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W Stoffel

Publications and source records attributed to W Stoffel.

At least 55 records · Page 3Linked to original sources
PubMed1995

Adhesive properties of proteolipid protein are responsible for the compaction of CNS myelin sheaths.

We have studied the molecular function of proteolipid protein (PLP), the main integral membrane protein of CNS myelin, by generating mice lacking PLP expression. Here, we demonstrate that these PLP-minus mice show no pleiotropism as mice carrying point mutations within the PLP gene. The expression of other myelin genes (myelin basic protein, MBP; myelin associated glycoprotein, MAG; UDP-galactose-ceramide galactosyl transferase, CGT) is unimpaired on the RNA level. Protein level immunofluorescence analysis by confocal microscopy reveals that in PLP-minus mice there is a complete absence of PLP, a scattered appearance of MBP, and MAG expressed more widely in regions lacking MBP staining, which may be a compensatory mechanism. In electron microscopy the myelin lamellae of the ensheathed CNS axons are loosely wrapped with wide extracellular spaces between turning loops. Intraperiod dense lines are missing. The lateral loops of the paranode form regular axoglial junctions. In PLP-minus mice axons form regular axoglial junctions. In PLP-minus mice axons with large diameters are loosely myelinated, whereas small axons remain unmyelinated. Functionally, the mutant mice show deficits in their locomotor activity. We propose that adhesion properties of the extracellular domains of PLP are responsible for the tight apposition of the plasma membrane processes of oligodendrocytes wrapping axons to form the compact myelin sheath.

Adhesiveness↗
PubMed1995

Rat and human glutamate transporter GLAST1 stable heterologous expression, biochemical and functional characterization.

Human embryonic kidney cell lines (HEK293) which express heterologously and permanently the human and rat GLAST1 (high affinity, Na(+)-dependent, CNS-specific L-glutamate transporter) have been established by the transfer of the two minigenes under the control of the cytomegalovirus promoter by electroporation. The transfected HEKh GLAST1 (human) and HEKrGLAST1 (rat) cell lines strongly express the glutamate uptake system which exhibits all biochemical and electrophysiological properties determined so far in the transiently expressing Xenopus oocyte system except the K+ dependence. These cell lines are a valuable tool for further biochemical, physiological, and pharmacological studies on this uptake system of the most important excitatory neurotransmitter.

Amino Acid Transport System X-AG↗
PubMed1994

Disruption of the compacted myelin sheath of axons of the central nervous system in proteolipid protein-deficient mice.

The isoproteins proteolipid protein (PLP) and DM20, the two major integral membrane proteins of central nervous system (CNS) myelin, are encoded by a single gene on the X chromosome and show a different developmental expression pattern. To investigate their functions in myelin structure and myelination, we produced transgenic mice carrying a targeted alteration of the X chromosome-linked Plp gene containing a deletion within exon III, mimicking DM20, and a neo cassette in reverse orientation within intron III. Here we show that the antisense integration of the neo cassette disrupts the expression of the Plp gene. The ultrastructure of the multilayer myelin sheath of all axons in the CNS of hemizygous male or homozygous female PLP/DM20-deficient mice is highly disordered. The apposition of the extracytoplasmic surfaces and thereby the intraperiod dense line is lacking. The disrupted assembly of the myelin sheath leads to a profound reduction of conductance velocities of CNS axons, impairments in neuromotor coordination, and behavioral changes.

Animals↗
PubMed1994

Human neutral amino acid transporter ASCT1: structure of the gene (SLC1A4) and localization to chromosome 2p13-p15.

Screening for cDNAs encoding proteins similar to the sodium-coupled glutamate transporter GLAST1 led to the isolation of a cDNA clone coding for a protein that turned out to be identical to the recently described neutral amino acid transporter ASCT1. The new member of the GLAST-related transporter family does not transport glutamate or aspartate but alanine, serine, cysteine, and threonine instead. The expressed sequence tag EST02446, a short cDNA sequence found in the course of a large-scale sequencing project of human brain-derived cDNAs, showed significant similarity to the eukaryotic glutamate transporter GLAST1 and was therefore used as probe in the search for further glutamate transporter cDNAs. Fragments of the cDNA were used for the isolation and characterization of human ASCT1 genomic clones. The ORF of 1572 bp encoding 524 amino acid residues is distributed over 8 exons, which span at least 40 kb of human chromosomal DNA. The ASCT1 gene locus was assigned to chromosome 2p13-p15 by chromosomal in situ suppression (CISS) studies. The gene structure is not related to any other previously characterized transporter gene. In contrast to the genes of the sodium-coupled nonglutamate neurotransmitter transporters, it shows no obvious correspondence between intron/exon structure and transmembrane organization. The transcription start site in human liver tissue was determined by primer extension analysis to be located 291 bp upstream of the initiating ATG codon. The DNA region immediately upstream of the transcription start lacks any TATA or CAAT boxes but contains several binding sites for the transcription factors Sp1 and Egr-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Human mitochondrial 3,2-trans-enoyl-CoA isomerase (DCI): gene structure and localization to chromosome 16p13.3.

A key enzyme in the mitochondrial beta-oxidation of unsaturated fatty acids is the 3,2-trans-enoyl-CoA isomerase (DCI; EC 5.3.3.8). It catalyzes the transformation of 3-cis and 3-trans intermediates arising during the stepwise degradation of all cis-, mono-, and polyunsaturated fatty acids to the 2-trans-enoyl-CoA intermediates. A genomic clone encoding the human DCI was isolated and characterized by use of the previously cloned human DCI cDNA. The entire gene encompasses approximately 12.5 kb, and the coding sequence is distributed over seven exons. One major and three minor transcription start sites were determined by primer extension analysis. In common with promoters of other housekeeping genes encoding mitochondrial proteins, the GC-rich, immediate 5'-flanking region of the DCI transcription initiation site lacks typical TATA and CAAT boxes; instead, two GC box consensus sequences are present. Introns 2 and 6 contain several Alu repetetive sequences. The human DCI gene locus was assigned to chromosome 16 by use of human-rodent somatic cell hybrids and to chromosome 16p13.3 by chromosomal in situ suppression hybridization studies.

Animals↗
PubMed1994

Topology of CNS myelin proteolipid protein: evidence for the nonenzymatic glycosylation of extracytoplasmic domains in normal and diabetic animals.

Myelin proteolipid protein (PLP), the main integral membrane protein in the central nervous system myelin, was labeled at the extracytoplasmic domains with the membrane impermeant reagents pyridoxal 5'-phosphate and tritiated borohydride. Lysine-217, located in the fourth hydrophilic domain of PLP, was found to be the major labeled residue, which defined this domain to be extracytoplasmic in agreement with our previously proposed topological model. The remarkably high reactivity in vitro of this residue as compared to all other lysines in PLP led us to investigate the possible modification of PLP in vivo by other carbonyl compounds. We demonstrate that PLP is the most highly nonenzymatically glycosylated membrane protein in murine and bovine brain. The degree of modification increases significantly under hyperglycemic conditions, as studied in diabetic mice. The majority of the glycosylation sites are also located at extracytoplasmic domains. The degree of nonenzymatic glycosylation of PLP may be related to late diabetic complications affecting the central nervous system.

Amino Acid Sequence↗
PubMed1994

Functional properties and substrate specificity of the cloned L-glutamate/L-aspartate transporter GLAST-1 from rat brain expressed in Xenopus oocytes.

The rat brain L-glutamate/L-aspartate transporter GLAST-1 is a member of a family of Na(+)-dependent high-affinity L-glutamate transporters proposed to be involved in the termination and modulation of excitatory neurotransmitter signals. Application of electrophysiological and radiotracer techniques on Xenopus oocytes expressing cloned GLAST-1 revealed that the apparent Km value of the transporter for L-glutamate and Na+ ions did not depend on voltage while the maximal transport rate increased with more negative potentials, indicative of a low-field access channel. The apparent Km value of the transporter for L-glutamate depends on the Na+ concentration, suggesting that substrate and ions are transported by GLAST-1 in a simultaneous manner. All of the L-glutamate uptake blockers tested either were substrates or did not affect the current induced by L-glutamate. The changes in the amplitude of the current induced by simultaneous application of two substrates can be interpreted by a competition for one binding site.

Amino Acid Transport System X-AG↗
PubMed1993

Ceramide UDPgalactosyltransferase from myelinating rat brain: purification, cloning, and expression.

Cerebrosides and sulfatides are major glycosphingolipids of the lipid bilayer of the myelin sheath assembled by oligodendrocytes and Schwann cells during myelination. Cerebrosides are synthesized by ceramide UDPgalactosyltransferase [CGT; 2-hydroxyacylsphinogosine 1-beta-galactosyl-transferase; UDPgalactose:2-(2-hydroxyacyl)sphingosine 1-beta-D-galactosyltransferase; UDPgalactose:2-(2-hydroxyacyl)sphingosine 1-beta-D-galactosyltransferase, EC 2.4.1.45] with UDPgalactose and ceramide as substrates. Here we describe a purification method from microsomes of myelinating rat brains that includes ion exchange, dye ligand, and lectin affinity chromatography. The enzyme was identified as a 64-kDa high-mannose glycoprotein. A CGT-specific cDNA clone was isolated from a rat brain cDNA library using CGT oligonucleotides derived from peptide sequences. The cDNA insert encodes a polypeptide of 541 amino acid residues with a molecular weight of 61,126. The polypeptide has three putative glycosylation sites and one hydrophobic domain at the C terminus. A 20-residue N-terminal signal sequence is lost during cotranslational translocation. Northern blot analysis demonstrates that CGT expression is restricted to brain tissue and is time dependent, correlating with myelin basic protein expression. In situ hybridization reveals that CGT expression is restricted to the oligodendrocyte-containing cell layers of cerebrum and cerebellum, which also express myelin basic protein. The amino acid sequence of CGT shows significant homology to mammalian UDPglucuronyltransferases, which suggests a common evolutionary origin of these enzymes.

Amino Acid Sequence↗
PubMed1993

Site-directed mutagenesis of putative active-site amino acid residues of 3,2-trans-enoyl-CoA isomerase, conserved within the low-homology isomerase/hydratase enzyme family.

During beta-oxidation of unsaturated fatty acids, mitochondrial 3,2-trans-enoyl-CoA isomerase (mECI) converts 3-cis- or 3-trans-enoyl-CoA intermediates into their 2-trans isomers. The cDNA-derived amino acid sequence of mECI shows weak but significant homologies to the peroxisomal trifunctional enzyme (pTFE), the alpha-subunit of the fatty acid degradation complex from Escherichia coli (FadB), the mitochondrial 2-enoyl-CoA hydratase (mECH), the naphthoate synthase encoded by the menB gene from Bacillus subtilis (MenB), and the 4-chlorobenzoyl-CoA dehalogenase from Pseudomonas sp. (CBDH). These proteins from the isomerase/hydratase enzyme family. Tyr-150, Arg-151, and Asp-211 of the mECI are the only amino acids with protic side chains conserved within the enzymes with isomerase activity (pTFE and FadB). These amino acids are exchanged in the remaining enzymes of the isomerase/hydratase family. Glu-165 is conserved in all enzymes with isomerase and/or hydratase activity (pTFE, FadB, and mECH). We argue that these amino acids are possibly involved in the proton transfer at the active site of mECI. To test this hypothesis, mECI was functionally expressed in E. coli. The recombinant enzyme (rmECI) exhibits the same specific activity as the enzyme from rat liver. Exchange of the candidate active-site amino acids by site-directed mutagenesis revealed that Tyr-150 is not involved in isomerase catalysis. The exchange of Arg-151 and Asp-211 leads to a reduced expression of the recombinant enzyme accompanied by a reduced specific activity. The replacement of Glu-165 by Gln leads to a strongly reduced enzymatic activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Molecular cloning and gene organization of the mouse mitochondrial 3,2-trans-enoyl-CoA isomerase.

3,2-trans-enoyl-CoA isomerase (mECI, E.C. 5.3.3.8) is the key enzyme of mitochondrial beta-oxidation of unsaturated fatty acids. A mouse cDNA clone spanning the entire coding region of mECI was isolated and sequenced. Subsequently, two overlapping genomic clones containing the complete mECI gene were isolated and characterized. The mouse mECI cDNA comprises an open reading frame of 867 bp, encoding a protein of 32 kDa. The mECI gene, spanning about 15 kb, consists of seven exons. Multiple transcription starts were determined by primer extension experiments. Knowledge of the gene organization and availability of genomic clones for mouse mECI will facilitate the study of unsaturated fatty acid metabolism in normal and pathological states.

Amino Acid Sequence↗
PubMed1993

Electrogenic L-glutamate uptake in Xenopus laevis oocytes expressing a cloned rat brain L-glutamate/L-aspartate transporter (GLAST-1).

The transport of L-glutamate into Xenopus laevis oocytes expressing the cloned L-glutamate/L-aspartate transporter (GLAST-1) from rat brain was studied using the voltage clamp technique. At a holding potential of -90 mV, a bath application of 100 microM L-glutamate induced an inward current (IGLAST) with an amplitude ranging from -5 to -30 nA. IGLAST did not require extracellular Ca2+, Mg2+, or Cl-, was larger at negative potentials, and did not reverse up to +80 mV. The current was dependent on external L-glutamate and Na+ with half-maximal amplitudes at 11 microM L-glutamate and 41 mM Na+. IGLAST saturated at 100 microM L-glutamate and 80 mM Na+. The Hill coefficient for Na+ and L-glutamate was 3.3 and 1.3, respectively, suggesting that 3 Na+ accompany the transport of 1 L-glutamate molecule. At low [Na+]o, IGLAST was enhanced by reducing [K+]o, an indication for the countertransport of K+. Reducing external pH from 7.4 to 6.0 did not change the amplitude of IGLAST. This argues against a glutamate/proton cotransport. The results provide evidence for GLAST-1 carrying out a high affinity, sodium-dependent L-glutamate transport with a proposed stoichiometry of 3 Na+, 1 L-glutamate-/1 K+.

Amino Acid Transport System X-AG↗
PubMed1993

A novel mutation in the proteolipid protein gene leading to Pelizaeus-Merzbacher disease.

Point mutations of the gene of human proteolipid protein (PLP) have been recognized as the molecular basis of one form of leukodystrophy, the X-chromosome-linked Pelizaeus-Merzbacher disease (PMD). We report the molecular analysis of four PMD patients in three unrelated families and describe a point mutation (G-->A transition) in exon V which leads to the substitution of Gly216 by a serine residue in a highly conserved extracytosolic domain and a Mae I RFLP. Molecular modelling with energy minimization indicates that this seemingly minor alteration of the amino-acid sequence induces a considerable conformational change and tight packing of the polypeptide chain apparently not compatible with the regular PLP function in oligodendrocytes. This mutation has been detected and characterized by PCR amplification of genomic DNA using intron and exon primers and the complete sequence analysis of the seven exons and a 300 bp promoter region of the PLP gene of two affected brothers. The sequence analysis of a PCR fragment representing exon V amplified from genomic DNA of different kindreds of the pedigree revealed the mother as the only carrier indicating that the mutation has occurred de novo in the mother's germline. PLP gene (including the 8.8 kb intron I) rearrangements have been excluded by Southern blot hybridization and overlapping PCR amplification of genomic DNA.

Base Sequence↗
PubMed1993

Characterization of a brain-specific Sp1-like activity interacting with an unusual binding site within the myelin proteolipid protein promoter.

The proteolipid protein (PLP) gene encodes the main integral protein of the myelin membrane of the central nervous system. The expression of the gene is regulated in a cell- and development-specific manner. Comparison of approximately 1.5 kb of the upstream noncoding region from man, mouse, and rat gene revealed an extensive sequence identity of about 95% between -250 and +100 (the most upstream transcription start site is defined as +1) but only about 50% identity further upstream. To define potential cis-acting elements in the promoter of the mouse PLP gene the upstream region was studied by transfection of C6 glioblastoma cells and CHO fibroblasts with various 5' deletion constructs fused to the reporter gene luciferase. We localized a promoter at position -184 to +90, which is active in both cell lines. Analysis of this region by DNase I foot-printing experiments and band shift analysis with nuclear extracts from myelinating brain, liver, C6, and CHO cells shows the binding of several different proteins to the promoter region. One brain-specific and two ubiquitous factors bound to the sequence AAGGGGAGGAG (DR1/2 box). This motif is also present in the upstream region of other myelin-specific genes and in some variants of the glia cell-specific virus JC. The factors bound with similar affinity to a Sp1-binding site. Therefore one of the ubiquitous factors seems to be Sp1 suggesting that Sp1 may play a role in the transcriptional regulation of the PLP gene. It has been shown that the DR1/2 box-binding factors are Zn(2+)-dependent. By Southwestern blotting it has been demonstrated that the DR1/2 box binds a protein of about 66 kDa that is enriched in brain.

Animals↗
PubMed1992

Proteolipid protein (PLP) of CNS myelin: positions of free, disulfide-bonded, and fatty acid thioester-linked cysteine residues and implications for the membrane topology of PLP.

Proteolipid protein (PLP), the major integral membrane protein of central nervous system myelin, contains 14 cysteine residues within its 276-residue polypeptide chain. We determined the state of all cysteine residues and localized four of them as free thiols at positions 24, 32, 34, and 168. Four cysteines are connected by disulfide bonds: Cys200-Cys219 and Cys183-Cys227. The remaining six cysteine residues at positions 5, 6, 9, 108, 138, and 140 are modified by long-chain fatty acids, mainly palmitic acid, in thioester linkage. The extreme hydrophobicity of PLP can therefore be explained by two structural features: a composition of approximately 50% apolar amino acid residues and a high degree of fatty acid acylation. A differential fluorescent-labeling technique was developed for the structural studies: the cysteine residues belonging to one of the three states were derivatized by N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonic acid (I-AEDANS) either directly (a), after thioester cleavage with hydroxylamine (b), or after disulfide cleavage with dithiothreitol (c). The protein was then proteolytically digested with thermolysin, and the labeled peptides were isolated by reversed-phase HPLC followed by sequence analysis. The results were further confirmed by determination of the fatty acid to protein stoichiometry. The structural data not only demand the revision of our concept of the membrane topology of PLP but will also promote more sophisticated studies on the mechanism of myelination and new functions of PLP.

Amino Acid Sequence↗
PubMed1992

Structure, expression, and functional analysis of a Na(+)-dependent glutamate/aspartate transporter from rat brain.

Transport systems specific for L-glutamate and L-aspartate play an important role in the termination of neurotransmitter signals at excitatory synapses. We describe here the structure and function of a 66-kDa glycoprotein that was purified from rat brain and identified as an L-glutamate/L-aspartate transporter (GLAST). A GLAST-specific cDNA clone was isolated from a rat brain cDNA library. The cDNA insert encodes a polypeptide with 543 amino acid residues (59,697 Da). The amino acid sequence of GLAST suggests a distinctive structure and membrane topology, with some conserved motifs also present in prokaryotic glutamate transporters. The transporter function has been verified by amino acid uptake studies in the Xenopus laevis oocyte system. GLAST is specific for L-glutamate and L-aspartate, shows strict dependence on Na+ ions, and is inhibited by DL-threo-3-hydroxy-aspartate. In situ hybridization reveals a strikingly high density of GLAST mRNA in the Purkinje cell layer of cerebellum, presumably in the Bergmann glia cells, and a less dense distribution throughout the cerebrum. These data suggest that GLAST may be involved in the regulation of neurotransmitter concentration in central nervous system.

Amino Acid Sequence↗
PubMed1992

PROFILEGRAPH: an interactive graphical tool for protein sequence analysis.

The computer program PROFILEGRAPH, a graphical interactive tool for the analysis of amino acid sequences, is described. The main task of the program is to integrate a variety of sliding-window methods into a single user-friendly shell. The program allows the user to combine any amino acid specific parameter with a selection of several possible types of analysis and to plot the resulting graph in one of several windows on the screen. It is also possible to calculate the moment of the amino acid specific parameter for a given secondary structure and to display both the absolute moment value and the moment angle relative to a reference residue. Also included are several utilities that facilitate visual analysis of protein primary structures like, for example, helical-wheel diagrams. It is possible to adapt the majority of published sliding-window analysis procedures for use with PROFILEGRAPH.

Algorithms↗
PubMed1992

Mouse apolipoprotein AI. cDNA-derived primary structure, gene organisation and complete nucleotide sequence.

Apolipoprotein AI, the dominant protein component of serum high density lipoprotein, is intimately involved in cholesterol homeostasis. Apo AI activates the lecithin-cholesterol acyltransferase within the HDL particle and functions as ligand for a putative HDL receptor--two properties, which render this apolipoprotein a key mediator in reversed cholesterol transport. A functional analysis of the apo AI gene demands the isolation of the mouse apo AI gene for expression as transgenes in different mutant forms in the mouse. Here we describe the isolation of a full length apo AI-specific mouse liver cDNA clone with the human cDNA (892 bp) and the derived amino-acid sequence coding a polypeptide of 264 amino-acid residues. It showed a 70.7% homology to the rat and 66% to the human apo AI sequence. With this cDNA as probe the mouse apo AI gene was isolated and its organization analysed. Four exons, three of which are coding sequences, are aligned similarly to the human gene. The gene embraces 1825 bp between the transcription start, and the poly(A)+ tail attached 62 bp downstream of the stop codon. The complete nucleotide sequence of the four exons and three introns of the mouse apo AI gene was determined and its homology compared with that of the rat and human gene. Extensive deletions and a strongly reduced homology of the three introns of the two genes are obvious.

Amino Acid Sequence↗