Gaucher disease mouse models: point mutations at the acid β-glucosidase locus combined with low-level prosaposin expression lead to disease variants
2005; Elsevier BV; Volume: 46; Issue: 10 Linguagem: Inglês
10.1194/jlr.m500202-jlr200
ISSN1539-7262
AutoresYing Sun, Brian Quinn, David P. Witte, Gregory A. Grabowski,
Tópico(s)Glycosylation and Glycoproteins Research
ResumoGaucher disease is a common lysosomal storage disease caused by a defect of acid β-glucosidase (GCase). The optimal in vitro hydrolase activity of GCase requires saposin C, an activator protein that derives from a precursor, prosaposin. To develop additional models of Gaucher disease and to test in vivo effects of saposin deficiencies, mice expressing low levels (4–45% of wild type) of prosaposin and saposins (PS-NA) were backcrossed into mice with specific point mutations (V394L/V394L or D409H/D409H) of GCase. The resultant mice were designated 4L/PS-NA and 9H/PS-NA, respectively. In contrast to PS-NA mice, the 4L/PS-NA and 9H/PS-NA mice displayed large numbers of engorged macrophages and nearly exclusive glucosylceramide (GC) accumulation in the liver, lung, spleen, thymus, and brain. Electron microscopy of the storage cells showed the characteristic tubular storage material of Gaucher cells. Compared with V394L/V394L mice, 4L/PS-NA mice that expressed 4–6% of wild-type prosaposin levels had ∼25–75% decreases in GCase activity and protein in liver, spleen, and fibroblasts.These results imply that reduced saposin levels increased the instability of V394L or D409H GCases and that these additional decreases led to large accumulations of GC in all tissues. These models mimic a more severe Gaucher disease phenotype and could be useful for therapeutic intervention studies. Gaucher disease is a common lysosomal storage disease caused by a defect of acid β-glucosidase (GCase). The optimal in vitro hydrolase activity of GCase requires saposin C, an activator protein that derives from a precursor, prosaposin. To develop additional models of Gaucher disease and to test in vivo effects of saposin deficiencies, mice expressing low levels (4–45% of wild type) of prosaposin and saposins (PS-NA) were backcrossed into mice with specific point mutations (V394L/V394L or D409H/D409H) of GCase. The resultant mice were designated 4L/PS-NA and 9H/PS-NA, respectively. In contrast to PS-NA mice, the 4L/PS-NA and 9H/PS-NA mice displayed large numbers of engorged macrophages and nearly exclusive glucosylceramide (GC) accumulation in the liver, lung, spleen, thymus, and brain. Electron microscopy of the storage cells showed the characteristic tubular storage material of Gaucher cells. Compared with V394L/V394L mice, 4L/PS-NA mice that expressed 4–6% of wild-type prosaposin levels had ∼25–75% decreases in GCase activity and protein in liver, spleen, and fibroblasts. These results imply that reduced saposin levels increased the instability of V394L or D409H GCases and that these additional decreases led to large accumulations of GC in all tissues. These models mimic a more severe Gaucher disease phenotype and could be useful for therapeutic intervention studies. Gaucher disease is an autosomal recessive trait and the most common lysosomal storage disorder (1Beutler E. Grabowski G.A. Gaucher disease.in: Scriver R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Basis of Inherited Disease, C. McGraw-Hill, New York2001: 3635-3668Google Scholar). The defective lysosomal hydrolysis of glucosylceramide (GC) in Gaucher disease is caused by mutations in the gene [human (GBA), mouse (gba)] encoding acid β-glucosidase (GCase), a membrane-associated lysosomal hydrolase (1Beutler E. Grabowski G.A. Gaucher disease.in: Scriver R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Basis of Inherited Disease, C. McGraw-Hill, New York2001: 3635-3668Google Scholar). More than 200 mutations at the GBA locus have been identified in Gaucher disease patients, and the resultant defective or deficient enzyme activities lead to variable phenotypes (1Beutler E. Grabowski G.A. Gaucher disease.in: Scriver R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Basis of Inherited Disease, C. McGraw-Hill, New York2001: 3635-3668Google Scholar, 2Zhao H. Grabowski G.A. Gaucher disease: perspectives on a prototype lysosomal disease.Cell. Mol. Life Sci. 2002; 59: 694-707Google Scholar). The accumulation of GC leads to enlargement of the liver and spleen, bone lesions, and central nervous system (CNS) manifestations in some variants (1Beutler E. Grabowski G.A. Gaucher disease.in: Scriver R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Basis of Inherited Disease, C. McGraw-Hill, New York2001: 3635-3668Google Scholar, 3Kolodny E.H. Ullman M.D. Mankin H.J. Raghavan S.S. Topol J. Sullivan J.L. Phenotypic manifestations of Gaucher disease: clinical features in 48 biochemically verified type 1 patients and comment on type 2 patients.Prog. Clin. Biol. Res. 1982; 95: 33-65Google Scholar, 4Adachi M. Wallace B.J. Schneck L. Volk B.W. Fine structure of central nervous system in early infantile Gaucher's disease.Arch. Pathol. 1967; 83: 513-526Google Scholar, 5Volk B.W. Wallace B.J. Adachi M. Infantile Gaucher's disease: electron microscopic and histochemical studies of a cerebral biopsy.J. Neuropathol. Exp. Neurol. 1967; 26: 176-177Google Scholar). The macrophage is the primary cell displaying GC accumulation; nonmacrophage parenchymal cells appear normal in liver, lung, bone marrow, and spleen in Gaucher disease (e.g., hepatocytes, granulocytes, and lymphocytes) (1Beutler E. Grabowski G.A. Gaucher disease.in: Scriver R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Basis of Inherited Disease, C. McGraw-Hill, New York2001: 3635-3668Google Scholar). The knock out of gba in the mouse leads to lethality in the newborn period (6Tybulewicz V.L.J. Tremblay M.L. LaMarca M.E. Willemsen R. Stubblefield B.K. Winfield S. Zablocka B. Sidransky E. Martin B.M. Huang S.P. et al.Animal model of Gaucher's disease from targeted disruption of the mouse glucocerebrosidase gene.Nature. 1992; 357: 407-410Google Scholar). Efforts to create animal models of Gaucher disease with a longer life span have included the gba "knock in" of the L444P mutation, but this also led to early death attributable, at least in part, to a defective skin permeability barrier (7Liu Y. Suzuki K. Reed J.D. Grinberg A. Westphal H. Hoffmann A. Doring T. Sandhoff K. Proia R.L. Mice with type 2 and 3 Gaucher disease point mutations generated by a single insertion mutagenesis procedure.Proc. Natl. Acad. Sci. USA. 1998; 95: 2503-2508Google Scholar). Additional mouse models were designed based on genotype/phenotype correlations in humans (8Xu Y.H. Quinn B. Witte D. Grabowski G.A. Viable mouse models of acid β-glucosidase deficiency: the defect in Gaucher disease.Am. J. Pathol. 2003; 163: 2093-2101Google Scholar), as summarized in Table 1. Homozygosity for N370S in humans results in less severe to asymptomatic phenotypes (1Beutler E. Grabowski G.A. Gaucher disease.in: Scriver R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Basis of Inherited Disease, C. McGraw-Hill, New York2001: 3635-3668Google Scholar, 9Tsuji S. Martin B.M. Barranger J.A. Stubblefield B.K. LaMarca M.E. Ginns E.I. Genetic heterogeneity in type 1 Gaucher disease: multiple genotypes in Ashkenazic and non-Ashkenazic individuals.Proc. Natl. Acad. Sci. USA. 1988; 85: 2349-2352Google Scholar, 10Charrow J. Andersson H.C. Kaplan P. Kolodny E.H. Mistry P. Pastores G. Rosenbloom B.E. Scott C.R. Wappner R.S. Weinreb N.J. et al.The Gaucher registry: demographics and disease characteristics of 1698 patients with Gaucher disease.Arch. Intern. Med. 2000; 160: 2835-2843Google Scholar). In comparison, homozygosity for the D409H allele is associated with early-onset, variable disease of the viscera and CNS (11Theophilus B. Latham T. Grabowski G.A. Smith F.I. Gaucher disease: molecular heterogeneity and phenotype-genotype correlations.Am. J. Hum. Genet. 1989; 45: 212-225Google Scholar, 12Eyal N. Wilder S. Horowitz M. Prevalent and rare mutations among Gaucher patients.Gene. 1990; 96: 277-283Google Scholar). These patients also can develop characteristic calcific abnormalities of the aortic valves and ascending aorta (13Pasmanik-Chor M. Laadan S. Elroy-Stein O. Zimran A. Abrahamov A. Gatt S. Horowitz M. The glucocerebrosidase D409H mutation in Gaucher disease.Biochem. Mol. Med. 1996; 59: 125-133Google Scholar). The V394L allele has been observed only in the heteroallelic state, and when the heteroallele is L444P, the phenotype includes visceral and CNS involvement (11Theophilus B. Latham T. Grabowski G.A. Smith F.I. Gaucher disease: molecular heterogeneity and phenotype-genotype correlations.Am. J. Hum. Genet. 1989; 45: 212-225Google Scholar). In contrast, N370S homozygosity in mice was lethal in the neonatal period, V394L homozygosity displayed minor phenotypic abnormalities up to 12 months, and D409H or D409V homozygotes displayed low-grade Gaucher cell formation and GC storage (8Xu Y.H. Quinn B. Witte D. Grabowski G.A. Viable mouse models of acid β-glucosidase deficiency: the defect in Gaucher disease.Am. J. Pathol. 2003; 163: 2093-2101Google Scholar). Visceral tissues from homozygous murine models of V394L and D409H showed ⩽10% of wild-type (WT) GCase activity. Neither model displayed CNS involvement (8Xu Y.H. Quinn B. Witte D. Grabowski G.A. Viable mouse models of acid β-glucosidase deficiency: the defect in Gaucher disease.Am. J. Pathol. 2003; 163: 2093-2101Google Scholar).TABLE 1Characteristics of humans and mice with GCase mutationsGenotypeHumanMouseReferencesD409H/D409HMajor visceral and CNS diseaseMinor visceral disease1Beutler E. Grabowski G.A. Gaucher disease.in: Scriver R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Basis of Inherited Disease, C. McGraw-Hill, New York2001: 3635-3668Google Scholar, 8Xu Y.H. Quinn B. Witte D. Grabowski G.A. Viable mouse models of acid β-glucosidase deficiency: the defect in Gaucher disease.Am. J. Pathol. 2003; 163: 2093-2101Google Scholar, 11Theophilus B. Latham T. Grabowski G.A. Smith F.I. Gaucher disease: molecular heterogeneity and phenotype-genotype correlations.Am. J. Hum. Genet. 1989; 45: 212-225Google Scholar, 12Eyal N. Wilder S. Horowitz M. Prevalent and rare mutations among Gaucher patients.Gene. 1990; 96: 277-283Google ScholarD409V/L444PaHomozygotes of D409V and V394L or V394L/null have not been observed in humans.Major visceral and CNS diseaseMinor visceral disease1Beutler E. Grabowski G.A. Gaucher disease.in: Scriver R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Basis of Inherited Disease, C. McGraw-Hill, New York2001: 3635-3668Google Scholar, 8Xu Y.H. Quinn B. Witte D. Grabowski G.A. Viable mouse models of acid β-glucosidase deficiency: the defect in Gaucher disease.Am. J. Pathol. 2003; 163: 2093-2101Google ScholarD409V/nullMajor visceral disease1Beutler E. Grabowski G.A. Gaucher disease.in: Scriver R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Basis of Inherited Disease, C. McGraw-Hill, New York2001: 3635-3668Google Scholar, 8Xu Y.H. Quinn B. Witte D. Grabowski G.A. Viable mouse models of acid β-glucosidase deficiency: the defect in Gaucher disease.Am. J. Pathol. 2003; 163: 2093-2101Google ScholarN370S/N370SVariable visceral diseaseDeath in neonatal period1Beutler E. Grabowski G.A. Gaucher disease.in: Scriver R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Basis of Inherited Disease, C. McGraw-Hill, New York2001: 3635-3668Google Scholar, 8Xu Y.H. Quinn B. Witte D. Grabowski G.A. Viable mouse models of acid β-glucosidase deficiency: the defect in Gaucher disease.Am. J. Pathol. 2003; 163: 2093-2101Google Scholar, 9Tsuji S. Martin B.M. Barranger J.A. Stubblefield B.K. LaMarca M.E. Ginns E.I. Genetic heterogeneity in type 1 Gaucher disease: multiple genotypes in Ashkenazic and non-Ashkenazic individuals.Proc. Natl. Acad. Sci. USA. 1988; 85: 2349-2352Google Scholar, 10Charrow J. Andersson H.C. Kaplan P. Kolodny E.H. Mistry P. Pastores G. Rosenbloom B.E. Scott C.R. Wappner R.S. Weinreb N.J. et al.The Gaucher registry: demographics and disease characteristics of 1698 patients with Gaucher disease.Arch. Intern. Med. 2000; 160: 2835-2843Google ScholarL444P/L444PVariable visceral and CNS phenotypesDeath in neonatal period1Beutler E. Grabowski G.A. Gaucher disease.in: Scriver R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Basis of Inherited Disease, C. McGraw-Hill, New York2001: 3635-3668Google Scholar, 7Liu Y. Suzuki K. Reed J.D. Grinberg A. Westphal H. Hoffmann A. Doring T. Sandhoff K. Proia R.L. Mice with type 2 and 3 Gaucher disease point mutations generated by a single insertion mutagenesis procedure.Proc. Natl. Acad. Sci. USA. 1998; 95: 2503-2508Google ScholarV394L/L444P or nullaHomozygotes of D409V and V394L or V394L/null have not been observed in humans.Severe visceral and CNS diseaseMinor visceral disease1Beutler E. Grabowski G.A. Gaucher disease.in: Scriver R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Basis of Inherited Disease, C. McGraw-Hill, New York2001: 3635-3668Google Scholar, 8Xu Y.H. Quinn B. Witte D. Grabowski G.A. Viable mouse models of acid β-glucosidase deficiency: the defect in Gaucher disease.Am. J. Pathol. 2003; 163: 2093-2101Google ScholarV394L/V394LaHomozygotes of D409V and V394L or V394L/null have not been observed in humans.Minor visceral disease8Xu Y.H. Quinn B. Witte D. Grabowski G.A. Viable mouse models of acid β-glucosidase deficiency: the defect in Gaucher disease.Am. J. Pathol. 2003; 163: 2093-2101Google ScholarCNS, central nervous system; GCase, acid β-glucosidase.a Homozygotes of D409V and V394L or V394L/null have not been observed in humans. Open table in a new tab CNS, central nervous system; GCase, acid β-glucosidase. Saposins (sphingolipid activator proteins) A, B, C, and D are lysosomal glycoproteins that are encoded by a single gene, termed prosaposin (14Collard M.W. Sylvester S.R. Tsuruta J.K. Griswold M.D. Biosynthesis and molecular cloning of sulfated glycoprotein 1 secreted by rat Sertoli cells: sequence similarity with the 70-kilodalton precursor to sulfatide/GM1 activator.Biochemistry. 1988; 27: 4557-4564Google Scholar, 15Nakano T. Sandhoff K. Stumper J. Christomanou H. Suzuki K. Structure of full-length cDNA coding for sulfatide activator, a co-β-glucosidase and two other homologous proteins: two alternate forms of the sulfatide activator.J. Biochem. (Tokyo). 1989; 105: 152-154Google Scholar, 16O'Brien J.S. Kretz K.A. Dewji N. Wenger D.A. Esch F. Fluharty A.L. Coding of two sphingolipid activator proteins (SAP-1 and SAP-2) by same genetic locus.Science. 1988; 241: 1098-1101Google Scholar, 17Rorman E.G. Scheinker V. Grabowski G.A. Structure and evolution of the human prosaposin chromosomal gene.Genomics. 1992; 13: 312-318Google Scholar). The saposins are essential to the optimal activity of glycosphingolipid hydrolases (18Furst W. Sandhoff K. Activator proteins and topology of lysosomal sphingolipid catabolism.Biochim. Biophys. Acta. 1992; 1126: 1-16Google Scholar, 19Grabowski G.A. Gatt S. Horowitz M. Acid β-glucosidase: enzymology and molecular biology of Gaucher disease.Crit. Rev. Biochem. Mol. Biol. 1990; 25: 385-414Google Scholar, 20Harzer K. Paton B.C. Christomanou H. Chatelut M. Levade T. Hiraiwa M. O'Brien J.S. Saposins (sap) A and C activate the degradation of galactosylceramide in living cells.FEBS Lett. 1997; 417: 270-274Google Scholar, 21Kishimoto Y. Hiraiwa M. O'Brien J.S. Saposins: structure, function, distribution, and molecular genetics.J. Lipid Res. 1992; 33: 1255-1267Google Scholar). In particular, saposin C is needed for full GCase function (22Qi X. Grabowski G.A. Molecular and cell biology of acid β-glucosidase and prosaposin.Prog. Nucleic Acid Res. Mol. Biol. 2001; 66: 203-239Google Scholar, 23Sandhoff K. Kolter T. Van Echten-Deckert G. Sphingolipid metabolism. Sphingoid analogs, sphingolipid activator proteins, and the pathology of the cell.Ann. N. Y. Acad. Sci. 1998; 845: 139-151Google Scholar, 24Sun Y. Qi X. Grabowski G.A. Saposin C is required for normal resistance of acid β-glucosidase to proteolytic degradation.J. Biol. Chem. 2003; 278: 31918-31923Google Scholar). Saposin C has at least two effects on GCase: 1) optimization of GCase hydrolysis of GC and other substrates; and 2) a protective effect for GCase against proteolytic digestion (24Sun Y. Qi X. Grabowski G.A. Saposin C is required for normal resistance of acid β-glucosidase to proteolytic degradation.J. Biol. Chem. 2003; 278: 31918-31923Google Scholar). This latter finding probably accounts for the reduction of GCase activity and protein levels in prosaposin-deficient mice and humans (24Sun Y. Qi X. Grabowski G.A. Saposin C is required for normal resistance of acid β-glucosidase to proteolytic degradation.J. Biol. Chem. 2003; 278: 31918-31923Google Scholar). In humans, deficiency of saposin C leads to a variant form of Gaucher disease with GC accumulation in macrophages and CNS (25Schnabel D. Schroder M. Sandhoff K. Mutation in the sphingolipid activator protein 2 in a patient with a variant of Gaucher disease.FEBS Lett. 1991; 284: 57-59Google Scholar). The proteolytic processing of prosaposin to the individual saposins occurs predominantly in acidified compartments, including the lysosome (26Leonova T. Qi X. Bencosme A. Ponce E. Sun Y. Grabowski G.A. Proteolytic processing patterns of prosaposin in insect and mammalian cells.J. Biol. Chem. 1996; 271: 17312-17320Google Scholar, 27Vielhaber G. Hurwitz R. Sandhoff K. Biosynthesis, processing, and targeting of sphingolipid activator protein (SAP) precursor in cultured human fibroblasts. Mannose 6-phosphate receptor-independent endocytosis of SAP precursor.J. Biol. Chem. 1996; 271: 32438-32446Google Scholar). Prosaposin has been found in a variety of body fluids (14Collard M.W. Sylvester S.R. Tsuruta J.K. Griswold M.D. Biosynthesis and molecular cloning of sulfated glycoprotein 1 secreted by rat Sertoli cells: sequence similarity with the 70-kilodalton precursor to sulfatide/GM1 activator.Biochemistry. 1988; 27: 4557-4564Google Scholar, 28Kondoh K. Hineno T. Sano A. Kakimoto Y. Isolation and characterization of prosaposin from human milk.Biochem. Biophys. Res. Commun. 1991; 181: 286-292Google Scholar). To establish additional viable Gaucher disease mouse models and evaluate the in vivo effect of saposin C on mutant GCases, mice were created by cross-breeding V394L or D409H homozygotes into the PS-NA line that expresses subnormal levels of a transgene encoding mouse prosaposin/saposins. The resulting mice developed large accumulations of GC in a variety of tissues as a more florid viable analog of human Gaucher disease with CNS involvement. The following were from commercial sources: 4-methylumbelliferyl-β-d-glucopyranoside (4MU-Glc; Biosynth AG); sodium taurocholate (Calbiochem, La Jolla, CA); mouse anti-β-actin monoclonal antibody, trypsin, trypsin inhibitor, and primulin (Sigma, St. Louis, MO); NuPAGE 4–12% Bis-Tris gel, NuPAGE MES SDS running buffer, William's solution, collagenase, Ca/Mg-free HBSS, DMEM, neurobasal medium, and B27 supplement (Invitrogen, Carlsbad, CA); rat anti-mouse CD68 monoclonal antibody (Serotec, Oxford, UK); M-PER Mammalian Protein Extraction Reagent and BCA Protein Assay Reagent (Pierce, Rockford, IL); [35S]cysteine/methionine protein labeling mix (DuPont-New England Nuclear Research Products, Boston, MA); Molecular Dynamics Storm 860 scanner, Hybond™-ECL™ nitrocellulose membrane, and ECL detection reagent (Amersham Biosciences, Piscataway, NJ); ABC Vectastain and Alkaline Phosphatase Kit II (black) (Vector Laboratory, Burlingame, CA); and BD TALON™ Metal Affinity Resin (BD Biosciences Clontech, Palo Alto, CA). The mouse prosaposin cDNA was cloned from a λZap mouse liver library (29Sun Y. Witte D.P. Grabowski G.A. Developmental and tissue-specific expression of prosaposin mRNA in murine tissues.Am. J. Pathol. 1994; 145: 1390-1398Google Scholar). It does not contain exon 8, which encodes amino acid QDQ in a saposin B region that appears nonessential (30Cohen T. Auerbach W. Ravid L. Bodennec J. Fein A. Futerman A.H. Joyner A.L. Horowitz M. The exon 8-containing prosaposin gene splice variant is dispensable for mouse development, lysosomal function, and secretion.Mol. Cell. Biol. 2005; 25: 2431-2440Google Scholar). The mouse prosaposin transgene was created by cloning mouse prosaposin cDNA (2.5 kb) downstream from the 3-phosphoglycerate kinase promoter in the pBluescript vector. The prosaposin knockout (PS−/−) mice containing this prosaposin transgene were generated previously and named PS-NA (31Sun Y. Qi X. Witte D.P. Ponce E. Kondoh K. Quinn B. Grabowski G.A. Prosaposin: threshold rescue and analysis of the "neuritogenic" region in transgenic mice.Mol. Genet. Metab. 2002; 76: 271-286Google Scholar). The V394L and D409H point mutations at the mouse GCase locus were introduced using the cre-lox-P system (8Xu Y.H. Quinn B. Witte D. Grabowski G.A. Viable mouse models of acid β-glucosidase deficiency: the defect in Gaucher disease.Am. J. Pathol. 2003; 163: 2093-2101Google Scholar). The crossing of V394L or D409H homozygotes with PS-NA resulted in 4L/PS-NA or 9H/PS-NA mice, respectively, with a genetic background of ∼50% FVB, 25% C57BL/6, and 25% 129SvEvBrd. D409V/D409V PS-NA mice were never observed from the appropriate crosses. After mutation validation of GCase by direct sequencing, the lox-P site was used as a marker for GCase point mutation genotyping. Multiplex PCR was performed to genotype the point mutations and prosaposin transgene. For GCase point mutation, primers mGC4996F (5′-CACAGATGTGTATGGCCATCGG-3′ in intron 8 of GBA) and mGC5387R (5′-CTGAAGTGGCCAAGATGGTAG-3′ in exon 9 of GBA) generated a 391 bp fragment from WT and a 485 bp fragment (391 bp + 94 bp lox-P junction sequence in intron 8) for the mutant alleles (8Xu Y.H. Quinn B. Witte D. Grabowski G.A. Viable mouse models of acid β-glucosidase deficiency: the defect in Gaucher disease.Am. J. Pathol. 2003; 163: 2093-2101Google Scholar). For the prosaposin transgene, primers CBC (5′-ATGAAGCTGGTGTCTGATGT-3′ in the saposin B region of the transgene) and PO8 (5′-CACAAATCCAGGATCCATCAC-3′ in the saposin D region of the transgene) yielded a 1 kb band. Reaction conditions were PCR buffer (Invitrogen) with 3 mM MgCl2. Reactions were cycled 30 times as follows: 94°C for 60 s, 58°C for 60 s, and 72°C for 90 s. The prosaposin knockout allele was genotyped as described (31Sun Y. Qi X. Witte D.P. Ponce E. Kondoh K. Quinn B. Grabowski G.A. Prosaposin: threshold rescue and analysis of the "neuritogenic" region in transgenic mice.Mol. Genet. Metab. 2002; 76: 271-286Google Scholar). Mice were euthanized in age-matched groups. Liver, lung, spleen, thymus, brain, and spinal cord were collected, fixed in 10% formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin and periodic acid-Schiff, then analyzed by light microscopy. Karnovsky's fixative was used for ultrastructural studies. For immunohistochemistry, frozen tissue sections fixed with 4% paraformaldehyde were incubated with rat anti-mouse CD68 monoclonal antibody (1:200 in PBS with 5% BSA) overnight at 4°C. Detection was performed using ABC Vectastain and Alkaline Phosphatase Kit II (black) according to the manufacturer's instruction. The slides were counterstained with methylene green. Tissue samples (∼100 mg wet weight) in water (0.6 ml) and methanol (2 ml) were homogenized (PowerGen 35; Fisher Scientific), then chloroform (1 ml) was added. Homogenates were shaken (15 min) and centrifuged (5 min at 1,000 g). Pellets were reextracted with water (0.7 ml) and chloroform-methanol (1:2, v/v; 3 ml). The combined extracts were centrifuged (10 min at 7,000 g). The supernatants were transferred to fresh tubes and the solvents evaporated under N2. Dried extracts were redissolved in chloroform-methanol-water (60:30:4.5, v/v/v; 15 ml) and desalted on Sephadex G-25 columns (32Wells M.A. Dittmer J.C. The use of Sephadex for the removal of nonlipid contaminants from lipid extracts.Biochemistry. 1963; 172: 1259-1263Google Scholar). Samples were then subjected to alkaline methanolysis (33Dreyfus H. Guerold B. Freysz L. Hicks D. Successive isolation and separation of the major lipid fractions including gangliosides from single biological samples.Anal. Biochem. 1997; 249: 67-78Google Scholar) and desalted. Relative proportions of lipids from these tissue samples were determined by thin-layer chromatography with borate-impregnated plates (10 cm2 Merck HPTLC silica gel 60; 200 μm) (34Igisu H. Takahashi H. Suzuki K. Abnormal accumulation of galactosylceramide in the kidney of twitcher mouse.Biochem. Biophys. Res. Commun. 1983; 110: 940-944Google Scholar). Plates were developed in chloroform-methanol-water (65:25:4, v/v/v). Lipids were visualized with primulin spray (100 mg/l in 80% acetone) and blue fluorescence scanning (Storm 860; Amersham Pharmacia Biotech). Tissues were homogenized in 1% sodium taurocholate and 1% Triton X-100, with 0.25% each in final assay mixtures. GCase activities were determined fluorometrically with 4MU-Glc (35Xu Y.H. Ponce E. Sun Y. Leonova T. Bove K. Witte D. Grabowski G.A. Turnover and distribution of intravenously administered mannose-terminated human acid β-glucosidase in murine and human tissues.Pediatr. Res. 1996; 39: 313-322Google Scholar). Some assay mixtures were preincubated in the presence and absence of the GCase irreversible inhibitor, conduritol B epoxide (1 mM, 40 min at 37°C). The substrate (4MU-Glc) was added, and the reactions were stopped after an additional incubation (30 min at 37°C) (36Grace M.E. Newman K.M. Scheinker V. Berg-Fussman A. Grabowski G.A. Analysis of human acid β-glucosidase by site-directed mutagenesis and heterologous expression.J. Biol. Chem. 1994; 269: 2283-2291Google Scholar). WT activities of control tissues were determined in parallel for all assays. WT levels were set as 100%. The coding region of mouse prosaposin cDNA was cloned into pET21a vector and overexpressed in Escherichia coli as described (37Qi X. Leonova T. Grabowski G.A. Functional human saposins expressed in Escherichia coli. Evidence for binding and activation properties of saposins C with acid β-glucosidase.J. Biol. Chem. 1994; 269: 16746-16753Google Scholar). The expressed mouse prosaposin contained a 6 His tag at the carboxyl terminus. Harvested cell pellets were dissolved in 8 M urea (pH 8.0). The lysates were clarified by centrifugation (10,000 g, 25 min). The supernatants were mixed with BD TALON™ Metal Affinity Resin (cobalt-based) and gently agitated for 20 min to allow the His tag prosaposin binding to resin. The resin was washed with 25 mM imidazole, and the prosaposin protein was eluted with 150 mM imidazole, dialyzed, and analyzed by 10% SDS-PAGE. The single band products were used to raise goat antiserum (Harlan Bioproducts for Science, Madison, WI) after a 90 day protocol with initial inoculation of antigen (1 mg) and three boosts (500 μg each). Rabbit anti-mouse saposin D was raised against recombinant mouse saposin D produced in E. coli using the pET21a system (37Qi X. Leonova T. Grabowski G.A. Functional human saposins expressed in Escherichia coli. Evidence for binding and activation properties of saposins C with acid β-glucosidase.J. Biol. Chem. 1994; 269: 16746-16753Google Scholar). Using immunoblot analyses, goat anti-mouse prosaposin antiserum detected mouse prosaposin and saposins A, B, C, and D. Rabbit anti-mouse saposin D antiserum detected mouse saposin D and prosaposin. Hippocampal neurons were isolated from 17.5 day mouse embryos. The dissected hippocampi were digested with trypsin solution (1 mg/ml in HBSS, 20 min) at room temperature, and the tissues were transferred into trypsin inhibitor solution (1 mg/ml in HBSS) for 5 min, followed by washing in 2 ml of ice-cold HBSS. The tissues were dissociated, and the cells were plated in DMEM with 10% fetal bovine serum plus penicillin and streptomycin in polyethyleneimine-coated flasks. After 3 h, the medium was replaced with neurobasal medium with B27 supplement. The hippocampal neurons were allowed to grow for 5 days and then subjected to labeling and immunoprecipitation. Hepatocytes were isolated from liver after perfusion with collagenase solution (0.2 mg/ml collagenase and 0.33 mM CaCl2 in HBSS). Liver tissues were minced into small pieces with scissors and suspended in William's solution. The liver suspension was washed twice with William's solution, layered over the Percoll, and centrifuged (2 min at 800 g). The resultant pellets were washed twice in William's solution, and hepatocytes were plated in DMEM with 10% fetal bovine serum and 1% glutamine as well as penicillin and streptomycin in a 10 cm dish. The fresh medium was replaced after 1 h. The hepatocytes were allowed to grow for 7 days and then subjected to labeling and immunoprecipitation. Mouse lung or tail fibroblasts were cultured in DMEM supplemented with 10% heat-activated fetal bovine serum as described previously (38Qi X. Kondoh K. Yin H. Wang M. Ponce E. Sun Y. Grabowski G.A. Ex vivo localization of the mouse saposin C activation region for acid β-glucosidase.Mol. Genet. Metab. 2002; 76: 189-200Google Scholar). The above cells (5–10 × 105 cells per flask) were labeled for 4 h with 127 μCi of [35S]cysteine/methionine protein labeling mix. The media were collected and treated with anti-mouse saposin D antibody to precipitate the prosaposin protein. The proteins were resolved by SDS-PAGE as described (31Sun Y. Qi X. Witte D.P. Ponce E. Kondoh K. Quinn B. Grabowski G.A. Prosaposin: threshold rescue and analysis of the "neuritogenic" region in transgenic mice.Mol. Genet. Metab. 2002; 76: 271-286Google Scholar). Tissues were homogenized in M-PER Mammalian Protein Extraction Reagent. Protein concentrations were estimated using BCA Protein Assay Reagent. Tissue extracts were separated on NuPAGE 4–12% Bis-Tris gels with NuPAGE MES SDS running buffer and electroblotte
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