Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Previous studies from our laboratory have demonstrated the presence of two integral proteins with glycosidase activity in the plasma membrane of Drosophila melanogaster spermatozoa and we have suggested that these enzymes might have a role in sperm-egg binding. In this study the glycosidases have been purified and characterized. We have evidenced the presence of three distinct enzymes, two beta-N-acetylhexosaminidase isoforms, named HEX 1 and HEX 2, and an alpha-mannosidase. The molecular size of the native enzymes estimated by gel filtration was 158 kDa for beta-hexosaminidases and 317 kDa for alpha-mannosidase. SDS-PAGE showed that HEX 1 and HEX 2 are dimers formed by subunits with different molecular sizes, whereas alpha-mannosidase consists of three subunits with different molecular weights. All the enzymes are terminally glycosylated. Characterization of the purified enzymes included their 4-methylumbelliferyl-substrate preferences, kinetic properties, inhibitor constants and thermal stability. On the basis of substrate specificity, kinetics and the results of inhibition studies, beta-hexosaminidases appear to differ from each other. HEX 1 and HEX 2 are similar to mammalian isoenzyme A and isoenzyme B, respectively. These findings represent the first report on the characterization of sperm proteins that are potentially involved in interactions with the egg in Insects.
Insect Biochem Mol Biol 2002 Aug
PMID:Purification and characterization of the plasma membrane glycosidases of Drosophila melanogaster spermatozoa. 1211 Mar

Asthma is a complex condition in which exposure to environmental antigens induces inflammatory reactions in the airway characterized by activation of mast cells and eosinophils. Mast cells are known to be the main effector cells in eliciting IgE-mediated allergic response. These cells secrete various substances that perpetuate inflammation and provoke airway smooth muscle (ASM) contraction. A newly recognized addition to the repertoire of FcepsilonRI-mediated signaling events is the activation of sphingosine kinase leading to the generation of the potent sphingolipid mediator, sphingosine-1-phosphate (S1P) from sphingosine. S1P secretion by the lung significantly increases after challenge with an allergen, adding this sphingolipid metabolite to the variety of mediators that are released during an allergic reaction [FASEB J. 15 (2001) 1212]. Indeed, similar to previous reports, we found that FcepsilonRI cross-linking not only increased cellular levels of S1P, it also markedly enhanced its secretion from rat basophilic leukemia RBL-2H3 cells. Moreover, S1P induced degranulation of RBL and bone marrow derived mast cells (BMMCs) cells as determined by hexosaminidase release. Treatment of BMMCs with the sphingosine kinase inhibitors, DL-threo-dihydrosphingosine and dimethylsphingosine, reduced IgE/Ag stimulated histamine release. RT-PCR analysis demonstrated that these mast cells express S1P receptors EDG-1 and EDG-5 but not EDG-3, EDG-6 or EDG-8 transcripts. Further studies are needed to determine whether IgE triggering results in transactivation of EDG-1 or EDG-5 present on mast cells and whether this is a critical event for mast cell activation.
Mol Immunol 2002 Sep
PMID:The roles of sphingosine-1-phosphate in asthma. 1221 90

The underlying mechanisms by which lead ions produce their deleterious effects prior to the onset of clinical symptoms are incompletely understood. This study aimed to assess lead-induced cell toxicity mechanisms by focusing on the effects of the metal on cell growth, DNA synthesis, cellular ATP, intracellular hexosaminidase activity and lysosomal function, and examine the possible cytoprotective role of fetal calf serum (FCS). Several human dermal cultured fibroblast lines were exposed to Pb (400 microM) for 1-6 days with 2, 5, and 10% FCS. The earliest toxic effect of Pb was significant inhibition of DNA synthesis after 24 h direct exposure; this harmful effect was not progressive during the first 3 days, but worsened clearly on the 4th day regardless of the FCS concentration. Atime-dependent depletion of intracellularATP content was also caused by ionic lead, thereby compromising the cell energy charge which precedes cell death. Fibroblast growth was progressively and significantly inhibited from day 2 onwards; the greatest noxious effect was observed in the presence of 2% FCS: 49% reduction in cell proliferation after 5 days. Lead salts produced loss of cell adhesion to the culture dish which worsened from the 2nd day and was more pronounced when FCS in growth medium was decreased. Toxic actions on lysosomal membrane integrity provoked a decrease in neutral red uptake (NRU) which was exposure time-dependent and more marked with 2% FCS. In contrast, increased relative NRU (to 20% at 4 days), suggestive of endocytosis-induced lysosome enlargement, was observed in Pb-exposed cells. Intracellular hexosaminidase activity was not negatively affected until 5 days after exposure to Pb salts. FCS had a significant cytoprotective effect on Pb-induced toxicity.
Mol Cell Biochem 2002 Aug
PMID:In vitro lead-induced cell toxicity and cytoprotective activity of fetal calf serum in human fibroblasts. 1223 86

N-Acetyl-beta-D-hexosaminidase (beta-HexNAc'ase) (EC 3.2.1.52) was purified from rice seeds (Oryza sativa L. var. Dongjin) using ammonium sulfate (80%) precipitation, Sephadex G-150, CM-Sephadex, and DEAE-Sephadex chromatography, sequentially. The activities were separated into 7 fractions (Fsub1;- F7sub7) by CM-Sephadex chromatography. Among them, F6 was further purified to homogeneity with a 13.0% yield and 123.3 purification-fold. The molecular mass was estimated to be about 52 kDa on SDS-PAGE and 37.4 kDa on Sephacryl S- 300 gel filtration. The enzyme catalyzed the hydrolysis of both p-nitrophenyl-N-acetyl-beta-D-glucosaminide (pNP-GlcNAc) and p-nitrophenyl-N-acetyl-beta-D-galactosaminide (pNPGalNAc) as substrates, which are typical properties of beta-HexNAc'ase. The ratio of the pNP-GlcNAc'ase activity to the pNP-GalNAc'ase activity was 4.0. However, it could not hydrolyze chitin, chitosan, pNP-beta-glucopyranoside, or pNP-beta-galactopyranoside. The enzyme showed K(M), V(max) and K(cat) for pNP-GlcNAc of 1.65mM, 79.49mM min(1), and 4.79 x 10(6) min(1), respectively. The comparison of kinetic values for pNPGlcNAc and pNP-GalNAc revealed that the two enzyme activities are associated with a single binding site. The purified enzyme exhibited optimum pH and temperature for pNPGlcNAc of 5.0 and 50 degrees C, respectively. The enzyme activity for pNP-GlcNAc was stable at pH 5.0-5.5 and 20-40 degrees C. The enzyme activity was completely inhibited at a concentration of 0.1 mM HgCl(2) and AgNO(3), suggesting that the intact thiol group is essential for activity. Chloramine T completely inhibited the activity, indicating the possible involvement of methionines in the mechanism of the enzyme.
J Biochem Mol Biol 2002 May 31
PMID:Purification and characterization of beta-N-acetylhexosaminidase from rice seeds. 1229 15

Measurement of the hydrolysis of specific fluorogenic substrates by spectrophotometry as well as the substrate activity-SDS-PAGE gel analysis of the chitinolytic activity in Aedes aegypti guts showed that both chitinase and beta-N-acetylglucosaminidase are present and physiologically active. Both enzymes were present even in guts from unfed insects, but the activities increased rapidly after feeding on blood or an artificial protein-free diet. Chitinase activity was predominantly of the 'endo'-type, reaching its maximum activity at 36 h and then declining to very low levels after the degradation of the peritrophic matrix (PM). Chitinase assay in gels after SDS-PAGE was a very sensitive method that allowed us to detect two chitinases with distinct molecular weights in the mosquito gut. Hydrolysis of a chitinase-specific substrate by chitinolytic activities in the mosquito guts was inhibited by allosamidin, a potent chitinase inhibitor. Allosamidin treatment led to the formation of an atypical thick PM, while the addition of exogenous chitinase completely blocked its formation. This chitinolytic system appears to operate both on the formation and degradation of the PM. Since the PM is involved in pathogen invasion, these results are important in facilitating a search for mechanisms that can block pathogen development in the mosquito vector.
Insect Biochem Mol Biol 2002 Dec
PMID:Presence of chitinase and beta-N-acetylglucosaminidase in the Aedes aegypti. a chitinolytic system involving peritrophic matrix formation and degradation. 1242 24

In secretory carrier membrane proteins (SCAMPs), the most conserved structural segment is between transmembrane spans 2 and 3, facing the cytosol. A synthetic peptide, CWYRPIYKAFR (E peptide), from this segment of SCAMP2 potently inhibits exocytosis in permeabilized neuroendocrine (PC12) cells. E peptide blocked discharge of (35)S-labeled secretogranin with the same structural selectivity and potency as observed for hexosaminidase secretion in mast cells. SCAMPs 1 and 2 are concentrated primarily on intracellular membranes in PC12 cells. Both, however, are found on plasma membranes, but neither is present on large dense-core vesicles. Yet, large dense-core vesicles marked by secretogranin attach to plasma membranes at foci containing SCAMP2 along with syntaxin1 and complexin at putative cell-surface docking/fusion sites. Regulated overexpression of SCAMP2 with point mutations in its E peptide but not of normal SCAMP2 caused dose-dependent inhibition of depolarization-induced secretion. The SCAMP2 mutants also inhibited secretion stimulated by elevated calcium. Inhibition was largely overcome by adding lysophosphatidylcholine to the medium at concentrations that do not otherwise affect secretion. Although overexpression of normal or mutant SCAMP2 slightly inhibits endocytosis, this effect does not appear to be related to the specific effect of the mutant SCAMP on stimulated exocytosis. Thus, SCAMP2 not only colocalizes with fusion sites but also appears to have an essential function in granule exocytosis through actions mediated by its E peptide-containing domain.
Mol Biol Cell 2002 Dec
PMID:Role of secretory carrier membrane protein SCAMP2 in granule exocytosis. 1247 51

In humans, two major beta-hexosaminidase isoenzymes exist: Hex A and Hex B. Hex A is a heterodimer of subunits alpha and beta (60% identity), whereas Hex B is a homodimer of beta-subunits. Interest in human beta-hexosaminidase stems from its association with Tay-Sachs and Sandhoff disease; these are prototypical lysosomal storage disorders resulting from the abnormal accumulation of G(M2)-ganglioside (G(M2)). Hex A degrades G(M2) by removing a terminal N-acetyl-D-galactosamine (beta-GalNAc) residue, and this activity requires the G(M2)-activator, a protein which solubilizes the ganglioside for presentation to Hex A. We present here the crystal structure of human Hex B, alone (2.4A) and in complex with the mechanistic inhibitors GalNAc-isofagomine (2.2A) or NAG-thiazoline (2.5A). From these, and the known X-ray structure of the G(M2)-activator, we have modeled Hex A in complex with the activator and ganglioside. Together, our crystallographic and modeling data demonstrate how alpha and beta-subunits dimerize to form either Hex A or Hex B, how these isoenzymes hydrolyze diverse substrates, and how many documented point mutations cause Sandhoff disease (beta-subunit mutations) and Tay-Sachs disease (alpha-subunit mutations).
J Mol Biol 2003 Apr 11
PMID:Crystal structure of human beta-hexosaminidase B: understanding the molecular basis of Sandhoff and Tay-Sachs disease. 1266 33

Sandhoff disease is a severe neurodegenerative disorder with visceral involvement caused by mutations in the HEXB gene coding for the beta subunit of the lysosomal hexosaminidases A and B. HEXB mutations result in the accumulation of undegraded substrates such as GM2 and GA2 in lysosomes. We evaluated the efficacy of cationic liposome-mediated plasmid gene therapy using the Sandhoff disease mouse, an animal model of a human lysosomal storage disease. The mice received a single intravenous injection of two plasmids, encoding the human alpha and beta subunits of hexosaminidase cDNAs. As a result, 10-35% of normal levels of hexosaminidase expression, theoretically therapeutic levels, were achieved in most visceral organs, but not in the brain, 3 days after injection with decreased levels by day 7. Histochemical staining confirmed widespread enzyme activity in visceral organs. Both GA2 and GM2 were reduced by almost 10% and 50%, respectively, on day 3, and by 60% and 70% on day 7 compared with untreated age-matched Sandhoff disease mice. Consistent with the biochemical results, a reduction in GM2 was observed in liver cells histologically as well. These initial findings support further development of the plasmid gene therapy against lysosomal diseases with visceral pathology.
J Mol Med (Berl) 2003 Mar
PMID:Plasmid-based gene transfer ameliorates visceral storage in a mouse model of Sandhoff disease. 1268 27

Human lysosomal beta-hexosaminidases are dimeric enzymes composed of alpha and beta-chains, encoded by the genes HEXA and HEXB. They occur in three isoforms, the homodimeric hexosaminidases B (betabeta) and S (alphaalpha), and the heterodimeric hexosaminidase A (alphabeta), where dimerization is required for catalytic activity. Allelic variations in the HEXA and HEXB genes cause the fatal inborn errors of metabolism Tay-Sachs disease and Sandhoff disease, respectively. Here, we present the crystal structure of a complex of human beta-hexosaminidase B with a transition state analogue inhibitor at 2.3A resolution (pdb 1o7a). On the basis of this structure and previous studies on related enzymes, a retaining double-displacement mechanism for glycosyl hydrolysis by beta-hexosaminidase B is proposed. In the dimer structure, which is derived from an analysis of crystal packing, most of the mutations causing late-onset Sandhoff disease reside near the dimer interface and are proposed to interfere with correct dimer formation. The structure reported here is a valid template also for the dimeric structures of beta-hexosaminidase A and S.
J Mol Biol 2003 May 02
PMID:The X-ray crystal structure of human beta-hexosaminidase B provides new insights into Sandhoff disease. 1270 24

The glycosylation of serine and threonine residues with beta-O-linked N-acetylglucosamine (O-GlcNAc) is an abundant posttranslational modification of nuclear and cytoplasmic proteins in multicellular eukaryotes. This highly dynamic glycosylation/deglycosylation of protein is catalyzed by the nucleocytoplasmic enzymes, UDP-G1cNAc: polypeptide O-beta-N-acetylglucosaminyltransferase (OGT)/O-beta-N-acetylglucosaminidase. OGT is required for embryonic stem cell viability and mouse ontogeny, thus O-GlcNAc is essential for the life of eukaryotes. The gene encoding O-GlcNAcase maps to a locus important to late-onset Alzheimer's disease. All known O-GlcNAc-modified proteins are also phosphoproteins that form reversible multimeric protein complexes. There is both a global and often site-specific reciprocal relationship between O-GlcNAc and O-phosphate in many cellular responses to stimuli. Thus, regulation of the protein-protein interaction(s) and/or protein function by dynamic glycosylation/phosphorylation has been hypothesized. In this chapter, we will review the current status of dynamic glycosylation/phosphorylation of several important regulatory proteins including c-Myc, estrogen receptors, Sp1, endothelial nitric oxide synthase, and beta-catenin. Various aspects of subcellular localization, association with binding partners, activity, and/or turnover of these proteins appear to be regulated by dynamic glycosylation/ phosphorylation in response to cellular signals or stages.
Prog Nucleic Acid Res Mol Biol 2003
PMID:Dynamic interplay between O-glycosylation and O-phosphorylation of nucleocytoplasmic proteins: a new paradigm for metabolic control of signal transduction and transcription. 1288 16


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