Combined saposin C and D deficiencies in mice lead to a neuronopathic phenotype, glucosylceramide and alpha-hydroxy ceramide accumulation, and altered prosaposin trafficking

Saposins (A, B, C and D) are approximately 80 amino acid stimulators of glycosphingolipid (GSL) hydrolases that derive from a single precursor, prosaposin. In both humans and mice, prosaposin/saposin deficiencies lead to severe neurological deficits. The CD-/- mice with saposin C and D combined deficiencies were produced by introducing genomic point mutations into a critical cysteine in each of these saposins. These mice develop a severe neurological phenotype with ataxia, kyphotic posturing and hind limb paralysis. Relative to prosaposin null mice ( approximately 30 days), CD-/- mice had an extended life span ( approximately 56 days). Loss of Purkinje cells was evident after 6 weeks, and storage bodies were present in neurons of the spinal cord, brain and dorsal root ganglion. Electron microscopy showed well-myelinated fibers and axonal inclusions in the brain and sciatic nerve. Marked accumulations of glucosylceramides and alpha-hydroxy ceramides were present in brain and kidney. Minor storage of lactosylceramide (LacCer) was observed when compared with tissues from the prosaposin null mice, suggesting a compensation in LacCer degradation by saposin B for the saposin C deficiency. Skin fibroblasts and tissues from CD-/- mice showed an increase of intracellular prosaposin, impaired prosaposin secretion, deficiencies of saposins C and D and decreases in saposins A and B. In addition, the deficiency of saposin C in CD-/- mice resulted in cellular decreases of acid beta-glucosidase activity and protein. This CD null mouse model provides a tool to explore the in vivo functional interactions of saposins in GSL metabolism and lysosomal storage diseases, and prosaposin's physiological effects.     

Chronological changes in prosaposin in the developing rat brain

Prosaposin is the precursor protein of four glycoproteins, saposins A, B, C, and D, which activate sphingolipid hydrolases in lysosomes. Besides this role, intact prosaposin is also known as a potent neurotrophic factor that prevents neuronal cell death and stimulates neurite outgrowth in in vivo and in vitro experiments. In the present study, we examined chronological changes in prosaposin immunoreactivity in the rat brain using immunofluorescence staining and Diaminobenzidine (DAB) immunohistochemistry. In the hippocampal regions CA1, CA3, and dentate gyrus, the strongest staining of prosaposin was observed on postnatal day 1. The prosaposin immunoreactivity then decreased gradually until postnatal day 28. But in the cerebral cortex, prosaposin staining intensity increased from postnatal day 1 to 14, then decreased until postnatal day 28. The prosaposin immunoreactivity co-localized with the lysosomal granules labeled by an anti-Cathepsin D antibody, indicating that prosaposin mainly localized in the lysosomes of the neurons. We also examined the chronological changes in prosaposin mRNA and its two alternatively spliced variants using in situ hybridization. We found that both the mRNA forms, especially the one without a nine-base insertion, increased significantly from embryonic day 15 to postnatal day 7, then decreased gradually until postnatal day 28. Abundant prosaposin expression in the perinatal stages indicates a potential role of prosaposin in the early development of the rat brain.     

An Asn > Lys substitution in saposin B involving a conserved amino acidic residue and leading to the loss of the single N-glycosylation site in a patient with metachromatic leukodystrophy and normal arylsulphatase A activity

Sphingolipid activator proteins are small glycoproteins required for the degradation of sphingolipids by specific lysosomal hydrolases. Four of them, called saposins, are encoded by the prosaposin gene, the product of which is proteolytically cleaved into the four mature saposin proteins (saposins A, B, C, D). One of these, saposin B, is necessary in the hydrolysis of sulphatide by arylsulphatase A where it presents the solubilised substrate to the enzyme. As an alternative to arylsulphatase A deficiency, deficiency of saposin B causes metachromatic leukodystrophy. We identified a previously undescribed mutation (N215K) in the prosaposin gene of a patient with metachromatic leukodystrophy but with normal arylsulphatase A activity and elevated sulphatide in urine. The mutation involves a highly conserved amino acidic residue and abolishes the only N-glycosylation site of saposin B.     

Ex vivo localization of the mouse saposin C activation region for acid beta-glucosidase

Saposin C is a biological activator of acid beta-glucosidase (GCase), the lysosomal hydrolase with activity towards glucosylceramide (GC). In addition, saposin C possesses a functional domain that determines the in vitro and ex vivo neuritogenic effects of prosaposin, the precursor of saposins A, B, C, and D. The domains for enzymatic activation and neuritogenic function segregate in vitro, respectively, to the carboxyl- and amino-terminal halves of human and mouse saposin C. A chimeric mouse saposin C(1-8)B(8-28)C(30-80) was created to obliterate the neuritogenic region by substituting amino acids 9-29 of saposin C with amino acids 8-28 of saposin B. This saposin showed normal in vitro enzymatic activation effects toward GCase, but no neuritogenic activity. An altered prosaposin was made to contain the chimeric saposin C region. Expression of this altered or wild-type prosaposin was driven by the PGK-1 promoter as a transgene in prosaposin knock-out mice. In cultured fibroblasts from such mice, expressed saposins localized to the lysosomal compartments. Metabolic lipid labeling using L-[3-(14)C]serine showed retention or clearance of GC in prosaposin deficient or transgene reconstituted cells, respectively. In addition, sulfatide catabolism, that requires saposin B and arylsulfatase, was also normalized in prosaposin KO cells reconstituted with the transgenes. These data show that the transgenic prosaposins were expressed and processed to functional saposins in fibroblasts. These results also show that the enzymatic activation domain is located at carboxyl-terminal half of saposin C and functions only in the context of the general saposin structure.     

Localization of prosaposin in rat cochlea

Prosaposin, the precursor of the sphingolipid hydrolase activator proteins called saposins A, B, C, and D, is abundant in the nervous system and muscles. Besides its role as the precursor of saposins, prosaposin is reported to function as a neurotrophic factor, initiating neural differentiation and preventing neuronal cell death in vivo and in vitro. In this study, we examined the localization and synthesis of prosaposin in the rat cochlea. Intense prosaposin immunoreactivity was observed in the organ of Corti, stria vascularis, and spiral ganglion. In an immuno-electron microscopic study, prosaposin immunoreactivity was found mainly in lysosomal granules of the cells in these regions. In the lysosome, prosaposin does not always colocalize with cathepsin D, but was localized mainly in the dark area of the lysosome. Prosaposin mRNA was observed in these same regions. Our results suggest that prosaposin plays a role in homeostasis in the peripheral auditory system.     

Non-neuronopathic Gaucher disease due to saposin C deficiency

Gaucher disease is generally caused by a deficiency of the lysosomal enzyme glucocerebrosidase. The degradation of glycosphingolipids requires also the participation of sphingolipid activator proteins. The prosaposin PSAP gene codes for a single protein which undergoes post-translational cleavage to yield four proteins named saposins A, B, C and D. Saposin (SAP-) C is required for glucosylceramide degradation, and its deficiency results in a variant form of Gaucher disease. In this report, we present clinical, biochemical, and molecular findings in a 36-year-old man and his 30-year-old sister with non-neuronopathic Gaucher disease due to SAP-C deficiency. Very high levels of chitotriosidase activity, chemokine CCL18, and increased concentration of glucosylceramide in plasma and normal beta-glucosidase activity in skin fibroblasts were observed in the patients. A molecular genetics study of the PSAP gene enabled the identification of one missense mutation, p.L349P, located in the SAP-C domain and another mutation, p.M1L, located in the initiation codon of the prosaposin precursor protein. The presented findings describe the first cases where the non-neuronopathic Gaucher disease has been definitely demonstrated to be a consequence of SAP-C deficiency. Three previously described cases in the literature displayed a Gaucher type 3 phenotype.     

Changes in expression of prosaposin in the rat facial nerve nucleus after facial nerve transection

Prosaposin is the precursor of saposins A, B, C and D, which are activators of sphingolipid hydrolases. In addition, unprocessed prosaposin functions as a neurotrophic factor in the central and peripheral nervous systems by acting to prevent neuronal apoptosis, to elongate neurites and to facilitate myelination. In this study, the expression pattern of prosaposin in the facial nerve nucleus after facial nerve transection was examined by immunohistochemistry and in situ hybridization. Prosaposin immunoreactivity in the neurons on the operated side facial nerve nucleus showed a biphasic pattern: it was significantly increased on day 3 after transection, decreased dramatically on day 7, started to increase gradually on day 14 and reached another peak on day 21 after transection. Significant increases in the levels of prosaposin mRNA were identified in the neurons on the operated side, suggesting that prosaposin was synthesized vigorously by the neurons themselves in the case of facial nerve transection. The diverse changes in prosaposin immunoreactivity during the process of facial nerve regeneration may reflect the diverse neurotrophic activities of prosaposin in facial motoneurons.     

Expression patterns in alternative splicing forms of prosaposin mRNA in the rat facial nerve nucleus after facial nerve transection

Prosaposin acts as a neurotrophic factor, in addition to its role as the precursor protein for saposins A, B, C, and D, which are activators for specific sphingolipid hydrolases in lysosomes. In rats, the prosaposin gene generates two alternative splicing forms of mRNA: Pro+9 containing a 9-base insertion and Pro+0 without. The expression of these mRNAs changes after brain injury. We examined the expression patterns of the alternative splicing forms of prosaposin mRNA in the rat facial nerve nucleus for 52 days following facial nerve transection. Pro+0 mRNA increased within 3 days of transection, peaked after 5-10 days, and remained significantly elevated for 21 days. In contrast, the expression of Pro+9 mRNA was constant throughout the regenerative period. Prosaposin mRNA expression increased not only in facial motoneurons, but also in microglia during facial nerve regeneration. Our findings indicate that the saposin B domain of prosaposin, which is the domain affected by alternative splicing, plays an important role in both neurons and microglia during neuroregeneration.     

Prosaptide exacerbates ischemia-induced behavioral deficits in vivo; an effect that does not involve mitogen-activated protein kinase activation

Prosaposin is a 517 amino acid membrane component and secreted protein(5,7,9) that is proteolytically cleaved to generate the four small glycoproteins; saposins A, B, C and D.(9,13,19) Prosaposin's ability to promote neurite outgrowth(31) and to protect neurons from programmed cell death(28) in vitro, as well as to rescue neurons from ischemia and other damage in vivo(11,12,15,25) implied that prosaposin was neurotrophic/neuroprotectant.(1,7,24,31) The neurotrophic sequence of prosaposin was isolated to smaller peptide fragments termed prosaptides(15,31) within the amino terminal portion of saposin C.(1,6,8,10,17,20,21,28) The proposed use of synthetic prosaptides as peripherally administered neuroprotective and/or neurotrophic therapeutic agents has stemmed from their ability to cross the blood-brain barrier,(27) as well as their reported neurotrophic activity in vitro.(15,23,31) Few studies, however, have attempted to characterize these peptides, presumably due to their reported instability following peripheral administration.(27) With the recent design of a stable 11-mer retro-inverso prosaptide,(15,31) it has become feasible to investigate the pharmacological effects of a stable version of these peptides in the validated rabbit spinal cord ischemia model that has been used extensively in the development of therapeutics to treat ischemic stroke.(4,14,16,18) Our results show not only that prosaptide was not neurotrophic/neuroprotectant in vivo, but rather it worsened ischemia-induced behavioral deficits.     

A mutation in the saposin A domain of the sphingolipid activator protein (prosaposin) gene results in a late-onset, chronic form of globoid cell leukodystrophy in the mouse

Sphingolipid activator proteins (saposins A, B, C and D) are small homologous glycoproteins derived from a common precursor protein (prosaposin) encoded by a single gene. They are required for in vivo degradation of sphingolipids with short carbohydrate chains. Six cysteines and one glycosylation site are strictly conserved in all four saposins. Total deficiency of all saposins and specific deficiency of saposin B or C are known among human patients. A mouse model of total saposin deficiency closely mimics the human disease. However, no specific saposin A or D deficiency is known. We introduced an amino acid substitution (C106F) into the saposin A domain by the Cre/loxP system which eliminated one of the three conserved disulfide bonds. Saposin A(-/-) mice developed slowly progressive hind leg paralysis with clinical onset at approximately 2.5 months and survival up to 5 months. Tremors and shaking, prominent in other myelin mutants, were not obvious until the terminal stage. Pathology and analytical biochemistry were qualitatively identical to, but generally much milder than, that seen in the typical infantile globoid cell leukodystrophy (GLD) in man (Krabbe disease) and in several other mammalian species, due to genetic deficiency of lysosomal galactosylceramidase (GALC) (EC 3.2.1.46). Thus, saposin A is indispensable for in vivo degradation of galactosylceramide by GALC. It should now be recognized that, in addition to GALC deficiency, genetic saposin A deficiency could also cause chronic GLD. Genetic saposin A deficiency might be anticipated among human patients with undiagnosed late-onset chronic leukodystrophy without GALC deficiency.     

Mutation in saposin D domain of sphingolipid activator protein gene causes urinary system defects and cerebellar Purkinje cell degeneration with accumulation of hydroxy fatty acid-containing ceramide in mouse

The sphingolipid activator proteins (saposins A, B, C and D) are small homologous glycoproteins that are encoded by a single gene in tandem within a large precursor protein (prosaposin) and are required for in vivo degradation of some sphingolipids with relatively short carbohydrate chains. Human patients with prosaposin or specific saposin B or C deficiency are known, and prosaposin- and saposin A-deficient mouse lines have been generated. Experimental evidence suggests that saposin D may be a lysosomal acid ceramidase activator. However, no specific saposin D deficiency state is known in any mammalian species. We have generated a specific saposin D(-/-) mouse by introducing a mutation (C509S) into the saposin D domain of the mouse prosaposin gene. Saposin D(-/-) mice developed progressive polyuria at around 2 months and ataxia at around 4 months. Pathologically, the kidney of saposin D(-/-) mice showed renal tubular degeneration and eventual hydronephrosis. In the nervous system, progressive and selective loss of the cerebellar Purkinje cells in a striped pattern was conspicuous, and almost all Purkinje cells disappeared by 12 months. Biochemically, ceramides, particularly those containing hydroxy fatty acids accumulated in the kidney and the brain, most prominently in the cerebellum. These results not only indicate the role of saposin D in in vivo ceramide metabolism, but also suggest possible cytotoxicity of ceramide underlying the cerebellar Purkinje cell and renal tubular cell degeneration.     

Nonciliated cells of the rat efferent ducts endocytose testicular sulfated glycoprotein-1 (SGP-1) and synthesize SGP-1 derived saposins

Sertoli cell sulfated glycoprotein-1 (SGP-1) is a heavily glycosylated and sulfated 70 kDa protein that is secreted into the lumen of the seminiferous tubule where it binds to spermatozoa. Recent light and electron microscope immunocytochemistry has suggested that the testicular SGP-1 detaches from the surface of spermatozoa in the lumen of the efferent ducts to be endocytosed within the endocytic apparatus of the epithelial nonciliated cells. The finding of SGP-1 mRNA together with anti-SGP-1 immunogold labeling of the lysosomal compartment suggest that these cells synthesize an efferent duct form of SGP-1. In the present study, a number of different experimental approaches (ligation, tunicamycin treatment and a combination of both) in combination with quantitative electron microscope immunogold labeling and Western blot analysis were performed in order to test this hypothesis. The number of gold particles and the profile area of the early (endosomes, pale multivesicular bodies) and late (dense multivesicular bodies, secondary lysosomes) endocytic apparatus were estimated in each of the experimental groups and expressed as the number of gold particles per μm² (labeling densities). The data revealed that ligation produced a significant reduction of anti-SGP-1 immunogold labeling of the early endocytic apparatus but not of the late endocytic apparatus. Tunicamycin treatment on the other hand produced a significant reduction of immunogold labeling of both the early and late endocytic apparatus. The combination of both treatments resulted in a more effective reduction of the labeling densities of these two endocytic compartments. These results thus indicate that the nonciliated cells of the efferent ducts are involved both in the endocytosis of the Sertoli-derived SGP-1 and in the synthesis of an efferent duct form of SGP-1 that is targeted from the Golgi apparatus to secondary lysosomes after its glycosylation. In order to determine the biosynthetic pathway of SGP-1 within the efferent ducts, an I. V. injection of ³⁵S-cysteine followed by immunoprecipitation and SDS-PAGE revealed that SGP-1 was initially biosynthesized as a 55 kDa protein. This protein appears to be post-translationally modified to a 65 kDa form after 1 hour, which preceded the appearance of the 70 kDa form, and smaller peptides of about 15 kDa characteristic of saposins after 3–4 hours. Western blot analysis of ligated efferent ducts showed an increase in the biosynthesis of the 70 kDa form of SGP-1 when compared to untreated controls, however, it has yet to be established if this protein is secreted or retained in an intracellular compartment. These results thus further substantiate our hypothesis on the endocytosis of the Sertoli-derived SGP-1 by the nonciliated cells and the synthesis of an efferent duct form of SGP-1 as well as provide evidence for the presence of 15 kDa saposins and its 65 kDa precursor in secondary lysosomes of these cells. © 1993 Wiley-Liss, Inc.     

Developmental and tissue-specific expression of prosaposin mRNA in murine tissues.

Prosaposin is a multifunctional locus in humans and mice that encodes in tandem and in the same reading frame four glycoprotein activators, or saposins, of lysosomal hydrolases. These ubiquitously expressed glycoproteins and the precursor, prosaposin, have been proposed to function in glycosphingolipid catabolic pathways and glycolipid transport. To characterize the temporal and spatial expression of the prosaposin locus, prenatal and postnatal mouse tissues were screened by in situ hybridization with a mouse antisense riboprobe for prosaposin. Prenatally, prosaposin mRNA was expressed differentially in the placenta and prominently in the decidua basalis and capsularis where expression was gestational age dependent. No other region of high-level expression was detectable in the prenatal mouse. In comparison, high-level differential expression of prosaposin was clearly evident postnatally in a variety of organs, including secretory epithelial cells of the choroid plexus, ependymal lining, upper trachea, esophagus, cortical tubules of the kidney, sertoli cells of the testes and epididymis. Discrete localization of prosaposin mRNA expression was also found in neurons of the cerebral cortex, cerebellar cortex, and lateral columns of the spinal cord as well as in hepatocytes of the mature liver. Very high levels of expression were found in specialized tissues including the Harderian glands and macrophages of lymph nodes, lungs, splenic tissue, and thymus. These studies indicate that the expression of the prosaposin locus, a presumed "housekeeping" gene, is under tissue- and cell-specific differential control. The spatial organization of expression suggests a role for this locus in the expression of glycosphingolipid-storage diseases.     

Mutational analysis in a patient with a variant form of Gaucher disease caused by SAP-2 deficiency

It is now clear that the lysosomal hydrolysis of sphingolipids requires both lysosomal enzymes and so-called sphingolipid activator proteins (SAPs). One gene, called prosaposin, codes for a precursor protein that is proteolytically cut into four putative SAPs. These four SAPs, of about 80 amino acids, share some structural features but differ somewhat in their specificity. Domain 3 of prosaposin mRNA contains the coding region for SAP-2, an activator of glucocerebrosidase. While most patients with Gaucher disease store glucosylceramide due to defects in glucocerebrosidase, a few patients store this lipid in the presence of normal enzyme levels. In this paper we describe the identification of a point mutation in domain 3 of a patient who died with this variant form of Gaucher disease. Polymerase chain reaction amplification was performed in the small amount of genomic DNA available using primers generated from the intronic sequence s surrounding domain 3. The patient was found to have a T-to-G substitution at position 1144 (counting from the A of ATG initiation codon) in half of the M13 recombinant clones. This changes the codon for cysteine₃₈₂ to glycine. His father and unaffected brother also had this mutation, but his mother did not. She was found to have half of the normal amount of mRNA for prosaposin in her cultured skin fibroblasts. Therefore, this child inherited a point mutation in domain 3 from his father and a deficiency of all four SAPs coded for by prosaposin from his mother.     

A novel mutation in the coding region of the prosaposin gene leads to a complete deficiency of prosaposin and saposins, and is associated with a complex sphingolipidosis dominated by lactosylceramide accumulation

A fatal infantile storage disorder with hepatosplenomegaly and severe neurological disease is described. Sphingolipids, including monohexosylceramides (mainly glucosylceramide), dihexosylceramides (mainly lactosylceramide), globotriaosyl ceramide, sulphatides, ceramides and globotetraosyl ceramide, were stored in the tissues. In general, cholesterol and sphingomyelin levels were unaltered. The storage process was generalized and affected a number of cell types, with histiocytes, which infiltrated a number of visceral organs and the brain, especially involved. The ultrastructure of the storage lysosomes was membranous with oligolamellar, mainly vesicular, profiles. Infrequently, there were Gaucher-like lysosomes in histiocytes. The neuropathology was severe and featured neuronal storage and loss with a massive depopulation of cortical neurons and pronounced fibrillary astrocytosis. There was a paucity of myelin and stainable axons in the white matter with signs of active demyelination. Immunohistochemical investigations indicated that saposins A, B, C and D were all deficient. The patient was homozygous for a 1 bp deletion (c.803delG) within the SAP-B domain of the prosaposin gene which leads to a frameshift and premature stop codon. In the heterozygous parents, mutant cDNA was detected by amplification refractory mutation analysis in the nuclear, but not the cytoplasmic, fraction of fibroblast RNA, indicating that the mutant mRNA was rapidly degraded. The storage process in the proband resembled that of a published case from an unrelated family. Saposins were also deficient in this case, leading to its reclassification as prosaposin deficiency, and her mother was found to be a carrier for the same c.803delG mutation. Both of the investigated families came from the same district of eastern Slovakia.     

Neurological deficits and glycosphingolipid accumulation in saposin B deficient mice

Saposin B derives from the multi-functional precursor, prosaposin, and functions as an activity enhancer for several glycosphingolipid (GSL) hydrolases. Mutations in saposin B present in humans with phenotypes resembling metachromatic leukodystrophy. To gain insight into saposin B's physiological functions, a specific deficiency was created in mice by a knock-in mutation of an essential cysteine in exon 7 of the prosaposin locus. No saposin B protein was detected in the homozygotes (B-/-) mice, whereas prosaposin, and saposins A, C and D were at normal levels. B-/- mice exhibited slowly progressive neuromotor deterioration and minor head tremor by 15 months. Excess hydroxy and non-hydroxy fatty acid sulfatide levels were present in brain and kidney. Alcian blue positive (sulfatide) storage cells were found in the brain, spinal cord and kidney. Ultrastructural analyses showed lamellar inclusion material in the kidney, sciatic nerve, brain and spinal cord tissues. Lactosylceramide (LacCer) and globotriaosylceramide (TriCer) were increased in various tissues of B-/- mice supporting the in vivo role of saposin B in the degradation of these lipids. CD68 positive microglial cells and activated GFAP positive astrocytes showed a proinflammatory response in the brains of B-/- mice. These findings delineate the roles of saposin B for the in vivo degradation of several GSLs and its primary function in maintenance of CNS function. B-/- provide a useful model for understanding the contributions of this saposin to GSL metabolism and homeostasis.     

鞘脂激活蛋白原的研究进展

鞘脂激活蛋白原（prosaposin）是一种多功能的糖蛋白。Prosaposin刚合成时其分子量为53kD，经过翻译后加工成为两种功能完全不同的蛋白：一种分子量为65kD，是4种鞘脂激活蛋白（sphingolipid activator protein，saposin）的前提物，在sortilin蛋白的作用下运送至溶酶体，由蛋白水解酶水解成为4种低分子量的鞘脂激活蛋白saposin A、B、C、D，其功能是作为鞘脂糖水解途径中所涉及到的溶酶体酶的激活剂，参与带有短的寡糖链的脂类的代谢；另一种分子量为70kD，prosaposin作为完整的糖基化的分泌蛋白出现，存在于一些生物体液中，例如精液、乳汁、脑脊液，并具备与鞘脂激活蛋白完全不同的功能。     

Prosaposin: threshold rescue and analysis of the "neuritogenic" region in transgenic mice

Prosaposin is the precursor of four glycoprotein activators (saposins) for lysosomal hydrolases. Intact prosaposin also has lipid transfer properties in vitro as well as neuritogenic effects ex vivo and in vivo. Such "neuritogenic" effects of saposin C were evaluated in vivo using transgenic mice with prosaposin cDNAs having normal (PS-N) or mutated neuritogenic region. The mutant prosaposin cDNA (PS-CBC) encoded a chimeric saposin C that contained the non-neuritogenic sequence of saposin B, but retained acid beta-glucosidase (GCase) activation effects. When driven by the PGK (3-phosphoglycerate kinase) promoter, transgene expression was highest in the cerebrum for any of the transgenes (range from 15% to 42% of wild-type). Low levels were in visceral tissues. Prosaposin knock-out (PS-/-) mice expressing N or CBC transgenes, even at low levels, had delayed onset of neurologic signs and neuropathology, and significant lengthening of life span (from 1.7- to 7-fold) with age dependent partial correction of GlcCer and LacCer accumulation in the brain. Neuropathologic progression and neuronal glycosphingolipid storage were related directly to the transgene expression levels in the brain. Purkinje cell loss was age dependent. Gross brain and neuronal organizations were indistinguishable in PS-/- mice with or without the various transgenes, albeit the phenotype appeared later in the mice with transgenes. These studies show the degree of neuropathologic manifestations in each transgenic line depended on expression level rather than on the nature of the transgene. These studies also show in vivo localization of the GCase activation region to the carboxy terminal half of saposin C and the lack of a significant gross trophic effect of saposin C on CNS organization in vivo.     

The role of the protein glycosylation state in the control of cellular transport of the amyloid beta precursor protein

The amyloid beta precursor protein can exist as both a membrane-bound and a secreted protein, with the former having the potential to generate the amyloid beta peptide present in the neuritic plaques which are characteristic of Alzheimer's disease. In this study, we have used a clone of the AtT20 mouse pituitary cell line which expresses high levels of the amyloid beta precursor protein to characterize the glycosylation state of the secreted and membrane-bound forms of the protein and to examine the role of post-translational modifications in protein processing. Lectin blot analysis of immunoprecipitated amyloid beta precursor protein demonstrated that the soluble form of the protein contains significant amounts of sialic acid, with the lectin staining being reduced in the particulate cellular fractions. Treatment of the cells with mannosidase inhibitors to interfere with the formation of complex-type N-linked glycans resulted in a decrease in secreted amyloid beta precursor protein and an increase in the level of the cellular form of the protein. The increase in amyloid beta precursor protein levels in the cellular fraction was accompanied by an increase in perinuclear staining. Furthermore, cells overexpressing the alpha2,6(N)-sialyltransferase enzyme also demonstrated an increase in amyloid beta precursor protein secretion. These results suggest that the presence of terminal sialic acid residues on complex-type N-glycans may be required for the optimal transport of the amyloid beta precursor protein from the Golgi to the cell membrane with the subsequent cleavage to generate the secreted form of the protein.     

Saposins facilitate CD1d-restricted presentation of an exogenous lipid antigen to T cells

Members of the CD1 family present antigenic lipids to T lymphocytes. CD1 molecules survey endocytic compartments for lipid antigens that are sorted into these vesicles after incorporation into the membrane bilayer, and extraction from the bilayer is likely to be a critical step for lipid association. We hypothesized that lysosomal saposins, which are cofactors required for sphingolipid degradation, might be involved in this process. Here we show that saposins, although not required for the autoreactive recognition of CD1d by natural killer T cells, are indispensable for the binding of an exogenous lipid antigen, alpha-galactosylceramide, to CD1d in the endocytic pathway. We suggest that saposins mobilize monomeric lipids from lysosomal membranes and facilitate their association with CD1d.