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Varki A, Cummings RD, Esko JD, et al., editors. Essentials of Glycobiology. 2nd edition. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2009.

Cover of Essentials of Glycobiology

Essentials of Glycobiology. 2nd edition.

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Chapter 41Genetic Disorders of Glycan Degradation


This chapter explores the degradation and turnover of glycans in lysosomes, especially with respect to human genetic disorders. Delineation of the dismantling of a few representative glycans illustrates features unique to different pathways. The degradation of oligomannosyl N-glycans removed from misfolded, newly synthesized glycoproteins is covered in Chapter 36.


Most glycans are degraded in lysosomes by highly ordered and specific pathways employing endo- and exoglycosidases, sometimes aided by noncatalytic proteins. Much of the insight for unraveling these complex pathways emerged from studies of rare human genetic disorders called lysosomal storage diseases. In each of these diseases, selected molecules accumulate in the lysosomes. Clever experiments combining enzymology with glycan structural analyses revealed the steps of the pathways and also unlocked the mechanism of lysosomal enzyme targeting as described in Chapter 30.

Lysosomes contain an estimated 50–60 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 lysosomal enzymes have higher pH optima that are closer to neutral. Exoglycosidases cleave the glycosidic linkage of terminal sugars from the nonreducing end of the glycan (i.e., the residue at the outermost end of the molecule at the extreme left of glycan structures as represented in most of the figures in this book). The exoglycosidases recognize only one monosaccharide (rarely two) in a specific anomeric linkage, but they are much less particular about the structure of the molecule beyond the terminal 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. Substitutions such as acetate, sulfate, or phosphate groups usually have to be removed prior to further degradation. Esterases cleave acetyl groups and specific sulfatases remove the sulfate groups on glycosaminoglycans 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.

Even though the lysosomal glycosidases carry out similar reactions, their amino acid sequences are only about 15–20% identical and at most are 40–45% similar 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 discussed in Chapter 30. Therefore, they share aspects of the recognition marker for assembly of the mannose-6-phosphate moiety and have variable affinities for mannose-6-phosphate receptors. The concentration of enzymes within the lysosome is difficult to determine, but proteinases such as cathepsins B, D, and L have been estimated to be present at levels of 1 mM. Glycosidases are probably present at much lower concentrations.


More than 45 inherited diseases are known that impair the lysosomal degradation of macromolecules. 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 listed in Table 41.1 have animal models.

TABLE 41.1

TABLE 41.1

Defects in glycoprotein degradation

Table 41.1, Table 41.2, and Table 41.3 also show some of the major clinical symptoms of diseases associated with the degradation of each type of glycoconjugate. Many of the diseases share overlapping symptoms, and yet each disease has unique features that allow it to be specifically diagnosed by experienced clinicians. 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 attenuated (milder) symptoms. The later onset forms may even affect organ systems different 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, except for a combination of null alleles, which have severe outcomes. Hypomorphic alleles have residual glycosidase activity, but their prognosis is also difficult. Complete absence of a lysosomal hydrolase is uniformly severe.

TABLE 41.2

TABLE 41.2

Defects in glycosaminoglycan degradation—the mucopolysaccharidoses

TABLE 41.3

TABLE 41.3

Defects in glycolipid degradation

It is not clear whether accumulating different types of undegraded material in a lysosome 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 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 predominates in connective tissue, which might explain the bone, joint, and skin problems in mucopolysaccharidosis (MPS) I, II, VI, and VII. Keratan sulfate is present in cartilage; therefore, MPS IV is largely a skeletal disease. GM2 ganglioside is abundant in neurons but not in other tissues; therefore, gangliosidosis is predominantly a brain disorder. The importance of glycogen for muscle explains the impact of Pompe disease on the heart and diaphragm, leading to rapid lethality in that disease.

In the balance between synthesis and degradation of multiple glycans, reducing synthesis somewhat by the use of an inhibitor helps to retard the accumulation of undegraded material in mouse models and reduces the pathology of disease.


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-acetyl-galactosamine (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 actually quite close to the known number of enzymes in the 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.

Lysosomal Degradation of Complex N-Glycans

Much of what we know about this pathway comes from analysis of products that accumulate in patients’ tissues or urine due to the absence of one of the degradative enzymes (Table 41.1). Structural analysis of monosaccharide-labeled glycoproteins during degradation in perfused rat liver also aided elucidation of the pathway. By conducting the latter studies in the presence of inhibitors of different lysosomal enzymes, a picture of simultaneous and independent bidirectional degradation of the protein and carbohydrate chains emerged (Figure 41.1). The relative degradation rates vary depending on structural and steric factors of the protein and the sugar chains. The accumulation of GlcNAcβ1-4GlcNAcβAsn in cells that cannot cleave the GlcNAcβAsn linkage clearly shows that degradation of the sugar chain does not require prior cleavage of the Asn. Much of the protein is probably degraded before N-glycan catabolism begins. Removal of core fucose (Fucα1–6GlcNAc) and probably any peripheral fucose residues linked to the outer branches of the chain (e.g., Fucα1–3GlcNAc) appears to be the first step in degradation because patients lacking this enzyme still have intact N-glycans bound to asparagine. Glycosylasparaginase (aspartyl-N-acetyl-β-D-glucosaminidase) then cleaves the GlcNAcβAsn bond, provided the α-amino group is not in peptide linkage. In rodents and primates, chitobiase (an endo-β-N-acetylglucosaminidase) removes the reducing N-acetylglucosamine, leaving the oligosaccharide with only one terminal N-acetylglucosamine. In many other species, splitting of the chitobiose linkage (GlcNAcβ1–4GlcNAc) uses the β-N-acetylhexosaminidase mentioned below as the last step in degradation. Either pathway appears to be effective, leaving the presence of chitobiase in some species unexplained. The oligosaccharide chain is then sequentially degraded by sialidases and/or α-galactosidase, followed by β-galactosidase, β-N-acetylhexosaminidase, and α-mannosidases. The remaining Manβ1–4GlcNAc is cleaved by β-mannosidase to mannose and N-acetylglucosamine or, in those species that do not have chitobiase, to chitobiose, which is then degraded by β-N-acetylhexosaminidase.

FIGURE 41.1. Degradation of complex N-glycans.


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...)

Lysosomal sialidase (neuraminidase), β-galactosidase, and a serine carboxypeptidase called protective protein/cathepsin A form a complex in the lysosome that is required for efficient degradation of sialylated glycoconjugates. Cathepsin A protects β-galactosidase from rapid degradation and also activates the sialidase precursor, but the protection does not depend on the catalytic activity of cathepsin A. Mutations in this protective protein lead to galactosialidosis in which the simultaneous deficiencies in β-galactosidase and sialidase are secondary effects of defective cathepsin A.

Other glycans that have GalNAcβ1–4GlcNAc, GlcAβ1–3Gal, or Galα1–3Gal on the outer branches must first have these residues removed by β-N-acetylhexosaminidase, β-glucuronidase, and α-galactosidase, respectively, prior to any further digestion of the underlying oligosaccharide chain.

Lysosomal Degradation of Oligomannosyl N-Glycans

Oligomannosyl N-glycans that enter the lysosome are hydrolyzed by an α-mannosidase to yield Manα1–6Manβ1–4GlcNAc (a common intermediate derived from hybrid and complex N-glycans). A second α1–6-specific mannosidase can cleave this linkage in humans and rats, but only on molecules that have a single core region N-acetylglucosamine (i.e., those generated by chitobiase cleavage). Finally, β-mannosidase completes the degradation. Oligomannosyl N-glycans derived from dolichol-linked precursors or misfolded glycoproteins (primarily Man5GlcNAc) are handled differently as described in Chapter 36.

Degradation of O-Glycans

Degradation of typical αGalNAc-initiated O-glycans has not been systematically studied. Many of the outer structures seen on N-glycans are also found on O-glycans (see Chapter 13); therefore, degradation of these glycans probably uses the same group of exoglycosidases as discussed above. The exception to this, of course, is the linkage region, GalNAcα-O-Ser/Thr. Patients with Schindler disease lack an α-N-acetylgalactosaminidase, which is specific for αGalNAc and will not cleave αGlcNAc. This same enzyme probably removes terminal α-GalNAc from blood group A–containing glycans (GalNAcα1–3Gal) and some glycolipids such as the Forsmann antigen (GalNAcα1–3GalNAcβ1–3Galα1–4Galβ1–4GlcβCer). Patients lacking α-N-acetylgalactosaminidase accumulate GalNAc-containing glycopeptides in their urine, but curiously they also accumulate more complex, extended glycopeptides containing N-acetylglucosamine, galactose, and sialic acid. The structures are the same as those found on some native glycoconjugates. Their production could result from a general slowdown of oligosaccharide degradation, or they may arise by reassembly of glycans on GalNAcα-O-Ser/Thr glycopeptides that accumulate. How could this happen? There are several possibilities. One is that some of the partially degraded glycopeptides enter a compartment (perhaps the Golgi) that contains the appropriate glycosyltransferases and nucleotide sugars. Monosaccharides are then added sequentially before the products exit the cell. In this way, the glycopeptides generated in the lysosome would be behaving like oligosaccharide primers (see Chapter 50). However, another possibility is that the concentration of GalNAcα-O-Ser/Thr is high enough in the lysosome that lysosomal glycosidases use them as acceptors in a series of transglycosylation reactions (see Chapter 49). Because lysosomal glycosidases form complexes to degrade glycoconjugates more efficiently, partially degraded substrates may be in a preferred location as acceptors in transglycosylation reactions.


The glycosaminoglycans (GAGs), including heparan sulfate (HS), chondroitin sulfate (CS), dermatan sulfate (DS), keratan sulfate (KS), and hyaluronan, are degraded in a highly ordered fashion. The first three are O-xylose-linked to core proteins. KS can be both N- and O-linked depending on the tissue source, whereas hyaluronan is made as a free glycan (see Chapter 15). 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 analysis of partially degraded fragments in the lysosomes of cells from patients with genetic defects in these pathways cause mucopolysaccharidoses (MPS). MPS disorders were critical to dissecting the degradation pathways (see Table 41.2). Note that there is a range of clinical severities and manifestations with mutations in the same gene. For instance, MPS I is clinically subdivided into Hurler, Hurler/Scheie, and Scheie syndromes, although the three disorders represent a continuum of the same disease. Hurler is the most severe form of the disease (Table 41.2). 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 mental retardation.


Hyaluronan (see Chapter 15) is the largest and most abundant GAG: A 70-kg person degrades 5 g of hyaluronan (molecular mass 107 daltons) 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 approximately 20 kD (about 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 41.2). The exoglycosidases are thought to participate in the degradation of the larger fragments as well as that of the di-and tetrasaccharide units.

FIGURE 41.2. Degradation of hyaluronan.


Degradation of hyaluronan. Hyaluronidase (an endoglycosidase) cleaves large chains into smaller fragments, each of which is then sequentially degraded from the nonreducing end. Symbol Key: Image symbol_key_small.jpg

Heparan Sulfate

HS (see Chapter 16) is first degraded by an endoglucuronidase, followed by a well-ordered sequential degradation. An example is shown in Figure 41.3. A terminal iduronic acid-2-sulfate must be desulfated by a specific iduronic acid-2-sulfatase to make the modified sugar a suitable substrate for α-iduronidase. If a glucuronic acid (GlcA)-2-sulfate were at this position, a GlcA-2-sulfatase would first remove the sulfate, followed by β-glucuronidase cleavage. The new terminal glucosamine sulfate (GlcNSO4) is the next sugar for cleavage, but this process requires two steps. Sulfate is first removed by N-sulfatase forming glucosamine, but this sugar cannot be cleaved by α-N-acetylglucosaminidase. The amino group must be N-acetylated by an N-acetyltransferase embedded in the lysosomal membrane in order to convert glucosamine to N-acetylglucosamine, which is then a suitable substrate for enzymatic cleavage. In the first step, acetyl CoA donates the acetyl group to a histidine residue in the cytoplasmic domain of the enzyme. The acetyl group then becomes available on the luminal side of the lysosomal membrane and is transferred at low pH to the amino group of glucosamine on the partially degraded HS chain. The terminal αGlcNAc can now be cleaved. Cleavage of the 2-sulfated glucuronic acid residue requires that the sulfate first be removed, followed by β-glucuronidase cleavage. If the next αGlcNAc is 6-O-sulfated, the sulfate is removed by a specific GlcNAc-6-sulfatase. Table 41.2 provides a list of the enzymatic defects in HS degradation and the disorders that result.

FIGURE 41.3. Degradation of heparan sulfate (HS).


Degradation of heparan sulfate (HS). An endo-β-glucuronidase first cleaves large chains into smaller fragments and each monosaccharide is then removed from the nonreducing end as described in the text. N- and O-sulfate groups must first be removed (more...)

Dermatan Sulfate and Chondroitin Sulfate

DS and CS are related GAG chains based on a repeating polymer of βGlcA and βGalNAc (see Chapter 16). Figure 41.4 shows that a combination of endoglycosidases, sulfatases, and exoglycosidases degrades DS in the lysosome. Iduronate-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 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 41.4. Degradation of dermatan sulfate and chondroitin sulfate.


Degradation of dermatan sulfate and chondroitin sulfate. Sequential degradation can proceed by two different routes. One route uses GalNAc-4-SO4 sulfatase followed by cleavage with β-N-acetylhexosaminidase A or B. The other route uses only β- (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.

Keratan Sulfate

KS is an N- or O-glycan with a heavily sulfated poly-N-acetyllactosamine chain. Mammalian cells do not have an endoglycosidase to break down KS (Figure 41.5). The sequential action of sulfatases and exoglycosidases is needed. Galactose-6-SO4 sulfatase is the same enzyme that desulfates GalNAc-6-SO4 in CS degradation. This hydrolysis is followed by β-galactosidase digestion, leaving a terminal GlcNAc-6-SO4. Desulfation by the sulfatase followed by cleavage with β-N-acetylhexosaminidase A or B eliminates GlcNAc-6-SO4. Alternatively, β-N-acetylhexosaminidase A can directly release GlcNAc-6-SO4, followed by desulfation of the monosaccharide.

FIGURE 41.5. Degradation of keratan sulfate.


Degradation of keratan sulfate. Sequential degradation of KS occurs from the nonreducing end like the degradation of DS and CS. The terminal GlcNAc-6-SO4 can be cleaved sequentially by a sulfatase and then by β-N-acetylhexosaminidase A or B or, (more...)

Linkage Region

Degradation of the core region of O-linked KS (skeletal type; type II) probably occurs by the same route as other O-glycans. The N-glycan corneal-type KS (type I) is probably degraded by the same set of enzymes used for N-glycan degradation. The more typical O-xylose-linked GAG chains (DS, CS, HS) all share a common core tetrasaccharide: GlcAβ1-3Galβ1–3Galβ1–4Xylβ-O-Ser. An endo-β-xylosidase has been detected in rabbit liver. The details of how the linkage regions of GAGs are degraded remain to be established.

Multiple Sulfatase Deficiency

A very rare human disorder is the multiple sulfatase deficiency (MSD). All sulfatases are casualties of this defect since they all undergo a posttranslational conversion of an active site cysteine residue to a 2-amino-3-oxopropionic acid for activity. In essence, the SH group of cysteine is replaced by a double-bonded oxygen atom that probably acts as an acceptor for cleaved sulfate groups. Loss of the enzyme carrying out that reaction leads to inactive sulfatases. This deficit affects GAG degradation and any other sulfated glycan such as sulfatides.


Glycosphingolipids (see Chapter 10) are degraded from the nonreducing end by exoglycosidases while they are still bound to the lipid moiety ceramide. Since glycosphingolipids have many of the same outer sugar sequences that are found in N- and O-glycans (see Chapter 13), many of the same glycosidases are used for their degradation (Figure 41.6). However, specialized hydrolases are needed for cleaving the glucose-ceramide and galactose-ceramide bonds and other linkages near the membrane. Besides these specific enzymes, additional noncatalytic sphingolipid activator proteins (SAPs or saposins) help to present the substrate to the enzyme for cleavage. Endoglycoceramidases have been reported in leeches and earthworms. Like PNGase that releases N-glycans from protein, these enzymes release the entire glycan chain from the lipid.

FIGURE 41.6. Degradation of glycosphingolipids.


Degradation of glycosphingolipids. Required activator proteins are shown in parentheses and individual enzymes are as shown in previous figures in this chapter. (Sap) saposin. Symbol Key: Image symbol_key_small.jpg

Specialized Issues for Glycolipid Degradation

Some exoglycosidases for glycoprotein and GAG chain degradation also degrade glycolipids, but others are unique to glycolipid degradation. Absence of these unique enzymes causes glycolipid storage diseases, which are listed in Table 41.3. Glucocerebrosidase, also called β-glucoceramidase, is specific for the degradation of the GlcβCer bond. The loss of this enzyme causes Gaucher’s disease.

A specialized β-galactosidase, β-galactoceramidase, hydrolyzes the bond between galactose and ceramide and can also cleave the terminal galactose from lactosylceramide. Loss of this enzyme produces Krabbe disease. Galactosylceramide is often found with a 3-sulfate ester (sulfatide) and a specific sulfatase (arylsulfatase A) is needed for its removal prior to β-galactosylceramidase action. Loss of this sulfatase causes metachromatic leukodystrophy and the accumulation of sulfatide. Glycolipids terminated with α-galactose residues are degraded by a specific α-galactosidase, the loss of which causes Fabry disease.

Activator Proteins

Sugars that lie too close to the lipid bilayer apparently have limited access to soluble lysosomal enzymes, and additional proteins called saposins are required to present the substrates to the enzymes. Two genes code for all known SAPs. These proteins can also be used to form complexes with more than one degradative enzyme for more efficient hydrolysis of short glycolipids close to the membrane. SAPs are derived from a 524-amino-acid precursor called prosaposin, which is processed into four homologous activator proteins each comprising about 80 amino acids: saposins A, B, C, and D. Despite the homology, each saposin has different properties. Saposin A and saposin C both help β-glucosyl- and β-galactosylceramidase degradation. Saposin B assists arylsulfatase A, α-galactosidase, sialidase, and β-galactosidase. The activation mechanism of SAPs is thought to be similar to that of the GM2 activator, which is described below. Saposins D and B also assist in sphingomyelin degradation by sphingomyelinase. Complete absence of prosaposin is lethal in humans, and deficiencies in saposin B and saposin C lead to defects that clinically resemble aryl sulfatase A deficiency (metachromatic leukodystrophy) and Gaucher’s disease. These symptoms might be predicted based on the enzymes that these saposins assist.

GM2 activator protein forms a complex with either GM2 or GA2 and presents them to β-N-acetylhexosaminidase A for cleavage of β-GalNAc. The activator protein binds a molecule of glycolipid, forming a soluble complex, which can be cleaved by the hexosaminidase. The resulting product is then inserted back into the membrane, and the activator presents the next GM2 molecule, and so on. Genetic loss of this activator protein causes the accumulation of GM2 and GA2, resulting in the AB variant of GM2 gangliosidosis.

Topology for Degradation and Roles of Lipids

Endocytic vesicles deliver membrane components to the lysosome for degradation and are often seen as intralysosomal multivesicular storage bodies (MVB). They appear like vesicles within the lysosome and are especially prominent in patients with glycolipid storage disorders. How do “vesicles within vesicles” form and why do we have them? MVBs serve an important function. The internal surface of the lysosomal membrane has a thick, degradation-resistant glycocalyx of integral and peripheral membrane proteins decorated with poly-N-acetyllactosamines, which protect the lysosomal membrane against destruction. However, these proteins would also shield incoming membranes from degradation if fusion simply occurred. By creating multiple internal membranes seen in typical MVBs, the target molecules are exposed to the soluble lysosomal enzymes, and digestion proceeds efficiently on the membrane surfaces of these internal vesicles.

MVB formation starts with inward budding of the limiting endosomal membrane. Lipids and proteins are sorted to either the internal or the limiting membrane. Ubiquitination of cargo proteins targets them to internal vesicles of MVBs, but ubiquitin-independent routes also exist. Intra-endosomal membranes and limiting endosomal membrane have different lipid and protein compositions. Membrane segregation and lipid sorting prepare the internal membranes for lysosomal degradation. During maturation of the internal membranes, cholesterol is continually stripped away (to <1%) and a negatively charged lipid bis(monoacylglycero)phosphate (BMP) increases up to 45%. This molecule is highly resistant to phospholipases. BMP also stimulates sphingolipid degradation on the inner membranes of acidic compartments. Since BMP is not present in the lysosomal outer membrane, the unique lipid profile ensures that degradation by hydrolases and membrane-disrupting SAPs occurs without digesting the lysosomal outer membrane.


Degradation of glycans is not always complete. Partially degraded or incomplete glycans on glycoproteins and glycosphingolipids at the cell surface can be internalized and then elongated within a functional Golgi compartment containing the sugar nucleotide and the appropriate glycosyltransferase. 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 could simply be salvage and repair mechanisms or might play an integral part in an unidentified physiological pathway.

Reglycosylation and Recycling of Glycoproteins

Experiments clearly show that the half-life of specific membrane proteins is longer than the half-life of their sugar chains. Terminal monosaccharides turn over faster than those near the reducing end of the glycan, suggesting that terminal sugars are removed by exoglycosidases. Cleavage may occur at the cell surface or when proteins are endocytosed in the course of normal membrane recycling. Because the mildly acidic late endosomes contain lysosomal enzymes with a fairly broad pH range, terminal sugars such as sialic acid can be cleaved. If the endocytosed proteins are not degraded in lysosomes, the proteins may encounter sialyltransferases in the Golgi, become resialylated, and appear again on the cell surface. A similar situation may occur if a protein has lost both sialic acid and galactose residues from its glycans. Some membrane proteins synthesized in the presence of oligosaccharide processing inhibitors can still reach the cell surface in an unprocessed form. Subsequent incubation in the absence of inhibitors leads to normal processing over time. The extent of processing and the kinetics depend on the protein and the cell line. Because Golgi enzymes are not distributed identically in all cells, general statements about how much “reprocessing” can occur are difficult to make. Most studies have monitored N-glycans, but membrane proteins with O-glycans probably behave similarly.

Glycosphingolipid Recycling

The majority of the newly synthesized glucosylceramide arrives at the cell surface by a Golgi-independent cytoplasmic pathway. Some of this material, as well as glucosylceramide generated by partial degradation of complex glycosphingolipids, can recycle to the Golgi. There, like glycoproteins, simple glycosphingolipids can serve as acceptors for the synthesis of longer sugar chains. In the lysosome, sphingosine and sphinganine are produced by complete degradation of glycosphingolipids and are reutilized. Together, these pathways, especially the latter, may account for the majority of complex glycolipid synthesis in many cells. Depending on the physiological state of the cell and synthetic demands, the de novo pathway starting from serine and palmitoyl CoA may account for only 20–30% of the total synthesis. Shuttling the recycled components through and among the various organelles appears to involve vimentin intermediate filaments. Mechanisms and details of this process are presently lacking, but these studies offer a physiological function for glycolipid transfer proteins that were purified from brain preparations many years ago (see Chapter 10).


Monosaccharide salvage is discussed in Chapter 4. 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.


There are several ways to prevent lysosomal degradation of glycoconjugates. One is by raising the intralysosomal pH. Another is to inactivate a particular glycosidase genetically or by using a specific inhibitor. Although proteolysis inhibitors may also be an effective treatment, these drugs will not completely prevent degradation,

Because nearly all lysosomal enzymes have acidic pH optima, degradation can be curtailed by increasing the intralysosomal pH. This process can be accomplished with chemicals such as ammonium chloride and chloroquine. These agents lower the rate of degradation, but may not completely block it. Some lysosomal enzymes have relatively broad pH optima, and a few of these have pH optima higher than the lysosome. It has also been suggested that lysosomes may not always remain at low pH. Some “lysosomal” enzymes are actually present and active in early and late endosomes, which have a higher pH than lysosomes.

Glycosylation inhibitors are covered in Chapter 50, 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 can block various enzymes when tested in enzymatic assays in vitro, the inhibitors may not be effective in cells or whole animals because the drugs may not enter the lysosome, and, even if they do, their concentration may be insufficient for efficacy.


As already discussed, genetic defects in lysosomal enzymes provided major insights into catabolic pathways and 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 (see Chapter 30). 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 complications caused by these products. Some estimates suggest that less than 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 less than 1% normal activity in fibroblasts but lived until 77 years of age.

These observations have led to two therapeutic approaches: enzyme replacement therapy (ERT) and substrate reduction therapy (SRT). Because accumulation of stored material depends on the relative rates of glycan synthesis and degradation, reducing a glycan’s synthetic rate by SRT can offset the effects of 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 was 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 seemed to be effective for patients with non-severe Gaucher’s disease, but their improvement was modest and the treatment caused significant side effects. Small preclinical trials of a few infantile Tay–Sachs disease patients did not arrest their neurological deterioration, but the treatment prevented, the development of macrocephaly. Even though SRT seems promising, the expected benefits to date have been minimal.

A recent development called enzyme enhancement therapy (EET) may provide another therapeutic approach. Here, the inhibitors of the enzymes behave as molecular chaperones to stabilize the mutated enzymes in the endoplasmic reticulum to prevent their misfolding and proteasomal degradation. For example, glucocerebrosidase can be stabilized en route to the lysosome, but the inhibitor dissociates at lysosomal pH generating an active enzyme. The overall increase in activity is small, but this small amount can have significant clinical benefits. Chemical chaperones should be able to treat other enzyme deficiencies. This is likely to be a promising approach for other lysosomal storage disorders, because some of the stabilizing molecules may be able to cross the blood-brain barrier.

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 quite substantial, and high doses do not work better than lower ones for improving visceral and hematological features. 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, making this therapy ineffective for treating neurological symptoms. In addition, patients sometimes develop antibodies against the injected human protein. ERT trials with other recombinant lysosomal enzymes have progressed. Thus, the enzyme (marketed as Cerezyme®) works very well for type I disease without neurological involvement but not for Gaucher’s with neurological involvement. In this disorder, the nonneurological form is more common.

Recombinant α-L-iduronidase for MPS I and recombinant α-galactosidase for Fabry disease were approved for clinical use in 2003. In MPS I, the enzyme works very well with the intermediate severity form (known as Hurler/Scheie), which was the focus group of the MPS I clinical trial. It is unclear whether the enzyme will have any effect on the neurological manifestations of the severe form (Hurler syndrome), which unfortunately is the form affecting the majority of MPS I patients. Recombinant N-acetylgalactosamine 4-sulfatase (arylsulfatase B; Naglazyme®) was approved for MPS VI, as was α-glucosidase (Myozyme®) for treating Pompe disease patients. Iduronate sulfatase (Elaprase®) is approved for treating Hunter syndrome. These results clearly validate this approach.

Gene therapy is also an option that is under study. The availability of both naturally occurring and engineered animals deficient in specific lysosomal enzymes offers appropriate systems to test the efficacy of corrective genes. Progression to human therapy may occur when successful gene therapy is more commonly used.


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