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Expression of the Three Alternative Forms of the Sphingolipid Activator Protein Precursor in Baby Hamster Kidney Cells and Functional Assays in a Cell Culture System

1996; Elsevier BV; Volume: 271; Issue: 14 Linguagem: Inglês

10.1074/jbc.271.14.8416

1083-351X

Margarete Henseler, Andreas Klein, Gereon J. Glombitza, Kunihiko Suziki, Konrad Sandhoff,

Phagocytosis and Immune Regulation

Sphingolipid activator proteins (SAPs) are non-enzymatic glycoproteins required for lysosomal degradation of various sphingolipids with short oligosaccharide chains by their respective exohydrolases. Four of these (SAP-A to SAP-D or saposins A to D) are derived from a common precursor by proteolytic processing. Alternative splicing of the SAP-precursor gene results in insertion of additional 6 or 9 bases of exon 8′ or 8, respectively, into the SAP-B coding region of the transcribed mRNAs.To examine the features of the three different SAP-precursor proteins (prosaposins), the respective cDNAs were stably expressed in baby hamster kidney cells. Pulse-chase experiments with transfected cells and endocytosis studies on human fibroblasts showed that synthesis, transport, and maturation of all SAP-precursor led to formation of the four mature SAPs (SAP-A to SAP-D).In order to determine the biological function of the three different SAP-B isoforms, SAP-precursor-deficient human fibroblasts were loaded with recombinant SAP-precursor proteins with or without 2- and 3-amino acid insertions, respectively, purified from the medium of the baby hamster kidney cells. They were found to stimulate at nanomolar concentrations the turnover of biosynthetically labeled ceramide, glucosylceramide, and lactosylceramide. Since the physiological function of SAP-B is to stimulate the degradation of sulfatide by arylsulfatase A (EC 3.1.6.1) and globotriaosylceramide by β-galactosidase (EC 3.2.1.23) loading studies with the respective exogenously labeled lipids on SAP-precursor-deficient fibroblasts were performed. Addition of different purified SAP-precursors to the medium of the lipid-loaded fibroblasts showed positive stimulation of the lipid degradation by all three SAP-B isoforms derived from the SAP-precursors. These findings establish that all three forms of the SAP-B can function as sulfatide/globotriaosylceramide activator. Sphingolipid activator proteins (SAPs) are non-enzymatic glycoproteins required for lysosomal degradation of various sphingolipids with short oligosaccharide chains by their respective exohydrolases. Four of these (SAP-A to SAP-D or saposins A to D) are derived from a common precursor by proteolytic processing. Alternative splicing of the SAP-precursor gene results in insertion of additional 6 or 9 bases of exon 8′ or 8, respectively, into the SAP-B coding region of the transcribed mRNAs. To examine the features of the three different SAP-precursor proteins (prosaposins), the respective cDNAs were stably expressed in baby hamster kidney cells. Pulse-chase experiments with transfected cells and endocytosis studies on human fibroblasts showed that synthesis, transport, and maturation of all SAP-precursor led to formation of the four mature SAPs (SAP-A to SAP-D). In order to determine the biological function of the three different SAP-B isoforms, SAP-precursor-deficient human fibroblasts were loaded with recombinant SAP-precursor proteins with or without 2- and 3-amino acid insertions, respectively, purified from the medium of the baby hamster kidney cells. They were found to stimulate at nanomolar concentrations the turnover of biosynthetically labeled ceramide, glucosylceramide, and lactosylceramide. Since the physiological function of SAP-B is to stimulate the degradation of sulfatide by arylsulfatase A (EC 3.1.6.1) and globotriaosylceramide by β-galactosidase (EC 3.2.1.23) loading studies with the respective exogenously labeled lipids on SAP-precursor-deficient fibroblasts were performed. Addition of different purified SAP-precursors to the medium of the lipid-loaded fibroblasts showed positive stimulation of the lipid degradation by all three SAP-B isoforms derived from the SAP-precursors. These findings establish that all three forms of the SAP-B can function as sulfatide/globotriaosylceramide activator. INTRODUCTIONThe physiological degradation of glycosphingolipids with short oligosaccharide chains is catalyzed by lysosomal exohydrolases in the presence of small, heat-stable glycoproteins, so called sphingolipid activator proteins (SAPs) ( 1The abbreviations used are: SAPsphingolipid activator proteinpBHE0pBHE6, and pBHE9, expression plasmid pBHE containing the human prosaposin cDNA without additional base pairs, with 6 base pairs, or 9 base pairs, respectivelyBHK cellsbaby hamster kidney cellsSAP-precursor0SAP-precursor6, and SAP-precursor9, proteins (SAP-precursor, 46- and 30 kDa-protein forms) purified from the medium of transfected BHK cellsMEMminimal essential mediumFCSfetal calf serumEndo Hendo-β-N-acetylglucosaminidase HCerceramide, N-acylsphingosineGlcCerglucosylceramide, Glcβ1-1CergalactosylceramideGalβ1-1CerLacCer, lactosylceramideGalβ1-4Glcβ1-1CerdigalactosylceramideGalα1-4Galβ1-1CersulfatideGal(3-sulfate)β1-1CerELISA, enzyme-linked immunosorbent assay. The abbreviated designations of the gangliosides are according to Svennerholm's nomenclature for ganglio series gangliosides (SvennerholmL.(1963) J. Neurochem.10, 613-623). To date there are different nomenclatures used throughout the literature for the sphingolipid activator proteins and their precursor protein: sphingolipid activator protein precursor = prosaposin or SAP-precursorsphingolipid activator proteins A-D: saposin A = SAP-A, saposin B = SAP-B, saposin C = SAP-C, and saposin D = SAP-D. In this article we use the designation SAP-precursor, SAP-A, SAP-B, SAP-Cand SAP-D for the corresponding proteins.) or saposins(1.Sandhoff K. Harzer K. F ü rst W. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The metabolic and Molecular Basis of Inherited Disease. 7th Ed. McGraw-Hill, New York1995: 2427-2441Google Scholar). Four of the five known activator proteins (SAP-A to SAP-D or saposins A-D) are derived from a common precursor polypeptide by proteolytic processing(2.Fürst W. Machleidt W. Sandhoff K. Biol. Chem. Hoppe-Seyler. 1988; 369: 317-328Google Scholar, 3.O'Brien J.S. Kretz K.A. Dewji N. Wenger D.A. Esch F. Fluharty A.L. Science. 1988; 241: 1098-1101Google Scholar). This precursor also called prosaposin was identified as a protein of 68 kDa in human skin fibroblasts by studying the synthesis and processing of SAP-C(4.Fujibayashi S. Wenger D.A. J. Biol. Chem. 1986; 261: 15339-15343Google Scholar). After modification of its N-linked oligosaccharides in the Golgi apparatus, a 73-kDa form is secreted into the culture medium. Processing to the mature polypeptides of 8-13 kDa takes place after the transport of the precursor to the acidic organelles of the cells.Although different activating properties for the SAPs (SAP-A to SAP-D) have been described, their physiological relevance has been established only for SAP-B and SAP-C(1.Sandhoff K. Harzer K. F ü rst W. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The metabolic and Molecular Basis of Inherited Disease. 7th Ed. McGraw-Hill, New York1995: 2427-2441Google Scholar). The absence of SAP-C in human patients with Gaucher disease shows the relevance of this activator protein for the degradation of glucosylceramide by glucosylceramidase (EC 3.2.1.45) (1.Sandhoff K. Harzer K. F ü rst W. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The metabolic and Molecular Basis of Inherited Disease. 7th Ed. McGraw-Hill, New York1995: 2427-2441Google Scholar, 5.Fürst W. Sandhoff K. Biochim. Biophys. Acta. 1992; 1126: 1-16Google Scholar). While SAP-A is known to stimulate the breakdown of several glycosphingolipids in vitro(6.Tayama M. Soeda S. Kishimoto Y. Martin B.M. Callahan J.W. Hiraiwa M. O'Brien J.S. Biochem. J. 1993; 290: 401-404Google Scholar, 7.Morimoto S. Martin B.M. Yamamoto Y. Kretz K.A. O'Brien J.S. Kishimoto Y. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 3389-3393Google Scholar), SAP-D has recently been shown to be involved in ceramide degradation in vivo(8.Klein A. Henseler M. Klein C. Suzuki K. Harzer K. Sandhoff K. Biochem. Biophys. Res. Commun. 1994; 200: 1440-1448Google Scholar). This was established in an in vivo test system, in which the breakdown of [14C]serine-labeled sphingolipids accumulating in human fibroblasts derived from patients with SAP-precursor deficiency (9.Schnabel D. Schröder M. Fürst W. Klein A. Hurwitz R. Zenk T. Weber J. Harzer K. Paton B.C. Poulos A. Suzuki K. Sandhoff K. J. Biol. Chem. 1992; 267: 3312-3315Google Scholar) was stimulated by addition of different SAPs (SAP-A to SAP-D) to the culture medium.SAP-B itself has capacity to bind several sphingolipids, but its genetic defect leads to an accumulation of sulfatides, globotriaosylceramide, digalactosylceramide, and ganglioside GM3 in a disorder classified as metachromatic leukodystrophy(1.Sandhoff K. Harzer K. F ü rst W. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The metabolic and Molecular Basis of Inherited Disease. 7th Ed. McGraw-Hill, New York1995: 2427-2441Google Scholar, 10.Fluharty A.L. Trends Genet. 1995; 7: 167-189Google Scholar).The SAP-precursor gene localized on the long arm of human chromosome 10 (10q21-22) (11.Kao F.-T. Law M.L. Hartz J. Jones C. Zhang X.-L. Dewji N. O'Brien J.S. Wenger D.A. Somatic Cell Mol. Genet. 1987; 13: 685-688Google Scholar) consists of at least 15 exons, four of which (exon 6-9) code for SAP-B. Exon 8 consists of only 9 base pairs (12.Holtschmidt H. Sandhoff K. Fürst W. Kwon H.Y. Schnabel D. Suzuki K. FEBS Lett. 1991; 280: 267-270Google Scholar) and can be spliced differentially, generating three different cDNAs(13.Nakano T. Sandhoff K. Stümper J. Christomanou H. Suzuki K. J. Biochem. (Tokyo). 1989; 105: 152-154Google Scholar, 14.Holtschmidt H. Sandhoff K. Kwon H.Y. Harzer K. Nakano T. Suzuki K. J. Biol. Chem. 1991; 266: 7556-7560Google Scholar): one containing all 9 bases of exon 8, one containing the downstream 6 bases of exon 8 (also called exon 8′) and one completely lacking this exon (Fig. 1). At the protein level, only SAP-B without the amino acids derived from exon 8 has been detected so far(10.Fluharty A.L. Trends Genet. 1995; 7: 167-189Google Scholar, 15.Fürst W. Schubert J. Machleidt W. Meyer H.E. Sandhoff K. Eur. J. Biochem. 1990; 192: 709-714Google Scholar). A recent report shows a differential tissue distribution of the mRNAs with or without exon 8 in different cell lines or tissues(16.Lamontagne S. Portier M. J. Biol. Chem. 1994; 269: 20528-20532Google Scholar). The multiple mRNA species were also detected in the mouse(17.Tsuda H. Sakiyama T. Endo H. Kitagawa T. Biochem. Biophys. Res. Commun. 1992; 184: 1266-1272Google Scholar). These findings prompt the question whether the different sequences within the SAP-B domain in the SAP-precursor have any biological significance. In the present report we describe the expression and processing of the three SAP-precursor forms in BHK cells stably transfected with the respective cDNAs. In addition, the stimulation of sulfatide and globotriaosylceramide degradation by the three SAP-B isoforms was tested in vivo in a cell culture system by adding purified recombinant precursor proteins (designated as SAP-precursor 0, 6, or 9) to the medium of lipid loaded human SAP-precursor-deficient fibroblasts.EXPERIMENTAL PROCEDURESMaterialsRestriction and modifying enzymes were purchased from Boehringer Mannheim (Mannheim, Federal Republic of Germany), Life Technologies, Inc. (Eggenstein, Federal Republic of Germany), and New England Biolabs (Schwalbach, Federal Republic of Germany). Peptide-N-glycanase F and Endo H were from New England Biolabs (Schwalbach, Federal Republic of Germany). Radiolabeled chemicals were obtained from Amersham Corp. (Braunschweig, Federal Republic of Germany). Culture media for bacterial and eukaryotic cells were from Life Technologies, Inc., Biochrom (Berlin, Federal Republic of Germany), Sigma (Deisenhofen, Federal Republic of Germany), and ICN (Meckenheim, Federal Republic of Germany). Fetal calf serum was purchased from Cytogen (Berlin, Federal Republic of Germany). Puromycin was from Sigma (Deisenhofen, Federal Republic of Germany) and G418 sulfate from Life Technologies, Inc. Protein A-Sepharose was obtained from Sigma, and protein G-agarose was from Calbiochem-Novabiochem (Bad Soden, Federal Republic of Germany).All other commercially available materials were obtained from the following suppliers: Sigma, Merck (Darmstadt, Federal Republic of Germany), Pharmacia LKB Biotechnologies (Freiburg, Federal Republic of Germany), and New England Biolabs.[3H]Sulfatide, which was labeled in the sphingoid moiety, and [3H]globotriaosylceramide labeled in the terminal galactosyl residue were synthesized in our laboratory according to published procedures(18.Schwarzmann G. Biochim. Biophys. Acta. 1978; 529: 106-114Google Scholar, 19.Suzuki Y. Suzuki K. J. Lipid Res. 1972; 13: 680-686Google Scholar).AntiseraHuman SAP-A and SAP-C were purified from spleen of a Gaucher patient essentially as described earlier(8.Klein A. Henseler M. Klein C. Suzuki K. Harzer K. Sandhoff K. Biochem. Biophys. Res. Commun. 1994; 200: 1440-1448Google Scholar, 20.Sano A. Radin N.S. Johnson L.L. Tarr G.E. J. Biol. Chem. 1988; 263: 19597-19601Google Scholar). Polyclonal anti-SAP-A and anti-SAP-C antisera were raised in rabbits by injection of 100 μg of pure protein in combination with Freund's complete adjuvant followed by three booster injections of 100 μg protein each in Freund's incomplete adjuvant given in 4-week intervals. The animals were bled 10 days after the final injection. The antisera had ELISA titer of 1:950 and 1:900 (half-maximal A405 of 3-fold serum dilutions on a 50-ng antigen coat) against SAP-A and SAP-C, respectively. Specificity of the antisera was assessed by ELISA, immunoblot and immunoprecipitation, and in the case of the anti-SAP-C antiserum by comparison with the rabbit anti-SAP-C antiserum described earlier(21.Christomanou H. Aignesberger A. Linke R.P. Biol. Chem. Hoppe-Seyler. 1986; 367: 879-890Google Scholar).The preparation of the goat anti-SAP-B and anti-SAP-D antisera has been described earlier(8.Klein A. Henseler M. Klein C. Suzuki K. Harzer K. Sandhoff K. Biochem. Biophys. Res. Commun. 1994; 200: 1440-1448Google Scholar).Cells and Cell CultureHuman skin fibroblasts and baby hamster kidney cells (BHK 21 ATCC CCC 10) were maintained at 37°C in 5% CO2 in Dulbecco's modified Eagle's medium supplemented with antibiotics, 4.5 mM NaHCO3 and 10% fetal calf serum (FCS) as described previously(22.Weitz G. Lindl T. Hinrichs U. Sandhoff K. Hoppe-Seyler's Z. Physiol. Chem. 1983; 364: 862-871Google Scholar). Transfected BHK cells were routinely cultured in medium additionally supplemented with 5 μg/ml puromycin. The fibroblasts from a SAP-precursor deficient human fetus have been described earlier(9.Schnabel D. Schröder M. Fürst W. Klein A. Hurwitz R. Zenk T. Weber J. Harzer K. Paton B.C. Poulos A. Suzuki K. Sandhoff K. J. Biol. Chem. 1992; 267: 3312-3315Google Scholar, 23.Harzer K. Paton B.C. Poulos A. Kustermann-Kuhn B. Roggendorf W. Grisar T. Popp M. Eur. J. Pediatr. 1989; 149: 31-39Google Scholar, 24.Bradová V. Smid F. Ulrich-Bott B. Roggendorf W. Paton B.C. Harzer K. Hum. Genet. 1993; 92: 143-152Google Scholar).Expression of the SAP-precursor Proteins in BHK CellsThe expression plasmid pBHE9 was constructed by placing the entire SAP-precursor cDNA (from nucleotide −9 to 1597, numbered from A of the ATG initiation codon) which contained the 9 base pairs of exon 8 into the XbaI and SmaI site downstream of the SV40 early promoter in the expression vector pBHE(25.Artelt P. Morelle C. Ausmeier M. Fitzek M. Hauser H. Gene (Amst.). 1988; 68: 213-219Google Scholar). The expression plasmid pBHE0 containing the SAP-precursor cDNA without exon 8 and the expression plasmid pBHE6 containing the cDNA with the 6 bases of exon 8′ were constructed by replacing nucleotides 543 to 797 (using the Bsu36I and BglII restriction sites on the precursor cDNAs) in the expression plasmid pBHE9 by the respective cDNA segments of the other SAP-precursor cDNAs. These cDNA segments were derived from partial cDNAs obtained by reverse transcriptase-polymerase chain reaction of total RNA isolated from human fibroblasts(9.Schnabel D. Schröder M. Fürst W. Klein A. Hurwitz R. Zenk T. Weber J. Harzer K. Paton B.C. Poulos A. Suzuki K. Sandhoff K. J. Biol. Chem. 1992; 267: 3312-3315Google Scholar). The correct cDNA sequence of each expression plasmids pBHE0, pBHE6, and pBHE9 was confirmed by DNA sequencing (26.Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Google Scholar).Stably transfected BHK cells were obtained by cotransfection of the expression plasmids and of the plasmids pSV2 pac and pSV2 neo conferring resistance to puromycin and G418 sulfate to the cells(27.Vara J.A. Portela A. Ortin J. Jimenez A. Nuleic Acids Res. 1986; 14: 4617-4624Google Scholar), as described previously(28.Wirth M. Bode J. Zettlmeissel G. Hauser H. Gene (Amst.). 1988; 73: 419-426Google Scholar). BHK cells transfected only with the expression plasmid pBHE were used as control.Processing and Endocytosis StudiesConfluent cultures of transfected BHK cells in 25-cm2 tissue culture flasks were incubated for 1 h in 1 ml of methionine-free modified Eagle's medium (MEM) containing 4% dialyzed and heat-inactivated FCS. The cells were labeled by incubation in 0.7 ml of this medium supplemented with 1.85 MBq of L-[35S]methionine (specific radioactivity > 37 TBq/mmol) for 1 h. The chase periods were started by addition of unlabeled methionine (final concentration 10 mM). For endocytosis studies, 0.7 ml of medium from transfected BHK cells labeled for 5 h with 2.5 MBq of L-[35S]methionine was added to 1.3 ml of culture medium of human SAP-precursor-deficient fibroblasts and the media were supplemented with unlabeled methionine to a final concentration of 10 mM. The fibroblasts were maintained for 24 h in this medium.Cell extracts were prepared with phosphate-buffered saline containing 1% Nonidet P-40, 10 mM EDTA, 2 mM phenylmethanesulfonyl fluoride, 1 mM pepstatin A and leupeptin each. Immunoprecipitations using the corresponding anti-SAP-antisera were performed as described previously(9.Schnabel D. Schröder M. Fürst W. Klein A. Hurwitz R. Zenk T. Weber J. Harzer K. Paton B.C. Poulos A. Suzuki K. Sandhoff K. J. Biol. Chem. 1992; 267: 3312-3315Google Scholar). Deglycosylation of the precipitated proteins with peptide-N-glycanase F and Endo H was performed according to the manufacturer's instructions. Immunoprecipitated proteins were analyzed by SDS-polyacrylamide gel electrophoresis using gradient gels (7-17%). The gels were impregnated with Amplify for fluorography and exposed to the Kodak X-Omat AR film.Isolation and Purification of Recombinant SAP-precursor ProteinsThe three different SAP-precursor differing in the additional amino acids encoded by exon 8 or 8′ were purified from the medium (Dif 1000-medium, 0.1% FCS) of the BHK cells transfected with the corresponding cDNA according to published procedures(29.Hiraiwa M. O'Brien J.S. Kishimoto Y. Galdzicka M. Fluharty A.L. Ginns E.I. Martin B.M Arch. Biochem. Biophys. 1993; 304: 110-116Google Scholar). The purification of the proteins was monitored by immunoblotting and ELISA.Metabolic Labeling of Sphingolipids by [14C]SerineHuman fibroblasts were preincubated for 24 h with MEM containing 0.6% FCS and SAP-precursor 0, 6, or 9 as indicated. Subsequently the medium was exchanged against medium supplemented with 0.3% FCS and [14C]serine (1 μCi/ml medium, 54 Ci/mol). After 24 h the medium was removed and replaced by a medium containing L-serine (185 nmol/ml medium), 0.6% FCS, and the same concentrations of the SAP-precursor 0, 6, or 9 as during the preincubation period. After 120 h of chase the cells were harvested by trypsinization.Sulfatide- and Globotriaosylceramide Loading StudiesSulfatide and globotriaosylceramide loading studies were performed by modifications of published procedures(30.Kudoh T. Wenger D.A. J. Clin. Invest. 1982; 70: 89-97Google Scholar, 31.Paton B.C. Schmid B. Kustermann-Kuhn B. Poulos A. Harzer K. Biochem. J. 1992; 285: 481-488Google Scholar). In brief, the fibroblasts were preincubated for 24 h with MEM containing 10% FCS and the purified SAP-precursor 0, 6, or 9 (0.5 mg/ml medium) as indicated. Then the medium was exchanged with MEM supplemented with 10% FCS and [3H]sulfatide (3.3 nmol/ml medium, 86 Ci/mol) or [3H]globotriaosylceramide (1.35 nmol/ml medium, 180 Ci/mol). After 48 h of loading, the cells were incubated for additional 120 h in MEM containing the same supplements as during the preincubation period. Subsequently the cells were harvested by trypsinization.Isolation of Cellular SphingolipidsCellular sphingolipids were isolated and identified as described previously(8.Klein A. Henseler M. Klein C. Suzuki K. Harzer K. Sandhoff K. Biochem. Biophys. Res. Commun. 1994; 200: 1440-1448Google Scholar). In brief, the lipids were extracted from the cell pellets with 7 ml of chloroform/methanol/water/pyridine (60:160:6:1 by volume) for 24 h at 50°C. Phospholipids were degraded by mild alkaline hydrolysis with methanolic NaOH (50 mM) for 2 h at 37°C. Then, the lipid extracts were desalted by reversed-phase chromatography(32.Kyrklund T. Lipids. 1987; 22: 274-277Google Scholar). For the [14C]serine-labeled lipids, samples were applied to TLC plates on the bases of equal amounts of radioactivity, while for the loading studies, samples were chromatographed on the basis of amounts of protein (estimated after homogenization of the cells). In both instances, the solvent system was chloroform/methanol/0.22% aqueous CaCl2 (60:35:8 by volume). All glycosphingolipids were identified from their RF values. Radioactive spots were quantified using a Fuji BAS 1000 phosphorimaging system and the TINA program (version 2.07). They were visualized by autoradiography.Other MethodsProteins were assayed with bovine serum albumin as standard using the bicinchoninic acid assay(33.Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Gocke N.M. Olson B.J. Klenk C.D. Anal. Biochem. 1985; 150: 76-85Google Scholar).Presentation of DataRadioactivity of individual sphingolipids is expressed as percentage of total radioactivity of the sphingolipid fraction. Total radioactivity is normalized to the same amount of cellular protein. All data are given as means of at least two separate experiments.RESULTSExpression and Maturation of the Three Different SAP-precursor Forms in BHK CellsIn order to follow synthesis and transport of the different SAP-precursor isoforms and their maturation in cell culture, stably transfected BHK cells were prepared. Metabolic labeling of transfected BHK cells with [35S]methionine and immunoprecipitation of the expressed proteins from the cell extracts with an anti-SAP-C antiserum revealed that after 1 h of pulse labeling a SAP-precursor of apparent 68 kDa was generated by all three cDNA forms (Fig. 2). Additionally processed forms of about 46 and 30 kDa were detectable. Within 2 h of chase there was significant biosynthesis of radiolabeled mature SAP-C with an apparent mass of 13 kDa. This protein was further processed to 11- to 8-kDa forms during an additional chase of 18 h. The major immunoprecipitable protein, secreted into the culture media, was the SAP-precursor with an apparent molecular mass of 73 kDa, which was already detectable after 1 h of pulse. In addition, processed proteins of molecular masses of 46 and 30 kDa were precipitable from the media, presumably identical with the tri- and disaposin forms described earlier in expression of a SAP-precursor in insects cells(27.Vara J.A. Portela A. Ortin J. Jimenez A. Nuleic Acids Res. 1986; 14: 4617-4624Google Scholar). During the pulse and chase periods more than 60% of the immunoprecipitable proteins were secreted into the medium of the transfected cells. Secretion of the 73-kDa forms started 30 min after a short pulse labeling of 5 min (data not shown). No cross-reacting material was precipitated from extracts of control BHK cells (Fig. 2, Co).Figure 2:Biosynthesis of the SAP-precursor isoforms in transfected BHK cells. BHK cells transfected either with the expression plasmids pBHE0, pBHE6, pBHE9, or only the vector pBHE without a cDNA insertion as a control (Co) were pulsed with [35S]methionine for 1 h and chased for the times indicated. SAP-precursors and SAP-C from the cell extracts and the media were immunoprecipitated using a rabbit anti-SAP-C antiserum and were analyzed by denaturing gel electrophoresis and fluoropgraphy. A faint band at 46 kDa, detectable only in the immunoprecipitations of the cell extracts and denoted by an asterisk, is caused by unspecific contaminations of immunoprecipitates.View Large Image Figure ViewerDownload (PPT)This labeling experiment indicated formation of similar polypeptides in BHK cells transfected with the different SAP-precursor cDNAs as described for in human fibroblasts(4.Fujibayashi S. Wenger D.A. J. Biol. Chem. 1986; 261: 15339-15343Google Scholar). The different amounts of immunoprecipitable proteins in the transfected BHK cells were due to the fact that the totality of BHK cells resistant to the antibiotics were used for pulse-chase experiments and that the selection leaves varying numbers of non-resistant cells.In order to assess correct processing of all three different recombinant SAP-precursor to the four mature SAPs (SAP-A to SAP-D) and to analyze their carbohydrate structures, the BHK transfectants were metabolically labeled for 5 h, and the cell extracts were subjected to immunoprecipitation with polyclonal antisera against the corresponding mature SAP-A to SAP-D (Fig. 3). All three SAP-precursors resulted in formation of mature SAP-A, SAP-B, SAP-C, and SAP-D, differing with respect to their carbohydrate types as analyzed previously(34.Yamashita K. Inui K. Totani K. Kochibe N. Furukawa M. Okada S. Biochemistry. 1990; 29: 3030-3039Google Scholar, 35.Ito K. Takahashi N. Takahashi A. Shimada I. Arata Y. O'Brien J.S. Kishimoto Y. Eur. J. Biochem. 1993; 215: 171-179Google Scholar). In our hands, mature SAP-A never yielded a clear band in its glycosylated form, but only a broad patch ranging from 14.5 to 29 kDa. Upon deglycosylation, however, a clear band comparible in size with deglycosylated mature SAP-B, SAP-C, and SAP-D was obtained. The carbohydrate chains of SAP-A were Endo H-resistant, presumably being of the complex type, whereas the carbohydrate moieties of SAP-C and SAP-D were predominantly of the Endo H-sensitive high mannose or hybrid type. It is noteworthy that each SAP-precursor generated a mature SAP-B with an Endo H-resistant carbohydrate chain.Figure 3:Immunoprecipitation and N-glycosylation analysis of all mature SAPs (SAP-A to SAP-D) from transfected BHK cells. The BHK cells transfected with the expression plasmids pBHE0, pBHE6, and pBHE9 were labeled with [35S]methionine for 5 h. The mature SAPs (SAP-A to SAP-D) were immunoprecipitated with the corresponding antisera as indicated and treated either with peptide-N-glycanase F (PNGase F) or Endo H as described under #x0201C;Experimental Procedures."View Large Image Figure ViewerDownload (PPT)Endocycosis of the SAP-precursor IsoformsTo confirm that human cells process the three SAP-precursors in the same manner as the BHK transfectants, BHK cells were labeled with [35S]methionine, and the medium containing the secreted proteins was added to human fibroblasts derived from a SAP-precursor-deficient fetus(9.Schnabel D. Schröder M. Fürst W. Klein A. Hurwitz R. Zenk T. Weber J. Harzer K. Paton B.C. Poulos A. Suzuki K. Sandhoff K. J. Biol. Chem. 1992; 267: 3312-3315Google Scholar). After a 24-h incubation the media and cell extracts were analyzed for immunoprecipitable SAP-B, SAP-C, and SAP-D with the corresponding antisera (Fig. 4). Some SAP-precursor was still detectable in the medium. About 50% of each of the different radiolabeled SAP-precursor forms had been internalized by the SAP-precursor deficient fibroblasts and processed to the mature SAP-A to SAP-D. Repeating the endocytosis experiment with normal human fibroblasts showed an uptake of about 30% of each prosaposin form and the same correct processing to the mature proteins (data not shown). Thus, no difference with regard to maturation was observed for any of the three SAP-precursors in human fibroblasts in comparison with BHK cells.Figure 4:Endocytosis of SAP-precursor isoforms by cultured human fibroblasts from a patient with SAP-precursor deficiency. Medium from [35S]methionine-labeled BHK cells transfected with the SAP-precursor cDNA isoforms (pBHE0, pBHE6, and pBHE9) or with the expression plasmid pBHE (Co) were added to the culture medium of unlabeled fibroblasts derived from a patient with SAP-precursor deficiency. After 24 h the fibroblasts were harvested and analyzed for internalized immunoprecipitable SAP-B, SAP-C, and SAP-D. Immunoprecipitations were carried out from the media with an anti-SAP-C antiserum and from the cell extracts with the antisera indicated. Asterisk, the faint bands visible in the control precipitation with the anti-SAP-D antiserum presumable arose from endogenous BHK cell SAP-precursor cross-reacting with this antiserum.View Large Image Figure ViewerDownload (PPT)Purification of the SAP-precursor IsoformsThe three different isoforms of the recombinant SAP-precursors were purified from the medium of transfected BHK cells by a two-step procedure as described for the purification of SAP-precursor expressed in insect cells(29.Hiraiwa M. O'Brien J.S. Kishimoto Y. Galdzicka M. Fluharty A.L. Ginns E.I. Martin B.M Arch. Biochem. Biophys. 1993; 304: 110-116Google Scholar). Affinity chromatography on a concanavalin A-Sepharose was performe