E. Defect in a protective protein
F. Defects in transport and trafficking of substrates
a. Cystinosis b. Salla disease c. Niemann-Pick disease type C
NPC1 or NPC2
a. Mucolipidosis IV
a group of diseases collectively referred to as the mucopolysaccharidoses.
When glycoproteins are degraded in the lysosome, breakdown of the glycan part requires a set of glycosi-dases. Degradation of the glycans involves their sequential 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 a-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 neuronal 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 pathomorphology features. In two subtypes the primary defect was found in a soluble lysosomal enzyme. The enzymes involved are protein thioesterase (PPT) and tripeptidyl-peptidase 1 (TPP1). The PPT enzyme removes fatty acids from several S-acetylated proteins. The function of TPP1, a serine protease, is the removal 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 lysosomal diseases.Another lysosomal enzyme, cathepsin D, is for instance involved in an ovine neurodegener-ative disease with ultrastructural features closely resembling human NCL. The human counterpart of this disease has not yet been identified.
Although the classification of lysosomal disorders according to storage compounds appears straightforward, it is important to realize that lysosomal enzymes are frequently specific not for a certain substrate, but for a component that may occur in different substrates. Thus, glycosidases are specific for particular 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, a-N-acetylgalactosaminidase, is involved in various pathways, so that this disease cannot be assigned unambiguously to any one of the above groups. It exerts an action in the catabolism of various glycoconjugates with terminal a-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 patients suffer predominantly from skeletal, orthopedic, craniofacial, and dental abnormalities.
Some enzymes require the presence of activator proteins or saposins for their catalytic function inside the lysosome. Examples are sphingomyelinase, aryl-sulfatase A, and a- and p-galactosidase. Since defects in activator proteins affect the degradation of sphin-golipids only, all activator protein deficiencies are lipidoses. The clinical signs and symptoms frequently 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 synthesis 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 biochemical 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 depends on a formylglycine residue (FGly) in the active center of all sulfatases. This amino acid residue is generated by a posttranslational modification from a cys-teine residue. Patients with multiple sulfatase deficiency have defects in the SUMF1 gene. The protein product of the SUMF1 gene is the FGiy-generating enzyme (=FGE) localized in the lumen of the endoplasmic reticulum. The function of this enzyme is to generate the formylglycine residue in the catalytic center of the sulfatases. When this modifying reaction is defective the sulfatases remain inactive. This causes accumulation of the various substrates. Therefore several compounds could be identified as storage material. The diagnosis can be established biochemically at the enzyme level by measuring various sulfatases in leukocytes, or preferably in fibroblasts.
Lysosomes have several structural proteins. Examples of such ubiquitous,highly glycosylated integral membrane proteins are LAMP1 and LAMP2 (LAMP = lysosome-associated membrane protein). They account 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 disease is characterized by lysosomal glycogen storage leading to cardiomyopathy and myopathy in patients with normal a-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.
Galactosialidosis is caused by a defect in cathepsin A. This protein has a dual function: it is not only a protease, but also a protective protein. It combines with neuraminidase and p-galactosidase in an early biosynthetic compartment. By virtue of this association the complex is correctly delivered to the lysosomes. In the lysosome, cathepsin A protects the neuraminidase and p-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 excreted 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 cytoplasm. Some molecules require specific carriers to leave the lysosome. Defects in such carriers lead to accumulation 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 involved 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 vesicular 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 subgroup 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 coordinate fashion with NPC1 protein.
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 understood 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 evidence for storage material in body fluids or tissues. Vacuolization may occur in peripheral white blood cells. The finding of sea blue histiocytes in bone marrow should also be followed up with a thorough workup 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 symptoms can occur in any decade of life, and even before birth. Hydrops fetalis has been observed as a presentation form in several lysosomal diseases. p-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 disease, 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 residual activity in leukocytes or fibroblasts.
In recent years numerous mutations of genes for proteins that are deficient in lysosomal storage diseases have been described, leading to a better under standing of the biochemical consequences of mutations. 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 biological 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 catalytic activity of the enzyme. In some cases a combination of these effects is the cause of enzyme deficiency. Missense mutations can influence the catalytic activity of the enzyme as badly as truncating mutations. When they occur in less relevant parts of the coding region they may allow residual activity, resulting in a milder clinical phenotype. It is not always possible to show a clear genotype-phenotype relation. 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 severe clinical symptoms, as they only have one X chromosome. However, female carriers (=XX) can also have clinical symptoms of the disease because of uneven X inactivation (lyonization).
To confirm a clinical suspicion of a lysosomal disease at the biochemical level various approaches can be used as diagnostic strategy. In some lysosomal diseases 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 glycoprotein degradation shown in Table 5.1. Thin layer chromatography of oligosaccharides is also diagnostic in infantile Pompe disease, where a tetraglucoside deriving from glycogen accumulates in the urine. In late-onset Pompe disease this tetraglucoside is generally 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 lysosomal 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.
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