Identity of the activator proteins for the enzymatic hydrolysis of sulfatide, ganglioside GM1, and globotriaosylceramide

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...

Section: Hydrolysis Of Liposomal Ganglioside Gm1 and Lactosylceramidecontrasting

confidence: 57%

“…In a micellar test system a V,, value of 230 nmol X h-' X (U GMl-P-galactosidase)-I and a K, of 6.6 pM for the sup-B-stimulated GM1 degradation by P-galactosidase was obtained [30].…”

Section: Saturation Of Sap-b With Gmi In a Liposomal Systemmentioning

confidence: 94%

Hydrolysis of lactosylceramide by human galactosylceramidase and GM1‐β‐galactosidase in a detergent‐free system and its stimulation by sphingolipid activator proteins, sap‐B and sap‐C Activator proteins stimulate lactosylceramide hydrolysis

Zschoche

Fürst

Schwarzmann

et al. 1994

“…Genetic defects of these lysosomal activator proteins result in sphingolipid storage diseases [4-lo]. While the human GM2 activator (SAP-3) specifically stimulates the hydrolysis of ganglioside GM2 and GA2 by hexosaminidase A [I 11, the sulfatide activator (SAP-I) furthers unspecifically the degradation of different glycosphingolipids in vitro [12,13]. In the test tube the sulfatide activator can stimulate the hydrolysis of sulfatides by arylsulfatase A, ganglioside GMl by f3-galactosidase, GbOse3-Cer by a-galactosidase A [12,14] and some gangliosides (Gola, GTlb, GM3) by lysosomal sialidase [15].…”

mentioning

confidence: 99%

Glycosphingolipid specifity of the human sulfatide activator protein

Vogel

Schwarzmann

Sandhoff

1991

Self Cite

The interaction of the sulfatide activator protein with different glycosphingolipids has been studied in detail. The following findings were made.1. 2. In the absence of degrading enzymes the activator protein acts in vitro as a glycosphingolipid transfer protein,transporting glycosphingolipids from donor to acceptor liposomes. Lipids having less than three hexoses, e.g. galactosylceramide, sulfatide and ganglioside GM3 were transferred at very slow rates, whereas complex lipids such as gangliosides GMZ, GMl and GDla were transferred much faster than the former. The transfer rate increased with increasing length of the carbohydrate chain of the lipid molecules.3. Both the acyl residue in the ceramide moiety and the nature of the carbohydrate chain are significant for recognition of the glycosphingolipids by the sulfatide activator protein. Apparently, both residues serve as an anchor and the longer they are the better they are recognized by the protein.4. In the absence of activator protein, degradation rates of sulfatide derivatives by arylsulfatase A, and of ganglioside GM1 derivatives by P-galactosidase, increase with decreasing length of acyl residues in their hydrophobic ceramide moiety. Addition of activator protein stimulates the degradation of only those GM1 and sulfatide derivatives that have long-chain fatty acids in their hydrophobic ceramide anchor.Glycosphingolipids are components of the outer leaflet of plasma membranes. They form cell-type-specific patterns on the surfaces of mammalian cells. These patterns change characteristically with differentiation and oncogenic transformation [l].Correspondence to K. Sandhoff, Institut fur Organische Chemie und Biochemie der Universitat Bonn, Gerhard-Domagk-Strasse 1, W-5300 Bonn 1, Federal Republic of Germany Abbreviations. Cer, ceramide or N-acylsphingoid; Sph, D-erythrosphingosine; NeuAc, N-acetylneuraminic acid; GalNAc, 2-acetamido-2-deoxy-~-galactose; GalCer, galactosylceramide; GlcCer, glucosylceramide; LacCer, lactosylceramide; sulfatide or 3'-O-sulfogalactosyl-ceramide; globotriaosylceramide or GbOse3Cer or Galcll + 4Galpl --f 4Glc/l1+ 1Cer; lysosulfatide or 3'-O-SUlfO-galaCtOSy1-Derythro-sphingosine; C,-sulfatide, derivative of sulfatide containing acyl chains with x C atoms (x = 2, 6, 18); G M~, I13NeuAc-LacCer or Gal(3+2clNeuAc)~l+4Glc~I +lCer; G M Z ,

“…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].…”

mentioning

confidence: 99%

The complete amino‐acid sequences of human ganglioside GM2 activator protein and cerebroside sulfate activator protein

Fürst

Schubert

Machleidt

et al. 1990

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]