Acid sphingomyelinase is a water-soluble, lysosomal glycoprotein that catalyzes the degradation of membrane-bound sphingomyelin into phosphorylcholine and ceramide. Sphingomyelin itself is an important component of the extracellular leaflet of various cellular membranes. The aim of the present investigation was to study sphingomyelin hydrolysis as a membrane-bound process. We analyzed the degradation of sphingomyelin by recombinant, highly purified acid sphingomyelinase in a detergent-free, liposomal assay system. In order to mimic the in vivo intralysosomal conditions as closely as possible a number of negatively charged, lysosomally occuring lipids including bis(monoacylglycero)phosphate and phosphatidylinositol were incorporated into substrate-carrying liposomes. Dolichol and its phosphate ester dolicholphosphate were also included in this study. Bis(monoacylglycero)phosphate and phosphatidylinositol were both effective stimulators of sphingomyelin hydrolysis. Dolichol and dolicholphosphate also significantly increased sphingomyelin hydrolysis. The influence of membrane curvature was investigated by incorporating the substrate into small (SUVs) and large unilamellar vesicles (LUVs) with varying mean diameter. Degradation rates were substantially higher in SUVs than in LUVs. Surface plasmon resonance experiments demonstrated that acid sphingomyelinase binds strongly to lipid bilayers. This interaction is significantly enhanced by anionic lipids such as bis(monoacylglycero)phosphate. Under detergent-free conditions only the sphingolipid activator protein SAP-C had a pronounced influence on sphingomyelin degradation in both neutral and negatively charged liposomes, catalyzed by highly purified acid sphingomyelinase, while SAP-A, -B and -D had no noticeable effect on sphingomyelin degradation.

Most soluble lysosomal enzymes require a mannose-6-phosphate recognition marker present on asparagine-linked oligosaccharides for proper targeting to lysosomes. We have determined the influence of the six potential N-linked oligosaccharide chains of human acid sphingomyelinase (ASM) on catalytic activity, targeting, and processing of the enzyme. Each N-glycosylation site was modified by site-directed mutagenesis and subsequently expressed in COS-1 cells. Evidence is presented that five of these sites are used. Elimination of the four N-terminal glycosylation sites does not disturb lysosomal targeting, processing, or enzymatic activity. However, removal of the two C-terminal N-glycosylation sites inhibits the formation of mature enzyme. Absence of glycosylation site five resulted in rapid cleavage of the primary translation product to an enzymatically inactive protein which accumulated inside the endoplasmic reticuIudGolgi, whereas deletion of glycosylation site six led to the formation of an inactive ASM precursor, also retained inside the endoplasmic reticuludGolgi. Our results also provide evidence that the site of early proteolytic cleavage of newly synthesized ASM must be located between the second and third glycosylation sites.

The lysosomal ␤-hexosaminidases are dimers composed of ␣ and ␤ subunits. ␤-Hexosaminidase A (␣␤) is a heterodimer, whereas hexosaminidase B (␤␤) and S (␣␣) are homodimers. Although containing a high degree of amino acid identity, each subunit expresses a unique active site that can be distinguished by a differential ability to hydrolyze charged substrates. The site on the ␤-subunit primarily degrades neutral substrates, whereas the ␣-subunit site is, in addition, active against sulfated substrates. Isozyme specificity is also exhibited with glycolipid substrates. Among human isozymes, only ␤-hexosaminidase A together with the G M2 activator protein can degrade the natural substrate, G M2 ganglioside, at physiologically significant rates. To identify the domains of the human ␤-hexosaminidase subunits that determine substrate specificity, we have generated chimeric subunits containing both ␣-and ␤-subunit sequences. The chimeric constructs were expressed in HeLa cells to screen for activity and then selected constructs were produced in the baculovirus expression system to assess their ability to degrade G M2 ganglioside in the presence of G M2 activator protein. Generation of activity against the sulfated substrate required the substitution of two noncontinuous ␣-subunit sequences (amino acids 1-191 and 403-529) into analogous positions of the ␤-subunit. Chimeric constructs containing only one of these regions linked to the ␤-subunit sequence showed either neutral substrate activity only (amino acids 1-191) or lacked enzyme activity entirely (amino acids 403-529). Neither the chimeras nor the wild-type subunits displayed activator-dependent G M2 -hydrolyzing activity when expressed alone. However, one chimeric subunit containing ␣ amino acids 1-191 fused with ␤ amino acids 225 to 556, when co-expressed with the wild-type ␣-subunit, showed activity comparable with that of recombinant ␤-hexosaminidase A formed by the co-expression of the ␣-and ␤-subunits. This result indicates that the ␤-subunit amino acids 225-556 contribute an essential function in the G M2 -hydrolyzing activity of ␤-hexosaminidase A.The human ␤-hexosaminidases (EC 3.2.1.52) are dimeric lysosomal enzymes composed of two subunits, ␣-and ␤, that share about 60% of their amino acid sequence (1, 2). The subunits are synthesized as precursors in the endoplasmic reticulum where amino-terminal signal peptides are removed, Nlinked glycosylation occurs, disulfide bonds are formed, and the subunits are folded and assembled. The subunits dimerize and form three isozymes; ␤-hexosaminidase A (␣␤), ␤-hexosaminidase B (␤␤), and ␤-hexosaminidase S (␣␣). Dimerization of the subunits is required for acquisition of enzymatic activity. When properly folded and assembled the enzymes are transferred to the Golgi apparatus for synthesis of the mannose 6-phosphate recognition marker. Mannose 6-phosphate receptors then target the enzymes to lysosomes where the precursor subunits are proteolytically processed to their mature forms (for reviews see Refs. 3 and 4).The ␤-hexo...

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