Mannose 6-phosphate/insulin-like growth factor II receptors have been characterized in hepatocyte... more Mannose 6-phosphate/insulin-like growth factor II receptors have been characterized in hepatocytes and Kupffer cells isolated from adult rat liver. Affinity labeling with [125I]insulin-like growth factor II revealed a protein of Mr 250,000 in both cell types. Labeling was inhibited by an antiserum against the mannose 6-phosphate/insulin-like growth factor II receptor. In Kupffer cells, [125I]insulin-like growth factor II was also cross-linked to a second protein of Mr 130,000. In both cell types, insulin-like growth factor II was 10 times more potent than insulin-like growth factor I in displacing [125I]insulin-like growth factor II from its receptor. The mannose 6-phosphate-specific uptake of [125I]arylsulfatase A via the mannose 6-phosphate/insulin-like growth factor II receptor was inhibited by insulin-like growth factor II and antibodies against the receptor, but was not affected by insulin-like growth factor I, insulin or transforming growth factor beta 1. Cell surface iodination followed by immunoprecipitation of the mannose 6-phosphate/insulin-like growth factor II receptor showed that expression of the mannose 6-phosphate/insulin-like growth factor II receptors at the plasma membrane was increased two-fold by insulin-like growth factor II. These results suggest that binding of insulin-like growth factor II to the mannose 6-phosphate/insulin-like growth factor II receptor blocks the binding and uptake of mannose 6-phosphate-containing lysosomal enzymes and may be directly involved in a co-ordinate regulation of ligand uptake from plasma into hepatocytes and Kupffer cells.
Sialic acid acetyl esterase (SIAE) removes acetyl moieties from the hydroxyl groups in position 9... more Sialic acid acetyl esterase (SIAE) removes acetyl moieties from the hydroxyl groups in position 9 and 4 of sialic acid. Recently a dispute has been opened on its association to autoimmunity. In order to get new insights on human SIAE biology and to clarify its seemingly contradictory molecular properties we combined in silico characterization, phylogenetic analysis and homology modeling with cellular studies in COS7 cells. Genomic and phylogenetic analysis revealed that in most tissues only the "long" isoform, originally referred to lysosomal sialic acid esterase (Lse), is detected. Using the homology modeling approach we predicted a model of SIAE 3D structure, which fulfills the topological features of SGNH-hydrolase family. In addition, the model and site directed mutagenesis experiments allowed the definition of the residues involved in catalysis. SIAE transient expression revealed that the protein is glycosylated and is active in vitro as an esterase with a pH optimum ...
Several mammalian sialidases have been cloned so far and here we describe the identification and ... more Several mammalian sialidases have been cloned so far and here we describe the identification and expression of a new member of the human sialidase gene family. The NEU4 gene, identified by searching sequence databases for entries showing homologies to the human cytosolic sialidase NEU2, maps in 2q37 and encodes a 484-residue protein. The polypeptide contains all the typical sialidase amino
GM1 ganglioside carrying a fluorescent fatty acid in substitution of the natural one, has been ad... more GM1 ganglioside carrying a fluorescent fatty acid in substitution of the natural one, has been administered to cultured Madin-Darby canine kidney (MDCK) cells for different pulse times (0.5–24 h), and its metabolic fate was followed. The fluorescent GM2, asialo-GM2, asialo-GM1 and ceramide were the only detectable metabolites. The complete absence of fluorescent GM3 is consistent with the presence in these
The pheochromocytoma cells are a well-known model for studying the nerve growth factor (NGF)-indu... more The pheochromocytoma cells are a well-known model for studying the nerve growth factor (NGF)-induced molecular changes during the differentiation process. The involvement of sphingomyelin (SM) was studied using the fluorescent analogue of ceramide, i.e. N-lissamine rhodaminyl-(12-aminododecanoyl) D-erythro-sphingosine (C12-LRh-Cer). This fluorescent analogue is metabolically active and can be used to follow the biosynthesis of SM in intact cells. NGF induces a
The NEU1 gene is the first identified member of the human sialidases, glycohydrolitic enzymes tha... more The NEU1 gene is the first identified member of the human sialidases, glycohydrolitic enzymes that remove the terminal sialic acid from oligosaccharide chains. Mutations in NEU1 gene are causative of sialidosis (MIM 256550), a severe lysosomal storage disorder showing autosomal recessive mode of inheritance. Sialidosis has been classified into two subtypes: sialidosis type I, a normomorphic, late-onset form, and sialidosis type II, a more severe neonatal or early-onset form. A total of 50 causative mutations are reported in HGMD database, most of which are missense variants. To further characterize the NEU1 gene and identify new functionally relevant protein isoforms, we decided to study its genetic variability in the human population using the data generated by two large sequencing projects: the 1000 Genomes Project (1000G) and the NHLBI GO Exome Sequencing Project (ESP). Together these two datasets comprise a cohort of 7595 sequenced individuals, making it possible to identify rare variants and dissect population specific ones. By integrating this approach with biochemical and cellular studies, we were able to identify new rare missense and frameshift alleles in NEU1 gene. Among the 9 candidate variants tested, only two resulted in significantly lower levels of sialidase activity (p<0.05), namely c.650T>C and c.700G>A. These two mutations give rise to the amino acid substitutions p.V217A and p.D234N, respectively. NEU1 variants including either of these two amino acid changes have 44% and 25% residual sialidase activity when compared to the wild-type enzyme, reduced protein levels and altered subcellular localization. Thus they may represent new, putative pathological mutations resulting in sialidosis type I. The in silico approach used in this study has enabled the identification of previously unknown NEU1 functional alleles that are widespread in the population and could be tested in future functional studies.
Tobacco smoking is responsible for death of many people each year and increases the risk of devel... more Tobacco smoking is responsible for death of many people each year and increases the risk of developing numerous disorders, particularly cardiovascular disease and cancer. Among the components of cigarette smoke, nicotine is known to excert proatherosclerotic, prothrombotic and proangiogenic effects on vascular endothelial cells. The current study was designed to investigate the mechanisms by which nicotine induces endothelial dysfunction and further to examine whether melatonin protects against nicotine-induced vasculopathy. Four groups of male rats (controls, melatonin-treated, nicotine treated [100 microg/mL in drinking water], and nicotine plus melatonin [5 mg/kg/day] treated) were used in this study. After 28 days all the animals were killed by decapitation and the aorta was removed. We evaluated the hydroxyproline content, and the different expression of proteins involved in several types of stress (ERK1/2), in fibrosis (TGF-beta1, NF-kappaB) and in recruitment of circulating leukocytes onto the vessel wall, including intercellular adhesion molecule-1 (ICAM-1) and vascular cellular adhesion molecule-1 (VCAM-1). These metabolic pathways are important in the development of nicotine-induced atherosclerosis and hypertension. Our results show that nicotine induces marked structural and functional alterations in the aorta. Nicotine receptor binding results in activation and phosphorylation of ERK 1/2. This enzyme, in turn, activates both TGF-beta1 and NF-kappaB; they stimulate respectively the synthesis of type I collagen, responsible of fibrosis, and moreover ICAM-1, VCAM-1 and reactive oxygen species. Based on these findings, melatonin is able to minimize the negative effects of nicotine by blocking the activation of ERK and the other signalling pathways in which this enzyme is involved.
The present report is a comparative study on physico-chemical properties and metabolism of three ... more The present report is a comparative study on physico-chemical properties and metabolism of three different Lissamine-Rhodamine (LRh) derivatives of ceramide (Cer), with twelve (C12–LRh–Cer), six (C6–LRh–Cer) acyl linker spacers or with the fluorescent probe directly linked to the sphingosine moiety (C0–LRh–Cer). Transfer kinetics of the LRh–ceramides from water dispersion, albumin complex and donor vesicles to acceptor liposomes followed a double-exponential curve. Among the three ceramides, C12–LRh–Cer showed the lowest mobility. All ceramides were taken up by human fibroblasts and, with the exception of C0–LRh–Cer which was not metabolised, fluorescent sphingomyelin (SM) was the only detectable metabolite. Despite its low incorporation, C12–LRh–Cer was converted to the corresponding C12–LRh–SM with an efficiency roughly three times higher than that of C6–LRh–Cer. ‘In vitro’ enzymatic assays using cell homogenate gave similar results. These data indicate that C12–LRh–Cer is the best mimic of the natural ceramide.
Mannose 6-phosphate/insulin-like growth factor II receptors have been characterized in hepatocyte... more Mannose 6-phosphate/insulin-like growth factor II receptors have been characterized in hepatocytes and Kupffer cells isolated from adult rat liver. Affinity labeling with [125I]insulin-like growth factor II revealed a protein of Mr 250,000 in both cell types. Labeling was inhibited by an antiserum against the mannose 6-phosphate/insulin-like growth factor II receptor. In Kupffer cells, [125I]insulin-like growth factor II was also cross-linked to a second protein of Mr 130,000. In both cell types, insulin-like growth factor II was 10 times more potent than insulin-like growth factor I in displacing [125I]insulin-like growth factor II from its receptor. The mannose 6-phosphate-specific uptake of [125I]arylsulfatase A via the mannose 6-phosphate/insulin-like growth factor II receptor was inhibited by insulin-like growth factor II and antibodies against the receptor, but was not affected by insulin-like growth factor I, insulin or transforming growth factor beta 1. Cell surface iodination followed by immunoprecipitation of the mannose 6-phosphate/insulin-like growth factor II receptor showed that expression of the mannose 6-phosphate/insulin-like growth factor II receptors at the plasma membrane was increased two-fold by insulin-like growth factor II. These results suggest that binding of insulin-like growth factor II to the mannose 6-phosphate/insulin-like growth factor II receptor blocks the binding and uptake of mannose 6-phosphate-containing lysosomal enzymes and may be directly involved in a co-ordinate regulation of ligand uptake from plasma into hepatocytes and Kupffer cells.
Sialic acid acetyl esterase (SIAE) removes acetyl moieties from the hydroxyl groups in position 9... more Sialic acid acetyl esterase (SIAE) removes acetyl moieties from the hydroxyl groups in position 9 and 4 of sialic acid. Recently a dispute has been opened on its association to autoimmunity. In order to get new insights on human SIAE biology and to clarify its seemingly contradictory molecular properties we combined in silico characterization, phylogenetic analysis and homology modeling with cellular studies in COS7 cells. Genomic and phylogenetic analysis revealed that in most tissues only the "long" isoform, originally referred to lysosomal sialic acid esterase (Lse), is detected. Using the homology modeling approach we predicted a model of SIAE 3D structure, which fulfills the topological features of SGNH-hydrolase family. In addition, the model and site directed mutagenesis experiments allowed the definition of the residues involved in catalysis. SIAE transient expression revealed that the protein is glycosylated and is active in vitro as an esterase with a pH optimum ...
Several mammalian sialidases have been cloned so far and here we describe the identification and ... more Several mammalian sialidases have been cloned so far and here we describe the identification and expression of a new member of the human sialidase gene family. The NEU4 gene, identified by searching sequence databases for entries showing homologies to the human cytosolic sialidase NEU2, maps in 2q37 and encodes a 484-residue protein. The polypeptide contains all the typical sialidase amino
GM1 ganglioside carrying a fluorescent fatty acid in substitution of the natural one, has been ad... more GM1 ganglioside carrying a fluorescent fatty acid in substitution of the natural one, has been administered to cultured Madin-Darby canine kidney (MDCK) cells for different pulse times (0.5–24 h), and its metabolic fate was followed. The fluorescent GM2, asialo-GM2, asialo-GM1 and ceramide were the only detectable metabolites. The complete absence of fluorescent GM3 is consistent with the presence in these
The pheochromocytoma cells are a well-known model for studying the nerve growth factor (NGF)-indu... more The pheochromocytoma cells are a well-known model for studying the nerve growth factor (NGF)-induced molecular changes during the differentiation process. The involvement of sphingomyelin (SM) was studied using the fluorescent analogue of ceramide, i.e. N-lissamine rhodaminyl-(12-aminododecanoyl) D-erythro-sphingosine (C12-LRh-Cer). This fluorescent analogue is metabolically active and can be used to follow the biosynthesis of SM in intact cells. NGF induces a
The NEU1 gene is the first identified member of the human sialidases, glycohydrolitic enzymes tha... more The NEU1 gene is the first identified member of the human sialidases, glycohydrolitic enzymes that remove the terminal sialic acid from oligosaccharide chains. Mutations in NEU1 gene are causative of sialidosis (MIM 256550), a severe lysosomal storage disorder showing autosomal recessive mode of inheritance. Sialidosis has been classified into two subtypes: sialidosis type I, a normomorphic, late-onset form, and sialidosis type II, a more severe neonatal or early-onset form. A total of 50 causative mutations are reported in HGMD database, most of which are missense variants. To further characterize the NEU1 gene and identify new functionally relevant protein isoforms, we decided to study its genetic variability in the human population using the data generated by two large sequencing projects: the 1000 Genomes Project (1000G) and the NHLBI GO Exome Sequencing Project (ESP). Together these two datasets comprise a cohort of 7595 sequenced individuals, making it possible to identify rare variants and dissect population specific ones. By integrating this approach with biochemical and cellular studies, we were able to identify new rare missense and frameshift alleles in NEU1 gene. Among the 9 candidate variants tested, only two resulted in significantly lower levels of sialidase activity (p<0.05), namely c.650T>C and c.700G>A. These two mutations give rise to the amino acid substitutions p.V217A and p.D234N, respectively. NEU1 variants including either of these two amino acid changes have 44% and 25% residual sialidase activity when compared to the wild-type enzyme, reduced protein levels and altered subcellular localization. Thus they may represent new, putative pathological mutations resulting in sialidosis type I. The in silico approach used in this study has enabled the identification of previously unknown NEU1 functional alleles that are widespread in the population and could be tested in future functional studies.
Tobacco smoking is responsible for death of many people each year and increases the risk of devel... more Tobacco smoking is responsible for death of many people each year and increases the risk of developing numerous disorders, particularly cardiovascular disease and cancer. Among the components of cigarette smoke, nicotine is known to excert proatherosclerotic, prothrombotic and proangiogenic effects on vascular endothelial cells. The current study was designed to investigate the mechanisms by which nicotine induces endothelial dysfunction and further to examine whether melatonin protects against nicotine-induced vasculopathy. Four groups of male rats (controls, melatonin-treated, nicotine treated [100 microg/mL in drinking water], and nicotine plus melatonin [5 mg/kg/day] treated) were used in this study. After 28 days all the animals were killed by decapitation and the aorta was removed. We evaluated the hydroxyproline content, and the different expression of proteins involved in several types of stress (ERK1/2), in fibrosis (TGF-beta1, NF-kappaB) and in recruitment of circulating leukocytes onto the vessel wall, including intercellular adhesion molecule-1 (ICAM-1) and vascular cellular adhesion molecule-1 (VCAM-1). These metabolic pathways are important in the development of nicotine-induced atherosclerosis and hypertension. Our results show that nicotine induces marked structural and functional alterations in the aorta. Nicotine receptor binding results in activation and phosphorylation of ERK 1/2. This enzyme, in turn, activates both TGF-beta1 and NF-kappaB; they stimulate respectively the synthesis of type I collagen, responsible of fibrosis, and moreover ICAM-1, VCAM-1 and reactive oxygen species. Based on these findings, melatonin is able to minimize the negative effects of nicotine by blocking the activation of ERK and the other signalling pathways in which this enzyme is involved.
The present report is a comparative study on physico-chemical properties and metabolism of three ... more The present report is a comparative study on physico-chemical properties and metabolism of three different Lissamine-Rhodamine (LRh) derivatives of ceramide (Cer), with twelve (C12–LRh–Cer), six (C6–LRh–Cer) acyl linker spacers or with the fluorescent probe directly linked to the sphingosine moiety (C0–LRh–Cer). Transfer kinetics of the LRh–ceramides from water dispersion, albumin complex and donor vesicles to acceptor liposomes followed a double-exponential curve. Among the three ceramides, C12–LRh–Cer showed the lowest mobility. All ceramides were taken up by human fibroblasts and, with the exception of C0–LRh–Cer which was not metabolised, fluorescent sphingomyelin (SM) was the only detectable metabolite. Despite its low incorporation, C12–LRh–Cer was converted to the corresponding C12–LRh–SM with an efficiency roughly three times higher than that of C6–LRh–Cer. ‘In vitro’ enzymatic assays using cell homogenate gave similar results. These data indicate that C12–LRh–Cer is the best mimic of the natural ceramide.
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Papers by Roberto Bresciani