Biomedical subjects

A Hasilik

Publications and source records attributed to A Hasilik.

At least 55 records · Page 3Linked to original sources

PubMed1990

Brefeldin A prevents uncovering but not phosphorylation of the recognition marker in cathepsin D.

Brefeldin A (BFA) has been shown to inhibit transiently the subcellular transport of cathepsin D (Oda & Nishimura (1989) Biochem. Biophys. Res. Commun. 163, 220-225). We studied the effect of this antibiotic on processing of the phosphorylated oligosaccharides in cathepsin D in human promonocytes U937. In the presence of the drug the phosphorylation of cathepsin D precursor continued at a diminished rate. The phosphorylated oligosaccharides in cathepsin D comprised mono- and bis-phosphorylated forms. The relative amounts of the two species were not changed in the presence of BFA. The uncovering of the phosphate groups and the proteolytic processing of the phosphorylated precursor were abolished. In an in vitro assay the uncovering enzyme, N-acetylglucosamine-1-phosphodiester N-acetylglucosaminidase was not inhibited by BFA. We suggest that this drug interrupts the traffic between the compartments containing N-acetylglucosaminyl phosphotransferase and N-acetylglucosamine-1-phosphodiester N-acetylglucosaminidase.

Biological Transport↗

PubMed1989

Calcitriol enhances transcriptional activity of lysozyme and cathepsin D genes in U937 promonocytes.

Calcitriol (1,25-dihydroxyvitamin D3) has been reported to enhance the rate of synthesis of lysozyme [Rouis, Thomopoulos, Louache, Testa, Heroy & Titeux, (1985) Exp. Cell Res. 157, 539-543] and of cathepsin D [Stein, Braulke, von Figura & Hasilik (1987) Biol. Chem. Hoppe-Seyler 368, 413-418] in human promonocytes U937. In this study we show hat the hormone enhances the steady-state levels of cathepsin D and lysozyme mRNAs. The enhancement of the latter but not of the former is strongly inhibited by cycloheximide. The degradation rates of cathepsin D and lysozyme mRNAs as revealed in the presence of actinomycin D are not significantly affected by the treatment of the cells with calcitriol. In nuclei prepared from calcitriol-treated cells, an enhanced rate of synthesis of cathepsin D and lysozyme mRNA precursors is observed. These results suggest that in human promonocytes the rate of the transcription of cathepsin D and lysozyme genes is enhanced in the presence of calcitriol and that the regulation by the hormone of the transcription of lysozyme gene involves a short-lived or an induced protein.

Calcitriol↗

PubMed1989

Immunoprecipitation of labeled antigens with Eupergit C1Z.

Eupergit C1Z, a nonporous sedimentable acrylic polymer is shown to be a convenient solid support for isolation of antigens with high purity from small amounts of detergent-solubilized cells. Several applications of Eupergit C1Z coupled with antibodies and anti-antibodies are described, and compared with two other methods of immunoprecipitation.

Acrylic Resins↗

PubMed1989

Synthesis and localization of myeloperoxidase protein in transfected BHK cells.

Processing and localization of myeloperoxidase was studied in nonmyeloid cells. For this purpose BHK cells were transfected with human myeloperoxidase cDNA. In the transfected cells a protein with mol wt of 85,000 was found, which reacted with the specific anti-human myeloperoxidase antiserum. In size and in sensitivity to endo-beta-N-acetylglucosaminidase H this protein resembled the myeloperoxidase precursor synthesized in human promyelocytes. Unlike in the promyelocytes, in BHK cells the 85,000-Da protein was not converted to 60,000- and 14,000-Da polypeptides of the mature enzyme. In Percoll gradients the protein was found predominantly in the light membrane fractions. Microscopic examination revealed a conspicuous immune reaction over the endoplasmic reticulum and nuclear membranes and a moderate labeling over lysosome-like organelles. Pulse-chase experiments indicated that the protein was slowly released from the endoplasmic reticulum; after 1 day the protein was found in similar amounts in cells and in the medium. The secreted protein contained at least one endo-beta-N-acetylglucosaminidase-resistant oligosaccharide. It is suggested that normal intracellular segregation of myeloperoxidase depends on a signal or component, which is not or incompletely expressed in BHK cells.

Animals↗

PubMed1988

Calmodulin antagonists increase the amount of mRNA for the low-density-lipoprotein receptor in skin fibroblasts.

The effects of calmodulin antagonists on the amount of LDL receptor (LDL-R) mRNA in cultured human fibroblasts was examined by hybridization with a fragment of LDL-R cDNA. In a 'Northern' blot the fragment hybridized to a 5.3-kilobase RNA, as expected for LDL-R mRNA. The concentration of this RNA was increased in preparations from cells that were treated with trifluoperazine or W-7 [N-(6-aminohexyl)-5-chloronaphthalene-1-sulphonamide]. The selectivity of the increase was established by using a probe for beta-actin mRNA. In dot-blot hybridization it was observed that the calmodulin antagonists cause 2-4-fold relative increase in the amount of LDL-R mRNA.

Actins↗

PubMed1988

Mannose 6-phosphate/insulin-like growth factor II receptor: distinct binding sites for mannose 6-phosphate and insulin-like growth factor II.

Pentamannosyl phosphate substituted bovine serum albumin (PMP-BSA) and insulin like growth factor II (IGF II) bind specifically to immobilized mannose 6-phosphate/insulin like growth factor II receptor. An excess of IGF II inhibited binding of PMP-BSA by less than or equal to 20%, and an excess of PMP-BSA inhibited binding of IGF II by less than or equal to 10%. Polyclonal antibodies against the receptor purified from human liver inhibited preferentially the binding of PMP-BSA, and a monocloncal antibody 2C2 inhibited only the binding of IGF II to the receptor. Similar results were obtained for binding of PMP-BSA and IGF II to human skin fibroblasts. These results suggest that the binding sites for mannose 6-phosphate and IGF II reside in different portions of the receptor.

Binding Sites↗

PubMed1988

Synthesis and stability of steroid sulfatase in fibroblasts from multiple sulfatase deficiency.

Multiple sulfatase deficiency is a lysosomal storage disorder, which can be divided into group I with severe and group II with moderate deficiencies in sulfatases. Antibodies raised against steroid sulfatase purified from human placenta were used to follow the biosynthesis and stability of this enzyme in multiple sulfatase-deficiency fibroblasts. Fibroblasts from both groups synthesized steroid sulfatase of apparently normal size and stability, while the apparent rate of enzyme synthesis and catalytic properties of steroid sulfatase were affected to a variable extent. Cell lines were observed, that synthesized normal amounts of steroid-sulfatase polypeptides, which were catalytically inactive, as well as cell lines that synthesized diminished amounts of catalytically active steroid sulfatase.

Cell Line↗

PubMed1988

Low temperature blocks transport and sorting of cathepsin D in fibroblasts.

The transport of newly synthesized cathepsin D in fibroblasts at 16-28 degrees C was compared to that at 37 degrees C. At 37 degrees C newly synthesized cathepsin D passes the trans Golgi within 30-60 min, becomes segregated from the secretory route into prelysosomal organelles within 1-2 h and processed to mature forms in dense lysosomes within 1.5-3 h after synthesis. The small fraction of cathepsin D that escapes transport into lysosomes is secreted within less than 2 h. At 16-28 degrees C the transport of cathepsin D to lysosomes is inhibited in a temperature-dependent manner. At 16-28 degrees C cathepsin D precursors are slowly transported to the trans Golgi. The cathepsin D precursors accumulate at a site that is in continuity with the secretory pathway and located within or distal of the trans Golgi and proximal to the site where cathepsin D precursors leave the secretory pathway as complexes with mannose 6-phosphate receptors. The arrest at this site is not complete. The receptor-dependent segregation of the cathepsin D precursors released from the block is impaired at less than or equal to 26 degrees C. The inhibition of segregation results in an increased, albeit retarded secretion of cathepsin D. The fraction of cathepsin D precursors that is segregated from the secretory pathway encounters a further low temperature block in prelysosomal organelles. There cathepsin D precursors are proteolytically processed to an intermediate form, which accumulates transiently.(ABSTRACT TRUNCATED AT 250 WORDS)

Cathepsin D↗

PubMed1987

Neutral endopeptidase-24.11 (enkephalinase). Biosynthesis and localization in human fibroblasts.

The biosynthesis, glycosylation and subcellular localization of the neutral endopeptidase-24.11 were studied in cultured human fibroblasts. The enzyme was synthesized as a precursor (Mr 88,000) containing four or five N-linked oligosaccharides. Within 1 h the synthesis-mature (Mr 94,000) endopeptidase-24.11 was formed and contained sialylated oligosaccharides. The half-life of endopeptidase-24.11 was 3.7 days and in the presence of 10 mM-NH4Cl it increased to 6 days. Mature endopeptidase-24.11 was solubilized with 0.2% saponin and partitioned into Triton X-114. In intact fibroblasts, endopeptidase-24.11 was accessible to antibodies and to neuraminidase even when the treatment was performed at 4 degrees C. The localization of endopeptidase-24.11 to the plasma membrane in cultured fibroblasts was further demonstrated by immunocytochemistry.

Cell Membrane↗

PubMed1987

Association of the precursor of cathepsin D with coated membranes. Kinetics and carbohydrate processing.

Specific anti-chlathrin antibodies were used to isolate clathrin-coated membranes from homogenates of metabolically labelled fibroblasts. The isolated membranes were extracted with detergents and cathepsin D was isolated from the extracts. The 53-kDa precursor of cathepsin D was transiently associated with the coated membranes with a maximum approximately 60 min after synthesis. At maximum about 4.0% of the precursor was recovered with the coated membranes and the associated precursor contained complex oligosaccharides. The proportion of complex oligosaccharides in the coated membrane-associated precursor did not differ from that in the total precursor. These data support the view that coated membranes are involved in the transport of cathepsin D between trans Golgi and a prelysosomal organelle.

Biological Transport↗

PubMed1987

Conditions for a reliable application of the lysoplate method in the determination of lysozyme.

Using the 'lysoplate' method, less than 1 ng lysozyme can be detected. However, the sensitivity of this method is strongly dependent on the quality of the diffusion medium and on the presence of substances in the samples that affect the interaction of lysozyme with the gel medium and thus its rate of diffusion. The activity of lysozyme in the 'lysoplate' assay can be titrated with a specific antibody. The titration is suitable for quantitative determination of lysozyme. A screening of serum samples for pathologically increased lysozyme content can simply be performed by assaying lysozyme in samples supplemented with a standard amount of anti-lysozyme antibody.

Antibodies↗

PubMed1987

Genetic heterogeneity of steroid sulfatase deficiency revealed with cDNA for human steroid sulfatase.

Three cDNA clones with inserts of 1.2-1.6 kb that reacted both with antibodies and oligonucleotides specific for steroid sulfatase were isolated from a human placental library in lambda gt11. The 5'-end of one of the inserts, STS-3, was sequenced and colinearity with the amino acid sequence of 3 peptides of steroid sulfatase encompassing 64 amino acids was demonstrated. STS-3 hybridized with 2.5, 4.6 and 6.3 kb species in poly(A)+RNA and with 2.5, 4 and 9 kb fragments of EcoRI digested human DNA. The frequency of the EcoRI fragments in DNA from females was approximately twice that in DNA from males. DNA from two patients with steroid sulfatase deficiency and X-linked ichthyosis did not hybridize with STS-3. DNA from a third patient showed a normal hybridization pattern. It is concluded that steroid sulfatase deficiency is a genetically heterogenous disorder.

Amino Acid Sequence↗

PubMed1987

Sulfated oligosaccharides in human lysosomal enzymes.

Cathepsin D, arylsulfatase A and the alpha-chain of beta-hexosaminidase are synthesized in human fibroblasts as sulfated polypeptides. The sulfate is added posttranslationally. Its half-life is less than one-tenth of that of the respective polypeptide chains. The sulfate residues were found on asparagine-linked oligosaccharides sensitive to endoglycosidase F and peptide: N-glycosidase F and resistant to endoglycosidase H. Inhibition of formation of complex type oligosaccharides by 1-deoxy-manno-nojirimycin prevented sulfation, indicating that the sulfate residues were added to complex type oligosaccharides.

Acetylglucosaminidase↗

PubMed1987

Synthesis and processing of alpha-galactosidase A in human fibroblasts. Evidence for different mutations in Fabry disease.

The synthesis and processing of the human lysosomal enzyme alpha-galactosidase A was examined in normal and Fabry fibroblasts. In normal cells, alpha-galactosidase A was synthesized as an Mr = 50,500 precursor, which contained phosphate groups in oligosaccharide chains cleavable by endoglucosaminidase H. The precursor was processed via ill-defined intermediates to a mature Mr 46,000 form. Processing was complete within 3-7 days after synthesis. In the presence of NH4Cl and in I-cell fibroblasts, the majority of newly synthesized alpha-galactosidase A was secreted as an Mr = 52,000 form. For comparison, the processing and stability of alpha-galactosidase A were examined in fibroblasts from five unrelated patients with Fabry disease, which is caused by deficient alpha-galactosidase A activity. In one cell line, synthesis of immunologically cross-reacting polypeptides was not detectable. In another, the synthesis, processing, and stability of alpha-galactosidase A was indistinguishable from that in normal fibroblasts. In a third Fabry cell line, the mutation retarded the maturation of alpha-galactosidase A. Finally, in two cell lines, alpha-galactosidase A polypeptides were synthesized that were rapidly degraded following delivery to lysosomes. These results clearly indicate that Fabry disease comprises a heterogeneous group of mutations affecting synthesis, processing, and stability of alpha-galactosidase A.

Ammonium Chloride↗

PubMed1987

Mr 46,000 mannose 6-phosphate specific receptor: its role in targeting of lysosomal enzymes.

Antibodies that block the ligand binding site of the cation-dependent mannose 6-phosphate specific receptor (Mr 46,000 MPR) were used to probe the function of the receptor in transport of lysosomal enzymes. Addition of the antibodies to the medium of Morris hepatoma 7777 cells, which express only the Mr 46,000 MPR, resulted in a decreased intracellular retention and increased secretion of newly synthesized lysosomal enzymes. In fibroblasts and HepG2 cells that express the cation-independent mannose 6-phosphate specific receptor (Mr 215,000 MPR) in addition to the Mr 46,000 MPR, antibodies against the Mr 46,000 MPR inhibited the intracellular retention of newly synthesized lysosomal enzymes only when added to the medium together with antibodies against the Mr 215,000 MPR. Morris hepatoma (M.H.) 7777 did not endocytose lysosomal enzymes, while U937 monocytes, which express both types of MPR, internalized lysosomal enzymes. The uptake was inhibited by antibodies against the Mr 215,000 MPR, but not by antibodies against the Mr 46,000 MPR. These observations suggest that Mr 46,000 MPR mediates transport of endogenous but not endocytosis of exogenous lysosomal enzymes. Internalization of receptor antibodies indicated that the failure to mediate endocytosis of lysosomal enzymes is due to an inability of surface Mr 46,000 MPR to bind ligands rather than its exclusion from the plasma membrane or from internalization.

Animals↗

PubMed1987

Is movement of mannose 6-phosphate-specific receptor triggered by binding of lysosomal enzymes?

Mannose 6-phosphate-specific receptors with an apparent molecular mass of 215,000 are present in fibroblasts at the cell surface and in intracellular membranes. The cell surface receptors mediate endocytosis of exogenous lysosomal enzymes and exchange with the intracellular receptors, which function in the sorting of endogenous lysosomal enzymes. In the present study, several methods independent of receptor ligands were designed in order to examine the exchange of receptors under conditions where receptor-ligand complexes do not dissociate (weak bases and monensin) or where receptor-ligand complexes are not formed due to absence of endogenous ligands as a result of inhibition of protein synthesis. Weak bases and monensin reduce the concentration of receptors at the cell surface by 20-30% and free cell surface receptors were replaced by occupied receptors. The latter continued to be exchanged with internal ligand-occupied receptors and the rates of the exchange were similar to the control values. The exchange of receptors between the cell surface and internal membranes was also not affected when the receptor ligands were depleted from the transport compartments by treating the cells with cycloheximide for up to 10 h. We conclude from these results that movement of mannose 6-phosphate-specific receptors along the endocytosis and sorting pathways is constitutive and not triggered by binding or dissociation of ligands.

Ammonium Chloride↗

PubMed1987

Lysosomal enzyme precursors in coated vesicles derived from the exocytic and endocytic pathways.

The molecular forms of two lysosomal enzymes, cathepsin C and cathepsin D, have been examined in lysosomes and coated vesicles (CVs) of rat liver. In addition, the relative proportion of these lysosomal enzymes residing in functionally distinct CV subpopulations was quantitated. CVs contained newly synthesized precursor forms of the enzymes in contrast to lysosomes where only the mature forms were detected. Exocytic and endocytic CV subpopulations were prepared by two completely different protocols. One procedure, a density shift method, uses cholinesterase to alter the density of CVs derived from exocytic or endocytic pathways. The other relies on electrophoretic heterogeneity to accomplish the CV subfractionation. Subpopulations of CVs prepared by either procedure showed similar results, when examined for their relative proportion of cathepsin C and cathepsin D precursors. Within the starting CV preparation, exocytic CVs contained approximately 80-90% of the total steady-state levels of these enzymes while the level in the endocytic population was approximately 10-13%. The implications of these findings are discussed with regard to lysosome trafficking.

Animals↗

PubMed1987

Mutations affecting transport and stability of lysosomal enzymes.

The biosynthesis, post-translational processing and receptor-mediated transport of lysosomal enzymes will be briefly summarized. Mutations affecting the transport or the stability of a lysosomal enzyme but not its catalytic properties can result in a lysosomal storage disorder. Mutations causing a loss of catalytic activity may in addition affect transport or stability. Such mutations should not be classified as transport or stability mutations. Prototypes for transport and stability mutations are I-cell disease and late onset forms of metachromatic leukodystrophy.

Carrier Proteins↗