The activator protein for glucosylceramide beta-glucosidase from guinea pig liver. Improved isolation method and complete amino acid sequence

beta-Glucosidase activator (SAP-2) is a family of heat-stable, acidic glycoproteins which stimulate enzymatic hydrolysis of glucosylceramide. In this study, we improved the purification method and found that SAP-2 is highly heterogeneous. A hot water extract of frozen guinea pig liver was fractionated by ammonium sulfate sedimentation, then chromatographed with DEAE-Sephacel, Sephadex G-75, and concanavalin A-Sepharose. A fraction binding to concanavalin A-Sepharose was purified further with a C4 high performance liquid chromatography reverse phase column. This yielded several peaks, the main one of which was studied. The specific activity of the purified SAP-2 was 35 units/micrograms (1 unit produces 50% stimulation of a basal glucosidase preparation). N-terminal amino acid sequencing showed that this preparation is a mixture of polypeptides differing in the presence or absence of one or two of the end amino acids. The complete amino acid sequence of the 81 residues in SAP-2 has been determined. Comparison of the sequence of guinea pig SAP-2 with the sequence of human sphingomyelinase activator revealed 58% homology and quite similar hydropathy profiles. Both proteins possess a highly hydrophilic region around Asn-22, which is glycosylated, and 6 cysteine residues, in oxidized form, located in the same positions. Comparison with the published nucleotide sequence for the precursor form of the human activator protein for sulfatide sulfatase (SAP-1) suggested that this activator also has a possibly glycosylated Asn and 6 Cys residues at similar positions, although the remainder of the molecule is somewhat different. Examination of another region of the precursor's nucleotide sequence, assuming a few changes in the identifications, revealed the presence of the sphingomyelinase activator. It appears that two or more activators are derived from a single precursor protein. Marked homologies were seen also with a lung surfactant protein and a sulfated glycoprotein from Sertoli cells.     

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.     

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.     

Sulfatide activator protein. Alternative splicing that generates three mRNAs and a newly found mutation responsible for a clinical disease

The sulfatide activator protein, also known as SAP-1, is derived from a gene that generates an mRNA coding for four homologous proteins. Its physiological function is to stimulate hydrolysis of sulfatide by arylsulfatase A in vivo. A genetic defect in the sulfatide activator results in a metabolic disorder similar to classical metachromatic leukodystrophy, which is itself caused by a genetic defect in arylsulfatase A. In a patient with sulfatide activator deficiency, a nucleotide transversion G722----C (counted from A of the initiation codon ATG) was found in the mRNA of the sulfatide activator precursor, resulting in the substitution of serine for Cys241 in the mature sulfatide activator. The remainder of the coding sequence was completely normal except for a polymorphism C to T in position 1389, which does not change the amino acid sequence. The patient produces at least three different forms of mRNA for the precursor. Two of them include a stretch of an additional 9 and 6 bases, respectively, within the sulfatide activator coding region. In normal individuals this stretch of additional bases has also been observed. This could be explained by the presence of a small 9-base pair exon which can be introduced, or not, by alternative splicing as a stretch of 9 or 6 bases into the mature mRNA. The shortest form of the mRNA yields an active sulfatide activator (Fürst, W., Schubert, J., Machleidt, W., Meier, H. E., and Sandhoff, K. (1990) Eur. J. Biochem. 192, 709-714).     

Degradation of membrane-bound ganglioside GM1. Stimulation by bis(monoacylglycero)phosphate and the activator proteins SAP-B and GM2-AP

According to our hypothesis (Fürst, W., and Sandhoff, K. (1992) Biochim. Biophys. Acta 1126, 1-16) glycosphingolipids of the plasma membrane are digested after endocytosis as components of intraendosomal and intralysosomal vesicles and membrane structures. The lysosomal degradation of glycosphingolipids with short oligosaccharide chains by acid exohydrolases requires small, non-enzymatic cofactors, called sphingolipid activator proteins (SAPs). A total of five activator proteins have been identified as follows: namely the saposins SAP-A, -B, -C, and -D, which are derived from the single chain SAP-precursor protein (prosaposin), and the GM2 activator protein. A deficiency of prosaposin results in the storage of ceramide and sphingolipids with short oligosaccharide head groups. The loss of the GM2 activator protein blocks the degradation of the ganglioside GM2. The enzymatic hydrolysis of the ganglioside GM1 is catalyzed by beta-galactosidase, a water-soluble acid exohydrolase. The lack of ganglioside GM1 accumulation in patients suffering from either prosaposin or GM2 activator protein deficiency has led to the hypothesis that SAPs are not needed for the hydrolysis of the ganglioside GM1 in vivo. In this study we demonstrate that an activator protein is required for the enzymatic degradation of membrane-bound ganglioside GM1 and that both SAP-B and the GM2 activator protein significantly enhance the degradation of the ganglioside GM1 by acid beta-galactosidase in a liposomal, detergent-free assay system. These findings offer a possible explanation for the observation that no storage of the ganglioside GM1 has been observed in patients with either isolated prosaposin or isolated GM2 activator deficiency. We also demonstrate that anionic phospholipids such as bis(monoacylglycero)phosphate and phosphatidylinositol, which specifically occur in inner membranes of endosomes and in lysosomes, are essential for the activator-stimulated hydrolysis of the ganglioside GM1. Assays utilizing surface plasmon resonance spectroscopy showed that bis(monoacylglycero)phosphate increases the binding of both beta-galactosidase and activator proteins to substrate-carrying membranes.     

Detection of a point mutation in sphingolipid activator protein-1 mRNA in patients with a variant form of metachromatic leukodystrophy

The lysosomal degradation of sulfatide requires the specific enzyme, arylsulfatase A, as well as a heat stable protein called sphingolipid activator protein-1 (SAP-1). While most patients with metachromatic leukodystrophy have defects in arylsulfatase A, some patients have defects in SAP-1. SAP-1 is coded for by a gene on human chromosome 10 that also codes for three other proposed SAP. Examination of the cDNA from two siblings with SAP-1 deficiency revealed a point mutation of nucleotide #650 (counting from the initiation ATG) which is in the SAP-1 coding domain. This C to T transition changed the codon from threonine (ACC) to one coding for isoleucine (ATC). This eliminated the only glycosylation site in mature SAP-1 and could explain the findings made at the protein level.     

The precursor of sulfatide activator protein is processed to three different proteins

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 (A1 activator) (Kleinschmidt, 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 A1 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. Sci. U.S.A. 84, 8652-8656). We could align the protein sequences of sulfatide activator, A1 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.     

Synthesis and processing of sphingolipid activator protein-2 (SAP-2) in cultured human fibroblasts

Sphingolipid activator proteins (SAP) are relatively small molecular weight proteins that stimulate the enzymatic hydrolysis of sphingolipids in the presence of specific lysosomal hydrolases. SAP-2 has previously been demonstrated to activate the hydrolysis of glucosylceramide, galactosylceramide, and, possibly, sphingomyelin. Using monospecific rabbit antibodies against human spleen SAP-2, the synthesis and processing of SAP-2 were studied in cultured human fibroblasts. When [35S]methionine was presented in the medium to control human cells for 4 h, five major areas of radiolabeling were found. These had apparent molecular weights of 73,000, 68,000, 50,000, 12,000, and 9,000. Further studies indicated that the major extracellular product in normal cells given NH4Cl along with the [35S]methionine and in medium from cultures from patients with I cell disease had an apparent molecular weight of 73,000. The Mr = 68,000 and 73,000 species can be converted to a species with an apparent molecular weight of 50,000 by the action of endoglycosidase F. After labeling cells for 1 h followed by a 1-h chase, the Mr = 12,000 and 9,000 species appear. Treatment of the immunoprecipitated mixture with endoglycosidase F resulted in conversion of these species to one band with an apparent molecular weight of 7,600. These studies indicate that this relatively low molecular weight protein is rapidly synthesized from a relatively large molecular weight highly glycosylated precursor.     

Sphingolipid activator proteins: proteins with complex functions in lipid degradation and skin biogenesis.

Sphingolipid activator proteins (SAPs or saposins) are essential cofactors for the lysosomal degradation of membrane-anchored sphingolipids. Four of the five known proteins of this class, SAPs A--D, derive from a single precursor protein and show high homology, whereas the fifth protein, GM2AP, is larger and displays a different secondary structure. Although the main function of all five proteins is assumed to lie in the activation of lipid degradation, their specificities and modes of action seem to differ considerably. It has recently been demonstrated that the action of the proteins is highly enhanced by the presence of acidic lipids in the target membranes. These results have some interesting implications for the topology of lysosomal degradation of lipids and may provide new insights into the function of these interesting proteins, which are ubiquitously expressed in the different tissues of the body. Recent studies indicated that the SAPs play an important role in the biogenesis of the epidermal water barrier, which has been demonstrated by the analysis of the skin phenotype displayed by SAP-knockout mice. The results obtained so far have led to some new insights into the formation of the epidermal water permeability barrier and may lead to a better understanding of this complex process.     

Lipid metabolism, atherogenesis and CD1-restricted antigen presentation

CD1 molecules are a family of major histocompatibility complex (MHC)-related glycoproteins that present lipid and glycolipid antigens to T cells. Interestingly, it has been demonstrated that CD1d-restricted T cells have a pathogenic role in atherosclerosis. Recent studies suggest an association between the cellular machinery that loads CD1 molecules with glycolipids and several key proteins in lipid metabolism. These proteins include the sphingolipid activator proteins (SAPs), microsomal triglyceride transfer protein (MTP) and apolipoprotein E (apoE). MTP and SAPs seem to be crucial for loading CD1d with lipids in the endoplasmic reticulum and endosomal compartments, respectively, whereas apoE facilitates efficient uptake and delivery of exogenous lipid antigens to CD1d in endosomal compartments. These studies reveal new and unexpected relationships between lipid metabolism and antigen presentation by CD1 molecules. Targeting this pathway of immune activation might have therapeutic potential for the treatment of chronic inflammatory diseases.     

Immunocytochemical localization of sphingolipid activator protein-1, the sulfatide/GM1 ganglioside activator, to lysosomes in human liver and colon

Sphingolipid activator proteins (SAP) stimulate the enzymatic hydrolysis of sphingolipids. The results of biochemical studies have suggested that SAP are located within lysosomes. In this study we sought immunocytochemical verification of the lysosomal location of SAP-1, a SAP that stimulates the hydrolysis of sulfatide and GM1 ganglioside. We stained adjacent sections of normal adult liver and colon for either SAP-1, by peroxidase-labeled antibodies, or acid phosphatase, by enzyme histochemistry. At the light microscopic level, SAP-1 and acid phosphatase were present in similar cells of the colonic lamina propria and hepatic sinusoids, and in similar supranuclear sites of colonic epithelial cells. By electron microscopy, SAP-1 was present in vesicular structures morphologically similar to those containing acid phosphatase. Thus, SAP-1 is present in lysosomes of several different kinds of cells in the normal human liver and colon.     

Immunocytochemical localization of sphingolipid activator protein-1, the sulfatide/GM1 ganglioside activator,to lysosomes in human liver and colon

Summary Sphingolipid activator proteins (SAP) stimulate the enzymatic hydrolysis of sphingolipids. The results of biochemical studies have suggested that SAP are located within lysosomes. In this study we sought immunocytochemical verification of the lysosomal location of SAP-1, a SAP that stimulates the hydrolysis of sulfatide and G_M1 ganglioside. We stained adjacent sections of normal adult liver and colon for either SAP-1, by peroxidase-labeled antibodies, or acid phosphatase, by enzyme histochemistry. At the light microscopic level, SAP-1 and acid phosphatase were present in similar cells of the colonic lamina propria and hepatic sinusoids, and in similar supranuclear sites of colonic epithelial cells. By electron microscopy, SAP-1 was present in vesicular structures morphologically similar to those containing acid phosphatase. Thus, SAP-1 is present in lysosomes of several different kinds of cells in the normal human liver and colon.     

Metabolism of GM1 ganglioside in cultured skin fibroblasts: anomalies in gangliosidoses,sialidoses, and sphingolipid activator protein (SAP,saposin) 1 and prosaposin deficient disorders

Summary Cultured skin fibroblasts from controls and patients with lysosomal storage diseases were loaded with G_M1 ganglioside that had been labelled with tritium in its ceramide moiety. After a 65-h or 240-h incubation, a large percentage of this ganglioside remained undegraded in G_M1 gangliosidoses, whereas in the other storage diseases studied, one of its metabolites accumulated by 2–4 fold relative to controls. Labelled G_M2 ganglioside accumulated in 4 variants of G_M2 gangliosidosis, whereas labelled G_M3 ganglioside accumulated in sialidosis, galactosialidoses and sphingolipid activator protein 1 (SAP-1, saposin B) and prosaposin (saposin A, B, C an D) deficient lipidoses. The reduced degradation of G_M3 ganglioside in the SAP-1 and prosaposin deficiencies was attributed to the deficient function of SAP-1. The prosaposin deficient cells also showed a reduced re-utilization of radioactive metabolites from G_M1 ganglioside (i.e. sphingosine and fatty acid) for phospholipid biosynthesis compared with fibroblasts from the SAP-1 deficient patient or normal controls. This anomaly was ascribed to the previously shown defect in ceramide degradation in prosaposin deficiency.     

Inactivation of sortilin (a novel lysosomal sorting receptor) by dominant negative competition and RNA interference

To assess the role of sortilin in the sorting and trafficking of sphingolipid activator proteins (SAPs) the function of sortilin was abolished by a dominant-negative mutant and by the use of RNAi. Mutant sortilin lacking the carboxyl-terminal region that contains the sorting signal abolished the trafficking of SAPs to the lysosomes. Both sortilin and SAPs were retained in the Golgi apparatus. The use of chemically synthesized siRNA effectively blocked the trafficking of SAPs to the lysosomes as well. Additionally, we created a stable COS-7 cell line transfected with the pSilencer 3.1 H1 neo vector containing a selected siRNA template oligonucleotide (small hairpin interference RNA) where the levels of sortilin were greatly suppressed. The elimination of sortilin by this method will permit to determine whether or not sortilin is involved in a general mechanism of lysosomal sorting that involves the trafficking of various soluble lysosomal proteins other than SAPs.     

Correction of sulfatide metabolism after transfer of prosaposin cDNA to cultured cells from a patient with SAP-1 deficiency.

The lysosomal removal of the sulfate moiety from sulfatide requires the action of two proteins, arylsulfatase A and sphingolipid activator protein-1 (SAP-1). Recently, patients have been identified who have a variant form of metachromatic leukodystrophy which is characterized by mutations in the gene coding for SAP-1, which is also called "prosaposin." All of the mutations characterized in these patients result in (a) deficient mature SAP-1, as determined by immunoblotting after SDS-PAGE of tissue and cell extracts, and (b) decreased ability of cultured skin fibroblasts to metabolize endocytosed [14C]-sulfatide. We now report the insertion of the full-length prosaposin cDNA into the Moloney murine leukemia virus-derived retroviral vector, pLJ, and the infection of cultured skin fibroblasts from a newly diagnosed and molecularly characterized patient with SAP-1 deficiency. The cultured cells infected with the prosaposin cDNA construct now show both production of normal levels of mature SAP-1 and completely normal metabolism of endocytosed [14C]-sulfatide. These studies demonstrate that the virally transferred prosaposin cDNA is processed normally and is localized within lysosomes, where it is needed for interaction between sulfatide and arylsulfatase A. In addition, normal as well as mutant sequences can now be found by allele-specific oligonucleotide hybridization of PCR-amplified genomic DNA by using exonic sequences as primers.     

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.     

Assignment of the gene for human sphingolipid activator protein-2 (SAP-2) to chromosome 10.

Sphingolipid activator protein-2 (SAP-2) has been found to stimulate the enzymatic hydrolysis of glucosylceramide, galactosylceramide, and sphingomyelin. When human skin fibroblast extracts were subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis followed by electroblotting and immunochemical staining using monospecific antibodies against SAP-2, two or three major bands with estimated mol. wts. of 9,000-10,000 were found. These antibodies did not crossreact with purified SAP-1, another activating protein, or with extracts of CHO-K1 cells. A series of 22 human/Chinese hamster ovary cell hybrids containing different human chromosomes were examined by this method. All eight hybrid clones containing human chromosome 10 were found to have crossreacting protein in this region. Other chromosomes could be excluded by this method. From these results, we conclude that the gene coding for human SAP-2 is located on chromosome 10.     

Biosynthesis and molecular cloning of sulfated glycoprotein 1 secreted by rat Sertoli cells: sequence similarity with the 70-kilodalton precursor to sulfatide/GM1 activator

Sulfated glycoprotein 1 (SGP-1) is one of the abundant proteins secreted by rat Sertoli cells. Pulse-chase labeling shows that SGP-1 is synthesized as a cotranslationally glycosylated 67-kilodalton (kDa) precursor which is posttranslationally modified to a 70-kDa form before secretion to the extracellular space. A plasmid cDNA library was constructed from immunopurified mRNA, and two overlapping clones coding for the entire protein coding sequence were isolated. The cDNAs represent 27 nucleotides of 5' noncoding sequence, 1554 nucleotides of coding sequence, and 594 nucleotides of 3' noncoding sequence. The derived SGP-1 sequence contains 554 amino acids and has a molecular weight of 61,123. Four potential N-glycosylation sites occur within the sequence. An internal region of SGP-1 shows 78% sequence identity with the 67 N-terminal amino acids described for human sulfatide/GM1 activator (SAP-1). Sequence comparisons suggest that SGP-1 is the precursor to sulfatide/GM1 activator; however, the secretion of the protein from Sertoli cells is distinct from the proteolytic processing and lysosomal compartmentalization which have been described for human fibroblasts. The presence of internal sequence similarity suggests that three additional binding sites may occur in SGP-1. Northern blots show similar levels of expression for the 2.6-kilobase SGP-1 mRNA in all tissues examined. The site of SGP-1 synthesis in testis was localized to Sertoli cells by immunofluorescence and in situ hybridization.