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Biomedical subjects

G A Grabowski

Publications and source records attributed to G A Grabowski.

At least 19 recordsLinked to original sources
PubMed2007

Prosaposin is a novel androgen-regulated gene in prostate cancer cell line LNCaP.

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.

Androgens↗
PubMed2005

Novel mutations in type 2 Gaucher disease in Chinese and their functional characterization by heterologous expression.

We investigated 10 unrelated Chinese patients with type 2 Gaucher disease and performed ex vivo expression for the novel mutations to characterize their functional defects. These patients were diagnosed by enzymatic assays and clinicopathologic features over the past five years in a national centre in China. Genomic DNA was sequenced by a two-stage PCR approach for mutations in the functional GBA gene. Novel mutations were expressed with baculovirus-transfected Sf21 cells. Six novel mutations were found (in traditional nomenclature): P122L, Y363C, N382K, L383R, L385P, and M416V. Review of reported mutations indicated clustering of type 2 mutations in three regions of the GBA gene. Expression of novel mutations revealed that the enzyme defect could arise from one of two mechanisms: loss of catalytic activity (Y363C and M416V) or enzyme instability (P122L and N382K).

Age of Onset↗
PubMed2002

Gaucher disease: Perspectives on a prototype lysosomal disease.

Gaucher disease is an autosomal recessive trait and the most common lysosomal storage disease. The pathogenesis evolves from the diminished activity of the lysosomal hydrolase, acid beta-glucosidase and the resultant accumulation of glucosylceramide within lysosomes. The pathogenic mechanisms are poorly understood. During the past 2 decades, progress has been made in understanding the biochemical basis and molecular biology of the disease, but more fundamental knowledge is required to relate these advances to the cell and whole body phenotypes. Despite this lack of understanding, enzyme replacement therapy has proved a successful and effective management for Gaucher disease. However, basic details of this therapeutic efficacy require elucidation. Here, we review the current state of the molecular pathogenesis and provide our perspective of some major issues for continued advances in this prototype lysosomal storage disease.

Animals↗
PubMed2001

Enzyme therapy for lysosomal acid lipase deficiency in the mouse.

Lysosomal acid lipase (LAL) is the critical enzyme for the hydrolysis of the triglycerides (TG) and cholesteryl esters (CE) delivered to lysosomes. Its deficiency produces two human phenotypes, Wolman disease (WD) and cholesteryl ester storage disease (CESD). A targeted disruption of the LAL locus produced a null (lal( -/-)) mouse model that mimics human WD/CESD. The potential for enzyme therapy was tested using mannose terminated human LAL expressed in Pichia pastoris (phLAL), purified, and administered by tail vein injections to lal( -/-) mice. Mannose receptor (MR)-dependent uptake and lysosomal targeting of phLAL were evidenced ex vivo using competitive assays with MR-positive J774E cells, a murine monocyte/macrophage line, immunofluorescence and western blots. Following (bolus) IV injection, phLAL was detected in Kupffer cells, lung macrophages and intestinal macrophages in lal( -/-) mice. Two-month-old lal( -/-) mice received phLAL (1.5 U/dose) or saline injections once every 3 days for 30 days (10 doses). The treated lal( -/-) mice showed nearly complete resolution of hepatic yellow coloration; hepatic weight decreased by approximately 36% compared to PBS-treated lal( -/-) mice. Histologic analyses of numerous tissues from phLAL-treated mice showed reductions in macrophage lipid storage. TG and cholesterol levels decreased by approximately 50% in liver, 69% in spleen and 50% in small intestine. These studies provide feasibility for LAL enzyme therapy in human WD and CESD.

Animals↗
PubMed2001

Differential membrane interactions of saposins A and C: implications for the functional specificity.

Saposins are small, heat-stable glycoprotein activators of lysosomal glycosphingolipid hydrolases that derive from a single precursor, prosaposin, by proteolytic cleavage. Three of these saposins (B, C, and D) share common structural features including a lack of tryptophan, a single glycosylation sequence, the presence of three conserved disulfide bonds, and a common multiamphipathic helical bundle motif. Saposin A contains an additional glycosylation site and a single tryptophan. The oligosaccharides on saposins are not required for in vitro activation functions. Saposins A and C were produced in Escherichia coli to contain single tryptophans at various locations to serve as intrinsic fluorescence reporters, i.e. as topological probes, for interaction with phospholipid membranes. Maximum emission shifts, aqueous and solid quenching, and resonance energy transfer were quantified by fluorescence spectroscopy. Amphipathic helices at the amino- and carboxyl termini of saposins A and C were shown to insert into the lipid bilayer to about five carbon bond lengths. In comparison, the middle region of saposins A or C were either embedded in the bilayer or solvent-exposed, respectively. Conformational changes of saposin C induced by phosphatidylserine interaction suggested the reorientation of functional helical domains. Differential interaction models are proposed for the membrane-bound saposins A and C. By site-directed mutagenesis of saposin A and C, their membrane topological structures were correlated with their activation effects on acid beta-glucosidase. These findings show that proper orientation of the middle segment of saposin C to the outside of the membrane surface is critical for its specific and multivalent interaction with acid beta-glucosidase. Such membrane interactions and orientations of the saposins determine the proximity of their activation and/or binding sites to lysosomal hydrolases or lipoid substrates.

Amino Acid Sequence↗
PubMed2001

Phenotypic correction of lipid storage and growth arrest in wolman disease fibroblasts by gene transfer of lysosomal acid lipase.

Wolman disease is a lethal lysosomal storage disease due to deficiency of lysosomal acid lipase (LAL). Wolman disease is characterized by pronounced hepatic involvement while neurological symptoms are uncommon, making Wolman disease an attractive candidate for liver-directed gene therapy. This study was performed to test the effects of gene replacement in fibroblasts lacking LAL, using a recombinant adenovirus encoding the human LAL cDNA (AdhLAL). Human fibroblasts from a Wolman disease patient were infected with AdhLAL and showed a dose-dependent increase in LAL protein and activity up to 5-fold above levels in control fibroblasts. Furthermore, 72 hr after infection with AdhLAL there was a dose-dependent correction of the severe lipid storage phenotype of Wolman disease fibroblasts. Electron microscopy confirmed significant correction of the lysosomal lipid storage in AdhLAL-infected Wolman disease fibroblasts at the ultrastructural level. Intravenous injection of AdhLAL into wild-type mice resulted in a 13.5-fold increase in hepatic LAL activity, and overexpression of LAL was not associated with toxic side effects. These data demonstrate high-level lysosomal expression of recombinant LAL in vitro and in vivo and show the feasibility of gene therapeutic strategies for the treatment of Wolman disease.

Adenoviridae↗
PubMed2001

Temporal and spatial expression of murine acid beta-glucosidase mRNA.

The hydrolysis of glucosylceramide (GC) to ceramide and glucose requires the action of the lysosomal enzyme, acid beta-glucosidase (GCase), encoded by gba in the mouse. Gaucher disease, an autosomal recessive disorder, results from the inherited deficiency of this enzyme. Although enzyme activity is present in all mammalian tissues, the patterns of mRNA expression have not been explored. In situ hybridization analyses of mouse embryonic, newborn, and adult tissues were conducted to evaluate the spectrum of gba mRNA expression. Signals were present in all tissues and cell types. Distinct patterns of differential expression were identified in specific tissues and cell types, and at defined developmental stages. Differential expression was first observed around E14 in the intestinal tract, kidneys, skeletal system, and skin. At E18, moderate intensity signals were in adipocytes of brown fat and pancreatic cells. Differential expression remained in skin, bone, and the GI tract postnatally. In the postnatal and adult animals increasing expression was observed throughout the CNS, esophageal epithelium, intestinal villi, pancreas, and thymus and lymph node capsular cells. These tissue-, cell-, and developmental stage-specific variations of the gba mRNA level indicate major developmentally regulated changes in the expression pattern of gba in the late gestational period and postnatally.

Animals↗
PubMed2001

In vivo roles of RORalpha and Sp4 in the regulation of murine prosaposin gene.

Prosaposin has a central role in intracellular glycosphingolipid catabolism and also has extracellular functions. 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 RORalpha bind to Sp1 and RORE sites within a 310-bp fragment directly upstream of the transcription start site. These transcription factors exhibit negative cooperativity in vitro for prosaposin expression. Mice deficient in RORalpha and Sp4 (Staggerer [Sg(-/-)] 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 Sg(-/-) 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 Sg(-/-) mice. These results indicate that Sp4 and RORalpha play minor and major roles, respectively, in regional expression of the prosaposin locus in the brain, whereas expression in the spinal cord is independent of RORalpha.

Animals↗
PubMed2001

Ectopic expression of alpha1,6 fucosyltransferase in mice causes steatosis in the liver and kidney accompanied by a modification of lysosomal acid lipase.

The alpha1,6 fucosyltransferase (alpha1,6 FucT) catalyzes the transfer of a fucose from GDP-fucose to the innermost GlcNAc residue of N-linked glycans via an alpha1,6 linkage. alpha1,6 FucT was overexpressed in transgenic mice under the control of a combined cytomegalovirus and chicken beta-actin promoter. Histologically numerous small vacuoles, in which lipid droplets had accumulated, were observed in hepatocytes and proximal renal tubular cells. Electron microscopic studies showed that the lipid droplets were membrane-bound and apparently localized within the lysosomes. Cholesterol esters and triglycerides were significantly increased in liver and kidney of the transgenic mice. Liver lysosomal acid lipase (LAL) activity was significantly lower in the transgenic mice compared to the wild mice, whereas LAL protein level, which was detected immunochemically, was increased, indicating that the specific activity of LAL was much lower in the transgenic mice. In all of the transgenic and nontransgenic mice examined, the activity of liver LAL was negatively correlated with the level of alpha1,6 FucT activity. As evidenced by lectin and immunoblot analysis, LAL was found to be more fucosylated in the transgenic mice, suggesting that the aberrant fucosylation of LAL causes an accumulation of inactive LAL in the lysosomes. Such an accumulation of inactive LAL could be a likely cause for a steatosis in the lysosomes of the liver and kidney in the case of the alpha1,6 FucT transgenic mice.

Animals↗
PubMed2001

Lysosomal acid lipase-deficient mice: depletion of white and brown fat, severe hepatosplenomegaly, and shortened life span.

Lysosomal acid lipase (LAL) is essential for the hydrolysis of triglycerides (TG) and cholesteryl esters (CE) in lysosomes. A mouse model created by gene targeting produces no LAL mRNA, protein, or enzyme activity. The lal-/- mice appear normal at birth, survive into adulthood, and are fertile. Massive storage of TG and CE is observed in adult liver, adrenal glands, and small intestine. The age-dependent tissue and gross progression in this mouse model are detailed here. Although lal-/- mice can be bred to give homozygous litters, they die at ages of 7 to 8 months. The lal-/- mice develop enlargement of a single mesenteric lymph node that is full of stored lipids. At 6;-8 months of age, the lal-/- mice have completely absent inguinal, interscapular, and retroperitoneal white adipose tissue. In addition, brown adipose tissue is progressively lost. The plasma free fatty acid levels are significantly higher in lal-/- mice than age-matched lal+/+ mice, and plasma insulin levels were more elevated upon glucose challenge. Energy intake was also higher in lal-/- male mice, although age-matched body weights were not significantly altered from age-matched lal+/+ mice. Early in the disease course, hepatocytes are the main storage cell in the liver; by 3;-8 months, the lipid-stored Kupffer cells progressively fill the liver. The involvement of macrophages throughout the body of lal-/- mice provide evidence for a critical nonappreciated role of LAL in cellular cholesterol and fatty acid metabolism, adipocyte differentiation, and fat mobilization.

Adipocytes↗
PubMed2000

Prosaposin: promoter analysis and central-nervous-system-preferential elements for expression in vivo.

The expression of prosaposin is temporally and spatially regulated at the transcriptional and post-translational levels. In vitro, the mouse prosaposin promoter contains functional RORE [retinoic acid-receptor-related orphan receptor alpha subunit (RORalpha)-binding element], Sp1 and U (unknown) sites within 310 bp directly 5' to the transcription start site and additional elements within 2400 bp 5' to the transcription start site. To elucidate promoter regions important to tissue-preferential expression in vivo, transgenic mice were created with 5'-flanking deletions of the prosaposin gene fused to a luciferase reporter. Nearly exclusive expression was observed in cerebrum, cerebellum and eyes of adult transgenic mice containing constructs with 234-310 bp of 5'-flanking DNA. This central nervous system (CNS) expression was due to the presence of RORE and overlapping Sp1 sites in this region. Internal deletion of RORE and the Sp1 cluster from the longer constructs with 2400 bp of 5'-flanking DNA significantly diminished expression in the CNS. The appearance of substantial visceral tissue (e.g. liver, spleen, lung, kidney, thymus and heart) expression was obtained with transgenic mice bearing constructs with 742-2400 bp of 5'-flanking DNA. The cellular localization of luciferase reporter-gene expression from these constructs corresponded closely with that for prosaposin. These results define important CNS and visceral regulatory regions in the promoter in vivo and may be sufficient to account for the majority of prosaposin's tissue-preferential expression.

Animals↗
PubMed2000

Reconstitution of TCP80/NF90 translation inhibition activity in insect cells.

Acid beta-glucosidase (GCase) is the enzyme deficient in Gaucher disease, an inherited metabolic prototype for enzyme and gene therapy. An 80-kDa mammalian cytoplasmic protein (TCP80/NF90) was discovered to interact with the GCase mRNA coding region and inhibit its translation in vitro and ex vivo. Human TCP80/NF90 is identical to NF90, an IL-2 enhancer protein, and MPP4, an M-phase phosphoprotein. The interaction of recombinant TCP80/NF90 with GCase mRNA was evaluated using the baculovirus/Sf9 insect cell system since these cells lack this protein. Purified recombinant and isolated mammalian cytoplasmic TCP80/NF90 had identical functions including binding of coding regions of selected RNAs and inhibition of their in vitro translation. Individual baculoviruses containing the human TCP80/NF90 cDNA (vSf9/TCP80) and GCase cDNA (vSf9/GCase) were used to co-infect Sf9 cells. The presence of preformed TCP80/NF90 significantly (>87%) inhibited wild-type GCase mRNA translation in these cells, but baculovirus containing a mutant GCase did not. Sf9 cells co-infected with vSf9/TCP80 showed a major reduction of GCase RNA polysome association. These results show that the multifunctional protein, TCP80/NF90, can function in vivo as a translation inhibitory protein and include alterations of mRNA binding to polysomes as a component of its mechanism of action.

Animals↗
PubMed2000

Fate and sorting of acid beta-glucosidase in transgenic mammalian cells.

Gaucher disease (GD) is associated with mutations at the acid beta-glucosidase (GCase) locus and the resultant defective activity of the enzyme product. GCase is a membrane-associated glycoprotein that requires detergents for extraction and phospholipid interfaces for full catalytic activity. Normal human fibroblasts and overexpressing transgenic cell lines were used to evaluate the intracellular disappearance, degradation, and secretion of human GCase, including GD fibroblasts and C2C12 cells transduced with MFG-GCase retrovirus and CHO cells stably transfected with the tetracycline transactivation conditional expression system (tet-CHO-GCase). Compared to HF, the disappearance of GCase from the transgenic cells was 12-30 times greater, and had degradative and secretory components. In tet-CHO-GCase cells the majority of GCase was secreted. Intracellular degradation occurred in compartments sensitive to monensin and brefeldin A, and the ALLN or leupeptin protease inhibitors, i.e., ER, Golgi, and lysosomes. In tet-CHO-GCase cells, GCase degradation and secretion rates were inversely related to expression level. Saponin permeabilization analyses of tet-CHO-GCase cells showed that a majority of GCase was soluble, with a rapid disappearance via secretion and degradation. A progressively increasing proportion of GCase became saponin insoluble with a t(1/2) = 2-3 h. Intracellular saponin-soluble and -insoluble GCases were degraded with t(1/2) approximately 2 and 14 h, respectively. Confocal microscopy showed colocalization of glycosylated or unglycosylated GCase with LAMP-2, an integral lysosomal membrane protein, to vesicular bodies. These studies show that GCase secretion was N-linked glycosylation dependent, whereas sorting to and membrane attachment in the lysosome were N-linked glycosylation independent.

Animals↗
PubMed2000

Saposins A, B, C, and D in plasma of patients with lysosomal storage disorders.

BACKGROUND: Early diagnosis of lysosomal storage disorders (LSDs), before the onset of irreversible pathology, will be critical for maximum efficacy of many current and proposed therapies. To search for potential markers of LSDs, we measured saposins A, B, C, and D in patients with these disorders. METHODS: Four time-delayed fluorescence immunoquantification assays were used to measure each of the saposins in plasma from 111 unaffected individuals and 334 LSD-affected individuals, representing 28 different disorders. RESULTS: Saposin A was increased above the 95th centile of the control population in 59% of LSD patients; saposins B, C, and D were increased in 25%, 61%, and 57%, respectively. Saposins were increased in patients from several LSD groups that in previous studies did not show an increase of lysosome-associated membrane protein-1 (LAMP-1). CONCLUSION: Saposins may be useful markers for LSDs when used in conjunction with LAMP-1.

Adolescent↗