Chapter 134

## Abstract

Abstract

1. The lysosomal degradation of sphingolipids with short hydrophilic head groups is dependent on small nonenzymic glycoproteins, called sphingolipid activator proteins (SAPs), including saposins (Saps) and the GM2 activator protein.

2. Two genes code for all established and putative SAPs known so far. One gene on human chromosome 5, consisting of 4 exons and 3 introns, encodes the GM2 activator protein; a second gene on chromosome 10, consisting of 15 exons and 14 introns, codes for the prosaposin, which is processed to four homologous activator proteins or saposins, Sap-A, Sap-B, Sap-C, and Sap-D.

3. The mature human GM2 activator consists of a polypeptide chain of 162 amino acids and carries one N-linked carbohydrate chain on Asn 32. It is a membrane-active protein that binds structurally related glycosphingolipids and stimulates their degradation by a specific interaction with β-hexosaminidase A. This activator is essential for the hydrolysis of ganglioside GM2 and glycolipid GA2 in vivo.

4. Four point mutations have been identified so far in the GM2 activator gene in AB variant of GM2 gangliosidosis. They result in premature degradation of the GM2 activator and cause a pronounced neuronal storage of ganglioside GM2 and glycolipid GA2. The clinical course and pathologic findings observed in AB variant closely resemble those of Tay-Sachs disease.

5. The prosaposin (Sap precursor), with a total of 524 amino acids and five N-glycosylation sites, contains four homologous domains, each of about 80 amino acids. A major proportion of the precursor pool is exported to the cell surface and then imported into the lysosomal compartment, where it is processed to the mature glycoproteins Sap-A, Sap-B, Sap-C, and Sap-D. The occurrence of prosaposin in body fluids and its neurotrophic properties indicate that its function is not restricted to being the precursor of the mature intracellular Saps.

6. In vitro, Sap-A stimulates glucosylceramidase and galactosylceramidase in the presence of detergents; this determination is supported by studies in a conditional knock-out mouse model.

7. Sap-B is a nonspecific glycolipid-binding protein that stimulates the hydrolysis of about 20 glycolipids by different enzymes, in particular the hydrolysis of sulfatide by arylsulfatase A.

8. So far, seven different mutations have been identified in the stretch of the prosaposin gene coding for Sap-B. They result in a loss of the mature Sap-B, causing the accumulation of sulfatide and some other sphingolipids. The clinical course of the disease resembles that of juvenile metachromatic leukodystrophy, with certain exceptions.

9. In vitro, Sap-C stimulates the hydrolysis of glucosylceramide by glucosylceramidase, galactosylceramide by galactosylceramidase, and sphingomyelin by acid sphingomyelinase. In vivo, Sap-C is essential for the degradation of glucosylceramide. The Sap-C glycoprotein factor has been proposed to bind to, and thereby activate, glucosylceramidase. It is a membrane-active protein that solubilizes lipids. Deficiency of Sap-C results in a juvenile variant of Gaucher disease, with pronounced accumulation of glucosylceramide in the reticuloendothelial system. Sap-C seems to have a role also in galactolipid breakdown.

10. Sap-D has a lipid membrane-disrupting activity and stimulates, along with Sap-C, the degradation of ceramides by acid ceramidase in vivo.

11. Prosaposin deficiency due to a homozygous mutation in the initiation codon was found in a boy who died at 16 weeks and his fetal sibling, and also in a recent independent, very similar patient. This, as well as two other homozygous prosaposin mutations in two further patients led to complete absence of prosaposin and, therefore, the four mature Saps, resulting in a generalized accumulation of ceramide, glucosylceramide, galactosylceramide, sulfatide, lactosylceramide, digalactosylceramide, ganglioside GM3, and so forth. The symptoms of the disease resemble those of Gaucher (type 2) more than those of Farber disease.

12. It is hypothesized that sphingolipid activator proteins are essential for the degradation of intralysosomal vesicles containing former components of the plasma membrane.

13. Intracellular routing of prosaposin and of the GM2 activator protein depends mainly on sorting by sortilin and is only in part dependent on mannose-6-phosphate residues. Their endocytosis occurs in a carbohydrate-independent manner. Cellular uptake of prosaposin and its lysosomal delivery are mediated by the low-density lipoprotein receptor-related protein (LRP).

14. Mice with disrupted genes for prosaposin and GM2 activator have been created as authentic models for known human diseases.

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