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

Dewji, Nazneen N.; Wenger, David A.; S, Fujibayashi; Donoviel, Michael S.; Esch, Frederick; Hill, Fred; O’Brien, John S.

doi:10.1016/s0006-291x(86)80518-6

Cited by 34 publications

(14 citation statements)

References 12 publications

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“…This activator protein may have even broader substrate specificity (9). The primary structure of saposin B was determined by sequencing a cDNA encoding it (10), and its physiological significance is underscored by the discovery of its absence in a variant form of metachromatic leukodystrophy (activator-deficient metachromatic leukodystrophy) (11). A second activator protein, called saposin C in this report and also previously designated by several different terms (12)(13)(14)(15), stimulates the hydrolysis of glucosylceramide by j-glucosylceramidase (EC 3.2.1.45) (12)(13)(14)(15) and that of galactosylceramide byB-galactosylceramidase (EC 3.2.1.46) (16).…”

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confidence: 99%

“…Antibodies against saposin A were prepared in a rabbit as described by Crowle (24) and purified with an affinity column that contained purified saposin A linked to an Affi-Gel 10-activated support as described by the manufacturer (Bio-Rad); anti-saposin A was eluted with 2 M MgCl2. Dot blots and immunoblots with the antibodies obtained in this manner were performed as described (10).…”

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confidence: 99%

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Saposin A: second cerebrosidase activator protein.

Morimoto

Martin

Yamamoto

et al. 1989

Proc. Natl. Acad. Sci. U.S.A.

130

112

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Saposin A, a heat-stable 16-kDa glycoprotein, was isolated from Gaucher disease spleen and purified to homogeneity. Chemical sequencing from its amino terminus and of peptides obtained by digestion with protease from Staphylococcus aureus strain V-8 demonstrated that saposin A is derived from proteolytic processing of domain 1 of its precursor protein, prosaposin. Processing of prosaposin (70 kDa) also generates three other previously reported saposin proteins, B, C, and D, from its second, third, and fourth domains. Similar to saposin C, saposin A stimulates the hydrolysis of 4-methylumbelliferyl ,B-glucoside and glucocerebroside by ,B-glucosylceramidase and of galactocerebroside by ,B-galactosylceramidase, mainly by increasing the maximal velocity of both reactions. Saposin A is as active as saposin C in these reactions. Saposin A has no significant effect on other sphingolipid and 4-methylumbelliferyl glycoside hydrolases tested. Saposin A has two potential glycosylation sites that appear to be glycosylated. After deglycosylation, saposin A had a subunit molecular mass of 10 kDa and was as active as native saposin A. However, reduction and alkylation abolished the activation. A three-dimensional model comparing saposins A and C reveals significant sequence homology between them, especially preservation of conserved acidic and basic residues in their middle regions. Each appears to possess a conformationally rigid hydrophobic pocket stabilized by three internal disulffide bridges, with amphipathic helical regions interrupted by helix breakers. (16). Unlike saposin B, saposin C appears to interact directly with both enzymes to stimulate activity (17). The primary structure of saposin C was determined by chemical sequencing of its amino acids (18,19) and by deducing its sequence from nucleotide sequencing of a cDNA encoding prosaposin, its precursor (20). Saposin C has been reported to be deficient in a single patient with a variant form of Gaucher disease (21).Recently, the complete nucleotide sequence of a cDNA encoding prosaposin, the precursor of saposins B and C, was elucidated (20). Prosaposin is a 511-amino acid glycoprotein, and examination of its amino acid sequence reveals four saposin-like domains, two of which are saposins B and C; these are flanked by two additional domains, which we call saposin A and saposin D. Each domain is approximately 80-amino acid residues long; has nearly identical placement of cysteine residues, glycosylation sites, and helical regions; and is flanked by proteolytic cleavage sites. Molecular models indicate that the proteins derived from each domain can fold to give rise to a conformationally rigid hydrophobic pocket held together by three disulfide bridges. Proteolytic cleavage of prosaposin at each domain boundary was predicted to give rise to four saposin proteins (20). We recently have isolated saposin D, the protein arising from domain four of prosaposin and demonstrated it to be a specific sphingomyelin phosphodiesterase (EC 3.1.4.12) activator (22). In this...

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confidence: 99%

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confidence: 99%

Saposin A: second cerebrosidase activator protein.

Morimoto

Martin

Yamamoto

et al. 1989

Proc. Natl. Acad. Sci. U.S.A.

130

112

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“…Methods for screening cDNA library with antibody, isolation of clones, and sequencing of pure SAP-1 protein have been described in detail in our previous publications (6,7). DNA Sequence Analysis.…”

Section: Methodsmentioning

confidence: 99%

“…Recently, we reported the cloning and nucleotide sequence of a cDNA encoding human SAP-1 mature protein (6). This cDNA was isolated from a Xgtll human hepatoma expression library (7) by using polyclonal antibodies raised against human SAP-1.…”

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confidence: 99%

Nucleotide sequence of cloned cDNA for human sphingolipid activator protein 1 precursor.

Dewji

Wenger

O’Brien

1987

Proc. Natl. Acad. Sci. U.S.A.

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Two cDNA clones encoding prepro-sphingolipid activator protein 1 (SAP-1) were isolated from a Xgtli human hepatoma expression library using polyclonal antibodies. These had inserts of =2 kilobases (X-S-1.2 and X-S-1.3) and both were both homologous with a previously isolated clone (X-S-1.1) for mature SAP-1. We report here the nucleotide sequence of the longer two EcoRI fragments of S-1.2 and S-1.3 that were not the same and the derived amino acid sequences of mature SAP-1 and its prepro form. The open reading frame encodes 19 amino acids, which are colinear with the aminoterminal sequence of mature SAP-1, and extends far beyond the predicted carboxyl terminus of mature SAP-1, indicating extensive carboxyl-terminal processing. The nucleotide sequence of cDNA encoding prepro-SAP-1 includes 1449 bases from the assigned initiation codon ATG at base-pair 472 to the stop codon TGA at base-pair 1921. The first 23 amino acids coded after the initiation ATG are characteristic of a signal peptide. The calculated molecular mass for a polypeptide encoded by 1449 bases is =53 kDa, in keeping with the reported value for pro-SAP-1. The data indicate that after removal ofthe signal peptide (23 amino acids) mature SAP-1 (78 amino acids) is generated by removing an additional 7 amino acids from the amino terminus and =373 amino acids from the carboxyl terminus. One potential glycosylation site was previously found in mature SAP-1. Three additional potential glycosylation sites are present in the processed carboxyl-terminal polypeptide, which we designate as P-2. The molecular mass of glycosylated pro-SAP-1 is estimated at -69 kDa, assuming glycosylation of all four sites. The value is close to the reported 70-kDa value for glycosylated pro-SAP-1. A computer search failed to reveal homology between P-2 and the sequence of any other protein; its function is uncertain. The 3' untranslated region is composed of 90 base pairs and is incomplete, since it does not contain a polyadenylylation site or a poly(A) tail.The sphingolipid activator protein 1 (SAP-1), also known as the sulfatide sulfatase activator (1), promotes the hydrolysis of sulfatide by arylsulfatase A. Genetic deficiency of this lysosomal glycoprotein in humans results in the storage of sulfatide and the clinical picture of metachromatic leukodystrophy (2-4). The disorder appears to be transmitted as an autosomal recessive trait.Human SAP-1 is known to undergo extensive processing during biosynthesis. It has been shown that the subunit molecular mass of pro SAP-i is '70 kDa, which after endoglycosidase F treatment gives a polypeptide with a molecular mass of 53 kDa; this precursor is then processed to mature SAP-1 with a subunit molecular mass of 8-11 kDa (5).Recently, we reported the cloning and nucleotide sequence of a cDNA encoding human SAP-1 mature protein (6). This cDNA was isolated from a Xgtll human hepatoma expression library (7) by using polyclonal antibodies raised against human SAP-1. This clone had an insert of -0.3 kilobase (kb) and was designated X...

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“…The first activator protein discovered by Mehl and Jatzkewitz in 1964 [3] is the sulfatide activator, also called SAP-1 [4] . The physiological significance of this activator was shown by the occurrence of a variant form of the sulfatide storage disease (metachromatic leukodystrophy) due to its deficiency 15 ' 61 . A second well documented activator is the GM 2 ganglioside activator protein i?…”

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confidence: 99%

Complete Amino-Acid Sequence of the Naturally Occurring A₂Activator Protein for Enzymic Sphingomyelin Degradation: Identity to the Sulfatide Activator Protein (SAP-1)

Kleinschmidt¹,

Christomanou²,

Braunitzer³

1988

Biological Chemistry Hoppe-Seyler

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The naturally occurring A 2 activator asparagine in position 21, as well as 2 mol of protein for enzymic sphingolipid degradation is characterized by complete amino-acid sequence and carbohydrate content. It consists of 79 amino-acid residues and has a molecular mass of 8.875 kDa. The polypeptide chain contains 2 mol of 7V-acetylglucosamine, bound to galactose and mannose per mol protein.The primary structure of the A 2 activator protein is identical to that of the sulfatide activator protein (SAP-1). Possible differences in the carbohydrate content are discussed.Vollständige Aminosäuresequenz des natürlich vorkommenden A2-Aktivatorproteins für den enzymatischen Sphingomyelin-Abb au: Identität mit dem Sulfatid-Aktivatorprotein

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

Cited by 34 publications

References 12 publications

Saposin A: second cerebrosidase activator protein.

Saposin A: second cerebrosidase activator protein.

Nucleotide sequence of cloned cDNA for human sphingolipid activator protein 1 precursor.

Complete Amino-Acid Sequence of the Naturally Occurring A₂Activator Protein for Enzymic Sphingomyelin Degradation: Identity to the Sulfatide Activator Protein (SAP-1)

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

Cited by 34 publications

References 12 publications

Saposin A: second cerebrosidase activator protein.

Saposin A: second cerebrosidase activator protein.

Nucleotide sequence of cloned cDNA for human sphingolipid activator protein 1 precursor.

Complete Amino-Acid Sequence of the Naturally Occurring A2Activator Protein for Enzymic Sphingomyelin Degradation: Identity to the Sulfatide Activator Protein (SAP-1)

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Complete Amino-Acid Sequence of the Naturally Occurring A₂Activator Protein for Enzymic Sphingomyelin Degradation: Identity to the Sulfatide Activator Protein (SAP-1)