29] Sphingolipid hydrolases and activator proteins

Abstract

Glycosphingolipids (GSLs) are amphiphilic constituents of eukaryotic plasma membranes. They contain a hydrophobic ceramide moiety that anchors them in the lipid bilayer and a hydrophilic, extracellular oligosaccharide chain. GSLs are involved in cell-type specific adhesion processes and can influence the activity of membrane-bound receptors and enzymes. Inherited defects within the GSL degradation pathway give rise to various human diseases, the sphingolipidoses. The constitutive degradation of GSLs occurs in the acidic compartments of the cell: endosomes and lysosomes. Cell surface-derived GSLs reach the lysosomal compartment by endocytic membrane flow, presumably on the surface of intraendosomal and intralysosomal vesicles. Here, the GSLs are cleaved into their building blocks by the stepwise action of hydrolytic enzymes. Glycosidases cleave off the sugar residues from the nonreducing end of the GSLs. In the case of GSLs with short oligosaccharide chains of less than four sugar residues, the degrading enzymes need the assistance of protein cofactors, the so-called “sphingolipid activator proteins.” The sphingolipid activator proteins known so far are derived from two genes. One gene on chromosome carries the information for the GM2 activator and the other gene on chromosome 10 for the SAP precursor, also called “prosaposin,” which is processed to four homologous proteins (also called “saposins”): SAP-A, SAP-B, SAP-C, and SAP-D. Mechanistically, the GM2 activator and SAP-B function by solubilizing the lipid substrate, thus making it accessible to the water-soluble enzyme, whereas the other SAPs seem to facilitate interaction of the respective hydrolase with the lipid in or at the membrane in an as yet less well-characterized way. This chapter focuses on experimental approaches to mimic the in vivo degradation of glycolipids as closely as possible. This is required to obtain reliable data on activator protein specificity and function.