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W Stoffel

Publications and source records attributed to W Stoffel.

At least 91 records · Page 5Linked to original sources
PubMed1988

Structural requirements of human preproapolipoprotein AI for translocation and processing studied by site-directed mutagenesis in vitro.

A full length human serum apolipoprotein AI (apo AI) cDNA clone was isolated from a human liver cDNA library. The EcoRI insertion fragment was cloned into expression vectors pDS5 and pDS12 for in vitro transcription and translation. The primary translation product is correctly translocated and the N-terminal signal sequence of the primary translation product of the wild type apo AI cleaved in the presence of dog pancreatic endoplasmic reticulum (ER) membranes releasing proapo AI. Ala-7 at the C-terminus of the signal sequence and Gln-1 of the prosequence were transposed by site-directed mutagenesis thus mutually exchanging the C-termini Gln-8-Ala-7 of the presequence and Gln-2-Gln-1 of the prosequence. The primary translation product of this mutated preproapo AI cDNA is correctly cotranslationally translocated into the lumen of the ER membranes and remains uncleaved by the signal peptidase. Deletion of the hexapeptide prosequence by site-directed mutagenesis in the preproapo AI cDNA led to a primary translation product which is cotranslationally translocated with processing to the mature apo AI polypeptide. We conclude that neither the proteolytic cleavage of the presequence nor the presence of the prosequence are structurally essential for the cotranslational translocation of apo AI. The amino-acid sequence bordering the cleavage site at the C-terminus of the presequence is without influence for the specificity of the signal peptidase.

Amino Acid Sequence↗
PubMed1988

Expression of the human serum apolipoprotein AI and AII genes in Xenopus laevis oocytes. Lipid-associated secretion of gene products.

The two major apolipoproteins of plasma high-density lipoproteins (HDL) are apolipoprotein AI (apo AI) and AII (apo AII). The apo AI and the correctly oriented apo CIII genes separated by 2.6 kb were obtained by fusion of two human lambda-genomic clones. The apo AII gene was isolated as a 3 kb clone. These apolipoprotein genes have been injected independently and together into Xenopus laevis oocytes and their expression studied. Both apolipoprotein genes were transcribed and translated into their preproforms and processed in Xenopus laevis oocytes to their proforms. They were secreted into the medium associated with newly synthesized phospholipids and neutral lipids as particles floating in the high-density lipoprotein range between 1.12 and 1.21 g/ml. Secreted apo AI is associated mainly with newly synthesized phosphatidylethanolamine and little triglyceride, apo AII with phosphatidylethanolamine, lysophosphatidylethanolamine and neutral lipids. Simultaneous injection of the apo AI and apo AII genes led to the secretion of both apoproteins which separated into two bands during CsCl-density gradient centrifugation. The heavier particles were associated with proapo AI and AII, phosphatidylethanolamine (greater than 90%) and traces of lysophosphatidylethanolamine as lipid components. Proapo AII was immunoprecipitated from the less dense fraction and found to be mainly associated with lysophosphatidylethanolamine. Radiolabelled newly synthesized apolipoproteins in secreted particles were characterized by immunoprecipitation after delipidation of the secreted lipoprotein particles. The oocyte-system proved very suitable for studies of the expression of serum apolipoprotein genes, the assembly of the apolipoproteins with specific lipids to lipoprotein particles and their secretion.

Animals↗
PubMed1987

Studies on the organization of the human apolipoprotein B 100 gene.

The organization of five exons of the 3' terminal end of the human apolipoprotein B 100 (apo B 100) gene 1906, 184, 115, 7572 and 374 bp long have been determined from two overlapping EMBL3 human genomic clones extending over 18 kb. They encode more than 70% of the apo B 100 amino-acid sequence. The introns between these five exons were sequenced revealing the common intron/exon splice junction sequences. The 7572 bp exon is the longest exon so far reported for mammalian genes with the proposed sequence coding for the LDL receptor binding site. Its possible relationship to apolipoprotein B 48 is discussed.

Apolipoprotein B-100↗
PubMed1986

[Regression of coronary sclerosis in familial hypercholesterolemia IIa by specific LDL apheresis].

The long-term effect of specific LDL plasma immunabsorption on the course of coronary arteriosclerosis was tested in ten patients with familial hypercholesteremia type IIa (seven females, three males; age range 15-57 years). Available were ECG, bicycle ergometry, hemodynamic and angiographic data. These tests were repeated seriatim in the course of the LDL immunabsorption. It was found that exercise tolerance markedly increased, ischemic ST-changes became less marked and the initial hemodynamic parameters remained unchanged. The morphology of the coronary arteries was influenced lastingly: among 22 stenoses only one progressed, there was no further progression in 11 stenoses and regression in ten. When there was generalized arteriosclerosis, progression occurred in two of 79 coronary artery segments, progression was arrested in 56 and regression was noted in 21 of 79 segments. These results demonstrate that specific LDL plasma immunabsorption for the elimination of LDL cholesterol favorably influences the course of coronary arteriosclerosis in patients with familial hypercholeremia type IIa. The findings support the lipid theory of atherosclerosis and coronary arteriosclerosis, at least in this special group of patients.

Adolescent↗
PubMed1986

Individual exons encode the integral membrane domains of human myelin proteolipid protein.

The gene encoding human proteolipid protein (PLP) was isolated from a human genomic library by hybridization with labeled DNA of a PLP-specific cDNA clone. The entire PLP gene spans approximately 17 kilobases. Restriction and sequence analysis revealed seven exons and six introns. The entire nucleotide sequences of the exons and of the exon-intron transitions were determined, and the intron lengths were measured. Exon I includes only ATGG of the translated region, the N-terminal methionine codon and G of glycine, the first amino acid of mature PLP. Each hydrophobic trans- and cis-membrane domain of PLP together with its adjacent hydrophilic sequence correlates closely with one exon of the gene except for the C-terminal transmembrane helix that is encoded by two exons. The amino acid sequence of human PLP derived from the nucleic acid sequence is highly conserved. Human and rat PLP are completely homologous, whereas only four amino acid residues are exchanged in bovine PLP sequence derived from protein sequencing and a partial cDNA clone. Homology search on the nucleic acid level among human, bovine, and rat brain PLPs indicates an unusually high homology in the coding regions. Hybridization analysis with DNA of human-rodent hybrid clones revealed that the gene encoding PLP segregates with human X chromosome in the region q13-q22.

Amino Acid Sequence↗
PubMed1986

Isolation, expression and characterization of a human apolipoprotein B 100-specific cDNA clone.

The isolation and characterization of a human apolipoprotein B 100-specific cDNA clone (lambda gt-B1) containing a 1321 base pairs (bp) spanning insert is described. It encodes the 3'-nontranslated 281 bp long region up to the polyadenylation site and 1040 bp of the C-terminal coding region of 345 amino-acid residues of human apo B 100 and the stop codon. The lambda gt-B1 cDNA clone has been isolated from a human hepatoma cDNA expression library by immunoscreening using affinity-purified polyclonal anti apo B 100 antibodies. The nucleotide sequence of the apo B 100 insert has been determined. A part of the polypeptide sequence derived from this nucleotide sequence was identical with the amino-acid sequence obtained by protein sequencing of a purified cyanogen bromide fragment of apo B 100. The fusion protein consisting of beta-galactosidase and the 345 amino-acid residue long C-terminus of apo B 100 had an apparent molecular mass of 148 kDa in NaDodSO4 polyacrylamide gel electrophoresis. In Northern blot hybridization analysis the insert of the apo B 100-cDNA clone hybridized to a 20 to 22 kb mRNA from adult human liver.

Amino Acid Sequence↗
PubMed1986

Cloned proteolipid protein and myelin basic protein cDNA. Transcription of the two genes during myelination.

cDNA clones of rat brain proteolipid protein (PLP), also named lipophilin, the major integral myelin membrane protein, and of myelin basic protein (MBP), the major extrinsic myelin protein, have been isolated from a rat brain cDNA library cloned into the PstI site of pBR322. Poly(A)+ RNA from actively myelinating 18-day-old rats has been reversely transcribed. Oligonucleotides synthesized according to the established amino-acid sequence of lipophilin and the nucleotide sequence of the small myelin basic protein of the N-terminal, the central and C-terminal region of their sequences were used as hybridization probes for screening. The largest insert in one of several lipophilin clones was 2,585 base pairs (bp) in length (pLp 1). It contained 521 bp of the C-terminal coding sequence and the complete 2,064 bp long non-coding 3' sequence. The myelin basic protein cDNA insert of clones pMBP5 and pMBP6 is 2,530 bp long and that of clones pMBP2 and pMBP3 640 bp. These clones were also characterized. pMBP2 was sequenced and used together with the lipophilin cDNA clones as hybridization probes to estimate the lipophilin and myelin basic protein mRNA levels of rat brain during the myelination period. The expression of the lipophilin and myelin basic protein genes during development of the myelin sheath appears to be strictly coordinated.

Animals↗
PubMed1985

Synthesis and processing of human serum apolipoprotein AII in vitro and in Hep G2 cells.

The synthesis and structure of the primary translation product of apo AII in a human liver poly(A+) mRNA primed cell-free system and its cotranslational modification was studied parallel to studies in vivo with Hep G2 cells, a human hepatoma cell line. The primary translation product is a preproprotein containing 100 amino acid residues, which is cleaved by the signal peptidase of endoplasmic reticulum to pro-apo AII with the loss of the N-terminal pre-sequence consisting of 18 amino acid residues. Hep G2 cells contain about equal amounts of the proform of apolipoprotein AII and of mature apo AII. Approximately in the same ratio pro- and mature apo AII are secreted into the medium. Determination of the partial amino-acid sequence by automated Edman degradation of the labelled prepro- and proforms of apo AII led to the segmentation of the N-terminus of the primary translation product, consisting of 23 amino acid residues, into the pre-sequence (18 residues) and the pro-sequence (5 residues) with terminal Arg-Arg-residues at the cleavage site to apo AII. We must therefore correct our previously postulated 17 and 6 residues containing segmentation. So far no information has been obtained in which compartment and at what stage of posttranslational events the dimerization occurs by formation of the single disulfide bond at position Cys6 in the mature apo AII structure, leading to the symmetrical molecule.

Amino Acid Sequence↗
PubMed1985

Processing of proapolipoprotein AI requires specific conformation.

Apolipoprotein AI of human high-density lipoproteins is secreted by hepatocytes as a proapolipoprotein with a N-terminal hexapeptide sequence (Arg-His-Phe-Trp-Gln-Gln-) which differs from the prosequence of rat apolipoprotein AI (Trp-Asp-Phe-Trp-Gln-Gln). The two proteins have in common the unusual cleavage site -Gln-Gln-Asp-Glu-. It is hydrolysed by a specific serum proteinase with the release of mature apo AI. We synthesized a model substrate for the study of the final processing of pro-apo AI by the serum proteinase. It is an undecapeptide embracing the human pro-hexapeptide sequence and the first five N-terminal residues of apo AI, covalently linked to a hydrophilic resin. The N-terminal arginine residue was 3H-labelled. [formula; see text] This sequence was not cleaved by human serum under the conditions under which rat serum processes the pro-form of apo AI secreted by rat hepatocytes. Pepsin and chymotrypsin fragmented the undecapeptide at sites characteristic for these proteinases. We conclude that the proteolytic cleavage at the specific site (-Gln-Gln-Asp-Glu-) requires the correct conformation in addition to the specific amino-acid sequence.

Amino Acid Sequence↗
PubMed1985

Amino-acid sequence of human and bovine brain myelin proteolipid protein (lipophilin) is completely conserved.

Proteolipid protein (PLP) was isolated from white matter of human brain by chloroform/methanol extraction and further purified by chromatography. Performic acid oxidation yielded a product homogeneous in NaDodSO4-polyacrylamide electrophoresis with a molecular mass of 30 kDa. The carboxymethylated PLP was chemically cleaved with cyanogen bromide into four fragments: CNBr I 22-24 kDa, CNBr II 5 kDa, CNBr III 1.4 kDa and CNBr IV 0.7 kDa. HBr/dimethylsulfoxide cleavage at tryptophan residues released four fragments: Trp I 14-16 kDa, Trp II 2.0 kDa, Trp III 5 kDa and Trp IV 7 kDa. Hydrophilic fragments were enriched in 50% formic acid (CNBr II, III, IV and Trp II and III), whereas hydrophobic peptides precipitated from this solvent were CNBr I, Trp I and IV. The fragments were separated by gel filtration with 90% formic acid as solvent and finally purified by gel permeation HPLC (Si 60 and Si 100) for automated liquid and solid-phase Edman degradation. Large fragments were further cleaved with different proteinases (trypsin, V8-proteinase, endoproteinase Lys-C and thermolysin). We used an improved strategy in the sequencing of the human proteolipid protein compared with our approach to the structural elucidation of bovine brain PLP. The amino-acid sequence of human PLP contains 276 residues, the same as found in bovine proteolipid protein. The two sequences proved to be identical. The possible importance of the conservative structure of this integral membrane protein is discussed.

Amino Acid Sequence↗
PubMed1984

Synthesis, transport, and processing of apolipoproteins of high density lipoproteins.

Cell biology methods have greatly influenced the elucidation of the biosynthetic pathways of apolipoproteins. In vitro and tissue culture systems allow the study, to a large extent, of the process of synthesis, intracellular processing, secretion, and extracellular processing of the major high density lipoprotein apoproteins apoA-I and A-II and also of a minor component, apoA-IV. Whereas the latter apoprotein is equipped only with a signal sequence, the primary translation products of apoA-I and apoA-II carry N-terminal extensions of preprosequence of 24 amino acids for apoA-I and 23 amino acid residues for apoA-II. The pro-form of apoA-I characterized by a hexapeptide extension is completely stable intracellularly and is secreted as such. The pro-form is further processed by a serum protease specific for an unusual -Gln-Gln-Asp-Glu-sequence site. Pro-apoA-II, a pentapeptide sequence, is partially processed intracellularly to its mature form and secreted together with the residual pro-form. The cleavage site of pro-apoA-II is characterized by two basic amino acid residues Arg-Arg, present also in other known pro-proteins. The biological function of the N-terminal pro-sequences and details of their final processing by the serum protease(s) have yet to be established.

Amino Acid Sequence↗
PubMed1984

Structure and molecular arrangement of proteolipid protein of central nervous system myelin.

Proteolipid protein (PLP) of central nervous system myelin is one of the most hydrophobic integral membrane proteins. It consists of a 276-residue-long polypeptide chain with five strongly hydrophobic sequences of 26, 30, 39, 12, and 36 residues, respectively, linked by highly charged hydrophilic sequences. Hyposmotically dissociated bovine myelin membranes were treated with trypsin. PLP was completely cleaved into smaller fragments, whereas basic myelin protein remained essentially unaltered. The proteins and tryptic peptides of myelin were separated after the removal of the short, water-soluble peptides into three large fragments of 11, 7.3, and 9.0 kDA, respectively. They were characterized by their molecular mass and NH2-terminal amino acid sequences, which proved that trypsin cleaved predominantly at Arg-97 yielding the 11-kDa fragment from Gly-1 through Arg-97, at Arg-126 releasing the 7.3-kDa fragment from Gly-127 through Lys-191, and at Lys-191 releasing the 9-kDa fragment from Thr-192 through Phe-276. We propose that PLP is integrated into the lipid bilayer of myelin with the NH2 terminus and three positively charged hydrophilic loops oriented toward the extracytosolic side of the membrane, whereas one strongly negative hydrophilic loop and the positively charged COOH terminus cover the cytosolic side of the lipid bilayer. Basic myelin protein remains protected against tryptic cleavage, which indicates its apposition to the cytosolic side of the membrane. These cleavage sites of trypsin support the suggested orientation of PLP in the myelin membrane and thereby extend our knowledge about the molecular arrangement of the components of this membrane. In demyelinating processes membrane desintegration could be initiated by proteolysis at the external surfaces of proteolipid protein in a similar way as described here.

Amino Acid Sequence↗
PubMed1983

A new synthesis of bis(diacylglycero)phosphate.

The chemical synthesis of bis(diacylglycero) phosphate previously named bisphosphatidic acid, starting with a diacylglycerol and phosphatidic acid, is described. The phosphodiester bond formation is catalyzed by triisopropylbenzenesulfonylchloride. This simple approach allows the preparation of saturated as well as unsaturated bis(diacylglycero)phosphate species in one step without the use of any protecting group. The methods used until now yield only mono-acid species, or mixed-acid unsaturated species after many steps involving the introduction and the removal of protecting groups. The synthetic products have been characterized by component analysis and NMR-techniques.

Catalysis↗
PubMed1983

Cell-free translation of human liver apolipoprotein AI and AII mRNA. Processing of primary translation products.

Human liver apolipoprotein AI and A II poly(A+) mRNA has been translated in the cell-free rabbit reticulocyte lysate system. The structures of the two primary translation products of these two main protein components of human serum high-density lipoprotein (HDL) have been characterized. The products of the synthesis in vitro are preproapolipoproteins. The signal sequence (pre-sequence) of the primary translation product of human apo AI mRNA consists of 18 amino acids, that of apo AII of 17 amino acids. The cotranslational translocation into dog microsomal vesicles is associated with the cleavage of these sequences by the signal peptidase releasing the proapolipoproteins AI and AII, both extended by an N-terminal hexapeptide. Preproapolipoprotein AII is synthesized in its monomeric form consisting of 100 amino acids. Pro-apo AII is present in the vesicles of the endoplasmic reticulum also as monomer. Sequencing of the radiolabelled signal sequences of both pre-forms revealed their strongly hydrophobic nature. Despite the high affinity of HDL-apolipoproteins for complex lipids their secretion requires these hydrophobic signal sequences for translocation. Internal recognition sequences in the native apoproteins are not responsible for the transmembrane transport.

Amino Acid Sequence↗
PubMed1983

Serum apolipoprotein AI synthesis in rat hepatocytes and its secretion as proform.

Rat hepatocytes in monolayer or suspension culture synthesize serum lipoprotein AI. It is secreted into the serum-free culture medium. Synthesis and secretion processes were studied in the presence of radiolabelled amino acids. The synthesis product of the hepatocytes and the secretion product from the medium were isolated by immunoprecipitation with a mono-specific rabbit antiserum against rat apolipoprotein AI. The intracellular and secreted products were homogeneous and identical in polyacrylamide gel electrophoresis but had reduced electrophoretic mobility as compared to native apolipoprotein AI. They were submitted to automated Edman degradation. They were present in their proform, the N-terminus of which is extended by a hexapeptide. In the presence of rat serum the proform is proteolytically transformed into the mature form of apolipoprotein AI.

Amino Acid Sequence↗
PubMed1983

The primary structure of bovine brain myelin lipophilin (proteolipid apoprotein).

The amino-acid sequence of bovine myelin lipophilin (proteolipid apoprotein, Folch-protein) has been completed. Lipophilin is a 276 amino acid residues containing, extremely hydrophobic membrane protein with molecular mass 30,000 Da. The sequence determination was based on automated Edman degradation of four tryptophan and four cyanogen bromide fragments and of proteolytic peptides of complete lipophilin as well as the fragments obtained by chemical cleavage. Four additional sequences were determined which led to the completion of the primary structure. Lipophilin is esterified at threonine-198 by long chain fatty acids (palmitic, stearic and oleic acid). The attachment site has been established at the same threonine residue in three different peptides isolated from thermolysinolytic, papainolytic and chymotrypsinolytic hydrolysates. This threonine residue is part of a hydrophilic segment of lipophilin. The covalent fatty acyl bond is being discussed together with important structural and functional properties of this membrane protein which can be derived from sequence information. New separation and purification methods of hydrophobic and hydrophilic polypeptides for this sequence determination (fractional solubilization, silica gel exclusion, high-performance liquid chromatography) had to be elaborated as indispensable tools. They are generally applicable to the structural analysis of hydrophobic membrane proteins. Four long (26, 29, 40 and 36 residues) and one medium long (12 residues) hydrophobic segments are separated by four predominantly positively and one negatively charged hydrophilic segments. On the basis of structural data a model for the membrane integration of lipophilin is proposed.

Amino Acid Sequence↗
PubMed1983

A serum proteinase converts proapolipoprotein AI secreted by rat hepatocytes to the mature apolipoprotein.

Apolipoprotein AI integrated into the high-density lipoprotein particle in serum was synthesized in the rat hepatocyte in the presence of radiolabelled amino acids and isolated from the cells in primary culture (suspension) as its proform, with the N-terminus extended by a hexapeptide segment. The primary secretion product is this proform which is only further proteolytically processed in the presence of the serum fraction with density higher than 1.21 g/ml. The secretion product and the proteolytically converted product were characterized by Edman degradation of their respective amino-acid sequences after radiolabelling with [3H]valine and [3H]phenylalanine, the positions of which are well established in the preproform and in the N-terminus of mature rat apolipoprotein AI. The products from the lysed cells and their culture medium were purified by immunoprecipitation, sodium dodecyl sulfate gradient gel electrophoresis and subsequent electroelution of the apo AI band. The proform sediments associated with a particle of density 1.16-1.20 g/ml. The serum proteinase which is inhibited by phenylmethanesulfonyl fluoride, but not by sulfhydryl reagents, is presumably a serine proteinase.

Animals↗