20 results on '"Heikoop JC"'
Search Results
2. Optimizing heterologous expression in dictyostelium: importance of 5' codon adaptation.
- Author
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Vervoort EB, van Ravestein A, van Peij NN, Heikoop JC, van Haastert PJ, Verheijden GF, and Linskens MH
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Chorionic Gonadotropin, beta Subunit, Human biosynthesis, Codon genetics, Enzyme-Linked Immunosorbent Assay, Follicle Stimulating Hormone biosynthesis, Genetic Techniques, Humans, Molecular Sequence Data, Peptide Chain Elongation, Translational, Recombinant Proteins biosynthesis, Ribosomes metabolism, Chorionic Gonadotropin, beta Subunit, Human genetics, Dictyostelium genetics, Follicle Stimulating Hormone genetics
- Abstract
Expression of heterologous proteins in Dictyostelium discoideum presents unique research opportunities, such as the functional analysis of complex human glycoproteins after random mutagenesis. In one study, human chorionic gonadotropin (hCG) and human follicle stimulating hormone were expressed in Dictyostelium. During the course of these experiments, we also investigated the role of codon usage and of the DNA sequence upstream of the ATG start codon. The Dictyostelium genome has a higher AT content than the human, resulting in a different codon preference. The hCG-beta gene contains three clusters with infrequently used codons that were changed to codons that are preferred by Dictyostelium. The results reported here show that optimizing the first 5-17 codons of the hCG gene contributes to 4- to 5-fold increased expression levels, but that further optimization has no significant effect. These observations suggest that optimal codon usage contributes to ribosome stabilization, but does not play an important role during the elongation phase of translation. Furthermore, adapting the 5'-sequence of the hCG gene to the Dictyostelium 'Kozak'-like sequence increased expression levels approximately 1.5-fold. Thus, using both codon optimization and 'Kozak' adaptation, a 6- to 8-fold increase in expression levels could be obtained for hCG.
- Published
- 2000
- Full Text
- View/download PDF
3. Random mutagenesis and screening of complex glycoproteins: expression of human gonadotropins in Dictyostelium discoideum.
- Author
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Linskens MH, Grootenhuis PD, Blaauw M, Huisman-de Winkel B, Van Ravestein A, Van Haastert PJ, and Heikoop JC
- Subjects
- Animals, Chorionic Gonadotropin biosynthesis, Dictyostelium genetics, Dose-Response Relationship, Drug, Follicle Stimulating Hormone biosynthesis, Genetic Testing, Glycoproteins biosynthesis, Humans, Recombinant Proteins biosynthesis, Chorionic Gonadotropin genetics, Follicle Stimulating Hormone genetics, Glycoproteins genetics, Mutagenesis
- Abstract
The soil amoeba Dictyostelium discoideum is a host cell that provides simple genetics in combination with complex protein synthesis. We show that the complex human heterodimeric gonadotropins can be produced and secreted by this organism. Furthermore, both follicle stimulation hormone and choriogonadotropin produced by D. dictyostelium bind to their human receptors and elicit a biological response comparable to the wild-type hormones. We also show that structure-function analysis using random mutagenesis and screening of recombinant glycoprotein hormones is feasible. Thus, expression of gonadotropins in D. dictyostelium opens the way to the engineering of potential new therapeutic analogues.
- Published
- 1999
- Full Text
- View/download PDF
4. Towards minimized gonadotropins with full bioactivity.
- Author
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Heikoop JC, Huisman-de Winkel B, and Grootenhuis PD
- Subjects
- Animals, CHO Cells, Chorionic Gonadotropin genetics, Cricetinae, Drug Design, Genes, Reporter, Humans, In Vitro Techniques, Luciferases genetics, Molecular Weight, Mutagenesis, Site-Directed, Peptide Fragments chemistry, Peptide Fragments genetics, Peptide Fragments pharmacology, Protein Conformation, Receptors, LH drug effects, Receptors, LH metabolism, Structure-Activity Relationship, Chorionic Gonadotropin chemistry, Chorionic Gonadotropin pharmacology
- Abstract
Gonadotropins are highly complex glycoprotein hormones consisting of two noncovalently associated subunits, which are heavily glycosylated. Using the X-ray structure of human choriogonadotropin and structure/activity relationships we aimed to design 'minimized' gonadotropins of reduced complexity. Our results show that it is possible to reduce the size of natural human choriogonadotropin by one-third of its molecular weight while retaining its wild-type biopotency. To our knowledge, such 'mini'-human choriogonadotropins represent the smallest gonadotropins described so far with an lutropin/choriogonadotropin receptor affinity and in vitro biological activity comparable with that of natural human choriogonadotropin. It provides an important step towards the structure/function-based design of small molecule drugs to the human gonadotropin receptors.
- Published
- 1999
- Full Text
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5. Expression of a bioactive, single-chain choriogonadotropin in Dictyostelium discoideum.
- Author
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Heikoop JC, Grootenhuis PD, Blaauw M, Veldema JS, Van Haastert PJ, and Linskens MH
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, CHO Cells, Chorionic Gonadotropin pharmacology, Cloning, Molecular, Cricetinae, Gene Expression genetics, Humans, Molecular Sequence Data, Protein Binding physiology, Protein Sorting Signals chemistry, Receptors, LH metabolism, Recombinant Proteins genetics, Signal Transduction physiology, Transformation, Genetic, Chorionic Gonadotropin genetics, Dictyostelium chemistry
- Abstract
Human choriogonadotropin (hCG) is a highly complex glycoprotein consisting of two non-covalently associated subunits. We aimed for the expression of a single-chain hCG in the soil amoebae Dictyostelium discoideum, a host which, in principle, provides simple genetics in combination with complex protein synthesis. To limit anticipated problems in mRNA translation, the first 30 bases of the coding sequence were altered to conform to the Dictyostelium preferred codon usage. We show that, immunologically, active single-chain hCG is indeed produced by Dictyostelium. Furthermore, this single-chain hCG is able to bind to the human luteinizing hormone/CG receptor and elicit a biological response. Its receptor-binding affinity is comparable to single-chain hCG produced by mammalian cells. We conclude that Dictyostelium is able to express bioactive highly complex heterologous glycoproteins.
- Published
- 1998
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6. Partially deglycosylated human choriogonadotropin, stabilized by intersubunit disulfide bonds, shows full bioactivity.
- Author
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Heikoop JC, van den Boogaart P, de Leeuw R, Rose UM, Mulders JW, and Grootenhuis PD
- Subjects
- Animals, CHO Cells, Chorionic Gonadotropin genetics, Cricetinae, Disulfides chemistry, Drug Stability, Glycosylation, Humans, In Vitro Techniques, Mutagenesis, Site-Directed, Receptors, LH drug effects, Receptors, LH genetics, Signal Transduction, Temperature, Chorionic Gonadotropin chemistry, Chorionic Gonadotropin pharmacology
- Abstract
Several studies indicate that in human choriogonadotropin the N-linked oligosaccharide at position 52 of the alpha-subunit is important for bioactivity. We have generated choriogonadotropin mutants in which the alpha52 glycosylation site is removed and the alpha and beta subunits are covalently linked by intersubunit disulfide bonds. These mutants display wild-type receptor binding and bioactivity. Furthermore, we show that removal of the alpha52 sugar leads to instability of heterodimeric choriogonadotropin. Therefore, we conclude that the alpha52 oligosaccharide of choriogonadotropin is not involved in signal transduction, but in the stability of the heterodimer.
- Published
- 1998
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7. Structure-based design and protein engineering of intersubunit disulfide bonds in gonadotropins.
- Author
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Heikoop JC, van den Boogaart P, Mulders JW, and Grootenhuis PD
- Subjects
- Animals, Biotechnology, CHO Cells, Chorionic Gonadotropin chemistry, Chorionic Gonadotropin genetics, Chorionic Gonadotropin metabolism, Cricetinae, Cystine chemistry, Disulfides chemistry, Drug Design, Drug Stability, Follicle Stimulating Hormone chemistry, Follicle Stimulating Hormone genetics, Gonadotropins metabolism, Humans, In Vitro Techniques, Luteinizing Hormone chemistry, Luteinizing Hormone genetics, Models, Molecular, Mutagenesis, Site-Directed, Protein Conformation, Protein Engineering, Receptors, LH metabolism, Signal Transduction, Temperature, Transfection, Gonadotropins chemistry, Gonadotropins genetics
- Abstract
Pairs of cystine residues were introduced in the alpha- and beta-subunits of human choriogonadotropin at positions with optimal geometries for the formation of disulfide bonds. Using the homology with luteinizing hormone and follicle stimulating hormone, similar mutations were carried out in these glycoprotein hormones. In nearly all mutants the corresponding disulfide bonds were formed leading to a non-natural, covalent linkage between the alpha- and beta-subunits. The mutants typically display wild-type receptor binding and bioactivity. The mutants with non-natural intersubunit disulfide bonds display enhanced thermostabilities relative to the corresponding heterodimeric glycoprotein hormones, rendering them candidates for long acting gonadotropins with enhanced shelf lives.
- Published
- 1997
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8. Evaluation of subunit truncation and the nature of the spacer for single chain human gonadotropins.
- Author
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Heikoop JC, van Beuningen-de Vaan MM, van den Boogaart P, and Grootenhuis PD
- Subjects
- Animals, Base Sequence, CHO Cells, Cricetinae, Gonadotropins genetics, Humans, Molecular Sequence Data, Mutation, Recombinant Proteins chemistry, Recombinant Proteins genetics, Structure-Activity Relationship, Gonadotropins chemistry, Protein Conformation
- Abstract
Three single chain gonadotropins were designed based on the three-dimensional-structure of human choriogonadotropin and structure/activity relationships of the glycoprotein hormones. In each single chain, the C-terminal end of the human choriogonadotropin beta subunit is connected via Ser-Gly repeats to the N-terminal end of the alpha subunit. In addition, two of the single chains have truncated subunits. The three mutants were expressed in CHO cells. In vitro binding of two of the three mutants to the human lutropin/choriogonadotropin receptor was found to be comparable to wild-type lutropin. In contrast, both the receptor binding and the in vitro bioactivity of the mutant with truncated alpha and beta subunits in which the beta:26-110 disulphide bond cannot be formed, are lowered relative to wild-type lutropin. The fact that this mutant still displays biological activity shows that the seat-belt arrangement proposed by Isaacs and coworkers [Lapthorn, A. J., Harris, D. C., Littlejohn, A., Lustbader, J. W., Canfield, R. E., Machin, K. J.. Morgan, F. J. & Isaacs, N. W. (1994) Nature 369, 455-461] is important but not essential for receptor binding and biological activity in the context of single chain gonadotropins. Single chains in which Ser-Gly spacers are combined with truncated subunits, provide an attractive approach towards the design and generation of novel, biologically active gonadotropins.
- Published
- 1997
- Full Text
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9. Expression of the human Dp 71 (apo-dystrophin-1) gene from a 760-kb DMD-YAC transferred to mouse cells.
- Author
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Heikoop JC, Hogervorst FB, Meershoek EJ, Grootscholten PM, den Dunnen JT, and van Ommen GJ
- Subjects
- Animals, Base Sequence, Chromosomes, Artificial, Yeast, DNA Primers genetics, DNA, Complementary genetics, Dystrophin genetics, Exons, Gene Expression Regulation, Humans, Hybrid Cells, In Situ Hybridization, Fluorescence, L Cells, Mice, Molecular Sequence Data, Muscular Dystrophies genetics, Polymerase Chain Reaction, Promoter Regions, Genetic, RNA genetics, RNA Splicing, Dystrophin analogs & derivatives, Gene Transfer Techniques
- Abstract
A 760-kb YAC was constructed by homologous recombination in yeast, containing the genes located in the distal portion of the DMD gene. The YAC was introduced in mouse LA-9 cells by PEG-mediated cell fusion. One transformant accommodated an intact DMD-YAC, i.e. a full copy of the DMD internal Dp 116, Dp 71 and Dp 40 genes (apo-dystrophin-2, -1 and -3, respectively). We have studied the expression of the various gene products derived from the introduced DMD-YAC. RT-PCR revealed expression of human Dp 71 but not of Dp 116 or Dp 40. Remarkably, differences were observed in processing of the 3' region of the endogenous mouse and the human transcripts, due to different splicing of exons 71 (absent in human and present in mouse transcript) and 78 (present in human and absent in mouse transcript). The splicing pattern of the human transcript is the same as that of the major Dp 71 (apo-dystrophin-1) product in human blood. The observed splicing differences may be caused by either species-specific exon use and/or by cis-acting factors, e.g. the upstream transcript composition, because we have no evidence for endogenous Dp 71 expression.
- Published
- 1995
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10. A simple and rapid method for separating co-cloned YACs.
- Author
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Heikoop JC, Steensma Y, van Ommen GJ, and den Dunnen JT
- Subjects
- Dystrophin genetics, Humans, Chromosomes, Artificial, Yeast, Cloning, Molecular methods
- Published
- 1994
- Full Text
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11. Cytoplasmic catalase and ghostlike peroxisomes in the liver from a child with atypical chondrodysplasia punctata.
- Author
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Espeel M, Heikoop JC, Smeitink JA, Beemer FA, De Craemer D, Van den Berg M, Hashimoto T, Wanders RJ, Schutgens RB, and Poll-The BT
- Subjects
- Child, Chondrodysplasia Punctata enzymology, Female, Fibroblasts enzymology, Histocytochemistry, Humans, Liver enzymology, Microscopy, Electron, Microscopy, Immunoelectron, Catalase metabolism, Chondrodysplasia Punctata pathology, Cytoplasm enzymology, Liver ultrastructure, Microbodies ultrastructure
- Abstract
In the liver biopsy from an 8.5-year-old girl with the biochemical characteristics of rhizomelic chondrodysplasia punctata (RCDP), but with normal limbs, normal catalase-containing peroxisomes were absent. Light microscopy after diaminobenzidine staining for catalase activity (the peroxisomal marker enzyme) and immunostaining against catalase protein indicated a cytosolic localization of the enzyme. By electron microscopy, rare and extremely large, irregularly shaped vesicles were found in the parenchymal cells. The three peroxisomal beta-oxidation enzymes (acyl-CoA oxidase, bi(tri)functional enzyme, and 3-ketoacyl-CoA thiolase) and alanine-glyoxylate aminotransferase were immunolocalized in these organelles. However, a weak to negative label was obtained after staining against catalase. Diaminobenzidine staining demonstrated a minimal catalase reaction product in some vesicles only. Morphometry revealed a corrected mean d-circle of 1.44 microns and a maximum d-circle of 2.767 microns (controls: 0.635 microns and 1.027 microns, respectively). Numerical, volume, and surface densities were reduced to 3%, 41%, and 17% of control values, respectively. The large size, irregular shape, and rarity of the organelles are morphologic features of peroxisomal "ghosts." It seems that in this patient, apart from the known peroxisomal defects in RCDP, catalase incorporation into the peroxisomes is impaired together with a normal proliferation (division) of the organelles. In the cultured skin fibroblasts from the patient, however, immuno-electron microscopy showed normal catalase-containing peroxisomes in apparently normal numbers.
- Published
- 1993
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12. Protein truncation test (PTT) to rapidly screen the DMD gene for translation terminating mutations.
- Author
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Roest PA, Roberts RG, van der Tuijn AC, Heikoop JC, van Ommen GJ, and den Dunnen JT
- Subjects
- Base Sequence, DNA Primers, Dystrophin biosynthesis, Genetic Techniques, Humans, Infant, Newborn, Male, Molecular Sequence Data, Muscular Dystrophies diagnosis, Polymerase Chain Reaction methods, Protein Biosynthesis, RNA, Messenger analysis, Transcription, Genetic, Dystrophin genetics, Muscular Dystrophies genetics, Peptide Chain Termination, Translational, Point Mutation, RNA, Messenger metabolism
- Abstract
We have developed a rapid and sensitive method to screen the Duchenne muscular dystrophy (DMD) mRNA for translation terminating mutations by a combination of RT-PCR (Reverse Transcription and Polymerase Chain Reaction) and in vitro transcription/translation applied to white blood cell mRNA. This technique was termed the protein truncation test (PTT). Here we demonstrate the detection of a point mutation in a DMD patient and his mother, a carrier. The PTT can also be used for carrier detection when no patient material is available, or in the case of spontaneous mutations. We developed a protocol to screen the total coding region of the DMD gene in 5-10 PTT reactions. Furthermore, PTT could be of diagnostic value in any disease where premature terminations form a substantial part of the total mutation spectrum.
- Published
- 1993
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13. Subcellular fractionation of cultured normal human melanocytes: new insights into the relationship of melanosomes with lysosomes and peroxisomes.
- Author
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Smit NP, van Roermund CW, Aerts HM, Heikoop JC, Van den Berg M, Pavel S, and Wanders RJ
- Subjects
- Cell Fractionation, Cell Line, Glucosylceramidase metabolism, Humans, L-Lactate Dehydrogenase metabolism, Melanocytes enzymology, Microscopy, Fluorescence, Microscopy, Immunoelectron, Monophenol Monooxygenase metabolism, Subcellular Fractions, beta-N-Acetylhexosaminidases metabolism, Lysosomes, Melanocytes ultrastructure, Microbodies
- Abstract
In order to obtain information on the disputed nature of melanosomes a comparison was made between the localization of melanosomal markers with those of other well-defined subcellular organelles such as lysosomes and peroxisomes. The distribution of marker enzymes was studied using two different density gradient systems, i.e., Percoll and Nycodenz. Furthermore, the subcellular localization of various types of antigens was analyzed using indirect immunofluorescence and immuno-electron microscopy. All methods revealed the existence of partial co-localization of melanosomal and lysosomal proteins and different localization of peroxisomal markers. The results suggest that melanosomes may share a common origin with lysosomal structures.
- Published
- 1993
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14. Autophagic degradation of peroxisomes in isolated rat hepatocytes.
- Author
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Luiken JJ, van den Berg M, Heikoop JC, and Meijer AJ
- Subjects
- Animals, Cells, Cultured, Clofibrate pharmacology, Liver cytology, Liver drug effects, Male, Rats, Rats, Inbred Strains, Autophagy, Liver metabolism, Microbodies metabolism
- Abstract
Degradation of the peroxisomal enzymes fatty acyl-CoA oxidase and catalase was studied in hepatocytes isolated from rats treated with clofibrate and from control rats. Hepatocytes were incubated in the absence of amino acids in order to ensure maximal flux through the autophagic pathway and in the presence of cycloheximide to inhibit protein synthesis. (1) Degradation of the two peroxisomal enzymes in hepatocytes from clofibrate-fed rats, but not in hepatocytes from control rats, was much faster than that of other intracellular enzymes. This increased degradation of the peroxisomal enzymes was almost completely prevented by 3-methyladenine, an inhibitor of macroautophagic sequestration. (2) The increased degradation of the peroxisomal enzymes was also inhibited by a long-chain (C16:0) and a very-long-chain (C26:0) fatty acid, but not by C12:0, a medium-chain fatty acid, or by C8:0, a short-chain fatty acid. These results provide direct evidence for the proposal that autophagic sequestration can be highly selective [(1987) Exp. Mol. Pathol. 46, 114-122]. It is concluded that preferential autophagy of peroxisomes is prevented when these organelles are supplied with their fatty acid substrates.
- Published
- 1992
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15. Genetic and biochemical heterogeneity in patients with the rhizomelic form of chondrodysplasia punctata--a complementation study.
- Author
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Heikoop JC, Wanders RJ, Strijland A, Purvis R, Schutgens RB, and Tager JM
- Subjects
- Acetyl-CoA C-Acyltransferase metabolism, Acyltransferases deficiency, Acyltransferases genetics, Acyltransferases metabolism, Cell Fusion, Cell Line, Chondrodysplasia Punctata metabolism, Humans, Immunoblotting, Microscopy, Fluorescence, Phytanic Acid metabolism, Plasmalogens biosynthesis, Transferases deficiency, Transferases genetics, Transferases metabolism, Alkyl and Aryl Transferases, Chondrodysplasia Punctata genetics, Genetic Complementation Test
- Abstract
The genetic relationship between 10 patients with clinical manifestations of rhizomelic chondrodysplasia punctata (RCDP) was studied by complementation analysis after somatic cell fusion. Biochemically, 9 out of the 10 patients were characterized by a partial deficiency of acyl-CoA: dihydroxyacetone phosphate acyltransferase (DHAP-AT) and an impairment of plasmalogen biosynthesis, phytanate catabolism and the maturation of peroxisomal 3-oxoacyl-CoA thiolase; 3-oxoacyl-CoA thiolase was strongly reduced in the peroxisomes of these patients. Fusion of fibroblasts from these 9 patients with Zellweger fibroblasts resulted in complementation as indicated by the restoration of DHAP-AT activity, plasmalogen biosynthesis, and punctate fluorescence after staining with a monoclonal antibody to peroxisomal thiolase. No complementation was observed after fusion of different combinations of the 9 RCDP cell lines, suggesting that they belong to a single complementation group. The tenth patient was characterized biochemically by a deficiency of DHAP-AT and an impairment of plasmalogen biosynthesis. However, maturation and localization of peroxisomal thiolase were normal. Fusion of fibroblasts from this patient with fibroblasts from the other 9 patients resulted in complementation as indicated by the restoration of plasmalogen biosynthesis. We conclude that mutations in at least two different genes can lead to the clinical phenotype of RCDP.
- Published
- 1992
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16. Turnover of peroxisomal vesicles by autophagic proteolysis in cultured fibroblasts from Zellweger patients.
- Author
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Heikoop JC, van den Berg M, Strijland A, Weijers PJ, Just WW, Meijer AJ, and Tager JM
- Subjects
- Adenine analogs & derivatives, Adenine pharmacology, Autophagy drug effects, Cell Line, Fibroblasts ultrastructure, Fluorescent Antibody Technique, Humans, Hydrolases analysis, Leupeptins pharmacology, Lysosomes enzymology, Membrane Proteins analysis, Microbodies chemistry, Autophagy physiology, Fibroblasts physiology, Microbodies physiology, Zellweger Syndrome physiopathology
- Abstract
Previous studies have shown that in fibroblasts from patients with the Zellweger syndrome (ZS) aberrant membrane structures are present which contain peroxisomal membrane proteins (Santos, M. J. et al., Science 239, 1536-1538 (1988)). In order to characterize these structures we have performed double labeling immunoelectron microscopy experiments using antisera directed against the 69 kDa peroxisomal integral membrane protein (PMP) and lysosomal hydrolases. The results indicate that at least 80% of the structures earlier referred to as 'peroxisomal ghosts' contain lysosomal hydrolases. In addition, we have studied the effect of culture of ZS fibroblasts in the presence of 3-methyladenine, an inhibitor of autophagy, on the intracellular distribution of the 69 kDa PMP. Immunofluorescence experiments showed that in the presence of 3-methyladenine there is an increase in fluorescent spots and a change in the distribution of the spots from mainly perinuclear to randomly distributed throughout the cytoplasm. Double labeling immunoelectron microscopy revealed that after culture in the presence of 3-methyladenine the 69 kDa PMP also accumulates mainly in compartments containing lysosomal hydrolases. In one ZS cell line we found that after culture in the presence of 3-methyladenine there was also an accumulation of structures which were as small as normal microperoxisomes. We conclude that in ZS fibroblasts the 69 kDa PMP is mainly present in lysosomal compartments, presumably degradative autophagic vacuoles. Furthermore, in ZS fibroblasts peroxisomes of apparently normal morphology may be synthesized, but they are degraded by autophagic proteolysis.
- Published
- 1992
17. Subcellular localisation and processing of non-specific lipid transfer protein are not aberrant in Rhizomelic Chondrodysplasia Punctata fibroblasts.
- Author
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Heikoop JC, Ossendorp BC, Wanders RJ, Wirtz KW, and Tager JM
- Subjects
- Acetyl-CoA C-Acetyltransferase metabolism, Blotting, Western, Fibroblasts enzymology, Humans, Microbodies enzymology, Oligopeptides metabolism, Protein Processing, Post-Translational, Carrier Proteins metabolism, Chondrodysplasia Punctata metabolism
- Abstract
The import into peroxisomes and maturation of peroxisomal 3-oxoacyl-CoA thiolase are impaired in patients with the Rhizomelic form of Chondrodysplasia Punctata (RCDP). Here we show by means of immunoblotting and subcellular fractionation that non-specific lipid transfer protein (nsLTP), another peroxisomal protein synthesised as a larger precursor, is localised in peroxisomes and is present as the mature protein in RCDP fibroblasts. Thus the component of the import machinery defective in RCDP is not required for the import of nsLTP into peroxisomes.
- Published
- 1992
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18. Peroxisomes of normal morphology but deficient in 3-oxoacyl-CoA thiolase in rhizomelic chondrodysplasia punctata fibroblasts.
- Author
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Heikoop JC, Van den Berg M, Strijland A, Weijers PJ, Schutgens RB, Just WW, Wanders RJ, and Tager JM
- Subjects
- Animals, Cell Line, Cells, Cultured, Humans, Immunoblotting, Microbodies ultrastructure, Microscopy, Fluorescence, Microscopy, Immunoelectron, Rats, Zellweger Syndrome, Acetyl-CoA C-Acetyltransferase deficiency, Chondrodysplasia Punctata enzymology, Fibroblasts enzymology, Microbodies enzymology
- Abstract
Rhizomelic Chondrodysplasia Punctata (RCDP) is an autosomal recessive disorder in which plasmalogen biosynthesis and phytanate catabolism are impaired. Peroxisomal structure and the intracellular localization of catalase, the 69 kDa peroxisomal integral membrane protein (PMP), and 3-oxoacyl-CoA thiolase were studied in cultured skin fibroblasts from control subjects and patients with RCDP. A punctate fluorescence pattern characteristic for peroxisomes was seen in control cells incubated with either anti-(catalase), anti-(69 kDa PMP) or anti-(3-oxoacyl-CoA thiolase). Incubation of mutant cells with anti-(catalase) or anti-(69 kDa PMP) resulted in the same pattern. However, when RCDP fibroblasts were incubated with a monoclonal anti-(3-oxoacyl-CoA thiolase) antibody no punctate fluorescence could be observed. Cryosections from control and RCDP cells were examined by electron microscopy using double immunogold labelling. RCDP fibroblasts contained structures indistinguishable from control peroxisomes, the membranes reacting with anti-(69 kDa PMP) and the matrix with anti-(catalase). However, the matrix of RCDP peroxisomes, unlike control peroxisomes, did not react with anti-(3-oxoacyl-CoA thiolase). We conclude that RCDP fibroblasts contain regularly shaped peroxisomes, comparable to control peroxisomes in number as well as in content of catalase and 69 kDa PMP. However, in RCDP peroxisomes the amount of 3-oxoacyl-CoA thiolase protein proved to be below the limit of detection.
- Published
- 1991
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19. Function of oligosaccharide modification in glucocerebrosidase, a membrane-associated lysosomal hydrolase.
- Author
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Van Weely S, Aerts JM, Van Leeuwen MB, Heikoop JC, Donker-Koopman WE, Barranger JA, Tager JM, and Schram AW
- Subjects
- Acetylglucosaminidase metabolism, Catalysis, Cathepsin D metabolism, Cells, Cultured, Centrifugation, Density Gradient, Chemical Phenomena, Chemistry, Physical, Enzyme Stability, Fibroblasts enzymology, Glycoside Hydrolases metabolism, Humans, Isoelectric Point, Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase, Molecular Weight, Neuraminidase deficiency, Neuraminidase metabolism, Placenta enzymology, Protein Precursors metabolism, Structure-Activity Relationship, beta-Galactosidase deficiency, beta-Galactosidase metabolism, beta-N-Acetylhexosaminidases deficiency, beta-N-Acetylhexosaminidases metabolism, Glucosidases metabolism, Glucosylceramidase metabolism, Lysosomes enzymology, Oligosaccharides metabolism, Protein Processing, Post-Translational
- Abstract
The nature and function of oligosaccharide modification in glucocerebrosidase, a membrane-associated lysosomal hydrolase, have been investigated in cultured human skin fibroblasts. Glucocerebrosidase is synthesised as a 62.5-kDa precursor with high-mannose-type oligosaccharide chains and an apparent native isoelectric point of 6.0-7.0. Subsequent processing of the oligosaccharide moieties to sialylated complex-type structures results in formation of 65-68-kDa forms of the enzyme with apparent native isoelectric points of 4.3-5.0. These forms are transported to lysosomes and subsequently modified by the sequential action of lysosomal exoglycosidases, finally resulting in a 59-kDa form with an isoelectric point near neutrality. The existence of oligosaccharide modification of the enzyme in the lysosomes is illustrated by the accumulation of different intermediate forms of glucocerebrosidase in mutant cell lines deficient in lysosomal exoglycosidases. The enzyme does not undergo proteolytic modification during maturation. The possible physiological relevance of the oligosaccharide modification of glucocerebrosidase in the lysosomes was investigated by studying the properties of the enzyme in fibroblasts deficient in lysosomal exoglycosidases, and also the properties of homogeneous pure glucocerebrosidase from placenta, modified in the oligosaccharide moieties by digestion in vitro with glycosidases. Modification of the oligosaccharide moieties of glucocerebrosidase had no significant effect on the catalytic activity of the enzyme as measured with either artificial or natural substrates in the presence of artificial or natural activators. There was also no effect of modification of the oligosaccharide chains on the intracellular stability of the enzyme or on its apparent hydrophobicity. We conclude that oligosaccharide modification of glucocerebrosidase in the lysosomes simply reflects further maturation of the enzyme in the lysosome and is of no importance to its function.
- Published
- 1990
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20. Rhizomelic chondrodysplasia punctata. Deficiency of 3-oxoacyl-coenzyme A thiolase in peroxisomes and impaired processing of the enzyme.
- Author
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Heikoop JC, van Roermund CW, Just WW, Ofman R, Schutgens RB, Heymans HS, Wanders RJ, and Tager JM
- Subjects
- Blotting, Western, Cell Compartmentation, Centrifugation, Density Gradient, Fibroblasts metabolism, Humans, Plasmalogens biosynthesis, Protein Processing, Post-Translational, Acetyl-CoA C-Acyltransferase deficiency, Acyltransferases deficiency, Chondrodysplasia Punctata enzymology, Microbodies enzymology
- Abstract
The rhizomelic form of chondrodysplasia punctata (RCDP) is a peroxisomal disorder characterized biochemically by an impairment of plasmalogen biosynthesis and phytanate catabolism. We have now found that the maturation of peroxisomal 3-oxoacyl-CoA thiolase is impaired in fibroblasts from RCDP patients. To establish the subcellular localization of the 3-oxoacyl-CoA thiolase precursor protein, cultured skin fibroblasts were fractionated on a continuous Nycodenz gradient. Only a small amount of 3-oxoacyl-CoA thiolase activity was present in the catalase-containing (peroxisomal) fractions of RCDP fibroblasts in comparison with control fibroblasts. Moreover, the amount of thiolase protein in immunoblots of the catalase-containing fractions was below the limit of detection. Finally, the beta-oxidation of [14C]palmitoyl-CoA was found to be reduced in these fractions. We conclude that the mutation in RCDP leads to a partial deficiency of 3-oxoacyl-CoA thiolase activity in the peroxisomes and, concomitantly, an impairment in the ability to convert the precursor of this protein to the mature form. The reduction of 3-oxoacyl-CoA thiolase activity results in a decrease in the rate of peroxisomal beta-oxidation of palmitoyl-CoA. However, the capacity of the peroxisomes to oxidize very-long-chain fatty acids must be sufficient to prevent excessive accumulation of these compounds in vivo.
- Published
- 1990
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