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The enzymic degradation of a number of sphingolipids in the lysosomes is stimulated by small acid glycoproteins named activator proteins. We purified and sequenced a new protein, called component C, which seems to be related to sulfatide activator and to a recently described activator of glucosylceramidase (A l activator) (Kiemschmidt, T., Christomanou, H. & Braunitzer, G. (1987) Biol. Chem. Hoppe-Seyler 368, 1571-1578. It consists of 78 amino acids and carries one carbohydrate chain at aparagine 20. Component C shows 21.5% sequence homology to sulfatide activator and 34.2% homology to A! activator. Structural similarities between these three proteins have also been detected.Recently the cDNA sequence of the sulfatide activator precursor has been published (Dewji, N.N., Wenger, D.A. & O'Brien, J.S. (1987) Proc. Natl. Acad. Sei. U.S.A. 84, 8652-8656).We could align the protein sequences of sulfatide activator, Aj activator and component C with that of this large precursor protein. After minor corrections of the DNA sequence we obtained total fit. Thus it seems that three different proteins are derived from the sulfatide activator precursor by proteolytic processing. Possible processing sites were found on the precursor at sites adjacent to the N-termini and C-termini of the mature proteins. The processing of sulfatide activator was studied by Fujibayashi and Wenger (Fujibayashi, S. & Wenger, D.A. (1986) Biochim. Biophys. Acta 875, 554-562). Their data support our assumption that processing occurs by simultaneous cleavage at all possible sites. Der Vorläufer des Sulfatidaktivatorproteins wird zu drei verschiedenen Proteinen prozessiertZusammenfassung: Der enzymatische Abbau einer Reihe von Sphingolipiden in den Lysosomen wird durch kleine, saure Glycoproteine, genannt Aktivatorproteine, stimuliert. Wir reinigten und sequenzierten ein neues Protein, das wir Komponente C nennen und das mit dem Sulfa-

Two exo-P-galactosidases are involved in the lysosomal degradation of glycosphingolipids : GM1-P-galactosidase (EC 3.2.1.23) and galactosylceramidase (EC 3.2.1.46). Analyses were performed with both enzymes, using lactosylceramides with varying acyl chain lengths as substrates that were inserted into unilamellar liposomes and naturally occurring sphingolipid activator proteins sup-B and sup-C, rather than detergents, to stimulate the reaction.While sup-B was a better activator for the reaction catalyzed by GM1-P-galactosidase, sup-C preferentially stimulated lactosylceramide hydrolysis by galactosylceramidase. The enzymic hydrolysis of liposome-integrated lactosylceramides was significantly dependent on the structure of the lipophilic aglycon moiety of the lactosylceramide decreasing with increasing length of its fatty acyl chain (C, > C, > C, > C, > Clo> C,J. However, in the presence of detergents the degradation rates were independent of the acyl chain length. Hydrolysis of liposomal lactosylceramide was compared with sup-B-stimulated hydrolysis of liposomal ganglioside GM1 by GM1-P-galactosidase and sup-C-stimulated degradation of liposomal galactosylceramide by galactosylceramidase.Kinetic and dilution experiments indicated that sup-B forms water-soluble complexes with both lactosylceramide and GM1. These complexes were recognized by GM1-P-galactosidase as optimal substrates in the same mode, as postulated for the hydrolysis of sulfatides by arylsulfatase A [Fischer, G. and Jatzkewitz, H. (1977) Biochim. Biophys. A m 481,561 -5721. GM1-P-galactosidase was more active on these complexes than on glycolipids (GM1 and lactosylceramides) still residing in liposomal membranes. On the other hand, dilution experiments indicated that degradation of galactosylceramide and lactosylceramide by galactosylceramidase proceeds almost exclusively on liposomal surfaces : both activators, sup-C and sup-B, stimulated the hydrolysis of lactosylceramide analogues with long acyl chains more than the hydrolysis of lactosylceramides with short acyl chains.Glycosphingolipids are primarily components of the outer leaflet of plasma membranes. Their biosynthesis occurs in the endoplasmic reticulum and Golgi compartment, their degradation mainly in lysosomes [l]. A recent hypothesis on Correspondence to K. Sandhoff, Institut fur Organische Chemie und Biochemie, Universitat Bonn, Gerhard-Domagk-Stral3e 1, D-53121 Bonn, GermanyAbbreviations. Cer, ceramide ; LacCer, lactosylceramide, Galbl4GlcP1-1 Cer ; C,-LacCer, N-acetyl-(1-0-lactosyl) sphingosine; C,-LacCer, N-butanoyl-(1-0-lactosyl) sphingosine ; C,-LacCer, N-hexanoyl-(1-0-lactosyl) sphingosine; C,-LacCer, N-octanoyl-(1-0-lactosyl) sphingosine; C,,-LacCer, N-decanoyl-(1-0-lactosyl) sphingosine; C,,-LacCer, N-octadecanoyL(1-0-lactosyl) sphingosine; GalCer, galactosylceramide, Galbl-1 Cer ; GM1, Galj?l-3GalNAcj?l4Gal(3-2aNeuAc)~1-4Glc/?l-l Cer ; sup-B, sulfatide activator protein or SAP-1 or saposin B, sphingolipid activator protein B; sup-C, co-p-glucosidase or SAP-2 or saposin C, sphing...

The complete amino-acid sequences of human ganglioside GM2 activator protein and cerebroside sulfate activator protein have been established by Edman degradation. The GM2 activator is composed of 162 amino acids, the first two serine residues being present in only 20% of the material. A single carbohydrate chain is Nglycosidically linked to Asn32. Three hydrophobic a-helices may contribute to its lipid-binding site. Three amino acids differ from those found by cDNA sequencing which may be due to a polymorphism. The cerebroside sulfate activator consists of 80 amino acids and carries one N-linked carbohydrate chain at Asn21. The C-terminal valine residue is lacking in about 80% of the material. In spite their similar functions, both activator proteins show no sequence or structural similarities.The degradation of sphingolipids in the lysosomes is accomplished by the sequential action of acidic hydrolases. To attack their membrane-bound substrates some of these enzymes need the assistance of small, non-enzymic cofactors, called activator proteins. Cerebroside sulfate activator (SAP-1) [l, 21 stimulates the breakdown of cerebroside sulFate, ganglioside GM1 and globotriaosylceramide by the respective hydrolases [3,4]. Ganglioside GM2 activator protein (SAP-3) is more specific, accelerating only the breakdown of ganglioside GM2 and glycolipid GA2 by fl-hexosaminidase A [5]. Both activator proteins extract single lipid molecules from membranes or micelles, bind them as 1 : 1 complexes and present them to the respective enzyme for hydrolysis [6, 71. GM2 activator protein is synthesized as a 24-kDa precursor protein which is processed to a mature form of some 22 kDa [8]. Cerebroside sulfate activator is a homodimer of 12-kDa polypeptide chains and is derived from a 70-kDa precursor protein [9]. Recently, we [lo, 111 and others [12]

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