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Biomedical subjects

L P Aggerbeck

Publications and source records attributed to L P Aggerbeck.

At least 19 recordsLinked to original sources
PubMed2001

Apolipoprotein B48 glycosylation in abetalipoproteinemia and Anderson's disease.

BACKGROUND & AIMS: Abetalipoproteinemia and Anderson's disease are hereditary lipid malabsorption syndromes. In abetalipoproteinemia, lipoprotein assembly is defective because of mutations in the microsomal triglyceride transfer protein. Here, we evaluated the intracellular transport of apolipoprotein B48 to localize the defect in Anderson's disease. METHODS: Asparagine-linked oligosaccharide processing of apolipoprotein B48 in normal and affected individuals was determined by the endoglycosidase H and F sensitivities of the protein after metabolic labeling of intestinal explants in organ culture. Cell ultrastructure was evaluated with electron microscopy. RESULTS: In Anderson's disease as in normal individuals, there was a time-dependent transformation of high mannose endoglycosidase H-sensitive oligosaccharides, of endoplasmic reticulum origin, to complex endoglycosidase H-resistant oligosaccharides, added in the Golgi network. In contrast, despite the translocation of apolipoprotein B48 into the endoplasmic reticulum in patients with abetalipoproteinemia and in biopsies treated with Brefeldin A, which blocks anterograde transport between the endoplasmic reticulum and the Golgi network, there was no transformation of endoglycosidase H-sensitive oligosaccharides. CONCLUSIONS: In abetalipoproteinemia and Anderson's disease, apolipoprotein B48 is completely translocated into the endoplasmic reticulum, but only in Anderson's disease is the protein transported to the Golgi apparatus. This suggests that Anderson's disease is caused by a post-Golgi cargo-specific secretion defect.

Abetalipoproteinemia↗
PubMed2000

The role of the microsomal triglygeride transfer protein in abetalipoproteinemia.

The microsomal triglyceride transfer protein (MTP) is a dimeric lipid transfer protein consisting of protein disulfide isomerase and a unique 97-kDa subunit. In vitro, MTP accelerates the transport of triglyceride, cholesteryl ester, and phospholipid between membranes. It was recently demonstrated that abetalipoproteinemia, a hereditary disease characterized as an inability to produce chylomicrons and very low-density lipoproteins in the intestine and liver, respectively, results from mutations in the gene encoding the 97-kDa subunit of the microsomal triglyceride transfer protein. Downstream effects resulting from this defect include malnutrition, very low plasma cholesterol and triglyceride levels, altered lipid and protein compositions of membranes and lipoprotein particles, and vitamin deficiencies. Unless treated, abetalipoproteinemic subjects develop gastrointestinal, neurological, ophthalmological, and hematological abnormalities.

Abetalipoproteinemia↗
PubMed2000

[Microsomal triglyceride transfer protein and abetalipoproteinemia].

Microsomal triglyceride transfer protein (MTP) is a dimeric protein complex consisting of protein disulfide isomerase and a unique 97 kDa subunit. In vitro, MTP accelerates the transport of triglyceride, cholesteryl ester, and phospholipid between vesicles. It was recently demonstrated that abetalipoproteinemia, a disease characterized as an inability to produce chylomicrons and very low density lipoproteins in the intestine and liver, respectively, is the result of a genetic absence of MTP. Downstream effects resulting from this defect, include very low plasma cholesterol and triglyceride levels, absence of plasma apolipoprotein B and a lipid malabsorption syndrome, leading to lipo-soluble vitamin deficiencies. A low fat diet is instituted to eliminate the diarrhea. In addition, a therapy with vitamins A and E is essential to prevent patients from developing secondary effects such as neuropathy, muscle weakness, and retinopathy.

Abetalipoproteinemia↗
PubMed1999

Mimicking lipid-binding-induced conformational changes in the human apolipoprotein E N-terminal receptor binding domain effects of low pH and propanol.

We studied the effects of n-propanol and pH on the structure of the apolipoprotein E3 N-terminal receptor binding domain, apo E3(1-191), to determine whether conditions similar to those occurring near lipid surfaces (decreased dielectric constant and pH) can mimic lipid-induced conformational changes in apo E3. The addition of 30% n-propanol, at pH 7, induces a conformational change in apo E3(1-191) as shown by changes in the intrinsic tryptophan fluorescence and by an increase in the Stokes radius of the majority of the protein from 3.0 to 4.1 nm, although the protein remains monomeric as shown by chemical cross-linking. These changes are accompanied by increased resistance to limited proteolysis with trypsin, chymotrypsin, subtilisin and endoproteinase glu-C, as is the case for apo E3(1-191) reconstituted into phospholipid/cholesterol lipid bicelles. Far and near UV circular dichroism showed that n-propanol increases the amount of calculated alpha-helical structure (42-65%) and alters the tertiary structure of the protein although not as much as when apo E3(1-191) is incorporated into lipid bicelles. In the absence of n-propanol, lowering the pH to 4.5 decreases the Stokes radius of the majority of the protein somewhat, with little effect upon the secondary and the tertiary structures. The addition of 30% n-propanol at pH 4.5 increases the Stokes radius of apo E3(1-191) from 2.2 to 5.0 nm, even more than at pH 7 (3.0-4.1 nm) although the protein still remains predominantly monomeric. There is increased resistance to limited proteolysis with endoproteinase glu-C. As assessed by far and near UV circular dichroism, the addition of 30% n-propanol at pH 4.5, in contrast to pH 7, markedly increases the alpha-helical structure and changes the tertiary structure of the protein similarly to that resulting from the incorporation of apo E3(1-191) into lipid bicelles. The results suggest that a combination of n-propanol and low pH in aqueous solutions may be useful as a simple model system for studying conformational changes in apo E3 similar to those, which occur upon interaction of the protein with lipids.

1-Propanol↗
PubMed1999

Anderson's disease: exclusion of apolipoprotein and intracellular lipid transport genes.

Anderson's disease is a rare, hereditary hypocholesterolemic syndrome characterized by chronic diarrhea, steatorrhea, and failure to thrive associated with the absence of apo B48-containing lipoproteins. To further define the molecular basis of the disease, we studied 8 affected subjects in 7 unrelated families of North African origin after treatment with a low-fat diet. Lipid loading of intestinal biopsies persisted, but the pattern and extent of loading was variable among the patients. Electron microscopy showed lipoprotein-like particles in membrane-bound compartments, the densities (0.65 to 7.5 particles/mu(2)) and the mean diameters (169 to 580 nm) of which were, in general, significantly larger than in a normal fed subject (0.66 particles/mu(2), 209 nm mean diameter). There were also large lipid particles having diameters up to 7043 nm (average diameters from 368 to 2127 nm) that were not surrounded by a membrane. Rarely, lipoprotein-like particles 50 to 150 nm in diameter were observed in the intercellular spaces. Intestinal organ culture showed that apo B and apo AIV were synthesized with apparently normal molecular weights and that small amounts were secreted in lipid-bound forms (density <1.006 g/mL). Normal microsomal triglyceride transfer protein (MTP) and activity were also detected in intestinal biopsies. Segregation analyses of 4 families excluded, as a cause of the disease, significant regions of the genome surrounding the genes for apo AI, AIV, B, CI, CII, CIII, and E, as were the genes encoding 3 proteins involved in intracellular lipid transport, MTP, and fatty acid binding proteins 1 and 2. The results suggest that a factor other than apoproteins and MTP are important for human intestinal chylomicron assembly and secretion.

Adult↗
PubMed1997

PCF11 encodes a third protein component of yeast cleavage and polyadenylation factor I.

Cleavage and polyadenylation factor I (CF I) is one of four factors required in vitro for yeast pre-mRNA 3'-end processing. Two protein components of this factor, encoded by genes RNA14 and RNA15, have already been identified. We describe here another gene, PCF11 (for protein 1 of CF I), that genetically interacts with RNA14 and RNA15 and which presumably codes for a third protein component of CF I. This gene was isolated in a two-hybrid screening designed to identify proteins interacting with Rna14 and Rna15. PCF11 is an essential gene encoding for a protein of 626 amino acids having an apparent molecular mass of 70 kDa. Thermosensitive mutations in PCF11 are synergistically lethal with thermosensitive alleles of RNA14 and RNA15. The Pcf11-2 thermosensitive strain shows a shortening of the poly(A) tails and a strong decrease in the steady-state level of actin transcripts after a shift to the nonpermissive temperature as do the thermosensitive alleles of RNA14 and RNA15. Extracts from the pcf11-1 and pcf11-2 thermosensitive strains and the wild-type strain, when Pcf11 is neutralized by specific antibodies, are deficient in cleavage and polyadenylation. Moreover, fractions obtained by anion-exchange chromatography of extracts from the wild-type strain contain both Pcf11 and Rna15 in the same fractions, as shown by immunoblotting with a Pcf11-specific antibody.

Amino Acid Sequence↗
PubMed1996

A novel abetalipoproteinemia genotype. Identification of a missense mutation in the 97-kDa subunit of the microsomal triglyceride transfer protein that prevents complex formation with protein disulfide isomerase.

The microsomal triglyceride transfer protein (MTP) is a heterodimer composed of the ubiquitous multifunctional protein, protein disulfide isomerase, and a unique 97-kDa subunit. Mutations that lead to the absence of a functional 97-kDa subunit cause abetalipoproteinemia, an autosomal recessive disease characterized by a defect in the assembly and secretion of apolipoprotein B (apoB) containing lipoproteins. Previous studies of abetalipoproteinemic patient, C.L., showed that the 97-kDa subunit was undetectable. In this report, [35S]methionine labeling showed that this tissue was capable of synthesizing the 97-kDa MTP subunit. Electrophoretic analysis showed two bands, one with a molecular mass of the wild type 97-kDa subunit and the other with a slightly lower molecular weight. Sequence analysis of cDNAs from additional intestinal biopsies showed this patient to be a compound heterozygote. One allele contained a perfect in-frame deletion of exon 10, explaining the lower molecular weight band. cDNAs of the second allele were found to contain 3 missense mutations: His297 --> Gln, Asp384 --> Ala, and Arg540 --> His. Transient expression of each mutant showed that only the Arg540 --> His mutant was non-functional based upon its inability to reconstitute apoB secretion in a cell culture system. The other amino acid changes are silent polymorphisms. High level coexpression in a baculovirus system of the wild type 97-kDa subunit or the Arg540 --> His mutant along with human protein disulfide isomerase showed that the wild type was capable of forming an active MTP complex while the mutant was not. Biochemical analysis of lysates from these cells showed that the Arg to His conversion interrupted the interaction between the 97-kDa subunit and protein disulfide isomerase. Replacement of Arg540 with a lysine residue maintained the ability of the 97-kDa subunit to complex with protein disulfide isomerase and form the active MTP holoprotein. These results indicate that a positively charged amino acid at position 540 in the 97-kDa subunit is critical for the productive association with protein disulfide isomerase. Of the 13 mutant MTP 97-kDa subunit alleles described to date, this is the first encoding a missense mutation.

Apolipoproteins B↗
PubMed1996

Ultrastructural immunogold labeling of lipid-laden enterocytes from patients with genetic malabsorption syndromes.

Intestinal biopsies from patients having genetic disorders of lipoprotein assembly and secretion, such as abetalipoproteinemia (ABL) or Anderson's disease (AD), contain large amounts of lipids which are accumulated in the enterocytes. Determination of the intracellular sites in which the lipids accumulate and to which apolipoproteins the lipids are bound would help to identify the defects in these diseases and further elucidate the mechanisms by which lipoprotein assembly and secretion occur normally. Ultrastructural immunogold labeling, however, is hampered by the poor preservation of the lipids accumulated in the enterocytes of these patients. We have used routine electron microscopy (fixation and ultra-thin sectioning) along with three methods for immunogold labeling of lipid-laden enterocytes: ultrathin cryosectioning, low temperature freeze substitution with embedding in Lowicryl K4M, and ultra-low temperature freeze substitution with embedding in Lowicryl HM20, to establish a protocol for investigating the intestinal tissue from these patients. Ultracryosectioning, while preserving the overall morphology of the lipid laden enterocytes, did not preserve the lipid content and the immunogold labeling of apolipoprotein B (ApoB) appeared dislocated. Freeze substitution and low temperature embedding in Lowicryl K4M, in contrast, appeared to better preserve the lipid and lipoprotein structures; however, the antigenicity of both apoAI and apoB appeared to be lost and no specific labeling could be obtained. Freeze substitution and embedding in Lowicryl HM20 best preserved the lipid and lipoprotein structures while maintaining apoprotein antigenicity. In conclusion, immunogold labeling of apolipoproteins on lipid structures in the lipid-laden enterocytes of patients with ABL and AD is best obtained by freeze substitution and embedding in Lowicryl HM20.

Abetalipoproteinemia↗
PubMed1993

Cytoplasmic and periplasmic production of human apolipoprotein E in Escherichia coli using natural and bacterial signal peptides.

To understand the toxicity of high levels of heterologous human serum apolipoprotein E (ApoE) in Escherichia coli, as well as to prepare a system for producing the structural domains of this protein, plasmids were constructed in which the coding sequence of the N-terminal domain or all of ApoE followed E. coli or human apolipoprotein signal peptides (SP) or the N-terminal eleven amino acids (f10) of the gene 10-encoded protein of phage T7. High levels of production of the 22-kDa N-terminal domain (22K) of ApoE were obtained either as an f10::22K fusion protein, or using the natural SP, or SP derived from the periplasmic protein, alkaline phosphatase (PhoA), or from the outer membrane protein A (OmpA). Microsequencing showed that the SP of sPhoA::22K and sOmpA::22K, but not sApoE::22K, were correctly processed and, in the former cases, the protein could be released from the cells by osmotic shock. The extent of maturation of sPhoA::22K depended upon the host strain; with JM109, about 50% of the protein was not processed. Microsequencing of the f10::22K fusion protein, which could easily be purified following lysis of the cells, showed that the N-terminal methionine had been removed in agreement with the length parameter rule. Although considerable levels of the f10::ApoE fusion protein could be produced in the cytoplasm, production was markedly less using the PhoA signal peptide and the protein was not easily isolated following osmotic shock. The recombinant protein was biologically active after reconstitution with lipids in spite of the N-terminal modifications introduced.(ABSTRACT TRUNCATED AT 250 WORDS)

Apolipoproteins E↗
PubMed1992

Absence of microsomal triglyceride transfer protein in individuals with abetalipoproteinemia.

Abetalipoproteinemia is a human genetic disease that is characterized by a defect in the assembly or secretion of plasma very low density lipoproteins and chylomicrons. The microsomal triglyceride transfer protein (MTP), which is located in the lumen of microsomes isolated from the liver and intestine, has been proposed to function in lipoprotein assembly. MTP activity and the 88-kilodalton component of MTP were present in intestinal biopsy samples from eight control individuals but were absent in four abetalipoproteinemic subjects. This finding suggests that a defect in MTP is the basis for abetalipoproteinemia and that MTP is indeed required for lipoprotein assembly.

Abetalipoproteinemia↗
PubMed1991

Protein disulfide isomerase appears necessary to maintain the catalytically active structure of the microsomal triglyceride transfer protein.

Protein disulfide isomerase (PDI) is a component of the microsomal triglyceride transfer protein (MTP) complex. This study was initiated to help elucidate the role of PDI in MTP. The 88-kDa polypeptide of MTP (88K) was dissociated from PDI by using chaotropic agents (NaClO4 and KSCN), low concentrations of a denaturant (guanidine hydrochloride) or a nondenaturing detergent (octyl glucoside). As assessed by fluorescence and circular dichroism spectroscopy, these three different approaches appeared to dissociate the components of MTP under mild, nondenaturing conditions. The dissociating agents were diluted or removed by dialysis, and the free PDI and 88K were further characterized. In all cases, the dissociation coincided with the loss of triglyceride transfer activity. The free 88-kDa polypeptide readily aggregated, suggesting that it is a hydrophobic peptide. Even in the presence of chaotropic agents, when 88K was not aggregated, transfer activity was not expressed. These results suggest that the association of PDI with 88K is necessary to maintain the catalytically active form of the triglyceride transfer protein and prevent the aggregation of 88K.

Animals↗
PubMed1991

Structural properties of the microsomal triglyceride-transfer protein complex.

The microsomal triglyceride-transfer protein (MTP), which catalyzes the transport of triglyceride and cholesteryl ester between membranes, is a complex composed of two proteins having apparent molecular weights of 58,000 and 88,000. The 58,000 molecular weight component of MTP has been identified as the multifunctional protein, protein disulfide isomerase (PDI). The multisubunit nature of MTP as well as the presence of PDI as one of the subunits distinguishes this protein from previously characterized lipid-transfer proteins. In this study, we have more clearly defined structural elements of MTP that may play important functional roles. The molecular weight of the transfer protein complex was determined to be 150,000 by sedimentation equilibrium experiments performed at three different speeds, suggesting that MTP is a complex of one PDI and one 88,000 molecular weight polypeptide (88K). Following SDS-polyacrylamide gel electrophoresis, the Coomassie Blue staining intensity of PDI in a known amount of MTP was compared to that of known amounts of a PDI standard. A 1 to 0.98-1.30 ratio of PDI to 88K was determined, confirming the 1:1 stoichiometry of MTP. The sedimentation coefficient (5.85) determined by analytical ultracentrifugation and the Stokes radius (47 A) determined by polyacrylamide gradient gel electrophoresis indicate that the 150,000 molecular weight MTP complex is asymmetric and/or has an unusually high water of hydration. PDI and 88K form a stable protein complex; there was no evidence of a dissociation-reassociation reaction occurring between the two components. Analysis of far-ultraviolet circular dichroism spectra revealed MTP has about 28% alpha-helical and 28% beta-structural content.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Crystallization and preliminary X-ray diffraction studies on the amino-terminal (receptor-binding) domain of human apolipoprotein E3 from serum very low density lipoproteins.

Human apolipoprotein E is a component of several classes of circulating plasma lipoproteins. In addition to binding lipids, this apolipoprotein, which is composed of two structural domains, mediates some lipoprotein-receptor interactions by binding to the low density lipoprotein receptor. The receptor-binding function, as well as some lipid-binding capability, is contained in the amino-terminal structural domain of apolipoprotein E. Thrombin-catalyzed hydrolysis of apolipoprotein E yields a fragment (residues 1 to 191) that has the same properties as, and seems to be a good model for, the amino-terminal domain. Crystals of this amino-terminal fragment suitable for high-resolution X-ray diffraction experiments have now been grown. The crystals belong to the orthorhombic space group P2(1)2(1)2(1) and have unit cell dimensions of a = 86.0 A, b = 40.9 A, and c = 53.3 A (1 A = 0.1 nm). This is the first human serum apolipoprotein to be crystallized.

Amino Acid Sequence↗
PubMed1988

Human apolipoprotein E3 in aqueous solution. I. Evidence for two structural domains.

The stability and structure of human apolipoprotein (apo) E3 in aqueous solution were investigated by guanidine HCl denaturation and limited proteolysis. The guanidine HCl denaturation curve, as monitored by circular dichroism spectroscopy, was biphasic; the two transition midpoints occurred at 0.7 and 2.5 M guanidine HCl, indicating that there are stable intermediate structures in the unfolding of apoE. Limited proteolysis of apoE with five enzymes demonstrated two proteolytically resistant regions, an amino-terminal domain (residues 20-165) and a carboxyl-terminal domain (residues 225-299). The region between them was highly susceptible to proteolytic cleavage. Because of their similarity to the proteolytically resistant regions, the amino-terminal (residues 1-191) and carboxyl-terminal (residues 216-299) thrombolytic fragments of apoE were used as models for the two domains. Guanidine HCl denaturation of the carboxyl- and amino-terminal fragments gave transition midpoints of 0.7 and 2.4 M guanidine HCl, respectively. The results establish that the two domains identified by limited proteolysis correspond to the two domains detected by protein denaturation experiments. Therefore, the thrombolytic fragments are useful models for the two domains. The free energies of denaturation calculated from the denaturation curves of intact apoE or the model domains were approximately 4 and 8-12 kcal/mol for the carboxyl- and amino-terminal domains, respectively. The value for the carboxyl-terminal domain is similar to those of previously characterized apolipoproteins, whereas the value for the amino-terminal domain is considerably higher and resembles those of soluble globular proteins. These studies suggest that, in aqueous solution, apoE is unlike other apolipoproteins in that it contains two independently folded structural domains of markedly different stabilities: an amino-terminal domain and a carboxyl-terminal domain, separated by residues that may act as a hinge region.

Algorithms↗
PubMed1988

Human apolipoprotein E3 in aqueous solution. II. Properties of the amino- and carboxyl-terminal domains.

Hydrodynamic, chromatographic, and spectroscopic techniques were used to study the aqueous solution properties of the two structural domains of human apolipoprotein (apo) E3. An amino-terminal thrombolytic fragment of apoE (22 kDa, residues 1-191) and a carboxyl-terminal thrombolytic fragment of apoE (10 kDa, residues 216-299) were used as models for the two domains. Sedimentation equilibrium ultracentrifugation showed that apoE and the 10-kDa model domain self-associated predominantly as tetramers. The 22-kDa model domain was primarily monomeric. Molecular weights calculated from the weight average sedimentation and diffusion coefficients or from the sedimentation coefficients and Stokes radii were in agreement with the sedimentation equilibrium results. Derived frictional coefficients suggest larger axial ratios and/or more extensive hydration for the apoE and the 10-kDa domain tetramers as compared with the 22-kDa domain. Proteolysis of apoE followed by high performance liquid chromatography showed rapid production of free 22-kDa domain, whereas the free 10-kDa domain appeared as a tetramer late in the course of the hydrolysis. Assessment by circular dichroism demonstrated that both model domains and apoE had over 54% alpha-helical content, which changed little in a detergent (octyl-beta-D-glucopyranoside) or lipid (dimyristoylphosphatidylcholine) environment. In contrast to the circular dichroism results, apoE and the 10-kDa domain showed a marked blue shift in the fluorescence maximum in a lipid environment. The results suggest that the self-association of apoE in solution as a tetramer is mediated by the carboxyl-terminal domain and that the amino- and carboxyl-terminal domains do not associate with one another. The amino-terminal domain is most likely compact and globular, whereas the carboxyl-terminal domain is probably elongated. The isolated model domains appear to have structures that are similar to those of the domains in the intact protein.

Algorithms↗