Functional characterization of the postulated intramolecular sphingolipid activator protein domain of human acid sphingomyelinase

Degradation of membrane-bound sphingomyelin to phosphorylcholine and ceramide is catalyzed by the water-soluble lysosomal acid sphingomyelinase (A-SMase). The presence of sphingolipid activator proteins (Saps: saposins A-D; GM2 activator) is not essential to mediate this reaction at the water-lipid interface in vivo . A hypothesis based on amino acid sequence alignments suggests that the enzyme possesses an N-terminal saposin-homologous domain, which may facilitate the enzymatic reaction at the interface. We mutated one homologous and three conserved amino acid residues of this domain and studied the activity of the variant enzymes using different sphingomyelin degradation assays. A variant with an exchange of a conserved amino acid residue, Pro153Ala, still exhibited enzyme activity of approximately 52% of normal in a detergent-containing micellar assay, but only 13% of normal in a detergent-free liposomal assay system, which suggests that the Sap-homologous domain fulfills membrane-disturbing functions. Addition of saposin C to the liposomal assay mixtures increased the Pro153Ala variant sphingomyelinase activity to 46% of normal, indicating that the variant saposin-like domain can be substituted by the presence of the sphingolipid activator protein. On the other hand, the addition of saposin C did not result in complete restoration of the variant activity. Thus, the Sap-like domain may also have another role, e.g., to stabilize the fold of acid sphingomyelinase, which cannot be compensated by the presence of saposin C or a detergent. Such an essential second function of the saposin-like domain as an integral part of acid sphingomyelinase is confirmed by our observation that the Lys118Glu, Cys120Ser and Cys131Ser variants were almost completely devoid of activity in the detergent-containing micellar assay system as well as in the liposomal assay system in the presence of saposin C.     

Secretion of sphingolipid hydrolase activator precursor, prosaposin

Sphingolipid hydrolases are activated by activator proteins or saposins. The precursor protein has been expected from the studies on the cDNA for saposins. Here we demonstrate that prosaposin occurs in various kinds of human secretory fluids such as cerebrospinal fluid, semen, milk, pancreatic juice, and bile. However, mature type saposins were not detected in these fluids. In human milk the amount of prosaposin changed during the lactating period; it became high in concentration within a few days after delivery, decreased during the transitional milk lactating stage, and then increased again toward the mature milk lactating stage. Prosaposin was released from human platelets in response to stimulation by thrombin, but mature saposins were not. From the time course of the release of prosaposin induced by thrombin and from the fact that weak platelet agonists, ADP, epinephrine, and collagen, did not cause the release of prosaposin, prosaposin secretion from platelets seemed to be from lysosome like granules. We postulate that some prosaposin works as a precursor for saposins in the lysosomes and the other serves as an extracellular protein with other specific roles.     

Molecular cloning of the sphingolipid activator protein-1 (SAP-1), the sulfatide sulfatase activator

A cDNA coding for SAP-1 was isolated from a lambda gt11 human hepatoma expression library using polyclonal antibodies raised against human SAP-1. Three positive clones were isolated with inserts of approximately 0.3 Kb (S1.1), 2 Kb (S1.2) and 2.2 Kb (S-1.3). The latter 2 contained an internal EcoRI site. All three clones cross-hybridized with one another, indicating sequence homology. The nucleotide sequence of S-1.1 was determined. Colinearity was established between 19 amino acids obtained by sequencing the amino terminus of pure SAP-1 and 57 bp from the 5' end of S-1.1. The open reading frame of S-1.1 coded for 67 amino acids. One glycosylation site was found 21 residues from the amino terminus, and no stop codons were found. S-1.1 codes for a mature polypeptide chain with a calculated molecular weight of 8955 daltons, corresponding to approximately 99% of mature SAP-1.     

Stimulation of lysosomal sphingomyelin degradation by sphingolipid activator proteins

Lysosomal breakdown of glycosphingolipids with short hydrophilic carbohydrate headgroups is achieved by the simultaneous action of specific hydrolases and sphingolipid activator proteins (SAPs). Activator proteins are considered to facilitate the enzyme/substrate interaction between water-soluble enzymes and membrane-bound substrates. Sphingomyelin, containing the small hydrophilic phosphorylcholine moiety, is hydrolysed by acid sphingomyelinase (acid SMase). Recent experimental data on the in vivo and in vitro role of activator proteins in sphingomyelin breakdown by acid SMase are reviewed. These data combined with the results using homogenous protein preparations as well as a liposomal assay system mimicking the physiological conditions suggest that lysosomal sphingomyelin degradation is not critically dependent on any of the known activator proteins. Moreover, evidence is provided that the assumed intramolecular activator domain of acid SMase and especially the presence of negatively charged lipids in the lysosomes are sufficient for sphingomyelin turnover.     

Physiological relevance of sphingolipid activator proteins in cultured human fibroblasts

The physiological degradation of several membrane-bound glycosphingolipids (GSLs) by water-soluble lysosomal exohydrolases requires the assistance of sphingolipid activator proteins (SAPs). Four of these SAPs are synthesized from a single precursor protein (prosaposin). Inherited deficiency of this precursor results in a rare disease in humans with an accumulation of ceramide (Cer) and glycolipids such as glucosylceramide and lactosylceramide (LacCer). In a previous study, we have shown that human SAP-D stimulates the lysosomal degradation of Cer in precursor deficient cells. In order to study the role of SAPs (or saposins) A-D in cellular GSL catabolism, we recently investigated the catabolism of exogenously added [(3)H]labeled ganglioside GM1, Forssman lipid, and endogenously [(14)C]labeled GSLs in SAP-precursor deficient human fibroblasts after the addition of recombinant SAP-A, -B, -C and -D. We found that activator protein deficient cells are still able to slowly degrade gangliosides GM1 and GM3, Forssman lipid and globotriaosylceramide to a significant extent, while LacCer catabolism critically depends on the presence of SAPs. The addition of either of the SAPs, SAP-A, SAP-B or SAP-C, resulted in an efficient hydrolysis of LacCer.     

Aldosterone-induced glycoproteins: further characterization

Aldosterone induces the synthesis of a group of glycoproteins (GP65,70) in toad urinary bladders which are potential effectors of the natriferic action of this hormone. The GP65,70 complex is composed of two molecular weight classes of proteins (Mr 65 and 70 kDa), each class being composed of several discrete proteins of varying isoelectric points (5.8-6.2). These proteins can be partially enriched (approximately 20-fold) using wheat germ agglutinin-sepharose affinity chromatography, are neuraminidase-resistant, and can be N-deglycosylated by endoglycosidase-H and N-glycanase. Treatment with N-glycanase leads to the appearance of a microheterogeneous group of proteins, all having the same Mr (approximately 40 kDa). From these studies it can be concluded that these particular aldosterone-induced proteins: (1) are heavily glycosylated, (2) contain multiple high mannose and hybrid oligosaccharides side chains, and (3) contain similar (if not identical) peptide backbones. Post-translational N-glycosylation accounts, at least in part, for their electrophoretic polymorphism (variation in Mr) but not for their electrophoretic microheterogeneity (variation in pI). The latter may reflect other types of post-translational modification (e.g. O-glycosylation, phosphorylation) or may be due to subtle differences in amino acid composition. The partial purification and biochemical characterization of GP65,70 should ultimately lead to a better understanding of the function of these putative "effectors" of aldosterone-stimulated Na+ transport.     

Analysis of the multiple forms of Gaucher spleen sphingolipid activator protein 2.

Gaucher spleen sphingolipid activator protein 2 was fractionated into concanavalin A binding- and non-binding fractions. These fractions each contained several bands on non-denaturing polyacrylamide gel electrophoresis (PAGE). The two fractions were further fractionated by electroblotting the proteins from preparative gels onto nitrocellulose, staining with Ponceau S to locate the bands of protein and then eluting the protein components from the nitrocellulose. A total of ten fractions, each containing only one or two major components, was collected. All of these subfractions activated beta-glucocerebrosidase and sphingomyelinase and most subfractions also activated beta-galactocerebrosidase. The structural relationship of the bands was investigated using endoglycosidase digestions. The results indicated that the two bands with the fastest mobility on non-denaturing PAGE did not contain any carbohydrate. The remaining bands showed only limited or partial digestion with endoglycosidase H and endoglycosidase D, but were readily hydrolysed with endoglycosidase F. The products of these digestions included bands with similar mobilities to the non-carbohydrate containing bands.     

Simultaneous deficiency of sphingolipid activator proteins 1 and 2 is caused by a mutation in the initiation codon of their common gene.

Sphingolipid activator proteins (SAPs) are small, nonenzymic glycoproteins that stimulate lysosomal degradation of various sphingolipids. SAP-1, SAP-2, and two additional potential activator proteins are derived from a common precursor by proteolytic processing. A severe case of sphingolipid storage disease that led to death within 16 weeks was attributed to a possible total deficiency of the SAPs generated by this gene (Harzer, K., Paton, B. C., Poulos, A., Kustermann-Kuhn, B., Roggendorf, W., Grisar, T., and Popp, M. (1989) Eur. J. Pediatr. 149, 31-39). Analysis of the SAP precursor cDNA from the patient and his fetal sibling showed an A to T transversion in the initiation codon. Allele-specific oligonucleotide hybridization revealed that both parents are heterozygous carriers for this mutation. In pulse-chase experiments using antisera raised against SAP-1 or SAP-2, no cross-reacting material could be detected in the patients' fibroblasts.     

Structure of the lysosomal sphingolipid activator protein 1 by homology with influenza virus neuraminidase

The sphingolipid activator protein 1 (SAP-1) increases the rate of hydrolysis of sphingolipids in the lysosome by apparently bringing together the substrate and the corresponding hydrolytic enzyme. This implies specific recognition of both the substrate and enzyme by SAP-1. However, binding domains in SAP-1 and recognition mechanisms involved are unknown. Amino acid sequence comparison of SAP-1 with influenza virus neuraminidase (EC 3.2.1.18, FLU NA) indicates that functional amino acid residues in or near the sialic acid binding site of FLU NA are also found at equivalent positions in the first 48 N-terminal amino acids of SAP-1. This region of homology allows to propose folding of the SAP-1 polypeptide chain by comparison with known crystallographic structure of FLU NA and identify a potential domain for lysosomal enzyme recognition through sialic acid binding. There is also a region of 10 amino acid residues near the C-terminal end of SAP-1 which has a strong propensity to form an alpha-helix with amphiphilic properties of lipid-binding helices. This domain in SAP-1 is probably responsible for the lipid(substrate)-binding function of SAP-1.     

Stimulation of acid sphingomyelinase activity by lysosomal lipids and sphingolipid activator proteins

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.     

鞘脂激活蛋白原:一种新型神经营养因子

鞘脂激活蛋白 (ｓｐｈｉｎｇｏｌｉｐｉｄａｃｔｉｖａｔｏｒｐｒｏ ｔｅｉｎ ,ｓａｐｏｓｉｎ)是包含鞘脂激活蛋白Ａ、Ｂ、Ｃ、Ｄ的一组热稳定糖蛋白 ,它们在溶酶体酶解鞘脂的过程中发挥着重要作用[1] 。鞘脂激活蛋白由其前体鞘脂激活蛋白原 (ｐｒｏｓａｐｏｓｉｎ)水解产生。最近的研究表明 ,除作为前体外 ,鞘脂激活蛋白原还具有较强的神经营养活性。本文就这一方面的研究进展作一综述。1 .鞘脂激活蛋白原的神经营养作用及其分布1 .1　作用的发现　　鞘脂激活蛋白原是在对鞘脂激活蛋白的研究中发现的。Ｋｏｎｄｏｎ等用免疫组织法发现 ,脑…     

Determination of saposin proteins (sphingolipid activator proteins) in human tissues

Saposins are small glycoproteins which are required for sphingolipid hydrolysis by lysosomal hydrolases. Each saposin (A, B, C, and D) stimulates a different enzymatic activity. A new simple HPLC method to determine the levels of saposins A, C, and D in tissue was developed. Tissues were homogenized in 20 vol of water, boiled, and centrifuged. The supernatant was lyophilized and redissolved in 5 ml of water. A 1.5-ml sample of the solution was applied to a reverse-phase HPLC column (C4 column) and eluted with an acetonitrile gradient. Most contaminants eluted from the column prior to the saposins, which were eluted later as a cluster of peaks. This cluster was collected and then analyzed by another HPLC system equipped with an AX-300 anion-exchange column using a NaCl gradient. Saposins D, A, and C eluted from the AX-300 column separately and in that order. Quantitation of the saposins was made by measuring the sizes of each peak. Standard curves made from pure saposins showed that quantification was linear over a range from 1 to 5 micrograms. Saposin B was measured by its stimulation activity on pure human liver GM1 ganglioside beta-galactosidase. Stimulation was linear up to 80 micrograms of saposin B. Application of this method to analysis of human tissues for their saposin content is presented.     

Cytochrome P-460 of Nitrosomonas europaea: further purification and further characterization

Cytochrome P-460 of Nitrosomonas europaea [Erickson, R.H. and Hooper, A.B. (1972) Biochim. Biophys. Acta 275, 231-244] was further purified to an electrophoretically homogeneous state. The cytochrome molecule was composed of three molecules of subunits with Mr of 17,300-18,500, and contained three atoms of iron, which seemed to be heme iron, and six cysteine residues, but did not contain nonheme iron or inorganic sulfide. The cytochrome showed absorption peaks at 460 and 688 nm with a broad shoulder at 635 nm in the reduced form. The ESR spectrum of ferricytochrome P-460 showed signals at g = 5.91, 5.63, and 1.99, indicating that the protein was a high spin hemoprotein. The heme of the cytochrome was not cleaved by the methods which were available for cleavage of heme c. The pyridine ferrohemochrome of the hemoprotein did not show the distinct alpha and beta peaks which are shown by the ferrohemochromes of many other cytochromes so far known. The N-terminal amino acid sequence of cytochrome P-460 differed from that of hydroxylamine oxidoreductase. Therefore, cytochrome P-460 did not seem to be the solubilized P-460 moiety of hydroxylamine oxidoreductase, in agreement with the finding by D.J. Miller et al. [J. Gen. Microbiol. 130, 3049-3054 (1984)]. However, cytochrome P-460 had several enzymatic activities which hydroxylamine oxidoreductase showed. Although most of the activities of the cytochrome were lower than the corresponding activities of the oxidoreductase, the hydroxylamine-cytochrome c-552 reductase activity of the cytochrome was about 5-times as high as that of the oxidoreductase.     

Identification of a novel sequence involved in lysosomal sorting of the sphingolipid activator protein prosaposin

Prosaposin is synthesized as a 53-kDa protein, post-translationally modified to a 65-kDa form and further glycosylated to a 70-kDa secretory product. The 65-kDa protein is associated to Golgi membranes and is targeted to lysosomes, where four smaller nonenzymatic saposins implicated in the hydrolysis of sphingolipids are generated by its partial proteolysis. The targeting of the 65-kDa protein to lysosomes is not mediated by the mannose 6-phosphate receptor. The Golgi apparatus appears to accomplish the molecular sorting of the 65-kDa prosaposin by decoding a signal from its amino acid backbone. This investigation deals with the characterization of the sequence involved in this process by deleting the saposin functional domains A, B, C, and D and the highly conserved N and C termini of prosaposin. The truncated cDNAs were subcloned into expression vectors and transfected to COS-7 cells. The destination of the mutated proteins was assessed by immunocytochemistry. Deletion of the C terminus did not interfere with the secretion of prosaposin but abolished its transport to lysosomes. Deletion of saposins and the N-terminal domain did not affect the lysosomal or secretory routing of prosaposin. A chimeric construct of albumin and the C terminus of prosaposin was not directed to lysosomes. However, albumin connected to the C terminus and one or more functional domains of prosaposin reached lysosomes, indicating that the C terminus and at least one saposin domain are required for this process. In summary, we are reporting a novel sequence involved in the targeting of prosaposin to lysosomes.     

Ecdysteroids in nemerteans: further characterization and identification

Ecdysteroids are a class of steroidal hormones thatare important in molting and reproduction inarthropods. These hormones have been recentlydetected in non-arthropodan groups, such assoft-bodied worms. To continue our efforts todetermine the presence of ecdysteroids in nemerteans,this study further documents the identification of20-hydroxyecdysone (20E), in Paranemertesperegrina, by gas chromatography/mass spectrometry(GC/MS). After C₁₈ Sep-Pak extraction,radioimmunoassay of the 40% and 80% methanolicfractions from an extract of 1000 worms indicated 98and 156 ng of ecdysteroids, respectively. Ecdysteroids of these two samples, as well as the 20Eand ecdysone standards, were N-trimethylsilyimidazole(TMSI)-derivatized before GC/MS analysis. Themethanolic samples contained a large number ofcompounds but only small, insignificant peaks in thearea where ecdysteroid standards eluted. However, thereconstruction ion current (RIC) chromatograms for m/z561 indicated the presence of 20E at the correctretention time of 12.48 min in the 40% methanolicfraction. Reanalysis of the samples under selectedion monitoring mode demonstrated the presence of 20Ein both the 40% and 80% fractions. These resultsdemonstrate conclusively the presence of an activeecdysteroid in the phylum Nemertea.     

Human placental sialidase: further purification and characterization

An acid sialidase [EC 3.2.1.18] has been purified from human placenta by means of successive procedures including extraction, Con A-Sepharose adsorption, ammonium sulfate precipitation, activation, p-aminophenyl thio-beta-D-galactoside-CH-Sepharose (PATG-Sepharose) affinity chromatography and high-performance liquid chromatography on a Shim pack Diol 300 column. The purified enzyme liberated sialic acid residues from sialooligosaccharides, sialoglycoproteins, and gangliosides. In particular, gangliosides GM3, GD1a, and GD1b were hydrolyzed much faster than alpha (2-3) and alpha (2-6)sialyllactoses, and sialoglycoproteins by the enzyme. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified enzyme gave five protein bands with molecular weight of 78,000 (78K), 64,000 (64K), 46,000 (46K), 30,000 (30K), and 20,000 (20K). Rabbit antisera were raised against 78K and 46K proteins, and the two antibodies were specifically reactive with the respective component on immunoblot analysis. Both anti-78K protein and anti-46K protein antisera could precipitate sialidase activity. It is likely that the 78K protein and 46K protein are sub-components which are essential for sialidase activity.     

The lysosomal trafficking of sphingolipid activator proteins (SAPs) is mediated by sortilin

Most soluble lysosomal proteins bind the mannose 6-phosphate receptor (M6P-R) to be sorted to the lysosomes. However, the lysosomes of I-cell disease (ICD) patients, a condition resulting from a mutation in the phosphotransferase that adds mannose 6-phosphate to hydrolases, have near normal levels of several lysosomal proteins, including the sphingolipid activator proteins (SAPs), GM2AP and prosaposin. We tested the hypothesis that SAPs are targeted to the lysosomal compartment via the sortilin receptor. To test this hypothesis, a dominant-negative construct of sortilin and a sortilin small interfering RNA (siRNA) were introduced into COS-7 cells. Our results showed that both the truncated sortilin and the sortilin siRNA block the traffic of GM2AP and prosaposin to the lysosomal compartment. This observation was confirmed by a co-immunoprecipitation, which demonstrated that GM2AP and prosaposin are interactive partners of sortilin. Furthermore, a dominant-negative mutant GGA prevented the trafficking of prosaposin and GM2AP to lysosomes. In conclusion, our results show that the trafficking of SAPs is dependent on sortilin, demonstrating a novel lysosomal trafficking.     

Mutation in the sphingolipid activator protein 2 in a patient with a variant of Gaucher disease

The lysosomal degradation of glucosylceramide requires the hydrolase, glucosylceramide-beta-glucosidase and a sphingolipid activator protein (Gaucher factor, SAP-2, saposin C). Genetic defects in either of these lysosomal proteins cause phenotypically similar disorders in man, the Gaucher disease. SAP-2 originates from a gene which generates a mRNA that codes for four homologous proteins. In a patient with an immunologically proven SAP-2 deficiency a G1154----T transversion (counted from A of the initiation codon ATG) was found in the mRNA of the SAP-2 precursor which results in the substitution of Phe for Cys385 in the mature SAP-2. The rest of the coding sequence remained entirely normal.