutated alleles. Predicting the disease severity (prognosis) from the specific mutation is generally difficult. Complete absence of a lysosomal hydrolase is uniformly severe. Hypomorphic alleles have variable residual glycosidase activity making their prognosis is difficult.

TABLE 44.1.

Defects in glycoprotein degradation

TABLE 44.2.

Defects in glycosaminoglycan (GAG) degradation—the mucopolysaccharidoses

TABLE 44.3.

Defects in glycolipid degradation

It is not clear whether accumulating different types of undegraded glycans leads to the different symptoms characteristic of each disease. There is no evidence that the stored material causes lysosomes to burst and spew their contents into the cytoplasm. Some leakage may occur into the cytoplasm or even out of cells, or the cell may sense an “engorged” lysosome. The pathology likely depends on the cell type and the cellular balance of synthesis and turnover rates. For instance, dermatan sulfate (DS), a GAG (Chapter 17), predominates in connective tissue, which might explain the bone, joint, and skin problems in mucopolysaccharidosis (MPS) I, II, VI, and VII. Keratan sulfate (KS), another GAG, is present in cartilage; therefore, MPS IV is largely a skeletal disease. Gangliosides (Chapter 11) are most abundant in neurons; therefore, gangliosidoses are predominantly brain disorders. The importance of glycogen for muscle explains the impact of Pompe disease on the heart and diaphragm, leading to rapid lethality in that disease. Given the balance between synthesis and degradation of multiple glycans, reducing synthesis somewhat by the use of an inhibitor may help to retard the accumulation of undegraded material and reduces the pathology of some diseases.

GLYCOPROTEIN DEGRADATION

The great majority of N- and O-glycans reaching the lysosome contain only six sugars linked in one or two anomeric configurations: β-N-acetylglucosamine (βGlcNAc), α/β-N-acetylgalactosamine (α/βGalNAc), α/β-galactose (α/βGal), α/β-mannose (α/βMan), α-fucose (αFuc), and α-sialic acid (αSia). Each linkage should theoretically require only one anomer-specific glycosidase, assuming that each glycosidase ignores the underlying glycan. This number is close to the known number of enzymes in most glycan degradation pathways. However, some linkages require a specific enzyme outside of this group. For example, β-N-acetylhexosaminidase cleaves both βGlcNAc and βGalNAc residues. Degradation of the GlcNAcβAsn and GalNAcαSer/Thr linkages also requires specific enzymes.

GLYCOSAMINOGLYCAN DEGRADATION

GAGs, including heparan sulfate (HS), chondroitin sulfate (CS), DS, KS, and hyaluronan, are degraded in a highly ordered fashion. The first three are O-xylose-linked to core proteins (Chapter 17), KS is N- or O-linked, and hyaluronan is a free glycan (Chapter 16). Some proteoglycans are internalized from the cell surface and the protein portion is degraded. The GAG chains are then partially cleaved by enzymes such as endo-β-glucuronidases or endohexosaminidases that clip at a few specific sites. Endoglycosidase cleavage creates multiple terminal residues that can be degraded by unique or overlapping sets of sulfatases and exoglycosidases. Structural analyses of partially degraded fragments in the lysosomes of cells from patients with MPS revealed both the degradation pathways and genetic defects responsible (Table 44.2). The clinical severities and manifestations vary even with mutations in the same gene. For instance, MPS I is clinically subdivided into Hurler (most severe), Hurler–Scheie, and Scheie syndromes, although the three disorders represent a continuum of the same disease (Table 44.2). Hurler–Scheie patients progress more slowly and die in early adulthood, whereas Scheie patients can survive to middle or old age. The milder forms of this disease do not cause intellectual disability.

Hyaluronan

Hyaluronan (Chapter 16) is the largest and most abundant GAG: a 70-kg person degrades 5 g of hyaluronan (molecular mass 10⁷ Da) per day. Degradation of hyaluronan involves a series of two endo-β-N-acetylhexosaminidases called hyaluronidases (Hyal-1 and Hyal-2), β-glucuronidase, and finally β-N-acetylhexosaminidase. Hyal-2 is active at low pH and is GPI-anchored on the cell surface. This enzyme associates both with hyaluronan in lipid rafts and with an Na⁺/H⁺ exchanger to create an acidic microenvironment. Cleavage generates fragments of ∼20 kDa (approximately 50 disaccharides), which are internalized, delivered to endosomes, and then finally to lysosomes, where the fragments are degraded by Hyal-1 into tetra- and disaccharides (Figure 44.2). The exoglycosidases are thought to participate in the degradation of the larger fragments as well as that of the di-and tetrasaccharide units. Mutations in HYAL-1 cause MPS IX.

FIGURE 44.2.

Degradation of hyaluronan and heparan sulfate. (Left) Hyaluronidase (an endoglycosidase) cleaves large chains into smaller fragments, each of which is then sequentially degraded from the nonreducing end. (Right) Degradation of heparan sulfate (HS). An (more...)

Dermatan Sulfate and Chondroitin Sulfate

DS and CS are related GAG chains based on a repeating polymer of βGlcA and βGalNAc (Chapter 17). Figure 44.3 shows that a combination of endoglycosidases, sulfatases, and exoglycosidases degrades DS in the lysosome. Iduronic acid-2-sulfatase is followed by α-iduronidase. The terminal GalNAc-4-SO₄ can be removed by either of two pathways. In the first pathway, a GalNAc-4-SO₄ sulfatase acts followed by β-N-acetylhexosaminidase A or B to remove N-acetylgalactosamine. In the second, β-N-acetylhexosaminidase A removes the entire GalNAc-4-SO₄ unit followed by sulfatase cleavage. Absence of the GalNAc-4-SO₄ sulfatase (MPS VI) is unique to the DS pathway. β-Glucuronidase cleaves the β-glucuronic acid residue and the process is repeated on the rest of the molecule.

FIGURE 44.3.

Degradation of chondroitin/dermatan sulfates (CS/DS) and keratan sulfate (KS). (Left) Only DS degradation is shown. After removal of sulfated iduronic acid in two steps, further sequential degradation can proceed by two different routes. One route (straight (more...)

To degrade CS, GalNAc-6-SO₄ sulfatase and GalNAc-4-SO₄ sulfatase work in combination with β-N-acetylhexosaminidase A or B and β-glucuronidase. Hyaluronidases can also degrade CS, but no CS-specific endoglycosidases have been found.

GLYCOSPHINGOLIPID DEGRADATION

Glycosphingolipids (Chapter 11) are degraded from the nonreducing end by exoglycosidases while they are still bound to the lipid moiety ceramide. Because glycosphingolipids share some of the same outer sugar sequences found in N- and O-glycans (Chapter 14), many of the same glycosidases are used for their degradation (Figure 44.4). However, specific hydrolases cleave the glucose–ceramide and galactose–ceramide bonds. Besides specific enzymes, noncatalytic sphingolipid activator proteins (SAPs or saposins) help to present the lipid substrates to enzymes for cleavage. Endoglycoceramidases from leeches and earthworms release the entire glycan chain from the lipid, much like PNGase releases N-glycans from proteins.

FIGURE 44.4.

Degradation of glycosphingolipids. Required activator proteins are shown in parentheses and individual enzymes are as shown in previous figures in this chapter. (Sap) saposin.

DEGRADATION AND RESYNTHESIS

Degradation of glycans is not always complete. Partially degraded or incomplete glycans on glycoproteins, glycopeptides, and glycosphingolipids can be internalized within a functional Golgi compartment containing sugar nucleotides and glycosyltransferases and then elongated. In the case of glycosphingolipids, this pathway makes a substantial contribution to total cellular synthesis, but in glycoproteins it probably makes a relatively small contribution. These processes can be salvage and repair mechanisms or may play an integral part in an unidentified physiological pathway.

SALVAGE OF MONOSACCHARIDES

Monosaccharide salvage is discussed in Chapter 5. Monosaccharides derived from glycan degradation are transported back into the cytoplasm using transporters specific for neutral sugars, N-acetylated hexoses, anionic sugars, sialic acid, and glucuronic acid. Turnover of glycans is high and the salvage of sugars can be quite substantial. There have been few studies comparing the actual contributions of exogenous monosaccharides, those salvaged from glycan turnover, and those generated from de novo synthesis. Cells probably differ in their preference for each source of monosaccharides. These differences may explain why monosaccharide therapy used to treat some glycosylation disorders is effective in certain cells and not others.

BLOCKING DEGRADATION

Lysosomal degradation of glycans can be blocked by raising the intralysosomal pH, by inactivating a particular glycosidase, by mutation, or by protein-specific inhibitors.

Lysosomal enzymes usually have acidic pH optima and adding ammonium chloride or chloroquine slows degradation by increasing the intralysosomal pH. However, some lysosomal enzymes have relatively broad pH optima, higher than the lysosome. Some active “lysosomal” enzymes can be found in early and late endosomes, which have a higher pH than lysosomes.

Glycosylation inhibitors are covered in Chapter 55 but some of these agents also inhibit specific lysosomal enzymes. For example, swainsonine blocks lysosomal α-mannosidase as well as the α-mannosidase II involved in glycoprotein processing. Sheep and cattle become neurologically deranged by eating food rich in swainsonine, which is also called locoweed. These temporary symptoms are likely induced because lysosomal α-mannosidase is inhibited. Undegraded oligosaccharides probably accumulate in affected animals. Although many inhibitors block various enzymes in vitro, they may not be effective in cells or whole animals because they may not enter the lysosome at sufficient concentrations.

THERAPY FOR LYSOSOMAL ENZYME DEFICIENCIES

Study of lysosomal enzyme defects provided major insights into catabolic pathways and showed their importance to human health. Mannose-6-phosphate targeting of lysosomal enzymes was discovered by showing that cocultivation of fibroblasts from patients with different storage diseases led to the disappearance of stored material in both types of cells. Each supplied the corrective factors (i.e., lysosomal enzymes) that the other cell lacked by delivery of secreted enzymes through cell-surface mannose-6-phosphate receptors (Chapter 33). Cross-correction highlights the fact that only a relatively small amount of enzyme may be needed to prevent accumulation of storage products and the resulting pathology. Some estimates suggest that <5% of normal β-N-acetylhexosaminidase activity may be sufficient to prevent pathological symptoms of Tay–Sachs disease. In fact, the index Scheie patient with α-L-iduronidase deficiency had <1% normal activity in fibroblasts and lived 77 years.

Current therapeutic approaches include enzyme replacement therapy (ERT), substrate reduction therapy (SRT), and enzyme enhancement therapy (EET). Gene replacement therapy is a promising future approach, but not yet clinically tested. Accumulation of stored undegraded material occurs because the rate of glycan synthesis is greater than its degradation at steady state. SRT reduces the glycan's synthetic rate to counter low glycosidase activity. This hypothesis was tested in a mouse model of Sandhoff disease by using N-butyldeoxynojirimycin, an inhibitor of glucosylceramide synthase, which catalyzes the first step in glycosphingolipid biosynthesis. When wild-type mice were treated with this compound, the amount of glycosphingolipids fell 50%–70% in all tissues without obvious pathological effects. When this compound was given to Sandhoff disease mice, the accumulation of GM2 in the brain was blocked and the amount of stored ganglioside reduced. Thus, reducing the synthesis of the primary precursor reduced the load of GM2 to levels that were degradable by the glycosidase-deficient mice. Because this compound inhibits the first biosynthetic step, theoretically this drug could reduce the accumulation of storage products in any glycolipid storage disorder.

In clinical trials, N-butyldeoxynojirimycin (miglustat, Zavesca) was effective for many patients with mild-to-moderate Gaucher's disease, although improvement varied as did side effects. Small trials in infantile Tay–Sachs disease patients did not arrest neurological deterioration, but prevented macrocephaly. Further clinical studies are likely to reveal the potential and limitations of SRT.

EET or pharmacological chaperone therapy (PCT) is based on the concept that inhibitors of an enzyme can act as molecular chaperones to stabilize the mutated enzymes in the endoplasmic reticulum (ER). When the enzyme reaches the lysosome, the inhibitor dissociates at low pH and the abundant stored material is gradually digested. The overall increase in activity is small, but it can have significant clinical benefits. Clinical trials are in progress for treating Fabry disease, Gaucher Type I, and Pompe disease, whereas preclinical trials are being performed for GM1 and GM2 gangliosidosis. A potential problem is that some inhibitors may not cross the blood–brain barrier, limiting their effectiveness in the central nervous system (CNS). The advent of high-throughput screening methods and large chemical libraries increase the potential of this approach. In addition, allosteric site binders can be selected that remain bound to the enzyme and any successful compound could be used in combination with ERT to further enhance activity.

ERT has been quite successful for treating Gaucher's disease. Injection of glucocerebrosidase carrying mannose-terminated N-glycans targets the enzyme to the macrophage/monocytes, which are the primary sites of substrate accumulation. In use now for many years, the added enzyme consistently improves patients' clinical features with minimal side effects. The cost of treatment is high, and high doses are no more effective than lower doses for improving visceral and hematological pathologies. The proven effectiveness makes glucocerebrosidase the favorite therapy for these patients, but terminal mannose residues cannot be used to target the enzyme to other cells or organs or to enable treatment of other lysosomal storage disorders. Insufficient infused enzyme crosses the blood–brain barrier, thus, the enzyme (Cerezyme) works very well for type I disease without neurological involvement but not for the less common Gaucher's with neurological involvement. In addition, patients sometimes develop antibodies against the injected human protein.

ERT trials with other recombinant lysosomal enzymes have progressed. Enzyme replacement therapy is available for Fabry disease, Pompe disease, and mucopolysaccharidosis types I, II, IVA, and VI and is under development for others. Recombinant α-galactosidase for Fabry disease works well and recombinant α-L-iduronidase for MPS I works very well with the intermediate severity form (Hurler–Scheie), but its effect is unclear for patients with the more common neurological form (Hurler syndrome). Recombinant N-acetylgalactosamine-4-sulfatase (arylsulfatase B; Naglazyme) was approved for MPS VI, as was α-glucosidase (Myozyme) for treating Pompe disease patients. Iduronic acid-2-sulfatase (Elaprase) is approved for treating Hunter syndrome. These results clearly validate this approach. The efficacy of most of these enzymes relies on the engineering of Man-6-P-containing glycans. This is one of the foremost examples of how the clinically motivated exploration of a disorder (I-cell disease) led to the discovery of Man-6-P-based targeting of lysosomal enzymes (Chapter 33)and the development of an entire industry targeting therapy for multiple diseases. The availability of animals deficient in specific lysosomal enzymes offers appropriate systems to test the efficacy of corrective genes, compounds, and enzymes.

ACKNOWLEDGMENTS

The authors appreciate helpful comments and suggestions from Michelle Dookwah, Chrissa Dwyer, and Bastian Ramms.

FURTHER READING

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