Apolipoprotein CII (apo CII) gene expression defect in an individual with familial apo CII deficiency

We have examined the expression of the apolipoprotein CII (apo CII) gene in an individual with familial apo CII (apo CII) deficiency. Total RNA was prepared from this patient's liver tissue and analysed in Slot Blot and Northern Blot experiments using a cloned apo CII cDNA as a probe. In this patient, there is at least a four-fold decrease in the level of apo CII mRNA, when compared to liver tissue from a control individual. The residual apo CII mRNA detected in this patient is of normal length. These results suggest that the failure to detect apo CII protein in this patient's serum is not due to a failure to transcribe or process apo CII mRNA, but probably to a defect in the translation of the apo CII message. This defect results in partial degradation of the apo CII message leading to the much reduced levels which we have observed.     

Polymorphisms in the apolipoprotein (apo) AI-CIII-AIV gene cluster: detection of genetic variation determining plasma apo AI,apo CIII and apo AIV concentrations

We have examined the associations between levels of plasma apolipoprotein (apo) AI, apo CIII and apo AIV and genetic variation in the apo AI-CIII-AIV gene cluster in 162 boys and young men from Belgium aged from 7 to 23 years. Genotypes were determined for six restriction enzymes XmnI, PstI, SstI, PvuIIA-CIII, PvuIIB-AIV and XbaI, and for the G to A substitution at -75 bp in the 5' region of the apo AI gene. The polymorphism most strongly associated with apo AI levels was the G to A substitution (P = 0.025, R2 x 100 = 3.6%) confirming previous observations. The polymorphism most strongly associated with apo CIII levels was that of PvuIIA-CIII (P = 0.023, R2 x 100 = 2.9%) in the apo CIII gene. This novel association must be interpreted with caution until it has been confirmed in an independent sample. The polymorphism associated with the largest effect on apo AIV levels was that detected with XbaI in the apo AIV gene, but this association was not statistically significant. Previously reported associations between the SstI polymorphism and triglyceride levels, and between the PstI polymorphism and apo AI levels, were weakly detected in the present sample. Our results show that variation associated with some of the polymorphisms in the apo AI-CIII-AIV cluster makes a small, but statistically significant, contribution to the determination of apo AI and apo CIII levels in this sample of young men and boys. These observations may, in part, explain reported associations between polymorphisms in this gene cluster, differences in plasma lipid and lipoprotein levels, and prevalence of coronary artery disease.     

There are two gene sequences for human apolipoprotein CI (apo CI) on chromosome 19, one of which is 4 kb from the gene for apo E

A cDNA probe corresponding to the mRNA sequence for apolipoprotein E (apo E) was used to screen two independently-constructed human genomic libraries. Two recombinants (lambda E-2, and lambda E2-1), isolated using the apo E cDNA probe, also contain part or all of the apo CI gene. Hybridisation studies using both apo E and apo CI cDNA probes show that these two genes are in the same orientation and separated by 4 kb.     

Familial apolipoprotein CII deficiency: A preliminary analysis of the gene defect in two independent families

Summary We have used a cDNA clone for human apolipoprotein CII (apo CII) to study the apo CII genes in two independent individuals with familial apo CII deficiency. With all the restriction enzymes so far used, gene fragments hybridising with apo CII cDNA are observed that are indistinguishable from normal samples. This demonstrates that in neither of these individuals is the defect due to a major deletion of DNA in or around the apo CII gene. We have used a common polymorphism of the apo CII gene detected with the enzyme TaqI to follow the inheritance of the gene in the families of these apo CII deficient individuals. The pattern of inheritance that we observe is consistent with the defect causing apo CII deficiency being in, or closely linked to the apo CII structural gene.     

Expression of normal and mutagenized apolipoprotein CII in procaryotic cells. Structure-function relationship

A full-length human apo CII cDNA clone has been constructed by completing the 5' end of an incomplete cDNA with a 44 bp long synthetic oligonucleotide. This apo CII cDNA insert was cloned into the pSP19 expression vector and transcribed and translated in vitro. Its N-terminal signal sequence (23 amino-acid residues) was accurately cleaved during cotranslational translocation through endoplasmic reticulum membranes to yield the mature apo CII. Mature apo CII was expressed on a preparative scale as fusion protein apo CII-beta-galactosidase with the full-length apo CII cDNA integrated into the pUR291 vector. Furthermore it was expressed in E. coli transformed with the pKK233-2 apo CII clone. The preform was accurately processed by the host cell. C-Terminal apo CII deletion mutants generated by partial Bal31 digestion of the pKK233-2 apo CII vector yielded well-defined truncated apo CII polypeptides on a preparative scale which allowed the determination of the polypeptide domain responsible for the activation of the serum lipoprotein lipase.     

Site-directed mutagenesis of an apolipoprotein E mutant, apo E5(Glu3----Lys) and its binding to low density lipoprotein receptors.

Apo E5(Glu3----Lys) is a naturally occurring apolipoprotein E (apo E) mutant found in patients with hyperlipoproteinemia and atherosclerosis. It has been shown to have a high affinity for low density lipoprotein (LDL) receptors. In this study, mutant apo E5 was produced by Chinese hamster ovary cells by means of an in vitro site-directed mutagenesis technique, and its LDL receptor binding activity was assessed. The apo E5 obtained from gene expression bound more readily to the LDL receptor than did plasma apo E3. The concentrations required for 50% competitive binding of 125I-labeled LDL to the LDL receptors were 58.9 ng/ml for plasma apo E3 and 25.7 ng/ml for the expressed apo E5. The expressed apo E5 displayed 229% normal binding. This result is highly consistent with that obtained with plasma apo E5, which showed 217% normal binding. Although the experimental apo E isoproteins contained more sialic acid than plasma apo E, the extent of sialylation had no effect on the receptor binding of apo E.     

Variation at the apolipoprotein (apo) AI-CIII-AIV gene cluster and apo B gene loci is associated with lipoprotein and apolipoprotein levels in Italian children.

We have used RFLPs of the apolipoprotein (apo) B gene and apo AI-CIII-AIV gene cluster to estimate the genetic contribution of variation at these loci to the variability of plasmid lipid, lipoprotein, and apolipoprotein levels in 209 children from Sezze in central Italy. The sample was randomly divided into group I (107 children) and group II (102 children). Four site polymorphisms (PvuII, XbaI, MspI, and EcoRI) of the apo B gene and five site polymorphisms (XmnI, PstI, SstI, PvuII-CIII, and PvuII-AIV) of the apo AI-CIII-AIV gene cluster were examined in group I children. After adjustment for gender, age, and body-mass index, polymorphisms at both gene loci (PvuII-B, PvuII-CIII, and PvuII-AIV) were associated with significant effects on the levels of plasma apo AI, apo B, or high-density lipoprotein-cholesterol. RFLPs that showed significant effects in group I were genotyped in group II. All three polymorphisms were associated with similar effects on apolipoprotein levels, though for all RFLPs the magnitude of the effects was smaller in the group II children and only statistically significant for the effect of the PvuII-B genotype on apo AI levels. In the total sample of 209 children 7.4% of the sample variance in apo AI levels was explained by variation associated with the apo B PvuII-B RFLP. In addition, the PvuII-B RFLP was associated with significant effects on plasma apo B levels and explained 5.7% of the sample variance. The PvuII-CIII and PvuII-AIV polymorphisms were both associated with differences in apo AI levels, explaining 3.7%-5.7% of the sample variance. Taken together, the three PvuII polymorphisms explained 17.7% of the phenotypic variance in apo AI levels. There was significant evidence for an effect of nonlinearity of the PvuII-CIII genotypes on apo AI levels, with the individuals heterozygous for the polymorphism having the highest apo AI levels. No evidence of interaction between genotype and gender, age, and body-mass index was shown by covariance analysis. The molecular explanation of this effect is unclear. Our data show that variation at both the apo AI-CIII-AIV and apo B loci are associated with lipoprotein and apolipoprotein levels in this sample of Italian children.     

ApoE genotype affects allele-specific apo[a] levels for large apo[a] sizes in African Americans: the Harlem-Basset Study

The genetic variability of apolipoprotein E (apoE) influences plasma lipoprotein levels, and allele frequencies differ between African Americans and Caucasians. As African Americans have higher lipoprotein [a] (Lp[a]) levels than Caucasians, we investigated the effects of the apoE gene on allele-specific apolipoprotein [a] (apo[a]) levels across ethnicity. We determined apo[a] sizes, allele-specific apo[a] levels (i.e., levels associated with alleles defined by size), and the apoE gene polymorphism in 231 African Americans and 336 Caucasians. African Americans, but not Caucasians, with the apo E2 genotype had lower levels of Lp[a] compared with those with the apo E4 genotype (9.6 vs. 11.2 nmol/l; P = 0.034, expressed as square root levels). Distribution of apo[a] alleles across apoE genotypes were similar between African Americans and Caucasians. Among African Americans with large apo[a], the allele-specific apo[a] level was significantly lower among epsilon2 carriers compared with epsilon3 or epsilon4 carriers (5.4 vs. 6.6 and 7.4 nmol/l, respectively; P < 0.005, expressed as square root levels). In contrast, there was no significant difference in allele-specific apo[a] levels across apoE genotypes among Caucasians. For large apo[a] sizes, apoE genotype contributed to the observed African American-Caucasian differences in allele-specific apo[a] levels.     

The isolation of a genomic clone containing the apolipoprotein CII gene and the detection of linkage disequilibrium between two common DNA polymorphisms around the gene

Summary We report the isolation of a genomic clone containing the apolipoprotein CII (apo CII) gene and 5 and 3 flanking sequences. A detailed restriction map of the gene has been constructed and DNA fragments that are unique, or low copy number sequences in the genome have been identified. One of these detects a common restriction fragment length polymorphism (RFLP) with the enzyme BglI. Marked linkage disequilibrium is observed between this RFLP and that detected with the apo CII cDNA clone, even though the two variable restriction enzyme sites are approximately 12 kb apart. However, the usefulness of the apo CII gene as a marker for linkage studies is increased by the use of both RFLPs.     

Association of methylenetetrahydrofolate (MTHFR) and apolipoprotein E (apo E) genotypes with homocysteine, vitamin and lipid levels in carotid stenosis

The aim of the study was to investigate the association between methylenetetrahydrofolate (MTHFR) genotypes and levels of homocysteine (Hcy), folate, vitamin B12 and lipids as well as the association between apolipoprotein E (apo E) genotypes and levels of lipids in a Croatian healthy control group and a group of patients with > 70% carotid stenosis (CS). The study included 98 Croats, 38 patients with > 70% carotid stenosis and 60 age- and sex-matched controls. The MTHFR and apo E genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), Hcy by enzyme immunoassay, vitamins by immunochemiluminiscence, and lipids by spectrophotometric method. There was no difference between control subjects and CS patients in the distribution of C677T MTHFR genotypes (p=0. 786) and alleles (p=0.904), however, differences in the frequencies of apo E genotypes (p=0.012) and alleles (p=0.029) were statistically significant. The odds ratio for apo E 3/4 genotype was 3.93 (95% CI 1.23-12.61). Hyperhomocysteinemia (> or =15 micromol/L) was found in 11% of CS patients and 5% of control subjects. Total cholesterol, triglycerides, vitamin B12 and folate were statistically different in "all MTHFR genotypes" (p<0.001, p<0.01, p=0.044 and p=0.036, respectively), and in TC/TT (p<0.001, p=0.003, p=0.030 and p=0.032, respectively) groups. The levels of total cholesterol, LDL cholesterol and triglycerides in the apo E 3/3, and total cholesterol in the apo E 3/4 group yielded statistical difference. An association was found of apo E 3/4 genotype but not of MTHFR genotypes with the risk of CS. MTHFR and apo E affect blood lipid levels, which was statistically confirmed. An association was also recorded between hyperhomocysteinemia and patients with CS. Vitamin status in CS showed a statistically verified association with TC/TT MTHFR genotype. In the group of patients with TC/TT MTHFR genotype, lower vitamin B12 and higher folate values were recorded. The results of multiple logistic analysis showed that there was no statistical significance of Hcy levels (OR 2.403, p=0.334) or conventional vascular risk factors such as smoking habit (OR 0.505, p=0.149), age (OR 1.048, p=0.087) or sex (OR 2.037, p=0.112) in predicting CS.     

The serum apo B and apo E in rats following cholesterol diet and thymus treatment

The serum levels of apo B and apo E in rats fed on a diet rich in cholesterol before and after thymus treatment were determined by the authors. The diet enriched with cholesterol increases the serum levels of apo E and of the large and small species of apo B. After treatment the large apo B and the small one strongly decrease, while apo E increases further. These data support the hypothesis that the dropping of total cholesterol, after thymus treatment, cannot be ascribed to apo E decrease but possibly to B-apoproteins.     

Apolipoprotein E1 Lys-146----Glu with type III hyperlipoproteinemia.

During the screening of samples obtained from 5 individuals with type III hyperlipidemia, we identified a variant of apolipoprotein (apo) E which exhibited a discrepancy in apo E phenotype showing the E3/E1 isoform on isoelectric focusing (IEF) analysis and E3/E3 on gene analysis. Sequence analysis of the DNA of the proband that was amplified by PCR and subcloned, revealed a single substitution of one lysine (AAG) for one glutamic acid (GAG) at position 146, thereby adding two negatively charged units to apo E3. This defect had been described only for apo E1 to date (Mann et al. (1989) Clin. Res. 37, 520A (abstract)). In this case, PCR-mediated site-directed mutagenesis was used to identify the structural alterations forming the abnormal E1 genotype in the proband's family. Purified apo E1 Lys-146----Glu showed less than 10% of binding activity to apo B, E receptor on human skin fibroblasts compared with apo E3. This substitution demonstrates that Lys-146 is essential for the binding of apo E to the receptor.     

Two coding regions closely linked to the rat apolipoprotein E gene: nucleotide sequences of rat apolipoprotein C-I and ECL cDNA.

Restriction fragments isolated from a 17-kb rat genomic DNA clone containing the gene for apolipoprotein (apo) E were radiolabeled and used to screen a rat liver cDNA library. A cDNA clone hybridizing to a 6-kb genomic DNA fragment was isolated and the nucleotide sequence of the cDNA insert determined. The sequence was homologous to the sequence for human apo C-I and was used to derive the corresponding amino acid sequence. Unlike human apo C-I, mature rat apo C-I contains histidine, lacks valine, and has alanine at the C terminus and aspartate as the N terminus. Screening the rat liver cDNA library with a radiolabeled 1.9-kb restriction fragment from the genomic DNA clone containing the rat apo E gene identified another cDNA clone (ECL cDNA). Nucleotide sequencing yielded a derived 75-amino-acid sequence for the ECL protein with a hydrophobicity profile similar to that of rat apo C-I. Northern analysis demonstrated a 0.50-kb band for ECL mRNA. The tissue-specific expression of the gene is similar to that of rat apo C-I. This study indicates that the rat apo C-I and ECL genes are closely linked, about 4.5 and 12 kb downstream of the apo E gene, respectively.     

T cell immunity to type II collagen in the biobreeding rat: the identification and characterization of RT1u-restricted T cell epitopes on alpha 1(II)

Susceptibility to experimental collagen-induced arthritis in rodents is dependent on MHC class II elements to bind peptides from the type II collagen (CII) molecule. Although a substantial body of data has been reported in mice defining these peptide Ags, little has been reported in rats. In this study, we investigate the locations and sequences of CII peptides, which are bound by RT1(u) molecules, expressed by diabetic-resistant, arthritis-susceptible Biobreeding rats, and, in turn, stimulate CII-specific T cells. By using overlapping and substituted peptide homologues of CII, we have identified and characterized an immunodominant and five subdominant epitopes on CII, which stimulate RT1(u)-restricted T cell proliferation. The immunodominant epitope, CII (186-192), contains a QGPRG core sequence, which was found in a subdominant epitope CII (906-916). Similar sequences containing single conservative substitutions were identified in three other epitopes. One, CII (263-272), contained a conservatively substituted R-->K substitution, whereas CII (880-889) and CII (906-916) contained nonconservative substitutions, i.e., P-->D and R-->M, respectively. Homologue peptides containing these sequences stimulated T cell proliferative responses, although less intensely than peptides containing CII (186-192). Substituting QGR residues in the QGPRG core with alanine, isoleucine, or proline reduced proliferation, as did substituting flanking E and G residues at the N terminus and E at the C terminus. Collectively, these data indicate that RT1(u)-restricted immunodominant and several subdominant epitopes on CII often share a QGPRG-like motif, with conservative substitutions present at either P or R positions. This motif is similar to one recognized by collagen-induced arthritis-susceptible HLA-DR1- and HLA-DR4-transgenic mice.     

Apolipoprotein E genotyping in Turkish myocardial infarction survivors and healthy controls

The apolipoprotein (Apo) E gene is known to be polymorphic. Three common alleles determine six phenotypes which can easily be detected by restriction fragment length polymorphism. We performed apo E genotyping in myocardial infarction survivors and healthy controls for the first time in the Turkish population. DNA was amplified by polymerase chain reaction (PCR) and the PCR product was digested with restriction enzymesHhaI to detect apo E2, E3, E4 and withTaqI to detect apo E1. Relative allele frequency for the patient group was found to be 0.91 for E3, 0.07 for E2, 0.02 for E4 and for the control group 0.875 for E3, 0.067 for E2, 0.058 for E4.     

Isolation and characterization of two sub-species of Lp(a), one containing apo E and one free of apo E.

Lipoprotein Lp(a) was isolated by immunoaffinity chromatography using anti apolipoprotein B and anti apolipoprotein (a) immunosorbents. Besides apolipoproteins (a) and B, this fraction was shown to contain apolipoproteins C and E. Therefore, it was decided to further purify this crude Lp(a) into particles containing apolipoprotein E and particles free of apo E, using chromatography with an anti apolipoprotein E immunosorbent. Lp(a), free of apolipoprotein E was cholesterol ester rich and triacylglycerol poor and was found mainly in the LDL size range. In contrast, Lp(a) containing apolipoprotein E was triacylglycerol rich and was distributed mainly in the VLDL and IDL size range. Binding of these two fractions, one containing apo E and one free of it, to the apo B/E receptor of HeLa cells was studied. Both fractions bound to the receptor but the one containing apo E had a better affinity than the one free of apo E. Further studies are needed to identify the clinical importance of these two different entities.     

Crystal structures of the apo and ATP bound Mycobacterium tuberculosis nitrogen regulatory PII protein

PII constitutes a family of signal transduction proteins that act as nitrogen sensors in microorganisms and plants. Mycobacterium tuberculosis (Mtb) has a single homologue of PII whose precise role has as yet not been explored. We have solved the crystal structures of the Mtb PII protein in its apo and ATP bound forms to 1.4 and 2.4 Å resolutions, respectively. The protein forms a trimeric assembly in the crystal lattice and folds similarly to the other PII family proteins. The Mtb PII:ATP binary complex structure reveals three ATP molecules per trimer, each bound between the base of the T‐loop of one subunit and the C‐loop of the neighboring subunit. In contrast to the apo structure, at least one subunit of the binary complex structure contains a completely ordered T‐loop indicating that ATP binding plays a role in orienting this loop region towards target proteins like the ammonium transporter, AmtB. Arg38 of the T‐loop makes direct contact with the γ‐phosphate of the ATP molecule replacing the Mg²⁺ position seen in the Methanococcus jannaschii GlnK1 structure. The C‐loop of a neighboring subunit encloses the other side of the ATP molecule, placing the GlnK specific C‐terminal 3₁₀ helix in the vicinity. Homology modeling studies with the E. coli GlnK:AmtB complex reveal that Mtb PII could form a complex similar to the complex in E. coli. The structural conservation and operon organization suggests that the Mtb PII gene encodes for a GlnK protein and might play a key role in the nitrogen regulatory pathway.     

Primary structure of the bovine analogues to human apolipoproteins CII and CIII. Studies on isoforms and evidence for proteolytic processing

Two major isoforms of the bovine analogue to human apolipoprotein (apo) CII were purified from plasma. They were both as effective as human apo CII in activating lipoprotein lipase. Amino acid sequencing revealed that one form contained 79 amino acid residues, and corresponded to human pro apo CII. The other form lacked the first six residues at its N-terminus. This was apparently due to cleavage of the -Gln-Asp- linkage in the sequence H2N-Ala-His-Val-Pro-Gln-Gln-Asp-Glu-, analogous to cleavages described for human apo AI and apo CII. Previous studies with human apo CII have shown that the ability to activate lipoprotein lipase resides in the C-terminal third of the molecule. This was highly conserved in the bovine analogue: of the 30 last residues, 21 are identical. Five residues in this part of human apo CII have been reported to be essential for activation of lipoprotein lipase. Only one of these, Tyr63, is present in the bovine sequence. The bovine structure contains a threonine at position 61, instead of serine in the human, and the four last residues are -Ser-Gly-Lys-Asp instead of the allegedly necessary -Lys-Gly-Glu-Glu. Three differently sialylated isoforms of the bovine analogue to human apolipoprotein CIII were also isolated and partially sequenced. All three lacked the first three N-terminal residues as compared to sequences from other species (man, dog and rat). Sequence differences were more pronounced at the ends than in the central parts of the apo CIII molecules.