Lysosomes and Lysosomal Disorders R.A. Wevers,V. Gieselmann

5.1 Lysosomal Biogenesis and Biochemical Functions

Lysosomes are hydrolase-rich organelles surrounded by membranes and with an acidic interior milieu.

They are present in almost all types of body cells.

Their number varies greatly, depending on cell type and function. They display considerable structural heterogeneity and appear in all shapes, sizes, and den- sities. They have been given their name because they are small bodies (soma = body) containing various enzymes that are hydrolytic (lysis = dissolution).

These hydrolases catalyze reactions in which macro- molecules and macromolecular structures are de- graded into smaller components. Among the more than 50 different lysosomal enzymes so far identified are proteases, nucleases, glycosidases, lipases, phos- pholipases, sulfatases, and phosphatases. The variety of enzymes enables the lysosome to digest almost all types of biological macromolecules, such as proteins, polysaccharides, lipids, and nucleic acids. The low- molecular components released are transported to the cytoplasm to be reused. For this purpose the lyso- somal membrane contains various transporters to translocate amino acids, sugars, and possibly nu- cleotides into the cytoplasm. The lysosomal mem- brane separates the hydrolytic enzymes from the cy- toplasm to prevent uncontrolled lysis of cytoplasmic components. The acidic interior of lysosomes pro- vides a favorable environment for the digestive activ- ities of the enzymes: oligomeric proteins dissociate into monomers, proteins dissociate away from the protecting membrane, and stabilizing complexes be- come split. The low pH is generated by a complex multisubunit ATP-dependent proton pump. Several subunits of this proton pump are found on the cy- tosolic side of the lysosomal membrane, and others are integral lysosomal membrane proteins. Further- more, the lysosomal membrane harbors various pro- teins, with highly glycosylated intralysosomal do- mains (e.g. LAMP1 and 2). The high carbohydrate content is thought to protect the lysosomal mem- brane from hydrolytic attack by the enzymes.

Lysosomal enzymes, along with secretory proteins and plasma membrane proteins, are synthesized on membrane-bound polyribosomes on the rough en- doplasmic reticulum. An important question is how proteins, which are destined for specific intracellular compartments, are targeted at their destination from

their site of synthesis. The signal that specifies the destination of each nascent protein resides in its se- quence or spatial structure. The cellular transport machinery recognizing these signals distributes the proteins to the diverse cellular compartments. Some signal peptides direct proteins specifically into the nucleus, mitochondria, or peroxisomes. Membrane, secretory, or lysosomal proteins are also sorted initially via signal peptides into the lumen of the endoplasmic reticulum. Here the lysosomal enzyme proteins undergo glycosylation, as do most of the secretory and plasma membrane proteins. The glyco- sylation step involves the transfer of a large oligosac- charide with high mannose content to selected as- paragine residues of the nascent protein. Subsequent- ly, the signal peptide is cleaved, the protein folds, and the processing of the asparagine-linked oligosaccha- ride begins. From the endoplasmic reticulum the pro- teins travel via vesicular transport to the Golgi appa- ratus. In the cis compartment of the Golgi complex, oligosaccharide side chains of lysosomal enzymes are phosphorylated and thus acquire mannose-6-phos- phate moieties. In contrast, oligosaccharide side chains of secretory and membrane proteins are trimmed and remodeled further to yield complex- type side chains. The synthesis of the mannose-6- phosphate residues is initiated by a phosphotrans- ferase, which specifically recognizes lysosomal en- zymes. Recognition does not occur by way of a signal peptide but is mediated by a spatial signal depending on the three-dimensional structure of the enzymes.

Given the structural diversity of lysosomal enzymes the precise nature of the signal shared by all enzymes is still a mystery. So far, only surface-located lysine residues seem to be an essential common component of this topogenic signal.

Phosphorylated lysosomal enzymes then proceed through the remainder of the Golgi complex (from cis- through medial to trans-Golgi). In the trans-Gol- gi network (TGN) they bind to mannose-6-phosphate receptors, which segregate the enzymes into distinct transport vesicles away from secretory and cell sur- face proteins. There are two mannose-6-phosphate receptors, which bind different but overlapping sets of enzymes. Once lysosomal enzymes are bound, the mannose-6-phosphate receptor–enzyme complexes are collected in clathrin-coated pits, which bud off to form coated vesicles. Most of these transport vesicles deliver the complexes to acidic early endosomes, but

Lysosomes and Lysosomal Disorders

R.A. Wevers, V. Gieselmann

Chapter 5

complexes do also arrive at late endosomes. The low pH in the endosomes causes lysosomal hydrolases and receptors to dissociate. After dissociation, man- nose-6-phosphate receptors are retrieved from this compartment and returned to the TGN, whereas lyso- somal enzymes are delivered to mature lysosomes.

Thus, receptors do not occur in lysosomes. Their ab- sence is an important histochemical feature of lyso- somes and differentiates them from late endosomes.

Mannose-6-phosphate receptors also cycle between the endosomal compartment and the plasma mem- brane. One of the receptors can bind lysosomal en- zymes at the plasma membrane and mediate their en- docytosis and subsequent delivery to lysosomes. This is probably a recapture mechanism, since depending on cell type, 5–40% of newly synthesized lysosomal enzymes escape receptor binding in the TGN and are secreted. A proportion of these enzymes bind to the mannose-6-phosphate receptors on the plasma mem- brane and are recaptured, internalized, and delivered to lysosomes. Sorting signals within the cytoplasmic tails of the receptors are crucial for their correct in- tracellular trafficking.

Although the mannose-6-phosphate recognition pathway is a major route for targeting soluble lysoso- mal enzymes, there is evidence for an alternative mechanism, independent of mannose-6-phosphate, and localizing soluble acid hydrolases to lysosomes.

Although it seems likely that this pathway is also re- ceptor mediated, attempts to demonstrate this recep- tor have so far been unsuccessful. Only in cases of activator proteins – see below – has the multiligand receptor sortilin been shown to be involved in lysoso- mal trafficking independent of mannose-6-phos- phate.

Lysosomal membrane glycoproteins travel the same route as soluble enzymes from the rough endo- plasmic reticulum via the Golgi apparatus and endo- somes to lysosomes. However, the transport of lyso- somal membrane glycoproteins to lysosomes is inde- pendent of the mannose-6-phosphate receptor sys- tem, depending rather on signals in their cytoplasmic portion. An example is the classic lysosomal marker enzyme acid phosphatase. It is synthesized as a trans- membrane precursor protein with a large luminal do- main and a short cytoplasmic tail. After reaching the TGN the enzyme precursor is repeatedly recycled be- tween the cell surface and the endosomal compart- ment before reaching the lysosome. After its delivery to the lysosome, acid phosphatase undergoes proteo- lytic processing of the membrane-anchoring domain, resulting in conversion to a soluble form. Sorting signals for this mannose-6-phosphate receptor-inde- pendent pathway reside in the short cytoplasmic tail of the acid phosphatase precursor.

In addition to oligosaccharide processing, lysoso- mal hydrolases are synthesized as pre-proenzymes,

and almost all undergo proteolytic processing. The pre-piece is the signal sequence, which is cleaved immediately after transport into the endoplasmic reticulum. With the exception of aspartylgluco- saminidase, which is already processed in the endo- plasmic reticulum, the pro-piece is cleaved later in en- dosomal compartments. Cleavage is completed after arrival of the enzymes in the lysosomes. For lysoso- mal proteases, cleavage of proenzymes is accompa- nied by activation of the enzymes. Prior to arrival in the lysosomes the pro-piece keeps the proteases in an inactive state. In lysosomal enzymes other than pro- teases, however, the biological significance of this proteolytic processing is poorly understood.

Some enzymes involved in the degradation of sphingolipids need the assistance of enzymatically inactive activator proteins for hydrolysis of their sub- strates. So far, five different activator proteins encod- ed by two different genes have been identified. One gene codes for the GM

ganglioside activator protein only, whereas the other encodes a precursor protein that harbors four different but homologous sphin- golipid activator proteins (SAPs). The mature SAPs A, B, C, and D – also called saposins – are generated from this precursor via proteolytic processing. They act on different enzymes and facilitate the degradation of various sphingolipids. They also differ in their mode of action. GM

-activator protein and SAP-B bind the lipid substrates and present them to the respective enzymes, whereas SAP-C activates the enzyme direct- ly.

Lysosomes are the final destination of endocytic,

autophagic and phagocytic routes. The endosomal

membranous network connects the lysosomes to the

Golgi apparatus and the plasma membrane. Early en-

dosomes start to accumulate internal membranes,

and as this accumulation proceeds they mature into

late endosomes. Since late endosomes are rich in lu-

minal membranes they are also referred to as multi-

vesicular bodies (MVBs) or multivesicular endo-

somes. Lipid and protein composition of these lumi-

nal membranes differs from that of early endosomes,

suggesting a specific partitioning event during their

generation. Thus, for some proteins it has been shown

that tagging with ubiquitin directs them through this

luminal compartment for lysosomal degradation,

whereas other proteins seem to be quite stable in

these membranes. This endocytotic lysosomal route

can also be used to terminate growth factor receptor

signaling, a process that is crucial for cellular regula-

tion. Thus, ligand activation of epidermal growth fac-

tor receptor does not only activate downstream sig-

naling pathways, but also induces endocytosis. Endo-

cytosed receptors may be cycled back to the plasma

membrane for continuous signaling or can be deliv-

ered to the lysosome for degradation, resulting in sig-

nal termination. Thus, the balance between recycling

and lysosomal delivery has a key role in regulation of the signal intensity of at least some tyrosine kinase receptors.

The MVBs/late endosomes can fuse homotypical- ly, but they also fuse with lysosomes, forming a hybrid organelle. The dense lysosomes can be regarded as storage granules of hydrolytic enzymes, which fuse with late endosomes to perform their hydrolytic task on the late endosome contents. During this process continuous condensation occurs to recover lyso- somes from this hybrid organelle.

Autophagy is the process by which the cell se- questers parts of its own cytoplasm, often containing entire organelles. In the first step, called autophagic sequestration, a cytoplasmic membrane, which is probably derived from the endoplasmic reticulum, envelops a region of cytoplasm in a closed vacuole called an autophagosome. Through fusion, the se- questered material is transferred to lysosomes. The lysosomal membrane protein LAMP2 seems to be essential for the maturation of autophagosomes. In normal cells this process is important because of its participation in cell renewal and turnover of worn- out cell constituents. In secretory cells there is a spe- cial kind of autophagy, called crinophagy. It occurs by way of direct fusion between secretory granules and lysosomes and results in the destruction of excess se- cretory material. Alternatively, in chaperone-mediat- ed autophagy proteins can be unfolded in the cyto- plasm and transported directly through the lysoso- mal membrane.

Finally, phagocytosis is the process by which cells internalize large particles, such as bacteria. Thus, phagocytosis is particularly active in neutrophils and macrophages. After internalization the interior of a phagosome initially resembles the extracellular mi- lieu. However, phagosomes may fuse with endosomes and slowly acquire the characteristics of late endo- somes and lysosomes. In this context it is important to note that lysosomes also generate peptides via hy- drolysis of phagocytosed material to load MHCII molecules. Thus, lysosomes have an essential role in the immune system, maintaining the health of cells and the body’s defense against foreign invaders.

Apart from the catabolic functions, lysosomes have also been shown to play an essential part in the repair of plasma membrane defects. In wounded cells lysosomes can fuse with the defective plasma mem- brane via a calcium-triggered exocytotic process.

Lysosomes can thus serve as a reservoir allowing for rapid provision of membrane lipids in the case of extended defects that cannot be compensated by lipid biosynthesis within an appropriate time period. This clearly demonstrates that lysosomes also have ana- bolic functions.

5.2 The Pathobiochemistry

of Lysosomal Disease in Humans More than 45 different lysosomal diseases are cur- rently known in man (Table 5.1). They can be caused by defects in the genes of individual lysosomal hydro- lases, activator proteins, transporters, lysosomal membrane proteins, or enzymes modifying lysoso- mal hydrolases. In general a profound deficiency with residual activity of the respective protein <5% of nor- mal is found in tissues of affected persons. Since un- degraded substrates cannot leave the lysosomes, or only very slowly, these organelles are converted into storage granules, which steadily increase in size and number.

Over the years several classes of lysosomal diseases have been unraveled. In Table 5.1 and in the text be- low we have classified the lysosomal diseases primar- ily on the basis of functional characteristics, resulting in seven main groups. Furthermore, we have taken ac- count of well-established clinical entities that are of- ten based on the nature of the accumulating com- pound.

5.2.1 Defects in Individual Hydrolases

The molecular basis of most currently known lysoso- mal diseases is a deficiency of an individual hydrolase in the lysosome. These hydrolases have a role in the catabolism of different molecular species, such as lipids, mucopolysaccharides, and glycoproteins. This has led to a classification according to the biochemi- cal nature of the substrate accumulating in a particu- lar disease (see also Table 5.1).

In many lysosomal diseases lipid species accumu- late. Examples of lipids that are involved are sphin- gomyelin (Niemann-Pick A and B), glucosylceramide (Gaucher), galactosylceramide and psychosine (Krabbe), globotriaosylceramide (Fabry), ceramide (Farber), sulfatides (metachromatic leukodystrophy), gangliosides (GM

and GM

gangliosidoses), choles- teryl esters, and triglycerides (Wolman). This group of diseases can be referred to collectively as the lipi- doses. Figure 5.1 illustrates the sequential action of the various enzymes involved in the pathway of lyso- somal lipid metabolism.

In a second subgroup acid mucopolysaccharides, also called glycosaminoglycans, accumulate. These consist of long chains of repeating disaccharide units.

Different subspecies exist, which are characterized by the composition of their repeating disaccharide units.

Heparan sulfate, dermatan sulfate, chondroitin sul- fate, keratan sulfate, and hyaluronan are representa- tives of the mucopolysaccharide family. The mu- copolysaccharides are degraded in the lysosome. De- fects in the enzymes involved in this catabolism cause

Chapter 5 Lysosomes and Lysosomal Disorders

68

Table 5.1. A review of the lysosomal storage disorders, with the defective enzymes and proteins in each A. Defects in individual lysosomal hydrolases

1. Lipidoses

a. Metachromatic leukodystrophy Arylsulfatase A b. Globoid cell leukodystrophy (Krabbe disease) Galactocerebrosidase

c. GM

gangliosidosis β-Galactosidase

d. GM

gangliosidoses:

Tay Sachs disease Hexosaminidase A

Sandhoff disease Hexosaminidase A and B

e. Gaucher disease β-Glucosidase

f. Fabry disease α-Galactosidase A

g. Farber disease Acid ceramidase

h. Niemann-Pick disease (types A and B) Sphingomyelinase i. Wolman disease and cholesterol ester storage disease Acid lipase 2. Mucopolysaccharidoses

a. Hurler disease and Scheie disease (I) α-Iduronidase

b. Hunter disease (II) Iduronate sulfatase

c. Sanfilippo disease (IIIA-D) a. Heparin sulfamidase b. N-Acetyl α-glucosaminidase c. α-Glucosaminide N-acetyltransferase d. N-Acetylglucosamine 6-sulfatase

d. Morquio disease (IV) Galactose 6-sulfate sulfatase

e. Maroteaux-Lamy disease (VI) Arylsulfatase B

f. Sly disease (VII) β-Glucuronidase

g. Hyaluronidase deficiency (IX) Hyaluronidase 3. Disorders of glycoprotein degradation

a. Sialidosis Neuraminidase

b. Fucosidosis α-Fucosidase

c. Mannosidosis ( α and β) α− and β-Mannosidase

d. Aspartylglycosaminuria Aspartylglucosaminidase

4. Glycogen storage disorders

a. Pompe disease α-Glucosidase

5. Neuronal ceroid lipofuscinoses

a. Infantile Finnish type NCL (Santavuori disease) Palmitoyl thioesterase b. Late-infantile NCL (Jansky-Bielschowsky disease) Tripeptidyl peptidase1 6. Non classifiable

a. Pyknodysostosis Cathepsin K

b. Schindler disease N-Acetyl α-galactosaminidase

B. Defects in activator proteins 1. Lipidoses

a. GM

activator protein deficiency GM

Activator protein

b. Saposin B deficiency Metachromatic leukodystrophy variant

c. Saposin C deficiency Gaucher disease variant

d. Prosaposin deficiency

C. Defects in the postsynthetic modification of lysosomal proteins 1. Mucolipidoses

a. I cell disease (mucolipidosis II) N-Acetylglucosaminylphosphotransferase b. Pseudo-Hurler polydystrophy (mucolipidosis III) N-Acetylglucosaminylphosphotransferase 2. Non-classifiable

a. Multiple sulfatase deficiency F

-Generating enzyme D. Defects in structural lysosomal proteins

1. Glycogen storage disorders

a. Danon disease LAMP2

E. Defect in a protective protein

a. Galactosialidosis Cathepsin A

F. Defects in transport and trafficking of substrates

a. Cystinosis Cystinosin

b. Salla disease Sialin

c. Niemann-Pick disease type C NPC1 or NPC2

G. Non-classifiable

a. Mucolipidosis IV Mucolipin 1

a group of diseases collectively referred to as the mu- copolysaccharidoses.

When glycoproteins are degraded in the lysosome, breakdown of the glycan part requires a set of glycosi- dases. Degradation of the glycans involves their se- quential action. If one of these enzymes is deficient various oligosaccharides deriving from the glycan accumulate in tissues and body fluids. The material accumulating in the lysosomes is disease specific. As a group, these diseases can be referred to as glycopro- tein degradation disorders.

The breakdown of glycogen, a glucose polymer, requires lysosomal α-glucosidase enzymatic activity.

Defects in this enzyme lead to Pompe disease with massive glycogen storage in muscle and liver in the infantile-onset form of the disease. This disease is one of the family of glycogen storage disorders.

A last group in this category is formed by the neu- ronal ceroid lipofuscinoses (NCL). To date, eight main genetic forms are generally accepted to occur in man.

The NCLs collectively constitute the most common group of neurodegenerative diseases in childhood, with an estimated total incidence in the U.S. of 1:12 500. All NCL forms share unifying pathomor- phologic features. In two subtypes the primary defect was found in a soluble lysosomal enzyme. The en- zymes involved are protein thioesterase (PPT) and tripeptidyl-peptidase 1 (TPP1). The PPT enzyme re- moves fatty acids from several S-acetylated proteins.

The function of TPP1, a serine protease, is the re- moval of N-terminal tripeptides from substrates with free amino termini. The in vivo substrates of PPT and TPP1 remain unknown. It may turn out that some of the remaining eight genetic NCL forms are also lyso- somal diseases.Another lysosomal enzyme, cathepsin D, is for instance involved in an ovine neurodegener- ative disease with ultrastructural features closely re- sembling human NCL. The human counterpart of this disease has not yet been identified.

Chapter 5 Lysosomes and Lysosomal Disorders 70

Fig. 5.1. Disorders in lysosomal

lipid metabolism

Although the classification of lysosomal disorders according to storage compounds appears straightfor- ward, it is important to realize that lysosomal en- zymes are frequently specific not for a certain sub- strate, but for a component that may occur in differ- ent substrates. Thus, glycosidases are specific for par- ticular sugar residues and the geometry of their linkage. The respective sugar and its linkage can occur both in lipids and in glycosaminoglycans.

Degradation of both is affected in the case of enzyme deficiency, and both lipids and glycosaminoglycans accumulate. Thus, in some diseases the classification is somewhat artificial.

In Schindler disease the defective enzyme, α-N- acetylgalactosaminidase, is involved in various path- ways, so that this disease cannot be assigned unam- biguously to any one of the above groups. It exerts an action in the catabolism of various glycoconjugates with terminal α-N-acetylgalactosaminyl residues.

Deficiency of the enzyme results in accumulation of glycopeptides, glycosphingolipids, and oligosaccha- rides in many tissues.

The defective enzyme in pyknodysostosis has been found in cathepsin K, a lysosomal protease. This enzyme is highly expressed in osteoclasts. The defect leads to a reduced capacity of this cell to remove organic bone matrix, thus causing defective bone growth and remodeling. This explains why the pa- tients suffer predominantly from skeletal, orthopedic, craniofacial, and dental abnormalities.

5.2.2 Defects in Activator Proteins

Some enzymes require the presence of activator pro- teins or saposins for their catalytic function inside the lysosome. Examples are sphingomyelinase, aryl- sulfatase A, and α- and β-galactosidase. Since defects in activator proteins affect the degradation of sphin- golipids only, all activator protein deficiencies are lipidoses. The clinical signs and symptoms frequent- ly resemble those found in patients in whom the same glycolipid accumulates as the result of deficiency of hydrolase activated by the respective saposins (e.g., saposin B deficiency causes a variant form of meta- chromatic leukodystrophy).

5.2.3 Defects in the Postsynthetic Modification of Lysosomal Proteins As outlined earlier, all soluble lysosomal enzymes are N-glycosylated and their oligosaccharide side chains receive mannose-6-phosphate residues, which are a lysosomal targeting signal, in the Golgi apparatus.

Defects in a phosphotransferase initiating the synthe- sis of mannose-6-phosphate residues result in a de-

fect in the targeting of lysosomal enzymes towards the lysosome. This causes an intracellular deficiency of many lysosomal enzymes. The diagnosis can be confirmed by abnormally high enzymatic activity of many lysosomal enzymes in blood plasma. Because of the mistargeting, these enzymes are directed out of the cell and end up in the blood plasma. Defects in the phosphotransferase cause mucolipidosis II (inclusion body or I cell disease) and III. Patients with mucolipi- doses II and III share clinical symptoms and bio- chemical characteristics with patients who have a mucopolysaccharidosis or a sphingolipidosis. Glyco- lipids as well as mucopolysaccharides accumulate in lysosomes in these diseases. Recently the primary defect in mucolipidosis IV was found to be in the MCOLN1 gene encoding for a protein, mucolipin 1.

The function of the protein has not yet been fully characterized, and this disorder is therefore nonclas- sifiable (group G in Table 5.1).

Among the lysosomal storage disorders multiple sulfatase deficiency is particularly interesting. In this disorder the activity of all lysosomal and nonlysoso- mal sulfatases is reduced. Since sulfate groups occur in many different molecules a complex mixture of compounds accumulates. The enzymatic activity de- pends on a formylglycine residue (F

) in the active center of all sulfatases. This amino acid residue is gen- erated by a posttranslational modification from a cys- teine residue. Patients with multiple sulfatase defi- ciency have defects in the SUMF1 gene. The protein product of the SUMF1 gene is the F

-generating en- zyme (=FGE) localized in the lumen of the endoplas- mic reticulum. The function of this enzyme is to gen- erate the formylglycine residue in the catalytic center of the sulfatases. When this modifying reaction is de- fective the sulfatases remain inactive. This causes ac- cumulation of the various substrates. Therefore sev- eral compounds could be identified as storage mater- ial. The diagnosis can be established biochemically at the enzyme level by measuring various sulfatases in leukocytes, or preferably in fibroblasts.

5.2.4 Defects in Structural Lysosomal Proteins

Lysosomes have several structural proteins. Examples of such ubiquitous, highly glycosylated integral mem- brane proteins are LAMP1 and LAMP2 (LAMP = lysosome-associated membrane protein). They ac- count for about 50% of the protein content of the lysosomal membrane. Recently the primary defect of Danon disease has been assigned to the LAMP2 gene.

This gene encodes LAMP2, which is thought to be a

structural protein in the lysosome. This X-linked dis-

ease is characterized by lysosomal glycogen storage

leading to cardiomyopathy and myopathy in patients

with normal α-glucosidase activity.It is not fully clear how a defect in this protein can lead to accumulation of glycogen. It can be anticipated that several new lysosomal diseases in this subgroup will be found in the future.

5.2.5 Defects in a Protective Protein

Galactosialidosis is caused by a defect in cathepsin A.

This protein has a dual function: it is not only a pro- tease, but also a protective protein. It combines with neuraminidase and β-galactosidase in an early biosynthetic compartment. By virtue of this associa- tion the complex is correctly delivered to the lyso- somes. In the lysosome, cathepsin A protects the neu- raminidase and β-galactosidase against rapid prote- olysis and inactivation. In the case of cathepsin A deficiency both enzymes are rapidly degraded and thus deficient. Sialyloligosaccharides accumulate in the lysosomes of affected patients and are also excret- ed in the urine.

5.2.6 Defects in Transport and Trafficking of Substrates

Lysosomal degradation of macromolecules leads to the formation of smaller molecules, which generally are exported from the lysosome towards the cyto- plasm. Some molecules require specific carriers to leave the lysosome. Defects in such carriers lead to ac- cumulation of the molecule involved within the lyso- some. Examples of such diseases are cystinosis and Salla disease. Cystinosis is characterized by intralyso- somal storage of the amino acid cystine and is caused by defective carrier-mediated transport of cystine across the lysosomal membrane. The protein in- volved is cystinosin. In Salla disease intralysosomal storage of sialic acid occurs, caused by a defect in its transport across the lysosomal membrane by the transporter sialin.

Niemann-Pick disease type C is a lipid trafficking disorder. The majority of patients have mutations in the gene coding for the NPC1 protein. The postulated role for this protein involves modulation of the vesic- ular trafficking of cholesterol and glycolipids. Several lipids (sphingomyelin, phospholipids, glycolipids, and unesterified cholesterol) are stored in excess in the liver and spleen of these patients. Foam cells or sea blue histiocytes may be found in many tissues of affected patients. Primarily, the diagnosis requires the demonstration of excess cholesterol in fibroblasts with the so-called filipin staining test. In a small sub- group of patients with Niemann-Pick type C disease there are mutations in another gene coding for the NPC2 protein, a soluble lysosomal enzyme with un-

known function that is thought to work in a coordi- nate fashion with NPC1 protein.

5.3 Clinical Features and Diagnosis

A full survey of clinical symptomatology of lysosomal diseases is beyond the scope of this chapter. A few characteristics or general features can clearly be un- derstood from the molecular basis of the diseases.

Storage material often gives rise to organomegaly, for instance of liver or spleen.Another characteristic that may occur is the loss of acquired mental or motor skills in the course of time, which is due to an increase in storage material with time. Some clinical features, such as a cherry red spot in the retina or downward gaze paralysis, may be highly suggestive for lysosomal disease, and in some cases even pathognomonic for a specific disease. The same holds in the case of evi- dence for storage material in body fluids or tissues.

Vacuolization may occur in peripheral white blood cells. The finding of sea blue histiocytes in bone mar- row should also be followed up with a thorough work- up for lysosomal diseases. As most cell types in the human body contain lysosomes, many tissues or cell types can be involved in lysosomal diseases. Often these diseases affect the CNS, resulting in neurode- generative disease. Others, such as Morquio and Pompe disease, leave the brain relatively unaffected.

In general, the lysosomal diseases are multisystem diseases. Pyknodysostosis is an example of a disease in which the molecular defect, the deficiency of cathepsin K, seems to interfere predominantly with the function of only one cell type. Dysfunction of the osteoclast causes the clinical features of this disease.

Most lysosomal diseases show significant clinical heterogeneity. The onset of clinical signs and symp- toms can occur in any decade of life, and even before birth. Hydrops fetalis has been observed as a presen- tation form in several lysosomal diseases. β-Glu- curonidase deficiency is an example of a disease that can present as early as this. The time of presentation can vary rather widely within one disease. Pompe dis- ease, for example, can have an early infantile onset: in such patients the course of the disease is invariably very severe and most of them die before the age of 6 months. Other patients with Pompe disease have adult-onset forms of the disease and have milder symptoms. The concept for our understanding of this variability in clinical presentation lies in the residual activity of the enzyme in a specific patient. However, with the methodology currently available it is not possible to predict the disease course from the resid- ual activity in leukocytes or fibroblasts.

In recent years numerous mutations of genes for proteins that are deficient in lysosomal storage dis- eases have been described, leading to a better under-

Chapter 5 Lysosomes and Lysosomal Disorders

72

standing of the biochemical consequences of muta- tions. Severe mutations that truncate the protein or shift the reading frame thereby alter the primary structure of the protein so that it has no residual bio- logical activity. Such mutations almost always result in a severe clinical phenotype. In lysosomal storage diseases missense mutations are the most frequent.

Often missense mutations lead to misfolded enzymes, which are not transported to the lysosomes but are retained and degraded in the endoplasmic reticulum.

Alternatively, defective enzymes may still be sorted properly but become rapidly degraded on arrival in the lysosome. Mutations can be located in the active center of an enzyme or indirectly influence the cat- alytic activity of the enzyme. In some cases a combi- nation of these effects is the cause of enzyme defi- ciency. Missense mutations can influence the catalyt- ic activity of the enzyme as badly as truncating muta- tions. When they occur in less relevant parts of the coding region they may allow residual activity, result- ing in a milder clinical phenotype. It is not always possible to show a clear genotype–phenotype rela- tion. Most lysosomal diseases have an autosomal recessive mode of inheritance. Few diseases have an X-chromosomal inheritance. Fabry, Danon, and Hunter diseases are examples. Males have more se- vere clinical symptoms, as they only have one X chro- mosome. However, female carriers (=XX ·

) can also have clinical symptoms of the disease because of un- even X inactivation (lyonization).

To confirm a clinical suspicion of a lysosomal dis- ease at the biochemical level various approaches can be used as diagnostic strategy. In some lysosomal dis- eases undegraded substrates can be found in the urine. Investigations of urine samples are therefore often used as a first step towards establishing the

diagnosis. An increased concentration of urinary mucopolysaccharides can be found in the mu- copolysaccharidoses. Subsequent electrophoresis of mucopolysaccharides will show which subtype of the mucopolysaccharides the patient cannot adequately degrade. Defects in mucopolysaccharide catabolism can affect the breakdown of heparan sulfate, der- matan sulfate, chondroitin sulfate, keratan sulfate, or hyaluronan. The result of electrophoresis will give a clue to the defective enzyme. Abnormal urinary oligosaccharides are present in the disorders of glyco- protein degradation shown in Table 5.1. Thin layer chromatography of oligosaccharides is also diagnos- tic in infantile Pompe disease, where a tetraglucoside deriving from glycogen accumulates in the urine. In late-onset Pompe disease this tetraglucoside is gener- ally not found in the urine and other techniques are necessary to establish the diagnosis. In some diseases, then, accumulating material cannot be detected in the urine, and for some diseases the techniques that would be required to diagnose them at the metabolite level are too time consuming. This is the case for the lipidoses, defects in activator proteins, and the NCLs, for instance. In such cases direct enzyme analysis in leukocytes is often used as a first step in establishing the diagnosis. Cultured skin fibroblasts can generally also be used to confirm the diagnosis. A further test that may be relevant to the demonstration of lysoso- mal involvement is measurement of the activity of chitotriosidase. This enzyme is secreted by activated macrophages. In several lysosomal diseases increased chitotriosidase activity can be used as a nonspecific diagnostic marker.

The chapters below discuss only those lysosomal

storage disorders that are accompanied by a white

matter disorder.