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Carlos R. Morales - One of the best experts on this subject based on the ideXlab platform.
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Immunolocalization of Prosaposin in the epididymis of neu3-/-neu4-/- mice.
2018Co-Authors: Regiana Oliveira, Louis Hermo, Alexey V. Pshezhetsky, Carlos R. MoralesAbstract:Initial segment in (A) shows immunoreaction of Prosaposin in wild type mice. (B) shows negative control. Immunoreaction of Prosaposin in the initial segment (C), caput (D), proximal (E) and distal (F-H) cauda regions of the epididymis of neu3-/-neu4-/- mice immunostained with an anti-Prosaposin antibody. In (C), small punctate reactive lysosomes (circles) are noted in the supranuclear area of P cells and in thin elongated processes of a BC. Large highly reactive cells (arrowheads) are seen at the base of the epithelium, as also demonstrated in (C inset). In (D), a narrow cell (NC) is reactive as well as several small (arrows) and large basally located cells (arrowheads). In (E-H) several large highly reactive cells (arrowheads) are noted at the base of the epithelium. In (E-G), P cells reveal infranuclear reactive L at their base (arrows). In (G and H) large vacuoles (V) are seen in the epithelium as well as basally located cells. In (G inset), P and clear (C) cells are reactive for Prosaposin. Scale bar: 20μm.
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Prosaposin: a protein with differential sorting and multiple functions.
Histology and histopathology, 2014Co-Authors: L. Carvelli, Yuan Libin, Carlos R. MoralesAbstract:In eukaryotes the delivery of newly synthesized proteins to their final destination is dependent on a series of functionally distinct compartments, including the endoplasmic reticulum and the Golgi apparatus, which plays a role in posttranslational modification, sorting and distribution of proteins. Most cargo is sorted within, and exits from, the trans-Golgi network (TGN). Proteins delivered to lysosomes include hydrolytic enzymes and nonenzymic activator proteins. They are directed away from the cell surface by their binding to mannose-6-phosphate receptors (MPR). However, in I-cell disease, in which the MPR pathway is disrupted, the nonenzymic sphingolipid activator protein, Prosaposin, continue to traffic to lysosomes. This observation led to discovery of a new lysosomal sorting receptor, sortilin. The targeting Prosaposin to the lysosomes results from the interaction of its C-terminus with sortilin. Deletion of the Cterminus did not interfere with its secretion, but abolished its transport to the lysosomes. Mutational analysis revealed that the first half of the Prosaposin Cterminus contains a motif required for its binding to sortilin and its transport to the lysosomes. Prosaposin can be also secreted to the extracellular space as oligomers. Extracellular Prosaposin showed to exert a variety of responses in nervous tissues including the activation of G protein-coupled receptors and ERK phosphorylation. Lastly, Prosaposin has been found to be expressed in other fluids of the body such as pancreatic juice, bile, cerebrospinal fluid, milk and seminal fluid, indicating that Prosaposin is not only a house keeping lysosomal protein but an essential factor in the development and maintenance of the nervous systems and other systems of the body.
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Copyright � American Society of Andrology Breakthroughs in Andrology Targeted Disruption of the Mouse Prosaposin Gene Affects the Development of the Prostate Gland and Other Male Reproductive
2013Co-Authors: Carlos R. Morales, Qing Zhao, Mohamed El-alfy, Kunihiko SuzukiAbstract:ABSTRACT: The Prosaposin gene encodes a 65–70 kilodalton (kd) protein, which is secreted or targeted to lysosomes. In lysosomes, Prosaposin is the precursor of 4 activator proteins, designated saposins A, B, C, and D, which promote by acidic hydrolases, the degradation of glycosphingolipids with short oligosaccharide chains. Mutations of the Prosaposin gene have been linked to several lysosomal storage disorders. An animal model was recently developed by creating a null allele in embryonic stem cells through gene targeting in order to investigate the phenotypic diversity of Prosaposin mutations, the involvement of this protein in lysosomal storage diseases, and to develop potential therapeutic approaches. Mutant homozygous mice die at 35–40 days of age and neurological disorders contribute to their early death. Secreted Prosaposin is present in milk and in cerebrospinal and seminal fluids. In the nervous system, Prosaposin exhibits a trophic activity
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Prosaposin sorting is mediated by oligomerization.
Experimental cell research, 2011Co-Authors: Libin Yuan, Carlos R. MoralesAbstract:Abstract The compartmental nature of eukaryotic cells requires sophisticated mechanisms of protein sorting. Prosaposin, the precursor of four sphingolipid activator proteins, is transported from the trans -Golgi network (TGN) to lysosomes as a partially glycosylated (65 kDa) protein with high-mannose/hybrid oligosaccharides. Prosaposin is also found in the extracellular space where it is secreted as a fully glycosylated (70 kDa) protein composed of complex glycans. Although the trafficking of Prosaposin to lysosomes is known to be mediated by sortilin, the mechanism of secretion of this protein is still unknown. In this study, we report that Prosaposin may covalently aggregate into oligomers. Our results demonstrate that while Prosaposin oligomers are secreted into the extracellular space, monomeric Prosaposin remains inside the cell bound to sortilin. We also found that deletion of the C-terminus of Prosaposin, previously shown to block its lysosomal transport, did not abolish its oligomerization and secretion. On the other hand, elimination of the N-terminus and of each saposin domain inhibited its oligomerization and resulted in its retention as a fully glycosylated protein. In conclusion, we are reporting for the first time that oligomerization of Prosaposin is crucial for its entry into the secretory pathway.
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The inactivation of the sortilin gene leads to a partial disruption of Prosaposin trafficking to the lysosomes.
Experimental cell research, 2009Co-Authors: Jibin Zeng, Jesse Racicott, Carlos R. MoralesAbstract:Lysosomes are intracellular organelles which contain enzymes and activator proteins involved in the digestion and recycling of a variety of cellular and extracellular substances. We have identified a novel sorting receptor, sortilin, which is involved in the lysosomal trafficking of the sphingolipid activator proteins, Prosaposin and GM 2 AP, and the soluble hydrolases cathepsin D, cathepsin H, and acid sphingomyelinase. Sortilin belongs to a growing family of receptors with homology to the yeast Vps10 protein, which acts as a lysosomal sorting receptor for carboxypeptidase Y. In this study we examined the effects of the sortilin gene inactivation in mice. The inactivation of this gene did not yield any noticeable lysosomal pathology. To determine the existence of an alternative receptor complementing the sorting function of sortilin, we quantified the concentration of Prosaposin in the lysosomes of the nonciliated epithelial cells lining the efferent ducts. These cells were chosen because they express sortilin and have a large number of lysosomes containing Prosaposin. In addition, the nonciliated cells are known to endocytose luminal Prosaposin that is synthesized and secreted by Sertoli cells into the seminiferous luminal fluids. Consequently, the nonciliated cells are capable of targeting both exogenous and endogenous Prosaposin to the lysosomes. Using electron microscope immunogold labeling and quantitative analysis, our results demonstrate that inactivation of the sortilin gene produces a significant decrease of Prosaposin in the lysosomes. When luminal Prosaposin was excluded from the efferent ducts, the level of Prosaposin in lysosomes was even lower in the mutant mice. Nonetheless, a significant amount of Prosaposin continues to reach the lysosomal compartment. These results strongly suggest the existence of an alternative receptor that complements the function of sortilin and explains the lack of lysosomal storage disorders in the sortilin-deficient mice.
John S. O'brien - One of the best experts on this subject based on the ideXlab platform.
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Regulation of gene expression in response to brain injury: enhanced expression and alternative splicing of rat Prosaposin (SGP-1) mRNA in injured brain.
Journal of neurotrauma, 2003Co-Authors: Masao Hiraiwa, Roberta Misasi, Jian Liu, Cui-ying Wang, Toyoaki Yamauchi, Isao Hozumi, Takashi Inuzuka, John S. O'brienAbstract:Prosaposin, the precursor of saposins or saps, is an injury-repair protein that acts on both neurons and glia. Previous studies identified the Prosaposin gene as one of differentially expressed genes following nerve injury. In the present study, we investigated expression of Prosaposin mRNA in injured brain utilizing rat models of focal cerebral ischemia and cortical stab wound in order to explore the significance of Prosaposin in nerve injury. In ischemic brain, the level of Prosaposin mRNA was elevated greater than 400% over controls within 5 days after ischemic insults. Importantly, this induction was accompanied by a 9-base splicing consistent with the alternative Exon-8 splicing of human Prosaposin mRNA. In normal brain, two Prosaposin mRNA species with and without the 9-base insertion were expressed at a ratio of 85:15; however, this equilibrium reverted to 5:95 following ischemic injury. Similar inductions were observed in stab wound brains. Immunohistochemical staining and in situ hybridization demonstrated an enhanced signal distribution of Prosaposin mRNA and injury-induced Prosaposin protein around the lesion. The data suggest the expression and processing of Prosaposin mRNA may be crucially regulated not only for cerebral homeostasis but also during nerve regenerative and degenerative processes.
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Prosaposin is immunolocalized to muscle and prosaptides promote myoblast fusion and attenuate loss of muscle mass after nerve injury
Muscle & Nerve, 2001Co-Authors: Mario Rende, Andrew P. Mizisin, Nigel A. Calcutt, W. Marie Campana, Emanuela Brizi, Rosario Donato, Carlo Provenzano, Rosalinda Bruno, Robert S. Garrett, John S. O'brienAbstract:Prosaposin is the precursor of the saposins and has both neurotrophic and myelinotrophic activity in vitro and in vivo. Using an antibody specific for the holoprotein, an immunocytochemical survey demonstrated intense staining of adult rat skeletal, cardiac, and smooth muscle cells. Prosaposin immunoreactivity in muscle appears dependent on innervation, as denervated adult rat skeletal muscles showed decreased immunostaining that returned to normal levels after reinnervation. TX14(A), a peptide derived from the neurotrophic sequence of Prosaposin, attenuated the decline in muscle mass loss following nerve injury induced by a constricting ligature. In vitro, both L6 myoblasts and primary chick-embryo myoblasts showed similar Prosaposin immunopositivity, mainly in myotubes. TX14(A) induced a threefold increase in L6 myoblast fusion during early stages of differentiation without affecting cell proliferation. The fusion process was decreased in vitro in a dose-dependent fashion by addition of a neutralizing anti-Prosaposin antibody. These data suggest that, in addition to neurotrophic and myelinotrophic activities, Prosaposin has myotrophic properties. © 2001 John Wiley & Sons, Inc. Muscle Nerve 24: 799–808, 2001
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Prosaposin is immunolocalized to muscle and prosaptides promote myoblast fusion and attenuate loss of muscle mass after nerve injury
Muscle & nerve, 2001Co-Authors: Mario Rende, Andrew P. Mizisin, Nigel A. Calcutt, W. Marie Campana, Emanuela Brizi, Rosario Donato, Carlo Provenzano, Rosalinda Bruno, Robert S. Garrett, John S. O'brienAbstract:Prosaposin is the precursor of the saposins and has both neurotrophic and myelinotrophic activity in vitro and in vivo. Using an antibody specific for the holoprotein, an immunocytochemical survey demonstrated intense staining of adult rat skeletal, cardiac, and smooth muscle cells. Prosaposin immunoreactivity in muscle appears dependent on innervation, as denervated adult rat skeletal muscles showed decreased immunostaining that returned to normal levels after reinnervation. TX14(A), a peptide derived from the neurotrophic sequence of Prosaposin, attenuated the decline in muscle mass loss following nerve injury induced by a constricting ligature. In vitro, both L6 myoblasts and primary chick-embryo myoblasts showed similar Prosaposin immunopositivity, mainly in myotubes. TX14(A) induced a threefold increase in L6 myoblast fusion during early stages of differentiation without affecting cell proliferation. The fusion process was decreased in vitro in a dose-dependent fashion by addition of a neutralizing anti-Prosaposin antibody. These data suggest that, in addition to neurotrophic and myelinotrophic activities, Prosaposin has myotrophic properties.
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Prosaposin treatment induces PC12 entry in the S phase of the cell cycle and prevents apoptosis: activation of ERKs and sphingosine kinase
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 2001Co-Authors: Roberta Misasi, Luisa Di Marzio, Antonio Pavan, Maria Grazia Cifone, Maurizio Sorice, W. Marie Campana, Sabrina Molinari, Giuseppe M. Pontieri, John S. O'brienAbstract:We report that Prosaposin treatment induced extracellular signal-regulated kinases (ERKs) and sphingosine kinase activity, increased DNA synthesis, and prevented cell apoptosis. Prosaposin treatmen...
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Secretion of Prosaposin, a multifunctional protein, by breast cancer cells.
Biochimica et biophysica acta, 1999Co-Authors: W M Campana, John S. O'brien, Masao Hiraiwa, S. PattonAbstract:Western blotting and immunodetection with three antibodies were used to probe conditioned media of breast cancer cells (MDA231, MDA435, MCF-7) for Prosaposin, a lysosomal protein that occurs in milk. It was readily detected in media from these cells, and from that of an sv40-transformed mammary epithelial cell, HBL100, but not from medium of human neural tumor cells (SK-N-MC). In cultures of MCF-7 cells, the Prosaposin pattern of secretion over time closely resembled that of procathepsin D, another lysosomal protein occurring in milk. Supplementing medium with 17beta-estradiol (0. 1-100 nM) dose dependently increased secretion of both proteins after 48 h without changes in cell viability. The influence of 17beta-estradiol on secretion could play a role in the trophic activity of Prosaposin in cellular differentiation and cell death protection. In concert with other lysosomal proteins in the tumor environment, such as procathepsin D, Prosaposin may be a factor in eliminating barriers to tumor metastasis by facilitating hydrolysis of membrane glycolipids. The number of milk proteins known to be secreted by breast cancer cells is growing. There is evidence that at least some of these may be secreted in an endocrine manner in the normal, non-lactating breast.
Masao Hiraiwa - One of the best experts on this subject based on the ideXlab platform.
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gpr37 protein trafficking to the plasma membrane regulated by Prosaposin and gm1 gangliosides promotes cell viability
Journal of Biological Chemistry, 2014Co-Authors: Ebba Gregorsson Lundius, Masao Hiraiwa, Vladana Vukojevic, Ellen Hertz, Nikolas Stroth, Andreas Cederlund, Lars Terenius, Per SvenningssonAbstract:The subcellular distribution of the G protein-coupled receptor GPR37 affects cell viability and is implicated in the pathogenesis of parkinsonism. Intracellular accumulation and aggregation of GPR37 cause cell death, whereas GPR37 located in the plasma membrane provides cell protection. We define here a pathway through which the recently identified natural ligand, Prosaposin, promotes plasma membrane association of GPR37. Immunoabsorption of extracellular Prosaposin reduced GPR37tGFP surface density and decreased cell viability in catecholaminergic N2a cells. We found that GPR37tGFP partitioned in GM1 ganglioside-containing lipid rafts in the plasma membrane of live cells. This partitioning required extracellular Prosaposin and was disrupted by lipid raft perturbation using methyl-β-cyclodextrin or cholesterol oxidase. Moreover, complex formation between GPR37tGFP and the GM1 marker cholera toxin was observed in the plasma membrane. These data show functional association between GPR37, Prosaposin, and GM1 in the plasma membrane. These results thus tie together the three previously defined components of the cellular response to insult. Our findings identify a mechanism through which the receptor's natural ligand and GM1 may protect against toxic intracellular GPR37 aggregates observed in parkinsonism.
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11. Individual saposins
2013Co-Authors: Yasuo Kishimoto, Masao Hiraiwa, John S, Saposin A. A, Saposin B. B, Saposin C. C, Saposin D. DAbstract:derived from a common precursor protein, Prosaposin. These mature saposins, as well as Prosaposin, activate several lysosomal hydrolases involved in the metabolism of various sphingolipids. All four saposins are structurally similar to one another including placement of six cysteines, a glycosylation site, and conserved prolines in identical positions. In spite of the structural similarities, the specificity and mode of activation of sphingolipid hydrolases differs among individual saposins. Saposins appear to be lysosomal proteins, exerting their action upon lysosomal hydrolases. Prosaposin is a 70 kDa glycoprotein containing four domains, one for each saposin, placed in tandem. Prosaposin is proteolytically processed to saposins A, B, C and D, apparently within lysosomes. However, Prosaposin also exists as an integral membrane protein not destined for lysosomal entry and exists uncleaved in many biological fluids such as seminal plasma, human milk, and cerebrospinal fluid, where it appears to have a different function. The physiological significance of saposins is underlined by their accumulation in tissues of lysosomal storage disease patients and the Occurrence of sphingolipidosis due to mutations in the Prosaposin gene. This review presents an overview of the occurrence, structure and function of these saposin proteins.-Kishimoto, Y., M. Hiraiwa, and J. S. O'Brien. Saposins: structure, function, distribution, and molecular genetics. J. Lipid Res. 1992. 33: 1255
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Neurotrophic signalling pathway triggered by Prosaposin in PC12 cells occurs through lipid rafts.
The FEBS journal, 2008Co-Authors: Maurizio Sorice, Masao Hiraiwa, Tina Garofalo, Luisa Di Marzio, Vincenzo Mattei, Sabrina Molinari, Vincenzo Tasciotti, Laura Ciarlo, Roberta MisasiAbstract:Prosaposin is a neurotrophic factor that has been demonstrated to mediate trophic signalling events in different cell types; it distributes to surface membranes of neural cells and also exists as a secreted protein in different body fluids. Prosaposin was demonstrated to form tightly bound complexes with a variety of gangliosides, and a functional role has been suggested for ganglioside–Prosaposin complexes. In this work, we provide evidence that exogenous Prosaposin triggers a signal cascade after binding to its target molecules on lipid rafts of pheochromocytoma PC12 cell plasma membranes, as revealed by scanning confocal microscopy and linear sucrose gradient analysis. In these cells, Prosaposin is able to induce extracellular signal-regulated kinase phosphorylation, sphingosine kinase activation, and consequent cell death prevention, acting through lipid rafts. These findings point to the role of lipid rafts in the Prosaposin-triggered signalling pathway, thus supporting a role for this factor as a new component of the multimolecular signalling complex involved in the neurotrophic response.
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Molecular mechanism for neuro-protective effect of Prosaposin against oxidative stress: Its regulation of dimeric transcription factor formation
Biochimica et biophysica acta, 2008Co-Authors: Takashi Ochiai, Masao Hiraiwa, Roberta Misasi, Yuka Takenaka, Yukako Kuramoto, Masakazu Kasuya, Kanemasa Fukuda, Masahiko Kimura, Hiroshi Shimeno, Shinji SoedaAbstract:Prosaposin triggers G-protein-coupled receptor (GPCR)-mediated protein kinase B (Akt)/extracellular signal-regulated kinase (ERK) phosphorylation cascades to exert its neurotrophic and myelinotrophic activity capable of preventing neural cell death and promoting neural proliferation and glial differentiation. In the present study, we investigated the down-stream neurotrophic signaling mechanism of Prosaposin by which rat pheochromocytoma (PC-12) cells are protected from cell death induced by oxidative stress. When PC-12 cells were exposed to H2O2, the cells underwent abrupt shrinkage followed by apoptosis. Prosaposin treatment at as low as 1 nM protected PC-12 cells from cell death by the oxidative stress with the activation of an ERK phosphorylation cascade. Simultaneously, Prosaposin blocked the oxidative stress induced-Akt phosphorylation that acts on the down-stream of caspase-3 activation. A MEK inhibitor, PD98059, or a phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, abolished the survival effect of Prosaposin on the oxidative stress-induced cell death. Furthermore, Prosaposin blocked the oxidative stress-induced phosphorylations of c-Jun N-terminal kinase (JNK) and p38 stress-activated protein kinase. We further investigated the effect of Prosaposin treatment on the phosphorylation of activating protein-1 (AP-1) complex components, c-Jun and activating transcription factor (ATF)-3. Western blot analysis demonstrated that Prosaposin treatment at 100 ng/ml decreased the levels of c-Jun and ATF-3 induced by H2O2 stimulation. Our results suggest that Prosaposin aids survival of PC-12 cells from oxidative stress not only by reducing the phosphorylation levels of JNK and p38, but also by regulating the c-Jun/AP-1 pathway.
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Prosaposin is a novel androgen regulated gene in prostate cancer cell line lncap
Journal of Cellular Biochemistry, 2007Co-Authors: Shahriar Koochekpour, Masao Hiraiwa, Ruoxiang Wang, Nathalie Delorme, G A Grabowski, Zoran Culig, Ardalan MinokadehAbstract:Androgen-regulated genes (ARG) are implicated in normal and neoplastic growth of the prostate. Recently, we reported genomic amplification and/or overexpression of a previously known neurotrophic factor, Prosaposin, in androgen-independent (AI) or metastatic prostate cancer (PCa) cells and tissues. Prosaposin and/or its known active molecular derivatives (e.g., saposin C) function as a pluripotent growth factor with diverse biological activities that favor malignant phenotypes in PCa cells. In addition, Prosaposin or saposin C upregulates androgen receptor (AR) and AR-target genes (i.e., prostate-specific antigen, Probasin) expression and activity in LNCaP cells. Here, we examined Prosaposin as an ARG. We report that DHT treatment of LNCaP cells increases Prosaposin expression. In addition, we demonstrate androgen-responsiveness of Prosaposin promoter and AR occupancy to a hormone-responsive element located in the proximal region of the Prosaposin promoter. Our data for the first time identify Prosaposin as an ARG. This observation, together with the pleiotropic growth factor activity of Prosaposin, might suggest a role for this molecule in AR-dependent progression of prostate cancer at its early or late AI-state. J. Cell. Biochem. 101: 631–641, 2007. © 2006 Wiley-Liss, Inc.
Gregory A. Grabowski - One of the best experts on this subject based on the ideXlab platform.
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In Vivo Roles of RORa and Sp4 in the Regulation of
2016Co-Authors: Murine Prosaposin Gene, Ying Sun, Peng Jin, Gregory A. GrabowskiAbstract:Prosaposin has a central role in intracellular glycosphingolipid catabolism and also has extracellular func-tions. This locus is regulated temporally and spatially. The highest mRNA expression occurs in the central nervous system (CNS) and reproductive system. In vitro, the CNS-expressed proteins Sp4 and RORa bind to Sp1 and RORE sites within a 310-bp fragment directly upstream of the transcription start site. These tran-scription factors exhibit negative cooperativity in vitro for Prosaposin expression. Mice deficient in RORa and Sp4 (Staggerer [Sg2 /2] and Sp4 knockout [Sp4 KO], respectively) containing selected Prosaposin promoter deletion transgenes were used in comparative expression studies to evaluate this negative cooperativity in vivo. Constructs containing the RORE or Sp1/U cluster alone were independently stimulatory. Deletion of the Sp1/U site led to a decrease in reporter activity only in the cerebellum of Sg2/2 mice. The deletion of RORE and Sp1/U sites did alter the increase of reporter activity in the brain and eye, but not in the spinal cord, of Sg2/2 mice. These results indicate that Sp4 and RORa play minor and major roles, respectively, in regional ex-pression of the Prosaposin locus in the brain, whereas expression in the spinal cord is independent of RORa
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Role of Sp Proteins and RORa in Transcription Regulation of
2016Co-Authors: Murine Prosaposin, Ying Sun, Peng Jin, Gregory A. GrabowskiAbstract:Prosaposin is the precursor of four low molecular weight sphingolipid-activating proteins (SAPs) or sa-posins. These four proteins function as intracellular ac-tivators of several lysosomal enzymes involved in the degradation of glycosphingolipids, and Prosaposin itself has neurite outgrowth effects. Expression of Prosaposin is regulated in a temporal and spatial manner with ex-pression in specific brain neurons and visceral cell types. Here a major regulatory fragment was character-ized within 310 bp 5 * to the transcription start site. Us-ing electrophoretic mobility shift assay (EMSA) and DNA footprinting, members of the Sp family (Sp1, Sp3, and Sp4), the orphan nuclear receptor (RORa), and an unknown transcription factor (U; TGGGGGAG) were shown to bind to this region. To evaluate the role of suc
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the role of udp glc glycoprotein glucosyltransferase 1 in the maturation of an obligate substrate Prosaposin
Journal of Cell Biology, 2010Co-Authors: Bradley R Pearse, Gregory A. Grabowski, Taku Tamura, Johan C Sunryd, Randal J Kaufman, Daniel N HebertAbstract:An endoplasmic reticulum (ER) quality control system assists in efficient folding and disposal of misfolded proteins. N-linked glycans are critical in these events because their composition dictates interactions with molecular chaperones. UDP-glucose:glycoprotein glucosyltransferase 1 (UGT1) is a key quality control factor of the ER. It adds glucoses to N-linked glycans of nonglucosylated substrates that fail a quality control test, supporting additional rounds of chaperone binding and ER retention. How UGT1 functions in its native environment is poorly understood. The role of UGT1 in the maturation of glycoproteins at basal expression levels was analyzed. Prosaposin was identified as a prominent endogenous UGT1 substrate. A dramatic decrease in the secretion of Prosaposin was observed in ugt1−/− cells with Prosaposin localized to large juxtanuclear aggresome-like inclusions, which is indicative of its misfolding and the essential role that UGT1 plays in its proper maturation. A model is proposed that explains how UGT1 may aid in the folding of sequential domain–containing proteins such as Prosaposin.
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Temporal gene expression profiling reveals CEBPD as a candidate regulator of brain disease in Prosaposin deficient mice.
BMC neuroscience, 2008Co-Authors: Ying Sun, David P. Witte, Li Jia, Michael T. Williams, Matt Zamzow, Huimin Ran, Brian Quinn, Bruce J. Aronow, Charles V. Vorhees, Gregory A. GrabowskiAbstract:Background Prosaposin encodes, in tandem, four small acidic activator proteins (saposins) with specificities for glycosphingolipid (GSL) hydrolases in lysosomes. Extensive GSL storage occurs in various central nervous system regions in mammalian Prosaposin deficiencies.
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Combined saposin C and D deficiencies in mice lead to a neuronopathic phenotype, glucosylceramide and α-hydroxy ceramide accumulation, and altered Prosaposin trafficking
Human molecular genetics, 2007Co-Authors: Ying Sun, Junko Matsuda, David P. Witte, Matt Zamzow, Huimin Ran, Brian Quinn, Gregory A. GrabowskiAbstract: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.
Klaus Harzer - One of the best experts on this subject based on the ideXlab platform.
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Model SV40-transformed fibroblast lines for metabolic studies of human Prosaposin and acid ceramidase deficiencies.
Clinica chimica acta; international journal of clinical chemistry, 1997Co-Authors: Martine Chatelut, Barbara C. Paton, Klaus Harzer, Yasuo Kishimoto, John S. O'brien, Helen Christomanou, Jean Feunteun, Marie-thérèse Pieraggi, Jean-pierre Basile, Jean-claude ThiersAbstract:Abstract Skin fibroblasts from patients with Farber disease (acid ceramidase deficiency) and from two siblings of the only known family affected with Prosaposin deficiency were transformed by transfection with a plasmid carrying the SV40 large T antigen. The Prosaposin-deficient transformed cell lines conserved their original metabolic defects, and in particular they were free of detectable immunoreactivity when using anti-saposin B and anti-saposin C antisera. Ultrastructurally, the cells contained heterogeneous lysosomal storage products. As found for their parental cell lines, the SV40-transformed fibroblasts exhibited deficient in vitro activities of lysosomal ceramidase and β-galactosylceramidase, but a normal activity of acid sphingomyelinase. As observed for SV40-transformed fibroblasts from Farber disease, degradation of radioactive glucosylceramide or low density lipoprotein-associated radiolabelled sphingomyelin by the Prosaposin-deficient cells in situ showed a clear impairment in the turnover of lysosomal ceramide. Ceramide storage in Prosaposin-deficient cells was also demonstrated by ceramide mass determination. In contrast to acid ceramidase deficient cells, both the accumulation of ceramide and the reduced in vitro activity of acid ceramidase in cells from Prosaposin deficiency could be corrected by addition of purified saposin D. The data confirm that Prosaposin is required for lysosomal ceramide degradation, but not for sphingomyelin turnover. The SV40-transformed fibroblasts will be useful for pathophysiological studies on human Prosaposin deficiency.
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Further evidence that human lysosomal sialidase is not derived from Prosaposin. Prosaposin biosynthesis and ganglioside sialidase studies in Prosaposin- and sialidase-deficient fibroblast lines.
Biological Chemistry Hoppe-Seyler, 1994Co-Authors: Barbara C. Paton, Klaus Harzer, Helge Renate Schneider-jakob, Jürgen Kopitz, Alt Poulos, Michael CantzAbstract:Human lysosomal sialidase has been considered by Potier et al. (Potier M., Lamontagne S., Michaud L., Tranchemontagne J. (1990) Biochem Biophys Res Commun 173, 449-456) to be a processing product of Prosaposin, the common precursor of the saposin proteins A, B, C, and D that function as activators in the lysosomal degradation of sphingolipids. We tested this hypothesis on cultured fibroblasts of patients with Prosaposin deficiency, a neurolipidosis caused by a complete lack of synthesis of the Prosaposin protein, by determining their lysosomal and, for comparison, their plasma membrane sialidase activities. Using both the natural substrate ganglioside GM3 and the synthetic compound 4-methylumbelliferyl neuraminate, we found the lysosomal sialidase activity in the Prosaposin-deficient cells to be in the normal control range; normal values were also found for the plasma membrane sialidase. In fibroblasts from patients with a genetic deficiency of the lysosomal sialidase (sialidosis), on the other hand, biosynthesis and processing of Prosaposin were unimpaired. Our findings therefore show no precursor/product relationship between Prosaposin and the lysosomal or the plasma membrane sialidase.
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Prosaposin deficiency: further characterization of the sphingolipid activator protein-deficient sibs
Human Genetics, 1993Co-Authors: V. Bradová, F Smid, W. Roggendorf, B Ulrich-bott, Barbara C. Paton, Klaus HarzerAbstract:Sphingolipid activator protein (SAP) deficiency, previously described in two sibs and shown to be caused by the absence of the common saposin precursor (Prosaposin), was further characterized by biochemical lipid and enzyme studies and by ultrastructural analysis. The 20 week old fetal sib had increased concentrations of neutral glycolipids, including mono-, di-, tri- and tetrahexosylceramide, in liver, kidney and cultured skin fibroblasts compared with the controls. Glucosylceramide and lactosylceramide were particularly elevated. The kidney of the affected fetus showed additional increases in the concentration of sulphatide, galactosylceramide and digalactosylceramide. Free ceramide was stored in the liver and kidney, and G_M3 and G_M2 gangliosides were elevated in the liver, but not the brain, of the fetus. Phospholipids, however, were normal in the affected fetus. In the liver biopsy of the propositus, who later died at 16 weeks of age, only a few lipids could be studied. Glucosylceramide, dihexosylceramide and ceramide were elevated in agreement with our previous study. Enzyme studies were undertaken using detergent free liposomal substrate preparations and fibroblast extracts. The sibs' β-glucocerebrosidase and β-galactocerebrosidase activities were clearly reduced, but their sphingomyelinase activities were normal. The normal activity of the latter enzyme and the almost normal tissue concentration of sphingomyelin in Prosaposin deficiency suggest that the Prosaposin derived SAPs are not required for sphingomyelinase activity in vivo. In keeping with the biochemical findings, skin biopsies from the sibs showed massive lysosomal storage with a vesicular and membranous ultrastructure. The function of SAPs in sphingolipid degradation and the role of SAPs for enzyme activity in vitro are discussed. In addition, the similarity in neutral glycolipid accumulations in Niemann Pick disease type C and in Prosaposin deficiency are noted. The phenotype of the Prosaposin deficient sibs resembled acute neuronopathic (type 2) Gaucher disease more than Farber disease in several aspects, but their genotype was unique.
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Prosaposin deficiency further characterization of the sphingolipid activator protein deficient sibs multiple glycolipid elevations including lactosylceramidosis partial enzyme deficiencies and ultrastructure of the skin in this generalized sphingolip
Human Genetics, 1993Co-Authors: V. Bradová, B Ulrichbott, F Smid, W. Roggendorf, Barbara C. Paton, Klaus HarzerAbstract:Sphingolipid activator protein (SAP) deficiency, previously described in two sibs and shown to be caused by the absence of the common saposin precursor (Prosaposin), was further characterized by biochemical lipid and enzyme studies and by ultrastructural analysis. The 20 week old fetal sib had increased concentrations of neutral glycolipids, including mono-, di-, tri- and tetrahexosylceramide, in liver, kidney and cultured skin fibroblasts compared with the controls. Glucosylceramide and lactosylceramide were particularly elevated. The kidney of the affected fetus showed additional increases in the concentration of sulphatide, galactosylceramide and digalactosylceramide. Free ceramide was stored in the liver and kidney, and GM3 and GM2 gangliosides were elevated in the liver, but not the brain, of the fetus. Phospholipids, however, were normal in the affected fetus. In the liver biopsy of the propositus, who later died at 16 weeks of age, only a few lipids could be studied. Glucosylceramide, dihexosylceramide and ceramide were elevated in agreement with our previous study. Enzyme studies were undertaken using detergent free liposomal substrate preparations and fibroblast extracts. The sibs' β-glucocerebrosidase and β-galactocerebrosidase activities were clearly reduced, but their sphingomyelinase activities were normal. The normal activity of the latter enzyme and the almost normal tissue concentration of sphingomyelin in Prosaposin deficiency suggest that the Prosaposin derived SAPs are not required for sphingomyelinase activity in vivo. In keeping with the biochemical findings, skin biopsies from the sibs showed massive lysosomal storage with a vesicular and membranous ultrastructure. The function of SAPs in sphingolipid degradation and the role of SAPs for enzyme activity in vitro are discussed. In addition, the similarity in neutral glycolipid accumulations in Niemann Pick disease type C and in Prosaposin deficiency are noted. The phenotype of the Prosaposin deficient sibs resembled acute neuronopathic (type 2) Gaucher disease more than Farber disease in several aspects, but their genotype was unique.
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Metabolism of G_M1 ganglioside in cultured skin fibroblasts: anomalies in gangliosidoses, sialidoses, and sphingolipid activator protein (SAP, saposin) 1 and Prosaposin deficient disorders
Human Genetics, 1992Co-Authors: B. Schmid, Barbara C. Paton, Konrad Sandhoff, Klaus HarzerAbstract:Cultured skin fibroblasts from controls and patients with lysosomal storage diseases were loaded with G_M1 ganglioside that had been labelled with tritium in its ceramide moiety. After a 65-h or 240-h incubation, a large percentage of this ganglioside remained undegraded in G_M1 gangliosidoses, whereas in the other storage diseases studied, one of its metabolites accumulated by 2–4 fold relative to controls. Labelled G_M2 ganglioside accumulated in 4 variants of G_M2 gangliosidosis, whereas labelled G_M3 ganglioside accumulated in sialidosis, galactosialidoses and sphingolipid activator protein 1 (SAP-1, saposin B) and Prosaposin (saposin A, B, C an D) deficient lipidoses. The reduced degradation of G_M3 ganglioside in the SAP-1 and Prosaposin deficiencies was attributed to the deficient function of SAP-1. The Prosaposin deficient cells also showed a reduced re-utilization of radioactive metabolites from G_M1 ganglioside (i.e. sphingosine and fatty acid) for phospholipid biosynthesis compared with fibroblasts from the SAP-1 deficient patient or normal controls. This anomaly was ascribed to the previously shown defect in ceramide degradation in Prosaposin deficiency.
