A truncated species of apolipoprotein B (B67) in a kindred with familial hypobetalipoproteinemia.

We describe a kindred in which the proband and 6 of his 12 children have hypobetalipoproteinemia. The plasma lipoproteins of the affected subjects contained a unique species of apolipoprotein (apo) B, apo B67, in addition to the normal species, apo B100 and apo B48. The size of apo B67 and immunochemical studies with a panel of apo B-specific antibodies indicated that apo B67 was a truncated species of apo B that contained approximately the amino-terminal 3,000-3,100 amino acids of apo B100. Sequencing of genomic apo B clones revealed that affected family members were heterozygous for a mutant apo B allele containing a single nucleotide deletion in exon 26 (cDNA nucleotide 9327). This frameshift mutation is predicted to result in the synthesis of a truncated apo B containing 3,040 amino acids. Apo B67 is present in low levels in the plasma but is easily detectable within the very low density lipoprotein and low density lipoprotein fractions. Examination of the proband's immediate family revealed seven normolipidemic subjects and seven subjects with hypobetalipoproteinemia. In the affected subjects, the mean total and low density lipoprotein cholesterol levels were 120 and 42 mg/dl, respectively. A significantly higher mean high density lipoprotein cholesterol level was found in the affected subjects (75 vs. 55 mg/dl). We hypothesize that the elevated high density lipoprotein cholesterol levels in subjects heterozygous for the apo B67 mutation may be metabolically linked to the low levels of apo B-containing lipoproteins in their plasma.

Familial hypobetalipoproteinemia caused by a mutation in the apolipoprotein B gene that results in a truncated species of apolipoprotein B (B-31). A unique mutation that helps to define the portion of the apolipoprotein B molecule required for the formation of buoyant, triglyceride-rich lipoproteins.

Apolipoprotein B-100 has a crucial structural role in the formation of VLDL and LDL. Familial hypobetalipoproteinemia, a syndrome in which the concentration of LDL cholesterol in plasma is abnormally low, can be caused by mutations in the apo B gene that prevent the translation of a full-length apo B-100 molecule. Prior studies have revealed that truncated species of apo B [e.g., apo B-37 (1728 amino acids), apo B-46 (2057 amino acids)] can occasionally be identified in the plasma of subjects with familial hypobetalipoproteinemia; in each of these cases, the truncated apo B species has been a prominent protein component of VLDL. In this report, we describe a kindred with hypobetalipoproteinemia in which the plasma of four affected heterozygotes contained a unique truncated apo B species, apo B-31. Apolipoprotein B-31 is caused by the deletion of a single nucleotide in the apo B gene, and it is predicted to contain 1425 amino acids. Apolipoprotein B-31 is the shortest of the mutant apo B species to be identified in the plasma of a subject with hypobetalipoproteinemia. In contrast to longer truncated apo B species, apo B-31 was undetectable in the VLDL and the LDL; however, it was present in the HDL fraction and the lipoprotein-deficient fraction of plasma. The density distribution of apo B-31 in the plasma suggests the possibility that the amino-terminal 1425 amino acids of apo B-100 are sufficient to permit the formation and secretion of small, dense lipoproteins but are inadequate to support the formation of the more lipid-rich VLDL and LDL particles.

Phenotypes of apolipoprotein B and apolipoprotein E after liver transplantation.

Apolipoprotein (apo) E and the two B apolipoproteins, apoB48 and apoB100, are important proteins in human lipoprotein metabolism. Commonly occurring polymorphisms in the genes for apoE and apoB result in amino acid substitutions that produce readily detectable phenotypic differences in these proteins. We studied changes in apoE and apoB phenotypes before and after liver transplantation to gain new insights into apolipoprotein physiology. In all 29 patients that we studied, the postoperative serum apoE phenotype of the recipient, as assessed by isoelectric focusing, converted virtually completely to that of the donor, providing evidence that greater than 90% of the apoE in the plasma is synthesized by the liver. In contrast, the cerebrospinal fluid apoE phenotype did not change to the donor's phenotype after liver transplantation, indicating that most of the apoE in CSF cannot be derived from the plasma pool and therefore must be synthesized locally. The apoB100 phenotype (assessed with immunoassays using monoclonal antibody MB19, an antibody that detects a two-allele polymorphism in apoB) invariably converted to the phenotype of the donor. In four normolipidemic patients, we determined the MB19 phenotype of both the apoB100 and apoB48 in the "chylomicron fraction" isolated from plasma 3 h after a fat-rich meal. Interestingly, the apoB100 in the chylomicron fraction invariably had the phenotype of the donor, indicating that the vast majority of the large, triglyceride-rich apoB100-containing lipoproteins that appear in the plasma after a fat-rich meal are actually VLDL of hepatic origin. The MB19 phenotype of the apoB48 in the plasma chylomicron fraction did not change after liver transplantation, indicating that almost all of the apoB48 in plasma chylomicrons is derived from the intestine. These results were consistent with our immunocytochemical studies on intestinal biopsy specimens of organ donors; using apoB-specific monoclonal antibodies, we found evidence for apoB48, but not apoB100, in donor intestinal biopsy specimens.

The wing in yeast heat shock transcription factor (HSF) DNA-binding domain is required for full activity.

The yeast heat shock transcription factor (HSF) belongs to the winged helix family of proteins. HSF binds DNA as a trimer, and additional trimers can bind DNA co-operatively. Unlike other winged helix-turn-helix proteins, HSF's wing does not appear to contact DNA, as based on a previously solved crystal structure. Instead, the structure implies that the wing is involved in protein-protein interactions, possibly within a trimer or between adjacent trimers. To understand the function of the wing in the HSF DNA-binding domain, a Saccharomyces cerevisiae strain was created that expresses a wingless HSF protein. This strain grows normally at 30 degrees C, but shows a decrease in reporter gene expression during constitutive and heat-shocked conditions. Removal of the wing does not affect the stability or trimeric nature of a protein fragment containing the DNA-binding and trimerization domains. Removal of the wing does result in a decrease in DNA-binding affinity. This defect was mainly observed in the ability to form the first trimer-bound complex, as the formation of larger complexes is unaffected by the deletion. Our results suggest that the wing is not involved in the highly co-operative nature of HSF binding, but may be important in stabilizing the first trimer bound to DNA.

Relationship of apolipoprotein E phenotypes to hypocholesterolemia.

PURPOSE: Persons with total cholesterol (TC) levels less than 130 mg/dL (less than 3.26 mmol/L) make up less than 1% of a healthy population. Causes of hypocholesterolemia include a diet very low in cholesterol and saturated fat, disease, genetic factors (including low apolipoprotein B-100 [apo B-100] and the apo E allele), and drug therapy. The purpose of this study was to determine the causes of hypocholesterolemia in a healthy Kaiser Foundation Health Plan (KFHP) population. PATIENTS AND METHODS: We conducted a dietary and health survey of 201 healthy hypocholesterolemic adults (range: 2.04 to 3.88 mmol/L [79 to 150 mg/dL]) and 200 matched control subjects with TC levels in the middle quintile of the population (range: 5.0 to 5.61 mmol/L [194 to 217 mg/dL]) who had routine health screening from 1983 through 1985. We did apo E phenotyping studies and lipid and apo A-1 and B-100 measurements in a subgroup of 45 hypocholesterolemic subjects (mean TC level: 3.26 mmol/L [126 mg/dL]) and in a comparison group of 49 unmatched volunteers (mean TC level: 5.04 +/- 0.75 mmol/L [195 +/- 29 mg/dL]). RESULTS: We found no differences in dietary intake or clinically significant medical illness between hypocholesterolemic and control subjects. In the hypocholesterolemic subgroup, we found an increased frequency of the apo E2 allele (epsilon 2) and a decreased frequency of the apo E4 allele (epsilon 4); the frequencies of the epsilon 2, epsilon 3, and epsilon 4 alleles were 33.3%, 63.3%, and 3.3%, respectively. The corresponding apo E allele frequencies in the comparison subgroup were 8.2%, 73.5%, and 18.4%, similar to those previously reported for the general population and significantly different from those found in the hypocholesterolemic subgroup (p < 0.0001). One hypocholesterolemic subject (a 46th patient) had a mutation in the apo B gene that resulted in the synthesis of a truncated species of apo B (apo B-46). CONCLUSION: Our study indicates that hypocholesterolemia in our KFHP urban population is usually not caused by diet or disease. Biochemical factors, including the increased frequency of the apo E-2 phenotype and the decreased frequency of the apo E-4 phenotype, are more important.

An ApaLI restriction site polymorphism is associated with the MB19 polymorphism in apolipoprotein B.

An apolipoprotein (apo) B-specific monoclonal antibody, MB19, detects a commonly occurring two-allele genetic polymorphism in human apoB (Young, S. G., S. J. Bertics, L. K. Curtiss, D. C. Casal, and J. L. Witztum. 1986. Proc. Natl. Acad. Sci. USA. 83: 1101-1105). Antibody MB19 binds to two different allotypes of apoB, MB19(1) and MB19(2), with high and low affinity, respectively. The epitope for antibody MB19 is located within apoB-100 thrombolytic fragment T4 (apoB-100 amino acid residues 1-1297). In this study, we examined the relationship of the MB19 polymorphism to a C----T nucleotide substitution at apoB cDNA nucleotide 421. This nucleotide substitution results in a Thr----Ile substitution at apoB-100 amino acid 71, and it changes an ApaLI restriction endonuclease site in the apoB gene. The nucleotide substitution was easily detectable by ApaLI digestion of a 141-base pair fragment of the apoB gene obtained by enzymatic amplification of genomic DNA. In 62 subjects, the MB19 phenotype, as determined by radioimmunoassays, correlated perfectly with the ApaLI restriction site polymorphism in the amplified DNA. The apoB allotype MB19(1) is associated with an Ile at residue 71 and the absence of the ApaLI site, whereas the apoB allotype MB19(2) is associated with a Thr at residue 71 and the presence of the ApaLI site. We conclude that the amino acid substitution at residue 71 probably accounts for the MB19 polymorphism in apoB.

Mutational analysis of the DNA-binding domain of yeast heat shock transcription factor.

Both randomized oligonucleotide cassette mutagenesis and site-directed mutagenesis have been used in combination with a yeast genetic screen to identify critical residues in the DNA-binding domain of heat shock transcription factor from Saccharomyces cerevisiae. Most of the surface residues in this highly conserved domain can be changed to alanine with no observable effect on function. Of nine critical residues identified in this screen, five are within helix alpha 3, previously designated as the probable DNA recognition helix in the crystal structure of the Kluyveromyces lactis protein. The other four residues may be involved in DNA-binding or protein-protein interactions.

The DNA-binding domain of yeast heat shock transcription factor independently regulates both the N- and C-terminal activation domains.

The expression of heat shock proteins in response to cellular stresses is dependent on the activity of the heat shock transcription factor (HSF). In yeast, HSF is constitutively bound to DNA; however, the mitigation of negative regulation in response to stress dramatically increases transcriptional activity. Through alanine-scanning mutagenesis of the surface residues of the DNA-binding domain, we have identified a large number of mutants with increased transcriptional activity. Six of the strongest mutations were selected for detailed study. Our studies suggest that the DNA-binding domain is involved in the negative regulation of both the N-terminal and C-terminal activation domains of HSF. These mutations do not significantly affect DNA binding. Circular dichroism analysis suggests that a subset of the mutants may have altered secondary structure, whereas a different subset has decreased thermal stability. Our findings suggest that the regulation of HSF transcriptional activity (under both constitutive and stressed conditions) may be partially dependent on the local topology of the DNA-binding domain. In addition, the DNA-binding domain may mediate key interactions with ancillary factors and/or other intramolecular regulatory regions in order to modulate the complex regulation of HSF's transcriptional activity.

Lipoproteins in pinnipeds: analysis of a high molecular weight form of apolipoprotein E.

We have examined the apolipoprotein content of the lipoproteins of several marine mammals by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Their apolipoprotein (apo) B-100, apoB-48, and apoA-I migrated to virtually the same position as the comparable human apolipoproteins. In cetaceans (bottlenose dolphins and killer whales), the molecular mass of the apoE was identical to that of human apoE (35 kDa). In contrast, in the lipoproteins of pinnipeds (harbor seals, sea lions, and walrus) there was no protein comparable in size to human apoE; however, there were two proteins in the 40- to 44-kDa range. The protein with the higher apparent molecular weight (44 kDa) was apoA-IV, as determined by NH2-terminal amino acid sequencing. Sequencing of the NH2-terminal 15 amino acids of the lower molecular weight protein (40-42 kDa) revealed no obvious homology with human apoE. However, a human apoE-specific monoclonal antibody, 1D7, bound to the 40- to 42-kDa protein, allowing us to identify that protein as apoE. Sequencing of sea lion apoE cDNA clones demonstrated that sea lion apoE is 311 amino acids in length, 12 residues longer than human apoE. All 12 additional residues are located in the NH2-terminal 31 amino acids, a region that has extremely low homology with the NH2-terminal portion of human apoE. The remainder of the sea lion apoE sequence is 74% homologous to human apoE. The sea lion apoE cDNA was expressed in Chinese hamster ovary (CHO) cells as well as CHO ldlD cells, a cell line that is deficient in O-glycosylation of proteins. The size of the sea lion apoE secreted by these two cell lines, compared with the apoE in sea lion plasma, indicated that the predominant form of apoE in sea lion plasma must be posttranslationally modified. Thus, our studies have demonstrated that the higher apparent molecular weight of pinniped (sea lion) apoE is due to a longer polypeptide chain as well as posttranslational modification of the protein.